Biology subjects

Cheng, A.

Publications and source records attributed to Cheng, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Bridging Gaps in Antibody Responses and Animal Welfare: Assessing Blood Collection Methods and Vaginal Immunity in Mice Immunized with Intranasal Gonococcal Vaccines

Assessing antibody titers and functional responses is essential for evaluating vaccine efficacy, yet the impact of blood collection methods on these immunological assessments remains unclear. Retro-orbital (RO) blood collection is commonly used but significant complications can occur. Increasingly, investigators have adopted alternative blood collection approaches, such as saphenous vein (SV) sampling to improve laboratory animal welfare. This study compared RO and SV sampling in the development of a Neisseria gonorrhoeae (Ng) vaccine, evaluating Adhesin Complex Protein (ACP) and multiple transferable resistance (Mtr) E protein (MtrE) as antigen candidates. Epitope mapping revealed that ACP and MtrE possess multiple, highly accessible B-cell and T-cell epitope clusters, reinforcing their immunological potential. Following intranasal immunization with rACP, rACP+CpG, and rMtrE+CpG, we assessed the specificity, magnitude, kinetics, and functional quality of immune responses elicited by the immunization regimens. Out of 45 comparisons, only eight significant differences were detected in antibody titers, while the human serum bactericidal assays revealed no differences between RO and SV in antigen-immunized groups. However, antibodies elicited by rACP alone or ACP+CpG in SV samples restored 30.05% and 75.2% of human lysozyme hydrolytic activity compared to 19.3 and 59.9 % in RO, respectively suggesting that SV sampling may be more reliable for assessing functional antibody responses. Beyond its immunological advantages, SV sampling reduces stress, minimizes ocular trauma, and improves animal welfare, making it a viable alternative to RO collection. Given its widespread use in vaccine research, standardizing SV sampling could improve data reliability, ethical compliance, and translational relevance in preclinical studies.

microbiology↗

bioRxiv2024

Annotating CryoET Volumes: A Machine Learning Challenge

Cryo-electron tomography (cryoET) has emerged as a powerful structural biology tool for understanding protein complexes in their native cellular environments. Presently, 3D volumes of cellular environments can be acquired in the thousands in a few days where each volume provides a rich and complex cellular landscape. Despite numerous innovations, localizing and identifying the vast majority of protein species in these volumes remains prohibitively difficult. Machine learning based methods provide an opportunity to automate the process of labeling and annotating cryoET volumes. Due to current bottlenecks in the annotation process, and a lack of large standardized datasets, training datasets for machine learning algorithms have been scarce. Here, we present a defined "phantom" sample, along with "ground truth" annotations, that will be the basis of a machine learning challenge to bring cryoET and ML experts together and spur creativity to address this annotation problem. We have also set up a cryoET data portal that provides additional diverse sets of annotated 3D volumes from cryoET experts across the world for the machine learning challenge.

cell biology↗

bioRxiv2024

Epigenomic analysis of hepatocellular carcinoma reveals aberrant cis-regulatory changes and dysregulated retrotransposons with prognostic potentials

Hepatocellular carcinoma (HCC) exhibits widespread epigenetic alterations, yet their impact on cis-regulatory elements (CREs) and retrotransposons remains poorly understood. Here, we present an integrated epigenomic and transcriptomic analysis of HCC tumors and matched tumor-adjacent normal tissues. We identified extensive DNA hypomethylation coupled with changes in histone modifications at partially methylated domains, CREs, and retrotransposons. These epigenetic aberrations were associated with dysregulated expression of genes involved in cell cycle regulation, immune response, and extracellular matrix organization. Notably, our findings revealed a novel mechanism for the transcriptional dysregulation of GPC3, a key HCC biomarker and immunotherapeutic target. We observed that GPC3 upregulation is driven by both the reactivation of a fetal liver super enhancer and hypomethylation of GPC3-associated CpG islands. Moreover, we found that DNA hypomethylation-driven aberrant expression of retrotransposons carries prognostic significance in HCC. Patients with high expression of a long non-coding RNA driven by a HERVE-int element exhibited more aggressive tumors, poorer clinical outcomes, and molecular features associated with favorable immunotherapy response. Together, our study provides a comprehensive resource for understanding the role of epigenetic dysregulation in HCC and identifies retrotransposon-associated transcripts as potential biomarkers.

