Proteobacteria impair anti-tumor immunity in the omentum by consuming arginine.

Gut microbiota influence anti-tumor immunity, often by producing immune-modulating metabolites. However, microbes consume a variety of metabolites that may also impact host immune responses. We show that tumors grow unchecked in the omenta of microbe-replete mice due to immunosuppressive Tregs. By contrast, omental tumors in germ-free, neomycin-treated mice or mice colonized with altered Schaedler's flora (ASF) are spontaneously eliminated by CD8+ T cells. These mice lack Proteobacteria capable of arginine catabolism, causing increases in serum arginine that activate the mammalian target of the rapamycin (mTOR) pathway in Tregs to reduce their suppressive capacity. Transfer of the Proteobacteria, Escherichia coli (E. coli), but not a mutant unable to catabolize arginine, to ASF mice reduces arginine levels, restores Treg suppression, and prevents tumor clearance. Supplementary arginine similarly decreases Treg suppressive capacity, increases CD8+ T cell effectiveness, and reduces tumor burden. Thus, microbial consumption of arginine alters anti-tumor immunity, offering potential therapeutic strategies for tumors in visceral adipose tissue.

Autism-linked UBE3A gain-of-function mutation causes interneuron and behavioral phenotypes when inherited maternally or paternally in mice.

The E3 ubiquitin ligase Ube3a is biallelically expressed in neural progenitors and glial cells, suggesting that UBE3A gain-of-function mutations might cause neurodevelopmental disorders irrespective of parent of origin. Here, we engineered a mouse line that harbors an autism-linked UBE3AT485A (T503A in mouse) gain-of-function mutation and evaluated phenotypes in animals that inherited the mutant allele paternally, maternally, or from both parents. We find that paternally and maternally expressed UBE3AT503A results in elevated UBE3A activity in neural progenitors and glial cells. Expression of UBE3AT503A from the maternal allele, but not the paternal one, leads to a persistent elevation of UBE3A activity in neurons. Mutant mice display behavioral phenotypes that differ by parent of origin. Expression of UBE3AT503A, irrespective of its parent of origin, promotes transient embryonic expansion of Zcchc12 lineage interneurons. Phenotypes of Ube3aT503A mice are distinct from Angelman syndrome model mice. Our study has clinical implications for a growing number of disease-linked UBE3A gain-of-function mutations.

Biomimetic cardiac tissue chip and murine arteriovenous fistula models for recapitulating clinically relevant cardiac remodeling under volume overload conditions.

Cardiovascular events are the primary cause of death among dialysis patients. While arteriovenous fistulas (AVFs) are the access of choice for hemodialysis patients, AVF creation can lead to a volume overload (VO) state in the heart. We developed a three-dimensional (3D) cardiac tissue chip (CTC) with tunable pressure and stretch to model the acute hemodynamic changes associated with AVF creation to complement our murine AVF model of VO. In this study, we aimed to replicate the hemodynamics of murine AVF models in vitro and hypothesized that if 3D cardiac tissue constructs were subjected to "volume overload" conditions, they would display fibrosis and key gene expression changes seen in AVF mice. Mice underwent either an AVF or sham procedure and were sacrificed at 28 days. Cardiac tissue constructs composed of h9c2 rat cardiac myoblasts and normal adult human dermal fibroblasts in hydrogel were seeded into devices and exposed to 100 mg/10 mmHg pressure (0.4 s/0.6 s) at 1 Hz for 96 h. Controls were exposed to "normal" stretch and experimental group exposed to "volume overload". RT-PCR and histology were performed on the tissue constructs and mice left ventricles (LVs), and transcriptomics of mice LVs were also performed. Our tissue constructs and mice LV both demonstrated cardiac fibrosis as compared to control tissue constructs and sham-operated mice, respectively. Gene expression studies in our tissue constructs and mice LV demonstrated increased expression of genes associated with extracellular matrix production, oxidative stress, inflammation, and fibrosis in the VO conditions vs. control conditions. Our transcriptomics studies demonstrated activated upstream regulators related to fibrosis, inflammation, and oxidative stress such as collagen type 1 complex, TGFB1, CCR2, and VEGFA and inactivated regulators related to mitochondrial biogenesis in LV from mice AVF. In summary, our CTC model yields similar fibrosis-related histology and gene expression profiles as our murine AVF model. Thus, the CTC could potentially play a critical role in understanding cardiac pathobiology of VO states similar to what is present after AVF creation and may prove useful in evaluating therapies.

