RNA sequencing (RNA-seq) has matured into a reliable and low-cost assay for transcriptome profiling and has been deployed across a range of systems. The computational tool space for the analysis of ...RNA-seq data has kept pace with advances in sequencing. Yet tool development has largely centered around the human transcriptome. While eukaryotic and prokaryotic transcriptomes are similar, key differences in transcribed units limit the transfer of wet-lab and computational tools between the two domains. The article by M. Chung, R. S. Adkins, J. S. A. Mattick, K. R. Bradwell, et al. (mSystems 6:e00917-20, 2021, https://doi.org/10.1128/mSystems.00917-20), demonstrates that integrating prokaryote-specific strategies into existing RNA-seq analyses improves read quantification. Unlike in eukaryotes, polycistronic transcripts derived from operons lead to sequencing reads that span multiple neighboring genes. Chung et al. introduce FADU, a software tool that performs a correction for such reads and thereby improves read quantification and biological interpretation of prokaryotic RNA sequencing.
The sourmash software package uses MinHash-based sketching to create "signatures", compressed representations of DNA, RNA, and protein sequences, that can be stored, searched, explored, and ...taxonomically annotated. sourmash signatures can be used to estimate sequence similarity between very large data sets quickly and in low memory, and can be used to search large databases of genomes for matches to query genomes and metagenomes. sourmash is implemented in C++, Rust, and Python, and is freely available under the BSD license at http://github.com/dib-lab/sourmash.
Genomes computationally inferred from large metagenomic data sets are often incomplete and may be missing functionally important content and strain variation. We introduce an information retrieval ...system for large metagenomic data sets that exploits the sparsity of DNA assembly graphs to efficiently extract subgraphs surrounding an inferred genome. We apply this system to recover missing content from genome bins and show that substantial genomic sequence variation is present in a real metagenome. Our software implementation is available at https://github.com/spacegraphcats/spacegraphcats under the 3-Clause BSD License.
Natural killer (NK) cells are key effectors of the innate immune system, but major differences between human and murine NK cells have impeded translation. Outbred dogs offer an important link for ...studies of NK biology and immunotherapy. We analyzed gene expression of putative NK populations from healthy dogs and dogs with naturally-occurring cancers examining differential gene expression across multiple conditions, including steady-state,
activation with cytokines and co-culture, and
activation with inhaled IL-15 in dogs receiving IL-15 immunotherapy. We also compared dog, mouse and human CD3-NKp46+ NK cells using a novel orthologous transcriptome. Distinct transcriptional profiles between NK populations exist between conditions and
versus
treatments. In cross-species analysis, canine NK cells were globally more similar to human NK cells than mice. These data define canine NK cell gene expression under multiple conditions and across species, filling an important gap in translational NK studies.
Prolonged human interactions and artificial selection have influenced the genotypic and phenotypic diversity among dog breeds. Because humans and dogs occupy diverse habitats, ecological contexts ...have likely contributed to breed-specific positive selection. Prior to the advent of modern dog-feeding practices, there was likely substantial variation in dietary landscapes among disparate dog breeds. As such, we investigated one type of genetic variant, copy number variation, in three metabolic genes: glucokinase regulatory protein (GCKR), phytanol-CoA 2-hydroxylase (PHYH), and pancreatic α-amylase 2B (AMY2B). These genes code for proteins that are responsible for metabolizing dietary products that originate from distinctly different food types: sugar, meat, and starch, respectively. After surveying copy number variation among dogs with diverse dietary histories, we found no correlation between diet and positive selection in either GCKR or PHYH. Although it has been previously demonstrated that dogs experienced a copy number increase in AMY2B relative to wolves during or after the dog domestication process, we demonstrate that positive selection continued to act on amylase copy number in dog breeds that consumed starch-rich diets in time periods after domestication. Furthermore, we found that introgression with wolves is not responsible for deterioration of positive selection on AMY2B among diverse dog breeds. Together, this supports the hypothesis that the amylase copy number expansion is found universally in dogs.
