IL-12 drives the differentiation of human T follicular regulatory cells.

T follicular regulatory (Tfr) cells can counteract the B cell helper activity of T follicular helper (Tfh) cells and hinder the production of antibodies against self-antigens or allergens. A mechanistic understanding of the cytokines initiating the differentiation of human regulatory T (Treg) cells into Tfr cells is still missing. Herein, we report that low doses of the pro-Tfh cytokine interleukin-12 (IL-12) drive the induction of a Tfr cell program on activated human Treg cells while also preserving their regulatory function. Mechanistically, we found that IL-12 led to STAT4 (signal transducer and activator of transcription 4) phosphorylation and binding to IL-12-driven follicular signature genes. Patients with inborn errors of immunity in the IL12RB1 gene presented with a strong decrease in circulating Tfr cells and produced higher levels of anti-actin autoantibodies in vivo. Overall, this study unveils IL-12 as an inducer of Tfr cell differentiation in vivo and provides an approach for the in vitro generation of human Tfr-like cells.

The hormonal control of parturition.

Parturition is a complex physiological process that must occur in a reliable manner and at an appropriate gestation stage to ensure a healthy newborn and mother. To this end, hormones that affect the function of the gravid uterus, especially progesterone (P4), 17ß-estradiol (E2), oxytocin (OT), and prostaglandins (PGs), play pivotal roles. P4 via the nuclear P4 receptor (PR) promotes uterine quiescence and for most of pregnancy exerts a dominant block to labor. Loss of the P4 block to parturition in association with a gain in prolabor actions of E2 are key transitions in the hormonal cascade leading to parturition. P4 withdrawal can occur through various mechanisms depending on species and physiological context. Parturition in most species involves inflammation within the uterine tissues and especially at the maternal-fetal interface. Local PGs and other inflammatory mediators may initiate parturition by inducing P4 withdrawal. Withdrawal of the P4 block is coordinated with increased E2 actions to enhance uterotonic signals mediated by OT and PGs to promote uterine contractions, cervix softening, and membrane rupture, i.e., labor. This review examines recent advances in research to understand the hormonal control of parturition, with focus on the roles of P4, E2, PGs, OT, inflammatory cytokines, and placental peptide hormones together with evolutionary biology of and implications for clinical management of human parturition.

dTrmt10A impacts Hsp70 chaperone m6A levels and the stress response in the Drosophila brain.

Chronic cellular stress has a profound impact on the brain, leading to degeneration and accelerated aging. Recent work has revealed the vital role of RNA modifications, and the proteins responsible for regulating them, in the stress response. In our study, we defined the role of CG14618/dTrmt10A, the Drosophila counterpart of human TRMT10A a N1-methylguanosine methyltransferase, on m6A regulation and heat stress resilience in the Drosophila brain. By m6A-IP RNA sequencing on Drosophila head tissue, we demonstrated that manipulating dTrmt10A levels indirectly regulates m6A levels on polyA + RNA. dTrmt10A exerted its influence on m6A levels on transcripts enriched for neuronal signaling and heat stress pathways, similar to the m6A methyltransferase Mettl3. Intriguingly, its impact primarily targeted 3' UTR m6A, setting it apart from the majority of Drosophila m6A-modified transcripts which display 5' UTR enrichment. Upregulation of dTrmt10A led to increased resilience to acute heat stress, decreased m6A modification on heat shock chaperones, and coincided with decreased decay of chaperone transcripts and increased translation of chaperone proteins. Overall, these findings establish a potential mechanism by which dTrmt10A regulates the acute brain stress response through m6A modification.

A ligation-independent sequencing method reveals tRNA-derived RNAs with blocked 3' termini.

Despite the numerous sequencing methods available, the vast diversity in size and chemical modifications of RNA molecules makes the capture of the full spectrum of cellular RNAs a difficult task. By combining quasi-random hexamer priming with a custom template switching strategy, we developed a method to construct sequencing libraries from RNA molecules of any length and with any type of 3' terminal modification, allowing the sequencing and analysis of virtually all RNA species. Ligation-independent detection of all types of RNA (LIDAR) is a simple, effective tool to comprehensively characterize changes in small non-coding RNAs and mRNAs simultaneously, with performance comparable to separate dedicated methods. With LIDAR, we comprehensively characterized the coding and non-coding transcriptome of mouse embryonic stem cells, neural progenitor cells, and sperm. LIDAR detected a much larger variety of tRNA-derived RNAs (tDRs) compared to traditional ligation-dependent sequencing methods, and uncovered the presence of tDRs with blocked 3' ends that had previously escaped detection. Our findings highlight the potential of LIDAR to systematically detect all RNAs in a sample and uncover new RNA species with potential regulatory functions.

