Biology subjects

Zhao, Z.

Publications and source records attributed to Zhao, Z..

At least 451 records · Page 25Linked to original sources

bioRxiv2020

Class A Penicillin-Binding Protein-mediated cell wall synthesis promotes structural integrity during peptidoglycan endopeptidase insufficiency

The bacterial cell wall is composed primarily of peptidoglycan (PG), a poly-aminosugar that is essential to sustain cell shape, growth and structural integrity. PG is synthesized by two different types of synthase complexes (class A Penicillin-binding Proteins [PBP]s/Lpos and Shape, Elongation, Division, Sporulation [SEDS]/class B PBP pairs) and degraded by autolytic enzymes to accommodate growth processes. It is thought that autolsyin activity (and particulary the activity of endopeptidases, EPs) is required for PG synthesis and incorporation by creating gaps that are patched and paved by PG synthases, but the exact relationship between autolysins and the separate synthesis machineries remains incompletely understood. Here, we have probed the consequences of EP depletion for PG synthesis in the diarrheal pathogen Vibrio cholerae. We found that EP depletion resulted in severe morphological defects, increased cell mass, a decline in viability, and continuing (yet aberrant) incorporation of cell wall material. Mass increase and cell wall incorporation proceeded in the presence of Rod system inhibitors, but was abolished upon inhibition of aPBPs. However, the Rod system remained functional (i.e., exhibited sustained directed motion) even after prolonged EP depletion, without effectively promoting cell elongation. Lastly, heterologous expression of an EP from Neisseria gonorrhoeae could fully complement growth and morphology of an EP-insufficient V. cholerae. Overall, our findings suggest that in V. cholerae, the Rod system requires endopeptidase activity (but not necessarily direct interaction with EPs) to promote cell expansion and substantial PG incorporation, whereas aPBPs are able to engage in sacculus construction even during severe EP insufficiency. ImportanceSynthesis and turnover of the bacterial cell wall must be tightly co-ordinated to avoid structural integrity failure and cell death. Details of this coordination are poorly understood, particularly if and how cell wall turnover enzymes are required for the activity of the different cell wall synthesis machines. Our results suggest that in Vibrio cholerae, one class of turnover enzymes, the endopeptidases, are required only for substantial PG incorporation mediated by the Rod system, while the aPBPs maintain structural integrity during endopeptidase insufficiency. Our results suggest that aPBPs are more versatile than the Rod system in their ability to recognize cell wall gaps formed by autolysins other than the major endopeptidases, adding to our understanding of the co-ordination between autolysins and cell wall synthases. A detailed understanding of autolysin biology may promote the development of antibiotics that target these essential turnover processes.

microbiology↗

bioRxiv2020

A chronic photocapacitor implant for noninvasive neurostimulation with deep red light

Implantable clinical neuroelectronic devices are limited by a lack of reliable, safe, and minimally invasive methods to wirelessly modulate neural tissue. Here, we address this challenge by using organic electrolytic photocapacitors (OEPCs) to perform chronic peripheral nerve stimulation via transduction of tissue-penetrating deep-red light into electrical signals. The operating principle of the OEPC relies on efficient charge generation by nanoscale organic semiconductors comprising nontoxic commercial pigments. OEPCs integrated on an ultrathin cuff are implanted, and light impulses at wavelengths in the tissue transparency window are used to stimulate from outside of the body. Typical stimulation parameters involve irradiation with pulses of 50-1000 s length (638 or 660 nm), capable of actuating the implant about 10 mm below the skin. We detail how to benchmark performance parameters of OEPCs first ex vivo, and in vivo using a rat sciatic nerve. Incorporation of a microfabricated zip-tie mechanism enabled stable, long-term nerve implantation of OEPC devices in rats, with sustained ability to non-invasively mediate neurostimulation over 100 days. OEPC devices introduce a high performance, ultralow volume (0.1 mm3), biocompatible approach to wireless neuromodulation, with potential applicability to an array of clinical bioelectronics.

neuroscience↗

bioRxiv2020

Single-cell Protein Atlas of Transcription Factors Reveals the Combinatorial Code for Spatiotemporal Patterning the C. elegans Embryo

