阅读说明：本技术 用于治疗或预防恶心的类胡萝卜素 (Carotenoids for use in the treatment or prevention of nausea ) 是由 加里·B·鲁夫昆 约提·阿马纳特·戈文丹 埃拉姆帕里蒂·贾亚马尼 于 2019-05-10 设计创作，主要内容包括：本公开提供了与恶心和/或呕吐的治疗有关的技术,并且在一些实施方式中特别涉及诱发的(例如化疗诱发的)恶心和/或呕吐。可替代地,或另外地,在一些实施方式中,提供了与一种或多种进食障碍(例如神经性厌食症)的治疗或预防有关的技术。本文还提供了用于口服递送的治疗组合物,所述治疗组合物包含治疗有效量的C50类胡萝卜素化合物和药学上可接受的载体。(The present disclosure provides techniques related to the treatment of nausea and/or vomiting, and in some embodiments specifically to induced (e.g., chemotherapy-induced) nausea and/or vomiting. Alternatively, or additionally, in some embodiments, techniques are provided relating to the treatment or prevention of one or more eating disorders (e.g., anorexia nervosa). Also provided herein are therapeutic compositions for oral delivery comprising a therapeutically effective amount of a C50 carotenoid compound and a pharmaceutically acceptable carrier.)

1. A method for treating nausea and/or vomiting in a subject, the method comprising: administering to a subject in need thereof a therapeutically effective amount of a C50 carotenoid compound.

2. The method of claim 1, wherein the subject has or is at risk of developing nausea and/or vomiting associated with chemotherapy or radiotherapy.

3. The method of claim 1 or2, wherein the subject has or is at risk of developing chemotherapy-induced nausea and vomiting (CINV) or radiotherapy-induced nausea and vomiting (RINV).

4. The method of claim 1, wherein the subject has or is at risk of developing postoperative nausea and vomiting (PONV).

5. The method of any one of claims 1-4, wherein said C50 carotenoid compound is selected from the group consisting of: decaprenylflavin, C50-astaxanthin, C50-beta-carotene, C50-carotene (n ═ 3) (16, 16-diisopentenyl phytoene), C50-zeaxanthin, C50-xanthophyll, C50-cryptoxanthin, sarcinaxanthin, sarprenoxanthin, acyclic C50 carotenoid bacterin, C50-canthaxanthin, C50-lycopene, C50-phytoene, and combinations thereof.

6. The method of claim 5, wherein said C50 carotenoid compound is decaprenylflavin.

7. The method of any one of claims 1-6, wherein the step of administering comprises administering a composition that is or comprises: (i) c50-carotenoid-compound-synthetic microorganism or component thereof, (ii) C50-carotenoid-compound-synthetic microorganism extract, (iii) extracted C50-carotenoid compound, or (iv) combinations thereof.

8. The method of claim 7, wherein said C50-carotenoid-compound-synthesizing microorganism is viable or viable.

9. The method of claim 8, wherein the step of administering comprises administering a sufficient amount of the microorganism to colonize a microbiome of the subject.

10. The method of any one of claims 7-9, wherein the composition comprises or is prepared from a culture of the microorganism.

11. The method of claim 10, wherein the microorganism is a strain found in nature.

12. The method of any one of claims 7-9 or 10, wherein the microorganism is an engineered microorganism.

13. The method of claim 12, wherein said engineered microorganism comprises genetic alterations relative to other comparable reference microorganisms such that said engineered microorganism produces said C50 carotenoid compound at absolute or relative levels different from said reference microorganism.

14. The method of any one of claims 1-13, wherein the step of administering comprises administering a composition comprising or delivering a synthetic C50 carotenoid compound.

15. The method of claim 7, wherein said C50-carotenoid-compound-synthetic microorganism is selected from the group consisting of Cockera rhizophila, Corynebacterium glutamicum, Arthrobacter research team, and combinations thereof.

16. A method for reducing anorexia in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a C50 carotenoid compound.

17. The method of claim 16, wherein the subject has or is at risk of developing nausea and vomiting associated with chemotherapy or radiation therapy.

18. The method of claim 16 or 17, wherein the subject has or is at risk of developing chemotherapy-induced nausea and vomiting (CINV) or radiotherapy-induced nausea and vomiting (RINV).

19. The method of claim 16, wherein the subject has or is at risk of developing postoperative nausea and vomiting (PONV).

20. The method of any one of claims 16-19, wherein said C50 carotenoid compound is selected from the group consisting of: decaprenylflavin, C50-astaxanthin, C50-beta-carotene, C50-carotene (n ═ 3) (16, 16-diisopentenyl phytoene), C50-zeaxanthin, C50-xanthophyll, C50-cryptoxanthin, sarcinaxanthin, sarprenoxanthin, acyclic C50 carotenoid bacterin, C50-canthaxanthin, C50-lycopene, C50-phytoene, and combinations thereof.

21. The method of claim 20, wherein said C50 carotenoid compound is decaprenylflavin.

22. The method of any one of claims 16-21, wherein the step of administering comprises administering a composition that is or comprises: (i) c50-carotenoid-compound-synthetic microorganism or component thereof, (ii) C50-carotenoid-compound-synthetic microorganism extract, (iii) extracted C50-carotenoid compound, or (iv) combinations thereof.

23. The method of claim 22, wherein said C50-carotenoid-compound-synthesizing microorganism is viable or viable.

24. The method of claim 23, wherein the step of administering comprises administering a sufficient amount of the microorganism to colonize a microbiome of the subject.

25. The method of any one of claims 22-24, wherein the composition comprises or is prepared from a culture of the microorganism.

26. The method of claim 25, wherein the microorganism is a strain found in nature.

27. The method of any one of claims 22-24, wherein the microorganism is an engineered microorganism.

28. The method of claim 27, wherein said engineered microorganism comprises genetic alterations relative to other comparable reference microorganisms such that said engineered microorganism produces said C50 carotenoid compound at absolute or relative levels different from said reference microorganism.

29. A method according to any one of claims 16-28, wherein said step of administering comprises administering a composition comprising or delivering a synthetic C50 carotenoid compound.

30. The method of claim 22, wherein said C50-carotenoid-compound-synthetic microorganism is selected from the group consisting of corynebacterium rhizophilum, corynebacterium glutamicum, arthrobacter research team, and combinations thereof.

31. A therapeutic composition for oral delivery comprising a therapeutically effective amount of a C50 carotenoid compound and a pharmaceutically acceptable carrier.

32. The therapeutic composition of claim 31 wherein the composition comprises a microorganism that synthesizes a C50 carotenoid compound.

33. The therapeutic composition of claim 32, wherein the microorganism is a cultured microorganism.

34. The therapeutic composition of claim 33, wherein the microorganism is an engineered microorganism.

35. The therapeutic composition of claim 34, wherein the engineered microorganism comprises genetic alterations relative to other comparable reference microorganisms such that the engineered microorganism produces the C50 carotenoid compound at absolute or relative levels that are different from the reference microorganism.

36. The therapeutic composition of any one of claims 31-34, which is a liquid, syrup, tablet, troche, fondant, capsule, powder, gel, or film.

37. The therapeutic composition of any one of claims 31-36, wherein the C50 carotenoid compound comprises at least 20 w/w% of the composition.

38. The therapeutic composition of any one of claims 31-37, wherein the C50 carotenoid compound is purified.

39. The therapeutic composition of any one of claims 31-38, wherein the C50 carotenoid compound has a chemical structure found in nature.

40. The therapeutic composition of any one of claims 31-39, wherein the C50 carotenoid compound is an analog of a reference C50 carotenoid compound found in nature.

41. The therapeutic composition of any one of claims 31-40, wherein the C50 carotenoid compound is selected from the group consisting of: decaprenylflavin, C50-astaxanthin, C50-beta-carotene, C50-carotene (n ═ 3) (16, 16-diisopentenyl phytoene), C50-zeaxanthin, C50-xanthophyll, C50-cryptoxanthin, sarcinaxanthin, sarprenoxanthin, acyclic C50 carotenoid bacterin, C50-canthaxanthin, C50-lycopene, C50-phytoene, and combinations thereof.

42. The therapeutic composition of claim 41, wherein said C50 carotenoid compound is decaprenylflavin.

43. The therapeutic composition of any one of claims 31-42, wherein the microorganism is viable or viable.

44. The therapeutic composition of any one of claims 31-43, wherein the microorganism has been killed.

45. The therapeutic composition of any one of claims 31-44, wherein the microorganism is selected from the group consisting of Cockera rhizophila, Corynebacterium glutamicum, Arthrobacter research team, and combinations thereof.

46. A method for manufacturing the therapeutic composition of any one of claims 31-45, the method comprising the steps of:

combining a pharmaceutically acceptable carrier with a C50 carotenoid compound; and

formulating said combination into said therapeutic composition.

47. The method of claim 46, wherein said combining step comprises combining said pharmaceutically acceptable carrier with a microorganism that synthesizes said C50 carotenoid compound.

48. The method of claim 46, wherein said combining step comprises combining said pharmaceutically acceptable carrier with a chemically synthesized C50 carotenoid compound.

49. A method for treating nausea and/or vomiting in a subject, the method comprising the steps of:

administering to a subject in need thereof: (i) c50-carotenoid-compound-synthetic microorganism or component thereof, (ii) C50-carotenoid-compound-synthetic microorganism extract, (iii) extracted C50-carotenoid compound, or (iv) combinations thereof.

50. The method of claim 49, wherein the subject has, or is at risk of developing, nausea and/or vomiting associated with chemotherapy or radiation therapy.

51. The method of claim 49 or 50, wherein the subject has or is at risk of developing chemotherapy-induced nausea and vomiting (CINV) or radiotherapy-induced nausea and vomiting (RINV).

52. The method of claim 49, wherein the subject has or is at risk of developing postoperative nausea and vomiting (PONV).

53. The method of any one of claims 49-52, wherein said C50 carotenoid compound-synthesizing microorganism is selected from the group consisting of Coccocus radiculosa, Corynebacterium glutamicum, Arthrobacter research team, and combinations thereof.

54. A method for alleviating anorexia in a subject, comprising the steps of:

administering to a subject in need thereof: (i) c50-carotenoid-compound-synthetic microorganism or component thereof, (ii) C50-carotenoid-compound-synthetic microorganism extract, (iii) extracted C50-carotenoid compound, or (iv) combinations thereof.

55. The method of claim 54, wherein the subject has or is at risk of developing nausea and vomiting associated with chemotherapy or radiation therapy.

56. The method of claim 54 or 55, wherein the subject has or is at risk of developing chemotherapy-induced nausea and vomiting (CINV) or radiotherapy-induced nausea and vomiting (RINV).

57. The method of claim 54, wherein the subject has or is at risk of developing postoperative nausea and vomiting (PONV).

58. The method of any one of claims 54-57, wherein said C50 carotenoid compound-synthesizing microorganism is selected from the group consisting of Coccocus radiculosa, Corynebacterium glutamicum, Arthrobacter research team, and combinations thereof.

Use of a C50 carotenoid compound for the treatment of nausea and/or vomiting in a subject in need thereof.

Use of C50-a carotenoid-compound-a synthetic microorganism for the treatment of nausea and/or vomiting in a subject in need thereof.

61. The use according to claim 60, wherein said C50 carotenoid compound-synthesizing microorganism is selected from the group consisting of Cockera rhizophila, Corynebacterium glutamicum, Arthrobacter research team, and combinations thereof.

Use of a C50 carotenoid compound for alleviating anorexia in a subject.

63. C50-Carotenoid-Compound-synthetic microorganism for use in alleviating anorexia in a subject.

64. The use according to claim 63, wherein said C50 carotenoid compound-synthesizing microorganism is selected from the group consisting of Cockera rhizophila, Corynebacterium glutamicum, Arthrobacter research team, and combinations thereof.

65. A method for assessing the anti-nausea and/or anti-vomiting activity of a carotenoid compound, said method comprising the steps of:

(i) contacting the system with the carotenoid compound;

(ii) determining whether the carotenoid compound alters a characteristic of the system, wherein the characteristic is associated with nausea and/or vomiting.

66. The method of claim 65, wherein the step of determining includes comparing the features before and after the step of contacting.

67. The method of claim 65, wherein the step of determining comprises comparing the features after the step of contacting a comparable reference.

68. The method of claim 67, wherein the comparable reference is a historical reference.

69. The method of claim 67, wherein the comparable reference is a negative control reference.

70. The method of claim 67, wherein the comparable reference is a positive control reference.

71. The method of any one of claims 65-70, wherein the system is or comprises C.

72. The method of any one of claims 65-71, wherein the characteristic is a level of anorexia.

73. The method of any one of claims 65-71, wherein the characteristic is the level or activity of a nucleic acid or protein or form thereof.

74. The method of claim 73, wherein the characteristic is or includes an aspect of xenobiotic detoxification response.

Background

Chemotherapy-induced nausea and vomiting (CINV) affect 70% -80% of patients undergoing chemotherapy and are the primary reason for discontinuation of cancer treatment.

Disclosure of Invention

The present disclosure provides techniques for treating and/or preventing certain diseases, disorders, and/or conditions, and also provides techniques for assessing one or more characteristics of agents useful in such treatment and/or prevention.

In some embodiments, the provided technology relates to the treatment of nausea and/or vomiting, and in some embodiments specifically to induced (e.g., chemotherapy-induced) nausea and/or vomiting. Alternatively, or additionally, in some embodiments, the provided technology relates to the treatment or prevention of one or more eating disorders (e.g., anorexia nervosa).

Nausea and/or vomiting are experienced with a variety of diseases and disorders, and in addition, nausea and/or vomiting may be caused by therapeutic treatments (e.g., chemotherapy). Nausea and/or vomiting, in any case, are generally considered to be unpleasant and undesirable. Thus, the subject is attempting to avoid nausea and/or vomiting; such avoidance may result, for example, in non-compliance with therapeutic treatment, for example, when the patient perceives that there is a correlation between such treatment and the experienced nausea and/or vomiting.

The present disclosure recognizes that certain biological pathways involved in the monitoring of and/or response to toxins (e.g., toxin detoxification pathways), particularly certain signaling pathways that transduce the detection of translational defects into induction of detoxification genes, are conserved in animal phylogeny and may also suppress aberrant human xenobiotic responses (xenobiotic responses); the present disclosure teaches that such agents may have therapeutic potential in the treatment of certain diseases, disorders or conditions (including in particular, for example, nausea and/or emesis, such as chemotherapy-induced nausea, which is a major problem in the treatment of cancer) and/or in eating disorders (such as anorexia nervosa).

Accordingly, the present disclosure provides model systems for characterizing agents for use as therapeutic agents described herein, such as c. Furthermore, the present disclosure describes the use of such systems for identifying and/or characterizing certain useful such agents.

The present disclosure teaches, among other things, that certain carotenoid compounds (e.g., certain C50 carotenoid compounds) can be used to treat and/or prevent diseases, disorders, and/or conditions (e.g., nausea and/or vomiting, e.g., induced nausea, such as chemotherapy-induced nausea) and/or one or more eating disorders (e.g., anorexia nervosa).

For example, the present disclosure provides that certain carotenoid compounds (e.g., certain C50 carotenoid compounds) inhibit detoxification pathways, including detoxification pathways that are activated in response to translational defects induced by mutations in toxins and/or translational components. The present disclosure recognizes that mammals, including humans, are generally well-tolerated as carotenoids, and teaches that they are an attractive class of compounds for therapeutic use as described herein (e.g., for the treatment and/or prevention of nausea and/or vomiting, and/or one or more eating disorders).

The present disclosure specifically recognizes that certain C50 carotenoid compounds are naturally produced by microorganisms (as reviewed by Hencke et al, "C50 Carotenoids: Occurence, biosyntheses, Glycosylation, and metabolism Engineering for the same over production", Chapter 5 of Bio-localization and Biotechnical Implementations, Ed. Singh, Wil & Sons, 2017). In some embodiments, delivery of a carotenoid compound (e.g., a C50 carotenoid compound) for therapeutic use as described herein can be accomplished by administering a composition that is or comprises a microorganism that produces one or more carotenoid compounds of interest, or an extract or purified component thereof. In some embodiments, such administration may be viable (e.g., microorganisms), and in certain embodiments may effect colonization of the administered microorganism in the recipient (e.g., as part of the recipient microorganism group).

The present disclosure recognizes that embodiments involving the administration of microorganisms, particularly viable (e.g., living) microorganisms, can provide certain advantages, such as, for example, reduced administration (e.g., reduced frequency of administration, duration of administration, total number of doses administered, volume and/or concentration of doses administered, and/or combinations thereof, etc.), reduced cost, long-term efficacy, and the like, among others.

However, one skilled in the art will understand, upon reading this disclosure, that delivery of a carotenoid compound (e.g., a C50 carotenoid compound) for use in treatment as described herein is not limited to delivery of a microorganism or even an extract and/or component thereof; rather, useful carotenoid compounds (e.g., as described herein) can be prepared in whole or in part by chemical synthesis and/or can be purified from a microbial source (e.g., cultured microbial cells, which can be naturally occurring and/or genetically engineered or otherwise engineered cells).

One of skill in the art will further appreciate that any of a variety of delivery routes and/or forms can be used to administer a composition that delivers (e.g., is or comprises a microorganism and/or an extract or component thereof and/or one or more pure carotenoid compounds, as described herein) a carotenoid compound useful as described herein. In many embodiments, the compositions are administered orally (e.g., by pills, tablets, capsules, powders, lozenges, syrups, elixirs, and the like). In some embodiments, oral administration is by a nutritional source (e.g., food or beverage).

One challenge associated with developing a useful treatment for nausea and/or vomiting is the lack of animal models. The present disclosure states that Caenorhabditis elegans (Caenorhabditis elegans) can provide an effective model for nausea and/or vomiting. The present disclosure provides, among other things, that caenorhabditis elegans can be used to characterize (e.g., screen) agents to assess their effect and/or usefulness in treating nausea and/or vomiting. For example, microbial toxins and virulence factors are often targeted to translation machinery. Caenorhabditis elegans responds to translational defects (such as may be caused by exposure to toxins and/or by mutations in translational components) by inducing detoxification and defense response genes. According to the present disclosure, agents that inhibit such induction are useful for treating nausea and/or vomiting, and assessment of such inhibition can be used to characterize (e.g., screen) such agents.

