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Bai, R.

Publications and source records attributed to Bai, R..

At least 19 recordsLinked to original sources

Multidimensional Mechanistic Study of Panax Notoginseng Saponins in the Treatment of Alcohol-Induced Osteonecrosis of the Femoral Head: Integrating Network Pharmacology, Molecular Dynamics Simulation and In Vivo Validation

BackgroundAlcohol-induced osteonecrosis of the femoral head (AIONFH) is an orthopedic disorder from chronic alcohol abuse, characterized by disrupted femoral head blood supply, osteocyte death and structural collapse. Current hip-preserving therapy is unsatisfactory, and most patients eventually require total hip arthroplasty. Panax Notoginseng Saponins (PNS), the core active component of Panax notoginseng, exerts pro-angiogenic and anti-osteocyte apoptosis effects, but its specific therapeutic mechanism remains unclear. ObjectiveThis study used network pharmacology, molecular dynamics simulation and animal experiments to identify PNSs active components, core targets and key pathways for AIONFH, verify its in vivo efficacy, and provide a scientific basis for clinical application. MethodsPNS active components, their targets and AIONFH-related targets were screened from databases; intersection targets constructed an interaction network, core targets were screened by three machine learning algorithms, with concurrent GO and KEGG analysis. Molecular docking was performed between core targets and PNS components; Gromacs 2022 conducted 100 ns simulation to evaluate complex stability. AIONFH rat models were grouped with 4-week intragastric intervention; pathology, immunofluorescence and PCR were used for detection. Results and DiscussionNetwork pharmacology identified 127 PNS targets and 18 intersections with 672 AIONFH targets. Six core targets (including FGF2, HSD11B1) were screened; KEGG indicated VEGF pathway as key. Ginsenoside Re bound HSD11B1 with the lowest binding energy (-12.4 kcal/mol), and 100 ns simulation confirmed complex stability. Animal experiments showed PNS improved trabecular structure and regulated osteocyte activity. PNS treats AIONFH via multi-component, multi-target mode, core mechanism being osteocyte apoptosis inhibition. Results and DiscussionNetwork pharmacology screening identified 127 potential targets of PNS, and 18 potential intersection targets were obtained by overlapping with 672 AIONFH-related targets. Six core targets including FGF2 and HSD11B1 were screened out by machine learning, and KEGG analysis indicated that the VEGF pathway and other pathways were the key signaling pathways for PNS action. Molecular docking showed that Ginsenoside Re had the lowest binding energy with HSD11B1 (-12.4 kcal/mol), and 100 ns molecular dynamics simulation confirmed the stable conformation of this complex. Animal experiments demonstrated that PNS could improve trabecular bone structure and regulate osteocyte activity. In summary, PNS exerts a therapeutic effect on AIONFH through a multi-component, multi-target and multi-pathway mode, with the core mechanism of inhibiting osteocyte apoptosis.

pharmacology and toxicology↗

Elimination of intramuscular immunoglobulin accumulation alleviates Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disorder due to loss of dystrophin, a cytoskeletal protein critical for muscle integrity and functionality. Despite recent therapeutic advances, there remains a significant unmet need for more effective and accessible therapeutics. Here, we discovered an early accumulation of immunoglobulin G (IgG) in the sarcolemma, which exacerbates tissue inflammation and disease progression. The IgG accumulation primarily resulted from ectopic localization of Fc{gamma}R1, a high-affinity IgG Fc receptor, on dystrophin-deficient myofibers. In two independent murine models, eliminating IgG accumulation via B cell depletion provided sustained benefit to alleviate DMD progression. Our findings uncover a novel disease accelerator in DMD and demonstrate the potential to target this mechanism as therapeutics for broader population of patients with DMD.

immunology↗

Lmna deficiency promotes EPHX2 nuclear translocation to ameliorate cardiac dysfunction in mice