During fermentation, Saccharomyces cerevisiae metabolizes sugars and other nutrients to obtain energy for growth and survival, while also modulating these activities in response to cell-environment ...interactions. Here, differences in S. cerevisiae gene expression were explored over a time course of fermentation and used to differentiate fermentations, using Pinot noir grapes from 15 unique sites. Data analysis was complicated by the fact that the fermentations proceeded at different rates, making a direct comparison of time series gene expression data difficult with conventional differential expression tools. This led to the development of a novel approach combining
iffusion
ping with continuous
ifferential
xpression analysis (termed DMap-DE). Using this method, site-specific deviations in gene expression were identified, including changes in gene expression correlated with the non-
yeast Hanseniaspora uvarum, as well as initial nitrogen concentrations in grape musts. These results highlight novel relationships between site-specific variables and Saccharomyces cerevisiae gene expression that are linked to repeated fermentation outcomes. It was also demonstrated that DMap-DE can extract biologically relevant gene expression patterns from other contexts (e.g., hypoxic response of Saccharomyces cerevisiae) and offers advantages over other data dimensionality reduction approaches, indicating that DMap-DE offers a robust method for investigating asynchronous time series gene expression data.
In this work, Saccharomyces cerevisiae gene expression was used as a biosensor to capture differences across and between fermentations of Pinot noir grapes from 15 unique sites representing eight American Viticultural Areas. This required development of a novel analysis method, DMap-DE, for investigation of asynchronous gene expression data. It was demonstrated that DMap-DE reveals biologically relevant shifts in gene expression related to cell-environment interactions in the context of hypoxia and fermentation. Using these data, it was discovered that gene expression by non-
yeasts and initial nitrogen content in grape musts are correlated with differences in gene expression among fermentations. These findings highlight important relationships between site-specific variables and gene expression that may be used to understand why foods and beverages, including wine, possess sensory characteristics associated with or derived from their place of origin.
Abstract
STUDY QUESTION
Does chronic hyperandrogenemia beginning at menarche, in the absence and presence of a western-style diet (WSD), alter ovarian and uterine structure-function in young adult ...rhesus monkeys?
SUMMARY ANSWER
Phenotypic alterations in ovarian and uterine structure/function were induced by exogenous testosterone (T), and compounded in the presence of a WSD (T+WSD).
WHAT IS KNOWN ALREADY
Hyperandrogenemia is a well-established component of PCOS and is observed in adolescent girls, indicating a potential pubertal onset of disease symptoms. Obesity is often associated with hyperandrogenemia and it is hypothesized that metabolic dysfunction exacerbates PCOS symptoms.
STUDY DESIGN, SIZE, DURATION
Macaque females (n = 40) near the onset of menarche (~2.5 years of age) were assigned to a 2 by 2 factorial cohort design. Effects on reproductive characteristics were evaluated after 3 years of treatment.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Rhesus macaques (Macaca mulatta) were fed either a normal balanced diet (n = 20) or a WSD (n = 20). Additionally, implants containing cholesterol (n = 20) or T (n = 20) were implanted subcutaneously to elevate serum T approximately 5-fold. This resulted in treatment groups of controls (C), T, WSD and T+WSD (n = 10/group). Vaginal swabbing was performed daily to detect menses. After 3 years of treatment, daily serum samples from one menstrual cycle were assayed for hormone levels. Ovarian structure was evaluated in the early follicular phase by 3D/4D ultrasound. Uterine endometrial size and ovarian/luteal vascular function was also evaluated in subgroups (n = 6/group) in the late follicular and mid-luteal phases by 3D/4D ultrasound and contrast-enhanced ultrasound, respectively. Expression of steroid hormone receptors and markers of decidualization and endometrial receptivity were assessed in endometrial biopsies at mid-luteal phase.