A high-resolution protein atlas is essential for understanding the molecular basis of biological processes. Using protein-fusion reporters and imaging-based single-cell analyses, we present a protein expression atlas of C. elegans embryogenesis encompassing 266 transcription factors (TFs) in nearly all (90%) lineage-resolved cells. Single-cell analysis reveals a combinatorial code and cascade that elucidate the regulatory hierarchy between a large number of lineage-, tissue-, and time-specific TFs in spatiotemporal fate patterning. Guided by expression, we identify essential functions of CEH-43/DLX, a lineage-specific TF, and ELT-1/GATA3, a well-known skin fate specifier, in neuronal specification; and M03D4.4 as a pan-muscle TF in converging muscle differentiation in the body wall and pharynx. Finally, systems-level analysis of TF regulatory state uncovers lineage- and time-specific kinetics of fate progression and widespread detours of the trajectories of cell differentiation. Collectively, our work reveals a single-cell molecular atlas and general principles underlying the spatiotemporal patterning of a metazoan embryo.

developmental biology↗

bioRxiv2020

Secondary single-cell transcriptomic analysis reveals common molecular signatures of cerebrovascular injury between traumatic brain injury and aging

Cerebrovascular injury is a common pathological feature of a spectrum of neurological disorders including traumatic brain injury (TBI), stroke, Alzheimers disease (AD), as well as aging. Vascular manifestations among these conditions are similar indeed, including the breakdown of the blood-brain barrier (BBB). However, whether there is a common molecular mechanism underlying the vascular changes among these conditions remains elusive. Here, we report secondary transcriptomic analysis on cerebrovascular cells based single-cell RNA-seq datasets of mouse models of mild TBI and aging, with a focus on endothelial cells and pericytes. We identify several molecular signatures commonly found between mTBI and aging vasculature, including Adamts1, Rpl23a, Tmem252, Car4, Serpine2, and Ndnf in endothelial cells, and Rps29 and Sepp1 in pericytes. These markers may represent the shared endophenotype of microvascular injury and be considered as cerebrovascular injury responsive genes. Additionally, pathway analysis on differentially expressed genes demonstrated alterations in common pathways between mTBI and aging, including vascular development and extracellular matrix pathways in endothelial cells. Hence, our analysis suggests that cerebrovascular injury triggered by different neurological conditions may share common molecular signatures, which may only be detected at the single-cell transcriptome level.

neuroscience↗

bioRxiv2020

Loss of O-GlcNAcylation on MeCP2 Thr 203 Leads to Neurodevelopmental Disorders

Mutations of the X-linked methyl-CpG-binding protein 2 (MECP2) gene in humans are responsible for most cases of Rett syndrome (RTT), an X-linked progressive neurological disorder. While genome-wide screens in clinical trials reveal several putative RTT-associated mutations on MECP2, their causative relevance regarding the functional regulation of MeCP2 on the etiologic sites at the protein level require more evidence. In this study, we demonstrate that MeCP2 is dynamically modified by O-linked-{beta}-N-acetylglucosamine (O-GlcNAc) at threonine 203 (T203), an etiologic site in RTT patients. Disruption of the O-GlcNAcylation of MeCP2 specifically at T203 impairs dendrite development and spine maturation in cultured hippocampal neurons, and disrupts neuronal migration, dendritic spine morphogenesis and dysfunction of synaptic transmission in the developing and juvenile mouse cerebral cortex. Mechanistically, genetic disruption of O-GlcNAcylation at T203 on MeCP2 decreases neuronal activity-induced induction of Bdnf transcription. Our study highlights the critical role of MeCP2 T203 O-GlcNAcylation in neural development and synaptic transmission potentially via BDNF.

neuroscience↗

bioRxiv2020

The production of within-family inequality: Insights and implications of integrating genetic data