BackgroundCardiovascular diseases are often associated with altered protein subcellular localization. As a major cause of inherited cardiomyopathy, LMNA deficiency could trigger nuclear envelope rupture and broadly impair the localization of nuclear and cytoplasmic proteins. Systemic approaches to identify, dissect and manipulate the localization of endogenous proteins are important for mechanistic and therapeutic investigation. MethodProximity proteomics of the nuclear lamina was performed specifically in cardiomyocytes in Lmna-deficient murine models. AAV-mediated Cas9-based gene silencing and subcellular gene upregulation were conducted via the nuclear localization signal (NLS) and nuclear export signal (NES). Cas9-based somatic mutagenesis was supplemented with the single-strand DNA templates of AAV to achieve robust homology-directed repair (HDR) and targeted NLS knock-in, which translocated cytoplasmic proteins into nuclei. ResultIn vivo proximity proteomics detected increased epoxide hydrolase 2 (EPHX2) in cardiomyocyte nuclei in mice carrying germline or cardiac-specific Lmna truncating variants. This phenotype was associated with ruptured nuclear envelope. Cas9-mediated Ephx2 knockout in cardiomyocytes ameliorated cardiac dysfunction in Lmna-deficient mice. Strikingly, overexpression of NLS-EPHX2, but not NES-EPHX2, also mitigated cardiac dysfunction. The cardiac protective EPHX2 substrates, epoxyeicosatrienoic acids (EETs), did not alter upon NLS-EPHX2 overexpression. By contrast, the Lmna-related DNA damage marker {gamma}-H2AX was reduced. The EPHX-D333A mutant lacking hydrolase activity recapitulated the effects of wildtype EPHX2 in nuclei. AAV-Cas9-based HDR achieved efficient NLS knock-in and EPHX2 nuclear translocation in more than 60% cardiomyocytes, which improved cardiac function. ConclusionLmna deficiency leads to the nuclear translocation of EPHX2, which ameliorated cardiac dysfunction in a hydrolase-independent manner. AAV-HDR-mediated somatic gene editing provides an efficient approach to manipulate the subcellular localization of endogenous proteins in cardiomyocytes in vivo. What is Known?O_LICardiovascular diseases are regulated by the changes in protein subcellular localization. In particular, LMNA-related cardiomyopathy is associated with nuclear rupture and impaired separation between nuclear and cytoplasmic proteins. C_LIO_LIEpoxide hydrolase 2 (EPHX2) is a cytoplasmic hydrolase that catalyzes the hydrolysis of cardioprotective epoxyeicosatrienoic acids (EETs) and aggravates an array of heart diseases including myocardial infarction and heart failure. C_LIO_LICRISPR/Cas9-mediated homology-directed repair (HDR) exhibits uniquely high gene editing efficiency in cardiomyocytes with an AAV-based DNA donor, which is suitable for somatic genetic knock-in of small DNA fragments in vivo. C_LI What New Information Does This Article Contribute?O_LILamin-targeted proximity proteomics specifically in cardiomyocytes uncovers novel proteins undergoing subcellular localization changes upon Lmna deficiency. C_LIO_LILmna deficiency leads to EPHX2 nuclear translocation that ameliorates cardiac dysfunction by both mechanisms of cytoplasmic reduction and nuclear induction. C_LIO_LIAAV-HDR-mediated knock-in provides a robust platform to manipulate subcellular localization of endogenous proteins specifically in cardiomyocytes in vivo. C_LI Cardiovascular diseases are often associated with altered protein subcellular localization, but systemic approaches to identify, study and manipulate subcellular localization remain incomplete. This study established an in vivo proximity proteomics approach to identify novel proteins undergoing localization changes relative to the nuclear lamina. In murine models of LMNA-related cardiomyopathy, this approach uncovered EPHX2, a classic cytoplasmic hydrolase that aggravates cardiovascular diseases, as a new protein that translocated into cell nucleus and exerted a cardiac protective effect. CRISPR/Cas9-based cardiomyocyte gene editing with an AAV-based DNA donor efficiently achieved NLS knock-in into the Ephx2 gene, promoted EPHX2 protein nuclear translocation and mitigated cardiac dysfunction with Lmna deficiency. These findings indicated a novel avenue to identify and manipulate the subcellular localization changes of endogenous proteins for basic and translational cardiology.

