We show that a citizen science, self-selected cohort shipping samples through the mail at room temperature recaptures many known microbiome results from clinically collected cohorts and reveals new ones. Of particular interest is integrating n = 1 study data with the population data, showing that the extent of microbiome change after events such as surgery can exceed differences between distinct environmental biomes, and the effect of diverse plants in the diet, which we confirm with untargeted metabolomics on hundreds of samples.

These results suggest that hyperandrogenism may play a critical role in altering the gut microbiome in women with PCOS.

Gut microbial diversity changes throughout the human life span and is known to be associated with host sex. We investigated the association of age, sex, and gut bacterial alpha diversity in three large cohorts of adults from four geographical regions: subjects from the United States and United Kingdom in the American Gut Project (AGP) citizen-science initiative and two independent cohorts of Colombians and Chinese. In three of the four cohorts, we observed a strong positive association between age and alpha diversity in young adults that plateaued after age 40 years. We also found sex-dependent differences that were more pronounced in younger adults than in middle-aged adults, with women having higher alpha diversity than men. In contrast to the other three cohorts, no association of alpha diversity with age or sex was observed in the Chinese cohort. The association of alpha diversity with age and sex remained after adjusting for cardiometabolic parameters in the Colombian cohort and antibiotic usage in the AGP cohort. We further attempted to predict the microbiota age in individuals using a machine-learning approach for the men and women in each cohort. Consistent with our alpha-diversity-based findings, U.S. and U.K. women had a significantly higher predicted microbiota age than men, with a reduced difference being seen above age 40 years. This difference was not observed in the Colombian cohort and was observed only in middle-aged Chinese adults. Together, our results provide new insights into the influence of age and sex on the biodiversity of the human gut microbiota during adulthood while highlighting similarities and differences across diverse cohorts. IMPORTANCE Microorganisms in the human gut play a role in health and disease, and in adults higher gut biodiversity has been linked to better health. Since gut microorganisms may be pivotal in the development of microbial therapies, understanding the factors that shape gut biodiversity is of utmost interest. We performed large-scale analyses of the relationship of age and sex to gut bacterial diversity in adult cohorts from four geographic regions: the United States, the United Kingdom, Colombia, and China. In the U.S., U.K., and Colombian cohorts, bacterial biodiversity correlated positively with age in young adults but plateaued at about age 40 years, with no positive association being found in middle-aged adults. Young, but not middle-aged, adult women had higher gut bacterial diversity than men, a pattern confirmed via supervised machine learning. Interestingly, in the Chinese cohort, minimal associations were observed between gut biodiversity and age or sex. Our results highlight the patterns of adult gut biodiversity and provide a framework for future research.

Women with polycystic ovary syndrome (PCOS) have reproductive and metabolic abnormalities that result in an increased risk of infertility, diabetes and cardiovascular disease. The large intestine contains a complex community of microorganisms (the gut microbiome) that is dysregulated in humans with obesity and type 2 diabetes. Using a letrozole-induced PCOS mouse model, we demonstrated significant diet-independent changes in the gut microbial community, suggesting that gut microbiome dysbiosis may also occur in PCOS women. Letrozole treatment was associated with a time-dependent shift in the gut microbiome and a substantial reduction in overall species and phylogenetic richness. Letrozole treatment also correlated with significant changes in the abundance of specific Bacteroidetes and Firmicutes previously implicated in other mouse models of metabolic disease in a time-dependent manner. Our results suggest that the hyperandrogenemia observed in PCOS may significantly alter the gut microbiome independently of diet.

Polycystic ovary syndrome (PCOS) pathophysiology is poorly understood, due partly to lack of PCOS animal models fully recapitulating this complex disorder. Recently, a PCOS rat model using letrozole (LET), a nonsteroidal aromatase inhibitor, mimicked multiple PCOS phenotypes, including metabolic features absent in other models. Given the advantages of using genetic and transgenic mouse models, we investigated whether LET produces a similar PCOS phenotype in mice. Pubertal female C57BL/6N mice were treated for 5 wk with LET, which resulted in increased serum testosterone and normal diestrus levels of estradiol, similar to the hyperandrogenemia and follicular phase estrogen levels of PCOS women. As in PCOS, ovaries from LET mice were larger, polycystic, and lacked corpora lutea versus controls. Most LET females were acyclic, and all were infertile. LET females displayed elevated serum LH levels and higher Lhb mRNA in the pituitary. In contrast, serum FSH and Fshb were significantly reduced in LET females, demonstrating differential effects on gonadotropins, as in PCOS. Within the ovary, LET females had higher Cyp17, Cyp19, and Fsh receptor mRNA expression. In the hypothalamus, LET females had higher kisspeptin receptor mRNA expression but lower progesterone receptor mRNA levels. LET females also gained more weight than controls, had increased abdominal adiposity and adipocyte size, elevated adipose inflammatory mRNA levels, and impaired glucose tolerance, mirroring the metabolic phenotype in PCOS women. This is the first report of a LET paradigm in mice that recapitulates both reproductive and metabolic PCOS phenotypes and will be useful to genetically probe the PCOS condition.

