Biology subjects

Zhou, H.

Publications and source records attributed to Zhou, H..

At least 235 records · Page 13Linked to original sources

bioRxiv2023

Profiling plasma extracellular vesicle-derived microRNAs for noninvasive diagnosis of alpha-1 antitrypsin deficiency associated liver disease

Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disease characterized by reduced circulating levels of alpha-1 antitrypsin (AAT) due to the retention of misfolded AAT in the hepatocytes. The toxic AAT aggregates in hepatocytes cause liver fibrosis, cirrhosis, and hepatocellular carcinoma. Most patients remain asymptomatic until the final stage in which liver transplantation becomes the only treatment. Timely diagnosis of AATD liver disease plays a critical role in intervention and finding curative solutions. Assessing the prevalence and severity of AATD liver disease remains challenging due to limitations in current methods. Liver biopsy, the gold standard for evaluating the hepatic AAT accumulation, the initiating stage for AATD liver disease, is hindered by invasiveness and sampling errors. To address these limitations, we conducted a study using unique and precious clinical samples. We analyzed plasma extracellular vesicle (EV) derived miRNAs and liver transcriptomes from AATD individuals to develop a sensitive and noninvasive diagnostic approach for AATD liver disease. In the testing stage of our study, we enrolled 17 AATD individuals with different stages of liver disease, as determined by liver biopsy, and 20 controls. We identified differential expression of 178 miRNAs within the AATD group compared to controls by miRNA sequencing. Among those miRNAs, we selected miR-223-3p, miR-23a-3p, miR-15b-5p, let-7a-5p, let-7f-5p, and miR-374a-5p for further validation in an independent cohort of 45 AATD individuals. Using a logistic model that combined three miRNAs, we achieved an AUC of 0.737 for detecting AATD liver disease. Adding a fourth miRNA into this model increased the AUC to 0.751. The changes in EV miRNAs are correlated to dysregulated expression of liver mRNAs in AATD individuals with different stages of liver disease. We propose that plasma-circulating EV exhibit distinct miRNAs in AATD individuals and could serve as clinically significant biomarkers for the early detection of AATD liver disease.

molecular biology↗

bioRxiv2023

XBB.1.5 Spike Protein COVID-19 Vaccine Induces Broadly Neutralizing and Cellular Immune Responses Against EG.5.1 and Emerging XBB Variants

Monovalent SARS-CoV-2 Prototype (Wuhan-Hu-1) and bivalent (Prototype + BA.4/5) COVID-19 vaccines have demonstrated a waning of vaccine-mediated immunity highlighted by lower neutralizing antibody responses against SARS-CoV-2 Omicron XBB sub-variants. The reduction of humoral immunity due to the rapid evolution of SARS-CoV-2 has signaled the need for an update to vaccine composition. A strain change for all authorized/approved vaccines to a monovalent composition with Omicron subvariant XBB.1.5 has been supported by the WHO, EMA, and FDA. Here, we demonstrate that immunization with a monovalent recombinant spike protein COVID-19 vaccine (Novavax, Inc.) based on the subvariant XBB.1.5 induces cross-neutralizing antibodies against XBB.1.5, XBB.1.16, XBB.2.3, EG.5.1, and XBB.1.16.6 subvariants, promotes higher pseudovirus neutralizing antibody titers than bivalent (Prototype + XBB.1.5) vaccine, induces SARS-CoV-2 spike-specific Th1-biased CD4+ T-cell responses against XBB subvariants, and robustly boosts antibody responses in mice and nonhuman primates primed with a variety of monovalent and bivalent vaccines. Together, these data support updating the Novavax vaccine to a monovalent XBB.1.5 formulation for the 2023-2024 COVID-19 vaccination campaign.

immunology↗

bioRxiv2023

Enriched atlas of lncRNA and protein-coding genes for the GRCg7b chicken assembly and its functional annotation across 47 tissues.