genomics↗

bioRxiv2024

The SP140-RESIST pathway regulates interferon mRNA stability and antiviral immunity

Type I interferons (IFN-Is) are essential for antiviral immunity but must be tightly regulated1-3. The conserved transcriptional repressor SP140 inhibits interferon beta (Ifnb1) expression via an unknown mechanism4,5. Here we report that SP140 does not directly repress Ifnb1 transcription. Instead, SP140 negatively regulates Ifnb1 mRNA stability by directly repressing the expression of a previously uncharacterized regulator we call RESIST (REgulated Stimulator of Interferon via Stabilization of Transcript, previously annotated as Annexin-2 Receptor). RESIST promotes Ifnb1 mRNA stability by counteracting Ifnb1 mRNA destabilization mediated by the Tristetraprolin (TTP) family of RNA-binding proteins and the CCR4-NOT deadenylase complex. SP140 localizes within nuclear bodies, punctate structures that play important roles in silencing DNA virus gene expression in the nucleus4. Consistent with this observation, we found that SP140 inhibits replication of the gammaherpesvirus MHV68. The antiviral activity of SP140 was independent of its ability to regulate Ifnb1. Our results establish dual antiviral and interferon regulatory functions for SP140. We propose that SP140 and RESIST participate in antiviral effector-triggered immunity6,7.

immunology↗

bioRxiv2024

Targeting NOTCH2-JAG1 juxtacrine signaling reverses macrophage-mediated tumor resistance to taxol

Taxanes are widely used in chemotherapy, but primary and acquired resistance limit the clinical efficacy. Studies have shown tumor interaction with macrophages in the tumor microenvironment (TME) plays a significant role in taxane resistance, yet therapeutic strategies that directly deplete or repolarize macrophages are challenging and with considerable risk of side effects. Here we uncovered that tumor-macrophage interaction can be selectively targeted by inhibiting post-mitotic NOTCH2-JAG1 juxtacrine signaling in the TME, which strongly sensitizes paclitaxel response. Using translatome profiling, we found significant NOTCH2 upregulation during paclitaxel-induced prolonged mitosis. NOTCH2 was subsequently activated in the post-mitotic G1 phase by JAG1 expressed on the neighboring macrophages and tumor cells, which promoted tumor cell survival and upregulated cytokines that recruited JAG1-expressing macrophages, thus generating a positive feedback loop that further enhanced the pro-tumor NOTCH2 activity. By targeting this NOTCH2-JAG1 axis using NOTCH2 shRNA or a pan-NOTCH inhibitor, macrophage recruitment and paclitaxel resistance were significantly attenuated in multiple mouse tumor models of ovarian cancer. Clinical samples from paired primary and recurrent ovarian cancer patients also showed significant correlation of higher NOTCH2 expression with worse prognosis. Our results thus point to combining NOTCH2 inhibitor with taxane as an effective therapeutic strategy to selectively disrupt tumor-macrophage interaction in the TME and overcome macrophage-mediated taxane resistance in NOTCH2-positive tumors.

cancer biology↗

bioRxiv2024

Targeting complement C3a receptor resolves mitochondrial hyperfusion and subretinal microglial activation in progranulin-deficient frontotemporal dementia

