Biology subjects

Deng, Y.

Publications and source records attributed to Deng, Y..

At least 199 records · Page 11Linked to original sources

bioRxiv2022

Two-Stage Multivariate Mendelian Randomization on Multiple Outcomes with Mixed Distributions

SO_SCPLOWUMMARYC_SCPLOWIn clinical research, it is of importance to study whether certain clinical factors or exposures have causal effects on clinical and patient reported outcomes like toxicities, quality of life, and self-reported symptoms, which can help improve patient care. Usually, such outcomes are recorded as multiple variables with different distributions. Mendelian randomization is a commonly used technique for causal inference with the help of genetic instrumental variables to deal with observed and unobserved confounders. Nevertheless, the current methodology of Mendelian randomization on multiple outcomes only focuses on one outcome at a time, meaning that it does not consider the correlation structure of multiple outcomes, which may lead to loss of statistical power. In situations with multiple outcomes of interest, especially when there are mixed correlated outcomes with multiple distributions, it is much more desirable to jointly analyze them with a multivariate approach. Some multivariate methods have been proposed to model mixed outcomes, however, they do not incorporate instrumental variables and cannot handle unmeasured confounders. To overcome the above challenges, we propose a two-stage multivariate Mendelian Randomization Method (MRMO), that can perform multivariate analysis on mixed outcomes using instrumental variables. We demonstrate that our proposed MRMO algorithm can gain power over the existing univariate method through simulation studies and a clinical application on a randomized Phase III clinical trial study on colorectal cancer patients.

genetics↗

bioRxiv2022

Rapid modeling of experimental molecular kinetics with simple electronic circuits instead of with complex differential equations

Kinetic modeling has relied on using a tedious number of mathematical equations to describe molecular kinetics in interacting reactions. The long list of differential equations with associated abstract variables and parameters inevitably hinders readers easy understanding of the models. However, the mathematical equations describing the kinetics of biochemical reactions can be exactly mapped to the dynamics of voltages and currents in simple electronic circuits wherein voltages represent molecular concentrations and currents represent molecular fluxes. For example, we theoretically derive and experimentally verify accurate circuit models for Michaelis-Menten kinetics. Then, we show that such circuit models can be scaled via simple wiring among circuit motifs to represent more and arbitrarily complex reactions. Hence, we can directly map reaction networks to equivalent circuit schematics in a rapid, quantitatively accurate, and intuitive fashion without needing mathematical equations. We verify experimentally that these circuit models are quantitatively accurate. Examples include i) different mechanisms of competitive, noncompetitive, uncompetitive, and mixed enzyme inhibition, important for understanding pharmacokinetics; ii) product-feedback inhibition, common in biochemistry; iii) reversible reactions, important in many metabolic pathways; and iv) translation and transcription dynamics in a cell-free system, which brings insight into the functioning of all gene-protein networks. We envision that circuit modeling and simulation could become a powerful scientific communication language and tool for quantitative studies of kinetics in biology.

bioengineering↗

bioRxiv2022

Multi-omics analyses of drug repurposing reveal Acebutolol and Amiloride for osteoporosis treatment

Osteoporosis is a metabolic bone disease that occurs during aging, characterized by low bone mineral density (BMD) and a high risk of trauma fracture. While current pharmacological interventions provide symptomatic benefits, they are unsatisfactory and have major side effects. In this study, we used multi-omics data and drug similarity to construct osteoporosis driver signaling networks (ODSN) and drug functional networks (DFN), respectively. By integrating ODSN and DFN with treatment transcriptional responses, we observed 8 drugs that demonstrated strong targeting effects on ODSN. Mendelian Randomization analysis determines the causal effect on BMD using cis-eQTLs of the drug targets and BMD GWAS data. The findings suggested Acebutolol and Amiloride may increase BMD, while Acenocoumarol, Aminocaproic acid and Armodafinil may enhance bone loss. Zebrafish experiments experimentally showed Acebutolol hydrochloride and Amiloride hydrochloride had significant protective effects on osteoporosis in zebrafish embryos induced by Dexamethasone. Also, Acenocoumarol reduced bone mineralization compared with the control group. The findings suggest that the hypertension drugs Acebutolol and Amiloride warrant further investigation in functional mechanistic experiments to evaluate their effectiveness for osteoporosis treatments.