Gene atlases for livestock are steadily improving thanks to new genome assemblies and new expression data improving the gene annotation. However, gene content varies across databases due to differences in RNA sequencing data and bioinformatics pipelines, especially for long non-coding RNAs (lncRNAs) which have higher tissue and developmental specificity and are harder to consistently identify compared to protein coding genes (PCGs). As done previously in 2020 for chicken assemblies galgal5 and GRCg6a, we provide a new gene atlas, lncRNA-enriched, for the latest GRCg7b chicken assembly, integrating "NCBI RefSeq", "EMBL-EBI Ensembl/GENCODE" reference annotations and other resources such as FAANG and NONCODE. As a result, the number of PCGs increases from 18,022 (RefSeq) and 17,007 (Ensembl) to 24,102, and that of lncRNAs from 5,789 (RefSeq) and 11,944 (Ensembl) to 44,428. Using 1,400 public RNA-seq transcriptome representing 47 tissues, we provided expression evidence for 35,257 (79%) lncRNAs and 22,468 (93%) PCGs, supporting the relevance of this atlas. Further characterization including tissue-specificity, sex-differential expression and gene configurations are provided. We also identifiend conserved miRNA-hosting genes with human counterparts, suggesting common function. The annotated atlas is available at www.fragencode.org/lnchickenatlas.html.

genomics↗

bioRxiv2023

Colchicine inhibited oxidative stress-induced endothelial cell senescence via blocking NF-κB and MAPKs: Implications in vascular diseases

Smoking, alcohol abuse, and hypertension are - among others the potential risk factors for cardiovascular diseases. These risk factors generate oxidative stress and cause oxidative stress-induced DNA damage, resulting in cellular senescence and senescence-associated secretory phenotype (SASP). The SASP factors in feed-forward response exacerbate inflammation and cause tissue remodeling, resulting in atherosclerotic plaque formation and rupture. Colchicine was used to ameliorate oxidative stress-induced senescence in human umbilical vein endothelial cells. Oxidative stress was quantified by reactive oxygen species (ROS) assay and oxidative stress-induced DNA damage was analyzed by 8-OHDG immunofluorescence staining. Endothelial cell senescence was visualized by {beta}-gal staining. The relative mRNA expression was quantified by qPCR and protein analysis was performed by Western blot. Colchicine inhibited ROS generation and mitigated oxidative stress-induced DNA damage. It dampened oxidative stress-induced endothelial cell senescence and improved the expression of DNA repair protein KU80 and aging marker Lamin B1. The drug attenuated the expression of senescence marker P21 at mRNA and protein levels. The pathway analysis showed that colchicine inhibited NF-{kappa}B and MAPKs pathways and subdued mTOR activation. Colchicine also attenuated mRNA expression of interleukin (IL)-1{beta}, IL-6, IL-8 MCP-1, ICAM-1, and E-selectin. Furthermore, colchicine reduced the mRNA and protein expression of matrix metalloproteinase (MMP-2). In summary, colchicine blocked oxidative stress-induced senescence and SASP by inhibiting the activation of NF-{kappa}B and MAPKs pathways.

molecular biology↗

bioRxiv2023

ADAR2 inhibits ER-targeted mRNA translation via regulating nucleolar assembly of signal recognition particle

Adenosine-to-inosine RNA editing is mediated by ADAR family proteins and plays critical roles in regulating gene expression. Unlike other ADAR family members, ADAR2 mostly resides in nucleolus. The exact roles of this peculiar cellular distribution of ADAR2 is still largely unknown. Here, our current study unexpectedly uncovered that nucleoli-localized ADAR2 is involved in negatively regulating ER-targeted mRNA translation, which is mediated by SRP. Importantly, this regulation is not archived through direct binding of mRNA by ADAR2 or dependent on A-to-I editing of 7SL RNA. Further analysis indicated that ADAR2 directly interacts with SRP protein components, especially SRP68, in a RNA-independent manner and probably represses the SRP assembly. Hence, we proved insights into the novel functions of ADAR2 and long-lasting enigma about the peculiar translocation of SRP proteins into nucleoli during SRP biogenesis.

biochemistry↗

bioRxiv2023

Competitive fitness and homologous recombination of SARS-CoV-2 variants of concern