pathology↗

HGS+ enlarged vesicular compartment serves as site for coronavirus assembly at later infection stage

The precise site of coronavirus assembly remains poorly characterized. In this study, we observed that coronavirus infection induces the host factor hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) to form distinct HGS enlarged vesicular compartments at later infection stage. Confocal and live-cell super-resolution microscopy showed that viral structural proteins colocalize with these HGS enlarged vesicular compartments. Correspondingly, APEX2-based electron microscopy (APEX2-EM) and immuno-EM analyses confirmed the presence of assembled virions within these unique HGS compartments, identifying them as sites of virion assembly. This was further supported by cryogenic correlated light and electron microscopy (cryo-CLEM), which captured ongoing virion formation occurring within HGS enlarged vesicles. Crucially, whole cell volume EM revealed that HGS deficiency abolishes these vesicular compartments and markedly reduces assembled virions. Lastly, we demonstrated that HGS vesicular compartments are rearranged from Golgi and endosome/lysosome by coronavirus infection. Together, these findings establish that coronavirus-induced HGS vesicular compartments function as essential platforms for virion assembly at later infection stage.

microbiology↗

Targeting the host factor HGS-viral membrane protein interaction in coronavirus infection

While current antivirals primarily target viral proteins, host-directed strategies remain underexplored. Here, we performed a genome-wide CRISPRi screening to identify the host protein, Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate (HGS), as essential for the pan-coronaviruses infection both in vitro and in vivo. Mechanistically, HGS directly interacts with the viral membrane (M) protein, facilitating its trafficking to the ER-Golgi intermediate compartment (ERGIC) for virion assembly. Conversely, HGS deficiency caused M retention in the ER, blocking assembly. Leveraging this interaction, we designed M-derived peptides and screened over 5,000 FDA-approved drugs, identifying riboflavin tetrabutyrate (RTB). Both the peptides and RTB bind HGS and disrupt its interaction with the M protein, leading to M retention in the ER and subsequent blockade of virion assembly. These agents demonstrated broad anti-pan-coronavirus activity in vitro and in vivo. Collectively, our findings establish HGS as a druggable host target and identify RTB as a promising broad-spectrum antiviral candidate.

microbiology↗

Measure Sodium Transport in Cells with NMR

Sodium ions (Na+) are fundamental to numerous physiological functions, such as maintaining electrolyte balance, enabling nerve impulse transmission, and facilitating muscle contraction. Dysregulation of Na+ transport across cell membranes is implicated in a range of health issues, including metabolic syndromes, neurological conditions, and cardiovascular diseases. However, current methods for assessing cellular Na+ activity often face limitations; they can be invasive or fail to capture dynamic changes. In this study, we introduce a non-invasive 23Na nuclear magnetic resonance (NMR) methodology designed to directly quantify the transport rate of sodium ions in living cells. Our technique integrates relaxation exchange spectroscopy (REXSY) with a multi-site exchange model, enabling the investigation of Na+ transport dynamics on a timescale of sub-seconds. A key advantage is its ability to differentiate between intracellular and extracellular Na+ pools based on the endogenous NMR relaxation difference, thereby avoiding the need for potentially disruptive exogenous reagents. Experiments conducted on human cell lines successfully demonstrated the techniques capacity to distinguish between various physiological states, such as when ion channels are pharmacologically blocked or activated. The resulting measurements of Na+ transport rates and intracellular Na+ fractions show a clear correlation with cellular metabolic activity, offering valuable quantitative markers for monitoring transmembrane ion dynamics in vitro.

