Biology subjects

Chen, G.

Publications and source records attributed to Chen, G..

At least 127 records · Page 7Linked to original sources

bioRxiv2024

De novo identification of protein domains in cryo-electron tomography maps from AlphaFold2 models

In situ cryo-electron tomography (cryo-ET) enables visualization of protein complexes within their native cellular environments. However, sub-tomogram averaging (STA) often fails to achieve resolutions sufficient for atomic model building based on side-chain information, hindering accurate protein identification and assignment. To address this challenge, we developed DomainSeeker, a computational workflow that integrates AlphaFold2-predicted structures with experimental data to identify protein domains in cryo-ET density maps. DomainSeeker partitions AlphaFold2-predicted structures into domains using clique analysis based on predicted aligned errors and then fits these domains into target densities segmented from input cryo-ET maps. Each domain-density fit is evaluated globally and locally to derive prior probabilities, which are subsequently integrated with complementary data, such as cross-linking mass spectrometry (XL-MS), through Bayesian inference to compute posterior probabilities. Applying DomainSeekers prior probability analysis to densities from mouse sperm microtubule doublets revealed substantially higher recognition accuracy and resolution robustness compared with existing approaches. Incorporating XL-MS data for yeast nuclear pore complex densities further enhanced both recognition performance and confidence. Finally, we implemented an interactive ChimeraX plugin to facilitate user access. Together, DomainSeeker overcomes a key bottleneck in in situ structural biology, providing a robust and accessible tool for visual proteomics to uncover the structural and functional organization of native cellular assemblies.

biophysics↗

bioRxiv2024

A novel and simple maneuver greatly improves rat liver transplantation

BackgroundOrthotopic rat liver transplantation (OLT) is preferentially used to study transplant immunology; it remains challenging due to higher complications associated with 26-minute anhepatic time ceiling, here we safely extended the anhepatic time to improve success of OLT. MethodsInitially we performed OLT including whole graft OLT from inbred Sprague Dawley (SD) rat to SD (whole graft group, control group, n=21) and 30 minute anhepatic time (AHT) group (AHT group, n=11) to master this procedure. For generalization of this maneuver, partial OLT was performed from 50% Lewis allograft to Brown Norway (BN) rat to induce tolerance (half graft group, n=28), Cyclosporine A was injected once daily for 10 days in this group. ResultsFor whole graft group, 30-day survival rate was 85.5% (18/21), the reasons of death were gas embolism due to the missed suturing in 2 cases, blood loss in 1 case. For AHT group, 30-day survival rate was 72.7% (8/11), one recipient died from respiratory failure intra-operatively; two deaths were biliary leakage in 14 days. There were no differences in survival between whole graft group and AHT group (p=0.368). For tolerance group, 30-day survival rate was 82.0% (11/61), and the causes of death were diverse and recorded. ConclusionThe anhepatic time can be extended to 30 minutes simply through the change of clamping the diaphragm, which facilitates LT and improves its success good for the basic research.

systems biology↗

bioRxiv2024

Anti-HIV-1 HSPC-based gene therapy with safety kill switch to defend against and attack HIV-1 infection

Hematopoietic stem/progenitor cell (HSPC)-based anti-HIV-1 gene therapy holds promise to provide life-long remission following a single treatment. Here we report a multi-pronged anti-HIV-1 HSPC-based gene therapy designed to defend against and attack HIV-1 infection. We developed a lentiviral vector capable of co-expressing three anti-HIV-1 genes. Two are designed to prevent infection, including a short-hairpin RNA (CCR5sh1005) to knock down HIV-1 co-receptor CCR5 and a membrane anchored HIV-1 fusion inhibitor (C46). The third gene is a CD4-based chimeric antigen receptor (CAR) designed to attack HIV-1 infected cells. Our vector also includes a non-signaling truncated human epidermal growth factor receptor (huEGFRt) which acts as a negative selection-based safety kill switch against transduced cells. Anti-HIV-1 vector-transduced human CD34+ HSPC efficiently reconstituted multi-lineage human hematopoietic cells in humanized bone marrow/liver/thymus (huBLT) mice. HIV-1 viral load was significantly reduced (1-log fold reduction, p <0.001) in transplanted huBLT mice. Anti-huEGFR monoclonal antibody Cetuximab (CTX) administration significantly reduced huEGFRt+ vector-modified cells (>4-fold reduction, p <0.01) in huBLT mice. These results demonstrate that our strategy is highly effective for HIV-1 inhibition, and that CTX-mediated negative selection can deplete anti-HIV-1 vector-modified cells in the event of unwanted adverse effects in huBLT mice.