SARS-CoV-2 variants continue to emerge and cocirculate in humans and wild animals. The factors driving the emergence and replacement of novel variants and recombinants remain incompletely understood. Herein, we comprehensively characterized the competitive fitness of SARS-CoV-2 wild type (WT) and three variants of concern (VOCs), Alpha, Beta and Delta, by coinfection and serial passaging assays in different susceptible cells. Deep sequencing analyses revealed cell-specific competitive fitness: the Beta variant showed enhanced replication fitness during serial passage in Caco-2 cells, whereas the WT and Alpha variant showed elevated fitness in Vero E6 cells. Interestingly, a high level of neutralizing antibody sped up competition and completely reshaped the fitness advantages of different variants. More importantly, single clone purification identified a significant proportion of homologous recombinants that emerged during the passage history, and immune pressure reduced the frequency of recombination. Interestingly, a recombination hot region located between nucleotide sites 22995 and 28866 of the viral genomes could be identified in most of the detected recombinants. Our study not only profiled the variable competitive fitness of SARS-CoV-2 under different conditions, but also provided direct experimental evidence of homologous recombination between SARS-CoV-2 viruses, as well as a model for investigating SARS-CoV-2 recombination. ImportanceSARS-CoV-2 variants or subvariants keep emerging and the epidemic strains keeps changing in humans and animals. The continued replacement of the epidemic strains was attributed to higher competitive fitness evolved by the newly appeared ones than the older ones, but which factors affect the final outcomes are still not entirely clear. In this study, we performed in vitro coinfection and serial passage with three VOCs and WT under different conditions. Our results showed that the competition outcomes of these viral strains varied in different cell lines or under different immune pressure, confirming the probable effects of these two factors for the competitive fitness of different SARS-CoV-2 viral strains. Meanwhile, strikingly, we found that coinfection and serial passage with different SARS-CoV-2 viral strains can mimic the recombination process of SARS-CoV-2 occurred in coinfection individual, indicating it is a novel model to investigate the SARS-CoV-2 recombination mechanism.

microbiology↗

bioRxiv2023

Association between the rifampicin resistance mutations and rifabutin susceptibility in Mycobacterium tuberculosis: a meta-analysis

Some rifampicin-resistant Mycobacterium tuberculosis (MTB) strains were susceptible to rifabutin (RFB) and may be amenable to treatment with RFB. We performed a meta-analysis of available cross-sectional studies to determine which RIF-resistance mutations were associated with rifabutin susceptibility. We identified studies through PubMed, Web of Science, Embase, and Cochrane Library up to June 1, 2023. Studies that met our criteria were those that investigated rpoB mutations and reported phenotypic drug susceptibility for RIF and RFB. The relationship between RIF-resistance mutations to RFB-susceptibility was evaluated using odds ratio (OR). Twenty-five studies comprised 4,333 clinical RIF-resistant MTB isolates from 21 different countries met our criteria for inclusion. Of these isolates, 21.00% (910/4333) were susceptible to RFB. We found seven RIF-resistance mutations were high confidence (OR>10) in predicting RFB-susceptibility, which were D435V, D435Y, D435F, H445L, L430R, S441L, and S441Q. Among strains carrying these mutations, 83.01% (435/524) were susceptible to RFB. The minimum inhibition concentrations (MICs) of these strains revealed that they had low MIC (D435V, D435F, H445L, and D435Y) or were susceptible (S441L) for RFB and exhibited a significant lack of correlation between MICs to RIF and RFB. Mutations such as H445C, H445G, H445N, L430P, and L452P showed a moderate confidence (5<OR[≤]10) in prediction of RFB-susceptibility. Of these mutants, 62.16% (69/111) were susceptible to RFB. The most common RIF-resistance mutations S450L, as well as S450W, were associated with RFB-resistance (OR<1). These results provide a theoretical basis for molecular detection of RFB-susceptible TB and alternative treatment with RFB in MDR/RR-TB patients.

microbiology↗

bioRxiv2023

Reprogramming method does not impact the neuronal differentiation potential of 16p11.2 deletion patient iPSCs