systems biology↗

bioRxiv2022

Spatial-CITE-seq: spatially resolved high-plex protein and whole transcriptome co-mapping

We present spatial-CITE-seq for high-plex protein and whole transcriptome co-mapping, which was firstly demonstrated for profiling 189 proteins and transcriptome in multiple mouse tissue types. It was then applied to human tissues to measure 273 proteins and transcriptome that revealed spatially distinct germinal center reaction in tonsil and early immune activation in skin at the COVID-19 mRNA vaccine injection site. Spatial-CITE-seq may find a range of applications in biomedical research.

genomics↗

bioRxiv2022

Deep Phenotyping and Lifetime Trajectories Reveal Limited Effects of Longevity Regulators on the Aging Process in C57BL/6J Mice

Current concepts regarding the biology of aging are based on studies aimed at identifying factors regulating natural lifespan. However, lifespan as a sole proxy measure for aging can be of limited value because it may be restricted by specific sets of pathologies, rather than by general physiological decline. Here, we employed large-scale phenotyping to analyze hundreds of phenotypes and thousands of molecular markers across tissues and organ systems in a single study of aging male C57BL/6J mice. For each phenotype, we established lifetime profiles to determine when age-dependent phenotypic change is first detectable relative to the young adult baseline. We examined central genetic and environmental lifespan regulators (putative anti-aging interventions, PAAIs; the following PAAIs were examined: mTOR loss-of-function, loss-of-function in growth hormone signaling, dietary restriction) for a possible countering of the signs and symptoms of aging. Importantly, in our study design, we included young treated groups of animals, subjected to PAAIs prior to the onset of detectable age-dependent phenotypic change. In parallel to our studies in mice, we assessed genetic variants for their effects on age-sensitive phenotypes in humans. We observed that, surprisingly, many PAAI effects influenced phenotypes long before the onset of detectable age-dependent changes, rather than altering the rate at which these phenotypes developed with age. Accordingly, this subset of PAAI effects does not reflect a targeting of age-dependent phenotypic change. Overall, our findings suggest that comprehensive phenotyping, including the controls built in our study, is critical for the investigation of PAAIs as it facilitates the proper interpretation of the mechanistic mode by which PAAIs influence biological aging. HighlightsO_LIPhenotyping at scale defines lifetime trajectories of age-dependent changes in C57BL/6J mice C_LIO_LICentral genetic and environmental lifespan regulators (putative anti-aging interventions; PAAIs) influence age-sensitive phenotypes (ASPs) often long before the appearance of age-dependent changes in these ASPs C_LIO_LICorresponding genetic variants in humans also have age-independent effects C_LIO_LIMany PAAI effects shift the baseline of ASPs rather than slowing their rate of change C_LI

physiology↗

bioRxiv2022

Hypomorphism of a novel long ERα isoform causes severe reproductive dysfunctions in female mice

Estrogen receptor alpha (ER)-mediated estrogen signaling play a pivotal role in both reproductive and non-reproductive functions. Transcriptional regulation of ER gene is highly complex, with multiple transcript variants being differentially produced across the tissues. However, tissue-specific variation and physiological specificity of the ER variants are not yet fully understood. In an attempt to generate a Cre-dependently restorable ER-null mice for functional investigation of the genetic sufficiency, we unexpectedly produced ER hypomorphic mice with biased downregulation of a previously unappreciated long ER isoform that is enriched in the female reproductive organs (uterus and ovaries) and the pituitary but minimally expressed in the brain. Female homozygous mutant mice were capable of pregnancy but displayed irregular estrus cycle and rarely maintained alive newborns without significant morphological and pathological changes in reproductive system and the disruption of body weight homeostasis, indicating the vital role of this long isoform in female reproductive function. Collectively, our results define a tissue-specifically enriched long ER isoform and its preferential role in female reproductive function over body weight homeostasis.

