Biology subjects

Wang, M.

Publications and source records attributed to Wang, M..

At least 451 records · Page 25Linked to original sources

bioRxiv2023

High-resolution dissection of human cell type-specific enhancers in cis and trans activities

The spatiotemporal specific gene expression is regulated by cell type-specific regulatory elements including enhancers, silencers and insulators etc. The massively parallel reporter assay (MPRA) methods like STARR-seq facilitate the systematic study of DNA sequence intrinsic enhancer activities in a large scale. However, when applied to human cells, it remains challenging to identify and quantify cell type-specific active enhancers in the genome-wide scale with high-resolution, due to the large size of human genome. In this study, we selected the H3K4me1 associated dinucleosome with the linker DNA sequences as candidate enhancer sequences in two different human cell lines and performed ChIP-STARR-seq to quantify the cell type-specific enhancer activities with high-resolution in a genome-wide scale. Furthermore, we investigated how the activity landscape of enhancer repository would change when transferred from native cells (cis activity) to another cell lines (trans activity). Using ChIP-STARR-seq of the candidate enhancers in native cells and another type of cells, we obtained enhancers cis activity maps and trans activity maps in two different cell lines. The cis and trans activity maps enabled us to identify cell type-specific active enhancers, with enrichment of motifs of differentially expressed TFs. Comparisons between the cis and trans activity maps revealed general consistent regulatory property with different levels of activity in the two cell types, suggesting the sequence intrinsic regulatory properties keep similar in different type of cells. This study provides a new perspective of sequence intrinsic enhancer activities in different types of cells.

genomics↗

bioRxiv2023

Genome-wide Perturbation Analysis Screening System for Exosomes-Related Genes Based on the CRISPR/Cas9 Platform

As reported previously, exosomes have significant impacts on the physiological and pathological state in vivo. Exosomes have been extensively studied as a drug delivery carrier and some exosomes drugs have been undergoing clinical research. The mechanisms of exosome production, transport, and secretion remain to be studied in depth. Thus, here, we developed a novel CRISPR-UMI-based gene screening system, with which we can reveal the exosomes biogenesis, transport, and uptake mechanisms on a genome-wide scale. This system consists of two parts: one is a gene knockout component; the other part is a unique molecular identifiers (UMI) labeling component that can label the exosomes produced by the knockout cells, in which each sgRNA corresponds to a specific UMI. In this way, by detecting the UMI loaded in the exosomes, we can trace the knockout gene. In this study, we first verified the function of each component using plasmids and lentiviruses respectively. Next, we simulated the infection of cells with lentiviral libraries using a single lentivirus to validate the functionality of the screening system. Finally, we constructed a CRISPR-UMI-based library targeting 15 genes (genes with clear effects on exosomes biogenesis) to further validate the reliability of the screening system. The development of this screening system is of indispensable importance for the in-depth study of the mechanisms of exosome production and secretion, as well as for the improvement of exosomes production and the advancement of exosomes industrialization.

bioengineering↗

bioRxiv2023

Non-invasive measurement of intra-tumoral fluid dynamics with localized convolutional function regression

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is a routine method to non-invasively quantify perfusion dynamics in tissues. The standard practice for analyzing DCE-MRI data is to fit an ordinary differential equation to each voxel. Recent advances in data science provide an opportunity to move beyond existing methods to obtain more accurate measurements of fluid properties. Here, we developed a localized convolutional function regression that enables simultaneous measurement of interstitial fluid velocity, diffusion, and perfusion in 3D. We validated the method computationally and experimentally, demonstrating accurate measurement of fluid dynamics in situ and in vivo. Applying the method to human MRIs, we observed tissue-specific differences in fluid dynamics, with an increased fluid velocity in breast cancer as compared to brain cancer. Overall, our method represents an improved strategy for studying interstitial flows and interstitial transport in tumors and patients. We expect that it will contribute to the better understanding of cancer progression and therapeutic response. One-Sentence SummaryA physics-informed computational method enables accurate and efficient measurement of fluid dynamics in individual patient tumors and demonstrates differences between tissues.