A major impediment to the actualization of the induced pluripotent stem cell (iPSC)-based personalized medicine revolution is the lack of widely accepted standard operating procedures (SOPs) across different groups and institutions. The various methods employed can include choice of starting materials, reprogramming agents, and culture conditions, with each of these factors hypothesized to influence the reprogramming efficiency and transcriptional identity of iPSCs. As such, we systematically compared iPSC reprogramming procedures using cells derived from the somatic cells of three patients with 16p11.2 deletion syndrome (16p11.2del) and found remarkable similarity among the different methods. FACS analysis revealed that regardless of somatic cell type (fibroblast, lymphocyte, erythroblast), route of reprogramming factor introduction (mRNA, Sendai virus, episome), donor sex, or facility (Rutgers, NYSCF), 16p11.2del patient iPSCs were viable as high purity cultures expressing pluripotency marker proteins. This observation was supported at the transcript level by qPCR analysis, which demonstrated the ability for the iPSCs to differentiate into all three embryonic germ cell lineages after 12 days in culture as embryoid bodies. NGN2-mediated differentiation of these iPSCs produced functional neurons that formed active synaptic networks as revealed by multi-electrode array (MEA) recordings. Importantly, no group-wise comparisons among the reprogramming methods yielded consistent statistically significant differences, indicating that these procedures are equally capable of producing pluripotent stem cells that can efficiently differentiate into mature, functional neurons. This work highlights the utility of these reprogramming methods and supports the use of differentially reprogrammed iPSCs for direct comparative studies of human neurodevelopment.

developmental biology↗

bioRxiv2023

The ChickenGTEx pilot analysis: a reference of regulatory variants across 28 chicken tissues

Chicken is a valuable model for understanding fundamental biology, vertebrate evolution and diseases, as well as a major source of nutrient-dense and lean-protein-enriched food globally. Although it is the first non-mammalian amniote genome to be sequenced, the chicken genome still lacks a systematic characterization of functional impacts of genetic variants. Here, through integrating 7,015 RNA-Seq and 2,869 whole-genome sequence data, the Chicken Genotype- Tissue Expression (ChickenGTEx) project presents the pilot reference of regulatory variants in 28 chicken tissue transcriptomes, including millions of regulatory effects on primary expression (including protein-coding genes, lncRNA and exon) and post-transcriptional modifications (alternative splicing and 3 untranslated region alternative polyadenylation). We explored the tissue-sharing and context-specificity of these regulatory variants, their underlying molecular mechanisms of action, and their utility in interpreting adaptation and genome-wide associations of 108 chicken complex traits. Finally, we illustrated shared and lineage-specific features of gene regulation between chickens and mammals, and demonstrated how the ChickenGTEx resource can further assist with translating genetic findings across species. One-Sentence SummaryThe ChickenGTEx provides a multi-tissue reference of regulatory variants for chicken genetics and genomics, functional genomics, precision breeding, veterinary medicine, vertebrate evolution and even human biomedicine.

genetics↗

bioRxiv2023

Myeloid-derived suppressor cells exacerbate poly(I:C)-induced lung inflammation in mice with renal injury and older mice

Viral pneumonia is a global health burden with a high mortality rate, especially in the elderly and in patients with underlying diseases. Recent studies have found that myeloid-derived suppressor cells (MDSCs) are abundant in these patient groups; however, their roles in the progression of viral pneumonia remain unclear. In this study, we observed a substantial increase in MDSCs in a mouse model of renal ischemia/reperfusion (I/R) injury and in older mice. When intranasal polyinosinic- polycytidylic acid (poly(I:C)) administration was used to mimic viral pneumonia, we found that mice with renal I/R injury exhibited more severe lung inflammation than sham mice when challenged with poly(I:C). In addition, MDSC depletion attenuated lung inflammation in mice with I/R injury. Similar results were obtained in older mice compared with those in young mice. Furthermore, we found that the adoptive transfer of in vitro-differentiated MDSCs exacerbated poly(I:C)-induced lung inflammation. Taken together, these experimental results suggest that the increased proportion of MDSCs in mice with renal I/R injury and in older mice exacerbates poly(I:C)-induced lung inflammation. These findings have important implications for the treatment and prevention of severe lung inflammation caused by viral pneumonia.

immunology↗

bioRxiv2023

The aryl hydrocarbon receptor regulates epidermal differentiation through transient activation of TFAP2A