Genome-wide DNA methylation study identifies significant epigenomic changes associated with internalized stigma in adults with non-specific chronic low back pain.

Non-specific chronic low back pain (cLBP) represents a common musculoskeletal condition with no identifiable cause. It cannot be diagnosed with conventional neuroimaging techniques such as computerized tomography (CT). The diagnostic uncertainty that characterizes non-specific cLBP can lead to stigmatizing responses from others that can become internalized Among individuals with non-specific cLBP, internalized stigma is associated with greater pain intensity and disability. Yet, no study has examined the biological mechanism linking high internalized stigma to worse outcomes in individuals with non-specific cLBP. We aimed to identify differentially methylated loci (DML), enrichment pathways, and associated network interactions among individuals with non-specific cLBP experiencing low vs. high internalized stigma. We examined DNA methylation in whole blood samples from 48 adults, ages 19-85, using reduced representation bisulfite sequencing (RRBS). After controlling for age, sex, race, and multiple testing, differentially methylated loci (DML) differed in adults with low vs. high internalized stigma by at least 10% and q < 0.01 in 3,665 CpG sites: 2,280 hypomethylated and 1,385 hypermethylated. Gene ontology (GO) analyses of the annotated genes from these sites revealed significant enrichment of 274 biological processes, 29 cellular components, and 24 molecular functions (adjusted p < 0.05). The top enriched molecular functions regulate protein binding and DNA binding of transcription factor activity. Pathway analyses indicated that many functional genomic pathways, including Hippo Signaling, Melanogenesis, and Pathways in Cancer, were enriched with differentially methylated genes. Also, there was a significant interaction between relevance pathways such as P53, mTOR, PI3K-Akt, and Wnt signaling pathways. These pathways have previously been associated with neuroinflammation, neurodegeneration, and stress-related conditions. Thus, findings point to possible stress-induced DNAm changes as the link between high levels of internalized stigma and worse outcomes in adults with non-specific cLBP.

Epigenome-wide DNA methylation profiling of conditioned pain modulation in individuals with non-specific chronic low back pain.

BACKGROUND: The pathoanatomic cause of chronic low back pain (cLBP) cannot be identified for up to 90% of individuals. However, dysfunctional processing of endogenous nociceptive input, measured as conditioned pain modulation (CPM), has been associated with cLBP and may involve changes in neuronal gene expression. Epigenetic-induced changes such as DNA methylation (DNAm) have been associated with cLBP. METHODS: In the present study, the relationship between CPM and DNAm changes in a sample of community-dwelling adults with nonspecific cLBP (n = 48) and pain-free controls (PFC; n = 50) was examined using reduced representation bisulfite sequencing. Gene ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were applied to identify key pathways involved in efficient versus deficient CPM. RESULTS: Based on CPM efficiency, we identified 6006 and 18,305 differentially methylated CpG sites (DMCs) with q values < 0.01 among individuals with cLBP and PFCs, respectively. Most of the DMCs were hypomethylated and annotated to genes of relevance to pain, including OPRM1, ADRB2, CACNA2D3, GNA12, LPL, NAXD, and ASPHD1. In both cLBP and PFC groups, the DMCs annotated genes enriched many GO terms relevant to pain processing, including transcription regulation by RNA polymerase II, nervous system development, generation of neurons, neuron differentiation, and neurogenesis. Both groups also enriched the pathways involved in Rap1-signaling, cancer, and dopaminergic neurogenesis. However, MAPK-Ras signaling pathways were enriched in the cLBP, not the PFC group. CONCLUSIONS: This is the first study to investigate the genome-scale DNA methylation profiles of CPM phenotype in adults with cLBP and PFCs. Based on CPM efficiency, fewer DMC enrichment pathways were unique to the cLBP than the PFCs group. Our results suggest that epigenetically induced modification of neuronal development/differentiation pathways may affect CPM efficiency, suggesting novel potential therapeutic targets for central sensitization. However, CPM efficiency and the experience of nonspecific cLBP may be independent. Further mechanistic studies are required to confirm the relationship between CPM, central sensitization, and nonspecific cLBP.

