Biology subjects

Pan, X.

Publications and source records attributed to Pan, X..

At least 91 records · Page 5Linked to original sources

bioRxiv2024

Dysfunctional S1P/S1PR1 signaling in the dentate gyrus drives vulnerability of chronic pain-related memory impairment

Memory impairment in chronic pain patients is substantial and common, and few therapeutic strategies are available. Chronic pain-related memory impairment has susceptible and unsusceptible features. Therefore, exploring the underlying mechanisms of its vulnerability is essential for developing effective treatments. Here, combining two spatial memory tests (Y-maze test and Morris water maze), we segregated chronic pain mice into memory impairment-susceptible and -unsusceptible subpopulations in a chronic neuropathic pain model induced by chronic constrictive injury of the sciatic nerve. RNA-seq analysis and gain/loss-of-function study revealed that S1P/S1PR1 signaling is a determinant for vulnerability to chronic pain-related memory impairment. Knockdown of the S1PR1 in the DG promoted a susceptible phenotype and led to structural plasticity changes of reduced excitatory synapse formation and abnormal spine morphology as observed in susceptible mice, while overexpression of the S1PR1 and pharmacological administration of S1PR1 agonist in the DG promoted an unsusceptible phenotype and prevented the occurrence of memory impairment, and rescued the morphological abnormality. Finally, GO enrichment analysis and biochemical evidence indicated that down-regulation of S1PR1 in susceptible mice may impair DG structural plasticity via interaction with actin cytoskeleton rearrangement-related signaling pathways including Itga2 and its downstream Rac1/Cdc42 signaling and Arp2/3 cascade. These results reveal a novel mechanism and provide a promising preventive and therapeutic molecular target for vulnerability to chronic pain-related memory impairment.

neuroscience↗

bioRxiv2024

Elucidation of the Aspergillus fumigatus tRNA-derived RNA repertoire from conidia and mycelium.

Aspergillus fumigatus is a ubiquitous filamentous fungus and dangerous human pathogen that produces a limited pool of small RNAs, consisting in large part of tRNA-derived RNAs (tDRs). Here, we improve our understanding of the small RNAs produced in conidia and mycelium of A. fumigatus using small RNA-sequencing of strains lacking RNA interference (RNAi) machinery and a cutting-edge tDR-sequencing approach. We find little evidence of small RNAs dependent on the canonical RNAi machinery under laboratory growth conditions, but reveal tDRs to be differentially abundant across fungal morphotypes, with specific fragments proving dominant in each assessed condition (e.g., Asp(GTC)-5tRH in conidia; His(GTG)-5tRH in mycelium). Consistent with the literature, we observed distinct patterns of tDRs from nuclear- and mitochondria-derived tDRs, which was confirmed for wild-type fungus with tDR-seq. By inducing canonical RNAi with overexpression of an inverted-repeat transgene, we determined A. fumigatus to produce predominantly 20-nt, 5 uridine-containing small RNAs from the transgene, a population reliant on Argonaute and Dicer-like proteins. Surprisingly, we found that overexpression of this double-stranded RNA (dsRNA) limited growth of both wild-type and RNAi-deficient strains, with a strain lacking the two A. fumigatus RNA-dependent RNA polymerase orthologs particularly vulnerable. Dicer-like- and Argonaute-double knockout strains with few detectable transgene-derived small RNAs were also susceptible to growth inhibition, suggesting excessive dsRNA limits growth in A. fumigatus. Ultimately, we have provided an improved description of small RNA biogenesis in A. fumigatus and uncovered an intriguing link between dsRNA metabolism and filamentous growth. IMPORTANCEThe mechanisms of small RNA biogenesis in fungi are diverse and multifactorial. Here, using a panel of RNA interference gene deletion strains and multiple small RNA sequencing approaches, we reveal limited input of the canonical RNAi machinery on the small RNA landscape of the human fungal pathogen, Aspergillus fumigatus under normal growth conditions. We identify specific patterns of tRNA-derived RNAs unique to fungal morphotypes, which may hold promise for future diagnostic efforts. We then probed the mechanism of double-stranded RNA processing by the organism after artificial induction, revealing a potential link between proper double-stranded RNA processing and fungal growth arrest, improving our understanding of the evolution of double-stranded RNA regulation in a ubiquitous mold.