Further, the disclosure demonstrates that certain carotenoid compounds (e.g., certain C50 carotenoid compounds) can inhibit caenorhabditis elegans translational deficiency monitoring and response pathways (e.g., xenobiotic detoxification responses against translational deficient caenorhabditis elegans); according to the present disclosure, such carotenoid compounds are useful for therapeutic applications, such as the treatment of nausea and/or vomiting. For example, the present disclosure describes, inter alia, that C50 carotenoid compounds produced by Kocuria rhizophila (Kocuria rhizophila) inhibit caenorhabditis elegans translational defect monitoring and response pathways. The present disclosure describes, among other things, genetic analyses that identify the biosynthetic pathway for such carotenoids as mediating inhibition of the caenorhabditis elegans translational toxin defense response. Furthermore, the present disclosure describes a cocklebur rhizophilus extract: (i) the repression of the xenobiotic detoxification response against translational defects of caenorhabditis elegans was reproduced (recapitulante); and (ii) restores the ability of carotenoid mutants of kocuria rhizophila to inhibit such detoxification responses against translational defects.

In addition, the disclosure states that other carotenoid compounds (e.g., C50 carotenoid compounds produced by other species) also inhibit caenorhabditis elegans translational deficiency monitoring and response pathways (e.g., xenobiotic detoxification responses against translational deficiency caenorhabditis elegans). For example, the present disclosure states that C50 carotenoid decaprenylflavin (decaprenoxanthin) produced by corynebacterium glutamicum (C. glutamicum) also inhibits the caenorhabditis elegans detoxification response, and that carotenoid biosynthesis mutants of corynebacterium glutamicum are deficient in this inhibition.

Further, the disclosure describes another species, Arthrobacter research team (Arthrobacter ariliatensis), which also produces C50 carotenoids (specifically decaprenitein), and also inhibits the caenorhabditis elegans detoxification response.

Without wishing to be bound by any particular theory, the present disclosure proposes that the carotenoid compounds described herein (e.g., C50 carotenoid compounds) suppress the induction of xenobiotic detoxification by inhibiting the caenorhabditis elegans bile acid signaling pathway, which transduces the detection of translational defects into the induction of detoxification genes. As described herein, suppression of translation monitoring by carotenoid compounds (e.g., C50 carotenoid compounds) disables the drug detoxification response, thereby increasing the efficacy of the translation inhibiting drug. Thus, in some embodiments, useful carotenoid compounds according to the present disclosure may be characterized by their ability to increase the efficacy of translational inhibitory drugs. For example, in some embodiments, useful carotenoid compounds are characterized by an increase in one or more characteristics of the effect of a translation inhibiting drug on caenorhabditis elegans when such carotenoid compound is contacted with caenorhabditis elegans in the presence of the translation inhibiting drug, relative to characteristics observed under otherwise comparable conditions in the absence of the carotenoid compound (e.g., the presence of the same translation inhibiting drug at the same concentration, etc.).

The present disclosure also shows that certain carotenoid compounds (e.g., certain C50 carotenoid compounds) inhibit the coupling of translational monitoring to anorexia commonly induced by translational inhibitory drugs in caenorhabditis elegans. Thus, in some embodiments, useful carotenoid compounds according to the present disclosure may be characterized by the effect of the compound on caenorhabditis elegans anorexia behavior in the presence of a related toxin. For example, in some embodiments, useful carotenoid compounds may be characterized by an alteration in one or more characteristics of the effect of a toxin on the feeding behavior of caenorhabditis elegans when such a compound is contacted with caenorhabditis elegans in the presence of a toxin targeting protein translation, relative to that observed under otherwise comparable conditions in the absence of the carotenoid compound (e.g., the same toxin present at the same concentration, etc.). In some embodiments, agents that exhibit an effect on anorexia behavior as described herein (e.g., carotenoid compounds such as C50 carotenoid compounds) may be particularly useful for treating one or more anorexia disorders, such as anorexia nervosa.

Accordingly, provided herein are methods for treating nausea and/or vomiting, or for alleviating anorexia, in a subject. The method comprises administering to a subject in need thereof a therapeutically effective amount of a C50 carotenoid compound. In some embodiments, the subject has, or is at risk of developing, chemotherapy-or radiotherapy-associated nausea and/or vomiting (e.g., chemotherapy-induced nausea and vomiting (CINV) or radiotherapy-induced nausea and vomiting (RINV)).

In some embodiments, the subject has or is at risk of developing postoperative nausea and vomiting (PONV).

In some embodiments, the C50 carotenoid compound is selected from the group consisting of: decaprenylflavin, C50-astaxanthin, C50-beta-carotene, C50-carotene (n ═ 3) (16, 16-diisopentenyl phytoene), C50-zeaxanthin, C50-broxanthin, C50-cryptoxanthin (nostoxanthin), sarcinaxanthin (sarcocinaxanthin), sarprenoxanthin, acyclic C50 carotenoid bacterin, C50-canthaxanthin, C50-lycopene, C50-phytoene, and combinations thereof. In some embodiments, the C50 carotenoid compound is decaprenylflavin.

In some embodiments, the step of administering comprises administering a composition that is or comprises: (i) c50-a carotenoid-compound-synthetic microorganism or a component thereof, (ii) an extract from C50-a carotenoid-compound-synthetic microorganism, (iii) an extracted carotenoid compound, or (iv) a combination thereof. In some embodiments, the C50-carotenoid-compound-synthesizing microorganism is viable or viable. In some embodiments, the step of administering comprises administering a sufficient amount of a microorganism to colonize a microbiome of the subject. In some embodiments, the composition comprises or is prepared from a culture of a microorganism. In some embodiments, the microorganism is a strain found in nature. In some embodiments, the microorganism is an engineered microorganism. In some embodiments, the engineered microorganism comprises a genetic alteration relative to an otherwise comparable reference microorganism such that the engineered microorganism produces the C50 carotenoid compound at an absolute or relative level that is different from that of the reference microorganism.

In some embodiments, the step of administering comprises administering a composition comprising or delivering a synthetic C50 carotenoid compound. In some embodiments, the C50-carotenoid-compound-synthetic microorganism is selected from the group consisting of Corynebacterium rhizophilum, Corynebacterium glutamicum (Corynebacterium glutamicum), arthrobacterium research team (Arthrobacter arieticus), and combinations thereof.

Also provided herein are therapeutic compositions for oral delivery comprising a therapeutically effective amount of a C50 carotenoid compound and a pharmaceutically acceptable carrier.

In some embodiments, the composition comprises a microorganism that synthesizes a C50 carotenoid compound. In some embodiments, the microorganism is a cultured microorganism. In some embodiments, the microorganism is an engineered microorganism. In some embodiments, the engineered microorganism comprises a genetic alteration relative to an otherwise comparable reference microorganism such that the engineered microorganism produces the C50 carotenoid compound at an absolute or relative level that is different from that of the reference microorganism. In some embodiments, the microorganism is viable or viable. In some embodiments, the microorganism has been killed. In some embodiments, the microorganism is selected from the group consisting of a cocklebur rhizophila, a corynebacterium glutamicum, an arthrobacter research team, and combinations thereof.

In some embodiments, the C50 carotenoid compound comprises at least 20 w/w% of the composition.

In some embodiments, the C50 carotenoid compound is purified.

In some embodiments, the C50 carotenoid compound has a chemical structure found in nature.

In some embodiments, the C50 carotenoid compound is an analog of a reference C50 carotenoid compound found in nature.

In some embodiments, the C50 carotenoid compound is selected from the group consisting of: decaprenylflavin, C50-astaxanthin, C50-beta-carotene, C50-carotene (n ═ 3) (16, 16-diisopentenyl phytoene), C50-zeaxanthin, C50-xanthophyll, C50-cryptoxanthin, sarcinaxanthin, sarprenoxanthin, acyclic C50 carotenoid bacterin, C50-canthaxanthin, C50-lycopene, C50-phytoene, and combinations thereof. In some embodiments, the C50 carotenoid compound is decaprenylflavin.

In some embodiments, the therapeutic composition is a liquid, syrup, tablet, troche (troche), gummy (gummy), capsule, powder, gel, or film.

Further, provided herein are methods for manufacturing therapeutic compositions. For example, the method may comprise the steps of: combining a pharmaceutically acceptable carrier with a C50 carotenoid compound; and formulating the above combinations into therapeutic compositions.

In some embodiments, the step of combining comprises combining a pharmaceutically acceptable carrier with a microorganism that synthesizes a C50 carotenoid compound.

In some embodiments, the step of combining comprises combining a pharmaceutically acceptable carrier with the chemically synthesized C50 carotenoid compound.

Also provided herein are methods for treating nausea and/or vomiting, or for alleviating anorexia in a subject, comprising the step of administering to a subject in need thereof: (i) c50-carotenoid-compound-synthetic microorganism or component thereof, (ii) C50-carotenoid-compound-synthetic microorganism extract, or (iii) extracted C50-carotenoid compound, or (iv) combination thereof

In some embodiments, the subject has, or is at risk of developing, nausea and/or vomiting associated with chemotherapy or radiotherapy.

In some embodiments, the subject has, or is at risk of developing, chemotherapy-induced nausea and vomiting (CINV) or radiotherapy-induced nausea and vomiting (RINV). In some embodiments, the subject has or is at risk of developing postoperative nausea and vomiting (PONV).

In some embodiments, the C50 carotenoid compound-synthesizing microorganism is selected from the group consisting of corynebacterium rhizophilum, corynebacterium glutamicum, arthrobacterium research team, and combinations thereof.

Further, provided herein is a C50 carotenoid compound for use in treating nausea and/or vomiting, or for reducing anorexia, in a subject in need thereof, and a C50 carotenoid compound for use in treating nausea and/or vomiting, or reducing anorexia, in a subject in need thereof.

Also provided is a C50-carotenoid-compound-synthetic microorganism for use in treating nausea and/or vomiting, or for alleviating anorexia, in a subject in need thereof, and a C50-carotenoid-compound-synthetic microorganism for use in treating nausea and/or vomiting, or alleviating anorexia, in a subject in need thereof.

In some embodiments, the C50 carotenoid compound-synthesizing microorganism is selected from the group consisting of corynebacterium rhizophilum, corynebacterium glutamicum, arthrobacterium research team, and combinations thereof.

Also provided herein are methods for assessing anti-nausea and/or anti-vomiting activity of a carotenoid compound. The method comprises the following steps: (i) contacting the system with a carotenoid compound; and (ii) determining whether the carotenoid compound alters a characteristic of the system, wherein said characteristic is associated with nausea and/or vomiting.

In some embodiments, the step of determining comprises comparing the features before and after the step of performing the contacting.

In some embodiments, the step of determining comprises comparing the features after the step of contacting with a comparable reference.

In some embodiments, the comparable reference is a historical reference.

In some embodiments, the comparable reference is a negative control reference.

In some embodiments, a comparable reference is a positive control reference.

In some embodiments, the system is or comprises caenorhabditis elegans.

In some embodiments, the characteristic is a level of anorexia.

In some embodiments, the characteristic is the level or activity of a nucleic acid or protein or form thereof.

In some embodiments, the feature is or includes an aspect of xenobiotic detoxification response.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of embodiments of the invention will be apparent from the following detailed description and drawings, and from the claims.

Drawings

FIGS. 1A-1E

A) Eft-3(q145) on feeding with wild type kocuria radicicola; pgp-5p:: GFP induction was significantly reduced in pgp-5p:: GFP animals, while the mutants of Cockera radicophila crtEb (e17), Cockera crtI (e10) and Cockera radicophila crtYe (e2) did not suppress GFP induction.

B) At eft-3(q 145); in the gfp animal, the feeding of the cocklebur rhizophilus obviously reduces pgp-5p: gfp induction; whereas in the C.rhizophilus mutant, pgp-5p:: gfp expression was unaffected. Unpaired t-test,. P < 0.01. Mean ± s.d are shown. The number of animals analyzed under each condition is shown above the bars. ns is eft-3(q145) fed with E.coli OP 50; pgp-5p: gfp not significant.

C) Discolouring phenotype of mutants of kocuria radicicola.

D) A schematic representation of mutations in carotenoid clusters in Cockera rhizophila.

E) The putative C50 carotenoid biosynthetic pathway in kocuria rhizophila.

FIGS. 2A-2G

A) Eft-3(q145) in a feed with wild type Corynebacterium glutamicum; pgp-5p:: GFP induction was significantly reduced in GFP animals, while mutant corynebacterium glutamicum Δ crtEb, Δ crtI, Δ crtY and Δ crtB did not suppress GFP induction.

B) Eft-3(q145) fed with wild type C.glutamicum, mutants Δ crtEb, Δ crtI, Δ crtY and Δ crtB; quantification of pgp-5p:: gfp expression in gfp animals. Unpaired t-test, P < 0.0001. Mean ± s.d are shown. The number of animals analyzed under each condition is shown above the bars. ns is eft-3(q145) fed with E.coli OP 50; pgp-5p: gfp not significant.

C) Eft-3(q145) on Arthrobacter feeding in wild type research team; pgp-5p:: gfp animal, pgp-5p:: gfp induction was significantly reduced.

D) TLC of the cocklebur bacilli extract showed an orange pigment.

E) HPLC of the C.rhizophilus extract showed the absorbance of the orange pigment. The insert shows the elution time and absorbance of different peaks of the extract.

F)750 μ g/ml of Cockerella radicophila extract inhibits eft-3(q 145); pgp-5p, in gfp animal, pgp-5p, in gfp animal.

G) Eft-3(q145) fed with wild type kocuria radicicola, kocuria radicicola crtEb (e17), kocuria radicicola crtI (e10) and kocuria radicicola crtEb (e6), including control extracts or kocuria radicicola extracts; quantification of pgp-5p:: gfp expression in gfp animals.

FIGS. 3A-3F

A) In animals fed wild type C.radicicola, pgp-5p:: GFP induction was significantly reduced in response to 10mg/ml hygromycin, while mutant C.radicicola crtEb (e17) or C.radicicola crtI (e10) did not suppress GFP induction.

B) Animals treated with carotenoid extracts of Corkerobacter radicophila were highly sensitive to hygromycin. Unpaired t-tests, P <0.0001 compared to wild-type worms fed with e.coli OP50 containing solvent extract and hygromycin. Mean ± s.d are shown. Data were collected from three independent experiments with at least 20 animals per condition. ns were not significant compared to the wild type fed with E.coli OP50 containing the solvent extract but no hygromycin.

C) Animals treated with carotenoid extracts of Corkerobacter radicophila were highly sensitive to emetine.

D) Animals treated with carotenoid extracts of Corkspora radicophila were highly sensitive to cisplatin.

E) Animals treated with carotenoid extracts of Corkerobacter radicophilus did not evade hygromycin compared to animals treated with control solvents and hygromycin.

F) Animals treated with carotenoid extracts of Corkspora radicicola did not evade cisplatin compared to animals treated with control solvents and cisplatin. Unpaired t-test, P <0.01, P < 0.0001. Mean ± s.d are shown. ns, not significant.

FIGS. 4A-4E

A) In animals fed either E.coli OP50 or Cockerella radicophila, which expresses ZIP-2:: mCherry in the intestine under the control of the vha-6 promoter, pgp-5p:: gfp was constitutively induced.

B) Supplementation of bile acids suppressed eft-3(q 145); pgp-5p:: defective in gfp activation induced by Corticillium rhizophilum in animals.

C) Bile acid pair eft-3(q 145); quantification of suppression of defective gfp activation is shown in FIG. 4B. Unpaired t-test, P < 0.0001. Mean ± s.d are shown. The number of animals analyzed under each condition is shown above the bars. ns is eft-3(q145) fed with E.coli OP 50; pgp-5p: gfp not significant.

D) At eft-3(q 145); pgp-5p:: in GFP animals, lbp-5RNAi, chc-1RNAi, fcho-1RNAi and dyn-1RNAi suppressed the kocuria rhizophila-induced pgp-5p:: GFP activation defect, while rme-1RNAi or rab-5RNAi did not suppress GFP induction.

E) A working model of how carotenoid extracts of kocuria rhizophila can suppress induction of xenobiotic detoxification responses.

FIGS. 5A-5G

A) At eft-3(q 145); in pgp-5p: gfp animals feeding of Corkerobacter radicicola inhibited the induction of pgp-5p: gfp, whereas in Corkerobacter radicicola mutants pgp-5p: gfp expression was unaffected.

B) At eft-3(q 145); pgp-5p:: gfp animals feeding Cork.radicophilus suppressed pgp-5p:: gfp induction within 12 hours of feeding.

C) Although animals fed vrs-2dsRNA and transferred to E.coli showed induction of gfp, pgp-5p:: gfp expression was reduced in animals transferred to plates of Corkerobacterium rhizophilus.

D) Although pgp-5p:: gfp expression was reduced in animals transferred to the C.rhizophilus plate, while pgp-5p:: gfp expression was shown in animals fed rpl-1dsRNA and transferred to E.coli.

E) At eft-3(q 145); in pgp-5p:: gfp animals feeding Corkerobacter radicophilus significantly reduced pgp-5p:: gfp induction, while in Corkerobacter radicophilus mutants pgp-5p:: gfp expression was unaffected.

F) The colony color of wild type kocuria radicicola was different from that of the six mutants.

G) Colony color observed for the mutant of kocuria rhizophila was screened from EMS.

FIGS. 6A-6C

A) At eft-3(q 145); pgp-5p:: gfp in animals, the mutant of Cockera rhizophila failed to suppress induction of pgp-5p:: gfp.

B) Discolouration phenotype observed from 23 genome sequencing mutants.

C) Discolouring phenotypes observed from wild type C.glutamicum, mutants Δ crtEb, Δ crtI, Δ crtY and Δ crtB.

FIG. 7

Bacterial operon constructs containing a putative gene cluster likely to produce decaprenylflavin.

FIG. 8

Alignment of CrtI proteins in different genera. Amino acid conservation between sequences found in different genera is shown.

FIG. 9

Alignment of CrtB proteins in different genera. Amino acid conservation between sequences found in different genera is shown.

FIG. 10 shows a schematic view of a

Alignment of CrtEb proteins in different genera. Amino acid conservation between sequences found in different genera is shown.

FIG. 11

An exemplary biochemical isolation of an extract containing decaisoprenol from Coccocus radicicola.

FIG. 12A-FIG. 12B-FIG. 12C

Spectrophotometric analysis of methanol extracts from: wild type kocuria radicicola, crtI (e10), and crtb (e6) (12A); wild type, crtEb (e17) and crthyf (e18) (12B); and wild type, crtEb (e17) and crtYe (e22) (12C).

FIGS. 13A-13E

A) The bacteria, Cockerella radicophila, did not induce hsp-4p:: gfp.

B) Wild type Cockerella radicophila and crtEb (e17) or crtI (e10) mutants did not induce hsp-6p:: gfp.

C) Wild type Coccocus radicicola and crtEb (e17), crtI (e10), crtYf (e2) or crtYe (e22) mutants induced clec-60p: gfp.

D) Wild type Cockerella radicophila and crtEb (e17) or crtI (e10) mutants did not induce F35E12.5p:: gfp.

E) At eft-3(q 145); pgp-5p:: in gfp animals feeding Cordycoccus radicophilus induced repression of pgp-5p:: gfp was reversible.