cell biology↗

A Fibroblast State Choreographs an Epithelial YAP-dependent Regenerative Program Essential to (Pre)malignancy via ECM-mediated Mechanotransduction

Chronic lung injury generates metaplasia which occasionally, but ominously, progresses to squamous dysplasia and squamous lung cancer. To identify mechanisms through which disrupted tissue homeostasis contributes to malignant initiation and progression, we used in vivo and in vitro heterotypic recombinant models of human bronchial epithelial cells (hBECs) and fibroblasts. We demonstrate that injury-associated TGF-{beta} signaling creates a fibroblast state dependent upon HSP47 upregulation. These fibroblasts accumulated collagen, thus elevating tissue stiffness and activating mechanosignaling that sustained YAP-dependent embryonic-like, pro-malignant activities in adjacent hBECs. This Stress/Tension-Instructive Fibroblast (STIF) state, exhibited by stressed fibroblasts in premalignant and malignant lesions across multiple cancer types, was sufficient to reprogram disease-free hBECs to metaplasia and to drive hBECs with compromised tumor suppressor function to dysplasia, yet could be inhibited and reversed. STIFs suffice to activate epithelial phenotypes reminiscent of oncogene-mediated cell transformation and induce (pre)malignancy via increased force transmission, providing novel targets for prevention. Statement of significanceTissue injury creates a regenerative pro-tumorigenic Stress/Tension-Instructive Fibroblast (STIF) state which is sufficient to activate a YAP-dependent, pre-malignant program to induce or unmask pre-cancerous phenotypes in epithelial cells through mechanotransduction. Inhibition of STIF activity or mechanosignaling prevents metaplasia and progression to dysplasia. HighlightsO_LITissue injury creates a pro-tumorigenic Stress/Tension-Instructive Fibroblast (STIF) state in multiple organs that precedes and persists through cancer C_LIO_LISTIF signaling alone, working through fibroblasts and not epithelial cells, is sufficient to activate embryonic-like plasticity and induce epithelial pre-cancerous metaplastic lesions C_LIO_LISTIFs program (pre)malignant phenotypes in adjacent epithelial cells through mechanosignaling by activating YAP prior to tumor formation C_LIO_LIInhibiting STIFs or mechanosignaling prevents/reverts metaplasia and prevents progression to dysplasia C_LI

cancer biology↗

Proteomic Analysis of Breast Cancer Subtypes Identifies Stromal Contributions that Dictate Aggressive Malignant Behavior

Breast cancer manifests as multiple subtypes with distinct patient outcomes and treatment strategies. Here, we optimized proteomic analysis of Formalin-Fixed Paraffin-Embedded (FFPE) specimens from patients diagnosed with five breast cancer subtypes, luminal A, luminal B, Her2, triple negative (TNBC) and metaplastic breast cancers (MBC), and from disease-free individuals undergoing reduction mammoplasty (RM). We identified and quantified [~]6,000 protein groups (with >2 peptides per protein) with significant changes in over 26% of proteins comparing each cancer subtype with control RM. Stringent statistical filters allowed us to deeply mine 576 significant conserved protein changes shared by all subtypes and protein changes unique to each subtype. The most aggressive subtype, MBC, revealed exacerbated stromal stress responses, as illustrated by a collagenolytic extracellular matrix (ECM) and immune participation biased towards neutrophils and eosinophils. Immunostaining of breast tissue sections confirmed differences across subtypes, in particular, a strong upregulation of SERPINH1, neutrophil-specific myeloperoxidase and eosinophil cationic protein in MBC. In summary, we present deep proteomic, digitalized protein abundance profiles, generated from FFPE breast cancer tissues, that revealed significant changes in ECM and cellular proteins. Statement of Significance of the StudyThis study is significant as it discovered deep proteomic signatures for the highly aggressive and malignant metaplastic breast cancer (MCB) which is now considered a fifth subtype based upon its remarkable intra-tumoral heterogeneity that illustrates its unique cell plasticity. To efficiently analyze formalin-fixed paraffin-embedded (FFPE) breast tissues from patients with different breast cancer subtypes and disease-free individuals, we optimized a novel workflow in which we combined paraffinization and Folch extraction. We identified and confidently quantified [~]6,000 protein groups. We were able to find robust changes in extracellular matrix (ECM) with cancer, even though no ECM enrichments were performed. Interestingly, despite the relatively small human cohort size (42 patients), distinct protein signatures emerged throughout all cancer subtypes - common and unique - with remarkable statistical significance for many cancer-relevant proteins and pathways. This Pilot study indicates the hypothesis that an altered stroma can dictate epithelial tumor cell fate. We also observed that MBC was characterized by an especially immunosuppressed tumor environment. We do note the limitation of the relatively small cohort size of our study, and in the future additional patient cohorts will be needed to further validate our findings.

