Biology subjects

Zhao, L.

Publications and source records attributed to Zhao, L..

At least 271 records · Page 15Linked to original sources

bioRxiv2021

Differential and synergistic effects of low birth weight and Western diet on skeletal muscle vasculature, mitochondrial lipid metabolism and insulin signaling in male guinea pigs

Low birth weight (LBW) offspring are at increased risk for developing insulin resistance, a key precursor in metabolic syndrome and type 2 diabetes mellitus. Altered skeletal muscle vasculature, extracellular matrix, amino acid and mitochondrial lipid metabolism, and insulin signaling are implicated in this pathogenesis. Using uteroplacental insufficiency (UPI) to induce intrauterine growth restriction (IUGR) and LBW in the guinea pig, we investigated the relationship between UPI-induced IUGR/LBW and later life skeletal muscle arteriole density, fibrosis, amino acid and mitochondrial lipid metabolism, markers of insulin signaling and glucose uptake, and how a postnatal high-fat, high-sugar "Western" diet (WD) modulates these changes. Muscle of 145-day-old male LBW glucose tolerant offspring displayed diminished vessel density and altered acylcarnitine levels. Disrupted muscle insulin signaling despite maintained whole-body glucose homeostasis also occurred in both LBW and WD-fed male lean offspring. Additionally, postnatal WD unmasked LBW-induced impairment of mitochondrial lipid metabolism as reflected by increased acylcarnitine accumulation. This study provides evidence that early markers of skeletal muscle metabolic dysfunction appear to be influenced by the in utero environment and interact with a high fat-sugar postnatal environment to exacerbate altered mitochondrial lipid metabolism promoting mitochondrial overload.

physiology↗

bioRxiv2021

Suppression of B-Cell Activation by Human Cord Blood-Derived Stem Cells (CB-SC) through the Galectin-9-Dependent Cell Contact Mechanism

BackgroundWe developed the Stem Cell Educator therapy among multiple clinical trials based on the immune modulations of multipotent cord blood-derived stem cells (CB-SC) on different compartments of immune cells such as T cells and monocytes/macrophages in diabetes and other autoimmune diseases. However, the effects of CB-SC on the B cells remained unclear. To better understand the molecular mechanisms underlying the immune education of CB-SC, we explored the modulations of CB-SC on human B cells. MethodsCB-SC were isolated from human cord blood units and confirmed by flow cytometry with different markers for their purity. B cells were purified by using anti-CD19 immunomagnetic beads from human peripheral blood mononuclear cells (PBMC). Next, the activated B cells were treated in the presence or absence of coculture with CB-SC for 7 days before undergoing flow cytometry analysis of phenotypic change with different markers. RT-PCR was utilized to evaluate the levels of galectin expressions with or without treatment of activated B cells in order to find the key galectin contributing to the B-cell modulation. ResultsFlow cytometry demonstrated that the proliferation of activated B cells was markedly suppressed in the presence of CB-SC, leading to the down-regulation of immunoglobulin productions from the activated B cells. Phenotypic analysis revealed that treatment with CB-SC increased the percentage of IgD+CD27- naive B cells, but decreased the percentage of IgD-CD27+ switched B cells. Transwell assay showed that the immune suppression of CB-SC on B cells was dependent on the manner of cell-cell contact via Gal-9 molecule, as confirmed by the blocking experiment with the anti-Gal-9 monoclonal antibody. Mechanistic studies demonstrated that both calcium levels of cytoplasm and mitochondria were down-regulated after the treatment with CB-SC, causing the decline of mitochondrial membrane potential in the activated B cells. Western blot exhibited that the levels of phosphorylated Akt and Erk1/2 signaling proteins in the activated B cells were also markedly reduced in the presence of CB-SC. ConclusionsCB-SC displayed multiple immune modulations on B cells through the Gal-9-mediated cell-cell contact mechanism and calcium flux/Akt/Erk1/2 signaling pathways. The data advances current understanding about the molecular mechanisms underlying the Stem Cell Educator therapy to treat autoimmune diseases in clinics.

immunology↗

bioRxiv2021

A distinctive ligand recognition mechanism by the human vasoactive intestinal polypeptide receptor 2