Fig. 14A-14D.

A) At eft-3(q 145); pgp-5p:: gfp animal, N-acetylcysteine, ascorbic acid, trolox or resveratrol did not suppress pgp-5p:: gfp.

B) At eft-3(q 145); pgp-5p:: gfp animal, β -carotene or astaxanthin did not suppress pgp-5p:: gfp.

C) At eft-3(q 145); pgp-5p:: gfp in animals, colibacillus expressing zeaxanthin, neurosporene, violaxanthin, delta-carotene or alpha-carotene did not suppress pgp-5p:: gfp.

D) In animals fed wild type Corkspora radicicola, pgp-5p:: gfp induction was significantly reduced in response to 10. mu.g/ml and 20. mu.g/ml hygromycin. In both animals fed E.coli OP50 and wild type Corkspora radicicola, pgp-5p:, gfp induction was normal in response to 50mg/ml hygromycin.

FIGS. 15A to 15E

A) In animals fed wild type Cockerella radicicola, pgp-5p:: gfp induction was significantly reduced in response to 2.5 μ g/ml or 6.25 μ g/ml of emidine. However, in animals treated with 12.5. mu.g/ml of emidine, the response in pgp-5p:: gfp induction was partially reduced in animals fed with Corticiella radicicola. In animals treated with 25. mu.g/ml of emidine, pgp-5p:, in both animals treated with E.coli OP50 or wild type Cockera rhizophila, induction of gfp was normal.

B) In animals fed wild type C.radicicola, pgp-5p:: GFP induction was significantly reduced in response to 6.25. mu.g/ml of emidine, while the mutant C.radicicola crtEb (e17) or C.radicicola crtI (e10) did not suppress GFP induction.

C) In animals fed wild type C.radicicola, pgp-5p:: GFP induction was significantly reduced in response to 1mM cisplatin, while mutant C.radicicola crtEb (e17), crtYe (e22) or crtI (e10) did not suppress GFP induction.

D) In the absence of hygromycin, the carotenoids themselves are not toxic to the worms.

E) Animals fed control extract or extract of kocuria rhizophila were sensitive to antimycin.

FIGS. 16A to 16C

A) The ZK892.4 and C24A3.4 proteins share sequence similarity.

B) At eft-3(q 145); inactivation of either ZK892.4 RNAi or C24A3.4RNAi in gfp animals did not suppress pgp-5 p-gfp induction, while the dual RNAi of ZK892.4 and C24A3.4 suppressed gfp induction.

C) chc-1, fcho-1, or lbp-5 did not induce pgp-5p:: gfp.

Definition of

Administration of: as used herein, the term "administering" generally refers to administering a composition to a subject or system to effect delivery of an agent to the subject or system. In some embodiments, the agent is or is contained in a composition; in some embodiments, the agent is produced by metabolism of the composition or one or more components thereof. One of ordinary skill in the art is aware of the various routes available for administration to a subject (e.g., a human) where appropriate. For example, in some embodiments, administration can be ophthalmic, oral, parenteral, topical, and the like. In some particular embodiments, administration can be bronchial (e.g., by bronchial instillation), buccal, dermal (which can be or include, for example, one or more of topical to dermal, intradermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraorgan-specific (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In many embodiments provided by the present disclosure, the administration is oral administration. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve administering a fixed number of doses. In some embodiments, administration may involve intermittent (e.g., multiple doses separated in time) and/or periodic (e.g., single doses separated by a common period of time) administration. In some embodiments, administration may involve continuous administration (e.g., perfusion) for at least a selected period of time.

The analogues: as used herein, the term "analog" refers to a substance that shares one or more specific structural features, elements, components, or parts with a reference substance. Typically, an "analogue" shows significant structural similarity to a reference substance, for example sharing a core structure or a common structure, but also differing in some discrete manner (discrete ways). In some embodiments, an analog is a substance that can be produced from a reference substance, for example, by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be produced by performing a synthetic method that is substantially similar to (e.g., shares multiple steps with) the synthetic method that produces the reference substance. In some embodiments, the analog is or can be generated by performing a synthetic method different from that used to generate the reference substance.

Carotenoid compounds: as used herein, the term "carotenoid compound" refers to a compound that is a member of a structurally diverse class of naturally occurring carotenoid pigments, and structural analogs thereof. In nature, carotenoid compounds are often synthesized from isoprenoid pathway intermediates. Carotenoids may be acyclic or cyclic, and may or may not contain oxygen, so in some embodiments, the term "carotenoid" may include both carotenes and xanthophylls. Many carotenoids have strong light absorption. Generally, carotenoids are hydrocarbon compounds having a conjugated polyene carbon skeleton, formally derived from the pentacarbon compound isopentenyl pyrophosphate. In some embodiments, the carotenoid compound may be a triterpene (C30 didecyl carotenoid), tetraterpene (C40 carotenoid), or other compound, for example, of length C35, C50, C60, C70, C80, or other lengths. In some embodiments, the carotenoid may have a length in excess of C200. Over 1000 different carotenoids have been identified in nature. Carotenoids include, but are not limited to: antheraxanthin, adonirubin, adonixanthin, astaxanthin, canthaxanthin, capsorubin (capsorubin), beta-cryptoxanthin, alpha-carotene, beta, psi-carotene, delta-carotene, epsilon-carotene, echinenone, 3-hydroxyechinenone, 3' -hydroxyechinenone, gamma-carotene, psi-carotene, 4-keto-gamma-carotene, zeta-carotene, alpha-cryptoxanthin, deoxyflexixanthin, diatoxanthine, 7, 8-didehydroantixanthin, didehydrolycopene, fucoxanthin (fucoxanthin), iso-carotene, beta-iso-carotene, lactucaxanthin (lactucaxanthin), lutein, lycopene, myxobactene, neoxanthin, astaxanthin, Alternarycin, hydroxycrycin, polymethine (peridinin), phytoene, rhodopsin glucoside, 4-keto-rubicin, siphonaxanthin (siphoraxanthin), fullerene (sphaidene), sphaidenone, spiroxanthin, torula, 4-keto-torula, 3-hydroxy-4-keto-torula, urate (uriolide), uric acid acetate (uriolide acetate), violaxanthin, zeaxanthin-beta-diglucoside, zeaxanthin and C30 carotenoid. Furthermore, carotenoid compounds include derivatives of these molecules, which may include hydroxyl-functional groups, methoxy-functional groups, oxo-functional groups, epoxy-functional groups, carboxyl-functional groups or aldehyde functional groups. For example, carotenoids include oxidized derivatives. Additionally, carotenoid compounds included include esters (e.g., glycoside esters, fatty acid esters) and sulfate derivatives (e.g., esterified xanthophylls).

Carotenoid modification: as used herein, the term "carotenoid modification" refers to a modification of a host organism that modulates the production of one or more carotenoids, as described herein. For example, a carotene producing modification may increase the level of production of one or more carotenoids and/or may alter the relative levels of production of different carotenoids. In principle, an inventive carotene producing modification may be any chemical, physiological, genetic or other modification that suitably alters the production of one or more carotenoids produced by an organism in a host organism as compared to the level produced in an otherwise identical organism that has not undergone the same modification. However, in most embodiments, the carotene producing modification will include a genetic modification that generally results in increased production of one or more selected carotenoids. In some embodiments, the selected carotenoid is one or more C50 carotenoid compounds.

Carotenoid biosynthetic polypeptides: the term "carotenoid biosynthetic polypeptide" refers to any polypeptide involved in the synthesis of one or more carotenoids. These carotenoid biosynthetic polypeptides include, for example: phytoene synthase, phytoene dehydrogenase (or desaturase), lycopene cyclase, carotenoid ketolase, carotenoid hydroxylase, astaxanthin synthase, carotenoid epsilon hydroxylase, lycopene cyclase (beta and epsilon subunits), carotenoid glucosyltransferase, and acyl-coa: diacylglycerol acyltransferase.

Carotenoid-compound-synthetic microorganism: as used herein, the phrase "carotenoid-compound-synthesizing microorganism" refers to a microorganism (e.g., algae, fungi, bacteria) that synthesizes one or more carotenoid compounds. In some embodiments, the carotenoid-compound-synthesizing microorganism may naturally synthesize one or more carotenoid compounds. In some embodiments, the carotenoid-compound-synthesizing microorganism comprises a carotene-producing modification. In some embodiments, a carotenoid-compound-synthesizing microorganism can be genetically modified (e.g., to have one or more genetic alterations) such that it synthesizes one or more carotenoids at absolute or relative levels that are different from an otherwise comparable reference microorganism that has not been so genetically modified (i.e., that is, does not contain genetic alterations). For example, in some embodiments, a carotenoid-compound-synthesizing microorganism has been genetically engineered to synthesize at least one carotenoid compound in the absence of synthesis by the genetically engineered microorganism. Alternatively, in some embodiments, the carotenoid-compound-synthesizing microorganism may have been genetically engineered such that the synthesis of one or more specific carotenoid compounds thereof may be at a higher level relative to the microorganism in the absence of the genetic engineering. In some embodiments, the higher level may be evaluated with reference to a threshold level; in some embodiments, the higher level may be assessed with reference to another compound (e.g., another carotenoid compound) also produced by the microorganism (prior to genetic engineering). In some particular embodiments, the carotenoid-compound-synthesizing microorganism may have been genetically modified to add or increase expression of one or more genes encoding carotenoid biosynthesis polypeptides. Alternatively, or in addition, in some embodiments, the carotenoid-compound-synthesizing microorganism may have been genetically modified to increase carbon flux through the carotenoid biosynthetic pathway (e.g., by reducing carbon transfer to one or more other biosynthetic or metabolic pathways). In some embodiments, the carotenoid-compound-synthesizing microorganism may synthesize one or more carotenoid compounds having a specific number of carbon units. For example, in some embodiments, a carotenoid-compound-synthetic microorganism may synthesize (either naturally or as a result of genetic modification) one or more C50 carotenoid compounds; such a microorganism may be referred to as a C50-synthetic microorganism.

Comparable to that: as used herein, the term "comparable" means that two or more reagents, entities, conditions, sets of conditions, etc., may be different from each other but sufficiently similar to allow comparisons between them to be made so that one skilled in the art will understand that a conclusion may reasonably be drawn based on the differences or similarities observed. In some embodiments, a comparable group of conditions, environments, individuals, or populations is characterized by a plurality of substantially identical features and one or a few different features. One of ordinary skill in the art will understand, in this context, what degree of identity is required in any given instance to consider two or more such agents, entities, circumstances, sets of conditions, etc. to be comparable. For example, one of ordinary skill in the art will appreciate that groups of environments, individuals, or populations are comparable to one another when they are characterized by a sufficient number and type of substantially identical features to ensure that the following conclusions are reasonable: under, or in different groups of environments, individuals or populations, differences in the results or observed phenomena obtained are caused by or indicative of changes in those different characteristics.

The preparation formulation is as follows: one skilled in the art will appreciate that the term "dosage form" may be used to refer to a physically discrete unit of an agent (e.g., a therapeutic agent) for administration to a subject. Typically, each such unit contains a predetermined amount of reagent. In some embodiments, such an amount is a unit dose (or a whole portion thereof) suitable for administration according to the following dosing regimen: the dosing regimen has been determined to correlate with a desired or beneficial result when administered to a relevant population (i.e., a therapeutic dosing regimen). One of ordinary skill in the art understands that the total amount of therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve the administration of multiple dosage forms.

The administration scheme is as follows: the skilled person will appreciate that the term "dosing regimen" may be used to refer to a group of unit doses (typically more than one) each administered to a subject, typically spaced apart by a period of time. In some embodiments, a given agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, the dosing regimen comprises a plurality of doses, each of the plurality of doses being spaced apart in time from the other doses. In some embodiments, the individual doses are spaced from each other by a period of time of the same length; in some embodiments, the dosing regimen comprises a plurality of doses and at least two different time periods separating the individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen have different amounts. In some embodiments, a dosage regimen comprises a first dose in an amount of the first dose, followed by one or more additional doses in an amount of the second dose that is different from the amount of the first dose. In some embodiments, a dosing regimen comprises a first dose in an amount of the first dose, followed by one or more additional doses in an amount of a second dose that is the same as the amount of the first dose. In some embodiments, the dosing regimen is correlated with a desired or beneficial result when administered in a relevant population.

Engineered: in general, the term "engineered" refers to an aspect of human operation. For example, a cell or organism is considered "engineered" if it has been manipulated to alter its genetic information (e.g., by introducing new genetic material that did not previously exist, e.g., by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or altering or removing previously existing genetic material, e.g., by substitution or deletion mutations, or by mating protocols). As understood by those skilled in the art and as is customary practice, progeny of an engineered polynucleotide or cell are often referred to as "engineered" even if the actual manipulation of the prior entity (prior entity) is performed.

Excipient: as used herein, an excipient refers to an inactive (e.g., non-therapeutic) agent that may be included in a pharmaceutical composition, e.g., to provide or contribute to a desired consistency or stabilization. In some embodiments, suitable pharmaceutical excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

Functional: as used herein, a "functional" biomolecule is a biomolecule that is in a form in which it exhibits its characterized properties and/or activities. Biomolecules may have two functions (i.e., bifunctional) or more functions (i.e., multifunctional).

Ameliorating, increasing, inhibiting or reducing: as used herein, the terms "improve," "increase," "inhibit," "decrease," or grammatical equivalents thereof refer to a value measured relative to a baseline or other reference. In some embodiments, an appropriate reference measurement may be or include a measurement in a particular system (e.g., a single individual) in the absence (e.g., before and/or after) of a particular reagent or treatment, or in the presence of an appropriate comparable reference reagent, under otherwise comparable conditions. In some embodiments, an appropriate reference measurement may be or include a measurement in a comparable system that is known or expected to respond in a particular manner in the presence of the relevant agent or treatment. In some embodiments, a suitable reference is a negative reference; in some embodiments, the appropriate reference is a positive reference.

Separating: as used herein, "isolated" refers to a substance and/or entity that has been (1) separated from at least some of its associated components at the time of its initial manufacture (whether in nature and/or in an experimental setting), and/or (2) designed, produced, prepared, and/or manufactured by man. In some embodiments, the isolated substance or entity may be enriched; in some embodiments, the isolated substance or entity may be pure. In some embodiments, isolated substances and/or entities may be isolated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they are originally associated. In some embodiments, the isolated agent has a purity of about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99%. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as understood by one of skill in the art, a substance may still be considered "enriched," "isolated," or even "pure" after combination with certain other components (e.g., such as one or more carriers or excipients, e.g., buffers, solvents, water, etc.); in such embodiments, the percent isolation or purity of the substance is calculated without including such carriers or excipients. Those skilled in the art are aware of various techniques for separating (e.g., enriching or purifying) substances or reagents (e.g., using one or more of fractionation, extraction, precipitation, or other separation).

The pharmaceutical composition comprises: as used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that, when administered to a relevant population, shows a statistically significant likelihood of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including administration forms suitable for: oral administration, such as drench (aqueous or non-aqueous solution or suspension), tablets (e.g. intended for buccal, sublingual and systemic absorption), boluses (bolus), powders, granules, ointments applied to the tongue, capsules, powders and the like. In some embodiments, the active agent can be or comprise a cell or a population of cells (e.g., a culture, such as a carotenoid compound-synthesizing microorganism); in some embodiments, the active agent can be or comprise an extract or component of a cell or group of cells (e.g., a culture). In some embodiments, the active agent may be or comprise an isolated, purified, or pure compound. In some embodiments, the active agent may have been synthesized in vitro (e.g., by chemical and/or enzymatic synthesis). In some embodiments, the active agent may be or comprise a natural product (whether isolated from its natural source or synthesized in vitro).

Pharmaceutically acceptable: as used herein, the term "pharmaceutically acceptable" as it may be used, for example, in reference to a carrier, diluent or excipient used in formulating a pharmaceutical composition as disclosed herein, means that the carrier, diluent or excipient is compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

A pharmaceutically acceptable carrier: as used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or excipient, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in the transport or transport of a compound of interest from one organ or portion of the body to another organ or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc powder; excipients, such as cocoa butter and suppository waxes (suppository waxes); oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; a pH buffer solution; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible materials for use in pharmaceutical formulations.

Prevention: as used herein, the term "prevention" refers to a delay in onset, and/or a reduction in frequency and/or severity of one or more symptoms of a particular disease, disorder or condition. In some embodiments, prevention is assessed on a population basis such that an agent is considered "preventing" a disease, disorder or condition if a statistically significant reduction in the development, frequency and/or intensity of one or more symptoms of the disease, disorder or condition is observed in a population predisposed to the disease, disorder or condition. In some embodiments, prevention may be considered complete, for example, when the onset of a disease, disorder, or condition has been delayed for a predetermined period of time.

Reference: as used herein, the reference describes a standard or control against which the comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, a reference or control is tested and/or assayed at substantially the same time as the test or assay of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, the reference or control is determined or characterized under conditions or environments comparable to those of the subject under evaluation, as understood by one of skill in the art. One skilled in the art will understand when sufficient similarity exists to justify a dependency and/or comparison on a particular possible reference or control. In some embodiments, the reference is a negative control reference; in some embodiments, the reference is a positive control reference.

Risk: as will be understood from the context, "risk" of a disease, disorder and/or condition refers to the likelihood that a particular individual will develop the disease, disorder and/or condition. In some embodiments, the risk is expressed as a percentage. In some embodiments, the risk is 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, up to 100%. In some embodiments, the risk is expressed as a risk relative to a risk associated with a reference sample or a reference sample set. In some embodiments, the reference sample or reference sample set has a known risk of a disease, disorder, condition, and/or event. In some embodiments, the reference sample or set of reference samples is from an individual comparable to the particular individual. In some embodiments, the relative risk is 0, 1,2, 3, 4, 5, 6, 7,8, 9, 10, or more.

Sample preparation: as used herein, the term "sample" generally refers to a specimen of material obtained or derived from a source of interest. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may be or include a cell or organism, such as a microorganism, a plant, or an animal (e.g., a human). In some embodiments, the source of interest is or includes a biological tissue or fluid. In some embodiments, the biological tissue or fluid may be or include amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculated semen, endolymph, exudate, feces, gastric acid, gastric fluid, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, inflammatory secretions (rheum), saliva, sebum, semen, serum, periderm scale, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and/or combinations or components thereof. In some embodiments, the biological fluid may be or include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymphatic fluid, and/or transcellular fluid. In some embodiments, the biological fluid may be or include a plant exudate. In some embodiments, a biological tissue or sample can be obtained, for example, by aspiration, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral swab, nasal swab, skin swab, or vaginal swab), scrape, surgery, wash, or lavage (e.g., bronchoalveolar wash or lavage, catheter wash or lavage, nasal wash or lavage, ocular wash or lavage, oral wash or lavage, uterine wash or lavage, vaginal wash or lavage, or other wash or lavage). In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from a source of interest by any suitable means. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing (e.g., by removing one or more components and/or by adding one or more reagents thereto) a primary sample. For example, filtration is performed using a semipermeable membrane. Such "processed samples" may include, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques (e.g., amplification or reverse transcription of nucleic acids, isolation and/or purification of certain components, etc.), and the like.

