Biology subjects

Hua, R.

Publications and source records attributed to Hua, R..

11 recordsLinked to original sources

bioRxiv2026

Targeting Macrophage Connexin 43 Hemichannels Suppresses Synovial Inflammation and Osteoarthritis

Osteoarthritis (OA) is the most common chronic joint disease and a leading cause of disability worldwide, yet no disease-modifying therapies are available. Although OA has traditionally been viewed as a cartilage-centered degenerative disease, growing evidence suggests that synovial inflammation is a critical driver of disease progression. However, the upstream mechanisms and actionable targets that govern this inflammatory response remain poorly defined. Connexin 43 (Cx43), a key regulator of musculoskeletal homeostasis that forms both gap junctions and hemichannels (HCs), is upregulated in osteoarthritic cartilage and synovium, but the specific contribution of Cx43 channels remains unclear. Here, we show that selective inhibition of Cx43 HCs attenuates OA progression in a murine post-traumatic OA model. Using adeno-associated virus (AAV)-mediated gene deletion, we found that Cx43 knockdown in joint tissues reduced cartilage erosion, synovial hyperplasia, subchondral bone sclerosis, and OA-associated pain. To dissect HC-specific functions, we developed a monoclonal antibody, Cx43(M1), that blocks Cx43 HCs without affecting junctional communication. Cx43(M1) preserved cartilage integrity, suppressed synovial inflammation, and alleviated pain behaviors, even when treatment was initiated after established cartilage damage. Mechanistically, the antibody preferentially localized to synovial macrophages, where it inhibited HC activity, reduced ATP and PGE2 release, and suppressed pro-inflammatory gene expression. Macrophage-specific Cx43 deletion recapitulated these protective effects, and Cx43 HC blockade in primary human OA synovial macrophages similarly inhibited inflammatory and catabolic responses. These findings identify synovial macrophage Cx43 HCs as upstream drivers of OA and support a paradigm shift from a predominantly chondrocyte-centered view toward synovial macrophage directed intervention.

immunology↗

bioRxiv2024

msCNVS: medium throughput single cell copy number variation sequencing with barcoded library construction free of preamplification toward clinical implementation

Single cell copy number variation sequencing (scCNV-seq) is valuable for genomic analysis of a variety of health and disease systems, yet the available methods either depend on either preamplification of the whole genome of each cell, special devices or untracable, which hinder scCNV-seq practice in clinics. Here we provide scalable multiplex scCNV-seq (msCNVS) that allows direct medium-throughput library construction for single cells, which are barcoded individually with Tn5 transposome in a microplate and immediately pooled for downstream processes, elevating the efficiency by orders of magnitude. An algorithm is developed on the distribution of segmented normalized read count density to identify the major peaks associating with integer copy numbers, further improving the accuracy for objective CNV calling. Here msCNVS faithfully distinguishes the unique CNV patterns of 5 cell lines. The individual msCNVS profiles of 70 individual K562 cells highly correlate with the profile of K562 bulk cells (R=0.90-0.98), and the triplicates of HeLa3 bulk cells correlated nearly perfect (R=0.99). The msCNVS CNV profiles of primary cell cultures of amniotic fluid are confirmed by G-banding karyotype analysis and chromosomal microarray analysis. Additionally, the coverage uniformity of msCNVS is superior to that of MDA and MALBAC and approaches what eMDA and DOP-PCR achieves. Furthermore, msCNVS detects CNV deletions in 2 stunted, abnormal blastocysts, one with CNV mosaicism, and uncovered variants of CNVs in circulated tumor cells, cancerous pleural effusion cells, and patient-derived xenograft nuclei. Thus, msCNVS promises a robust, reliable and highly efficient approach to genomic testing of precious and rare cells like those typically obtained in reproductive and cancer clinics. One-Sentence SummaryBy early pooling of multiple single cells individually barcoded with Tn5 transposome, msCNVS enables efficient CNV profiling of rare clinic samples