MAIN RESULTS AND THE ROLE OF CHANCE
Approximately 90% of menstrual cycles appeared ovulatory with no differences in frequency or duration between groups. Serum estradiol (E2) levels during the early follicular phase were greatest in the T alone group, but reduced in T+WSD (P < 0.02). Serum LH was elevated in the T group (P < 0.04); however, there were no differences among groups in FSH levels (P > 0.13). Ovarian size at menses tended to be greater in the WSD groups (P < 0.07) and antral follicles ≥1 mm were more numerous in the T+WSD group (P < 0.05). Also, females in T and T+WSD groups displayed polycystic ovarian morphology (PCOM) at greater frequency than C or WSD groups (P < 0.01). Progesterone (P4) levels during the luteal phase were reduced in the T+WSD group compared to C and T groups (P < 0.05). Blood volume (BV) and vascular flow (VF) within the corpus luteum was reduced in all treatment groups compared to C (P < 0.01, P = 0.03), with the WSD alone group displaying the slowest BV and VF (P < 0.05). C and WSD groups displayed endometrial glands at mid-luteal phase with low estrogen receptor 1 (ESR1) and progesterone receptor (PGR) mRNA and immunohistochemical staining in the functionalis zone, but appreciable PGR in the stroma. In contrast, T and T+WSD treatment resulted in glands with less secretory morphology, high ESR1 expression in the glandular epithelium and low PGR in the stroma. Endometrial levels of TIMP3 and MMP26 mRNA and immunostaining were also decreased in the T and T+WSD groups, whereas AR expression was unchanged.
LARGE SCALE DATA
None.
LIMITATIONS, REASONS FOR CAUTION
Females are young adults, so effects could change as they reach prime reproductive age. The T level generated for hyperandrogenemia may be somewhat greater than the 3-4-fold increase observed in adolescent girls, but markedly less than those observed in male monkeys or adolescent boys.
WIDER IMPLICATIONS OF THE FINDINGS
Alterations to ovarian and uterine structure-function observed in T and, in particular, T+WSD-treated female macaques are consistent with some of the features observed in women diagnosed with polycystic ovary syndrome (PCOS), and suggest impaired fertility.
STUDY FUNDING/COMPETING INTEREST(S)
Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) of the National Institutes of Health (NIH) under Award Number P50HD071836 (to RLS). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Additional funding was provided by Office of the Director, NIH under Award Number P51OD011092 (Support for National Primate Research Center). Authors declare no competing interests.
metabolism produces ethanol and other compounds during the fermentation of grape must into wine. Thousands of genes change expression over the course of a wine fermentation, allowing
to adapt to and ...dominate the fermentation environment. Investigations into these gene expression patterns previously revealed genes that underlie cellular adaptation to the grape must and wine environments, involving metabolic specialization and ethanol tolerance. However, the majority of studies detailing gene expression patterns have occurred in controlled environments that may not recapitulate the biological and chemical complexity of fermentations performed at production scale. Here, an analysis of the
RC212 gene expression program is presented, drawing from 40 pilot-scale fermentations (150 liters) using Pinot noir grapes from 10 California vineyards across two vintages. A core gene expression program was observed across all fermentations irrespective of vintage, similar to that of laboratory fermentations, in addition to novel gene expression patterns likely related to the presence of non-
microorganisms and oxygen availability during fermentation. These gene expression patterns, both common and diverse, provide insight into
biology critical to fermentation outcomes under industry-relevant conditions.
This study characterized
RC212 gene expression during Pinot noir fermentation at pilot scale (150 liters) using industry-relevant conditions. The reported gene expression patterns of RC212 are generally similar to those observed under laboratory fermentation conditions but also contain gene expression signatures related to yeast-environment interactions found in a production setting (e.g., the presence of non-
microorganisms). Key genes and pathways highlighted by this work remain undercharacterized, indicating the need for further research to understand the roles of these genes and their impact on industrial wine fermentation outcomes.