bioengineering↗

bioRxiv2023

Ancient farmer and steppe pastoralist-related founding lineages contributed to the complex landscape of episodes in the diversification of Chinese paternal lineages

Ancient DNA advances have reported the complex genetic history of Eurasians, but how the knowledge of ancient subsistence strategy shifts and population movements influenced the fine-scale paternal genetic structure in East Asia has not been assessed. Here, we reported one integrated Y-chromosome genomic database of 15,530 people, including 1753 ancient people and newly-reported 919 individuals genotyped using our recently-developed targeted sequencing YHSeqY3000 panel, to explore Chinese genomic diversity, population evolutionary tracts and their genetic formation mechanism. We identified four major ancient technological innovations and population movements that shaped the landscape of Chinese paternal lineages. First, the expansion of millet farmers and early East Asians from the Yellow River Basin carrying the major O2/D subclades promoted the formation of the Sino-Tibetan peoples major composition and accelerated the Tibetan Plateaus permanent occupation. Second, rice farmers dispersal from the Yangtze River Valley carrying O1 and some sublineages of O2 contributed significantly to Tai-Kadai, Austronesian, Hmong-Mien, Austroasiatic people and southern Han Chinese. Third, Siberian-related paternal lineages of Q and C originated and boomed from Neolithic hunter-gatherers from the Mongolian Plateau and the Amur River Basin and significantly influenced the gene pools of northern Chinese. Fourth, western Eurasian-derived J, G and R lineages initially spread with Yamnaya steppe pastoralists and other proto-Indo-European people and further widely dispersed via the trans-Eurasian cultural communication along the Eurasian Steppe and the ancient Silk Road, remaining genetic trajectories in northwestern Chinese. Our work provided comprehensive modern and ancient genetic evidence to illuminate the impact of population interaction from the ancient farmer or herder-based societies on the genetic diversity patterns of modern people, revised our understandings of ancestral sources of Chinese paternal lineages, underscored the scientific imperative of the large-scale genomic resources of dense spatiotemporal underrepresented sampling populations to understand human evolutionary history.

genetics↗

bioRxiv2023

Population genomic analyses reveal extensive genomic regions within selective sweeps associated with adaptation and demographic history of a wheat fungal pathogen

Plant pathogens can quickly evolve in response to the changing host and environment, resulting in destructive epidemics. The fungus Puccinia striiformis f. sp. tritici (Pst), causing wheat stripe rust disease worldwide, is one such pathogen. In the arms race between pathogens and hosts, changes in host populations exerted continuous selective forces on the dynamics of pathogens. However, the footprints of these selection forces and the demography of Pst remain poorly explored, limited our understanding the evolutionary processes that shape the spread and evolution of pathogens. In this study, we revealed several features of worldwide Pst populations through population genomic analyses. There might be limited gene flow between Pst populations from China and other countries. A slower rate of linkage disequilibrium decay was detected comparing to rust fungi with known sexual reproduction. Furthermore, we detected extensive hard and soft sweeps associated with Pst adaptation. Genes within the selective sweeps were enriched in secreted proteins and effectors and showed functions related to pathogenicity or virulence, temperature tolerance, and fungicide resistance implying that Chinese Pst populations suffered positive selection pressures from host and abiotic factors. Moreover, demographic history indicated Pst populations experienced strong bottlenecks at the beginning of the wheat domestication around 10,000 years ago and during modern agriculture around 100 years ago, suggesting that the crop domestication and breeding programs could continuously contribute to the decline of pathogen effective population sizes. Our results provided insights into the evolution of the Pst genome and highlighted the role of modern agriculture on pathogen demography. Author SummaryWheat stripe rust, as a pathogen of wheat, is capable of inducing significant yield reduction on a global scale. Traditional epidemiological investigations have elucidated the causative agent behind this disease, the wheat stripe rust pathogen, as an organism exhibiting swift adaptive evolution in response to its environment. However, the precise mechanisms underpinning its adaptive strategies and the ensuing alterations in population dynamics remain relatively unexplored. In our research, we have undertaken an analysis encompassing wheat stripe rust samples throughout the world. Employing population genomics methods, we have sought to discern the genomic footprints corresponding to the adaptive process within the pathogen. These genomic footprints have illuminated the operation of positive selection pressures caused by both the host plant and the ecological context. Concurrently, it have provided insights into the population size that the wheat stripe rust pathogen has undergone over millennia. Notably, its change in population size revealed the demographic history associated with wheat domestication.