The integration of genetic data within large-scale social and health surveys provides new opportunities to test long standing theories of parental investments in children and within-family inequality. Genetic predictors, called polygenic scores, allow novel assessments of young childrens abilities that are uncontaminated by parental investments, and family-based samples allow indirect tests of whether childrens abilities are reinforced or compensated. We use over 16,000 sibling pairs from the UK Biobank to test whether the relative ranking of siblings polygenic scores for educational attainment is consequential for actual attainments. We find strong evidence of compensatory processes, on average, where the association between genotype and phenotype of educational attainment is reduced by over 20% for the higher-ranked sibling compared to the lower-ranked sibling. These effects are most pronounced in high socioeconomic status areas. We find no evidence that similar processes hold in the case of height or for relatives who are not full biological siblings (e.g. cousins). Our results provide a new use of polygenic scores to understand processes that generate within-family inequalities and also suggest important caveats to causal interpretations the effects of polygenic scores using siblingdifference designs.

genetics↗

bioRxiv2020

Precise Localization and Dynamic Distribution of Japanese Encephalitis Virus in theBrain Nuclei of Infected Mice

Japanese encephalitis virus (JEV) is a pathogen that causes severe vector-borne zoonotic diseases, thereby posing a serious threat to human health. Although JEV is potentially neurotropic, its pathogenesis and distribution in the host have not been fully elucidated. In this study, an infected mouse model was established using a highly virulent P3 strain of JEV. Immunohistochemistry and in situ hybridization, combined with anatomical imaging of the mouse brain, were used to dynamically localize the virus and construct three-dimensional (3D) images. Consequently, onset of mild clinical symptoms occurred in some mice at 84h post JEV infection, while most mice displayed typical neurological symptoms at 144h post infection. Moreover, brain pathology revealed typical changes associated with non-suppurative encephalitis, which lasted up to 192h. The earliest detection of viral antigen was achieved at 72h post infection, in the thalamus and medulla oblongata. At 144h post infection, the positive viral antigen signals were mainly distributed in the cerebral cortex, olfactory area, basal ganglia, thalamus, and brainstem regions in mice. At 192h post infection, the antigen signals gradually decreased, and the localization of JEV tended to concentrate in the cerebrum and thalamus, while no viral antigen was detected in the brain at 504h post infection. In this model, the viral antigen was first expressed in the reticular thalamic nucleus (Rt), at a consistent concentration. The expression of the viral antigen in the hippocampal CA2 region, the anterior olfactory nucleus, and the deep mesencephalic nucleus was high and persistent. The 3D images showed that viral signals were mostly concentrated in the parietal cortex, occipital lobe, and hippocampus, near the mid-sagittal plane. In the early stages of infection in mice, a large number of viral antigens were detected in denatured and necrotic neurons, suggesting that JEV directly causes neuronal damage. From the time of its entry, JEV is widely distributed in the central nervous system thereby causing extensive damage.Author summary There are many theories regarding the mechanism of entry of the Japanese encephalitis virus (JEV) into the nervous system. The inflammation cascade effect, resulting from the virus entering the central nervous system (CNS), is a major cause of brain injury in JEV patients. In this study, we found that the earliest point at which viral antigen was detected in the brain tissues following peripheral infection of JEV was at 72h. The virus was located in the nerve nuclei of the thalamus and medulla oblongata and, subsequently, viral antigens were found in the anterior olfactory nucleus. At 96h post infection, the virus was extensively distributed in the brain tissue, and at 144h-192h the viral antigen was widely distributed and highly concentrated. The viral concentration detected in the ventromedial thalamic nucleus (VM), deep mesencephalic nucleus (DpMe), and motor trigeminal nucleus (Mo5) remained high throughout the experiment. The hypertrophic nerve nuclei of JEV include the early anterior olfactory (AO) nucleus and the late hippocampal CA2 region. In the early stages of viral infection (72-144h post infection), the changes in viral antigen concentration and mortality rate were consistent. It was hypothesized that this stage represents the activation of viral proliferation and brain inflammation.View Full Text

pathology↗

bioRxiv2020

Multidimensional Chromatin Regulation of Cell Lineage Differentiation in a Metazoan Embryo