Activated by physiologically important peptide hormones, class B1 G protein-coupled receptors (GPCRs) modulate key physiological functions and serve as valuable drug targets for many diseases. Among them, vasoactive intestinal polypeptide receptor 2 (VIP2R) is the last member whose full-length 3-dimensional structure has yet to be determined. VIP2R, expressed in the central and peripheral nervous systems and involved in a number of pathophysiological conditions, is implicated in pulmonary arterial hypertension, autoimmune and psychiatric disorders. Here, we report the cryo-electron microscopy structure of the human VIP2R bound to its endogenous ligand PACAP27 and the stimulatory G protein. Different from all reported peptide-bound class B1 GPCR structures, the N-terminal -helix of VIP2R adopts a unique conformation that deeply inserts into a cleft between PACAP27 and the extracellular loop 1, thereby stabilizing the peptide-receptor interface. Its truncation or extension significantly decreased VIP2R-mediated cAMP accumulation. Our results provide additional information on peptide recognition and receptor activation among class B1 GPCRs and may facilitate the design of better therapeutics.

molecular biology↗

bioRxiv2021

Mct11 deficiency alters hepatic glucose metabolism and energy homeostasis

Genetic variation at the SLC16A11 locus contributes to the disproportionate impact of type 2 diabetes (T2D) on Latino populations. We recently demonstrated that T2D risk variants reduce SLC16A11 liver expression and function of MCT11, the monocarboxylate transporter encoded by the SLC16A11 gene. Here, we show that SLC16A11 expression within the liver is primarily localized to the low oxygen pericentral region, and that T2D risk variants disrupt oxygen-regulated SLC16A11 expression in human hepatocytes. Under physiologic oxygen conditions, MCT11 deficiency alters hepatocyte glucose metabolism, resulting in elevated intracellular lactate and a metabolic shift toward triacylglycerol accumulation. We also demonstrate an impact of Mct11 deficiency on glucose and lipid metabolism in Slc16a11 knockout mice, which display physiological changes that are observed in individuals with T2D. Our findings provide mechanistic insight into how SLC16A11 disruption impacts hepatic energy metabolism and T2D risk, and highlight MCT11-mediated regulation of lactate levels as a potential therapeutic target.

genetics↗

bioRxiv2021

Frontal cortex activity underlying the production of diverse vocal signals during social communication in marmoset monkeys

Vocal communication is essential for social behaviors in humans and many non-human primates. While the frontal cortex has been shown to play a crucial role in human speech production, its role in vocal production in non-human primates has long been questioned. Recent studies have shown activation in single neurons in the monkey frontal cortex during vocal production in relatively isolated environment. However, little is known about how the frontal cortex is engaged in vocal production in ethologically relevant social context, where different types of vocal signals are produced for various communication purposes. Here we studied single neuron activities and local field potentials (LFP) and in the frontal cortex of marmoset monkeys while the animal engaged in vocal exchanges with other conspecifics in a social environment. Marmosets most frequently produced four types of vocalizations with distinct acoustic structures, three of which were typically not produced in isolation. We found that both single neuron activities and LFP were modulated by the production of each of the four call types. Moreover, the neural modulations in the frontal cortex showed distinct patterns for different call types, suggesting a representation of vocal signal features. In addition, we found that theta-band LFP oscillations were phase-locked to the phrases of twitter calls, which indicates the coordination of temporal structures of vocalizations. Our results suggested important functions of the marmoset frontal cortex in supporting the production of diverse vocalizations in vocal communication.

neuroscience↗

bioRxiv2021

Vapor cannabis exposure generationally affects male reproductive functions in mice

This study was performed to examine whether vapor exposure to cannabis plant matter negatively impacts male reproductive functions and testis development in mice. Adult CD-1 male mice (F0) were exposed to air (control) or 200 mg of vaporized cannabis plant matter 3x/day over a 10 day period. Subsequently, F0 males were bred with drug naive CD-1 females to generate F1 males, and F1 offspring were used to generate F2 males. Cannabis vapor exposure decreased sperm count and/or motility in F0 and F1 males and disrupted the progression of germ cell development, as morphometric analyses exhibited an abnormal distribution of the stages of spermatogenesis in F0 males. Although plasma levels of testosterone were not affected by cannabis exposure in any ages or generations of males, dysregulated steroidogenic enzymes, Cyp11a1 and Cyp19a1, were observed in F0 testis. In the neonatal testis from F1 males, while apoptosis was not altered, DNA damage and DNMT1, but not DNMT3A and DNMT3B, were increased in germ cells following cannabis exposure. In contrast, the alterations of DNA damage and DNMT1 expression were not observed in F2 neonatal males. These results suggest that cannabis vapor exposure generationally affects male reproductive functions, probably due to disruption of spermatogenesis in the developing testis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/457271v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1a6782borg.highwire.dtl.DTLVardef@1b5e42org.highwire.dtl.DTLVardef@c94d78org.highwire.dtl.DTLVardef@1fd269d_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LICannabis vapor exposure to adult males impairs sperm counts, motility and spermatogenesis. C_LIO_LICannabis vapor exposure to F0 males causes reduced sperm counts in F1, but not in F2 males. C_LIO_LIPlasma testosterone levels are not affected in F0, F1 and F2 males by cannabis exposure. C_LIO_LICannabis increases DNA damage and the expression of DNA methyltransferase (DNMT) 1 in the F1 neonatal testis. C_LI