Ensheathing glia promote increased lifespan and healthy brain aging.

Glia have an emergent role in brain aging and disease. In the Drosophila melanogaster brain, ensheathing glia function as phagocytic cells and respond to acute neuronal damage, analogous to mammalian microglia. We previously reported changes in glia composition over the life of ants and fruit flies, including a decline in the relative proportion of ensheathing glia with time. How these changes influence brain health and life expectancy is unknown. Here, we show that ensheathing glia but not astrocytes decrease in number during Drosophila melanogaster brain aging. The remaining ensheathing glia display dysregulated expression of genes involved in lipid metabolism and apoptosis, which may lead to lipid droplet accumulation, cellular dysfunction, and death. Inhibition of apoptosis rescued the decline of ensheathing glia with age, improved the neuromotor performance of aged flies, and extended lifespan. Furthermore, an expanded ensheathing glia population prevented amyloid-beta accumulation in a fly model of Alzheimer's disease and delayed the premature death of the diseased animals. These findings suggest that ensheathing glia play a vital role in regulating brain health and animal longevity.

Mettl3-dependent m6A modification attenuates the brain stress response in Drosophila.

N6-methyladenosine (m6A), the most prevalent internal modification on eukaryotic mRNA, plays an essential role in various stress responses. The brain is uniquely vulnerable to cellular stress, thus defining how m6A sculpts the brain's susceptibility may provide insight to brain aging and disease-related stress. Here we investigate the impact of m6A mRNA methylation in the adult Drosophila brain with stress. We show that m6A is enriched in the adult brain and increases with heat stress. Through m6A-immunoprecipitation sequencing, we show 5'UTR Mettl3-dependent m6A is enriched in transcripts of neuronal processes and signaling pathways that increase upon stress. Mettl3 knockdown results in increased levels of m6A targets and confers resilience to stress. We find loss of Mettl3 results in decreased levels of nuclear m6A reader Ythdc1, and knockdown of Ythdc1 also leads to stress resilience. Overall, our data suggest that m6A modification in Drosophila dampens the brain's biological response to stress.

Intrinsic maturation of sleep output neurons regulates sleep ontogeny in Drosophila.

The maturation of sleep behavior across a lifespan (sleep ontogeny) is an evolutionarily conserved phenomenon. Mammalian studies have shown that in addition to increased sleep duration, early life sleep exhibits stark differences compared with mature sleep with regard to sleep states. How the intrinsic maturation of sleep output circuits contributes to sleep ontogeny is poorly understood. The fruit fly Drosophila melanogaster exhibits multifaceted changes to sleep from juvenile to mature adulthood. Here, we use a non-invasive probabilistic approach to investigate the changes in sleep architecture in juvenile and mature flies. Increased sleep in juvenile flies is driven primarily by a decreased probability of transitioning to wake and characterized by more time in deeper sleep states. Functional manipulations of sleep-promoting neurons in the dorsal fan-shaped body (dFB) suggest that these neurons differentially regulate sleep in juvenile and mature flies. Transcriptomic analysis of dFB neurons at different ages and a subsequent RNAi screen implicate the genes involved in dFB sleep circuit maturation. These results reveal that the dynamic transcriptional states of sleep output neurons contribute to the changes in sleep across the lifespan.

Skin Fibrosis and Recovery Is Dependent on Wnt Activation via DPP4.