Mutations in progranulin (GRN) cause frontotemporal dementia (GRN-FTD) due to deficiency of the pleiotropic protein progranulin. GRN-FTD exhibits diverse pathologies including lysosome dysfunction, lipofuscinosis, microgliosis, and neuroinflammation. Yet, how progranulin loss causes disease remains unresolved. Here, we report that non-invasive retinal imaging of GRN-FTD patients revealed deficits in photoreceptors and the retinal pigment epithelium (RPE) that correlate with cognitive decline. Likewise, Grn-/- mice exhibit early RPE dysfunction, microglial activation, and subsequent photoreceptor loss. Super-resolution live imaging and transcriptomic analyses identified RPE mitochondria as an early driver of retinal dysfunction. Loss of mitochondrial fission protein 1 (MTFP1) in Grn-/- RPE causes mitochondrial hyperfusion and bioenergetic defects, leading to NF-kB-mediated activation of complement C3a-C3a receptor signaling, which drives further mitochondrial hyperfusion and retinal inflammation. C3aR antagonism restores RPE mitochondrial integrity and limits subretinal microglial activation. Our study identifies a previously unrecognized mechanism by which progranulin modulates mitochondrial integrity and complement-mediated neuroinflammation.

neuroscience↗

bioRxiv2024

Analyzing RNA-Seq Data from Chlamydia with Super Broad Transcriptomic Activation: Challenges, Solutions, and Implications for Other Systems

MotivationRNA sequencing (RNA-Seq) offers profound insights into the complex transcriptomes of diverse biological systems. However, standard differential expression analysis pipelines based on DESeq2 and edgeR encounter challenges when applied to the immediate early transcriptomes of Chlamydia spp., obligate intracellular bacteria. These challenges arise from their reliance on assumptions that do not hold in scenarios characterized by extensive transcriptomic activation and limited repression. Standard analyses using unique chlamydial RNA-Seq reads alone identify nearly 300 upregulated and about 300 downregulated genes, significantly deviating from actual RNA-Seq read trends. ResultsBy incorporating both chlamydial and host reads or adjusting for total sequencing depth, the revised normalization methods each detected over 700 upregulated genes and 30 or fewer downregulated genes, closely aligned with observed RNA-Seq data. Further validation through qRT-PCR analysis confirmed the effectiveness of these adjusted approaches in capturing the true extent of transcriptomic activation during the immediate early phase of chlamydial infection. While the strategies employed are developed in the context of Chlamydia, the principles of flexible and context-aware normalization may inform adjustments in other systems with unbalanced gene expression dynamics, such as bacterial spore germination. Availability and implementationThe code for reproducing the presented bioinformatic analysis is available at https://zenodo.org/records/11201379.

bioinformatics↗

bioRxiv2024

Stabilizing mammalian RNA thermometer confers neuroprotection in subarachnoid hemorrhage