The aryl hydrocarbon receptor (AHR) is an evolutionary conserved environmental sensor identified as indispensable regulator of epithelial homeostasis and barrier organ function. Molecular signaling cascade and target genes upon AHR activation and their contribution to cell and tissue function are however not fully understood. Multi-omics analyses using human skin keratinocytes revealed that, upon ligand activation, AHR binds open chromatin to induce expression of transcription factors (TFs), e.g., Transcription Factor AP-2 (TFAP2A), as a swift response to environmental stimuli. The terminal differentiation program including upregulation of barrier genes, filaggrin and keratins, was mediated by TFAP2A as a secondary response to AHR activation. The role of AHR-TFAP2A axis in controlling keratinocyte terminal differentiation for proper barrier formation was further confirmed using CRISPR/Cas9 in human epidermal equivalents. Overall, the study provides novel insights into the molecular mechanism behind AHR-mediated barrier function and potential novel targets for the treatment of skin barrier diseases.

cell biology↗

bioRxiv2023

Recognition of centromere-specific histone Cse4 by the inner kinetochore Okp1-Ame1 complex

Successful mitosis depends on the timely establishment of correct chromosomal attachments to microtubules. The kinetochore, a modular multiprotein complex, mediates this connection by recognizing specialized chromatin containing a histone H3 variant called Cse4 in budding yeast and CENP-A in vertebrates. Structural features of the kinetochore that enable discrimination between Cse4/CENP-A and H3 have been identified in several species. How and when these contribute to centromere recognition and how they relate to the overall structure of the inner kinetochore are unsettled questions. We have determined the crystal structure of a Cse4 peptide bound to the essential inner kinetochore Okp1-Ame1 heterodimer from budding yeast. The structure and related experiments show in detail an essential point of Cse4 contact and provide information about the arrangement of the inner kinetochore.

cell biology↗

bioRxiv2023

Monocyte Adhesion and Transmigration Through Endothelium Following Cardiopulmonary Bypass Shearing is Mediated by IL-8 Signaling

BackgroundThe use of cardiopulmonary bypass (CPB) can induce sterile systemic inflammation that contributes to morbidity and mortality, especially in children. Patients have been found to have increased expression of cytokines and transmigration of leukocytes during and after CPB. Previous work has demonstrated that the supraphysiologic shear stresses present during CPB are sufficient to induce proinflammatory behavior in non-adherent monocytes. The interactions between shear stimulated monocytes and vascular endothelial cells have not been well studied and have important translational implications. MethodsTo test the hypothesis that non-physiological shear stress experienced by monocytes during CPB affects the integrity and function of the endothelial monolayer via IL-8 signaling pathway, we have used an in vitro CPB model to study the interaction between THP-1 monocyte-like cells and human neonatal dermal microvascular endothelial cells (HNDMVECs). THP-1 cells were sheared in polyvinyl chloride (PVC) tubing at 2.1 Pa, twice of physiological shear stress, for 2 hours. Interactions between THP-1 cells and HNDMVECs were characterized after coculture. ResultsWe found that sheared THP-1 cells adhered to and transmigrated through the HNDMVEC monolayer more readily than static controls. When co-culturing, sheared THP-1 cells also disrupted in the VE-cadherin and led to reorganization of cytoskeletal F-actin of HNDMVECs. Treating HNDMVECs with IL-8 resulted in upregulation of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) while also increasing the adherence of non-sheared THP-1 cells. Preincubating HNDMVECs with Reparixin, an inhibitor of CXCR2/IL-8 receptor inhibited sheared THP-1 cell adhesion to the HNDMVECs. ConclusionsThese results suggested that IL-8 not only increases the endothelium permeability during monocyte migration, but also affects the initial adhesion of monocytes in a CPB setup. This study revealed a novel mechanism of post-CPB inflammation and will contribute to the development of targeted therapeutics to prevent and repair the damage to neonatal patients. HighlightsO_LIShear stress in a CPB-like environment promoted the adhesion and transmigration of monocytes to and through endothelial monolayer. C_LIO_LITreating endothelial monolayer with sheared monocytes led to disruption of VE-cadherin and reorganization of F-actin. C_LIO_LIInteraction between sheared monocytes resulted in a significant increase of IL-8 release. C_LIO_LIInhibiting IL-8 receptor prevented sheared monocyte adhesion, while IL-8 promoted naive monocyte adhesion. C_LI

cell biology↗

bioRxiv2023

Haplotype-resolve genome assembly and resequencing provide insights into the origin and domestication of modern rose