Subject: as used herein, the term "subject" refers to an individual to whom the provided treatment is administered. In some embodiments, the subject is a mammal, e.g., a mammal that is experiencing or susceptible to a disease, disorder, or condition as described herein; in some embodiments, the subject is a human or non-human veterinary subject, such as a simian, cat, dog, monkey, or pig. In some embodiments, the subject is a human. In some embodiments, the patient is suffering from or susceptible to one or more diseases, disorders, or conditions as described herein. In some embodiments, the patient exhibits one or more symptoms of one or more diseases, disorders, or conditions as described herein. In some embodiments, the patient has been diagnosed with one or more diseases, disorders, or conditions as described herein. In some embodiments, the disorder or condition is or includes nausea and/or vomiting, and/or one or more anorexia disorders. In some embodiments, the subject is suffering from or susceptible to cancer, or is present with one or more tumors. In some embodiments, the subject is receiving or has received certain therapies to diagnose and/or treat a disease, disorder, or condition. In some embodiments, the subject has received a therapy (e.g., chemotherapy, radiation therapy, and/or surgery) that induces nausea and/or vomiting.

Reduction of symptoms: according to the present invention, a "symptom is reduced" when one or more symptoms of a particular disease, disorder or condition are reduced in magnitude (e.g., intensity, severity, etc.) and/or frequency. For the purposes of clarity, delay in the onset of a particular symptom is considered to be one form of reducing the frequency of that symptom.

The treatment scheme comprises the following steps: as the term is used herein, "treatment regimen" refers to a dosing regimen, the administration of which in a relevant population can be correlated with a desired or beneficial therapeutic result.

A therapeutically effective amount of: as used herein, a therapeutically effective amount refers to an amount that produces the desired effect for which it is administered. In some embodiments, the term refers to an amount sufficient to treat a disease, disorder, and/or condition when administered according to a therapeutic dosing regimen to a population suffering from or susceptible to such a disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and/or severity and/or delays onset of one or more symptoms of a disease, disorder, and/or condition. One of ordinary skill in the art will appreciate that the term "therapeutically effective amount" does not actually require successful treatment in a particular individual. Rather, a therapeutically effective amount can be an amount that provides a particular desired pharmacological response in a large number of subjects when administered to a patient in need of such treatment. In some embodiments, reference to a therapeutically effective amount may refer to an amount as measured in one or more specific tissues (e.g., tissues affected by a disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). One of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent or therapy can be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective agent can be formulated and/or administered in multiple doses, e.g., as part of a dosing regimen.

Treatment/treatment: as used herein, the term "treatment" refers to any administration of a therapy that partially or completely alleviates, ameliorates, alleviates, inhibits, delays the onset of, reduces the severity of, and/or reduces the incidence of one or more symptoms, characteristics, and/or causes of a particular disease, disorder, and/or condition. In some embodiments, such treatment may be directed to subjects that do not exhibit signs of the associated disease, disorder, and/or condition and/or subjects that exhibit only early signs of the disease, disorder, and/or condition. Alternatively, or in addition, such treatment may be directed to a subject exhibiting one or more determined signs of the associated disease, disorder, and/or condition. In some embodiments, the treatment may be for a subject who has been diagnosed with the associated disease, disorder, and/or condition. In some embodiments, treatment may be directed to a subject known to have one or more susceptibility factors statistically associated with an increased risk of development of the associated disease, disorder, and/or condition.

Detailed Description

Caenorhabditis elegans, a nematode, monitors for defects in essential cellular activity caused by, for example, microbial toxins, and responds by activating a cell-monitoring activated detoxification and defense (cSADD) response (Melo and Ruvkun, 2012). The abundant RNA and proteins of the ribosome, as well as other proteins that mediate translation of mRNA into proteins, can be targets of microbial toxins and virulence factors. The activity of these translational components is monitored and the reduction in protein synthesis caused by toxins or mutations can be detected. The reduced assay is coupled to a signal transduction cascade to activate xenobiotic detoxification and behavioral responses, such as anorexia coupled to p38MAPK, bZIP/ZIP-2 transcription factors and bile acid-like biosynthetic pathways (Melo and Ruvkun, 2012; Dunbar et al, 2012; Govindan et al, 2015). By monitoring the reduction in core cell function (rather than the molecular structure of unknown toxins), caenorhabditis elegans can detect unexpected pathogens and toxins. Many components of this signaling cascade (e.g., MAP kinase and bile acid biosynthetic pathways) are conserved among animals; the present disclosure recognizes that the toxin monitoring and response of the cSADD system may be applicable to animals other than caenorhabditis elegans, and further teaches that caenorhabditis elegans may be used as a model system to characterize agents that modulate this system, and may be useful for certain therapeutic applications as described herein.

The present disclosure further recognizes that animal defense strategies similar or homologous to the caenorhabditis elegans cSADD system may drive the evolution of bacterial strategies to thwart these defense responses; there are many examples of such evolutionary arms competition between hosts and pathogens. The present disclosure also recognizes that commensal bacteria may also attempt to silence such animal defense responses to establish a benign or symbiotic relationship.

The present disclosure further recognizes that microorganisms (e.g., bacteria) synthesize a variety of compounds with significant biological activity, including compounds that target ribosomes and/or related translation factors (Berdy et al, 2005); such microorganisms have proven to be a source of production for drugs or drug candidate compounds. The present disclosure (i) teaches useful therapeutic applications of agents that can inhibit certain detoxification pathways; (ii) systems are provided for assessing one or more characteristics of agents associated with inhibition of such detoxification pathways; and (iii) identify potential sources of such agents (e.g., microorganisms such as bacteria, which in some embodiments may be commensal microorganisms).

For example, the present disclosure provides that certain bacterial strains (e.g., strains that produce certain carotenoid compounds, particularly certain C50 carotenoid compounds) can be good sources of useful agents (e.g., certain useful carotenoid compounds) according to the present disclosure. In addition, the present disclosure provides techniques for assessing one or more relevant characteristics of such agents.

In a microbial repression screen of various bacteria for the following activities, potential activities were identified from wild-type C.rhizophilus: in C.elegans with genetically induced translational defects, this activity abrogates the activation of xenobiotic detoxification genes (Govindan et al, 2015). Genetic analysis of Cockerella radicophila was used to show that C50 carotenoid compounds can suppress the caenorhabditis elegans translational toxin defense response.

As shown herein, the C50 carotenoid compound decaisoprenol producing cocklebia radicicola inhibited caenorhabditis elegans cSADD translation and DNA damaging toxin defense responses. Mutants in the C50 carotenoid biosynthetic pathway of Cockerella radicicola failed to inhibit this C.elegans xenobiotic detoxification response (FIGS. 1A-1C). Extracts of c.radicophilus suppressed this xenobiotic detoxification response of c.elegans, and also restored the ability of carotenoid mutants of c.radicophilus to achieve this suppression (fig. 2B & 2C). The cocklebox extract also suppressed the induction of the caenorhabditis elegans xenobiotic detoxification pathway in response to targeted translation of the toxin (fig. 3A, fig. 15C). The Cockerobacter rhizophilus C50 carotenoid compound also suppresses the response to RNA interference inactivation of other genes, such as the core ribosomal protein or the aminoacyl tRNA synthetase required for protein synthesis. The C50 carotenoid from c.glutamicum also inhibited the induction of the caenorhabditis elegans xenobiotic detoxification pathway in response to translational defects, indicating that the regulation of caenorhabditis elegans surveillance by the C50 carotenoid is not restricted to one particular bacterial strain.

The present disclosure teaches that certain carotenoid compounds (e.g., certain C50 carotenoid compounds) can be useful for a variety of therapeutic applications, including, in particular, treating nausea and/or vomiting (including, in particular, induced nausea and/or vomiting, such as chemotherapy-induced nausea and/or vomiting). Carotenoids are most studied in photosynthetic bacteria and plants, where they are the accessory light-absorbing component in photosynthesis (Edge et al, 1997). In the photosynthetic chlorophyll cluster, carotenoids can absorb light energy and transfer it to the photosynthetic electron transport system, and then create a pH gradient because the reduction potential of photon-pumped electrons (photon-pumped electrons) causes the physical movement of various iron-sulfur and heme proteins, thereby moving protons through the lipid bilayer. Common among photosynthetic bacteria is the strong absorption of visible wavelengths and the lipid solubility of these pigments. These bacteria are highly pigmented because carotenoids are very abundant and have a conjugated double bond system that delocalizes electrons to very large resonance-stable potential wells, with orbital transitions at much lower energies than standard biochemical bonds. Carotenoids are also known antioxidants in photosynthesis. However, as shown herein, the antioxidant activity of carotenoids does not account for the suppression of drug detoxification responses in animals, since other antioxidants do not suppress caenorhabditis elegans monitoring.

The C50 carotenoid compound can influence animal monitoring of translational components by simple changes in membrane fluidity. As shown herein, the C50 carotenoid compound suppressed the induction of caenorhabditis elegans xenobiotic detoxification responses by inhibiting the bile acid biosynthesis pathway. Although bile acids have traditionally been considered as fat emulsifiers to aid digestion, several recent studies have found that bile acids are important signaling molecules in metabolic and immunological pathways. Bile acid signaling may be involved in the induction of a drug detoxification response or CINV in humans. In caenorhabditis elegans monitoring of translation and DNA damage responses, the lipid solubility and abundance of carotenoids may contribute to their anti-bile acid signaling function.

The cSADD response can induce not only xenobiotic detoxification, but also anorexia. As many toxins are derived from bacterial pathogens that may be associated with or cause food decay, aversion to food is an appropriate animal response. Aversion to food associated with induction of xenobiotic detoxification and bacterial immune pathways is likely to be an animal program derived from this evolutionary history. The present disclosure provides the following: chemotherapy-induced nausea and vomiting (CINV) responses in humans may be associated with these xenobiotic aversion programs. Interestingly, cisplatin used to block DNA replication has a high emetogenic potential in cancer patients. Emetine (Emetine) is an antibiotic that targets eukaryotic protein synthesis, and as the name suggests, is also highly emetic. The two drugs can induce heterotypic biomass detoxification response in caenorhabditis elegans and also induce strong anorexia. The pgp-5ABC transporter gene is strongly induced by toxins causing DNA damage, and also by chemically very different toxins that inhibit translation (but not by various toxins that target other pathways such as mitochondria or ER) (Govindan et al, 2015). Thus, the present disclosure teaches that caenorhabditis elegans anorexia is a good model for studying the underlying mechanisms of human CINV.

The serotonin pathway is involved in caenorhabditis elegans anorexia and human CINV (Melo and Ruvkun, 2012). Liver drug detoxification and clearance are key issues in cancer chemotherapy; faster amplification of ABC transporters to eliminate the drug is often observed in drug-resistant cancer patients. Carotenoid extracts of Cockerella radicophila caused a high sensitivity to heterotypic biomass as follows: xenobiotics that target protein translation, and xenobiotics that cause DNA damage by suppressing the induction of drug detoxification pathways. In addition, the C50 carotenoid compound of Corkspora radicicola suppressed anorexia induced by emetogenic toxin, emetine or cisplatin.

As a therapy for treating and/or reducing the risk of nausea and/or vomiting (e.g., induced nausea and/or vomiting, such as CINV, RINV, etc.), C50 carotenoid compounds, such as decaprenylflavin, C50-astaxanthin, C50-beta-carotene, C50-carotene (n ═ 3) (16, 16-diisopentenyl phytoene), C50-zeaxanthin, C50-milbexanthin, C50-cryptoxanthin, sarprenoxanthin, acyclic C50 carotenoid bacterin, C50-canthaxanthin, C50-lycopene, C50-phytoene, may be used according to the present disclosure. Alternatively, or in addition, the present disclosure provides that such C50 carotenoid compounds are useful as therapies for treating and/or reducing the risk of one or more anorexia (e.g., anorexia nervosa). Still further, the present disclosure provides systems for assessing one or more characteristics of agents associated with the usefulness of the therapies described herein (i.e., for treating, and/or reducing the risk of, nausea and/or vomiting and/or anorexia), and also indicates that useful such agents include compounds produced by certain microorganisms (e.g., bacteria), including certain commensal microorganisms. Using such a system, the present disclosure describes the usefulness of certain carotenoid compounds (e.g., C50 carotenoid compounds), including carotenoid compounds produced by various bacterial strains (e.g., synthesized by bacterial enzymes). It will be understood by those of skill in the art reading this disclosure that various other compounds, including various other carotenoid compounds, are produced by microorganisms and/or can be (and/or have been) made by chemical synthesis and evaluated for their activity (e.g., as embodied in the systems exemplified therein). Thus, it will be understood by those of ordinary skill in the art reading this disclosure that a variety of chemical agents are provided, including specifically carotenoid compounds and exemplified by the C50 carotenoid, which are readily evaluated and/or used as therapeutic agents as described herein.

Treatment/management method

The methods provided by the present disclosure include methods for treating certain diseases, disorders, and conditions. In some embodiments, the related diseases, disorders and conditions may be or include nausea and/or vomiting, and/or certain anorexia. In some embodiments, nausea and/or vomiting that may be treated as described herein may be associated with one or more of the following: motion sickness, pregnancy (e.g., pregnancy vomiting or hyperemesis gravidarum), pain, emotional stress, gallbladder disease, heart attack, concussion or brain injury (e.g., brain tumors), binge eating, gallbladder disease, infection, ulcer, gastroparesis, intestinal obstruction, appendicitis, infection (e.g., viral infection), and the like.

In some embodiments, nausea and/or vomiting that may be treated as described herein may be induced nausea and/or vomiting, for example induced by ingestion of toxins or other exposure to toxins (e.g., food poisoning, drug-induced nausea and/or vomiting, intoxication, etc.). In some embodiments, the induced nausea and/or vomiting that may be treated as described herein may be nausea and/or vomiting associated with chemotherapy or radiotherapy. In some embodiments, the induced nausea and/or vomiting that may be treated as described herein may be CINV or Radiotherapy Induced Nausea and Vomiting (RINV); generally, at least three types of emesis are associated with the use of chemotherapeutic agents, namely acute emesis, delayed emesis, and anticipatory emesis. In some embodiments, the induced nausea and/or vomiting that may be treated as described herein may be or include, for example, post-operative nausea and vomiting (PONV).

In general, the treatment methods provided by the present disclosure involve administering to a subject in need or determined to be in need of such treatment a therapeutically effective amount of a carotenoid compound as described herein.

In some embodiments, the methods of treatment provided herein are prophylactic or preventative, e.g., the methods may be administered to a subject prior to exhibiting significant symptoms and/or prior to exposure to a particular expected predisposition associated with nausea and/or vomiting (e.g., chemotherapy, radiation therapy, surgery, or other treatment (e.g., drug treatment)).

In some embodiments, the methods of treatment provided herein are therapeutic, e.g., the methods can be administered to a subject after significant symptoms of nausea and/or vomiting develop (e.g., during or after at least one episode of nausea or vomiting).

In preferred embodiments, the provided methods of treatment are administered to a subject that is a mammal (e.g., a mammal experiencing a disease, disorder, or condition as described herein); in some embodiments, the subject is a human or non-human veterinary subject, such as a simian, cat, dog, monkey, or pig.

In many embodiments, "treating" or "treatment" relates to ameliorating at least one symptom of a disease, disorder, or condition associated with nausea. Generally, nausea leads to anorexia, loss of appetite and/or reduced caloric intake and may lead to weight loss; in some embodiments, administration of a therapeutically effective amount of a carotenoid compound described herein can cause a reduction in nausea, vomiting and/or anorexia. Alternatively or additionally, in some embodiments, administration of a therapeutically effective amount of a carotenoid compound as described herein may effect a restoration of appetite, and/or a restoration or near normal caloric intake, a reduction, cessation or slowing of weight loss, weight gain/gain, and/or a reduction in the frequency, duration or severity of current or future episodes of nausea, vomiting, anorexia and/or anorexia.

In some embodiments, the method may comprise administering a therapeutically effective amount of a carotenoid compound, e.g., a subject about to receive chemotherapy, radiation therapy, or other treatment associated with nausea and vomiting, before, during (e.g., simultaneously with), or after administration of a treatment expected to be associated with nausea and/or vomiting.

In some embodiments, a subject receiving a treatment described herein may be receiving and/or may have received other treatment (e.g., chemotherapy, radiation therapy, surgery, etc.), such as other treatment that may induce emesis or that may be aimed at treating one or more symptoms or features of a disease, disorder, or condition as described herein, such that the carotenoid therapy is administered in combination with such other treatment to treat the associated disease, disorder, or condition.

Carotenoid compositions

The present disclosure provides, among other things, compositions comprising or otherwise delivering a carotenoid compound (e.g., a C50 carotenoid compound) to a subject suffering from or susceptible to one or more diseases, disorders, or conditions described herein.

Those skilled in the art know that more than 750 carotenoid compounds have been previously identified. Carotenoids are generally classified by the number of 5-carbon isoprenoid units such that their carbon skeleton lengths differ. See, e.g., Bio-segmentation and Biotechnical innovations, pp.107-126.doi:10.1002/9781119166191. Henke et al in ch5, (2017). C50 Carotenoids: Occurence, biosyntheses, glycation, and metabolism Engineering for the same over production; growth in Carotenoid Research, (2018) doi: 10.5772/Fernandes in interchopen.79542, introductive channel: Carotenoids-A Brief Overview on Its Structure, biosyntheses, Synthesis, and Applications; mezzomo et al, chemistry Volume 2016:1, (2016) cartenoids Functionality, Sources, and Processing by Supercritical Technology, A Review.

In some embodiments, carotenoid compounds useful according to the present disclosure have a relatively long isoprene backbone, for example having a length in the range of from about 45 to about 60 carbon atoms. In some embodiments, useful carotenoid compounds as described herein may have an isoprene backbone of 50 carbons in length, i.e., may be a C50 carotenoid compound, such as decaisoprenol or other C50 carotenoids, such as C50-astaxanthin (also known as decaisoprenylastaxanthin; see Milon et al, helv.chim.acta 69, 12-24(1986), fubayashi et al, Nat commu.2015; 6:7534), or C50-beta-carotene (also known as decaisoprenyl-beta-carotene) (see, e.g., Karrer et al, helv.chim.acta34, 28-33 (1951); Furubayashi et al, Nat commu.2015, 6: 7534); 16, 16' -diisopentenyl phytoene (Umeno et al, J Bacteriol, 186, 1531-1536 (2004)); c50-carotene (n ═ 3) (16, 16-diisopentenyl phytoene), C50-zeaxanthin, C50-brokelvin, and C50-cryptoxanthin (US 20140170700); sarcinaxanthin, sarprenoxanthin, acyclic C50 carotenoid-lycopene, C50-canthaxanthin, C50-lycopene, C50-phytoene (Li et al, Scientific Reports, Vol. 9, article No.: 2982 (2019)). See also Pfander, Pure and Applied Chemistry, 66 (10-11): 2369-2374(1994).