microbiology↗

bioRxiv2024

Multivalent Targeting of Blood-Brain Barrier LRP1 for Neurovascular Recovery Therapy for Alzheimer s Disease

The blood-brain barrier (BBB) is a highly selective permeability barrier that safeguards the central nervous system (CNS) from potentially harmful substances while regulating the transport of essential molecules. Its dysfunction is increasingly recognized as a pivotal factor in the pathogenesis of Alzheimers disease (AD), contributing to the accumulation of amyloid-{beta} (A{beta}) plaques. We propose an AD therapeutic strategy targeting the BBB low-density lipoprotein receptor-related protein 1 (LRP1). We show that a multivalent scaffold with LRP1-specific peptide modulates the A{beta} transport at the BBB. We show via detailed experimentation on AD model mice that this intervention markedly reduces A{beta} deposits and prevents cognitive decline. This study marks a new approach to drug design, combining multivalent targeting with the regulation of membrane trafficking through advanced molecular engineering. Crucially, the therapeutic effects emerge from the multivalent nature of our proposed system. Furthermore, our findings underscore the paramount significance of the BBB in AD pathogenesis, particularly emphasizing the critical role of LRP1-mediated A{beta} clearance in mitigating disease progression.

neuroscience↗

bioRxiv2024

Secondary deficiency of neuraminidase 1 contributes to CNS pathology in neurological mucopolysaccharidoses via hypersialylation of brain glycoproteins

Mucopolysaccharidoses (MPS) are lysosomal storage diseases caused by defects in catabolism of glycosaminoglycans. MPS I, II, III and VII are associated with lysosomal accumulation of heparan sulphate and manifest with neurological deterioration. Most of these neurological MPS currently lack effective treatments. Here, we report that, compared to controls, neuraminidase 1 (NEU1) activity is drastically reduced in brain tissues of neurological MPS patients and in mouse models of MPS I, II, IIIA, IIIB and IIIC, but not of other neurological lysosomal disorders not presenting with heparan sulphate storage. We further show that accumulated heparan sulphate disrupts the lysosomal multienzyme complex of NEU1 with cathepsin A (CTSA), {beta}-galactosidase (GLB1) and glucosamine-6-sulfate sulfatase (GALNS) necessary to maintain enzyme activity, and that NEU1 deficiency is linked to partial deficiencies of GLB1 and GALNS in cortical tissues and iPSC-derived cortical neurons of neurological MPS patients. Increased sialylation of N-linked glycans in brain samples of human MPS III patients and MPS IIIC mice implicated insufficient processing of brain N-linked sialylated glycans, except for polysialic acid, which was reduced in the brains of MPS IIIC mice. Correction of NEU1 activity in MPS IIIC mice by lentiviral gene transfer ameliorated previously identified hallmarks of the disease, including memory impairment, behavioural traits, and reduced levels of the excitatory synapse markers VGLUT1 and PSD95. Overexpression of NEU1 also restored levels of VGLUT1-/PSD95-positive puncta in cortical neurons derived from iPSC of an MPS IIIA patient. Together, our data demonstrate that heparan sulphate-induced secondary NEU1 deficiency and aberrant sialylation of glycoproteins implicated in synaptogenesis, memory, and behaviour constitute a novel pathological pathway in neurological MPS spectrum crucially contributing to CNS pathology. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/587986v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@3905c3org.highwire.dtl.DTLVardef@1a9672corg.highwire.dtl.DTLVardef@b4911forg.highwire.dtl.DTLVardef@a3b190_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Heterologous HSPC transplantation rescues neuroinflammation and ameliorates peripheral manifestations in the mouse model of lysosomal transmembrane enzyme deficiency, MPS IIIC.