microbiology↗

bioRxiv2024

NAD+ - and EVA1-C-dependent reversal of neurological deficits is mediated by differential alternative RNA splicing in tauopathic animal models

Aberrant alternative splicing (ASEs) is an aging hallmark to Alzheimers Disease (AD). Although NAD+ and related metabolites can slow down AD, NAD+ on ASEs in AD remain unclear. Mouse transcriptomic data revealed NR-induced ASEs, focusing on the Eva1-C locus. AI-based algorithms predicted EVA1-C protein structures and protein-protein interactions. AD postmortem brain samples and tauopathy models including transgenic mice and worm was used for validation. NAD+ abundance/metabolic status modulates ASEs and the expression of EVA1-C isoforms, which in turn regulate the interaction with BAG-1 and HSP70 proteins. Importantly, EVA1-C is dramatically reduced in 20 Braak 5/6 AD patients compared to cognitive normal humans in different brain regions. NAD+ metabolism modulates abundance of specific mRNA isoforms, and that ASEs influence disease progression in model tauopathies and potentially AD. These results could facilitate future development of NAD+-based splice-switching therapeutics for AD. TeaserUnveiling the Link Between NAD+ Metabolism and Alzheimers Disease: Discovering the Role of Alternative RNA Splicing in Disease Progression and Potential Therapeutic Targets

neuroscience↗

bioRxiv2024

Abnormalities of the liver have big impacts on orthotopic liver transplantation

BackgroundOrthotopic rat liver transplantation (OLT) is widely used in basic research; normal liver anatomy and structures are attributable to its success while its deformities complicate to have a negative on OLT. MethodsFor tolerance induction project, we performed OLT from Lewis to Brown Norway (BN) rat as chronic rejection model and encountered two anatomical deformities of recipient rats: of 47 liver transplantations, accessory liver lobe occurred to 4 cases and bifurcations of liver outflow to 5 cases in BN rats. ResultsFor the accessory liver lobe, we discontinued OLT for one case with a big accessory liver lobe; two rats died from pneumothorax upon separation; and succeed in one case with the small lobe. For two vein outflows of the liver, we succeeded in OLT due to its reconstruction in one case but the recipient died one week later, and succeeded in 1 case after one small orifice was sutured, we failed in 3 cases due to thrombosis following OLT. For 38 rats with normal livers, only 4 rats failed to survive LT. There were significant differences in OLT success (p<0.01) ConclusionLiver abnormal anatomy has negative impact on OLT, giving a clue to that pre-transplant comprehensive screening is significant and beneficial clinically.

physiology↗

bioRxiv2024

Kinesin Autoinhibition Requires Elbow Phosphorylation

Kinesin motor proteins facilitate microtubule-based transport by converting chemical energy into mechanic forces, but this activity is autoinhibited until cargo is loaded. Regulatory mechanisms underlying this autoinhibitory conformation are not well understood. Here, we show that a NEver in mitosis Kinase NEKL-3 directly phosphorylates a flexible elbow region between two coiled coil domains connecting the motor head and tail of an intraflagellar transport kinesin, OSM-3. The phosphor dead (PD) mutation, but not phosphor mimic (PM) mutation, induce constitutive motility of OSM-3 in vitro. Using knock-in animals, we discovered that both PD and PM mutations shorten the C. elegans sensory cilia. The constitutively active OSM-3PD fails to enter cilia and abnormally accumulates in neurites, mimicking another hyperactive mutation, OSM-3G444E. Conversely, OSM-3PM enters cilia but moves at a reduced speed, indicating an inhibitory role of elbow phosphorylation in kinesin motility. These findings highlight the crucial role of elbow phosphorylation in regulating kinesin autoinhibition.

cell biology↗

bioRxiv2024

Genomic and evolutionary evidence for drought adaptation of grass allopolyploid Brachypodium hybridum