word count: 203 68 3 Main text word count: 3280 69 70 Abstract: Although much work has linked the human microbiome to specific phenotypes and 71 lifestyle variables, data from different projects have been challenging to integrate and the extent 72 of microbial and molecular diversity in human stool remains unknown. Using standardized 73 protocols from the Earth Microbiome Project and sample contributions from over 10,000 citizen-74 scientists, together with an open research network, we compare human microbiome specimens 75 primarily from the USA, UK, and Australia to one another and to environmental samples. Our 76 results show an unexpected range of beta-diversity in human stool microbiomes as compared to 77 environmental samples, demonstrate the utility of procedures for removing the effects of 78 overgrowth during room-temperature shipping for revealing phenotype correlations, uncover 79 new molecules and kinds of molecular communities in the human stool metabolome, and 80 examine emergent associations among the microbiome, metabolome, and the diversity of plants 81 that are consumed (rather than relying on reductive categorical variables such as veganism, 82 which have little or no explanatory power). We also demonstrate the utility of the living data 83 resource and cross-cohort comparison to confirm existing associations between the microbiome 84 and psychiatric illness, and to reveal the extent of microbiome change within one individual 85 during surgery, providing a paradigm for open microbiome research and education. 86 87Importance: We show that a citizen-science, self-selected cohort shipping samples through the 88 mail at room temperature recaptures many known microbiome results from clinically collected 89 cohorts and reveals new ones. Of particular interest is integrating n=1 study data with the 90 population data, showing that the extent of microbiome change after events such as surgery can 91 4 exceed differences between distinct environmental biomes, and the effect of diverse plants in the 92 diet which we confirm with untargeted metabolomics on hundreds of samples. 93 94 Introduction 95The human microbiome plays a fundamental role in human health and disease. While 96 many studies link microbiome composition to phenotypes, we lack understanding of the 97 boundaries of bacterial diversity within the human population, and the relative importance of 98 lifestyle, health conditions, and diet, to underpin precision medicine or to educate the broader 99 community about this key aspect of human health. 100 We launched the American Gut Project (AGP; http://americangut.org) in November of 101 2012 as a collaboration between the Earth Microbiome Project (EMP) (1) and the Human Food 102 Project (HFP; http://humanfoodproject.com/) to discover the kinds of microbes and microbiomes 103 "in the wild" via a self-selected citizen-scientist cohort. The EMP is tasked with characterizing 104 the global microbial taxonomic and functional diversity, and the HFP is focused on 105 understanding microbial diversity a...

The precise interplay of hormonal influences that governs gonadotropin hormone production by the pituitary includes endocrine, paracrine and autocrine actions of hypothalamic gonadotropin-releasing hormone (GnRH), activin and steroids. However, most studies of hormonal regulation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the pituitary gonadotrope have been limited to analyses of the isolated actions of individual hormones. LHβ and FSHβ subunits have distinct patterns of expression during the menstrual/estrous cycle as a result of the integration of activin, GnRH, and steroid hormone action. In this review, we focus on studies that delineate the interplay among these hormones in the regulation of LHβ and FSHβ gene expression in gonadotrope cells and discuss how signaling cross-talk contributes to differential expression. We also discuss how recent technological advances will help identify additional factors involved in the differential hormonal regulation of LH and FSH.

A systematic study of selectively modified, 36-mer hammerhead ribozymes has resulted in the identification of a generic, catalytically active and nuclease stable ribozyme motif containing 5 ribose residues, 29 -30 2-O-Me nucleotides, 1-2 other 2-modified nucleotides at positions U4 and U7, and a 3-3-linked nucleotide "cap." Eight 2-modified uridine residues were introduced at positions U4 and/or U7. From the resulting set of ribozymes, several have almost wild-type catalytic activity and significantly improved stability. Specifically, ribozymes containing 2-NH 2 substitutions at U4 and U7, or 2-C-allyl substitutions at U4, retain most of their catalytic activity when compared to the all-RNA parent. Their serum half-lives were 5-8 h in a variety of biological fluids, including human serum, while the all-RNA parent ribozyme exhibits a stability half-life of only ϳ0.1 min. The addition of a 3-3-linked nucleotide "cap" (inverted T) did not affect catalysis but increased the serum half-lives of these two ribozymes to >260 h at nanomolar concentrations. This represents an overall increase in stability/activity of 53,000 -80,000-fold compared to the all-RNA parent ribozyme.Trans-acting ribozymes exert their activity in a highly specific manner and are therefore not expected to be detrimental to non-targeted cell functions. Because of this specificity, the concept of exploiting ribozymes for cleaving a specific target mRNA transcript is now emerging as a therapeutic strategy in human disease and agriculture (Cech, 1992;Bratty et al., 1993). For ribozymes to function as therapeutic agents, they may be introduced exogenously or produced endogenously in the target cells. In the former case, the chemically modified ribozyme must maintain its catalytic activity while also being stable to nucleases. A major advantage of chemically synthesized ribozymes is that site-specific modifications may be introduced at any position in the molecule. This approach provides flexibility in designing ribozymes that are catalytically active and stable to nucleases. In this manuscript we show that using this site-specific, chemical modification strategy, ribozymes can be designed that have wild-type catalytic activity and are not cleaved by nucleases.A variety of selective and uniform structural modifications have been applied to oligonucleotides to enhance nuclease resistance (Uhlmann and Peyman, 1990;Beaucage and Iyer, 1993;Milligan et al., 1993). Improvements in the chemical synthesis of RNA (Scaringe et al., 1990;Wincott et al., 1995) have led to the ability to similarly modify ribozymes containing the hammerhead ribozyme core motif Yang et al., 1992) (Fig. 1). Yang et al. (1992) demonstrated that 2Ј-O-Me modification of a ribozyme at all positions except G5, G8, A9, A15.1, and G15.2 (see numbering scheme in Fig. 1) led to a catalytically active molecule having a greatly decreased k cat value in vitro, but a 1000-fold increase in nuclease resistance over that of an all-RNA ribozyme when tested in a yeast extract. In another study (Paolella...