cancer biology↗

Optimization of gene knockout approaches and practical solutions to sgRNA selection challenges in hPSCs with inducible Cas9 system

RationaleCRISPR/Cas9 has been extensively used to knock out genes, allowing the study of genetic loss-of-function in human pluripotent stem cells (hPSCs). However, the current use of the Cas9-sgRNA plasmid or iCas9 system for gene editing in hPSCs has resulted in limited and inconsistent editing efficiency, as well as labor-intensive work. Additionally, identifying single-guide RNAs (sgRNAs) with high cleavage efficiency and distinguishing them from ineffective ones, which efficiently induce frameshift INDELs (Indels and Deletions) but fail to eliminate target proteins expression, are major challenges in gene knockout experiments. MethodsThis study addresses above issues using an optimized doxycycline-induced spCas9-expressing hPSCs (hPSCs-iCas9) system. We initially developed this system by optimizing a number of parameters to maximize INDELs introducing efficiency in hPSCs-iCas9 cells. The INDELs determined by this system were then compared to predicted scores from three cleavage efficiency scoring algorithms to validate the algorithms accuracy and consistency. Furthermore, we conducted gene knockout using a set of sgRNAs targeting different exons of the ACE2 gene to achieve approximately 80% INDELs for each targeting locus. Western blotting was then performed to detect ACE2 protein expression levels, enabling the identification of potentially ineffective sgRNAs. ResultsSeveral critical factors, including cell tolerance to nucleofection stress, sgRNA stability, nucleofection frequency, and the cell-to-sgRNA ratio, were found to have significant impact on editing efficiency in hPSCs-iCas9. Fine-tuning these parameters markedly improved this efficiency, resulting in up to 93% INDELs for single gene knockout. The three scoring algorithms exhibited significant differences or even conflicts in scoring cleavage efficiency. Through comparing experimental observations to predicted scores, we discovered that the Benchling algorithm outperformed the other two in terms of accuracy and consistency. Furthermore, a sgRNA targeting exon 2 of ACE2 gene was quickly identified as ineffective, as evidenced by the edited cells pool containing 80% INDELs while ACE2 protein expression retained unchanged detected by Western blot. ConclusionThe findings of this study offer valuable insights into the optimal design of gene knockout experiments in hPSCs and provide practical solutions to sgRNA selection challenges for gene editing.

cell biology↗

GABA-ergic inhibition in human MT predicts visuo-spatial intelligence mediated by reverberation with frontal cortex