physiology↗

bioRxiv2022

Gut microbiota-derived tryptamine impairs insulin sensitivity

Gut-microbiota plays a pivotal role in development of type 2 diabetes (T2D), yet the molecular mechanism remains elusive. Here, we show that tryptamine, a microbial metabolite of tryptophan, impairs glucose tolerance and insulin sensitivity. Tryptamine presents a higher level in monkeys with spontaneous diabetes and human with T2D and positively correlated with the glucose tolerance. In parallel, tryptamine level was suppressed by dietary fibers intervention in T2D subjects and negatively correlated with improvement of glucose tolerance. The inhibitory effect of tryptamine on insulin signaling as shown was dependent on a trace amine-associated receptor 1 (TAAR1)-extracellular signal-regulated kinase (ERK) signaling axis. Monoassociation of T2D-associated tryptamine-producing bacteria Ruminococcus gnavus impairs insulin sensitivity in pseudo germ-free mice. Our findings indicate gut microbiota-derived tryptamine contributes to the development of insulin resistance in T2D and may serve as a new target for intervention. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

microbiology↗

bioRxiv2022

Peptidoglycan recognition protein S2 is crucial for activation the Toll pathway against Israeli acute paralysis virus infection in honey bee Apis mellifera

Although honey bee responses to pathogens have been systematically described in the past decades, antiviral signalling pathways mechanisms are not thoroughly characterized. To decipher direct antiviral roles of an immune pathway, we firstly used the infectious clone of Israeli acute paralysis virus (IAPV) to screen 42 immune genes involved in mTOR, MAPK, Toll, Endocytosis, Jak-STAT pathway and homeobox protein, heat shock protein, as well as antimicrobial peptides (AMPs), and found that Toll pathway was a potential predominant immune pathway in Apis mellifera. Consistent with this, only dsRNA-PGRP-S2 treated A. mellifera significantly exhibited impaired activation of Toll pathway, promoting susceptibility to the IAPV infection. Finally, immunofluorescence results confirmed that the Toll pathway was initiated by peptidoglycan recognition protein S2 (PGRP-S2) interacting with Toll protein. Co-immunoprecipitation findings also further preliminarily confirmed PGRP-S2 directly interacting with viral capsid protein IAPV-VP3 to induce the activation of the Toll pathway in A. mellifera. These findings highlight that the Toll pathway is demanded efficient inhibitions of IAPV replication as a specific antiviral pathway in A. mellifera, and PGRP-S2, acting as a pattern recognition receptor, could be a new approach for control of the viral disease. Author summaryHoney bee viruses, particularly IAPV, had been implicated in the colony decline with a global distribution resulting in insufficient pollination services. However, little is known about the antiviral mechanism of honey bee. In this study, we found that the Toll pathway was required for A. mellifera against IAPV infection and initiated by PGRP-S2. We also confirmed that dsRNA-PGRP-S2 treated A. mellifera exhibited impaired Toll pathway activation and promoted susceptibility to the IAPV infection. As a result, we employed co-immunoprecipitation technique to identify the interaction between the PGRP-S2 with Toll. Moreover, it was found the PGRP-S2 directly recognized IAPV-VP3 to activate the immune pathway against IAPV infection. Our work provides novel evidence that honey bees own a specific antiviral immune pathway and suggests that targeting PGRP-S2 could be a new approach for controlling the viral disease.

immunology↗

bioRxiv2022

Neuronal miR-138 represses HSV-2 lytic infection by regulating viral and host genes with mechanistic differences compared to HSV-1