microbiology↗

bioRxiv2023

Von Willebrand Factor Synergizes with Tumor-Derived Extracellular Vesicles to Promote Gastric Cancer Metastasis

Cells of gastric cancer invade local tissue extensively and also metastasize through the circulation to remote organs. Patients with metastasized gastric cancer have poor outcomes. Cancer cells are known to release extracellular vesicles (EVs) that contribute to cancer progression, but how cancer cell-derived EVs promote cancer growth and metastasis remains poorly understood. We have recently reported that levels of circulating gastric cancer cell-derived EVs (gcEVs) and the adhesive ligand von Willebrand factor (VWF) are associated with cancer metastasis and poor prognosis of patients, but the underlying mechanism of this gcEV-VWF interaction was not known. Here we report results from a study designed to investigate the synergistic action of VWF and gcEVs in vitro and in mouse models. We showed that VWF in cancer-bearing mice was hyperadhesive and became microvesicle-bound. EV-bound VWF mediated the adhesion of gcEVs to the endothelium to disrupt endothelial integrity and facilitate the transendothelial migration of cancer cells and pulmonary metastasis. Reducing VWF adhesive activity by the metalloprotease ADAMTS-13 or promoting gcEV clearance by the scavenging factor lactadherin prevented pulmonary metastasis in mice. These results highlight the synergistic action of gcEVs and VWF in promoting gastric cancer metastasis and identifying new targets for its prevention. Key point: Author contributionsO_LIHyperadhesive VWF becomes microvesicle-bound to induce endothelial leakage and promote the pulmonary metastasis of gastric cancer in mice. C_LIO_LIReducing VWF activity by ADAMTS-13 and accelerating microvesicle clearance by lactadherin reduces pulmonary metastasis of gastric cancer. C_LI

cancer biology↗

bioRxiv2023

The evolution of autonomy from two cooperative specialists in the fluctuating environments

From microbes to humans, organisms perform numerous tasks for their survival, including food acquisition, migration, and reproduction. A complex biological task can be performed by either an autonomous organism or by cooperation among several specialized organisms. However, it remains unclear how autonomy and cooperation evolutionarily switch. For example, it remains unclear whether and how a group of cooperative specialists can evolve into an autonomous organism. Here, we address this question by experimentally evolving a mutualistic microbial consortium composed of two specialists that cooperatively degrade naphthalene. We observed that autonomous genotypes capable of performing the entire naphthalene degradation pathway evolved from two cooperative specialists and came to dominate the microbial community. This evolutionary transition was driven by the horizontal gene transfer between the two specialists. However, this evolution was exclusively observed to occur in the fluctuating environment supplied with either naphthalene or pyruvate, where mutualism and competition between the two specialists alternated. The naphthalene-supplied environment exerts selective pressure that favors the expansion of autonomous genotypes but was associated with both low cell density and low coexistence levels. In contrast, the pyruvate-supplying environment promoted the coexistence and cell density of the cooperative specialists, thereby increasing the likelihood of horizontal gene transfer. Using a mathematical model, we quantitatively demonstrate that environmental fluctuations facilitate the evolution of autonomy through HGT when the relative growth rate and carrying capacity of the cooperative specialists allow enhanced coexistence and higher cell density in the competitive environment. Together, our results demonstrate that mutualistic cooperation can evolve into autonomous organisms through direct genetic exchange under specific conditions, including the alternation of mutualism and competition in fluctuating environments in a manner frequently occurring in nature.

evolutionary biology↗

bioRxiv2023

Dual-specificity protein phosphatase 6 (DUSP6) overexpression reduces amyloid load and improves memory deficits in male 5xFAD mice