Mammals tightly regulate their core body temperature, yet how cells sense and respond to small temperature changes at the molecular level remains incompletely understood. Here, we discover a significant enrichment of RNA G-quadruplex (rG4) motifs around splice sites of cold-repressed exons. These thermosensing RNA structures, when stabilized, mask splice sites, reducing exon inclusion. Focusing on cold-induced neuroprotective RBM3, we demonstrate that rG4s near splice sites of a cold-repressed poison exon are stabilized at low temperatures, leading to exon exclusion. This enables evasion of nonsense-mediated decay, increasing RBM3 expression at cold. Additionally, increasing intracellular potassium concentration stabilizes rG4s and enhances RBM3 expression, leading to RBM3-dependent neuroprotection in a mouse model of subarachnoid hemorrhage. Our findings unveil a mechanism how mammalian RNAs directly sense temperature and potassium perturbations, integrating them into gene expression programs. This opens new avenues for treating diseases arising from splicing defects and disorders benefiting from therapeutic hypothermia. HighlightsO_LIrG4s are enriched near splice sites of cassette exons repressed upon cold shock C_LIO_LIrG4s act as RNA thermometers in mammals by controlling accessibility of splice sites C_LIO_LIrG4 stability mediates temperature-dependent RBM3 expression ex vivo and in vivo C_LIO_LIStabilizing rG4s in RBM3 exon 3a reduces brain damage in subarachnoid hemorrhage C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/594628v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@e1d625org.highwire.dtl.DTLVardef@b22d21org.highwire.dtl.DTLVardef@1b0faeaorg.highwire.dtl.DTLVardef@294ac1_HPS_FORMAT_FIGEXP M_FIG C_FIG Here is a refined model of RNA G-quadruplexes acting as evolutionarily conserved thermo- and potassium sensors, modulating alternative splicing in mammals. RNA G-quadruplexes (rG4s) function as reversible temperature sensors, impacting alternative splicing dynamics. In low temperatures or high potassium conditions, rG4s can mask surrounding splice sites, rendering these sites inaccessible, thereby promoting exon skipping. Conversely, at high temperatures or under low potassium conditions, rG4s become destabilized, allowing splice sites to be exposed, and facilitating efficient exon inclusion. RBM3, a well-known cold-induced protein with neuroprotective functions, harbors a poison exon with rG4s around splice sites, that, upon inclusion, triggers NMD (non-sense mediated decay) of the RBM3 mRNA. Under low temperatures or high potassium conditions, rG4s shield the splice sites, leading to poison exon skipping and increased RBM3 expression. Stabilization of these rG4s through increased K+ promotes poison exon skipping, enabling escape from NMD, and ultimately elevating RBM3 expression. Notably, 4-AP, a clinically used pan voltage-gated potassium channel blocker, protects against neuronal damage in a subarachnoid hemorrhage mouse model in an RBM-dependent manner. (ISS: intronic splicing silencer, ESE: exon splicing enhancer, ACA: anterior cerebral artery, MCA: middle cerebral artery, PPA: pterygopalatine artery, ICA: internal carotid artery, ECA: external carotid artery, CCA: common carotid artery)

molecular biology↗

bioRxiv2024

An inferotemporal coding strategy robust to partial object occlusion

Object coding in primate ventral pathway cortex progresses in sparseness/compression/efficiency, from many orientation signals in V1, to fewer 2D/3D part signals in V4, to still fewer multi-part configuration signals in AIT (anterior inferotemporal cortex).1-11 This progression could lead to individual neurons exclusively selective for unique objects, the sparsest code for identity, especially for highly familiar, important objects.12-18 To test this, we trained macaque monkeys to discriminate 8 simple letter-like shapes in a match-to-sample task, a design in which one-to-one coding of letters by neurons could streamline behavior. Performance increased from chance to >80% correct over a period of weeks, after which AIT neurons showed clear learning effects, with increased selectivity for multi-part configurations within the trained alphabet shapes. But these neurons were not exclusively tuned for unique letters based on training, since their responsiveness generalized to different, non-trained shapes containing the same configurations. This multi-part configuration coding limit in AIT is not maximally sparse, but it could explain the robustness of primate vision to partial object occlusion, which is common in the natural world and problematic for computer vision. Multi-part configurations are highly diagnostic of identity, and neural signals for various partial object structures can provide different but equally sufficient evidence for whole object identity across most occlusion conditions.

neuroscience↗

bioRxiv2024

Multifaceted roles of cohesin in regulating transcriptional loops

Cohesin is required for chromatin loop formation. However, its precise role in regulating gene transcription remains largely unknown. We investigated the relationship between cohesin and RNA Polymerase II (RNAPII) using single-molecule mapping and live-cell imaging methods in human cells. Cohesin-mediated transcriptional loops were highly correlated with those of RNAPII and followed the direction of gene transcription. Depleting RAD21, a subunit of cohesin, resulted in the loss of long-range (>100 kb) loops between distal (super-)enhancers and promoters of cell-type-specific genes. By contrast, the short-range (<50 kb) loops were insensitive to RAD21 depletion and connected genes that are mostly housekeeping. This result explains why only a small fraction of genes are affected by the loss of long-range chromatin interactions due to cohesin depletion. Remarkably, RAD21 depletion appeared to up-regulate genes located in early initiation zones (EIZ) of DNA replication, and the EIZ signals were amplified drastically without RAD21. Our results revealed new mechanistic insights of cohesins multifaceted roles in establishing transcriptional loops, preserving long-range chromatin interactions for cell-specific genes, and maintaining timely order of DNA replication.