Modern rose (Rosa hybrida) is a recently formed interspecific hybrid and has become one of the most important and widely cultivated ornamentals. Here, we report the haplotype-resolved chromosome-scale genome assembly of the tetraploid R. hybrida Samantha and a genome variation map of 233 Rosa accessions involving various wild species, and old and modern cultivars. The two subgenomes of Samantha show no significant global bias in gene loss but substantial expression bias, and homoeologous exchanges are frequently observed between subgenomes. Population genomic and genomic composition analyses reveal the contributions of wild Rosa species to modern roses, and highlight that R. odorata and its derived cultivars contribute more to modern roses than R. chinensis Old Blush. Furthermore, selective sweeps during modern rose breeding associated with major agronomic traits, including continuous flowering, floral organ identity, flower senescence, and disease resistance, are identified. This study provides insights into the genetic basis of modern rose origin and breeding history, and offers unprecedented genomic resources for rose improvement.

genomics↗

bioRxiv2023

Oxaliplatin-induced cardiotoxicity in mice is connected to the changes in energy metabolism in the heart tissue

Oxaliplatin is a platinum-based alkylating chemotherapeutic agent used for cancer treatment. At high cumulative dosage, the negative effect of oxaliplatin on the heart becomes evident and is linked to a growing number of clinical reports. The aim of this study was to determine how chronic oxaliplatin treatment causes the changes in energy-related metabolic activity in the heart that leads to cardiotoxicity and heart damage in mice. C57BL/6 male mice were treated with a human equivalent dosage of intraperitoneal oxaliplatin (0 and 10 mg/kg) once a week for eight weeks. During the treatment, mice were followed for physiological parameters, ECG, histology and RNA sequencing of the heart. We identified that oxaliplatin induces strong changes in the heart and affects the hearts energy-related metabolic profile. Histological post-mortem evaluation identified focal myocardial necrosis infiltrated with a small number of associated neutrophils. Accumulated doses of oxaliplatin led to significant changes in gene expression related to energy related metabolic pathways including fatty acid (FA) oxidation, amino acid metabolism, glycolysis, electron transport chain, and NAD synthesis pathway. At high accumulative doses of oxaliplatin, the heart shifts its metabolism from FAs to glycolysis and increases lactate production. It also leads to strong overexpression of genes in NAD synthesis pathways such as Nmrk2. Changes in gene expression associated with energy metabolic pathways can be used to develop diagnostic methods to detect oxaliplatin-induced cardiotoxicity early on as well as therapy to compensate for the energy deficit in the heart to prevent heart damage. Significance StatementThis study uncovers the detrimental impact of chronic oxaliplatin treatment on heart metabolism in mice, linking high accumulative dosages to cardiotoxicity and heart damage. By identifying significant changes in gene expression related to energy metabolic pathways, the findings pave the way for the development of diagnostic methods to detect oxaliplatin-induced cardiotoxicity at an early stage. Furthermore, these insights may inform the creation of therapies that compensate for the energy deficit in the heart, ultimately preventing heart damage and improving patient outcomes in cancer treatment.

pharmacology and toxicology↗

bioRxiv2023

Projectome-defined subtypes and modular intra-hypothalamic subnetworks of peptidergic neurons

The hypothalamus plays a vital role in coordinating essential neuroendocrine, autonomic, and somatomotor responses for survival and reproduction. While previous studies have explored population-level projections of hypothalamic neurons, the specific innervation patterns of individual hypothalamic axons remain unclear. To understand the organization of hypothalamic axon projections, we conducted a comprehensive reconstruction of single-cell projectomes from 7,180 mouse hypothalamic neurons expressing specific neuropeptides. Our analysis identified 31 distinct subtypes based on projectome-defined characteristics, with many exhibiting long-range axon collateral projections to multiple brain regions. Notably, these subtypes selectively targeted specific subdomains within downstream areas, either unilaterally or bilaterally. Furthermore, we observed that individual peptidergic neuronal types encompassed multiple projectome-defined subtypes, explaining their diverse functional roles. Additionally, by examining intra-hypothalamic axon projections, we uncovered six modular subnetworks characterized by enriched intramodular connections and distinct preferences for downstream targets. This modular organization of the intra-hypothalamic network likely contributes to the coordinated organization of hypothalamic outputs. In summary, our comprehensive projectome analysis reveals the organizational principles governing hypothalamic axon projections, providing a framework for understanding the neural circuit mechanisms underlying the diverse and coordinated functions of the hypothalamus.