Fibrosis is the life-threatening, excessive accumulation of the extracellular matrix and is sometimes associated with a loss of lipid-filled cells in the skin and other organs. Understanding the mechanisms of fibrosis and associated lipodystrophy and their reversal may reveal new targets for therapeutic intervention. In vivo genetic models are needed to identify key targets that induce recovery from established fibrosis. Wnt signaling is activated in animal and human fibrotic diseases across organs. Here, we developed a genetically inducible and reversible Wnt activation model and showed that it is sufficient to cause fibrotic dermal remodeling, including extracellular matrix expansion and shrinking of dermal adipocytes. Upon withdrawal from Wnt activation, Wnt-induced fibrotic remodeling was reversed in mouse skin-fully restoring skin architecture. Next, we demonstrated CD26/ DPP4 is a Wnt/ß-catenin-responsive gene and a functional mediator of fibrotic transformation. We provide genetic evidence that the Wnt/DPP4 axis is required to drive fibrotic dermal remodeling and is associated with human skin fibrosis severity. Remarkably, DPP4 inhibitors can be repurposed to accelerate recovery from established Wnt-induced fibrosis. Collectively, this study identifies Wnt/DPP4 axis as a key driver of extracellular matrix homeostasis and dermal fat loss, providing therapeutic avenues to manipulate the onset and reversal of tissue fibrosis.

Genome annotation with long RNA reads reveals new patterns of gene expression and improves single-cell analyses in an ant brain.

BACKGROUND: Functional genomic analyses rely on high-quality genome assemblies and annotations. Highly contiguous genome assemblies have become available for a variety of species, but accurate and complete annotation of gene models, inclusive of alternative splice isoforms and transcription start and termination sites, remains difficult with traditional approaches. RESULTS: Here, we utilized full-length isoform sequencing (Iso-Seq), a long-read RNA sequencing technology, to obtain a comprehensive annotation of the transcriptome of the ant Harpegnathos saltator. The improved genome annotations include additional splice isoforms and extended 3' untranslated regions for more than 4000 genes. Reanalysis of RNA-seq experiments using these annotations revealed several genes with caste-specific differential expression and tissue- or caste-specific splicing patterns that were missed in previous analyses. The extended 3' untranslated regions afforded great improvements in the analysis of existing single-cell RNA-seq data, resulting in the recovery of the transcriptomes of 18% more cells. The deeper single-cell transcriptomes obtained with these new annotations allowed us to identify additional markers for several cell types in the ant brain, as well as genes differentially expressed across castes in specific cell types. CONCLUSIONS: Our results demonstrate that Iso-Seq is an efficient and effective approach to improve genome annotations and maximize the amount of information that can be obtained from existing and future genomic datasets in Harpegnathos and other organisms.

Kr-h1 maintains distinct caste-specific neurotranscriptomes in response to socially regulated hormones.

Behavioral plasticity is key to animal survival. Harpegnathos saltator ants can switch between worker and queen-like status (gamergate) depending on the outcome of social conflicts, providing an opportunity to study how distinct behavioral states are achieved in adult brains. Using social and molecular manipulations in live ants and ant neuronal cultures, we show that ecdysone and juvenile hormone drive molecular and functional differences in the brains of workers and gamergates and direct the transcriptional repressor Kr-h1 to different target genes. Depletion of Kr-h1 in the brain caused de-repression of "socially inappropriate" genes: gamergate genes were upregulated in workers, whereas worker genes were upregulated in gamergates. At the phenotypic level, loss of Kr-h1 resulted in the emergence of worker-specific behaviors in gamergates and gamergate-specific traits in workers. We conclude that Kr-h1 is a transcription factor that maintains distinct brain states established in response to socially regulated hormones.

Quantifying procedural pain associated with office gynecologic tract sampling methods.

OBJECTIVE: Emerging technologies may enable detection of endometrial cancer with methods that are less invasive than standard biopsy methods. This study compares patient pain scores among 3 office gynecologic tract sampling methods and explores their potential determinants. METHODS: A prospective study including 3 sampling methods (tampon, Tao brush (TB), endometrial biopsy (EB)) was conducted between December 2015 and August 2017 and included women ≥45 years of age presenting with abnormal uterine bleeding, postmenopausal bleeding, or thickened endometrial stripe. Patients rated pain after each sampling procedure using a 100-point visual analog scale (VAS). RESULTS: Of 428 enrolled, 190 (44.39%) patients underwent all 3 sampling methods and reported a VAS score for each. Nearly half were postmenopausal (n = 93, 48.9%); the majority were parous (172, 90.5%) of which 87.8% had at least one vaginal delivery. Among the 190 patients, the median (IQR) pain score was significantly lower for sampling via tampon (0 [0,2]) compared to TB (28 [12, 52]) or EB (32 [15, 60]) (both p < 0.001, Wilcoxon signed rank test). Among women who underwent tampon sampling, age and pain scores showed a weak positive correlation (Spearman rank correlation, r = 0.14; p = 0.006); EB sampling was associated with a weak inverse correlation between parity and pain scores (r = -0.14; p = 0.016). CONCLUSION: Gynecologic tract sampling using a tampon had significantly lower pain than both EB and TB. Pain with tampon sampling was positively correlated with age and pain with EB sampling was inversely correlated with parity. Pain scores for TB and EB were not significantly related to age, menopausal status, or BMI.

Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency.

Active DNA demethylation via ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of the role of TETs in chromatin reprogramming. Here, we describe the first application of biochemically engineered TET mutants that unlink 5mC oxidation steps, examining their effects on somatic cell reprogramming. We show that only TET enzymes proficient for oxidation to 5fC/5caC can rescue the reprogramming potential of Tet2-deficient mouse embryonic fibroblasts. This effect correlated with rapid DNA demethylation at reprogramming enhancers and increased chromatin accessibility later in reprogramming. These experiments demonstrate that DNA demethylation through 5fC/5caC has roles distinct from 5hmC in somatic reprogramming to pluripotency.

Social reprogramming in ants induces longevity-associated glia remodeling.

In social insects, workers and queens arise from the same genome but display profound differences in behavior and longevity. In Harpegnathos saltator ants, adult workers can transition to a queen-like state called gamergate, which results in reprogramming of social behavior and life-span extension. Using single-cell RNA sequencing, we compared the distribution of neuronal and glial populations before and after the social transition. We found that the conversion of workers into gamergates resulted in the expansion of neuroprotective ensheathing glia. Brain injury assays revealed that activation of the damage response gene Mmp1 was weaker in old workers, where the relative frequency of ensheathing glia also declined. On the other hand, long-lived gamergates retained a larger fraction of ensheathing glia and the ability to mount a strong Mmp1 response to brain injury into old age. We also observed molecular and cellular changes suggestive of age-associated decline in ensheathing glia in Drosophila.

Infectious Hepatitis in Pregnancy.

Infectious hepatitis in pregnancy is clinically significant in both the acute and chronic phases. Here, we review the perinatal implications of chronic hepatitis B and C and acute hepatitis A and E. Familiarity with screening, transmission, diagnosis, and management of infectious hepatitis is of ongoing importance during obstetric care, as these diseases are endemic in much of the world. Pregnancy and interpregnancy care provide opportunities to prevent infection and transmission of hepatitis.

Mapping Native R-Loops Genome-wide Using a Targeted Nuclease Approach.

R-loops are three-stranded DNA:RNA hybrids that are implicated in many nuclear processes. While R-loops may have physiological roles, the formation of stable, aberrant R-loops has been observed in neurological disorders and cancers. Current methods to assess their genome-wide distribution rely on affinity purification, which is plagued by large input requirements, high noise, and poor sensitivity for dynamic R-loops. Here, we present MapR, a method that utilizes RNase H to guide micrococcal nuclease to R-loops, which are subsequently cleaved, released, and identified by sequencing. MapR detects R-loops formed at promoters and active enhancers that are likely to form transient R-loops due to the low transcriptional output of these regulatory elements and the short-lived nature of enhancer RNAs. MapR is as specific as existing techniques and more sensitive, allowing for genome-wide coverage with low input material in a fraction of the time.

lncRedibly versatile: biochemical and biological functions of long noncoding RNAs.

Long noncoding RNAs (lncRNAs) are transcripts that do not code for proteins, but nevertheless exert regulatory effects on various biochemical pathways, in part via interactions with proteins, DNA, and other RNAs. LncRNAs are thought to regulate transcription and other biological processes by acting, for example, as guides that target proteins to chromatin, scaffolds that facilitate protein-protein interactions and complex formation, and orchestrators of phase-separated compartments. The study of lncRNAs has reached an exciting time, as recent advances in experimental and computational methods allow for genome-wide interrogation of biochemical and biological mechanisms of these enigmatic transcripts. A better appreciation for the biochemical versatility of lncRNAs has allowed us to begin closing gaps in our knowledge of how they act in diverse cellular and organismal contexts, including development and disease.

High-Quality Genome Assemblies Reveal Long Non-coding RNAs Expressed in Ant Brains.