Metagenomics is a powerful method for interpreting the ecological roles and physiological capabilities of mixed microbial communities. Yet, many tools for processing metagenomic data are neither ...designed to consider eukaryotes nor are they built for an increasing amount of sequence data. EukHeist is an automated pipeline to retrieve eukaryotic and prokaryotic metagenome-assembled genomes (MAGs) from large-scale metagenomic sequence data sets. We developed the EukHeist workflow to specifically process large amounts of both metagenomic and/or metatranscriptomic sequence data in an automated and reproducible fashion. Here, we applied EukHeist to the large-size fraction data (0.8-2,000 µm) from Tara Oceans to recover both eukaryotic and prokaryotic MAGs, which we refer to as TOPAZ (Tara Oceans Particle-Associated MAGs). The TOPAZ MAGs consisted of >900 environmentally relevant eukaryotic MAGs and >4,000 bacterial and archaeal MAGs. The bacterial and archaeal TOPAZ MAGs expand upon the phylogenetic diversity of likely particle- and host-associated taxa. We use these MAGs to demonstrate an approach to infer the putative trophic mode of the recovered eukaryotic MAGs. We also identify ecological cohorts of co-occurring MAGs, which are driven by specific environmental factors and putative host-microbe associations. These data together add to a number of growing resources of environmentally relevant eukaryotic genomic information. Complementary and expanded databases of MAGs, such as those provided through scalable pipelines like EukHeist, stand to advance our understanding of eukaryotic diversity through increased coverage of genomic representatives across the tree of life.IMPORTANCESingle-celled eukaryotes play ecologically significant roles in the marine environment, yet fundamental questions about their biodiversity, ecological function, and interactions remain. Environmental sequencing enables researchers to document naturally occurring protistan communities, without culturing bias, yet metagenomic and metatranscriptomic sequencing approaches cannot separate individual species from communities. To more completely capture the genomic content of mixed protistan populations, we can create bins of sequences that represent the same organism (metagenome-assembled genomes MAGs). We developed the EukHeist pipeline, which automates the binning of population-level eukaryotic and prokaryotic genomes from metagenomic reads. We show exciting insight into what protistan communities are present and their trophic roles in the ocean. Scalable computational tools, like EukHeist, may accelerate the identification of meaningful genetic signatures from large data sets and complement researchers' efforts to leverage MAG databases for addressing ecological questions, resolving evolutionary relationships, and discovering potentially novel biodiversity.
Purpose
To investigate the impact of chronically elevated androgens in the presence and absence of an obesogenic diet on oocyte quality in the naturally selected primate periovulatory follicle.
...Methods
Rhesus macaques were treated using a 2-by-2 factorial design (
n
= 10/treatment) near the onset of menarche with implants containing either cholesterol (C) or testosterone (T, 4–5-fold increase above C) and a standard or “Western-style” diet alone (WSD) or in combination (T+WSD). Following ~ 3.5 years of treatment, females underwent controlled ovulation (COv,
n
= 7–10/treatment) cycles, and contents of the naturally selected periovulatory follicle were aspirated. Follicular fluid (FF) was analyzed for cytokines, chemokines, growth factors, and steroids. RNA was extracted from luteinizing granulosa cells (LGCs) and assessed by RNA-seq.
Results
Only healthy, metaphase (M) I/II-stage oocytes (100%) were retrieved in the C group, whereas several degenerated oocytes were recovered in other groups (33–43% of T, WSD, and T+WSD samples). Levels of two chemokines and one growth factor were reduced (
p
< 0.04) in FF of follicles with a MI/MII oocyte in WSD+T (CCL11) or T and WSD+T groups (CCL2 and FGF2) compared to C and/or WSD. Intrafollicular cortisol was elevated in T compared to C follicles (
p
< 0.02). Changes in the expression pattern of 640+ gene products were detected in LGC samples from follicles with degenerated versus MI/MII-stage oocytes. Pathway analysis on RNAs altered by T and/or WSD found enrichment of genes mapping to steroidogenic and immune cell pathways.
Conclusions
Female primates experiencing hyperandrogenemia and/or consuming a WSD exhibit an altered intrafollicular microenvironment and reduced oocyte quality/competency, despite displaying menstrual cyclicity.
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