How chromatin dictates cell differentiation is an intriguing question in developmental biology. Here, a reporter gene integrated throughout the genome was used as a sensor to map the chromatin activity landscape in lineage-resolved cells during C. elegans embryogenesis. Single-cell analysis of chromatin dynamics across critical dimensions of cell differentiation was performed, including lineage, tissue, and symmetry. During lineage progression, chromatin gradually diversifies in general and exhibits switch-like changes following specific cell division, which is predictive of anterior-posterior fate asymmetry. Upon tissue differentiation, chromatin of cells from distinct lineages converge to tissue-specific states but retain "memory" of each cells lineage history, which contributes to intra-tissue heterogeneity. However, cells with a morphologically left-right symmetric organization utilize a predetermination chromatin strategy to program analogous regulatory states in early progenitor cells. Additionally, chromatin co-regulation drives the functional coordination of the genome. Collectively, this work reveals the role of multidimensional chromatin regulation in cell differentiation.

developmental biology↗

bioRxiv2020

INSCT: Integrating millions of single cells using batch-aware triplet neural networks

Efficient integration of heterogeneous and increasingly large single cell RNA sequencing (scRNA-seq) data poses a major challenge for analysis and in particular, comprehensive atlasing efforts. Here, we developed a novel deep learning algorithm to overcome batch effects using batch-aware triplet neural networks, called INSCT ("Insight"). Using simulated and real data, we demonstrate that INSCT generates an embedding space which accurately integrates cells across experiments, platforms and species. Our benchmark comparisons with current state-of-the-art scRNA-seq integration methods revealed that INSCT outperforms competing methods in scalability while achieving comparable accuracies. Moreover, using INSCT in semi-supervised mode enables users to classify unlabeled cells by projecting them into a reference collection of annotated cells. To demonstrate scalability, we applied INSCT to integrate more than 2.6 million transcriptomes from four independent studies of mouse brains in less than 1.5 hours using less than 25 gigabytes of memory. This feature empowers researchers to perform atlasing scale data integration in a typical desktop computer environment. INSCT is freely available at https://github.com/lkmklsmn/insct. HighlightsO_LIINSCT accurately integrates multiple scRNA-seq datasets C_LIO_LIINSCT accurately predicts cell types for an independent scRNA-seq dataset C_LIO_LIEfficient deep learning framework enables integration of millions of cells on a personal computer C_LI

bioinformatics↗

bioRxiv2020

Comparison of SARS-CoV-2 spike protein binding to human, pet, farm animals, and putative intermediate hosts ACE2 and ACE2 receptors

The emergence of a novel coronavirus, SARS-CoV-2, resulted in a pandemic. Here, we used recently released X-ray structures of human ACE2 bound to the receptor-binding domain (RBD) of the spike protein (S) from SARS-CoV-2 to predict its binding to ACE2 proteins from different animals, including pets, farm animals, and putative intermediate hosts of SARS-CoV-2. Comparing the interaction sites of ACE2 proteins known to serve or not serve as receptor allows to define residues important for binding. From the 20 amino acids in ACE2 that contact S up to seven can be replaced and ACE2 can still function as the SARS-CoV-2 receptor. These variable amino acids are clustered at certain positions, mostly at the periphery of the binding site, while changes of the invariable residues prevent S-binding or infection of the respective animal. Some ACE2 proteins even tolerate the loss or the acquisition of N-glycosylation sites located near the S-interface. Of note, pigs and dogs which are not or not effectively infected, respectively, have only a few changes in the binding site have relatively low levels of ACE2 in the respiratory tract. Comparison of the RBD of S of SARS-CoV-2 with viruses from bat and pangolin revealed that the latter contains only one substitution, whereas the bat virus exhibits five. However, ACE2 of pangolin exhibit seven changes relative to human ACE2, a similar number of substitutions is present in ACE2 of bats, raccoon, and civet suggesting that SARS-CoV-2 may not especially adapted to ACE2 of any of its putative intermediate hosts. These analyses provide new insight into the receptor usage and animal source/origin of SARS-COV-2. IMPORTANCESARS-CoV-2 is threatening people worldwide and there are no drugs or vaccines available to mitigate its spread. The origin of the virus is still unclear and whether pets and livestock can be infected and transmit SARS-CoV-2 are important and unknown scientific questions. Effective binding to the host receptor ACE2 is the first prerequisite for infection of cells and determines the host range. Our analysis provides a framework for the prediction of potential hosts of SARS-CoV-2. We found that ACE2 from species known to support SARS-CoV-2 infection tolerate many amino acid changes indicating that the species barrier might be low. However, the lower expression of ACE2 in the upper respiratory tract of some pets and livestock means more research and monitoring should be done to explore the animal source of infection and the risk of potential cross-species transmission. Finally, the analysis also showed that SARS-CoV-2 may not specifically adapted to any of its putative intermediate hosts.