genomics↗

bioRxiv2024

Mechanical cues of extracellular matrix determines tumor innervation

Peripheral tumors can establish local autonomic and sensory nerve networks, termed as tumor innervation (TIN), to support tumorigenesis and metastasis. While nerve dependence in cancers is well-established, the mechanisms governing TIN remain unclear. Here, we report that extracellular matrix (ECM) stiffness, a major mechanical abnormality in the tumor microenvironment (TME), is an essential contributor of TIN. In preclinical models, reducing lysyl oxidase-mediated ECM crosslinking lowers tissue stiffness and TIN in pancreatic cancer, while inflammation-induced matrix stiffening boosts the hyperinnervation of the pancreatic precursor lesions. Mechanistically, {beta}1-containing integrins sense the mechanical cues exerted by ECM stiffness, and the translational co-activator YAP1 acts as an essential nuclear relay to induce the expression of neurotropic genes, particularly brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). 3D imaging of the whole cleared pancreas reveals that blockade of mechanosensor integrin {beta}1 or pharmacological inhibition of the mechanotransducer YAP1 effectively reduces TIN. In clinical settings, tumor samples with a dense, crosslinked, and stiffened ECM exhibit significant TIN. In summary, these findings identify ECM stiffness as an important driver of TIN and suggest that targeting integrin {beta}1/YAP1-dependent mechanotransduction may counteract TIN.

neuroscience↗

bioRxiv2023

Comparative chemical genomics in Babesia species identifies the alkaline phosphatase phoD as a novel determinant of resistance

Babesiosis is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of Babesia parasites. The diversity of Babesia parasites, coupled with the lack of potent inhibitors necessitates the discovery of novel conserved druggable targets for the generation of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of novel and conserved targets. CCG relies on parallel in vitro evolution of resistance in independent populations of evolutionarily-related Babesia spp. (B. bovis and B. divergens). We identified a potent antibabesial inhibitor from the Malaria Box, MMV019266. We were able to select for resistance to this compound in two species of Babesia, achieving 10-fold or greater resistance after ten weeks of intermittent selection. After sequencing of multiple independently derived lines in the two species, we identified mutations in a single conserved gene in both species: a membrane-bound metallodependent phosphatase (putatively named PhoD). In both species, the mutations were found in the phoD-like phosphatase domain, proximal to the predicted ligand binding site. Using reverse genetics, we validated that mutations in PhoD confer resistance to MMV019266. We have also demonstrated that PhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of PhoD alter the sensitivity to MMV019266 in the parasite: overexpression of PhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting PhoD is a resistance mechanism. Together, we have generated a robust pipeline for identification of resistance loci, and identified PhoD as a novel determinant of resistance in Babesia species. HighlightsO_LIUse of two species for in vitro evolution identifies a high confidence locus associated with resistance C_LIO_LIResistance mutation in phoD was validated using reverse genetics in B. divergens C_LIO_LIPerturbation of phoD using function genetics results in changes in the level of resistance to MMV019266 C_LIO_LIEpitope tagging reveals localization to the ER/apicoplast, a conserved localization with a similar protein in diatoms C_LIO_LITogether, phoD is a novel resistance determinant in multiple Babesia spp. C_LI

molecular biology↗

bioRxiv2023

A parabrachial-hypothalamic parallel circuit governs cold defense in mice

Thermal homeostasis is vital for mammals and is controlled by brain neurocircuits. Remarkable advances have been made in understanding how neurocircuits centered in the hypothalamic preoptic area (POA), the brains thermoregulation center, control warm defense, whereas mechanisms by which the POA regulates cold defense remain unclear. Here, we confirmed that the pathway from the lateral parabrachial nucleus (LPB) to the POA, is critical for cold defense. Parallel to this pathway, we uncovered that a pathway from the LPB to the dorsomedial hypothalamus (DMH), namely the LPB[->]DMH pathway, is also essential for cold defense. Projection-specific blockings revealed that both pathways provide an equivalent and cumulative contribution to cold defense, forming a parallel circuit. Specifically, activation of the LPB[->]DMH pathway induced strong cold-defense responses, including increases in thermogenesis of brown adipose tissue (BAT), muscle shivering, heart rate, and physical activity. Further, we identified a subpopulation of somatostatin+ neurons in the LPB that target the DMH to promote BAT thermogenesis. Therefore, we reveal a parabrachial-hypothalamic parallel circuit in governing cold defense in mice. This not only enables resilience to hypothermia but also provides a scalable and robust network in heat production, reshaping our understanding of how neural circuits regulate essential homeostatic behaviors.

neuroscience↗

bioRxiv2022

ClpP/ClpX deficiency impairs mitochondrial functions and mTORC1 signaling during spermatogenesis and meiosis