ZHX2 promotes HIF1α oncogenic signaling in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease, which warrants the critical need to identify new therapeutic targets. We show that Zinc Fingers and Homeoboxes 2 (ZHX2) is amplified or overexpressed in TNBC cell lines and patients. Functionally, depletion of ZHX2 inhibited TNBC cell growth and invasion in vitro, orthotopic tumor growth, and spontaneous lung metastasis in vivo. Mechanistically, ZHX2 bound with hypoxia-inducible factor (HIF) family members and positively regulated HIF1α activity in TNBC. Integrated ChIP-seq and gene expression profiling demonstrated that ZHX2 co-occupied with HIF1α on transcriptionally active promoters marked by H3K4me3 and H3K27ac, thereby promoting gene expression. Among the identified ZHX2 and HIF1α coregulated genes, overexpression of AP2B1, COX20, KDM3A, or PTGES3L could partially rescue TNBC cell growth defect by ZHX2 depletion, suggested that these downstream targets contribute to the oncogenic role of ZHX2 in an accumulative fashion. Furthermore, multiple residues (R491, R581, and R674) on ZHX2 are important in regulating its phenotype, which correspond with their roles on controlling ZHX2 transcriptional activity in TNBC cells. These studies establish that ZHX2 activates oncogenic HIF1α signaling, therefore serving as a potential therapeutic target for TNBC.

Transcriptomic analyses of gastrulation-stage mouse embryos with differential susceptibility to alcohol.

Genetics are a known contributor to differences in alcohol sensitivity in humans with fetal alcohol spectrum disorders (FASDs) and in animal models. Our study profiled gene expression in gastrulation-stage embryos from two commonly used, genetically similar mouse substrains, C57BL/6J (6J) and C57BL/6NHsd (6N), that differ in alcohol sensitivity. First, we established normal gene expression patterns at three finely resolved time points during gastrulation and developed a web-based interactive tool. Baseline transcriptional differences across strains were associated with immune signaling. Second, we examined the gene networks impacted by alcohol in each strain. Alcohol caused a more pronounced transcriptional effect in the 6J versus 6N mice, matching the increased susceptibility of the 6J mice. The 6J strain exhibited dysregulation of pathways related to cell death, proliferation, morphogenic signaling and craniofacial defects, while the 6N strain showed enrichment of hypoxia and cellular metabolism pathways. These datasets provide insight into the changing transcriptional landscape across mouse gastrulation, establish a valuable resource that enables the discovery of candidate genes that may modify alcohol susceptibility that can be validated in humans, and identify novel pathogenic mechanisms of alcohol. This article has an associated First Person interview with the first author of the paper.

MicroRNA-29 is an essential regulator of brain maturation through regulation of CH methylation.

Although embryonic brain development and neurodegeneration have received considerable attention, the events that govern postnatal brain maturation are less understood. Here, we identify the miR-29 family to be strikingly induced during the late stages of brain maturation. Brain maturation is associated with a transient, postnatal period of de novo non-CG (CH) DNA methylation mediated by DNMT3A. We examine whether an important function of miR-29 during brain maturation is to restrict the period of CH methylation via its targeting of Dnmt3a. Deletion of miR-29 in the brain, or knockin mutations preventing miR-29 to specifically target Dnmt3a, result in increased DNMT3A expression, higher CH methylation, and repression of genes associated with neuronal activity and neuropsychiatric disorders. These mouse models also develop neurological deficits and premature lethality. Our results identify an essential role for miR-29 in restricting CH methylation in the brain and illustrate the importance of CH methylation regulation for normal brain maturation.