bioinformatics↗

bioRxiv2020

Identification of Candidate COVID-19 Therapeutics using hPSC-derived Lung Organoids

Summary ParagraphThe SARS-CoV-2 virus has caused already over 3.5 million COVID-19 cases and 250,000 deaths globally. There is an urgent need to create novel models to study SARS-CoV-2 using human disease-relevant cells to understand key features of virus biology and facilitate drug screening. As primary SARS-CoV-2 infection is respiratory-based, we developed a lung organoid model using human pluripotent stem cells (hPSCs) that could be adapted for drug screens. The lung organoids, particularly aveolar type II cells, express ACE2 and are permissive to SARS-CoV-2 infection. Transcriptomic analysis following SARS-CoV-2 infection revealed a robust induction of chemokines and cytokines with little type I/III interferon signaling, similar to that observed amongst human COVID-19 pulmonary infections. We performed a high throughput screen using hPSC-derived lung organoids and identified FDA-approved drug candidates, including imatinib and mycophenolic acid, as inhibitors of SARS-CoV-2 entry. Pre- or post-treatment with these drugs at physiologically relevant levels decreased SARS-CoV-2 infection of hPSC-derived lung organoids. Together, these data demonstrate that hPSC-derived lung cells infected by SARS-CoV-2 can model human COVID-19 disease and provide a valuable resource to screen for FDA-approved drugs that might be repurposed and should be considered for COVID-19 clinical trials.

microbiology↗

bioRxiv2020

Rapid adaptation of SARS-CoV-2 in BALB/c mice: Novel mouse model for vaccine efficacy

Coronavirus disease 2019 (COVID-19) threatens global public health and economy. In order to develop safe and effective vaccines, suitable animal models must be established. Here we report the rapid adaption of SARS-CoV-2 in BALB/c mice, based on which a convenient, economical and effective animal model was developed. Specifically, we found that mouse-adapted SARS-CoV-2 at passage 6 (MACSp6) efficiently infected both aged and young wild-type BALB/c mice, resulting in moderate pneumonia as well as inflammatory responses. The elevated infectivity of MACSp6 in mice could be attributed to the substitution of a key residue (N501Y) in the receptorbinding domain (RBD). Using this novel animal model, we further evaluated the in vivo protective efficacy of an RBD-based SARS-CoV-2 subunit vaccine, which elicited highly potent neutralizing antibodies and conferred full protection against SARS-CoV-2 MACSp6 challenge. This novel mouse model is convenient and effective in evaluating the in vivo protective efficacy of SARS-CoV-2 vaccine. SummaryThis study describes a unique mouse model for SARS-CoV-2 infection and confirms protective efficacy of a SARS-CoV-2 RBD subunit vaccine.

microbiology↗

bioRxiv2020

Intrinsic, dynamic and effective connectivity among large-scale brain networks modulated by oxytocin

The neuropeptide oxytocin is a key modulator of social-emotional behavior and its intranasal administration can influence the functional connectivity of brain networks involved in the control of attention, emotion and reward reported in humans. However, no studies have systematically investigated the effects oxytocin on dynamic or directional aspects of functional connectivity. The present study employed a novel computational framework to investigate these latter aspects in 15 oxytocin-sensitive regions using data from randomized placebo-controlled between-subject resting state functional MRI studies incorporating 200 healthy subjects. Results showed that oxytocin extensively modulated effective connectivity both between and within emotion, reward, salience and social cognition processing networks and their interactions with the default mode network, but had no effect on the frequency of dynamic changes. Top-down control over emotional processing regions such as the amygdala was particularly affected. Oxytocin effects were also sex-dependent, being more extensive in males. Overall, these findings suggest that modulatory effects of oxytocin on both within- and between-network interactions may underlie its functional influence on social-emotional behaviors, although in a sex-dependent manner. Furthermore, they demonstrate a useful approach to determining pharmacological influences on resting state effective connectivity and support oxytocins potential therapeutic use in psychiatric disorders.