Caseinolytic protease proteolytic subunit (ClpP) and caseinolytic protease X (ClpX) are mitochondrial matrix peptidases that activate mitochondrial unfolded protein response to maintain protein homeostasis in the mitochondria. However, the role of ClpP and ClpX in spermatogenesis remains largely unknown. In this study, we demonstrated the importance of ClpP/ClpX for meiosis and spermatogenesis with two conditional knockout (cKO) mouse models. We found that ClpP/ClpX deficiency reduced mitochondrial functions and quantity in spermatocytes, affected energy supply during meiosis and attenuated zygotene-pachytene transformation of the male germ cells. The dysregulated spermatocytes finally underwent apoptosis resulting in decreased testicular size and vacuolar structures within the seminiferous tubules. We found mTORC1 pathway was over-activated after deletion of ClpP/ClpX in spermatocytes. Long-term inhibition of the mTORC1 signaling via rapamycin treatment in vivo partially rescue spermatogenesis. The data reveal the novel roles of ClpP and ClpX in regulating meiosis and spermatogenesis. One-Sentence SummaryClpP/ClpX is required for maintaining mitochondrial functions in spermatocytes during meiosis and spermatogenesis.

developmental biology↗

bioRxiv2022

Connexin 43 Hemichannels Regulate Mitochondrial ATP Generation, Mobilization, and Mitochondrial Homeostasis against Oxidative Stress

Oxidative stress is a major risk factor that causes osteocyte cell death and bone loss. Prior studies primarily focus on the function of cell surface expressed Cx43 channels. Here, we reported a new role of mitochondrial Cx43 (mtCx43) and hemichannels (HCs) in modulating mitochondria homeostasis and function in bone osteocytes under oxidative stress. In osteocyte MLO-Y4 cells, the translocation of Cx43 to mitochondria was increased under H2O2-induced oxidative stress. H2O2 increased the mtCx43 level accompanied by elevated mitochondrial Cx43 HC activity determined by dye uptake assay. Cx43 knockdown (KD) by CRISPR-Cas9 lentivirus system resulted in impairment of mitochondrial function, primarily manifested as decreased ATP production. Cx43 KD had reduced intracellular reactive oxidative species (ROS) levels and mitochondrial membrane potential. Additionally, live-cell imaging results demonstrated that the proton flux was dependent upon mtCx43 HCs because its activity was specifically inhibited by an antibody targeting Cx43 C-terminus. The co-localization and interaction of mtCx43 and ATP synthase subunit F (ATP5J2) were confirmed by fluorescence resonance energy transfer (FRET) and a protein pull-down assay. Together, our study suggests that mtCx43 hemichannels regulate mitochondrial ATP generation by mediating K+, H+, and ATP transfer across the mitochondrial inner membrane and the interaction with mitochondrial ATP synthase, leading to enhancing the protection capacity of osteocytes against oxidative insults.

cell biology↗

bioRxiv2022

Underlying beneficial effects of Rhubarb on constipation-induced inflammation, disorder of gut microbiome and metabolism

BackgroundAlthough constipation is a common syndrome and a worldwide health problem. Constipation patients are becoming younger with a 29.6% overall prevalence in the children, which has captured great attention because of its epigenetic rejuvenation and recurrent episodes. Despite the usage of rhubarb to relieve constipation, novel targets and genes involved in target-relevant pathways with remarkable functionalities should still be sought after. Materials and methodsWe established a reliable constipation model in C57B/6N male mice using intragastric administration diphenoxylate and the eligible subjects received 600mg/25g rhubarb extraction to ameliorate constipation. Resultant constipation was morphological and genetically compared with the specimen from different groups. ResultsThe constipation mice exhibited thicker muscle layers, improved content of cytokines, including IL-17 and IL-23, and lower content of IL-22. The bacterial abundance and diversity varied tremendously. Notably, the alterations were reversed after rhubarb treatment. Additionally, SCFA and MLCFA were significantly influenced by constipation accompanied by enhanced expressions of SCFA receptors, GPR41 and GPR43. ConclusionThis thesis has provided an insight that rhubarb promoted the flexibility of collagen fiber, reduced pro-inflammatory cytokines and enhanced anti-inflammatory cytokines, and maintained intestinal microflora balance with potential effects on affecting the metabolism of fatty acids and polyamines.