Climate change increases the frequency and severity of drought worldwide, threatening the environmental resilience of cultivated grasses. However, the genetic diversity in many wild grasses could contribute to the development of climate-adapted varieties. Here, we elucidated the impact of polyploidy on drought response using allotetraploid Brachypodium hybridum (Bh) and its progenitor diploid species B. stacei (Bs). Our findings suggest that progenitor species genomic legacies resulting from hybridization and whole-genome duplications conferred greater ecological adaptive advantages to Bh over Bs. Genes related to stomatal regulation and immune response from S-subgenomes were under positive selection during speciation, underscoring their evolutionary importance in adapting to environmental stresses. Biased expression in polyploid subgenomes [B. stacei-type (Bhs) and B. distachyon-type (Bhd)] significantly influenced differential gene expression, with the dominant subgenome exhibiting more differential expression. B. hybridum adapted a drought escape strategy characterized by higher photosynthetic capacity and lower WUEi than Bs, driven by a highly correlated co-expression network involving genes in the circadian rhythm pathway. In summary, our study showed the influence of polyploidy on ecological and environmental adaptation and resilience in model Brachypodium grasses. These insights hold promise for informing the breeding of climate-resilient cereal crops and pasture grasses.

evolutionary biology↗

bioRxiv2024

A new stegosaur (Dinosauria: Ornithischia) from the Upper Jurassic Qigu Formation of Xinjiang, China and a revision on Chinese stegosaurs phylogeny

Stegosaurs are a small but iconic clade of ornithischian dinosaurs. They and their sister taxa, the ankylosaurs, formed the clade Eurypoda which means broad-footed. Here, we describe a stegosaur from the Upper Jurassic Qigu Formation of Xinjiang, China, based on an associated partial skeleton that includes axial, pectoral girdle, pelvic girdle, limb and armor elements. It can be diagnosed as a new taxon, Angustungui, based on numerous autapomorphies. Some morphologies of Angustungui are more similar to the taxa from Europe, Africa and North America than to those from Asia. Our phylogenetic analysis recovers it as the sister taxon of Loricatosaurus. More importantly, the narrow and claw-shaped ungual of Angustungui proves that Eurypoda, at least stegosaur, has claw-shaped unguals. Besides, we revised the character scores for Chinese stegosaurs based on observations of the specimens.

paleontology↗

bioRxiv2024

GABAergic neuron dysregulation in a human neurodevelopmental model for major psychiatric disorders

"GABA dysfunction" is a major hypothesis for the biological basis of schizophrenia with indirect supporting evidence from human post-mortem brain and genetic studies. Patient-derived induced pluripotent stem cells (iPSCs) have emerged as a valuable platform for modeling psychiatric disorders, and previous modeling has revealed glutamatergic synapse deficits. Whether GABAergic synapse properties are affected in patient-derived human neurons and how this impacts neuronal network activity remain poorly understood. Here we optimized a protocol to differentiate iPSCs into highly enriched ganglionic eminence-like neural progenitors and GABAergic neurons. Using a collection of iPSCs derived from patients of psychiatric disorders carrying a Disrupted-in-Schizophrenia 1 (DISC1) mutation and their unaffected family member, together with respective isogenic lines, we identified mutation-dependent deficits in GABAergic synapse formation and function, a phenotype similar to that of mutant glutamatergic neurons. However, mutant glutamatergic and GABAergic neurons contribute differentially to neuronal network excitability and synchrony deficits. Finally, we showed that GABAergic synaptic transmission is also defective in neurons derived from several idiopathic schizophrenia patient iPSCs. Transcriptome analysis further showed some shared gene expression dysregulation, which is more prominent in DISC1 mutant neurons. Together, our study supports a functional GABAergic synaptic deficit in major psychiatric disorders.

neuroscience↗

bioRxiv2024

Antibodies against the capsid induced after intracranial AAV administration limits second administration in a dose dependent manner