Chd8 haploinsufficiency impairs early brain development and protein homeostasis later in life.

BACKGROUND: Chromodomain helicase DNA-binding protein 8 (Chd8) is a high-confidence risk gene for autism spectrum disorder (ASD). However, how Chd8 haploinsufficiency impairs gene expression in the brain and impacts behavior at different stages of life is unknown. METHODS: We generated a mutant mouse line with an ASD-linked loss-of-function mutation in Chd8 (V986*; stop codon mutation). We examined the behavior of Chd8 mutant mice along with transcriptional changes in the cerebral cortex as a function of age, with a focus on one embryonic (E14.5) and three postnatal ages (1, 6, and 12 months). RESULTS: Chd8V986*/+ mutant mice displayed macrocephaly, reduced rearing responses and reduced center time in the open field, and enhanced social novelty preference. Behavioral phenotypes were more evident in Chd8V986*/+ mutant mice at 1 year of age. Pup survival was reduced in wild-type x Chd8V986*/+ crosses when the mutant parent was female. Transcriptomic analyses indicated that pathways associated with synaptic and neuronal projections and sodium channel activity were reduced in the cortex of embryonic Chd8V986*/+ mice and then equalized relative to wild-type mice in the postnatal period. At 12 months of age, expression of genes associated with endoplasmic reticulum (ER) stress, chaperone-mediated protein folding, and the unfolded protein response (UPR) were reduced in Chd8V986*/+ mice, whereas genes associated with the c-MET signaling pathway were increased in expression. LIMITATIONS: It is unclear whether the transcriptional changes observed with age in Chd8V986*/+ mice reflect a direct effect of CHD8-regulated gene expression, or if CHD8 indirectly affects the expression of UPR/ER stress genes in adult mice as a consequence of neurodevelopmental abnormalities. CONCLUSIONS: Collectively, these data suggest that UPR/ER stress pathways are reduced in the cerebral cortex of aged Chd8V986*/+ mice. Our study uncovers neurodevelopmental and age-related phenotypes in Chd8V986*/+ mice and highlights the importance of controlling for age when studying Chd8 haploinsufficient mice.

Identification of BBOX1 as a Therapeutic Target in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease. Because of its heterogeneity and lack of hormone receptors or HER2 expression, targeted therapy is limited. Here, by performing a functional siRNA screening for 2-OG-dependent enzymes, we identified gamma-butyrobetaine hydroxylase 1 (BBOX1) as an essential gene for TNBC tumorigenesis. BBOX1 depletion inhibits TNBC cell growth while not affecting normal breast cells. Mechanistically, BBOX1 binds with the calcium channel inositol-1,4,5-trisphosphate receptor type 3 (IP3R3) in an enzymatic-dependent manner and prevents its ubiquitination and proteasomal degradation. BBOX1 depletion suppresses IP3R3-mediated endoplasmic reticulum calcium release, therefore impairing calcium-dependent energy-generating processes including mitochondrial respiration and mTORC1-mediated glycolysis, which leads to apoptosis and impaired cell-cycle progression in TNBC cells. Therapeutically, genetic depletion or pharmacologic inhibition of BBOX1 inhibits TNBC tumor growth in vitro and in vivo. Our study highlights the importance of targeting the previously uncharacterized BBOX1-IP3R3-calcium oncogenic signaling axis in TNBC. SIGNIFICANCE: We provide evidence from unbiased screens that BBOX1 is a potential therapeutic target in TNBC and that genetic knockdown or pharmacologic inhibition of BBOX1 leads to decreased TNBC cell fitness. This study lays the foundation for developing effective BBOX1 inhibitors for treatment of this lethal disease.This article is highlighted in the In This Issue feature, p. 1611.

A conditional mouse expressing an activating mutation in NRF2 displays hyperplasia of the upper gastrointestinal tract and decreased white adipose tissue.