pharmacology and toxicology↗

bioRxiv2021

Constitutive signal bias mediated by the human GHRHR splice variant 1

Alternative splicing of G protein-coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates Gs while SV1 selectively couples to {beta}-arrestins. Based on the cryo-electron microscopy structures of SV1 in the apo state or in complex with the Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs vs. {beta}-arrestins. Suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias towards {beta}-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation. Significance StatementThe mechanism of functional changes induced by alternative splicing of GHRHR is largely unknown. Here, we demonstrate that GHRH-elicited signal bias towards {beta}-arrestin recruitment is constitutively mediated by SV1. The cryo-electron microscopy structures of SV1 and molecular dynamics simulations reveal the different functionalities between GHRHR and SV1 at the near-atomic level, i.e., the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs vs. {beta}-arrestins. Our findings provide valuable insights into functional diversity of class B1 GPCRs which may aid in the design of better therapeutic agents against certain cancers.

molecular biology↗

bioRxiv2021

Stromal-cell deletion of STAT3 protects mice from kidney fibrosis by inhibiting pericytes trans-differentiation and migration

Signal transducer and activator of transcription 3 (STAT3) is a key transcription factor implicated in the pathogenesis of kidney fibrosis. Although tubular Stat3 deletion in tubular epithelial cells is known to protect mice from kidney fibrosis, the exact function of STAT3 in stromal cells remains unknown. We utilized stromal-cell Stat3 knock-out (KO) mice, CRISPR and pharmacologic activators and inhibitors of STAT3 to investigate its function in pericyte-like cells. STAT3 is phosphorylated in tubular epithelial cells in acute kidney injury whereas its activation expanded to interstitial cells in chronic kidney disease in mice and humans. Stromal cell-specific deletion of Stat3 protects mice from folic acid- and aristolochic acid-induced kidney fibrosis. Mechanistically, STAT3 directly regulates the inflammatory pathway, differentiation of pericytes into myofibroblasts. Specifically, STAT3 activation leads to an increase in migration and profibrotic signaling in genome-edited pericyte-like cells, 10T1/2. Conversely, Stat3 KO or blocking STAT3 function inhibits detachment, spreading, migration, and profibrotic signaling. Furthermore, STAT3 binds to Collagen1a1 promoter of fibrotic mouse kidneys and in pericyte-like cells. Together, our study identifies a previously unknown function of STAT3 in stromal cells that promotes kidney fibrosis and may have therapeutic value in fibrotic kidney disease. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/456872v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@35dcbforg.highwire.dtl.DTLVardef@2fc055org.highwire.dtl.DTLVardef@fc0d92org.highwire.dtl.DTLVardef@1def2e6_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

A Cell Culture Model of BK Polyomavirus Persistence, Genome Recombination, and Reactivation

BK Polyomavirus (BKPyV) is a small non-enveloped DNA virus that establishes a ubiquitous, asymptomatic, and lifelong persistent infection in at least 80% of the worlds population. In some immunosuppressed transplant recipients, BKPyV reactivation causes polyomavirus-associated nephropathy and hemorrhagic cystitis. We report a novel in vitro model of BKPyV persistence and reactivation using a BKPyV natural host cell line. In this system, viral genome loads remain constant for various times post-establishment of persistent infection, during which BKPyV undergoes extensive random genome recombination. Certain recombination events result in viral DNA amplification and protein expression, resulting in production of viruses with enhanced replication ability. ImportanceBK polyomavirus (BKPyV) generally establishes a persistent subclinical infection in healthy individuals but can cause severe disease in transplant recipients. While an in vitro model to study acute replication exists, no practical model with which to study BKPyV persistence is currently available. We established a BKPyV persistence model in cell culture. Our model reveals that the virus can persist for varying periods of time before random recombination of the viral genome leads to enhanced replication.