The person skilled in the art is aware of various techniques for producing carotenoid compounds. See, e.g., J.chemistry, Mezzomo et al, volume 2016:1, (2016) cartenoids Functionality, Sources, and Processing by Supercosmetic Technology, AReview. In some embodiments, the carotenoid compound may be isolated from an organism (e.g., a plant or microorganism) that produces the carotenoid compound. In some such embodiments, such plants or microorganisms may have been developed and/or cultivated by humans. In some embodiments, the plant or microorganism may be a natural plant or microorganism. In some embodiments, the plant or microorganism may be an engineered plant or microorganism, for example a plant or microorganism engineered to synthesize a plant or microorganism as a carotenoid-compound-as described herein.

Those skilled in the art are aware of various plant and/or microbial sources that have been or can be engineered into carotenoid-compound-synthetic plants or microorganisms as described herein. See, for example, WO 2016/102342.

In some embodiments, the carotenoid compound may be isolated from a plant or microbial source (e.g., from a culture or cultivar thereof). The skilled person is aware of various techniques for treating plant and/or microbial cells or tissues, e.g. to prepare extracts thereof and/or to isolate components and/or compounds thereof therefrom.

Alternatively, or additionally, in some embodiments, the carotenoid compound may be prepared partially or wholly in vitro (e.g., by chemical and/or enzymatic synthesis, or a combination thereof), and may optionally be further isolated and/or purified as is known in the art.

Various techniques are known in the art which can be used to prepare extracts from cells or organisms which produce the relevant carotenoid compounds, and/or to isolate extracts, components or compounds from such cells or organisms, or to dispose of an in vitro carotenoid synthesis system (e.g., to isolate and/or purify one or more carotenoid compounds from an in vitro carotenoid synthesis system). Such techniques may include, for example, one or more of organic extraction, vacuum concentration, chromatography, and the like, to name a few examples.

Those skilled in the art know that the end of lycopene is modified by cyclization or oxidation to synthesize various lower (shorter carbon chain) carotenoids, such as alpha-carotene, beta-carotene, gamma-carotene, delta-carotene, epsilon-carotene, lutein, zeaxanthin, canthaxanthin, fucoxanthin, astaxanthin, antheraxanthin and violaxanthin. Higher (more carbon atoms) carotenoids can be prepared, for example, using in vitro methods or in vivo or ex vivo methods, for example, by natural or genetically modified organisms. For example, the C50 carotenoid can be synthesized in vitro by adding two molecules of dimethylallyl pyrophosphate (DMAPP) to the C (2) and C (2') of the corresponding C40 carotenoid, or extracted from an organism (e.g., wild-type or engineered) that synthesizes the C50 carotenoid (e.g., C50-carotenoid-compound-synthesizing microorganism). See, e.g., Milon et al, Helv.Chim.acta 69, 12-24 (1986); karrer et al, Helv.Chim.acta34, 28-33 (1951); furubayashi et al, Nat Commun.2015, 6: 7534; tobias and Arnold, Biochim biophysis acta.2006feb, 1761 (2): 235 to 46 parts of; henke et al (2017, 6, 14 days.) A Carotenoid Production by Corynebacterium The Workhorse of Industrial Amino Acid Production as Host for Production of a Broad Spectrum of C40 and C50 Carotenoids, Dragon J.Cvetkovic and Goran S.Nikolic, Intech Open, DOI:10.5772/67631, available from: com/books/carotenoids/carotenoid-production-by-corynebacterium-the-work-of-induced-amino-acid-production-as-host-f; bio-localization and Biotechnical implementions, pp.107-126.doi 10.1002/9781119166191. Henke et al in ch5, (2017). C50 Carotenoids: Occurence, biosyntheses, glycation, and Metabolic Engineering for the same production; growth in Carotenoid Research, (2018) doi: 10.5772/Fernandes in interchopen.79542, introductive channel: Carotenoids-A Brief Overview on Its Structure, biosyntheses, Synthesis, and applications; heider et al, Appl Microbiol Biot 2014; 98(10) 4355e 68; wang et al, Biotechnol Adv 2007; 25(3) 211e 22; niu et al, Synthetic and Systems Biotechnology 2(2017)167e 175; li et al, Scientific Reports, Vol.9, article No.: 2982 (2019); US 20050260699; US 20140170700; US 20040091958; US20090197321 and the like.

In some embodiments, one or more carotenoid compounds for use according to the present disclosure may be provided as a purified or substantially purified molecule, or less purified (e.g., enriched) extract, of a carotenoid-producing organism.

In some embodiments, a formulation that is or comprises one or more carotenoid compounds is incorporated or otherwise used to produce a pharmaceutical composition as described herein that delivers the carotenoid compounds to a subject upon administration thereto.

In some embodiments, a carotenoid formulation or carotenoid composition (e.g., a pharmaceutical composition comprising or delivering a carotenoid compound) comprises at least 20 w/w%, 30 w/w%, 40 w/w%, 50 w/w%, 60 w/w%, 70 w/w%, 80 w/w%, 90 w/w%, 95 w/w%, 97 w/w%, or 99 w/w% of the carotenoid compound.

In some embodiments, the composition for use according to the present disclosure is a pharmaceutical composition, e.g., for oral administration. Pharmaceutical compositions generally comprise an active agent (e.g., a carotenoid compound, such as a C50 carotenoid compound or a source thereof) and a pharmaceutically acceptable carrier. Certain exemplary pharmaceutically acceptable carriers include, for example, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

In some embodiments, a pharmaceutical composition for use according to the present disclosure may include, and/or may be administered in combination with, one or more supplemental active compounds; in certain embodiments, such supplemental active agents may include ginger, curcumin, probiotics (e.g., probiotic bacterial strains of one or more of the following genera: lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium), yeast (Saccharomyces), Enterococcus (Enterococcus), Streptococcus (Streptococcus), Pediococcus (Pediococcus), Leuconostoc (Leuconostoc), Bacillus (Bacillus) and/or escherichia coli (see Fijan, Int J Environ Res Public health, 2014 5 months, 11 (5)): 4745 + 4767); prebiotics (non-digestible food ingredients that help support the growth of probiotics, for example, fructans, such as Fructooligosaccharides (FOS) and inulin; galactans, such as Galactooligosaccharides (GOS); the dietary fiber is added into the feed for the food, such as resistant starch, pectin, beta-glucan and xylo-oligosaccharides (Hutkins et al, Curr Opin biotechnol.2016feb; 37: 1-7)) and combinations thereof.

Pharmaceutical compositions are generally formulated to be compatible with their intended route of administration. Examples of routes of administration include oral administration.

Methods of formulating suitable pharmaceutical compositions are known in The art, see, e.g., Remington, The Science and Practice of Pharmacy, 21 st edition, 2005; and books in the Drugs and the Pharmaceutical Sciences a Series of Textbooks and monggrams (Dekker, NY) Series. Oral compositions typically comprise an inert diluent or an edible carrier. To name a few examples, in some embodiments, the oral formulation may be or include a syrup, liquid, tablet, troche, fondant, capsule, such as a gelatin capsule, powder, gel, film, and the like.

In some embodiments, pharmaceutically compatible binders and/or auxiliary materials may be included as part of the pharmaceutical composition. In some particular embodiments, the pharmaceutical composition may comprise, for example, any one or more of the following inactive ingredients or compounds of similar nature: a binder, such as microcrystalline cellulose, gum tragacanth or gelatin; excipients, such as starch or lactose; disintegrants, for example alginic acid, Primogel or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. In some embodiments, the composition may be taken as is or sprinkled on or mixed into food or liquid (e.g., water).

In some embodiments, a carotenoid composition that can be administered to a subject as described herein can be or comprise an ingestible item (e.g., a food or beverage) that comprises (e.g., is supplemented with) one or more carotenoids. In some embodiments, useful compositions may comprise one or more carotenoid compounds at levels above that found in its components, and/or above that typically present in the relevant food or beverage, for example to provide carotenoid levels sufficient to provide a therapeutic effect as described herein.

In some embodiments, the food may be or include one or more of the following: bars, candies, baked goods, cereals, savory snacks, pasta, chocolate and other solid foods, as well as liquid or semi-solid foods, including yogurt, soup and stew, and beverages, such as smoothies, milkshakes, juices and other carbonated or non-carbonated beverages. In some embodiments, a food is provided that already comprises a carotenoid therein; in some embodiments, the food is prepared by the subject by admixing carotenoids.

The compositions may be contained in a kit, container, package, or dispenser with instructions for administration or instructions for use in the methods described herein.

One of skill in the art having read this disclosure will appreciate that, in some embodiments, a carotenoid composition as described herein can be or comprise one or more cells, tissues or organisms (e.g., plant or microbial cells, tissues or organisms) that produce (e.g., have produced and/or are producing) a compound of interest. In some embodiments, such cells, tissues or organisms may have previously produced the relevant carotenoids; in some embodiments, such cells, tissues or organisms are producing carotenoid compounds.

In some embodiments, the carotenoid composition may comprise cells, tissues and/or organisms that have been killed (e.g., heat killed). Alternatively, in some embodiments, the carotenoid composition may comprise living or viable cells, tissues and/or organisms.

In some embodiments, the treatment methods described herein involve the administration of one or more viable or viable carotenoid-compound-synthesizing cells, tissues or organisms. In some such embodiments, the cell, tissue, or organism is a microbial cell and is administered according to a protocol that obtains a population of the subject's microbiome with the administered cell.

In some embodiments, a carotenoid composition as described herein comprises one or more cell cultures and/or supernatants or precipitates thereof, and/or a powder formed therefrom, and/or a carotenoid composition as described herein is formulated by using one or more cell cultures and/or supernatants or precipitates thereof, and/or a powder formed therefrom.

One skilled in the art will appreciate that in some embodiments, techniques for preparing a carotenoid composition and/or formulation, and/or for preparing one or more carotenoid compositions (particularly for preparing a pharmaceutical composition) may include one or more steps of evaluating or characterizing a compound, formulation or composition (e.g., as part of quality control). In some embodiments, the analyzed material is discarded if it does not meet predetermined specifications for a relevant assessment. In some embodiments, if such analyzed material does meet predetermined specifications, it continues to be processed as described herein.

Method of identification and/or characterization

The present disclosure provides, among other things, systems that allow for the assessment of one or more reagent characteristics relevant to the usefulness described herein. In some embodiments, techniques for identifying and/or characterizing an agent as described herein may involve comparison to an appropriate reference (e.g., to a positive control reference and/or to a negative control reference). In some embodiments, the reference may be or include a historical reference; in some embodiments, the reference may be or include a contemporaneous reference (concordance).

In some embodiments, the provided techniques can be used to screen test agents, for example, which can be or comprise one or more polypeptides, peptides, inorganic or organic macromolecules or small molecules, or compositions comprising or delivering them, in order to identify agents useful in methods as described herein. Alternatively, or additionally, in some embodiments, the provided techniques may be used to characterize one or more agents, for example during development and/or commercialization of such agents or pharmaceutically acceptable compositions thereof.

As used herein, "small molecule" refers to small organic or inorganic molecules having a molecular weight of less than about 3,000 daltons. Typically, the small molecules may have a molecular weight of less than 3,000 daltons (Da). The small molecule can be, for example, at least about 100Da to about 3,000Da (e.g., about 100Da to about 3,000Da, about 100Da to about 2500Da, about 100Da to about 2,000Da, about 100Da to about 1,750Da, about 100Da to about 1,500Da, about 100Da to about 1,250Da, about 100Da to about 1,000Da, about 100Da to about 750Da, about 100Da to about 500Da, about 200Da to about 1500Da, about 500Da to about 1000Da, about 300Da to about 1000Da, or about 100Da to about 250 Da).

One skilled in the art will appreciate that in some embodiments, particularly in screening embodiments, the provided techniques can be used to identify (e.g., screen) and/or characterize a variety of agents. In some embodiments, such plurality is or includes a reasonably comparable agent (e.g., one or more specific small molecule compounds and various analogs thereof); in some embodiments, the plurality of agents is or includes a plurality of natural products (e.g., carotenoid compounds, such as C50 carotenoid compounds) and/or one or more analogs thereof. In some embodiments, the plurality of agents is or comprises a combinatorial library of small molecule compounds. Combinatorial techniques suitable for synthesizing Small molecules are known in the art, for example, as exemplified by Obrecht and Villalgord, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compounds Libraries, Pergamon-Elsevier Science Limited (1998), and include techniques such as "Mixed and pool" or "Parallel" synthetic techniques, Solid and liquid phase techniques, and coding techniques (see, e.g., Czarnik, Curr. Opin. chem. Bio.1:60-6 (1997)). In addition, many small molecule libraries are commercially available. Many suitable small molecule test compounds are listed in U.S. patent No.6,503,713, which is incorporated by reference herein in its entirety.

In some embodiments, the provided techniques can be used to screen and/or evaluate a variety of agents that encompass a variety of functions (e.g., charge, aromaticity, hydrogen bonding, flexibility, size, side chain length, hydrophobicity, and rigidity).

In some embodiments, a library screened as described herein can comprise multiple types of test compounds. In some embodiments, a given library can include a set of structurally related or unrelated test compounds. In some particular embodiments, the library may comprise a set of peptide or peptidomimetic molecules. In some embodiments, the library may comprise carotenoid compounds, such as naturally occurring or synthetic carotenoids, for example C35, C40, C45, C50, C55 or C60 carotenoids.

In some embodiments, the provided techniques are used to assess groups of reagents that are related to each other by systematically varying the structure of the first reagent; in some such examples, the first agent may be or comprise a compound of known activity (e.g., a carotenoid compound, such as a C50 carotenoid compound).

In some embodiments, the techniques provided herein utilize or generate correlations (i.e., structure-function relationships) between structural features and the presence or absence (or level) of a biological activity of interest. In some cases, the structure-function relationship may be defined empirically; in some embodiments, the structure-function relationship may be defined by using computer modeling and/or analytical prediction methods.

In some embodiments, an anorexia assay as described herein is used. In some embodiments, the test sample is or is derived from (e.g., a sample is taken from) an in vivo model of a disorder described herein. For example, a test compound is applied to a test sample comprising one or more caenorhabditis elegans organisms, and one or more effects of the test compound are evaluated. For example, test compounds can be tested for their ability to combat anorexia in the presence of chemotherapeutic agents or other nausea-inducing stimuli. Alternatively, the effect on a reporter gene (e.g., ABC transporter gene fusion pgp-5p:: GFP) can be assessed by detecting changes in GFP fluorescence. Those skilled in the art will appreciate that other reporter genes can be readily used.

In some embodiments, a compound can be screened by the methods described herein to determine whether it can reduce nausea (e.g., have anti-nausea activity), vomiting (e.g., have anti-vomiting activity), and/or anorexia in a system (e.g., an animal model such as caenorhabditis elegans). In some embodiments, compounds determined to reduce nausea (e.g., having anti-nausea activity), vomiting (e.g., having anti-vomiting activity), and/or anorexia in a system (e.g., an animal model such as caenorhabditis elegans) may be considered candidate compounds. Candidate compounds that have been screened, e.g., in a system having features associated with nausea, vomiting and/or anorexia (e.g., in vivo models of diseases, disorders or conditions associated with nausea and/or vomiting, such as caenorhabditis elegans) and determined to have a desired effect on nausea, vomiting and/or anorexia may be considered candidate therapeutics. In some embodiments, candidate therapeutic agents may be tested in a larger animal model or clinical setting. Once a candidate therapeutic agent is screened in a clinical setting, the candidate therapeutic agent can be a therapeutic agent. Candidate compounds, candidate therapeutic agents and therapeutic agents may optionally be optimized and/or derivatized and formulated with physiologically acceptable excipients to form pharmaceutical compositions.

A compound can be evaluated by the methods described herein to determine whether it can reduce nausea (e.g., have anti-nausea activity), vomiting (e.g., have anti-vomiting activity), and/or anorexia in a system (e.g., an animal model such as caenorhabditis elegans). In some embodiments, the present disclosure provides methods for evaluating a compound (e.g., a carotenoid) to determine its ability to reduce nausea (e.g., anti-nausea activity), vomiting (e.g., anti-vomiting activity), and/or anorexia in a system (e.g., an animal model, such as caenorhabditis elegans). In some embodiments, the method of evaluating a compound to determine its ability to reduce nausea (e.g., anti-nausea activity), vomiting (e.g., anti-vomiting activity), and/or anorexia in a system (e.g., an animal model such as caenorhabditis elegans) is part of an assay (e.g., release test, stability test, efficacy test, etc.) performed for approval or maintenance approval by a regulatory agency (e.g., the united states food and drug administration, the european drug administration, etc.). In some embodiments, a method of evaluating a compound (e.g., a carotenoid) to determine its ability to reduce nausea (e.g., anti-nausea activity), vomiting (e.g., anti-vomiting activity), and/or anorexia in a system (e.g., an animal model such as caenorhabditis elegans) is part of a manufacturing process. In some embodiments, the assessment may be performed as part of a screening method. In some embodiments, the system may be an animal system. In some embodiments, the animal system is a model system, such as caenorhabditis elegans, cat, dog, ape, or pig.

The compounds evaluated to determine reduction of nausea (e.g., with anti-nausea activity), vomiting (e.g., with anti-vomiting activity) and/or anorexia in a system (e.g., an animal model such as caenorhabditis elegans) can be systematically varied, e.g., using rational design, to optimize binding affinity, avidity, specificity or another parameter. Such optimizations can also be screened and/or evaluated using the methods described herein. In some embodiments, the method comprises screening a first library of compounds using one or more steps known in the art and/or described herein, e.g., identifying one or more hits in the library, subjecting the hits to systematic structural changes to create a second library of compounds structurally related to the hits, screening the second library using the methods described herein, or a combination thereof. Thus, in one embodiment, the method comprises screening a first library of compounds using one or more steps known in the art and/or described herein, e.g., identifying one or more hits in the library, subjecting the hits to systematic structural changes to create a second library of compounds structurally related to the hits, screening the second library using the methods described herein, or a combination thereof.