Recombinant adeno-associated virus (rAAV) is a widely used viral vector for gene therapy. However, a limitation of AAV-mediated gene therapy is that patients are typically dosed only once. In this study, we investigated the possiblility to deliver multiple rounds of AAV through intracerebral injections in the mouse brain. We discovered a dose-dependent modulation of the second round AAV infection by the first round AAV injection in the brain-wide scales besides the injection region. High-dose AAV infection increases chemokines CXCL9 and CXCL10 to recruit the parenchymal infiltration of lymphocytes. Surprisingly, the blood-brain-barrier was relatively intact. Brain-wide dissection discovered the likely rountes of the infiltrated lymphocytes through perivascular space and ventricles. Further analysis using B-cell depleted mice revealed that B lymphocytes, but not T lymphocytes, played a critical role in inhibiting the second round AAV infection. Strategies against neutralizing antibodies had limited effects, while reducing the dosage for the first injection or switching the second AAV to a different serotype appeared to be more effective in antagonizing the first round AAV inhibition. Together, these results suggest that mammalian brains are not immunoprivileged for AAV infection, but multiple rounds of AAV gene therapy are still possible if designed carefully with proper doses and serotypes.

immunology↗

bioRxiv2024

Engineering a Cell-Based Orthogonal Ubiquitin Transfer Cascade for Profiling the Substrates of RBR E3 Parkin

The E3 ubiquitin (UB) ligase Parkin utilizes a Ring-Between-Ring (RBR) domain to mediate the transfer of UB to its substrates to regulate diverse cellular functions, including mitochondrial quality control, cell cycle progression, metabolism programming, and the establishment of synaptic functions. Mutations affecting the E3 ligase activity of Parkin are associated with cancer and Parkinsons disease (PD). An essential role of Parkin is to synthesize UB chains on the surface of damaged mitochondria to initiate mitophagy. Still, it is not clear how Parkin carries out other biological functions through the ubiquitination of its downstream targets in the cell. We hypothesized that a comprehensive substrate profile of Parkin would facilitate the discovery of ubiquitination pathways underpinning its multifaceted roles in cell regulation and reveal mechanistic linkages between Parkin malfunction and disease development. Here, we used phage display to assemble an orthogonal ubiquitin transfer (OUT) cascade of Parkin that can exclusively deliver an engineered UB mutant (xUB) to Parkin and its substrates in living cells. We then generated a substrate profile of Parkin by purifying xUB-conjugated proteins from cells and identifying them by proteomics. The OUT screen identified Parkin substrates involved in DNA replication, protein translation, intracellular protein transport, and rhythmic regulation. Based on previous literature implicating alterations in membrane vesicle trafficking in PD, we verified Parkin-catalyzed ubiquitination of Rab GTPases (Rab1a, Rab5a, Rab5c, Rab7a, Rab8a, Rab10, an Rab13) as well as CDK5, with reconstituted ubiquitination reactions in vitro and in cells. We also found chemical-induced stimulation of mitophagy enhanced Parkin-mediated ubiquitination of Rab proteins. These findings demonstrate that the OUT cascade of Parkin can serve as an empowering tool for identifying Parkin substrates to elucidate its cellular functions.

biochemistry↗

bioRxiv2024

A 15-Gene prognostic signature with TFAP2B functioning in Platinum Resistance of Ovarian Carcinoma

Resistance to platinum is the main challenge in the chemotherapy of ovarian cancer (OV). Therefore, developing a response signature to platinum is essential for the precision therapy of OV. Existing quantitative signatures of platinum are susceptible to batch effects and sequencing platform variations. To address this, we developed a transcriptome-based platinum signature, named PRSM, consisting of 15 genes, based on within-sample prognostic and relative expression ordering of genes, to predict individual responses to platinum in OV. The PRSM model demonstrated superior classification accuracy compared to previous quantitative signatures. Resistant samples classified by PRSM exhibited poorer overall survival, lower SNV neoantigen load, tumor mutational burden, and distinct methylation patterns compared to sensitive samples. Pathway analysis revealed the activation of MYC targets V2 and oxidative phosphorylation in resistant tumors. Single-cell analysis highlighted the roles of NK and epithelial cells in resistance. Among the 15 core genes, five (TFAP2B, KRT81, PAGE1, CRNN, UGT2B17) were linked to poor prognosis, with TFAP2B having the highest contribution to PRSM. Overexpression of TFAP2B in A2780 cells enhanced cisplatin sensitivity, while in A2780cis cells, it inhibited growth. In brief, our findings provide a multi-dimensional view of platinum resistance in ovarian cancer, introducing a robust predictive model and identifying potential therapeutic targets.

pharmacology and toxicology↗

bioRxiv2024

A statistical-based method for the construction and analysis of gene network：application to bacteria