microbiology↗

bioRxiv2021

Structural basis for the therapeutic advantage of dual and triple agonists at the human GIP, GLP-1 or GCG receptors

Glucose homeostasis, regulated by glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and glucagon (GCG) is critical to human health. Several multi-targeting agonists at GIPR, GLP-1R or GCGR, developed to maximize metabolic benefits with reduced side-effects, are in clinical trials to treat type 2 diabetes and obesity. To elucidate the molecular mechanisms by which tirzepatide, a GIPR/GLP-1R dualagonist, and peptide 20, a GIPR/GLP-1R/GCGR triagonist, manifest their superior efficacies over monoagonist such as semaglutide, we determined cryo-electron microscopy structures of tirzepatide-bound GIPR and GLP-1R as well as peptide 20-bound GIPR, GLP-1R and GCGR The structures reveal both common and unique features for the dual and triple agonism by illustrating key interactions of clinical relevance at the atomic level. Retention of glucagon function is required to achieve such an advantage over GLP-1 monotherapy. Our findings provide valuable insights into the structural basis of functional versatility and therapeutic supremacy of tirzepatide and peptide 20.

molecular biology↗

bioRxiv2021

Deficiency in Lyst function leads to accumulation of secreted proteases and predisposition to mechanic stress-induced retinal detachment

Chediak-Higashi syndrome, caused by mutations in the Lysosome Trafficking Regulator (Lyst) gene, is a recessive hypopigmentation disorder characterized by albinism, neuropathies, neurodegeneration, and defective immune responses, with enlargement of lysosomes and lysosome-related organelles. Although recent studies have suggested that Lyst mutations impair the regulation of sizes of lysosome and lysosome-related organelle, the underlying pathogenic mechanism of Chediak-Higashi syndrome is still unclear. Here we show striking evidence that deficiency in LYST protein function leads to accumulation of photoreceptor outer segment phagosomes in retinal pigment epithelial cells, and reduces adhesion between photoreceptor outer segment and retinal pigment epithelial cells in a mouse model of Chediak-Higashi syndrome. In addition, we observe elevated levels of cathepsins, matrix metallopeptidase (MMP) 3 and oxidative stress markers in the retinal pigment epithelium of Lyst mutants. Previous reports showed that impaired degradation of photoreceptor outer segment phagosomes causes elevated oxidative stress, which could consequently lead to increases of cysteine cathepsins and MMPs in the extracellular matrix. Taken together, we conclude that the loss of LYST function causes accumulation of phagosomes in the retinal pigment epithelium and elevation of several extracellular matrix-remodeling proteases through oxidative stress, which may, in turn, reduce retinal adhesion. Our work reveals previously unreported pathogenic events in the retinal pigment epithelium caused by Lyst deficiency, which may place Chediak-Higashi syndrome patients at increased risk for retinal detachment. The same pathogenic events may be conserved in other professional phagocytic cells, such as macrophages in the immune system, contributing to overall Chediak-Higashi syndrome pathology.

genetics↗

bioRxiv2021

A transmissible γδ intraepithelial lymphocyte hyperproliferative phenotype is associated with the intestinal microbiota and confers protection against acute infection.

Intraepithelial lymphocytes expressing the {gamma}{delta} T cell receptor ({gamma}{delta} IELs) serve as a first line of defense against luminal microbes. Although the presence of an intact microbiota is dispensable for {gamma}{delta} IEL development, several microbial factors contribute to the maintenance of this sentinel population. However, whether specific commensals influence population of the {gamma}{delta} IEL compartment under homeostatic conditions has yet to be determined. We identified a novel {gamma}{delta} IEL hyperproliferative phenotype that arises early in life and is characterized by expansion of multiple V{gamma} subsets. Horizontal transfer of this hyperproliferative phenotype to mice harboring a phenotypically normal {gamma}{delta} IEL compartment was prevented following antibiotic treatment, thus demonstrating that the microbiota is both necessary and sufficient for the observed increase in {gamma}{delta} IELs. Further, we identified a group of unique gut bacteria represented by 5 amplicon sequence variants (ASV) which are strongly associated with {gamma}{delta} IEL expansion. Using intravital microscopy, we find that hyperproliferative {gamma}{delta} IELs also exhibit increased migratory behavior leading to enhanced protection against bacterial infection. These findings reveal that transfer of a specific group of commensals can regulate {gamma}{delta} IEL homeostasis and immune surveillance, which may provide a novel means to reinforce the epithelial barrier.