BackgroundDual specificity protein phosphatase 6 (DUSP6) was recently identified as a key hub gene in a causal network that regulates late-onset Alzheimers disease. Importantly, decreased DUSP6 levels are correlated with an increased clinical dementia rating in human subjects, and DUSP6 levels are additionally decreased in the 5xFAD amyloidopathy mouse model. MethodsAAV5-DUSP6 or AAV5-GFP (control) were stereotactically injected into the dorsal hippocampus (dHc) of female and male 5xFAD or wild type mice to overexpress DUSP6 or GFP. Spatial learning memory of these mice was assessed in the Barnes maze, after which hippocampal tissues were isolated for downstream analysis. ResultsBarnes maze testing indicated that DUSP6 overexpression in the dHc of 5xFAD mice improved memory deficits and was associated with reduced amyloid plaque load, A{beta}1-40 and A{beta}1-42 levels, and amyloid precursor protein processing enzyme BACE1, in male but not in female mice. Microglial activation and microgliosis, which are increased in 5xFAD mice, were significantly reduced by dHc DUSP6 overexpression in both males and females. Transcriptomic profiling of female 5xFAD hippocampus revealed upregulated expression of genes involved in inflammatory and extracellular signal-regulated kinase (ERK) pathways, while dHc DUSP6 overexpression in female 5xFAD mice downregulated a subset of genes in these pathways. A limited number of differentially expressed genes (DEGs) (FDR<0.05) were identified in male mice; gene ontology analysis of DEGs (p<0.05) identified a greater number of synaptic pathways that were regulated by DUSP6 overexpression in male compared to female 5xFAD. Notably, the msh homeobox 3 gene, Msx3, previously shown to regulate microglial M1/M2 polarization and reduce neuroinflammation, was one of the most robustly upregulated genes in female and male wild type and 5xFAD mice overexpressing DUSP6. ConclusionsIn summary, our data indicate that DUSP6 overexpression in dHc reduced amyloid deposition and memory deficits in male but not female 5xFAD mice, whereas reduced neuroinflammation and microglial activation were observed in both males and females. The sex-dependent regulation of synaptic pathways by DUSP6 overexpression, however, correlated with the improvement of spatial memory deficits in male but not female 5xFAD.

neuroscience↗

bioRxiv2023

YY1 Binding to Regulatory Elements that Lack Enhancer Activity Promotes Locus Folding and Gene Activation

Enhancers activate their cognate promoters over huge distances but how enhancer/promoter interactions become established is not completely understood. There is strong evidence that cohesin-mediated loop extrusion is involved but this does not appear to be a universal mechanism. Here, we identify an element within the mouse immunoglobulin lambda (Ig{lambda}) light chain locus, HSC{lambda}1, that has characteristics of active regulatory elements but lacks intrinsic enhancer or promoter activity. Remarkably, knock-out of the YY1 binding site from HSC{lambda}1 reduces Ig{lambda} transcription significantly and disrupts enhancer/promoter interactions, even though these elements are >10 kb from HSC{lambda}1. Genome-wide analyses of mouse embryonic stem cells identified 3503 similar YY1-bound, putative genome organizing elements that lie within CTCF/cohesin loop boundaries but that lack intrinsic enhancer activity. We suggest that such elements play a fundamental role in locus folding and in facilitating enhancer/promoter interactions. HighlightsHow long-range enhancer-promoter interactions are established is not fully understood An element in the lambda light chain locus, HSC{lambda}1, lacks intrinsic enhancer activity Removal of YY1 binding from HSC{lambda}1 disrupts neighbouring enhancer/promoter contacts Genome-wide analyses detect similar elements that lack enhancer or promoter activity We propose these elements aid locus folding and nearby enhancer-promoter interactions O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/554459v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@562875org.highwire.dtl.DTLVardef@7a1a85org.highwire.dtl.DTLVardef@634652org.highwire.dtl.DTLVardef@19ddfda_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