genomics↗

bioRxiv2024

Using RNA-targeting CRISPR-Cas13 and engineered U1 systems to reduce ABCA4 splice variants in Stargardt disease

Dysregulation of the alternative splicing process results in aberrant mRNA transcripts, leading to dysfunctional proteins or nonsense-mediated decay that cause a wide range of mis-splicing diseases. Development of therapeutic strategies to target the alternative splicing process could potentially shift the mRNA splicing from disease isoforms to a normal isoform and restore functional protein. As a proof of concept, we focus on Stargardt disease (STGD1), an autosomal recessive inherited retinal disease caused by biallelic genetic variants in the ABCA4 gene. The splicing variants c.5461-10T>C and c.4773+3A>G in ABCA4 cause the skipping of exon 39-40 and exon 33-34 respectively. In this study, we compared the efficacy of different RNA-targeting systems to modulate these ABCA4 splicing defects, including four CRISPR-Cas13 systems (CASFx-1, CASFx-3, RBFOX1N-dCas13e-C and RBFOX1N-dPspCas13b-C) as well as an engineered U1 system (ExSpeU1). Using a minigene system containing ABCA4 variants in the human retinal pigment epithelium ARPE19, our results show that RBFOX1N-dPspCas13b-C is the best performing CRISPR-Cas system, which enabled up to 80% reduction of the mis-spliced ABCA4 c.5461-10T>C variants and up to 78% reduction of the ABCA4 c.4773+3A>G variants. In comparison, delivery of a single ExSpeU1 was able to effectively reduce the mis-spliced ABCA4 c.4773+3A>G variants by up to 84%. We observed that the effectiveness of CRISPR-based and U1 splicing regulation is strongly dependent on the sgRNA/snRNA targeting sequences, highlighting that optimal sgRNA/snRNA designing is crucial for efficient targeting of mis-spliced transcripts. Overall, our study demonstrated the potential of using RNA-targeting CRISPR-Cas technology and engineered U1 to reduce mis-spliced transcripts for ABCA4, providing an important step to advance the development of gene therapy to treat STGD1.

molecular biology↗

bioRxiv2024

The Nrf2/Id2 Axis in Vascular Smooth Muscle Cells: Novel Insights into the Interplay between Vascular Calcification and Aging

OBJECTIVEVascular calcification (VC) significantly contributes to cardiovascular morbidity and mortality and escalates with age. However, effective pharmaceutical interventions are lacking, and the molecular mechanisms linking aging to VC remain elusive. This study explores the role of nuclear factor erythroid 2-related factor 2 (NRF2) in VC, specifically focusing on the interplay between vascular senescence and oxidative stress (OS). APPROACH AND RESULTSUsing a chronological aging mouse model, we noted a significant decline in the expression and activity of Nrf2 in the aortas of aged mice, coinciding with increased medial VC. Administering NRF2 activators effectively reduced this calcification. In the vascular smooth muscle cell (VSMC)-specific Nrf2 knockout (Nrf2SMCKO) models, created using adenine and Vitamin D, there was an increase in calcium deposition within the aortic medial layer, alongside heightened VSMC senescence and OS. Furthermore, NRF2 knockout exacerbated VC in aortic rings and primary VSMCs in high-phosphate conditions, while Nrf2 overexpression in VSMCs inhibited calcium deposition by alleviating cell senescence and OS. RNAseq analysis of the aortas from Nrf2SMCKO and control mice highlighted a significant downstream regulator of Inhibitor of DNA Binding 2 (ID2). Our results show that reduced NRF2 levels lead to increased VSMC senescence, characterized by heightened p16 expression and diminished ID2 expression. Inhibiting ID2 negated NRF2s protective effects against VSMC senescence and VC. CONCLUSIONThis study emphasizes the critical role of NRF2 dysfunction in the nexus of vascular senescence, OS, and VC. It proposes the NRF2-ID2 axis in VSMCs as a promising therapeutic target for reducing VC and mitigating age-related cardiovascular diseases. HighlightsO_LIDecline in NRF2 activity is linked to increased vascular calcification (VC) and aging. C_LIO_LIVSMC-specific Nrf2 knockout causes a remarkable exacerbation of arterial calcification. C_LIO_LINRF2 alleviates VC by inhibiting oxidative stress and VSMC senescence. C_LIO_LIID2 contributes to the protective role of NRF2 in VSMC senescence and VC. C_LI