Bacteria play a crucial role in environmental conservation, human health, and medicine. Whether in the gut or the soil, bacterial genomes are rich repositories of resources, such as exploring potential drugs and biopesticides. However, our ability to develop new therapies and deepen our understanding of the bacterial world is hindered by the largely unknown functions of bacterial genes. In this study, we proposed a method of gene network construction and analysis based on a Gaussian Graphical Model (GGM) and random sampling strategy to infer direct interactions at the genomic level in bacteria. Using Vibrio cholerae and Staphylococcus aureus as examples, we integrated partial correlation-based gene co-expression data with gene regulatory and essentiality information extracted from public databases to construct more comprehensive gene networks. Networks built upon bacterial different phenotypes, such as biofilm formation, flagellar assembly, and stress response, demonstrate the effectiveness of this method in deciphering unknown gene functions, uncovering new phenotype-associated factors, and identifying their corresponding interactions, thus providing new targets for experimental validation by researchers. Additionally, we extended this method to 14 bacteria, including 13 pathogens, supporting the investigation of gene functions and pathways at the genomic level in these bacteria. More importantly, for other species, this method of gene network construction can be easily implemented, provided that sufficient transcriptome sequencing samples are available.

systems biology↗

bioRxiv2024

Endogenous retroviruses activate MARCO-mediated inflammatory response to block retroviral infection

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections and can profoundly affect the host antiviral innate immune response, although the mechanisms by which these changes occur are largely unknown. Here we report that chicken-specific ERVs exert genetic resistance to exogenous retrovirus infection. Mechanistically, chicken-specific ERVs activated the scavenger receptor MARCO (macrophage receptor with collagenous structure)-mediated TLR3-IL-1{beta} inflammatory response in macrophages. Under the presence of MARCO, macrophages response to viral infection through inducing TLR3-IL-1{beta} inflammatory response. Conversely, lack of MARCO increased the viral replication levels and attenuated the antiviral inflammatory response. MARCO-mediated ligand delivery enhances TLR3-IL-1{beta} antiviral response, and IL-1{beta} expression is responsible for viral inhibition. Restoring MARCO or IL-1{beta} expression overcomes viral infection in macrophages. Our study provides new insights into the molecular mechanisms underlying the host defense against exogenous retroviruses infection and may have important implications for the development of novel therapeutic strategies against retroviruses infection.

immunology↗

bioRxiv2024

A flexible high-precision photoacoustic retinal prosthesis

Retinal degenerative diseases of photoreceptors are a leading cause of blindness with no effective treatment. Retinal prostheses aim to restore sight by stimulating residual retinal cells. Here, we present a photoacoustic retinal stimulation technology. We designed a polydimethylsiloxane and carbon-based flexible film that converts near-infrared laser pulses into a localized acoustic field with 56-{micro}m lateral resolution, allowing precise stimulation of mechanosensitive retinal cells. This photoacoustic stimulation robustly and locally modulated retinal ganglion cell activity in both wild-type and degenerated ex vivo rat retinae. In animals subretinally implanted with a millimeter-sized photoacoustic film, pulsed laser stimulation generated neural modulation along the visual pathway to the superior colliculus, as measured by functional ultrasound imaging. The biosafety of the film was confirmed by the absence of short-term adverse effects , while local thermal increases were measured below 1 {degrees}C. These findings demonstrate the potential of photoacoustic stimulation for high-acuity visual restoration in blind patients.

neuroscience↗

bioRxiv2024

Genomic signatures and demographic history of the widespread and critically endangered yellow-breasted bunting

Population declines may have long-term genetic consequences, including genetic erosion and inbreeding depression, which could affect species evolutionary potential and increase their risk of extinction. Small populations are more vulnerable to genetic threats than common species, but even species with large populations can also be at risk of extinction. The yellow-breasted bunting (Emberiza aureola) is a common and widespread songbird in the northern Palearctic regions, but its population has drastically declined by around 90% throughout the past 30 years, leading to an upgrade of its conservation status to critically endangered. In this study, we identified three populations within this species using whole-genome resequencing data, but the genetic differentiation between populations was shallow. These populations underwent similar population fluctuations but differed in the extent of population decline, resulting in lower genetic diversity and more homozygous deleterious mutations in a population comprising individuals on islands. The ancient demographic history was mainly associated with the climate, while recent population declines are likely due to human activities. Our results suggest that the yellow-breasted bunting population before the recent population collapse faced relatively low genetic threats and had high evolutionary potential. However, we should be vigilant about the genetic threats faced by this species as our sampling time occurred at the onset of its recent global population collapse. This study provides valuable genetic information for appropriate conservation management of the yellow-breasted bunting and sheds light on the extinction risks and genetic consequences common species face.