pharmacology and toxicology↗

bioRxiv2022

Identification of Circular Dorsal Ruffles as Signal Platforms for the AKT pathways in Glomerular Podocytes

Circular dorsal ruffles (CDRs) are rounded membrane ruffles induced by growth factors to function as precursors of the large-scale endocytosis called macropinocytosis. In cell line systems, CDR/macropinocytosis regulate the AKT-mTORC1 pathway, a canonical growth factor signaling. However, it is not known if this mechanism occurs in tissues. Here, utilizing ultra-high-resolution scanning electron microscopy, we report that CDRs are expressed in glomerular podocytes ex vivo and in vivo. Biochemical and imaging analysis revealed that AKT phosphorylation is localized to CDRs upstream of mTORC1 activation in podocyte cell line and isolated glomeruli, indicating that CDRs function as signal platforms for AKT-mTORC1 pathway in podocytes to maintain the kidney function. Because mTORC1 has critical roles in the podocyte metabolism and the aberrant activation of mTORC1 triggers podocytopathies, these results suggest that targeting CDR formation would be a potential therapeutic approach for the diseases.

cell biology↗

bioRxiv2021

Gene losses may contribute to subterranean adaptations in naked mole-rat and blind mole-rat

The naked mole-rats (Heterocephalus glaber, NMRs) and the blind mole-rats (Spalax galili, BMRs) are representative subterranean rodents that evolved many extraordinary traits, including hypoxia tolerance, longevity and cancer resistance. Although a batch of candidate loci responsible for these intriguing traits have been uncovered by genomic studies, many of them are limited to functional modifications of intact genes and little is known about the contributions of other genetic makeups. Here, to address this issue, we focused on gene losses (unitary pseudogenes) and systematically analyzed gene losses in NMRs and BMRs, as well as their respective terrestrial relatives, guinea pigs and rats, in a genome-wide scale. 167, 139, 341 and 112 pseudogenes were identified in NMRs, BMRs, guinea pigs and rats, respectively. Functional enrichment analysis identified 4 shared and 2 species-specific enriched functional groups (EFGs) in subterranean lineages. The pseudogenes in these EFGs might be associated with either regressive (e.g. visual system) or adaptive (e.g. altered DNA damage response) traits. In addition, several pseudogenes including TNNI3K and PDE5A, might be associated with their specific cardiac features observed in subterranean linages. Furthermore, we observed 20 convergent gene losses in NMRs and BMRs. Given that the functional investigations of these genes are generally scarce, we provided functional evidence that independent loss of TRIM17 in NMRs and BMRs might be beneficial for neuronal survival under hypoxia, supporting the positive role of eliminating TRIM17 function in hypoxia adaptation. We also demonstrated that pseudogenes, together with positively selected genes, reinforced subterranean adaptations cooperatively. Overall, our study provides new insights into the molecular underpinnings of subterranean adaptations and highlights the importance of gene losses in mammalian evolution.

evolutionary biology↗

bioRxiv2020

Exploiting toxin internalization receptors to enhance delivery of proteins to lysosomes for enzyme replacement therapy

Lysosomal storage diseases are a group of over 70 inherited genetic diseases caused by a defect or deficiency in a lysosomal protein. Enzyme replacement therapy, in which a functional copy of the defective enzyme is injected either systemically or directly into the brain of affected individuals, has proven to be an effective strategy for treating certain lysosomal storage diseases; however, the inefficient uptake of recombinant enzymes into cells and tissues via the low-affinity mannose-6-phosphate receptor prohibits broader utility of replacement therapy. Here, to improve the efficiency and efficacy of lysosomal enzyme uptake, we exploited the strategy used by diphtheria toxin to enter into the endo-lysosomal network of cells by creating a chimera between the receptor-binding fragment of diphtheria toxin and the lysosomal hydrolase TPP1. We show that the targeted TPP1 chimera binds with high affinity to target cells and is delivered into lysosomes with much greater efficiency than TPP1 alone. Further, we demonstrate efficient and durable uptake of the chimera in vivo following intracerebroventricular injection in mice lacking TPP1. Targeting the highly efficient diphtheria toxin internalization pathway represents a novel approach for improving the efficacy and utility of enzyme replacement therapy for treating lysosomal storage diseases.

pharmacology and toxicology↗