neuroscience↗

bioRxiv2023

Theoretical framework and experimental solution for the air-water interface adsorption problem in cryoEM

As cryogenic electron microscopy (cryoEM) gains traction in the structural biology community as a method of choice for determining atomic structures of biological complexes, it has been increasingly recognized that many complexes that behave well under conventional negative-stain electron microscopy tend to have preferential orientation, aggregate or simply mysteriously "disappear" on cryoEM grids, but the reasons for such misbehavior are not well understood, limiting systematic approaches to solving the problem. Here, we have developed a theoretical formulation that explains these observations. Our formulation predicts that all particles migrate to the air-water interface (AWI) to lower the total potential surface energy -- rationalizing the use of surfactant, which is a direct solution to reducing the surface tension of the aqueous solution. By conducting cryogenic electron tomography (cryoET) with the widely-tested sample, GroEL, we demonstrate that, in a standard buffer solution, nearly all particles migrate to the AWI. Gradual reduction of the surface tension by introducing surfactants decreased the percentage of particles exposed to the surface. By conducting single-particle cryoEM, we confirm that applicable surfactants do not damage the biological complex, thus suggesting that they might offer a practical, simple, and general solution to the problem for high-resolution cryoEM. Application of this solution to a real-world AWI adsorption problem with a more challenging membrane protein, namely, the ClC-1 channel, has led to its first near-atomic structure using cryoEM.

biophysics↗

bioRxiv2023

N6-methyladenosine demethylase FTO serves as an indicator for predicting prognosis and immunotherapy response in individuals with gastric cancer

BackgroundN6-methyladenosine (m6A) RNA methylation is the most common chemical decoration in mammalian RNAs which exerts vital effects on numerous cellular processes. Recently, m6A regulators have been validated to participate in promoting immune evasion and act as prognostic factors in various cancers. Nevertheless, the predictive abilities of m6A regulators for the prognosis and immunotherapy response in gastric cancer (GC) remain indistinct. MethodsHerein, The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx) database, The Human Protein Atlas (HPA), and a clinical GC cohort were applied for differential expression analysis, correlation analysis, survival analysis, and hazard model construction. Consensus clustering analysis was performed to authenticate the PD-L1 (CD274) expression, stemness features, immune cell infiltration, and tumor microenvironment (TME) in GC individuals. Furthermore, protein-protein interaction, immunotherapy response prediction, and drug susceptibility prediction were performed, respectively. Additionally, tissue microarray (TMA), immunohistochemical staining, western blot assay, Transwell assay, and flow cytometry assay were adopted to evaluate the protein expression, the prognostic value, and the influence of FTO on GC malignant phenotypes as well as the expression of PD-L1. ResultsIn agreement with the majority of m6A regulators, FTO was overexpressed and predicted poor prognosis in GC. Based on consensus clustering analysis, two independent subgroups (G1/G2) were identified. Notably, FTO was upregulated in the G1 subgroup. Meanwhile, the infiltration level of CD8+ T cells was strikingly decreased while the stemness features were enhanced in the G1 subgroup. More importantly, FTO was negatively correlated with microsatellite instability (MSI) and tumor mutation burden (TMB). Furthermore, immune checkpoint blockade (ICB) response prediction indicated that patients with upregulated FTO showed high tumor immune dysfunction and exclusion (TIDE) scores. Subsequently, FTO was confirmed to be related to multiple immune checkpoints, particularly PD-L1. Specifically, FTO was dramatically upregulated in GC cell lines and clinical cancer samples. Functional experiments illustrated that FTO acted as an oncogene to facilitate malignant phenotypes. Notably, PD-L1 was remarkably downregulated after RNA interference-mediated knockdown of FTO. ConclusionFTO can aggravate GC malignant phenotypes. More importantly, it could be utilized to predict the long-term prognosis and the immunotherapy response in GC individuals. However, larger trials should be performed to verify the prediction accuracy.

cancer biology↗