The prevailing opinion emphasizes fronto-parietal network (FPN) is key in mediating general fluid intelligence (gF). Meanwhile, recent studies show that human MT complex (hMT+), located at the occipito-temporal border and involved in 3D perception processing, also plays a key role in gF. However, the underlying mechanism is not clear, yet. To investigate this issue, our study targets visuo-spatial intelligence, which is considered to have high loading on gF. We use ultra-high field magnetic resonance spectroscopy (MRS) to measure GABA/glutamate concentrations in hMT+ combining resting-state fMRI functional connectivity (FC), behavioral examinations including hMT+ perception suppression test and gF subtest in visuo-spatial component. Our findings show that both GABA in hMT+ and frontal-hMT+ functional connectivity significantly correlate with the performance of visuo-spatial intelligence. Further, serial mediation model demonstrates that the effect of hMT+ GABA on visuo-spatial gF is fully mediated by the hMT+ frontal FC. Together our findings highlight the importance in integrating sensory and frontal cortices in mediating the visuospatial component of general fluid intelligence.

neuroscience↗

Human disturbance increases spatiotemporal associations among mountain forest terrestrial mammal species

Spatial and temporal associations between sympatric species underpin biotic interactions, structure ecological assemblages, and sustain ecosystem functioning and stability. However, the resilience of interspecific spatiotemporal associations to human activity remains poorly understood, particularly in mountain forests where anthropogenic impacts are often pervasive. Here we applied context-dependent Joint Species Distribution Models to a systematic camera-trap survey dataset from a global biodiversity hotspot in eastern Himalaya to understand how prominent human activities in mountain forests influence species associations within terrestrial mammal communities. We obtained 10,388 independent detections of 17 focal species (12 carnivores and five ungulates) from 322 stations over 43,163 camera days of effort. We identified a higher incidence of positive associations in habitats with higher levels of human modification (87 %) and human presence (83 %) compared to those located in habitats with lower human modification (64 %) and human presence (65 %) levels. We also detected a significant reduction of pairwise encounter time at increasing levels of human disturbance, corresponding to more frequent encounters between pairs of species. Our findings indicate that human activities can push mammals together into more frequent encounters and associations, which likely influences the coexistence and persistence of wildlife, with potential far-ranging ecological consequences.

ecology↗

Expression and molecular regulation of key genes during adipogenesis of neural crest stem cells

Neural crest stem cells (NCSCs) are pluripotent stem cells derived from the "fourth germ layer". Similar to mesenchymal stem cells (MSCs), NCSCs can differentiate into a variety of cell types, such as adipocytes. However, the mechanism of NCSCs adipogenesis remains unclear. Previously, we have revealed that primary cells have neural crest and stem cell properties and can differentiate into adipocytes. Therefore, in this study, the differentially expressed genes (DEGs) of NCSCs at specific time points of adipogenesis were predicted by mRNA sequencing, the key genes of adipogenesis were predicted by principal component analysis, heat map, GO and KEGG enrichment analysis, and the expression of DEGs was verified by RT-qPCR. RNA interference (RNAi) technology was used to inhibit the expression of DEGs, and RT-qPCR and Western blot were used to explore the regulatory mechanism between DEGs. Sequencing results indicated a possible regulatory relationship between C/EBP, C/EBP{beta}, C/EBP{delta} and PPAR{gamma}. The results of RT-qPCR were consistent with those of mRNA sequencing. Combined with RT-qPCR and western blot results, we found that C/EBP{beta} and PPAR{gamma} regulated the transcription of C/EBP during NCSCs adipogenesis, and C/EBP{beta} and PPAR{gamma} formed positive feedback loop.

developmental biology↗

Vascular-water-exchange MRI (VEXI) enables the detection of subtle BBB breakdown in Alzheimer's disease without MRI contrast agent