molecular biology↗

bioRxiv2023

Interpretable Online Network Dictionary Learning for Inferring Long-Range Chromatin Interactions

Dictionary learning (DL), implemented via matrix factorization (MF), is commonly used in computational biology to tackle ubiquitous clustering problems. The method is favored due to its conceptual simplicity and relatively low computational complexity. However, DL algorithms produce results that lack interpretability in terms of real biological data. Additionally, they are not optimized for graph-structured data and hence often fail to handle them in a scalable manner. In order to address these limitations, we propose a novel DL algorithm called online convex network dictionary learning (online cvxNDL). Unlike classical DL algorithms, online cvxNDL is implemented via MF and designed to handle extremely large datasets by virtue of its online nature. Importantly, it enables the interpretation of dictionary elements, which serve as cluster representatives, through convex combinations of real measurements. Moreover, the algorithm can be applied to data with a network structure by incorporating specialized subnetwork sampling techniques. To demonstrate the utility of our approach, we apply cvxNDL on 3D-genome RNAPII ChIA-Drop data with the goal of identifying important long-range interaction patterns (long-range dictionary elements). ChIA-Drop probes higher-order interactions, and produces data in the form of hypergraphs whose nodes represent genomic fragments. The hyperedges represent observed physical contacts. Our hypergraph model analysis has the objective of creating an interpretable dictionary of long-range interaction patterns that accurately represent global chromatin physical contact maps. Through the use of dictionary information, one can also associate the contact maps with RNA transcripts and infer cellular functions. To accomplish the task at hand, we focus on RNAPII-enriched ChIA-Drop data from Drosophila Melanogaster S2 cell lines. Our results offer two key insights. First, we demonstrate that online cvxNDL retains the accuracy of classical DL (MF) methods while simultaneously ensuring unique interpretability and scalability. Second, we identify distinct collections of proximal and distal interaction patterns involving chromatin elements shared by related processes across different chromosomes, as well as patterns unique to specific chromosomes. To associate the dictionary elements with biological properties of the corresponding chromatin regions, we employ Gene Ontology (GO) enrichment analysis and perform multiple RNA coexpression studies. Availability and ImplementationThe code and test datasets are available at:\ https://github.com/rana95vishal/chromatin_DL/ Author summaryWe introduce a novel method for dictionary learning termed online convex Network Dictionary Learning (online cvxNDL). The method operates in an online manner and utilizes representative subnetworks of a network dataset as dictionary elements. A key feature of online cvxNDL is its ability to work with graph-structured data and generate dictionary elements that represent convex combinations of real data points, thus ensuring interpretability. Online cvxNDL is used to investigate long-range chromatin interactions in S2 cell lines of Drosophila Melanogaster obtained through RNAPII ChIA-Drop measurements represented as hypergraphs. The results show that dictionary elements can accurately and efficiently reconstruct the original interactions present in the data, even when subjected to convexity constraints. To shed light on the biological relevance of the identified dictionaries, we perform Gene Ontology enrichment and RNA-seq coexpression analyses. These studies uncover multiple long-range interaction patterns that are chromosome-specific. Furthermore, the findings affirm the significance of convex dictionaries in representing TADs cross-validated by imaging methods (such as 3-color FISH (fluorescence in situ hybridization)).

genomics↗

bioRxiv2023

Effects of intensified training with insufficient recovery on joint level and single muscle fibre mechanical function: The role of myofibrillar Ca2+ sensitivity