immunology↗

bioRxiv2021

The framework of plant regeneration in duckweed (Lemna turonifera) comprises genetic transcript regulation and cyclohexane release

Regeneration is important for vegetative propagation of excellent variety, detoxification and the obtain of transgenic plant, but plant regeneration is time-consuming. Here, we found that duckweed regeneration could be enhanced by regenerating callus. Firstly, Genetic transcript regulation has been applied to study the molecular mechanism controlling regeneration. Auxin related genes have been significantly down-regulated in regenerating callus. Cytokinin signal pathway genes have been up-regulated in regenerating callus. Secondly, volatile organic compounds release has been analysised by gas chromatography/mass spectrum during the stage of plant regeneration, and 11 kinds of unique volatile organic compounds in the regenerating callus were increased. Among them, cyclohexane treatment enhanced duckweed regeneration by initiating root. Moreover, Auxin signal pathway genes were down-regulated in callus treated by cyclohexane. All together, these results provide novel mechanistic insights into how regenerating callus promotes duckweed regeneration. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/453434v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@1c28166org.highwire.dtl.DTLVardef@bd86cforg.highwire.dtl.DTLVardef@dd9802org.highwire.dtl.DTLVardef@26ba1c_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2021

A pair of transporters controls mitochondrial Zn2+ levels to maintain mitochondrial homeostasis

Zn2+ is required for the activity of many mitochondrial proteins, which regulate mitochondrial dynamics, apoptosis and mitophagy. However, it is not understood how the proper mitochondrial Zn2+ level is achieved to maintain mitochondrial homeostasis. Using Caenorhabditis elegans, we reveal here that a pair of mitochondrion-localized transporters controls the mitochondrial level of Zn2+. We demonstrate that SLC-30A9/ZnT9 is a mitochondrial Zn2+ exporter. Loss of SLC-30A9 leads to mitochondrial Zn2+ accumulation, which damages mitochondria, impairs animal development and shortens the life span. We further identify SLC-25A25/SCaMC-2 as an important regulator of mitochondrial Zn2+ import. Loss of SLC-25A25 suppresses the abnormal mitochondrial Zn2+ accumulation and defective mitochondrial structure and functions caused by loss of SLC-30A9. Moreover, we reveal that the endoplasmic reticulum contains the Zn2+ pool from which mitochondrial Zn2+ is imported. These findings establish the molecular basis for controlling the correct mitochondrial Zn2+ levels for normal mitochondrial structure and functions. SummaryZn2+ is a trace ion essential for the function of many mitochondrial proteins. It is not known how mitochondrial Zn2+ levels are regulated. Ma at al. identify transporters that mediate mitochondrial Zn2+ export and import to maintain mitochondrial homeostasis.

cell biology↗

bioRxiv2021

An improved auxin-inducible degron system for fission yeast

Conditional degron technologies, which allow a protein of interest to be degraded in an inducible manner, are important tools for biological research, and are especially useful for creating conditional loss-of-function mutants of essential genes. The auxin-inducible degron (AID) technology, which utilizes plant auxin signaling components to control protein degradation in non-plant species, is a widely used small-molecular-controlled degradation method in yeasts and animals. However, the currently available AID systems still have room for further optimization. Here, we have improved the AID system for the fission yeast Schizosaccharomyces pombe by optimizing all three components: the AID degron, the small-molecule inducer, and the inducer-responsive F-box protein. We chose a 36-amino-acid sequence of the Arabidopsis IAA17 protein as the degron and employed three tandem copies of it to enhance efficiency. To minimize undesirable side effects of the inducer, we adopted a bulky analog of auxin, 5-adamantyl-IAA, and paired it with the F-box protein OsTIR1 that harbors a mutation (F74A) at the auxin-binding pocket. 5-adamantyl-IAA, when utilized with OsTIR1-F74A, is effective at concentrations thousands of times lower than auxin used in combination with wild-type OsTIR1. We tested our improved AID system on 10 essential genes and achieved inducible lethality for all of them, including ones that could not be effectively inactivated using a previously published AID system. Our improved AID system should facilitate the construction of conditional loss-of-function mutants in fission yeast.

genetics↗

bioRxiv2021

Dynamic surveillance of mosquitoes and their viromes in Wuhan during 2020.