CASi: A multi-timepoint scRNAseq data analysis framework

Single-cell RNA sequencing (scRNA-seq) technology has been widely used to study the differences in gene expression at the single cell level, providing insights into the research of cell development, differentiation, and functional heterogeneity. Various pipelines and workflows of scRNA-seq analysis have been developed but few considered multi-timepoint data specifically. In this study, we develop CASi, a comprehensive framework for analyzing multiple timepoints scRNA-seq data, which provides users with: (1) cross-timepoint cell annotation, (2) detection of potentially novel cell types emerged over time, (3) visualization of cell population evolution, and (4) identification of temporal differentially expressed genes (tDEGs). Through comprehensive simulation studies and applications to a real multi-timepoint single cell dataset, we demonstrate the robust and favorable performance of the proposal versus existing methods serving similar purposes.

bioinformatics↗

bioRxiv2023

Cooperation between bacteriocytes and endosymbionts drives function and development of symbiotic cells in mussel holobionts

Symbiosis drives the adaptation and evolution in multicellular organisms. Modeling the function and development of symbiotic cells/organs in holobionts is yet challenging. Here, we surveyed the molecular function and developmental trajectory of bacteriocyte lineage in non-model deep-sea mussels by constructing a high-resolution single-cell expression atlas of gill tissue. We show that mussel bacteriocytes optimized immune processes to facilitate recognition, engulfment, and elimination of endosymbionts, and interacted with them intimately in sterol, carbohydrate, and ammonia metabolism. Additionally, the bacteriocytes could arise from three different stem cells as well as bacteriocytes themselves. In particular, we showed that the molecular functions and developmental process of bacteriocytes were guided by the same set of regulatory networks and dynamically altered regarding to symbiont abundance via sterol-related signaling. The coordination in the functions and development of bacteriocytes and between the host and symbionts underlies the interdependency of symbiosis, and drives the deep-sea adaptation of mussels.

ecology↗

bioRxiv2023

Distinct mechanisms by which antioxidant transcription factors Nrf1 and Nrf2 as drug targets contribute to the anticancer efficacy of Cisplatin on hepatoma cells

Cisplatin (cis-Dichlorodiamineplatinum[II], CDDP) is generally accepted as a platinum-based alkylating agent type of the DNA-damaging anticancer drug, which is widely administrated in clinical treatment of many solid tumors. The pharmacological effect of CDDP is mainly achieved by replacing the chloride ion (Cl-) in its structure with H2O to form active substances with the strong electrophilic properties and then react with any nucleophilic molecules, primarily leading to genomic DNA damage and subsequent cell death. In this process, those target genes driven by the consensus electrophilic and/or antioxidant response elements (EpREs/AREs) in their promoter regions are also activated or repressed by CDDP. Thereby, we here examined the expression profiling of such genes regulated by two principal antioxidant transcription factors Nrf1 and Nrf2 (both encoded by Nfe2l1 and Nfe2l2, respectively) in diverse cellular signaling responses to this intervention. The results demonstrated distinct cellular metabolisms, molecular pathways and signaling response mechanisms by which Nrf1 and Nrf2 as the drug targets differentially contribute to the anticancer efficacy of CDDP on hepatoma cells and xenograft tumor mice. Interestingly, the role of Nrf1, rather than Nrf2, is required for the anticancer effect of CDDP, to suppress malignant behavior of HepG2 cells by differentially monitoring multi-hierarchical signaling to gene regulatory networks. To our surprise, it was found there exists a closer relationship of Nrf1 than Nrf2 with DNA repair, but the hyperactive Nrf2 in Nrf1-/-cells manifests a strong correlation with its resistance to CDDP, albeit their mechanistic details remain elusive.

cell biology↗

bioRxiv2023

The longitudinal single-cell panorama of cynomolgus monkey ovary throughout lifespan revealed a conserved ovarian clock between primates