Intense exercise training with insufficient recovery is associated with reductions in neuromuscular performance. However, it is unclear how single muscle fibre mechanical function and myofibrillar Ca2+ sensitivity contribute to these impairments. We investigated the effects of overload training on joint-level neuromuscular performance and cellular-level mechanical function. Fourteen athletes (4 female, 10 male) underwent a 3-week intensified training protocol consisting of [~]140% of their regular training hours with three additional high-intensity training sessions per week. Neuromuscular performance of the knee extensors was assessed via maximum voluntary contraction (MVC) force, electrically evoked twitch contractions, and a force-frequency relationship. Muscle biopsies were taken from the vastus lateralis to assess single fibre mechanical function. Neither MVC force nor twitch parameters were altered following intensified training (all p>0.05), but a rightward shift in the force-frequency curve was observed with a 6-27% reduction in force at low-frequencies (5-20Hz, all p<0.05). In single fibres, maximal force output was not reduced following intensified training, but there was a rightward shift in the force-pCa curve driven by a 6% reduction in Ca2+ sensitivity as indicated by a lower pCa50 value (i.e., higher [Ca2+]) across fibre types (Pre=6.477{+/-}0.157, Post=6.088{+/-}0.480, p<0.05). These data indicate intensified training leads to impaired Ca2+ sensitivity at the single fibre level, which in part explains impaired neuromuscular function at the joint level during lower frequencies of activation. This is an important consideration for athletes, as performance is often assessed at maximal levels of activation, and these underlying impairments in force generation may be less obvious. New & NoteworthyIntense exercise training with insufficient recovery leads to impaired muscle contractile performance. These impairments often manifest at lower frequencies of muscle stimulation, termed prolonged low-frequency force depression. Impaired myofibrillar calcium sensitivity has been suggested as a potential mechanism of prolonged low-frequency force depression. Our work shows that impaired calcium sensitivity of single muscle fibres coincided with joint level prolonged low-frequency force depression after intense exercise training with insufficient recovery.

physiology↗

bioRxiv2023

Border-forming wound repair astrocytes

Central nervous system (CNS) lesions become surrounded by neuroprotective borders of newly proliferated reactive astrocytes. Fundamental features of these cells are poorly understood. Here, we show that 90% of border-forming astrocytes derive from proliferating Aldh1l1-expressing local astrocytes, and 10% from Pdgfra-expressing oligodendrocyte progenitors in mice. Temporal transcriptome analysis, snRNAseq and immunohistochemistry showed that after CNS injury, local mature astrocytes dedifferentiated, proliferated, and became transcriptionally reprogrammed to permanently altered new functional states, with persisting downregulation of molecules associated with astrocyte-neuron interactions, and upregulation of molecules associated with wound healing, microbial defense, and interactions with stromal and immune cells. Our findings show that at CNS injury sites, local mature astrocytes proliferate and adopt canonical features of essential wound repair cells that persist in adaptive states and are the predominant source of neuroprotective borders that re-establish CNS integrity by separating neural parenchyma from stromal and immune cells as occurs throughout the healthy CNS.

neuroscience↗

bioRxiv2023

Requirement of GrgA for Chlamydia infectious progeny production, optimal growth, and efficient plasmid maintenance