Determining that an evaluated compound that reduces nausea (e.g., has anti-nausea activity), vomiting (e.g., has anti-vomiting activity) and/or anorexia in a system (e.g., an animal model such as caenorhabditis elegans) can be considered a candidate therapeutic compound for reducing nausea, vomiting and/or anorexia as described herein. A variety of techniques for determining the structure of a compound may be used in the methods described herein, such as NMR, mass spectrometry, gas chromatography equipped with an electron capture detector, fluorescence and absorption spectroscopy. The present disclosure provides the following: compounds that reduce nausea (e.g., have anti-nausea activity), emesis (e.g., have anti-emesis activity), and/or anorexia in a system (e.g., by the methods described herein) are identified as useful in methods of treating, preventing, or delaying the development or progression of a disease, disorder, and/or condition described herein. The present disclosure provides the following: compounds that reduce nausea (e.g., with anti-nausea activity), vomiting (e.g., with anti-vomiting activity), and/or anorexia in a system (e.g., by the methods described herein) are identified for use in methods of treating nausea, vomiting, and/or anorexia as described herein.

Compounds evaluated to determine reduction of nausea (e.g., anti-nausea activity), vomiting (e.g., anti-vomiting activity), and/or anorexia in a system (e.g., caenorhabditis elegans) can be evaluated in a second system (e.g., a large animal such as a ape, monkey, cat, dog, pig, etc.). Animals can be monitored for changes that occur due to the presence of the compound (e.g., reduced nausea or anorexia). In some embodiments, the system is a human, e.g., a human with CINV or RINV, and the parameter is reduced severity, frequency, or duration of nausea, vomiting, and/or anorexia.

Examples

The embodiments provided herein are illustrated in the following examples, which do not limit the scope of the disclosure or claims.

Materials and methods

The following materials and methods were used in the following examples.

Bacterial strains

N2 Bristol is the wild type strain used.

The following strains and mutant alleles were used:

SJ4100[zcIs13[hsp-6：：GFP]，

WE5172[ajIsI(pgp-5：：gfp)X]，

JG16[eft-3(q145)/hT2[bli-4(e937)let-？(q782)qIs48](I；III)；ajIsl(pgp-5：：gfp)X]，

JG20[ajls1(pgp-5：：gfp)；agEX(pvha-6：：mcherry：：zip-2+myo-2：：gfp)]，

AY101[acls101[F35E12.5P：：GFP+rol-6(su10006)]

SJ4005[zcIs4[hsp-4：：GFP]V]，

SJ4100(zcls13[hsp-6：：GFP])

growth and treatment of the microorganisms used

The 16S ribosomal sequence was amplified and sequenced using specific primers to identify the microorganism. LB medium and plates were used for the cultivation of Coccocus radicicola, Arthrobacter research team and Corynebacterium glutamicum and mutants thereof. 500ml of overnight culture was inoculated onto SK culture plates and incubated at room temperature for 2 days before starting the experiment. For experiments involving wild type cocklebur rhizophila and mutants, wild type research team arthrobacter and corynebacterium glutamicum and mutants, synchronized L1 larval stage animals were grown in escherichia coli OP50 inoculated plates until the L4 larval stage or first day of adulthood and washed at least five times in M9 buffer before being transferred to appropriate bacterial diet.

Drug treatment

Hygromycin diluted to the desired concentration in M9 solution was added to the NGM plate containing e.coli OP50 bacteria. Stock solutions of emetine or cisplatin were diluted in M9 and the desired concentration was added to NGM plates containing e.coli OP50 bacteria. 750 μ g/ml of the extract of Corkspora radicicola was added to NGM plates containing E.coli OP50 bacteria, containing the appropriate concentrations of hygromycin or cisplatin or emidine. For xenobiotic experiments, synchronized L1 stage animals were dropped onto drug-containing plates and scored after 4 days.

RNAi assay

For RNAi assays, synchronized L1 larval stage animals of the appropriate genotype were fed with the appropriate RNAi clones until they reached the first day of adulthood. Subsequently, the RNAi-treated animals were washed at least five times in M9 to remove e.coli and transferred to a cocklebox rhizophila or e.coli OP50 inoculation plate.

Microscopic examination

The nematodes were mounted on agar pads and images were taken using a Zeiss AXIO Imager Z1 microscope equipped with a Zeiss AXIO cam HRm camera and Axiovision 4.6(Zeiss) software. All fluorescence images displayed in the same graphic panel were collected together using the same exposure time. The image was converted to an 8-bit image, thresholded and quantized using ImageJ. Statistical significance was determined using student's t-test. Low-magnification brightfield and GFP fluorescence images were acquired using Zeiss AxioZoom V16 equipped with a Hammamatsu Orca flash 4.0 digital camera and using Axiovision ZEN software.

Multiple alignment of protein sequences

Multiple alignments were performed using Clustal Omega software.

Mutant screening of cocklebur bacteria EMS

Mutagenesis was performed by treating overnight cultures of C.radicicola in PBS for 45 minutes at 37 deg.C with 50mM EMS. Serial dilutions of the mutagenized cultures of c.radicophilus were plated on LB medium plates, and approximately 2000 colonies of the mutagenized bacteria were picked and grown in LB solution. Before starting the experiment, 500ml of overnight culture was inoculated onto SK culture plates and incubated at room temperature for two days. Eft-3(q145) of synchronized L1 larval stages; pgp-5p gfp animals were grown in E.coli OP50 inoculation plates until the larval stage of L4 or the first day of adult, and washed at least five times in M9 buffer before transfer to the bacterial food of the C.rhizophilus mutant. For GFP induction, plates were visually screened after 24 hours.

Identification of EMS-induced mutations by Whole genome sequencing

Genomic DNA extraction, library preparation, Illumina MiniSeq Sequencing and biological information were all performed at the Sequencing Center (Sequencing Center) located in Fort Collins, Colorado.

Isolation of carotenoids from Cockerella radicophila

Carotenoids were isolated from C.rhizophilus as described (Giuffrida et al, 2016) with the following modifications. After centrifugation at 4000RPM for 15min, the cultures of c.radicicola grown in LB solution were washed with an equal volume of water. After centrifugation to remove water, an equal volume of acetone was added and centrifuged again at 4000RPM for 15 min. After removal of acetone, the samples were wrapped in aluminum foil and protected from light, and the bacterial pellet was extracted with methanol in a water bath at 65 ℃. The sample was extracted several times with methanol until all cells were destained (bleached). The supernatant was filtered through Whatman filter paper No 1. To the methanol extract was added two volumes of 15% sodium chloride and after mixing an equal volume of hexane was added. The yellow carotenoids were separated from the methanol-salt mixture and accumulated in the hexane fraction. The hexane fraction was removed and washed with water at least 3 times. The hexane fraction was evaporated and the resulting carotenoid precipitate was dissolved in methanol.

High performance liquid chromatography

Crude methanol extracts were separated on an Agilent Eclipse Plus c 184.6 x 250mm column (with a particle size of 5 microns) using Agilent 1200HPLC equipped with a diode array detector, autosampler, column oven, solvent degasser and binary pump. The mobile phase comprises (A) water and (B) methanol, and the flow rate is 2 mL/min. The column was pre-equilibrated with 90% B at 40 ℃ before sample injection. After injection, the column was washed isocratically at 90% B for 5min, then ramped up to 100% B within 5 min. The absorbance spectrum of the eluent is monitored from 300nm to 700 nm.

Pull-out (Pull down) experiments with rat liver

To identify carotenoid-binding proteins, the protocol of Pilbrow et al (2014) was used with the following modifications. The protein extract was obtained by cutting about 10 g of adult rat liver into small pieces and homogenizing using Tissue-in T-PER Tissue protein extraction reagent containing Roche protease inhibitor (Roche protease inhibitor). The extract was defatted using methanol-chloroform. Approximately 10mg of carotenoid from Coccocus radicicola were incubated with 1 g of protein extract for one hour at 22 ℃. Unbound carotenoids were removed by size exclusion chromatography using Bio-spin P-6(6K MWCO). The carotenoid-binding protein extract was supplied to a DEAE anion exchange agarose resin column, which was pre-equilibrated at 4 ℃ with anion exchange buffer A (0.05M sodium hydrogen phosphate, pH 8.0). The sample was allowed to flow through the column under gravity and the column was washed with anion exchange buffer a. The protein was eluted with 0.5ml volumes of 0.5M NaCl. The yellow fraction was collected, dialyzed to remove salts, and concentrated using a Vivaspin centrifugation concentration column. Yellow fractions were separated on 4% -12% native PAGE and visible orange bands were excised and subjected to mass spectrometry for identification of proteins.

Putative carotenoid biosynthesis clusters from microorganisms the putative carotenoid biosynthesis clusters of the following bacteria are very similar and of similar size to the genome of the bacteria rhizophilus show the same organization:

leifsonia xyli (Lxx15630, Lxx15620, Lxx15610, Lxx15600, Lxx15590 and Lxx15580) (Monteiro-Vitrello et al, 2004), Microbacterium rubrum (Microbacterium testaceum) (MTES _3133, MTES _3132, MTES _3131, MTES _3130, MTES _3129 and MTES _3128) (Morohoshi et al, 2011), Cellvibrio gilvus (Celgi _1516, Celgi _1515, Celgi _1514, Celgi _1513, Celgi _1512 and Celgi _1511) (Christopherson et al, 2013), Cellulomonas faecalis (Cellulomonas) (Celluodenfi _3171, Celf _3170, Celf _3169, Celf _3168, Cellsf _3168, and Cellsberg _ 319) (Jhav _ 0335089, Jhav _ 039, Jydenrea _ 0855, Skynrea _ 0855, Jordan _ 319, Jocke _ RS # 3, Jocke _ 0855, Skynrea, Jocke _033 _0339, and the like) (Mycole _ 085, Skynrv _033 _ 0855, Skynrea _ 0855, Skyd _ 088, Hsancombinations thereof) (My _ 088, Hsancombinations thereof, Hsanillustrating, Hsancombinations of fungi) (Polyporus, 12780, Hsancombinations of Myoden _ 085, III, 3, III, D9V29_ RS08885 and D9V29_ RS08890), Euonymus micranthoides (Mycetocola midauisis) (BM197_ RS02470, BM197_ RS02475, BM197_ RS02480, BM197_ RS02485, BM197_ RS02490 and BM197_ RS02495), psychrobacterium chillatum (Cryobabacter psychroloerans) (BLQ39_ RS02180, BLQ39_ RS02185, BLQ39_ RS02190, BLQ39_ RS02195, BLQ39_ RS02200 and BLQ39_ RS 05), psychrophilum north benthamatum (Subticola boreus) (B7R21_ RS 95, B7R21_ RS02700, B7R21_ RS02705, B7R21_ RS 02772, B21, HS2RS 07055, RS07002 _ RS 3655, RS3655, RS3672, RS07055, 3655, 21, 3655 RS 3655, 21, 3655, 21, 3655, 3675, 368 RS21, 3655, 3675, 368 RS 3655, 21, 368 RS21, 3655, 21, 368, 3655, 21, 3655, 368, 21, 3655, 21, and 368. multidrug-RS 368. multidrug-3. multidrug-RS 0708, and 21 (HSbloodr 21, and 21, and 21. multidrug-3. multidrug-RS 0708. multidrug-21, and 21 (HSbloodr 0708. multidrug-RS0708. multidrug-RS 0708. multidot, B4U78_ RS09540 and B4U78_ RS 09545);

plant Bacillus H (plantabacter sp.H53) (A4X _ RS18565, A4X _ RS18570, A4X _ RS18575, A4X _ RS18580, A4X _ RS18585 and A4X _ RS18590), Bacillus pumilus (Curtobacterium sp.) (ASF _ RS14315, ASF _ RS14320, ASF _ RS14325, ASF _ RS14330, ASF _ RS14335 and ASF _ RS14340), Microtricola pindaiensis (GY _ RS04745, GY _ RS04750 MBR GY _ RS04755, RS 378_ RS04760, GY _ RS04765 and ASF 04770), Geobacillus (Frondihbecome bits anse sp.) (EDF _ RS08000, EDF _ RS08005, EDF _ RS 378, EDF _ RS08010, SABDRS 08015, SAM _ 0800606060606060620, SAM _ RS 06220, SANDRIBEN0629620, SANDERN 0629620, SANDERN 062969, SANDERN 0629620, SANDERN 062969, SANDERN 0629620, SANDERS 969, SANDERN 062969, SANDERN 0629620, SANDERN 062969, SANDERS, SANDERN 062969, SANDERN 250, and SANDERN 062969, SANDERN 250, SANDERN 0629620, SANDERN 250, SANDERN # and SANDERN 250, SANDERN # ASF, BN2404_ RS04375, BN2404_ RS04380, BN2404_ RS04385, BN2404_ RS04390 and BN2404_ RS04395), Cryobacillus plunkundans (GY21_ RS00565, GY21_ RS00570, GY21_ RS00575, GY21_ RS00580, GY21_ RS00585 and GY21_ RS00590), Microbacterium oxydans (Microbacterium oxydans) (RN51_ RS07325, RN51_ RS07330, RN51_ RS07335, RN51_ RS07340, RN51_ RS07345 and RN51_ RS07350), Arthrobacter luteus (Arthroteluteolus) (AL3_ RS02570, AL 5_ RS02575, AL 5802580, RN 3_ RS02585, CJ 03028 13458, CJ 03028 _ RS028 03028 028 03028 35028 03028 35028 028 03028 35028, CJ 03028, CJ 03028 48 _ RS028, CJ 03028 0358, CJ — 028 03028 48 _ RS028 48, CJ 48 RS028 48, CJ 03028 48 RS028 48, CJ 48 RS028 48 _ RS028 48, CJ 48 RS028 48, CJ and CJ 03028 48 RS028 03028 48, CJ 03028 48 RS028 03028 48 _ RS028 48, CJ 03028 48, CJ 48 _ RS028 03028 48, CJ 03028 48 _ RS028 48, CJ 03028 48, CJ 48 _ RS028 48, CJ and CJ 03028 48 _ RS028, CJ and CJ 48 _ RS028 48, CJ 48 _ RS028 48, CJ 48 _ RS028 48, CJ _ RS028 48, CJ 48 _ RS028 and CJ 48 _ RS028 and CJ 48, CJ 48 _ RS028 and CJ 48 _ RS028 48 _ RS028 03028 _ RS028 48, CJ 48 _ RS028 and CJ 48 _ RS028 03028 48, CJ 03028 48 _ RS028 03028 _ RS028 48, CJ and CJ 48, CJ _ RS028 48 _ RS028 and CJ 48 _ RS 028;