Blood-brain barrier (BBB) impairment is an important pathophysiological process in Alzheimers disease (AD) and a potential biomarker for early diagnosis of AD. However, most current neuroimaging methods assessing BBB function need the injection of exogenous contrast agents (or tracers), which limits the application of these methods in a large population. In this study, we aim to explore the feasibility of vascular water exchange MRI (VEXI), a diffusion-MRI-based method to assess the BBB permeability to water molecules without using a contrast agent, in the detection of the BBB breakdown in AD. We tested VEXI on a 3T MRI scanner on three groups: AD patients (AD group), mild cognitive impairment (MCI) patients due to AD (MCI group), and the age-matched normal cognition subjects (NC group). Interestingly, we find that VEXI can detect the BBB permeability to water molecules increase in MCI and this BBB breakdown happens specifically in the hippocampus. This BBB breakdown gets worse and extends to more brain regions (orbital frontal cortex and thalamus) from MCI group to the AD group. Furthermore, we find that the BBB breakdown of these three regions detected by VEXI is correlated significantly with impairment of respective cognitive domains independent of age, sex and education. These results suggest VEXI is a promising method to assess the BBB breakdown in AD. HighlightsO_LIThe vascular water exchange MRI (VEXI) is a contrast-agent-free method to assess BBB permeability C_LIO_LIBBB breakdown happens specifically in the hippocampus, orbital frontal cortex, and thalamus in AD C_LIO_LIBBB breakdown detected by VEXI is significantly correlated with cognitive dysfunction C_LI

neuroscience↗

Data-Independent Acquisition and Quantification of Extracellular Matrix from Human Lung in Chronic Inflammation-Associated Carcinomas

Early events associated with chronic inflammation and cancer involve significant remodeling of the extracellular matrix (ECM), which greatly affects its composition and functional properties. Using lung squamous cell carcinoma (LSCC), a chronic inflammation-associated cancer (CIAC), we optimized a robust proteomic pipeline to discover potential biomarker signatures and protein changes specifically in the stroma. We combined ECM enrichment from fresh human tissues, data-independent acquisition strategies, and stringent statistical processing to analyze Tumor and matched adjacent histologically normal ( Matched Normal) tissues from patients with LSCC. Overall, 1,802 protein groups were quantified with at least two unique peptides, and 56% of those proteins were annotated as extracellular. Confirming dramatic ECM remodeling during CIAC progression, 529 proteins were significantly altered in the Tumor compared to Matched Normal tissues. The signature was typified by a coordinated loss of basement membrane proteins and small leucine-rich proteins. The dramatic increase in the stromal levels of SERPINH1/heat shock protein 47, that was discovered using our ECM proteomic pipeline, was validated by immunohistochemistry (IHC) of Tumor and Matched Normal tissues, obtained from an independent cohort of LSCC patients. This integrated workflow provided novel insights into ECM remodeling during CIAC progression, and identified potential biomarker signatures and future therapeutic targets. Statement of significance of the studyThe extracellular matrix (ECM) is a complex scaffolding network composed of glycoproteins, proteoglycans and collagens, which binds soluble factors and, most importantly, significantly impacts cell fate and function. Alterations of ECM homeostasis create a microenvironment promoting tumor formation and progression, therefore deciphering molecular details of aberrant ECM remodeling is essential. Here, we present a multi-laboratory and refined proteomic workflow, featuring i) the prospective collection of tumor and matched histologically normal tissues from patients with lung squamous cell carcinoma, ii) the enrichment for ECM proteins, and iii) subsequent label-free data-independent acquisition (DIA)-based quantification. DIA is a powerful strategy to comprehensively profile and quantify all detectable precursor ions contained in the biological samples, with high quantification accuracy and reproducibility. When combined with very stringent statistical cutoffs, this unbiased strategy succeeded in capturing robust and highly confident proteins changes associated with cancer, despite biological variability between individuals. This label-free quantification workflow provided the flexibility required for ongoing prospective studies. Discussions with clinicians, surgeons, pathologists, and cancer biologists represent an opportunity to interrogate the DIA digitalized maps of the samples for newly formulated questions and hypotheses, thus gaining insights into the continuum of the disease and opening the path to novel ECM-targeted therapies.

cancer biology↗

Thinking like a structural biologist: A pocket-based 3D molecule generative model fueled by electron density