Mosquitoes are medically important arthropod vectors and harbor a great variety of viruses. The population density, species and virome of mosquitoes varies according to geography and climate. To investigate the dynamic changes in the species composition and diversity of mosquitoes and their viromes in Wuhan, China, a total of 2,345 adult mosquitoes collected from different habitats including an urban residential area, two hospitals, a scenic area, and a pig farm in a rural region from April to October 2020 were subjected to morphological identification, RT-qPCR and metagenomic sequencing. The results indicated that the dominant presence of Culex mosquitoes was observed in both urban regions (90.32%, 1538/1703) and the pig farm (54.98%, 353/642). Viromes of Culex showed dynamic changes during the collection time. Several viruses, such as Culex flavivirus, Alphamesonivirus 1, Hubei mosquito virus 2 and Hubei mosquito virus 4, had seasonal changes and unimodal increases or declines. Other viruses, such as Wuhan mosquito virus 6, Hubei virga-like virus 2 and Zhejiang mosquito virus 3, were stable in all collected Culex and should be potential members of "core viromes". This study improves the understanding of the dynamic composition of mosquitoes and the viromes they carry and provides useful information for informing mosquito control and mosquito-borne disease prevention strategies.

microbiology↗

bioRxiv2021

Functional analysis of Mmd2 and related PAQR genes during sex determination in mice

Sex determination in eutherian mammals is controlled by the Y-linked gene Sry, which drives the formation of testes in male embryos. Despite extensive study, the genetic steps linking Sry action and male sex determination remain largely unknown. Here, we focused on Mmd2, a gene that encodes a member of the progestin and adipoQ receptor (PAQR) family. We show that Mmd2 is expressed during the sex-determining period in XY but not XX gonads, specifically in the Sertoli cell lineage which orchestrates early testis development. Analysis of knockout mice deficient in Sox9 and Sf1 revealed that Mmd2 operates downstream of these known sex-determining genes. However, when we used CRISPR to ablate Mmd2 in the mouse, fetal testis development appeared to progress normally. To determine if other genes might have compensated for the loss of Mmd2, we identified the closely related PAQR family members Paqr8 and Mmd as also being expressed during testis development. We used CRISPR to generate mouse strains deficient in Paqr8 and Mmd, but both knockout lines appeared phenotypically normal and fertile. Finally, we generated Mmd2;Mmd and Mmd2;Paqr8 double-null embryos and again observed normal testis development. These results may reflect functional redundancy among these factors. Our findings highlight the difficulties involved in identifying genes with a functional role in sex determination and gonadal development through expression screening and loss-of-function analyses of individual candidate genes, and may help to explain the paucity of genes in which variations have been found to cause human disorders/differences of sex development.

developmental biology↗

bioRxiv2021

Profiling chromatin accessibility in formalin-fixed paraffin-embedded samples

Archived formalin-fixed paraffin-embedded (FFPE) samples are the global standard format for preservation of the majority of biopsies in both basic research and translational cancer studies, and profiling chromatin accessibility in the archived FFPE tissues is fundamental to understanding gene regulation. Accurate mapping of chromatin accessibility from FFPE specimens is challenging because of the high degree of DNA damage. Here, we first showed that standard ATAC-seq can be applied to purified FFPE nuclei but yields lower library complexity and a smaller proportion of long DNA fragments. We then present FFPE-ATAC, the first highly sensitive method for decoding chromatin accessibility in FFPE tissues that combines Tn5-mediated transposition and T7 in vitro transcription. The FFPE-ATAC generates high-quality chromatin accessibility profiles with 500 nuclei from a single FFPE tissue section, enables the dissection of chromatin profiles from the regions of interest with the aid of hematoxylin and eosin (H&E) staining, and reveals disease-associated chromatin regulation from the human colorectal cancer FFPE tissue archived for more than 10 years. In summary, the approach allows decoding of the chromatin states that regulate gene expression in archival FFPE tissues, thereby permitting investigators, to better understand epigenetic regulation in cancer and precision medicine.

genomics↗