Herpes simplex virus 2 (HSV-2) establishes latent infection in dorsal root ganglion (DRG) neurons after productive (lytic) infection in peripheral tissues. A neuron-specific microRNA, miR-138, favors HSV-1 latency by repressing viral ICP0, and host Oct-1 and Foxc1 genes, yet the role of miR-138 in HSV-2 infection was unknown. The ICP0 mRNAs of HSV-1, HSV-2 and chimpanzee herpesvirus each have one to two canonical miR-138 binding sites. The sites are 100% conserved in 308 HSV-1 and 300 HSV-2 published sequences of clinical isolates. In co-transfection assays, miR-138 repressed HSV-2 ICP0 expression through the seed region and surrounding interactions that are different from HSV-1. An HSV-2 mutant with disrupted miR-138 binding sites on ICP0 showed increased ICP0 expression in Neuro-2a cells. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation confirmed miR-138 binding to HSV-2 ICP0, and identified UL19 and UL20 as additional targets, whose expression was repressed by miR-138 during co-transfection. In Neuro-2a cells, transfected miR-138 and its antagomir decreased and increased HSV-2 replication, respectively, and knockout experiment showed that miR-138s host targets OCT-1 and FOXC1 were important for HSV-2 replication. In primary mouse DRG neurons, both ICP0 and FOXC1 positively regulated HSV-2 replication, but both overexpressed and endogenous miR-138 suppressed HSV-2 replication primarily by repressing ICP0 expression. Thus, miR-138 can suppress HSV-2 neuronal replication through multiple viral and host pathways. These results reveal functional similarities and mechanistic differences in how miR-138 regulates HSV-1 and HSV-2 infection and indicate an evolutionary advantage of using miR-138 to repress lytic infection in neurons. ImportanceHerpes simplex virus 1 (HSV-1) and HSV-2 are closely related viruses with major differences. Both viruses establish latency in neurons from which they reactivate to cause disease. A key aspect of HSV latency is repression of productive infection in neurons. Based on previous work with HSV-1, we investigated the role of a neuron-specific microRNA, miR-138, in HSV-2 infection, and established it as a repressor of HSV-2 productive infection in neuronal cells. This repression is mediated mainly by targeting viral ICP0 and host Foxc1 mRNAs, but other pathways also contribute. Despite functional conservation of the role of miR-138 between HSV-1 and HSV-2, many molecular mechanisms differ including how miR-138 represses ICP0 expression and miR-138 targeting of HSV-2 but not HSV-1 UL19 and UL20. To our knowledge, this study provides the first example of host microRNA regulation of HSV-2 infection.

microbiology↗

bioRxiv2022

Genome-wide in vivo CRISPR screens identify GATOR1 as a TP53 induced tumour suppressor

Identifying tumor suppressor genes is predicted to inform on the development of novel strategies for cancer therapy. To identify new lymphoma driving processes that cooperate with oncogenic MYC (which is abnormally highly expressed in [~]70% of human cancers) we have used a genome-wide CRISPR knockout screen in E{micro}-Myc;Cas9 transgenic hematopoietic stem and progenitor cells in vivo. We discovered that loss of any of the GATOR1 complex components - NPRL3, DEPDC5, NPRL2 - significantly accelerated c-MYC-driven lymphoma development in mice. Low expression of the GATOR1 complex genes correlated with poor survival outcomes for human patients with high MYC- expressing cancers. Murine lymphomas lacking GATOR1 were highly sensitive to mTOR inhibitors as a single agent therapy, both in vitro and in vivo. These findings identify inhibition of mTORC1 as a potent tumor suppressive mechanism in c-MYC-driven lymphomagenesis and suggest a new avenue for therapeutic intervention in GATOR1-deficient lymphomas through mTOR inhibition. Key PointsO_LIIn vivo CRISPR/Cas9 whole genome knockout screens identified the GATOR1 complex as a potent suppressor of c-MYC-driven lymphomagenesis. C_LIO_LIGATOR1 deficiency in MYC-driven lymphomas confers sensitivity to mTOR inhibition, suggesting a novel therapeutic approach. C_LI

cancer biology↗

bioRxiv2022

OCTA observation of changes in macular vessel density in diabetic patients and its correlation with diabetic retinopathy staging

ObjectiveTo investigate the association between disease progression and OCTA vessel density and other indices in patients with diabetic retinopathy. MethodsParticipants were selected with the following criteria: 63 patients (100 eyes) diagnosed with type 2 diabetes mellitus, which included 44 patients (72 eyes) with diabetic retinopathy and 19 patients (28 eyes) with type 2 diabetes mellitus and non-diabetic retinopathy (NDR), who were seen at the Eye Hospital China Academy of Chinese Medical Sciences from September 2020 to July 2021. All patients underwent OCTA examination, and FAZ, PERIM, AI, FD, SVD, DVD and other indices were counted. Results(1) The correlation coefficients of SVD, paracentric SVD, DVD, paracentric DVD and DR processes were: -0.525, -0.586, -0.323, and -0.424 (P< 0.05), respectively, and all were moderately negatively correlated. (2) The correlation coefficients of FAZ and PERIM with DR process were: -0.031, 0.084 (P>0.05), respectively, and not correlated. The correlation coefficients of AI and FD with DR process were: 0.307, -0.459 (P<0.05), and with moderate positive and negative correlations, respectively. (3) The correlation coefficients of FAZ, PERIM, AI and FD with age were: -0.124, -0.052, 0.113, -0.170 (P>0.05), and no correlation, respectively. ConclusionThe disease progression of DR was moderately correlated with OCTA superficial vessel density and deep vessel density; and moderately correlated with AI and FD. OCTA could assist in the assessment of DR disease progression.