Ovarian function is critical for female fertility and impacts reproductive longevity. It is of great importance to accurately predict the aging process within the ovary for fertility assessment and disease diagnosis. However, cell metrics for evaluating the ovarian aging rate are still in urgent need, and molecular insights into ovarian development and dysfunction during the primate life course are also limited. Here, we reported large-scale ovarian cell atlas of consecutive development of cynomolgus monkeys across 22 years with 20 time points, covering the foetal, newborn, prepubertal, pubertal, adult, perimenopausal and menopausal stages. We characterized and validated distinct molecular signatures of each cluster of cells within primate ovaries, and uncovered a previously undocumented RHOXF1-positive oocyte type during primordial follicle assembly in primates. Furthermore, the constitution and developmental trajectories of primate germ cells, granulosa cells and stromal / theca cells were also elucidated, and their precursors were identified. More importantly, dynamics of cellular compositions were unravelled through the ovarian development, featured by granulosa, epithelial, stromal, and immune cells that showed strong temporal heterogeneity spanning lifetime, whilst referred to the key function during the corresponding stages. Based on the correlations of each cell type with age and stage-specific molecular dynamics, we further constructed a transcriptomic ovarian clock which could perceive an effective biological age prediction of the ovary and further applied to humans. The findings reveal granulosa, epithelial, and stromal cells as the highest performance predictors of ovarian biological age, while highlighting the crucial role of AGE-RAGE and Relaxin signaling pathways in regulating ovarian aging. Our work not only provide valuable resource for obtaining insights into the development, aging and dysfunction of key organs, but also establish a transcriptomic clock to predict biological ovarian aging thus to be potential clinical implementation in future.

developmental biology↗

bioRxiv2023

Wiskott-Aldrich Syndrome Protein Regulates Nucleolar Organization and Function in Innate Immune Response

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disorder caused by the dysfunction of the WAS protein (WASP). Using an isogenic macrophage model derived from genome edited induced pluripotent stem cells we demonstrated that WASP functions in the nucleolus, which plays important roles in immune regulation. The absence of WASP resulted in smaller and misshapen nucleoli, decreased fibrillar center territory, and impaired ribosomal RNA (rRNA) transcription. The nucleolar and rRNA phenotypes were confirmed in WAS patient samples. Furthermore, WASP interacts with nucleolar proteins, including nucleophosmin 1 (NPM1) and fibrillarin (FBL). NPM1 deficiency is known to cause elevated cytokine expression following lipopolysaccharide (LPS) stimulation. Consistently, WASP deficient cells displayed lower levels of NPM1 and a heightened inflammatory cytokine response to LPS, which was rescued by overexpressing NPM1. Together, our research provides novel insights into the critical role of WASP in nucleolar function and the modulation of inflammatory cytokine production.

cell biology↗

bioRxiv2023

Pretrainable Geometric Graph Neural Network for Antibody Affinity Maturation

Increasing the binding affinity of an antibody to its target antigen is a crucial task in antibody therapeutics development. This paper presents a pretrainable geometric graph neural network, GearBind, and explores its potential in in silico affinity maturation. Leveraging multi-relational graph construction, multi-level geometric message passing and contrastive pretraining on mass-scale, unlabeled protein structural data, GearBind outperforms previous state-of-the-art approaches on SKEMPI and an independent test set. A powerful ensemble model based on GearBind is then derived and used to successfully enhance the binding of two antibodies with distinct formats and target antigens. ELISA EC50 values of the designed antibody mutants are decreased by up to 17 fold, and KD values by up to 6.1 fold. These promising results underscore the utility of geometric deep learning and effective pretraining in macromolecule interaction modeling tasks.

bioinformatics↗

bioRxiv2023

Rapid and unbiased enrichment of extracellular vesicles via meticulously engineered peptide

Extracellular vesicles (EVs) have garnered significant attention in biomedical applications, particularly as biomarkers and therapeutic agents for cancer diagnosis and treatment. However, the rapid, efficient, and unbiased separation of EVs from complex biological fluids remains a challenge due to their heterogeneity and low abundance concentration in biofluids. Herein, we report a novel approach to reconfigure and modify an artificial insertion peptide for the rapid isolation of EVs in 20 min with [~] 80% recovery. By inserting the peptide into the phospholipid bilayer of EVs, our method enables the unbiased isolation of EVs. Moreover, our approach demonstrates exceptional anti-interference capability and achieves a high purity of EVs comparable to standard ultracentrifugation and other methods. Importantly, we show that the isolated EVs could be directly applied for downstream protein and nucleic acids analyses, including proteomics analysis, exome sequencing analysis, as well as the detection of EGFR and KRAS gene mutation in clinical plasma samples. Our approach offers new possibilities for utilizing EVs in cancer diagnostics through liquid biopsy, as well as in various other biomedical applications.