neuroscience↗

bioRxiv2020

The scRNA-seq expression profiling of the receptor ACE2 and the cellular protease TMPRSS2 reveals human organs susceptible to COVID-19 infection

BackgroundCOVID-19 caused by SARA-CoV-2 is a disaster sweeping over 200 countries, and more than 2,150,000 people are suffering from the disease and 140,000 people died. ACE2 is a receptor protein of SARS- CoV-2, and TMPRSS2 promotes virus proliferation and transmission. Some patients developed multiple organ dysfunction syndromes other than lungs. Therefore, studying the viral susceptibility of other organs is important for a deeper understanding of viral pathogenesis. MethodsThe advantage of scRNA-seq data is the identification of cell types by clustering the gene expression of cells. ACE2 and TMPRSS2 are highly expressed in AT2 of lungs, we compared the ACE2 and TMPRSS2 expression levels of cell types from 31 organs, with AT2 of lungs to evaluate the risk of the viral infection using scRNA-seq data. FindingsFor the first time, we found the brain, gall bladder, and fallopian tube are vulnerable to COVID-19 infection. Besides, the nose, heart, small intestine, large intestine, esophagus, testis and kidney are also identified to be high-risk organs with high expression levels of ACE2 and TMPRSS2. Moreover, the susceptible organs are grouped into three risk levels based on the TMPRSS2 expression. As a result, the respiratory system, digestive system and reproductive system are at the top-risk level to COVID-19 infection. InterpretationThis study provides evidence for COVID-19 infection in the human nervous system, digestive system, reproductive system, respiratory system, circulatory system and urinary system using scRNA-seq data, which helps for the clinical diagnosis and treatment of patients.

bioinformatics↗

bioRxiv2020

Studies on trans-sutural distraction osteogenesis-related genes based on transcriptome sequencing

Trans-sutural distraction osteogenesis (TSDO) is an important approach to improve mid-face hypoplasia. In recent years, many studies have been carried out on physical mechanisms of TSDO; however, its specific cytological and molecular mechanisms are still unclear. In this study, we performed transcriptome sequencing analysis in Sprague Dawley rats at 1 and 2 weeks after suture osteogenesis and compared RNA expression levels between experimental and control groups. At one week, enrichment pathways were mainly up-regulated in muscle- and bone-related pathways. By contrast, pathways of the immune system showed a state of inhibition and down-regulation, especially for B cells; the main immune pathways showed significant down-regulation. However, two weeks later, the experimental group showed positive up-regulation of the pathways related to DNA synthesis and replication, cell cycle, and chromosome replication. At the same time, the immune pathways that were down-regulated in the first week were up-regulated in the second week. In other words, the up-regulated muscle- and bone-related pathways show opposite trends. The expression of bone- and myogenesis-related transcriptome was up-regulated and the immune-related pathways were down-regulated in the experimental group at 1 week. At 2 weeks, the pathways related to bone- and muscle were down-regulated, while those related to cell cycle regulation and DNA replication were up-regulated. These results suggest that musculoskeletal-related molecules may play an important role during suture osteogenesis at 1 week, and immune regulation may be involved in this process; however, at 2 weeks, molecules related to cell proliferation and replication may be a major role.

bioinformatics↗

bioRxiv2020

Microvascular Injury in Mild Traumatic Brain Injury Accelerates Alzheimer-like Pathogenesis in Mice