Chlamydia, an obligate intracellular bacterial pathogen, has a unique developmental cycle involving the differentiation of invading elementary bodies (EBs) to noninfectious reticulate bodies (RBs), replication of RBs, and redifferentiation of RBs into progeny EBs. Progression of this cycle is regulated by three sigma factors, which direct the RNA polymerase to their respective target gene promoters. We hypothesized that the Chlamydia-specific transcriptional regulator GrgA, previously shown to activate {sigma}66 and {sigma}28, plays an essential role in chlamydial development and growth. To test this hypothesis, we applied a novel genetic tool known as dependence on plasmid-mediated expression (DOPE) to create Chlamydia trachomatis with conditional GrgA-deficiency. We show that GrgA-deficient C. trachomatis RBs have a growth rate that is approximately half of the normal rate and fail to transition into progeny EBs. In addition, GrgA-deficient C. trachomatis fail to maintain its virulence plasmid. Results of RNA-seq analysis indicate that GrgA promotes RB growth by optimizing tRNA synthesis and expression of nutrient-acquisition genes, while it enables RB-to-EB conversion by facilitating the expression of a histone and outer membrane proteins required for EB morphogenesis. GrgA also regulates numerous other late genes required for host cell exit and subsequent EB invasion into host cells. Importantly, GrgA stimulates the expression of {sigma}54, the third and last sigma factor, and its activator AtoC, and thereby indirectly upregulating the expression of {sigma}54-dependent genes. In conclusion, our work demonstrates that GrgA is a master transcriptional regulator in Chlamydia and plays multiple essential roles in chlamydial pathogenicity. IMPORTANCEHallmarks of the developmental cycle of the obligate intracellular pathogenic bacterium Chlamydia are the primary differentiation of the infectious elementary body (EB) into the proliferative reticulate body (RB) and the secondary differentiation of RBs back into EBs. The mechanisms regulating these transitions remain unclear. In this report, we developed an effective novel strategy termed DOPE that allows for the knockdown of essential genes in Chlamydia. We demonstrate that GrgA, a Chlamydia-specific transcription factor, is essential for the secondary differentiation and optimal growth of RBs. We also show that GrgA, a chromosome-encoded regulatory protein, controls the maintenance of the chlamydial virulence plasmid. Transcriptomic analysis further indicates that GrgA functions as a critical regulator of all three sigma factors that recognize different promoter sets at developmental stages. The DOPE strategy outlined here should provide a valuable tool for future studies examining chlamydial growth, development, and pathogenicity.

microbiology↗

bioRxiv2023

Targeted Protein O-GlcNAcylation Using Bifunctional Small Molecules

Protein O-linked {beta}-N-acetylglucosamine modification (O-GlcNAcylation) plays a crucial role in regulating essential cellular processes. The disruption of O-GlcNAcylation homeostasis has been linked to various human diseases, including cancer, diabetes, and neurodegeneration. However, there are limited chemical tools for protein- and site-specific O-GlcNAc modification, rendering the precise study of O-GlcNAcylation challenging. To address this, we have developed first-in-class heterobifunctional small molecules, named O-GlcNAcylation targeting chimeras (OGTACs), which enable protein-specific O-GlcNAcylation in cells. OGTACs promote O-GlcNAcylation of proteins such as BRD4, CK2, and EZH2 in cellulo by recruiting O-GlcNAc transferase (OGT), with temporal and magnitude control. Mass spectrometry data revealed that OGTACs induced site-selective O-GlcNAcylation of BRD4. Overall, OGTACs represent a promising approach for inducing protein-specific O-GlcNAcylation, thus enabling functional dissection and offering new directions for O-GlcNAc-targeting therapeutic development.

cell biology↗

bioRxiv2023

Natural constraints explain working memory capacity limitations in sensory-cognitive models

The limited capacity of the brain to retain information in working memory has been well-known and studied for decades, yet the root of this limitation remains unclear. Here we built sensory-cognitive neural network models of working memory that perform tasks using raw visual stimuli. Contrary to intuitions that working memory capacity limitation stems from memory or cognitive constraints, we found that pre-training the sensory region of our models with natural images imposes sufficient constraints on models to exhibit a wide range of human-like behaviors in visual working memory tasks designed to probe capacity. Examining the neural mechanisms in our model reveals that capacity limitation mainly arises in a bottom-up manner. Our models offer a principled and functionally grounded explanation for the working memory capacity limitation without parameter fitting to behavioral data or much hyperparameter tuning. This work highlights the importance of developing models with realistic sensory processing even when investigating memory and other high-level cognitive phenomena.

neuroscience↗