bioengineering↗

bioRxiv2023

An inhibitor/anti-inhibitor system controls the activity of lytic transglycosylase MltF in Pseudomonas aeruginosa

Most bacterial cell envelopes contain a cell wall layer made of peptidoglycan. The synthesis of new peptidoglycan is critical for cell growth, division and morphogenesis, and is also coordinated with peptidoglycan hydrolysis to accommodate the new material. However, the enzymes that cleave peptidoglycan must be carefully controlled to avoid autolysis. In recent years, some control mechanisms have begun to emerge, although there are many more questions than answers for how most cell wall hydrolases are regulated. Here, we report a novel cell wall hydrolase control mechanism in Pseudomonas aeruginosa, which we discovered during our characterization of a mutant sensitive to the overproduction of a secretin protein. The mutation affected an uncharacterized Sel1-like repeat protein encoded by the PA3978 locus. In addition to the secretin-sensitivity phenotype, PA3978 disruption also increased resistance to a {beta}-lactam antibiotic used in the clinic. In vivo and in vitro analysis revealed that PA3978 binds to the catalytic domain of the lytic transglycosylase MltF and inhibits its activity. {Delta}PA3978 mutant phenotypes were suppressed by deleting mltF, consistent with them having been caused by elevated MltF activity. We also discovered another interaction partner of PA3978 encoded by the PA5502 locus. The phenotypes of a {Delta}PA5502 mutant suggested that PA5502 interferes with the inhibitory function of PA3978 towards MltF, and we confirmed that activity for PA5502 in vitro. Therefore, PA3978 and PA5502 form an inhibitor/anti-inhibitor system that controls MltF activity. We propose to name these proteins Ilt (inhibitor of lytic transglycosylase) and Lii (lytic transglycosylase inhibitor, inhibitor). IMPORTANCEA peptidoglycan cell wall is an essential component of almost all bacterial cell envelopes, which determines cell shape and prevents osmotic rupture. Antibiotics that interfere with peptidoglycan synthesis have been one of the most important treatments for bacterial infections. Peptidoglycan must also be hydrolyzed to incorporate new material for cell growth and division, and to help accommodate important envelope-spanning systems. However, the enzymes that hydrolyze peptidoglycan must be carefully controlled to prevent autolysis. Exactly how this control is achieved is poorly understood in most cases, but is a highly active area of current research. Identifying hydrolase control mechanisms has the potential to provide new targets for therapeutic intervention. The work here reports the important discovery of a novel inhibitor/anti-nhibitor system that controls the activity of a cell wall hydrolase in the human pathogen Pseudomonas aeruginosa, and which also affects resistance to an antibiotic used in the clinic.

microbiology↗

bioRxiv2023

Endothelial SARS-CoV-2 infection is not the underlying cause of COVID19-associated vascular pathology in mice

Endothelial damage and vascular pathology have been recognized as major features of COVID-19 since the beginning of the pandemic. Two main theories regarding how Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) damages endothelial cells and causes vascular pathology have been proposed: direct viral infection of endothelial cells or indirect damage mediated by circulating inflammatory molecules and immune mechanisms. However, these proposed mechanisms remain largely untested in vivo. Here, we utilized a set of new mouse genetic tools1 developed in our lab to test both the necessity and sufficiency of endothelial human angiotensin-converting enzyme 2 (hACE2) in COVID19 pathogenesis. Our results demonstrate that endothelial ACE2 and direct infection of vascular endothelial cells does not contribute significantly to the diverse vascular pathology associated with COVID-19.

pathology↗