Activation of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or NRF2) transcription factor is a critical and evolutionarily conserved cellular response to oxidative stress, metabolic stress, and xenobiotic insult. Deficiency of NRF2 results in hypersensitivity to a variety of stressors, whereas its aberrant activation contributes to several cancer types, most commonly squamous cell carcinomas of the esophagus, oral cavity, bladder, and lung. Between 10% and 35% of patients with squamous cell carcinomas display hyperactive NRF2 signaling, harboring activating mutations and copy number amplifications of the NFE2L2 oncogene or inactivating mutations or deletions of KEAP1 or CUL3, the proteins of which co-complex to ubiquitylate and degrade NRF2 protein. To better understand the role of NRF2 in tumorigenesis and more broadly in development, we engineered the endogenous Nfe2l2 genomic locus to create a conditional mutant LSL-Nrf2E79Q mouse model. The E79Q mutation, one of the most commonly observed NRF2-activating mutations in human squamous cancers, codes for a mutant protein that does not undergo KEAP1/CUL3-dependent degradation, resulting in its constitutive activity. Expression of NRF2 E79Q protein in keratin 14 (KRT14)-positive murine tissues resulted in hyperplasia of squamous cell tissues of the tongue, forestomach, and esophagus, a stunted body axis, decreased weight, and decreased visceral adipose depots. RNA-seq profiling and follow-up validation studies of cultured NRF2E79Q murine esophageal epithelial cells revealed known and novel NRF2-regulated transcriptional programs, including genes associated with squamous cell carcinoma (e.g. Myc), lipid and cellular metabolism (Hk2, Ppard), and growth factors (Areg, Bmp6, Vegfa). These data suggest that in addition to decreasing adipogenesis, KRT14-restricted NRF2 activation drives hyperplasia of the esophagus, forestomach, and tongue, but not formation of squamous cell carcinoma. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Putative modulation of the gut microbiome by probiotics enhances preference for novelty in a preliminary double-blind placebo-controlled study in ferrets.

BACKGROUND: Increasing evidence suggests a causal relationship between the gut microbiome and psychiatric illnesses. In particular, autism spectrum disorder is associated with gastrointestinal symptoms and alterations in the gut microbiome. Administration of probiotics is a commonly used strategy by caregivers of people with neurodevelopmental illness. However, evidence for successful improvement in gut microbiome and (behavioral) symptoms has been lacking. RESULTS: Here, we use a novel ferret model of maternal immune activation to show that high-dose probiotic administration in a placebo-controlled study design causes changes in the gut microbiome in the form of a transient increase in the administered bacterial species. In contrast, we found no differences in baseline microbiome composition or changes induced by probiotic administration between animals exposed in utero to maternal immune activation and control animals. However, the relative presence of several bacterial species correlated with an increased preference for novelty (object and conspecific). Intriguingly, several of the hits in this screen are species that have previously emerged in the literature as being associated with autism and anxiety. CONCLUSIONS: Together, our results suggest that high-dose probiotic interventions may be beneficial for the adjunct treatment of psychiatric illnesses. Placebo-controlled clinical trials in humans are urgently needed.

Unique and Shared Roles for Histone H3K36 Methylation States in Transcription Regulation Functions.

Set2 co-transcriptionally methylates lysine 36 of histone H3 (H3K36), producing mono-, di-, and trimethylation (H3K36me1/2/3). These modifications recruit or repel chromatin effector proteins important for transcriptional fidelity, mRNA splicing, and DNA repair. However, it was not known whether the different methylation states of H3K36 have distinct biological functions. Here, we use engineered forms of Set2 that produce different lysine methylation states to identify unique and shared functions for H3K36 modifications. Although H3K36me1/2 and H3K36me3 are functionally redundant in many SET2 deletion phenotypes, we found that H3K36me3 has a unique function related to Bur1 kinase activity and FACT (facilitates chromatin transcription) complex function. Further, during nutrient stress, either H3K36me1/2 or H3K36me3 represses high levels of histone acetylation and cryptic transcription that arises from within genes. Our findings uncover the potential for the regulation of diverse chromatin functions by different H3K36 methylation states.

Transcriptome-Wide Regulation of Key Developmental Pathways in the Mouse Neural Tube by Prenatal Alcohol Exposure.