neuroscience↗

bioRxiv2022

Anti-seed PNAs targeting multiple oncomiRs for brain tumor therapy

Glioblastoma (GBM) is one of the most lethal malignancies in the United States with poor survival and high recurrence rates, suggesting the need for approaches targeting the most important molecular drivers of tumor growth. Here, we aimed to simultaneously target oncomiRs 10b and 21, which have been reported to drive the aggressive growth and invasiveness of GBM. We designed short (8-mer bases) gamma-({gamma})-modified peptide nucleic acids (s{gamma}PNAs), which target the seed region of oncomiRs 10b and 21 with high affinity. We entrapped these anti-miR s{gamma}PNAs in nanoparticles (NPs) formed from a block copolymer of poly(lactic acid) and hyperbranched polyglycerol (PLA-HPG); the NPs were also functionalized with aldehydes to produce bioadhesive NPs. We have previously shown that these bioadhesive NPs (BNPs) produce superior transfection efficiency, with a tropism for tumor cells. The s{gamma}PNA BNPs showed superior anti-miR efficacy in comparison to the regular full length PNA BNPs in vitro. When combined with temozolomide, s{gamma}PNA BNPs administered via convention-enhanced delivery (CED) inhibited the growth of intracranial tumors and significantly improved the survival of animals (>120 days). RNA sequencing analysis revealed the role of vascular endothelial growth factor A (VEGFA) and integrin beta 8 (ITGB8), direct targets of both miR-10b and miR-21, in mediating the tumor growth. Hence, we established that BNPs loaded with anti-seed s{gamma}PNAs targeting multiple oncomiRs is a promising approach to improve the treatment of GBM, with a potential to personalize treatment based on tumor specific oncomiRs. SummaryO_ST_ABSTargeting oncomiRs 21 and 10b to improve GBM survivalC_ST_ABSGlioblastoma (GBM) is an aggressive malignant disorder with high recurrence rates and poor survival. Here, we aimed to simultaneously inhibit two aberrant oncomiRs--miR 21 and miR 10b--which have been previously associated with GBM invasiveness and progression. We synthesized short, gamma-modified peptide nucleic acids (s{gamma}PNA) targeted to the miR seed regions and loaded the s{gamma}PNAs into bioadhesive nanoparticles (BNPs). When the s{gamma}PNA-BNPs were added to cultured tumor cells, we observed significant reduction of target oncomiRs and increase of apoptosis in vitro. When delivered in vivo by convection-enhanced delivery, s{gamma}PNA BNPs dramatically increased the survival in two orthotopic (intracranial) mouse models of GBM. Moreover, the combination of s{gamma}PNA BNPs with temozolomide (TMZ) increased the survival of mice with GBM beyond the planned endpoint (120 days) with significant improvements in histopathology. The proposed strategy of s{gamma}PNA BNP with TMZ provides an alternative, promising approach for treatment of GBM.

cancer biology↗

bioRxiv2022

In vivo correction of cystic fibrosis mediated by PNA nanoparticles

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We sought to correct the multiple organ dysfunction of the F508del CF-causing mutation using systemic delivery of peptide nucleic acid gene editing technology mediated by biocompatible polymeric nanoparticles. We confirmed phenotypic and genotypic modification in vitro in primary nasal epithelial cells from F508del mice grown at air-liquid interface and in vivo in F508del mice following intravenous delivery. In vivo treatment resulted in a partial gain of CFTR function in epithelia as measured by in situ potential differences and Ussing chamber assays and correction of CFTR in both airway and GI tissues with no off-target effects above background. This is the first report of systemic gene editing for CF. Our data suggest that systemic delivery of PNA NPs designed to correct CF-causing mutations is a viable option to ameliorate the disease in multiple affected organs.