Ants are an emerging model system for neuroepigenetics, as embryos with virtually identical genomes develop into different adult castes that display diverse physiology, morphology, and behavior. Although a number of ant genomes have been sequenced to date, their draft quality is an obstacle to sophisticated analyses of epigenetic gene regulation. We reassembled de novo high-quality genomes for two ant species, Camponotus floridanus and Harpegnathos saltator. Using long reads enabled us to span large repetitive regions and improve genome contiguity, leading to comprehensive and accurate protein-coding annotations that facilitated the identification of a Gp-9-like gene as differentially expressed in Harpegnathos castes. The new assemblies also enabled us to annotate long non-coding RNAs in ants, revealing caste-, brain-, and developmental-stage-specific long non-coding RNAs (lncRNAs) in Harpegnathos. These upgraded genomes, along with the new gene annotations, will aid future efforts to identify epigenetic mechanisms of phenotypic and behavioral plasticity in ants.

Wnt/ß-catenin Signaling Pathway Regulates Specific lncRNAs That Impact Dermal Fibroblasts and Skin Fibrosis.

Wnt/ß-catenin signaling is required for embryonic dermal fibroblast cell fate, and dysregulation of this pathway is sufficient to promote fibrosis in adult tissue. The downstream modulators of Wnt/ß-catenin signaling required for controlling cell fate and dermal fibrosis remain poorly understood. The discovery of regulatory long non-coding RNAs (lncRNAs) and their pivotal roles as key modulators of gene expression downstream of signaling cascades in various contexts prompted us to investigate their roles in Wnt/ß-catenin signaling. Here, we have identified lncRNAs and protein-coding RNAs that are induced by ß-catenin activity in mouse dermal fibroblasts using next generation RNA-sequencing. The differentially expressed protein-coding mRNAs are enriched for extracellular matrix proteins, glycoproteins, and cell adhesion, and many are also dysregulated in human fibrotic tissues. We identified 111 lncRNAs that are differentially expressed in response to activation of Wnt/ß-catenin signaling. To further characterize the role of mouse lncRNAs in this pathway, we validated two novel Wnt signaling- Induced Non-Coding RNA (Wincr) transcripts referred to as Wincr1 and Wincr2. These two lncRNAs are highly expressed in mouse embryonic skin and perinatal dermal fibroblasts. Furthermore, we found that Wincr1 expression levels in perinatal dermal fibroblasts affects the expression of key markers of fibrosis (e.g., Col1a1 and Mmp10), enhances collagen contraction, and attenuates collective cell migration. Our results show that ß-catenin signaling-responsive lncRNAs may modulate dermal fibroblast behavior and collagen accumulation in dermal fibrosis, providing new mechanistic insights and nodes for therapeutic intervention.

The Neuropeptide Corazonin Controls Social Behavior and Caste Identity in Ants.

Social insects are emerging models to study how gene regulation affects behavior because their colonies comprise individuals with the same genomes but greatly different behavioral repertoires. To investigate the molecular mechanisms that activate distinct behaviors in different castes, we exploit a natural behavioral plasticity in Harpegnathos saltator, where adult workers can transition to a reproductive, queen-like state called gamergate. Analysis of brain transcriptomes during the transition reveals that corazonin, a neuropeptide homologous to the vertebrate gonadotropin-releasing hormone, is downregulated as workers become gamergates. Corazonin is also preferentially expressed in workers and/or foragers from other social insect species. Injection of corazonin in transitioning Harpegnathos individuals suppresses expression of vitellogenin in the brain and stimulates worker-like hunting behaviors, while inhibiting gamergate behaviors, such as dueling and egg deposition. We propose that corazonin is a central regulator of caste identity and behavior in social insects.

Novel approaches to early detection of endometrial cancer.

PURPOSE OF REVIEW: Detection of endometrial cancer at an early stage leads to better oncologic outcomes. To date, a screening test for endometrial cancer does not exist. RECENT FINDINGS: Evolving knowledge of molecular changes involved in endometrial cancer carcinogenesis paired with sensitive and high-throughput technological advancements are a promising combination that can be leveraged to detect tumor DNA and proteins. These molecular biomarkers can be identified in biospecimens collected via minimally invasive and noninvasive approaches. Exploiting lower genital tract secretions as a biospecimen also allows for patient self-sampling. SUMMARY: Successful development of a screening test for endometrial cancer using self-collected lower genital tract biospecimens has the potential to increase accessibility to care and improve patient compliance.