genomics↗

bioRxiv2024

Dysregulated Airway Host Defense in Hyper IgE Syndrome due to STAT3 Mutations

RationaleHyper IgE syndrome (STAT3-HIES), also known as Jobs syndrome, is a rare immunodeficiency disease typically caused by dominant-negative STAT3 mutations. STAT3-HIES syndrome is characterized by chronic pulmonary infection and inflammation, suggesting impairment of pulmonary innate host defense. ObjectivesTo identify airway epithelial host defense defects consequent to STAT3 mutations that, in addition to reported mutant STAT3 immunologic abnormalities, produce pulmonary infection. MethodsSTAT3-HIES sputum was evaluated for biochemical/biophysical properties. STAT3-HIES excised lungs were harvested for histology; bronchial brush samples were collected for RNA sequencing and in vitro culture. A STAT3-HIES-specific mutation (R382W), expressed by lentiviruses, and a STAT3 knockout, generated by CRISPR/Cas9, were maintained in normal human bronchial epithelia under basal or inflammatory (IL1{beta}) conditions. Effects of STAT3 deficiency on transcriptomics, and epithelial ion channel, secretory, antimicrobial, and ciliary functions were assessed. Measurements and Main ResultsMucus concentrations and viscoelasticity were increased in STAT3-HIES sputum. STAT3-HIES excised lungs exhibited mucus obstruction and elevated IL1{beta} expression. STAT3 deficiency impaired CFTR-dependent fluid and mucin secretion, inhibited expression of antimicrobial peptides, cytokines, and chemokines, and acidified airway surface liquid at baseline and post-IL1{beta} exposure in vitro. Notably, mutant STAT3 suppressed IL1R1 expression. STAT3 mutations also inhibited ciliogenesis in vivo and impaired mucociliary transport in vitro, a process mediated via HES6 suppression. Administration of a {gamma}-secretase inhibitor increased HES6 expression and improved ciliogenesis in STAT3 R382W mutant cells. ConclusionsSTAT3 dysfunction leads to multi-component defects in airway epithelial innate defense, which, in conjunction with STAT3-HIES immune deficiency, contributes to chronic pulmonary infection.

cell biology↗

bioRxiv2024

Sleep defined by arousal threshold reveals decreases in corticocortical functional correlations independently from the conventional sleep stages

Reports of sleep-specific brain activity patterns have been constrained by assessing brain function as it related to the conventional polysomnographic sleep stages. This limits the variety of sleep states and underlying activity patterns that one can discover. The current study used all-night functional MRI sleep data and defined sleep behaviorally with auditory arousal threshold (AAT) to characterize sleep depth better by searching for novel neural markers of sleep depth that are neuroanatomically localized and temporally unrelated to the conventional stages. Functional correlation values calculated in a four-min time window immediately before the determination of AAT were entered into a linear mixed effects model, allowing multiple arousals across the night per subject into the analysis, and compared to models with sleep stage to determine the unique relationships with AAT. These unique relationships were for thalamocerebellar correlations, the relationship between the right language network and the right "default-mode network dorsal medial prefrontal cortex subsystem," and the relationship between thalamus and ventral attention network. These novel neural markers of sleep depth would have remained undiscovered if the data were merely analyzed with the conventional sleep stages. Significance StatementThe original classification of sleep into distinct stages used behavioral characteristics. With the proliferation of new techniques, the first experiments might have been to perform correlations with arousal threshold. These experiments have never been performed, either with functional MRI or with any other modern technique. The amount of communication between brain regions as measured by all-night functional magnetic resonance imaging was correlated with arousal threshold. This revealed novel neural markers of sleep depth that would have remained undiscovered if the data were merely analyzed with the conventional stages. This expands our understanding of sleep and its functions beyond the constraints imposed by the conventional stages.

neuroscience↗