bioengineering↗

bioRxiv2021

A subset of Memory B-derived antibody repertoire from 3-dose vaccinees is ultrapotent against diverse and highly transmissible SARS-CoV-2 variants, including Omicron

Omicron, the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising unprecedented concerns about the effectiveness of antibody therapies and vaccines. We examined whether sera from individuals who received two or three doses of inactivated vaccine, could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2/60) and 95% (57/60) for 2- and 3-dose vaccinees, respectively. For three-dose recipients, the geometric mean neutralization antibody titer (GMT) of Omicron was 15, 16.5-fold lower than that of the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in 3-dose vaccinees, half of which recognize the receptor binding domain (RBD) and show that a subset of them (24/163) neutralize all SARS-CoV-2 variants of concern (VOCs), including Omicron, potently. Therapeutic treatments with representative broadly neutralizing mAbs individually or antibody cocktails were highly protective against SARS-CoV-2 Beta infection in mice. Atomic structures of the Omicron S in complex with three types of all five VOC-reactive antibodies defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to one major class of antibodies bound at the right shoulder of RBD through altering local conformation at the binding interface. Our results rationalize the use of 3-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are a rational target for a universal sarbecovirus vaccine. One sentence summaryA sub-set of antibodies derived from memory B cells of volunteers vaccinated with 3 doses of an inactivated SARS-CoV-2 vaccine work individually as well as synergistically to keep variants, including Omicron, at bay.

microbiology↗

bioRxiv2021

Transcriptomic Profiling Reveals Cancer-Associated Fibroblasts as Potential Targets for the Prognosis and Treatment of Cervical Squamous Cell Carcinoma

Effective treatment of advanced invasive cervical cancer remains challenging nowadays. Herein, single-nucleus RNA sequencing (snRNA-seq) and SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq) technology are used to investigate the immunological microenvironment of cervical squamous cell carcinoma (CSCC), a major type of cervical cancers. The expression levels of most immune checkpoint genes in tumor and inflammation areas of CSCC were not significantly higher than those in the non-cancer samples except for LGALS9 and IDO1. Stronger signals of CD56+ NK cells and immature dendritic cells are found in the hypermetabolic tumor areas, while more eosinophils, immature B cells, and Treg cells are found in the hypometabolic tumor areas. Moreover, a cluster of cancer-associated fibroblasts (CAFs) are identified around some tumors, which highly expressed ACTA2, POSTN, ITGB4, and FAP. The CAFs might support the growth and metastasis of tumors by inhibiting lymphocyte infiltration and remodeling the tumor extracellular matrix. Furthermore, CAFs are associated with poorer survival probability in CSCC patients and might be present in a small fraction ([~]20%) of advanced cancer patients. Collectively, these findings might enhance understanding of the CSCC immunological microenvironment and shed some light on the treatment of advanced CSCC.

cancer biology↗

bioRxiv2021

Integrative interactome and ubiquitinome analyses reveal multiple regulatory pathways targeted by ICP0 in HSV-1 infected neuronal cells