We report for the first time the use of experimental electron density (ED) as training data for the generation of drug-like three-dimensional molecules based on the structure of a target protein pocket. Similar to a structural biologist building molecules based on their ED, our model functions with two main components: a generative adversarial network (GAN) to generate the ligand ED in the input pocket and an ED interpretation module for molecule generation. The model was tested on three targets including kinase (HPK1), protease (Covid19-3CL), and nuclear receptor (VDR), and evaluated with a reference dataset composed of over 8,000 compounds that have their activities reported in the literature. The evaluation examined the chemical validity, chemical space distribution-based diversity, and similarity with reference active compounds concerning the molecular structure and pocket-binding mode. Our model can reproduce classical active compounds and can also generate novel molecules with similar binding modes as active compounds, making it a promising tool for library generation supporting high-throughput virtual screening. Our model is available as an online service to academic users via https://edmg.stonewise.cn/#/create.

bioinformatics↗

Evidence for oligodendrocyte progenitor cell heterogeneity in the adult mouse brain.

White matter loss has been described as a common occurrence in Alzheimers disease (AD) patients for multiple decades. However, it remains unclear why oligodendrocyte progenitor cells (OPCs) fail to repair myelin deficits in these patients. Here, we show that clusterin, a risk factor for late-onset AD, is produced by OPCs and inhibits their differentiation into oligodendrocytes. Specifically, we demonstrate that a unique subset of OPCs produces clusterin. We show that phagocytosis of debris, including amyloid beta (A{beta}) and myelin, drives the upregulation of clusterin in OPCs. We confirm, in vivo, that A{beta} oligomers drive clusterin upregulation and that OPCs phagocytose A{beta}. Furthermore, we show that clusterin is a potent inhibitor of OPC differentiation and prevents the production of myelin proteins. Finally, we demonstrate that clusterin inhibits OPC differentiation by significantly reducing the production of IL-9 by OPCs. Our data reveals that clusterin may be responsible for the lack of myelin repair observed in AD and is a promising therapeutic target for AD-associated cognitive decline.

neuroscience↗

Magnetic Resonance measurements of sub-cellular membrane structures in live and fixed neural tissue

We develop magnetic resonance (MR) methods for measuring real-time changes of tissue microstructure and membrane permeability of live and fixed neural tissue. Diffusion and exchange MR measurements are performed using the large static gradient produced by a single-sided permanent magnet. Using tissue delipidation methods, we show that water diffusion is restricted solely by lipid membranes. Most of the diffusion signal can be assigned to water in tissue which is far from membranes. The remaining 25% can be assigned to water restricted on length scales of roughly a micron or less, near or within membrane structures at the cellular, organelle, and vesicle levels. Diffusion exchange spectroscopy measures water exchanging between membrane structures and free environments at 100 s-1.

biophysics↗

Extensive adaptive immune response of AAVs and Cas proteins in non-human primates

The CRISPR-mediated Cas system is the most widely used tool in gene editing and gene therapy for its convenience and efficiency. Delivery of the CRISPR system by adeno-associated viruses (AAVs) is currently the most promising approach to gene therapy. However, pre-existing adaptive immune responses against CRISPR nuclease (PAIR-C) and AAVs has been found in human serum, indicating that immune response is a problem that cannot be ignored, especially for in vivo gene correction. Non-human primates (NHPs) share many genetic and physiological traits with human, and are considered as the bridge for translational medicine. However, whether NHPs have same PAIR-C status with human is still unknown. Here, macaques (rhesus and cynomolgus), including normal housed and CRISPR-SpCas9 or TALENs edited individuals, were used to detect PAIR-C which covered SaCas9, SpCas9, AsCas12a and LbCas12a. Dogs and mice were also detected to expand the range of species. In addition, pre-existing adaptive antibodies to AAV8 and AAV9 were performed against macaques of different ages. The results showed that adaptive immunity was pre-existing in the macaques regardless of Cas proteins and AAVs. These findings indicate that the pre-existing adaptive immune of AAV-delivered CRISPR construction and correction system should be concerned for in vivo experiments.

immunology↗