BACKGROUND: Early gestational alcohol exposure is associated with severe craniofacial and CNS dysmorphologies and behavioral abnormalities during adolescence and adulthood. Alcohol exposure during the formation of the neural tube (gestational day [GD] 8 to 10 in mice; equivalent to4th week of human pregnancy) disrupts development of ventral midline brain structures such as the pituitary, septum, and ventricles. This study identifies transcriptomic changes in the rostroventral neural tube (RVNT), the region of the neural tube that gives rise to the midline structures sensitive to alcohol exposure during neurulation. METHODS: Female C57BL/6J mice were administered 2 doses of alcohol (2.9 g/kg) or vehicle 4 hours apart on GD 9.0. The RVNTs of embryos were collected 6 or 24 hours after the first dose and processed for RNA-seq. RESULTS: Six hours following GD 9.0 alcohol exposure (GD 9.25), over 2,300 genes in the RVNT were determined to be differentially regulated by alcohol. Enrichment analysis determined that PAE affected pathways related to cell proliferation, p53 signaling, ribosome biogenesis, and immune activation. In addition, over 100 genes involved in primary cilia formation and function and regulation of morphogenic pathways were altered 6 hours after alcohol exposure. The changes to gene expression were largely transient, as only 91 genes identified as differentially regulated by prenatal alcohol at GD 10 (24 hours postexposure). Functionally, the differentially regulated genes at GD 10 were related to organogenesis and cell migration. CONCLUSIONS: These data give a comprehensive view of the changing landscape of the embryonic transcriptome networks in regions of the neural tube that give rise to brain structures impacted by a neurulation-stage alcohol exposure. Identification of gene networks dysregulated by alcohol will help elucidate the pathogenic mechanisms of alcohol's actions.

USP37 promotes deubiquitination of HIF2α in kidney cancer.

Clear cell renal cell carcinoma (ccRCC) is characterized by loss of tumor suppressor Von Hippel Lindau (VHL) function, which leads to accumulation of hypoxia inducible factor α (including HIF1α and HIF2α). HIF2α was previously reported to be one of the major oncogenic drivers in ccRCC, however, its therapeutic targets remain challenging. Here we performed a deubiquitinase (DUB) complementary DNA (cDNA) library binding screen and discovered that ubiquitin-specific peptidase 37 (USP37) is a DUB that binds HIF2α and promotes HIF2α deubiquitination. As a result, USP37 promotes HIF2α protein stability in an enzymatically dependent manner, and depletion of USP37 leads to HIF2α down-regulation in ccRCC. Functionally, USP37 depletion causes decreased cell proliferation measured by MTS, two-dimensional (2D) colony formation as well as three-dimensional (3D) anchorage- independent growth. USP37 is also essential for maintaining kidney tumorigenesis in an orthotopic xenograft model and its depletion leads to both decreased primary kidney tumorigenesis and spontaneous lung metastasis. Our results suggest that USP37 is a potential therapeutic target in ccRCC.

Genome-wide Screening Identifies SFMBT1 as an Oncogenic Driver in Cancer with VHL Loss.

von Hippel-Lindau (VHL) is a critical tumor suppressor in clear cell renal cell carcinomas (ccRCCs). It is important to identify additional therapeutic targets in ccRCC downstream of VHL loss besides hypoxia-inducible factor 2α (HIF2α). By performing a genome-wide screen, we identified Scm-like with four malignant brain tumor domains 1 (SFMBT1) as a candidate pVHL target. SFMBT1 was considered to be a transcriptional repressor but its role in cancer remains unclear. ccRCC patients with VHL loss-of-function mutations displayed elevated SFMBT1 protein levels. SFMBT1 hydroxylation on Proline residue 651 by EglN1 mediated its ubiquitination and degradation governed by pVHL. Depletion of SFMBT1 abolished ccRCC cell proliferation in vitro and inhibited orthotopic tumor growth in vivo. Integrated analyses of ChIP-seq, RNA-seq, and patient prognosis identified sphingosine kinase 1 (SPHK1) as a key SFMBT1 target gene contributing to its oncogenic phenotype. Therefore, the pVHL-SFMBT1-SPHK1 axis serves as a potential therapeutic avenue for ccRCC.