Mucopolysaccharidosis III type C (MPS IIIC) is an untreatable neuropathic lysosomal storage disease caused by a genetic deficiency of the lysosomal N-acetyltransferase, HGSNAT, catalyzing a transmembrane acetylation of heparan sulfate. HGSNAT is a transmembrane enzyme uncapable of free diffusion between the cells and their cross-correction which limits development of therapies based on the enzyme replacement and gene correction. Since our previous work identified neuroinflammation as a hallmark of the CNS pathology in MPS IIIC, we tested whether it can be corrected by replacement of activated brain microglia with neuroprotective macrophages/microglia derived from a heterologous HSPC transplant. Eight-week-old MPS IIIC (HgsnatP304L) mice were transplanted with HSPC from congenic wild type mice after myeloablation with Busulfan and studied by behaviour test battery staring from the age of 6 months. At the age of [~]8 months, mice were sacrificed to study pathological changes in the brain, heparan sulfate storage and other biomarkers of the disease. We found that the treatment corrected several behaviour deficits including hyperactivity and reduction of socialization, but not a memory decline. It also improved several features of CNS pathology such as microastroglyosis, expression of pro-inflammatory cytokine IL-1{beta}, and accumulation of misfolded amyloid aggregates in cortical neurons. At the periphery, the treatment delayed development of terminal urinary retention, potentially increasing longevity, and reduced blood levels of heparan sulfate. However, we did not observe correction of lysosomal storage phenotype in neurons and heparan sulfate brain levels. Together, our results demonstrate that neuroinflammation in a neurological lysosomal storage disease, caused by defects in a transmembrane enzyme, can be effectively ameliorated by replacement of microglia bearing the genetic defect with the cells from normal healthy donor. They also suggest, that heterologous HSPC transplant, if used together with other methods, such as chaperone therapy or substrate reduction therapy, may constitute an effective combination therapy for MPS IIIC and other disorders with a similar etiology.

genetics↗

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

Spike Neural Network of Motor Cortex Model for Arm Reaching Controlling

Motor cortex modeling is crucial for understanding movement planning and execution. While interconnected recurrent neural networks have successfully described the dynamics of neural population activity, most existing methods utilize continuous signal-based neural networks, which do not reflect the biological spike neural signal. To address this limitation, we propose a recurrent spike neural network to simulate motor cortical activity during an arm-reaching task. Specifically, our model is built upon integrate-and-fire spiking neurons with conductance-based synapses. We carefully designed the interconnections of neurons with two different firing time scales - "fast" and "slow" neurons. Experimental results demonstrate the effectiveness of our method, with the models neuronal activity in good agreement with monkeys motor cortex data at both single-cell and population levels. Quantitative analysis reveals a correlation coefficient 0.89 between the models and real data. These results suggest the possibility of multiple timescales in motor cortical control.

neuroscience↗

bioRxiv2023

High-resolution ultrastructure and morphology of marine ammonia-oxidizing archaea Nitrosopumilus maritimus SCM1 revealed by Cryo-ET and STEM-EDS

Nitrososphaerota, formerly known as Thaumarchaeota, constitute a diverse and widespread group of ammonia-oxidizing archaea (AOA) inhabiting ubiquitously in marine and terrestrial environments, playing a pivotal role in global nitrogen cycling. Despite their significance in Earths ecosystems, the cellular organization of AOA remains largely unexplored, leading to a significant question unanswered of how the machinery of these organisms underpins metabolic functions. In this study, we combined chromatic-aberration-corrected cryo-electron tomography (cryo-ET), scanning transmission electron microscopy (STEM), and energy dispersive X-ray spectroscopy (EDS) to unveil the cellular organization and elemental composition of Nitrosopumilus maritimus SCM1, a representative member of marine Nitrososphaerota. Our tomograms show the native ultrastructural morphology of SCM1 and one to several dense storage granules in the cytoplasm. STEM-EDS analysis identifies two types of storage granules in which one type is possibly composed of polyphosphate, while the other type is polyhydroxyalkanoate. Collectively, these findings provide visual evidence for the resilience of AOA in the vast oligotrophic marine environment.

microbiology↗

bioRxiv2023

Internal Dynamics Interact with Proprioceptive Feedback During Movement Execution in an RNN Model of Motor Cortex