microbacterium Paraoxydans (Microbacterium paraoxydans) (SAMN04489809_1122, SAMN04489809_1123, SAMN04489809_1124, SAMN04489809_1125, SAMN04489809_1126 and SAMN04489809_1127), Agrobacterium subbetticus (H521_ RS21795, H521_ RS21800, H521_ RS0106365, H521_ RS0106370, H521_ RS21805 and H521_ RS0106380), Arthrobacter crystallopoiides (Arthrobacter crystaphylopoides) (D477_ RS18370, D477_ RS18375, D477_ RS18380, D477_ RS 477_ 18385, D _ RS18390, D477_ RS18395), Georgenesporanyaya (DSZ 7_ RS 3535048, D477_ RS031 _ 0418385, D _ RS 350413835, KR64048, KR 640435, KR 64048, KR RS13815, KR 64048, KR 0335048, KR 640435, KR RS 35033503350335048, KR RS13815, KR 64048, KR RS 3503640420, KR RS 35035, KR RS 350364048, KR RS 3515, KR RS 350364048, KR RS 35035, KR RS 353515, KR RS 350364048, KR RS 3515, KR RS 350364048, KR RS 3515, KR RS 350364048 and KR RS 3515, KR RS 350364048, KR RS 3503640435, KR RS 3515, KR RS 350364048, KR RS 3515, KR RS 350364048, KR RS 3515, KR RS 350364048, KR RS 35035, KR RS 350364048, KR RS 3515, KR RS 350364048, KR RS 35035, KR RS 350364048, KR RS 3515, KR RS 350364048 and KR RS 350364048, KR RS 35035, KR RS 3515, KR RS 64048, KR RS 350364048, KR RS 3515, KR RS 35035, KR RS 3515, KR RS 35035, KR RS 350364048 and KR RS 64048 and KR RS 35035, KR RS 64048, KR RS 3515, KR RS 64048, KR RS 35035, KR RS 64048, KR RS 350364048, KR RS 3515, KR RS 35035, KR RS 3515, KR RS 64048 and KR RS 64048, KR RS 35035, KR RS 64048, KR RS 3515, KR RS 64048, KR RS 35035, KR RS 3515, KR RS 64048 and KR RS 3515, KR RS 64048 and KR RS 3515, KR RS, Agrobacterium cerinus (BUR _ RS12060, BUR _ RS12065, BUR _ RS12070, BUR _ RS12075, BUR _ RS12080 and BUR _ RS12085), Agrogrisella grassland (Agreiia pratensis) (B9Y _ RS06900, B9Y _ RS06905, B9Y _ RS06910, B9Y _ RS06915, B9Y _ RS06920 and B9Y _ RS06925), Microbacterium laeviformis (Microbacterium laeviformes) (OR221_3062, OR221_ 0313, OR221_3064, OR221_3065, OR221_3066 and OR221_ 0310310310317), Arthrobacter tristearnebacterium (Arthrobacter stanebrancidinii) (CVV _17785, CVV _17790, CVV _17780, CVV _17800 and CVV 1930335, CVbacterium 0319335, CVQ _ RS 0335, ADthrobacter sakupfrogans (ADiavor 0335, ADthrobacter sayides), Microbacterium strain 19335, ADthrobacter sayingrs 0335, ADthrobacter sayingrs 19335, ADthrobacter saxatilis 0335, CVn RS19335, and ADthrobacter sayingrs 0335) (CViazu _ RS19335, ADthrobacter sayingrs 0335, ADthrobacter sayingrs 19335, CV19335, ADthrobacter sayingrs 0319335, ADthrobacter sayingrs 031031031031031 and ADyingrs 19335), Microbacterium sp AOP _ RS09035, AOP _ RS09040, AOP _ RS09045, AOP _ RS09050 and AOP _ RS09055), Pleurotus ostreatus (ZHIHengliuella halolorans) (CUR _ RS12685, CUR _ RS12690, CUR _ RS12695, CUR _ RS12700, CUR _ RS12705 and CUR _ RS12710), Paenigliticum antarcticas (BN2261_ RS08280, BN2261_ RS08285, BN2261_ RS08290, BN2261_ RS08295, BN2261_ RS 915, BN 22622622622600 and BN2261_ RS08305), Pleurotus citrinopileatus (Janibacterium meloronis) (EEW _ RS00715, EEW _ RS00720, EEW _ RS00725, EEW _ RS00730, EEW _ RS 0635 and CVW 06060600740), Microbacterium arborescens (EERS 02250, CVRS 02250, CVjerseus 0239, CVjerseus 02355, CVjerusabungium sakus 0279, CVjerusabunyas 0279, CVjerusabuns 0279, CVjerusabunia sakus 0279, CVjerusabunyas 0279, CVtoprs 007028rs 0070285, CVjerusabunia 19, CVjerusabunia sakus 0279, CVtoprius 0275, CVtoprs 0279, and CVtoprs 0275, CVtoprs 028rs 19, CVtoprs 19, CVtopus 0275, CVtoprs 19, CVtopus 0275, CVtopus sakurB _ RS 028rs 19, CVtopus sakurZ _ RS 028rs 19, CVtopus 0275, CVtopus sakurB, CVtoprs 19, CVtopus 0275, CVtopus sakurB _ RS 028rs 19, CVtopus sakurB _ RS 0285, CVtopus sakurB, CVtopus sakurZ _ RS 0285, CVtopus 0275, CVtopus 19, CVtopus sakurB, CVtopus 0275, CVtopus sakurB, CVtope RS 028RS 0285, CVtopus 0275, CVtopus 19, CVtopus sakurB, CVtopus sakurZ _ RS 0285, CVtopus sakurZ _ RS 028RS 0285, CVtopus sakurK _ RS 0285, CVtopus sakurB, CVtopus sakurK # and CVtopus 19, CVtopus sakurB, CVtopus sakurK _ RS 028RS 0285, CVtope, Samtricola pindarriensis (GY24_ RS04745, GY24_ RS04750, GY24_ RS04755, GY24_ RS04760, GY24_ RS04765 and GY24_ RS04770), Microbacterium indicum (H576_ RS15860, H576_ RS0112930, H _ RS0112935, H576_ RS0112940, H576_ RS0112945 and H576_ RS15865), homoneinimononsp (DL891_ RS01870, DL891_ RS01875, DL891_ RS01880, DL891_ RS01890 and DL891_ RS 0189895), cryobacter sphaeroides (sambuccopsorallinum) (SAMN 162 11068, SAMN 11011074, sambuc 11011005276, sambuc 16280, sambuc 16205203, 16205276, 16245, 16286880, 16286876, 1624178 and 16205280), 16203, 1625, 16203, 1625 and 16203, 1625, 16203, 1625, 16203, 162five, 1625, 162five, and 0528, five, ten, five, ten, five, ten, five, ten, five, ten, five, ten, five, ten, five, ten, five, ten, five, ten, five, ten, five, ten, xylomyces sambucus (BLS _ RS13650, BLS _ RS13655, BLS _ RS13660, BLS _ RS13665, BLS _ RS13670 and BLS _ RS13675), Krasilnikoviella flava (B5Y _ RS20515, B5Y _ RS20520, B5Y _ RS20525, B5Y _ RS20530, B5Y _ RS20535 and B5Y _ RS20540), Actinothera siderophyllum (Actinothera ferrorae) (N866_01505, N866_ RS 866_01515, N866_, N _ ubiA), Lysinimicbium soli (AOM _ RS11780, M _ RS11785, M _ RS11790, AOM _ RS11795, AOM _ RS _ KR-RS-KR-and AOM-RS 11899, Sp _ AOK _ AORS 13835, PRO _ RS13835, Prochlora _ RS 082-RS 138300, Klauspiceus RS 1387, Klausura RS 0980, Kl _ RS-KR-RS-O-KR-O-RS-O-K-O-K-O-R-K-O-K-R-K-O-K-R-O-K-O-K-R-O-K-O-K-O-K-O-K-O-K-O-R-O-R-O-R-O-R-O-R-O-R-O-R-O-R-O-R-, RM50_ RS01680, RM50_ RS01685, RM50_ RS01690, RM50_ RS01695 and RM50_ RS01700), Acritices phytoseiuli (C501_ RS0107225, C501_ RS0107230, C501_ RS0107235, C501_ RS0107240, C501_ RS0107245 and C501_ RS0107250), Leuconobacter muraum (AMS67_ RS10795, 67_ RS10800, AMS67_ RS10805, AMS67_ RS10810, AMS67_ RS10815 and AMS67_ RS10820), Microbacterium avium beijing (Ornithicum parabikingense) (K330_ RS19765, K330_ RS0107130, K330_ RS 70, K330_ RS 75, K _ 19771330 _ RS 7145, Citrobacter 7113713713713748, TRIAARI 2513755, TRI _ RS 01013748, Morus _ RS 13748, Morus _ RS 01013748, Moraxe _ RS 13755, Morchella _ RS 01013755, and TRI _ RS 2570) (research team AARI 2513755, TRI RS 010RS 13755, TRI RS 2570, TRI _ RS 13750, TRI RS 01020). The decaisoprenoid flavin-producing gene cluster in Corynebacterium glutamicum (cg0723, cg0721, cg0720, cg0719, cg0718 and cg0717) (Kalinowski et al, 2003) and Corynebacterium efficiens (HMPREF0290_1086, HMPREF0290_1088, HMPREF0290_1089, HMPREF0290_1090, HMF 0290_1091 and HMF 0290_1092) (Nishio et al, 2003) is similar in size and tissue to that of Corynebacteria rhizophila, except for the unrelated gene cg0722 inserted between crtE and crtB in Corynebacterium glutamicum or HMPREF0290_1088 in C efficiens. In addition, in Corynebacterium glutamicum, additional carotenoid clusters (NCgl0600, NCgl0598, NCgl0597, NCgl0596, NCgl0595, and NCgl0594) are also present in the genome. In the species dermatophytes (Kytococcus sedentarius), the genes responsible for carotenoid production (Kseed _13840, Kseed _13830, Kseed _13820, Kseed _13810, Kseed _13800) are arranged in the same cluster, while Kseed _16070, which encodes a geranylgeranyl pyrophosphate synthase, is located elsewhere in the genome (Sims et al, 2009).

In Brevibacterium micheliae (Brevibacterium mcbrellineri), the genes responsible for carotenoid production (HMPREF0183_0793, HMPREF0183_0794, HMPREF0183_0795, HMPREF0183_0796 and HMPREF0183_0797) are located in the same cluster, while HMPREF0183_0437 encoding polyprenyl synthetase is located elsewhere in the genome.

In Beutenbergia calvernae, the genes responsible for carotenoid production (Bcav _3492, Bcav _3491, Bcav _3490, Bcav _3489, Bcav _3488) are located in the same cluster, while Bcav _0970, which encodes a polyprenyl synthetase, is located elsewhere in the genome (Land et al, 2009).

In Brachybacterium faecium (Bfae _04470, Bfae _04440, Bfae _04430, Bfae _04420, Bfae _04410 and Bfae _04400) (Lapidus et al, 2009), carotenoid biosynthetic clusters are similar in size and organization to the bacteria cockleborum rhizophilus except for the insertion of two unrelated genes, Bfae _04460 and Bfae _04450, between Bfae _04470 and Bfae _ 04440.

In Cellulomonas flavigena (Cfla _2888, Cfla _2889, Cfla _2890, Cfla _2891 and Cfla _2892) (Abt et al, 2010), all genes responsible for carotenoid production are present except Cfla _2893 (which may be a pseudogene due to a frameshift mutation).

Decaprenylflavins is the first C50 carotenoid found from Flavobacterium dehydrogenans (now known as Agrobacterium mesenulus (Liaaen-Jensen et al, 1968)).

Various bacteria are known to produce decaprenylflavins, including: agrobacterium Miylanica (Agromyces mediolanus) (Liaaen-Jensen et al, 1968), Aureobacterium (Aureobacterium sp.) (Fukuoka et al, 2004), Arthrobacter glacialis (Arpin et al, 1975), Arthrobacter research team (Arthrobacter arihatensis) (Sutthiwong et al, 2014), Cellulomonas bizotea (Weeks et al, 1980), Citrioccus sp and Corynebacterium glutamicum (Krubakik et al, 2001).

Example 1 monitoring of translational defects by caenorhabditis elegans is inhibited by bacterial carotenoids

Of the approximately 500 caenorhabditis elegans xenobiotics detoxification genes (e.g., caenorhabditis elegans ABC transporter gene pgp-5), a specific set of cytochrome p450, ABC transporter, UDP-glycosyltransferase genes can be induced for expression by toxin, RNAi, or by mutation suppression of ribosomal proteins, tRNA synthetases and other genes involved in translation (Govindan et al, 2015). Even if the translational defect is restricted to the germ line, for example in the caenorhabditis elegans eft-3(q145) mutant (mutation in the germ line isoform of translational elongation factor-1, where translation in somatic and somatic development is normal, but translation in germ cells is disabled and germ cells do not proliferate), the ABC transporter gene in the intestinal tract is fused pgp-5p:: expression of gfp is strongly induced when animals are fed benign E.coli OP50 (FIG. 5A). To C.elegans eft-3(q 145); pgp-5p: gfp feeding Corkerobacter radicophilus (but not E.coli) disrupted the normal induction of pgp-5p: gfp (FIGS. 5A & 5B). The bacteria, Cockerella radicophila, are gram-positive cocci of the phylum Actinomycetes, which is a clade rich in the biosynthetic pathway of drugs. The species Cockera rhizophilus are found in various niches, including the soil and the caenorhabditis elegans gut microbiome from a natural population of nematodes from orchards (Felix et al, 2010). The rhizophilus species are normal inhabitants of the skin and mucous membranes of humans and animals, but may be associated with human infections.

To establish that cocklebur bacteria can suppress monitoring of a range of translational defects, RNAi-induced xenobiotic detoxification responses to other ribosomal proteins were tested. Concurrent larval stages of L1 pgp-5p: gfp animals were fed with E.coli expressing either RPL-1dsRNA or VRS-2dsRNA, which inhibited the production of C.elegans RPL-1 ribosomal protein and VRS-2tRNA synthetase. When these animals reached adulthood, they were transferred to plates containing E.coli OP50 or plates inoculated with C.rhizophilus and scored for GFP induction after 24 hours. In animals fed vrs-2dsRNA or rpl-1dsRNA and transferred to plates containing E.coli OP50, pgp-5p:: gfp was induced. In contrast, in animals fed rpl-1dsRNA or vrs-2dsRNA and transferred to a C.rhizophilus plate, pgp-5p:: gfp expression was disrupted (FIG. 5C & FIG. 5D). Since Cockerobacter does not suppress the induction of mitochondrial stress response or endoplasmic reticulum stress response, suppression of the monitoring pathway by Cockerobacter is specific for translation stress (Govindan et al, 2015).

In order to identify a mutant strain of C.elegans (eft-3(q145)) that is responsible for inhibiting the pgp-5p:: gfp-induced C.radicophila pathway, a mutant strain of C.radicophila that is defective in inhibiting pgp-5p:: gfp-induction was forward genetically screened for a strain carrying a translational genetic defect (C.elegans). Eft-3(q145) was fed about 2000 individual strains of Corkspora radicicola that grew normally on bacterial LB plates after EMS mutagenesis; pgp-5p, gfp animal. These 2000 individual wells of different mutant strains of Coccocus radicicola were screened at eft-3(q 145); pgp-5p:: gfp animal failed to suppress pgp-5p:: gfp-induced mutant bacterial strain. Six mutant strains of the cocklebur rhizophilus are identified to fail to reach eft-3(q 145); pgp-5p:: gfp was induced in gfp animals (FIG. 5A & FIG. 5E). Visual inspection showed that all of these mutant strains were deficient in colony pigmentation compared to wild type cocklebur i (fig. 5F). Wild type cocklebox is yellow and six mutant colonies are red or white or orange. Using this discolouring phenotype, we visually screened about 500,000 bacterial colonies generated by EMS mutagenesis to screen for mutants having a discolouring phenotype. We isolated 71 discoloured mutants (fig. 5G). For eft-3(q 145); GFP-5 p GFP animals tested 71 mutants generated in the same EMS mutagenesis along with 25 control non-color-changing mutants and scored for GFP induction. All 71 discolouring mutants failed to suppress pgp-5p:: gfp induction in the C.elegans eft-3(q145) mutant, while 25 normally coloured strains suppressed pgp-5p:: gfp induction (FIG. 6A; FIG. 1A-FIG. 1C).

Genomic sequencing of the mutants of C.radicophilus that failed to suppress pgp-5p:: gfp-induced mutagenesis of 23 EMS in the eft-3(q145) mutant showed that each mutant carried a mutation in one of the six carotenoid biosynthesis cluster genes (FIG. 6B; FIG. 1C-FIG. 1E). Carotenoids are yellow to red pigments produced by the terpene biosynthetic pathway. The genome of kocuria rhizophila contains an operon encoding the predicted carotenoid (crt) biosynthesis genes (Takarada et al, 2008). These include crtE (KRH _ 20850; encoding GGPP synthase), crtB (KRH _ 20840; encoding phytoene synthase), crtI (KRH _ 20830; encoding phytoene desaturase), crtEb (KRH _ 20800; encoding lycopene elongase), crtYe (KRH _ 20820; encoding C)₅₀Carotenoid epsilon cyclase) and crthyf (KRH _ 20810; code C₅₀Carotenoid epsilon cyclase) (FIG. 1C-FIG. 1E). Based on homology, it was predicted that the reactions catalyzed by GGPP synthase CrtE, phytoene synthase CrtB and phytoene desaturase CrtI mediate steps in lycopene production (Klassen et al, 2010; Krubasik et al, 2001). CrtEb and CrtYe/f cyclases catalyze C₅₀The biosynthesis of carotenoids from lycopene. C₅₀Carotenoids are rare in nature and very few of them are characterized (Krubasik et al, 2001 a; Krubasik et al, 2001 b; Norgard et al, 1970; Tao et al, 2007; Netzer et al, 2010).

Although there are many microorganisms containing CrtEb and CrtYe/f genes (FIG. 7), the only genetic and biochemical genes to date areWell characterized C₅₀The carotenoid is decaisoprenol from Corynebacterium glutamicum (Heider et al, 2012). In C.glutamicum, the enzymes CrtEb, CrtYe and CrtYf convert lycopene to C₅₀The carotenoid decaprenylflavin (Krubasik et al, 2001 a; Krubasik et al, 2001b) (FIG. 1E). The reaction proceeds in two steps: CrtEb catalysis C₄₀Elongation of acyclic lycopene to acyclic C₅₀The carotenoid Ranunculin (Krubasik et al, 2001 a; Krubasik et al, 2001 b). The products of CrtYe and CrtYf combine to form C₅₀Cyclase, then catalyse C₅₀The carotenoid Ranunculin is converted to decaprenylflavin (Krubasik et al, 2001 a; Krubasik et al, 2001 b). The bacteria of the species Cockera rhizophila crtYe and crtYf have 38% and 34% identity with the bacteria of the species Corynebacterium glutamicum crtYe and crtYf, respectively. Thus, the yellow pigment produced by Coccocus radicophilus may belong to decaprenylflavin C₅₀-subfamily.

From the genetic screen of cocklebur bacteria, multiple mutations were obtained in crtI encoding phytoene desaturase, including six missense mutations (E21, E11, E23, E14, E5 and E13) and two nonsense mutations (E15 and E10) (fig. 1C-fig. 1E). CrtI catalyzes the conversion of colorless phytoene to red lycopene. All these crtI mutants were white colonies (fig. 6B; fig. 1C), similar to the corynebacterium glutamicum Δ crtI mutant (Heider et al, 2012) (fig. 6C), and therefore may lack lycopene synthesis. Six missense mutations are located in highly conserved residues, suggesting that they may be important for protein function (FIG. 8). E4, E6 and E8 are missense mutations in the crtB gene, which encodes phytoene synthase (fig. 1D & fig. 1E). All of these missense mutations are located in highly conserved residues, suggesting that they may be important for protein function (FIG. 9). These mutants produced white bacterial colonies (FIG. 6B; FIG. 1C) (Heider et al, 2012) as observed in the C.glutamicum Δ crtB mutant (FIG. 6C). Four mutations were obtained in crtEb; two nonsense mutations (e16 and e17) and two missense mutations (e3 and e19) were obtained in the highly conserved residues (FIG. 1D; FIG. 10). Mutations in crtEb may be defective in the conversion of lycopene to fisetin (fig. 1E). These mutants formed reddish colonies (fig. 6; fig. 1B), as observed in the corynebacterium glutamicum Δ crtEb mutant, which was probably due to the accumulation of lycopene (rather than ranunculin) (Heider et al, 2012) (fig. 6C). e17 is an early termination mutation in crtEb that is expected to produce a truncated protein of only 13 amino acids (fig. 10). Mutations in crtYe and crtYf are defective in the last step; homologues of these genes catalyze the synthesis of decaprenylflavins. These mutants produced reddish to orange colonies (FIG. 6B). Interestingly, the corynebacterium glutamicum Δ crtY mutant accumulated ranunculin and also appeared pale orange to red (Heider et al, 2012) (fig. 6C).

Since Corynebacterium glutamicum ATCC13032 is known to produce decaprenylflavins, it was found that the strain at eft-3(q 145); pgp-5p:: gfp animals it was tested whether feeding Corynebacterium glutamicum ATCC13032 represses pgp-5p:: gfp induction. Eft-3(q 145); pgp-5p:: gfp animal feeding wild type Corynebacterium glutamicum ATCC13032 suppressed pgp-5p:: gfp induction (FIG. 2A & FIG. 2B). In Corynebacterium glutamicum ATCC13032, the carotenoid gene cluster CrtE-cg0722-CrtBIYeYfEb mediated decaisoprenol biosynthesis (Heider et al, 2012). Whether the deletion mutant of C.glutamicum ATCC13032 in crtY, crtEb, crtI and crtB could suppress eft-3(q 145); pgp-5p:: gfp induction in gfp animals, these mutants are known to be deficient in decaprenol production (Heider et al, 2012). Unlike the same strain grown on wild-type C.glutamicum, animals fed with. DELTA.crtY,. DELTA.crtEb,. DELTA.crtI,. DELTA.crtB C.glutamicum exhibited normal pgp-5p:: gfp induction (FIGS. 2A & 2B).

Arthrobacter, a research team on colored bacteria, is known to produce decaisoprenoid flavins (Monnet et al, 2010); experiments tested whether Arthrobacter would suppress eft-3(q145) by the feeding research team; pgp-5p:: gfp induction in gfp animals. Eft-3(q 145); pgp-5p:: gfp Arthrobacter, animal feeding study team, also repressed pgp-5p:: gfp expression (FIG. 2C). Thus, corynebacterium glutamicum, arthrobacter research team, or coobacter rhizophilus produced colored carotenoids that mediated repression of translational monitoring of induction of ABC transporter detoxification responses.