Herpes simplex virus 1 (HSV-1) is a neurotropic virus that can undergo both productive and latent infection in neurons. ICP0 is an HSV-1 E3 ubiquitin ligase crucial for productive infection and reactivation from latency. However, its targets have not been systematically investigated in neuronal cells. After confirming the importance of ICP0 in HSV-1 neuronal replication using an ICP0-null virus, we identified many ICP0-interacting proteins in infected neuronal and non-neuronal cells by mass-spectrometry-based interactome analysis. Co-immunoprecipitation assays validated ICP0 interactions with ACOT8, C1QBP, OTUD4, SNX9 and VIM in both Neuro-2a and 293T cells. Overexpression and knockdown experiments showed that SNX9 restricted replication of the ICP0-null but not wild-type virus in Neuro-2a cells. Ubiquitinome analysis by immunoprecipitating the trypsin digested ubiquitin reminant followed by mass spectrometry identified numerous candidate ubiquitination substrates of ICP0 in infected Neuro-2a cells, among which OTUD4 and VIM were novel substrates confirmed to be ubiquitinated by transfected ICP0 in Neuro-2a cells despite no evidence of their degradation by ICP0. Expression of OTUD4 was induced independently of ICP0 during HSV-1 infection. Overexpressed OTUD4 enhanced type I interferon expression during infection with the ICP0-null but not wild-type virus. In summary, by combining two proteomic approaches followed by confirmatory and functional experiments, we identified and validated multiple novel targets of ICP0 in neuronal cells, and revealed potential restrictive activities of SNX9 and OTUD4 as well as ICP0-dependent antagonism of these activities. Author SummaryHerpes simplex virus 1 (HSV-1) establishes latent infection in neurons. ICP0 is known for its critical role in antagonizing cellular restrictive functions thereby initiating productive infection. It has been demonstrated to be important for both acute infection and reactivation from latency in neurons. However, little is known about its targets in neuronal cells. Here we combined two proteomic approaches, interactome and ubiquitinome analyses, to integratively identify interaction partners and substrates of ICP0 in HSV-1 infected neuronal cells. The results identified many novel targets as well as confirming previously reported ones. We also further validated some of the binding interactions and ubiquitin modifications. Functional studies revealed that the ICP0-interacting protein SNX9 restricted HSV-1 replication and the ICP0 substrate OTUD4 was induced to enhance type I interferon expression during HSV-1 neuronal infection. Moreover, the activities of these proteins appeared to be antagonized by ICP0-dependent mechanisms. This study provided comprehensive insight into ICP0 targets in neuronal cells and might prompt further investigation into the newly identified targets of ICP0.

microbiology↗

bioRxiv2021

CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

It has recently been shown that CFIm25, a canonical mRNA 3 processing factor, could play a variety of physiological roles through its molecular function in the regulation of mRNA alternative polyadenylation (APA). Here, we used CRISPR/Cas9-mediated gene editing approach in human embryonic stem cells (hESCs) for CFIm25, and obtained three gene knockdown/mutant cell lines. CFIm25 gene editing resulted in higher proliferation rate and impaired differentiation potential for hESCs, with these effects likely to be directly regulated by the target genes, including the pluripotency factor rex1. Mechanistically, we unexpected found that perturbation in CFIm25 gene expression did not significantly affect cellular mRNA 3 processing efficiency and APA profile. Rather, we provided evidences that CFIm25 may impact RNA polymerase II (RNAPII) occupancy at the body of transcribed genes, and promote the expression level of a group of transcripts associated with cellular proliferation and/or differentiation. Further study indicated that CFIm25 association with LEO1, an RNAPII associated factor, might contribute to the effect. Taken together, these results reveal novel mechanisms underlying CFIm25s modulation in determination of cell fate, and provide evidence that the process of mammalian gene transcription may be regulated by an mRNA 3 processing factor.

molecular biology↗

bioRxiv2021

Evaluation of methods for the inference of ancestral recombination graphs

The ancestral recombination graph (ARG) is a structure that describes the joint genealogies of sampled DNA sequences along the genome. Recent computational methods have made impressive progress towards scalably estimating whole-genome genealogies. In addition to inferring the ARG, some of these methods can also provide ARGs sampled from a defined posterior distribution. Obtaining good samples of ARGs is crucial for quantifying statistical uncertainty and for estimating population genetic parameters such as effective population size, mutation rate, and allele age. Here, we use standard neutral coalescent simulations to benchmark the estimates of pairwise coalescence times from three popular ARG inference programs: ARGweaver, Relate, and tsinfer+tsdate. We compare 1) the true coalescence times to the inferred times at each locus; 2) the distribution of coalescence times across all loci to the expected exponential distribution; 3) whether the sampled coalescence times have the properties expected of a valid posterior distribution. We find that inferred coalescence times at each locus are most accurate in ARGweaver, and often more accurate in Relate than in tsinfer+tsdate. However, all three methods tend to overestimate small coalescence times and underestimate large ones. Lastly, the posterior distribution of ARGweaver is closer to the expected posterior distribution than Relates, but this higher accuracy comes at a substantial trade-off in scalability. The best choice of method will depend on the number and length of input sequences and on the goal of downstream analyses, and we provide guidelines for the best practices.

genomics↗