Midtrimester microbial DNA variations in maternal serum of women who experience spontaneous preterm birth.

Objectives: To evaluate if midtrimester maternal serum contains microbial DNA and whether it differs between women with spontaneous preterm birth (SPTB) and those delivering at term.Study design: In this retrospective case-control study, we identified 20 healthy nulliparas with SPTB at 24-33 weeks of a nonanomalous singleton in 2014. Each case was matched by race/ethnicity to a control delivering at 39-40 weeks. Serum samples, collected at 15-20 weeks and stored at -80 C, were thawed and DNA extracted. PCR with primers targeting the 16S rDNA V4 region were used to prepare an amplicon library, sequenced using Illumina MiSeq, and analyzed using quantitative insight into microbial ecology (QIIME). Taxonomy was assigned using Ribosomal Database program (RDP) Classifier (threshold 0.8) against a modified Greengenes database. Differences in number of observed species, microbial alpha-diversity and beta-diversity, and taxa level analyses were undertaken.Results: All 40 samples were included. Women with SPTB had more unique observed species (p = .046) and higher mean alpha-diversity by Shannon index (but not Chao1 or Simpson) (p = .024). Microbial composition was different between groups by Bray-Curtis clustering (p = .03) but not by weighted (p = .13) or unweighted Unifrac (p = .11). Numerous taxa in the Firmicutes, Proteobacteria, and Actinobacteria phyla differed between groups (p < .05).Conclusions: SPTB is associated with distinct microbial DNA changes detected in midtrimester maternal serum.

Primary Cilia Signaling Promotes Axonal Tract Development and Is Disrupted in Joubert Syndrome-Related Disorders Models.

Appropriate axonal growth and connectivity are essential for functional wiring of the brain. Joubert syndrome-related disorders (JSRD), a group of ciliopathies in which mutations disrupt primary cilia function, are characterized by axonal tract malformations. However, little is known about how cilia-driven signaling regulates axonal growth and connectivity. We demonstrate that the deletion of related JSRD genes, Arl13b and Inpp5e, in projection neurons leads to de-fasciculated and misoriented axonal tracts. Arl13b deletion disrupts the function of its downstream effector, Inpp5e, and deregulates ciliary-PI3K/AKT signaling. Chemogenetic activation of ciliary GPCR signaling and cilia-specific optogenetic modulation of downstream second messenger cascades (PI3K, AKT, and AC3) commonly regulated by ciliary signaling receptors induce rapid changes in axonal dynamics. Further, Arl13b deletion leads to changes in transcriptional landscape associated with dysregulated PI3K/AKT signaling. These data suggest that ciliary signaling acts to modulate axonal connectivity and that impaired primary cilia signaling underlies axonal tract defects in JSRD.

Prolyl hydroxylase substrate adenylosuccinate lyase is an oncogenic driver in triple negative breast cancer.

Protein hydroxylation affects protein stability, activity, and interactome, therefore contributing to various diseases including cancers. However, the transiency of the hydroxylation reaction hinders the identification of hydroxylase substrates. By developing an enzyme-substrate trapping strategy coupled with TAP-TAG or orthogonal GST- purification followed by mass spectrometry, we identify adenylosuccinate lyase (ADSL) as an EglN2 hydroxylase substrate in triple negative breast cancer (TNBC). ADSL expression is higher in TNBC than other breast cancer subtypes or normal breast tissues. ADSL knockout impairs TNBC cell proliferation and invasiveness in vitro and in vivo. An integrated transcriptomics and metabolomics analysis reveals that ADSL activates the oncogenic cMYC pathway by regulating cMYC protein level via a mechanism requiring ADSL proline 24 hydroxylation. Hydroxylation-proficient ADSL, by affecting adenosine levels, represses the expression of the long non-coding RNA MIR22HG, thus upregulating cMYC protein level. Our findings highlight the role of ADSL hydroxylation in controlling cMYC and TNBC tumorigenesis.