Carotenoids (such as decaprenylflavins) are lipophilic molecules that localize to the cell membrane and can be easily extracted in non-polar solvents. Cultures of cocklebox were extracted with such solvents (fig. 11). TLC analysis of the extract showed the presence of yellow-orange pigment (figure 2D). HPLC analysis of methanol extracts from C.rhizophilus combined at least six different components (FIG. 11; FIG. 2E). These peaks were designated peak 1(5.4min), peak 2(6.3min), peak 3(6.5min), peak 4(7.2min), peak 5(7.8min) and peak 6(8.7min) according to the time of elution (fig. 2E). The absorption spectra of the eluted peaks show absorption maxima at 420nm, 440nm and 470nm, which are similar to the published absorption spectra of decaisoprenol from Arthrobacter (Arthrobacter) bacteria (Giuffrida et al, 2016; Sutthiwong et al, 2014).

To analyze the production of carotenoids in different mutants of kocuria rhizophila, carotenoids were extracted from crtI (e10), crtEb (e17), crtYe (e22) and crtYf (e18) as nonsense mutant alleles and crtB (e6) as missense mutants. Spectrophotometric analysis of methanol extracts from wild type cocklebur rhizophilus showed an absorption maximum between 415nm and 425nm, whereas crtEb (e17) extracts showed an absorption maximum between 445nm and 455nm (fig. 12). The methanol extracts of the cocklebox creb (e17) and crtYe (e22) mutants showed similar absorption spectra, while extracts from the cocklebox cre (e10) and crtb (e6) showed no absorption at all. The methanol extract from crthy (e18) showed two separate absorption peaks, one at about 400nm and the other smaller at about 500 nm.

Crude methanol extracts (containing carotenoids) from wild type cocklebur rhizophilus were also tested at eft-3(q 145); pgp-5p: ability to suppress GFP induction in GFP animals. Eft-3(q145) fed with E.coli with control methanol extract; pgp-5p:: animals fed with E.coli supplemented with wild type Cockerella radicophila extract showed significantly reduced pgp-5p:: gfp expression (FIG. 2F). Methanol extracts of wild-type kocuria rhizophila as extracts can rescue the suppression of caenorhabditis elegans monitoring defects of carotenoid biosynthesis mutants of kocuria rhizophila: when eft-3(q145) is fed with a mutant of kocuria radicicola crtEb (e17), crtB (e6) or crtI (e10) supplemented with a wild type extract of kocuria radicicola; pgp-5p GFP was not induced in GFP animals, whereas GFP expression was induced by the C.elegans eft-3 mutation in animals fed with the control extract (FIG. 2G).

A simple explanation for the failure of carotenoid mutants of C.radicicola to suppress pgp-5p:: gfp in translation-deficient C.elegans mutants is that these pigmentation mutants of C.radicicola may induce pgp-5p:: gfp even in the background of wild-type C.elegans. To test this possibility, wild-type C.elegans carrying the pgp-5p:: gfp fusion gene was fed with Cockera rhizophilus crtEb (e17), crtYe (e22), crtYf (e18), crtB (e6) or crtI (e10) mutants. The results show that wild type Cocotomycota radicicola and the carotenoid mutants do not induce pgp-5p:: gfp (FIG. 3A). Another possible explanation is that Cork. rhizophilus feeding may elicit other stress responses from C.elegans, thereby "dispersing" the animal's monitoring of translation in some way. The induction of stressed additional GFP fusion reporter genes was tested in wild-type and various mutants of cocklebur bacteria. hsp-4p:: gfp and hsp-6p:: gfp are endoplasmic reticulum Unfolded Protein Responses (UPR) respectively^ER) And mitochondrial Unfolded Protein Response (UPR)^mito) The reporter gene of (Yoneda et al, 2004; calfon et al, 2002). clec-60 is a C-type lectin/CUB domain protein induced by the gram-positive pathogens staphylococcus aureus (s. aureus) and m.nematophilum (O' Rourke et al, 2006). F35E12.5p GFP is a CUB domain protein induced by Yersinia pestis (Y. pestis), M.nematophilum, and Pseudomonas aeruginosa (P.aeruginosa) (O' Rourke et al, 2006; Troemel et al, 2006; Bolz et al, 2010). Wild type Coccocus radicicola and carotenoid mutants are used to feed caenorhabditis elegans hsp-4p, GFP, hsp-6p, GFP, F35E12.5p, GFP and clec-60p, GFP. Cockerobacterium rhizophilus does not induce hsp-4p:: gfp expression (FIG. 13A). Similarly, wildThe H.rhizophilus or carotenoid mutants did not induce hsp-6p:: gfp expression (FIG. 13B). The wild type Cockerella radicophila or carotenoid mutant did not induce F35E12.5p:: GFP (FIG. 13D). However, the wild type of Kocuria radicicola or the carotenoid mutant induced clec-60:GFP (FIG. 13C), which was induced by gram-positive bacteria. Since Cockerella radicophila is also a gram positive bacterium, induction of clec-60 is likely to be an immune response to the pathogen.

To ascertain whether the effect of Corkspora radicicola feeding on pgp-5p:: gfp-induced repression is reversible, eft-3(q145) fed with Corkspora radicicola will be used after different times; pgp-5p: gfp animals were transferred to E.coli OP50 plates. GFP expression recovered within 12 hours of transfer (fig. 13E).

Since the C50 carotenoids have multiple conjugated double bonds, they may possess antioxidant activity (Edge et al, 1997). However, for a variety of reasons, the ROS quenching properties of carotenoids are unlikely to be responsible for repression of pgp-5p:: gfp induction. First, pgp-5p:: gfp is not induced by oxidative stress (Govindan et al, 2015). Secondly, it was tested in a C.elegans translation-deficient mutant whether the known antioxidant can suppress pgp-5p:: gfp induction. Eft-3(q145) grown on E.coli OP50 with N-acetylcysteine, ascorbic acid, trolox or resveratrol; gfp animals were treated and screened after 50 hours at 20 ℃. Eft-3(q145) treated with a blank-treated (mock-treated); there was no significant difference in pgp-5p:: gfp induction in antioxidant treated animals compared to gfp animals (FIG. 14A). Third, the ability of commercially available carotenoids to suppress pgp-5p:: gfp induction in C.elegans translation-deficient mutants was tested. Eft-3(q145) grown on e.coli OP50 with β -carotene or astaxanthin; gfp animals were treated and screened after 50 hours at 20 ℃. Eft-3(q145) treated with a blank control; there was no significant difference in pgp-5p:: gfp induction in animals fed with this antioxidant compared to animals fed with gfp (FIG. 14B). Finally, eft-3(q145) is paired with E.coli expressing zeaxanthin, neurosporene, violaxanthin, delta-carotene or alpha-carotene; pgp-5p: GFP animals were fed and screened for GFP induction. Eft-3(q145) treated with a blank control; pgp-5p:: gfp animals there was no significant difference in pgp-5p:: gfp induction in animals fed with carotenoid expressing e.coli (figure 14C). None of these carotenoids are the C50 carotenoid.

The cocklebur rhizophila also suppresses the detoxification response to translational inhibitory drugs. Hygromycin is a bacterially produced antibiotic (from Streptomyces hygroscopicus) that inhibits translation and induces xenobiotic detoxification in c. Although hygromycin at 10. mu.g/ml induced pgp-5p:: gfp expression in animals fed E.coli OP50, animals fed Corticiella radicophila and hygromycin at 10. mu.g/ml failed to induce pgp-5p:: gfp (FIG. 3A; FIG. 14D). However, at high concentrations of hygromycin, both pgp-5p:: gfp were induced in animals fed E.coli OP50 and C.rhizophilus (FIG. 14D). In contrast, pgp-5p fed with Cockera rhizophilus crtI (e10) or crtEb (e 17). GFP animals did not affect GFP induction in response to hygromycin treatment (FIG. 3A). Similar results were obtained with emetine which blocks protein synthesis by binding to the 40S subunit of the ribosome. 6.25. mu.g/ml of emistine induced pgp-5p:: gfp expression in animals fed E.coli OP50 (FIG. 15A & FIG. 15B); however, animals fed with Cordymus radicophilus and 6.25. mu.g/ml of emidine failed to induce pgp-5p:: gfp. However, at high concentrations of emitidine, both pgp-5p:: gfp were induced in animals fed E.coli OP50 and C.rhizophilus (FIG. 15A). In contrast, pgp-5p fed Cockera rhizophilus crtI (e10) or crtEb (e17) animals did not affect GFP induction in response to emetine treatment (FIG. 15B). In response to cisplatin-induced genotoxic stress, pgp-5p:: gfp was activated, which interfered with DNA replication. Although 1mM cisplatin induced pgp-5p:: gfp expression in animals fed E.coli OP50, animals fed C.radicophilus and 1mM cisplatin failed to induce pgp-5p:: gfp (FIG. 15C). In contrast, pgp-5p:, fed Cockera rhizophilus crtI (e10), crtEb (e17) or crtYe (e22) GFP animals did not affect GFP induction in response to cisplatin treatment (FIG. 15C).

Since the carotenoid of kocuria rhizophila suppressed induction of xenobiotic detoxification genes of caenorhabditis elegans by translational defects, the ability of the carotenoid of kocuria rhizophila to increase sensitivity of caenorhabditis elegans to translational inhibitors was evaluated. While > 80% of wild type animals fed E.coli OP50 and 10. mu.g/ml hygromycin reached adulthood within four days, < 10% of animals fed E.coli OP50 and 10. mu.g/ml hygromycin and the carotenoid extract of C.rhizophilus reached adulthood within four days (FIG. 3B; FIG. 15D). In the absence of hygromycin, the carotenoids themselves were not toxic to the worms (FIG. 3B; FIG. 15D). Similar results were obtained with emetine: animals treated with the extract of Corkspora radicophila were highly sensitive to emetine compared to animals fed with the control extract (FIG. 3C). Furthermore, animals treated with the extract of cocklebur bacilli were highly sensitive to cisplatin compared to animals fed with the control extract (fig. 3D). The xenobiotic hypersensitive phenotype is unlikely to be a universal phenomenon, since carotenoid extracts of Cockerella radicicola do not alter the sensitivity of the animal against antimycin (mitochondrial toxicant) (FIG. 15E).

Anorexia behavior of caenorhabditis elegans is induced when animals are exposed to xenobiotics or inactivation of essential genes (Melo and Ruvkun, 2012). Exposure of animals to hygromycin (ribosomal translation inhibitor) or cisplatin (DNA replication inhibitor) causes a strong anorexia. While about 40% of animals exposed to 25 μ g/ml hygromycin showed anorexia behavior, about 15% of animals exposed to 25 μ g/ml hygromycin and the carotenoid extract of kocuria rhizophila showed aversion (fig. 3E). Similar results were obtained with cisplatin: approximately 50% of animals exposed to 1mM cisplatin exhibited aversive behavior, while < 20% of animals exposed to 1mM cisplatin and the carotenoid extract of C.radicophilus exhibited anorexia (FIG. 3F).

Example 2 C.radicophila translational inhibition of C.elegans pathway analysis

To assess how carotenoids of Coccocus radiculoides inhibit induction of the heterotypic biomass detoxification response pathway, a genetic profiling was performed with a series of C.elegans mutations that disrupt or activate the signal transduction pathway used for translational monitoring at various steps (Govindan et al, 2015). The zip-2/bZIP transcription factor is required for induction of gfp expression in response to translational repression and represents the last step in transcription induction (Govindan et al, 2015). Even in the absence of translational inhibition, overexpression of ZIP-2:: mCherry under an gut-specific promoter was sufficient to induce pgp-5p:: gfp expression in wild-type C.elegans (FIG. 4A). Whether feeding Cork. rhizophilus affected pgp-5p:: gfp induction was tested in ZIP-2:: mCherry overexpression strains. Gfp-5 p gfp induction was similar in animals fed with e.coli OP50 and kocuria radicicola (fig. 4A), indicating that the carotenoid of kocuria radicicola disrupts the monitoring pathway components upstream (or in parallel) of the ZIP-2bZIP transcription factor.

Induction of caenorhabditis elegans xenobiotics detoxification genes by translational inhibition is dependent on the bile acid signaling pathway (Govindan et al, 2015). Caenorhabditis elegans with genetic defects in bile acid biosynthesis fail to activate pgp-5p:: gfp (which responds to eft-3(q145), RNAi of translational components, or inhibition of translated G418 drugs), but mammalian bile acids can reactivate this signal (Govindan et al, 2015). Although feeding of C.radicophilus inhibited pgp-5p:: GFP induction in eft-3(q145) animals, addition of exogenous mammalian bile acids reactivated GFP expression even in the presence of wild-type C.radicophilus (FIGS. 4B & 4C). Thus, the carotenoids of the bacteria, kocuria rhizophila, function upstream of the caenorhabditis elegans translational monitoring and response pathway or in the bile acid signaling step.

To determine the mechanistic pathways by which carotenoids of kocuria rhizophila might modulate the bile acid signaling pathway, we performed the chery-selected RNAi screen for caenorhabditis elegans homologs of eukaryotic genes that mediate carotenoid binding or transport (table 1). In this screen, we used E.coli pair eft-3(q 145); pgp-5p fed gfp animals expressing dsRNA for caenorhabditis elegans homologues of a carotenoid binding or transport protein. When these animals reached adulthood, they were transferred to a Cockera rhizophilus inoculation plate and scored for GFP induction after 24 hours. In animals fed with dsRNA negative control and transferred to a C.radicophilus plate, pgp-5p:: gfp expression was disrupted. Similar pgp-5p:: decreased gfp expression was also found in animals fed 23 other dsRNA (Table 1). However, we found that eft-3(q 145); pgp-5p:: gfp expression was not repressed in pgp-5p:: gfp animals (FIG. 4D). lbp-5 encodes an intracellular fatty acid binding protein that is expected to function as a transporter for hydrophobic molecules such as lipids and steroid hormones (Xu et al, 2014).

Table 1 is directed to eft-3(q145) in response to Cockerella radicophila; known carotenoid-binding protein genes tested for suppression of gfp expression in gfp animals

+, GFP open; -, GFP off

Furthermore, to identify eukaryotic cellular targets of carotenoids of the species of cocklebur bacilli, a pull-out assay was performed using rat liver cell extracts. The liver was selected for the identification of carotenoid-binding proteins for several reasons: first, the liver is the main site of xenobiotic detoxification. Second, the liver is the site of bile acid biosynthesis. Third, the liver has a known carotenoid transport system. Fourth, a large amount of tissue is readily available, which is not feasible in C.elegans. To identify carotenoid-binding proteins, protein extracts from rat liver were incubated with carotenoids from c.rhizophilus and unbound carotenoids were removed using size exclusion chromatography. The carotenoid-bound protein extract is subjected to anion exchange chromatography and the protein fraction is eluted. The yellow fractions (indicating the presence of carotenoids) were pooled, concentrated and desalted. The color of the concentrated peak was orange-yellow, indicating that the presence of carotenoids was analyzed on native PAGE, and the visible orange-yellow band was excised and subjected to mass spectrometry to identify proteins. 48 proteins were identified by mass spectrometry (Table 2). Interestingly, one of the identified proteins was FABP1 (fatty acid binding protein 1), which is a homolog of C.elegans LBP-5. Among the proteins identified by mass spectrometry are PAK2(p21 protein kinase), which is a homologue of caenorhabditis elegans PAK-1, which we previously identified as pgp-5p:, in response to translational inhibition, a hit in the genome-wide RNAi screen required for gfp induction (Govindan et al, 2015). To determine eft-3(q 145); whether any of these 48 proteins was required for gfp induction or not was performed for pgp-5p:: pgp-5p in gfp, and RNAi screening of C.elegans homologs of these rat proteins was performed. Feeding eft-3(q145) Escherichia coli; pgp-5p: gfp animal, said E.coli expressing dsRNA for caenorhabditis elegans homologues of the respective carotenoid binding protein. When these animals reached adulthood, they were screened to determine if they disrupted GFP induction. In this screen, most of the carotenoid-binding proteins, except pak-1RNAi, did not block eft-3(q 145); pgp-5p:: gfp induction in gfp animals (Table 2). Interestingly, one of the identified carotenoid-binding proteins is AMACR (α -methylacyl-coa racemase), which is required for bile acid biosynthesis (Autio et al, 2014). In caenorhabditis elegans, C24A3.4 and ZK892.4 are homologs of AMACR (fig. 16A). Although either C24A3.4 or ZK892.4 RNAi alone did not repress eft-3(q 145); pgp-5p:: gfp induction in gfp animals, but the dual RNAi of C24A3.4 and ZK892.4 suppressed pgp-5p:: gfp induction (FIG. 16B).

To determine whether any of the 48 C.elegans proteins were required for carotenoid-induced repression of pgp-5 p:. gfp-induced repression by C.rhizophilus, RNAi screens were performed on C.elegans homologs of these proteins. When these animals reached adulthood, they were transferred to a Cockera rhizophilus inoculation plate and scored for GFP induction after 24 hours. In animals fed dsRNA negative control and transferred to a C.rhizophilus plate, pgp-5p:: gfp expression was disrupted (Table 2). In the animals fed, a similar reduction in pgp-5p:: gfp expression was found in most dsRNA animals (Table 2); however, in animals treated with chc-1 or fcho-1 or lbp-5RNAi, pgp-5p:: gfp induction was not disrupted (FIG. 4D). chc-1 encodes a caenorhabditis elegans clathrin heavy chain ortholog, while FCHo-1 encodes a caenorhabditis elegans homolog of the fer/Cip4 homeodomain (FCHo) family protein containing only the F-BAR domain. Both CHC-1 and FCHO-1 mediate endocytic transport (Grant et al, 2006). An alternative model is that chc-1 or fcho-1 or lbp-5RNAi itself may induce pgp-5p:: gfp expression even in the absence of ribosome stress; however, RNAi of these genes did not induce pgp-5p:: gfp expression (FIG. 16C).

Without wishing to be bound by theory, based on these findings, a model was proposed for how carotenoids of kocuria rhizophila inhibited the xenobiotic detoxification response (fig. 4E). Carotenoids released from the bacteria, kocuria rhizophila enter the caenorhabditis elegans gut by clathrin-mediated endocytosis. Within the intestinal cytoplasm, the carotenoids released from endocytic vesicles are subsequently bound by LBP-5 and delivered to the peroxisomes, where they inhibit bile acid biosynthesis by binding to AMACR. In addition, carotenoids also inhibit PAK-1 to disrupt the upregulation of xenobiotic-induced detoxification responses.

Table 2 RNAi response to c.radicophila pair repression eft-3(q145) of the list of mass-characterized proteins, and their c.elegans homologs; influence of gfp expression in gfp animals

+, GFP open; -GFP is off; no entry as untested

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Other embodiments

It is to be understood that while embodiments of the present invention have been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.