IntroductionTraumatic brain injury (TBI) is considered as the most robust environmental risk factor for Alzheimers disease (AD). Besides direct neuronal injury and neuroinflammation, vascular impairment is also a hallmark event of the pathological cascade after TBI. However, the vascular connection between TBI and subsequent AD pathogenesis remains underexplored. MethodsWe established a closed-head mild TBI (mTBI) model in mice with controlled cortical impact, and examined the time courses of microvascular injury, blood-brain barrier (BBB) dysfunction, gliosis and motor function impairment in wild type C57BL/6 mice. We also determined the brain clearance of {beta}-amyloid, as well as amyloid pathology and cognitive functions after mTBI in the 5xFAD mouse model of AD. ResultsmTBI induced microvascular injury with BBB breakdown, pericyte loss and cerebral blood flow reduction in mice, which preceded gliosis. mTBI also impaired brain amyloid clearance via the vascular pathways. More importantly, mTBI accelerated amyloid pathology and cognitive impairment in the 5xFAD mice. DiscussionOur data demonstrated that microvascular injury plays a key role in the pathogenesis of AD after mTBI. Therefore, restoring vascular functions might be beneficial for patients with mTBI, and potentially reduce the risk of developing AD.

neuroscience↗

bioRxiv2020

Predicting Metastasis with a Novel Biophysical Cell-Adhesion Force Technique

Metastasis is widely accepted to be responsible for approximately 90% of all cancer deaths. Current research on metastasis prediction often centers on gene sequencing; however, these analyses must account for the complexity of gene regulation and rely on comprehensive datasets. To investigate the process from a simpler, non-genomic angle, some studies indicate differences in cell adhesion force, an important physical process in metastasizing cells. However, cell adhesion force methods tend to focus on cell population approaches and therefore have their drawbacks in cost or efficiency, rendering them impractical outside a research setting. In this work, we test a novel and inexpensive bead-pipette assay to investigate the adhesion forces of non-metastatic NIH3T3 cells and mutated RasV12 cells, a metastatic model cell line. Control cells and RasV12 cells were evaluated with wound healing, spreading area, and focal adhesion (FA) analysis assays. Then cells were tested by the novel bead-pipette assay, which uses a fibronectin-coated bead and a glass micropipette to measure cell adhesion force using Hookes law. The RasV12 cells had faster migration, polarized cell shape, and smaller FA area than control cells. The RasV12 cells also exerted higher adhesion forces than control cells and a potential force threshold was determined for distinguishing metastatic cells through a Receiver Operating Characteristic (ROC) curve. An ROC curve was computed for all other assays and the bead-pipette assay was shown to perform higher as a classifier than other assays. The RasV12 cells had increased metastatic potential compared to control. The novel bead-pipette assay showed potential as a classifier for determining metastasizing cells from non-metastatic cells. With further work, it may serve as a clinical diagnostic tool for cancer patients or as a testbed to be used in the development of anti-metastatic drugs.

biophysics↗

bioRxiv2020

Immune factor of bacterial origin protects ticks against host microbial commensals

Hard ticks are blood-feeding arthropods that carry and transmit microbes to their vertebrate hosts1. Tick-borne disease cases have been on the rise over the last several decades, drawing much-needed attention to the molecular interplay between transmitted pathogens and their human hosts. However, far less is known about how ticks control their own microbes, which is critical for understanding how zoonotic transmission cycles persist. We previously found that ticks horizontally acquired an antimicrobial toxin gene from bacteria known as domesticated amidase effector 2 (dae2)2. Here we show that this effector from the tick vector Ixodes scapularis (Dae2Is) has structurally and biochemically diverged from ancestral bacterial representatives, expanding its antibacterial targeting range to include host skin microbes. Disruption of dae2Is increases the burden of skin-associated staphylococci within I. scapularis and adversely affects tick fitness, suggesting resistance of host microbes may be important for the parasitic blood-feeding lifestyle. In contrast, Dae2Is has no intrinsic ability to kill Borrelia burgdorferi, the tick-borne bacterium of Lyme disease. Our observations suggest that ticks have evolved to tolerate their own symbionts while resisting host skin commensals, which we discover are natural opportunistic pathogens of ticks. This work moves our understanding of vector biology beyond a human-centric view: just as tick commensals are pathogenic to humans, so too do our commensals pose a threat to ticks. These observations illuminate how a complex and mirrored set of interkingdom interactions between blood-feeding vectors, their hosts, and associated microbes can ultimately lead to disease.

evolutionary biology↗