Proprioceptive feedback provides the information about the state of the body, which is critical in motor control. However, the contribution of proprioceptive feedback to motor cortical activity during voluntary movement execution is unclear. Here, we built an recurrent neural network model of motor cortex that receives proprioceptive feedback, and optimized it to control a virtual arm to perform a delayed-reach task. Simulated neural activity is similar to real data, indicating that our model captures the motor cortical dynamics. We further disrupted recurrent connectivity and proprioceptive feedback to dissect their contribution, and found that internal dynamics dominate in neural population activity, while proprioceptive feedback controls movement termination. Moreover, proprioceptive feedback improves the networks robustness against noisy initial conditions. We further investigated the relative importance of the components in proprioceptive feedback and found that the feedback of hand velocity contributes most to the similarity between simulation and real data. Finally, we show that our motor cortex model can be implemented in the sensorimotor system, demonstrating our models biological plausibility. In summary, motor command may arise from the intersection between recurrent dynamics in motor cortex and proprioceptive feedback.

neuroscience↗

bioRxiv2023

Integration of multi-source gene interaction networks and omics data with graph attention networks to identify novel disease genes

The pathogenesis of diseases is closely associated with genes, and the discovery of disease genes holds significant importance for understanding disease mechanisms and designing targeted therapeutics. However, biological validation of all genes for diseases is expensive and challenging. In this study, we propose DGP-AMIO, a computational method based on graph attention networks, to rank all unknown genes and identify potential novel disease genes by integrating multi-omics and gene interaction networks from multiple data sources. DGP-AMIO outperforms other methods significantly on 20 disease datasets, with an average AUROC and AUPR exceeding 0.9. The superior performance of DGP-AMIO is attributed to the integration of multiomics and gene interaction networks from multiple databases, as well as triGAT, a proposed GAT-based method that enables precise identification of disease genes in directed gene networks. Enrichment analysis conducted on the top 100 genes predicted by DGP-AMIO and literature research revealed that a majority of enriched GO terms, KEGG pathways and top genes were associated with diseases supported by relevant studies. We believe that our method can serve as an effective tool for identifying disease genes and guiding subsequent experimental validation efforts.

bioinformatics↗

bioRxiv2023

Cross-modal Generation of Hit-like Molecules via Foundation Model Encoding of Gene Expression Signatures

Designing de novo molecules with specific biological activity is an essential task since it holds the potential to bypass the exploration of target genes, which is an initial step in the modern drug discovery paradigm. However, traditional methods mainly screen molecules by comparing the desired molecular effects within the documented experimental results. The data set limits this process, and it is hard to conduct direct cross-modal comparisons. Therefore, we propose a solution based on cross-modal generation called GexMolGen (Gene Expression-based Molecule Generator), which generates hit-like molecules using gene expression signatures alone. These signatures are calculated by inputting control and desired gene expression states. Our model GexMolGen adopts a "first-align-then-generate" strategy, aligning the gene expression signatures and molecules within a mapping space, ensuring a smooth cross-modal transition. The transformed molecular embeddings are then decoded into molecular graphs. In addition, we employ an advanced single-cell large language model for input flexibility and pre-train a scaffold-based molecular model to ensure that all generated molecules are 100% valid. Empirical results show that our model can produce molecules highly similar to known references, whether feeding in- or out-of-domain transcriptome data. Furthermore, it can also serve as a reliable tool for cross-modal screening.

bioinformatics↗

bioRxiv2023

Distinct functions of the kinesin-9 family in mammalian motile cilia

Ciliary beat and intraflagellar transport (IFT) depend on dynein and kinesin motors. Kinesin-9 family members Kif6 and Kif9 are implicated in ciliary motilities across protists and mammals. How they function and whether they act redundantly, however, remain unclear. Here, we show that they play distinct roles in mammals. Kif6 forms puncta that move bidirectionally without or with IFT-B particles along axonemes, whereas Kif9 is immobilized on ciliary central apparatus. Only Kif6 binds to and glides microtubules, and the activities are self-inhibited. Kif6 deficiency in mice impairs directional ciliary beat across ependymal tissues and cerebrospinal fluid flow, resulting in severe hydrocephalus and high mortality, whereas Kif9 deficiency induces mild hydrocephalus without obviously defective ciliary beat and life span. Both Kif6-/- and Kif9-/- males are infertile but show respectively oligozoospermia with poor sperm motility and defective forward motion of sperms. These results suggest Kif6 as a motile cilia-specific IFT motor and Kif9 as a central apparatus regulator.

cell biology↗

bioRxiv2023

DeepSipred: A deep-learning-based approach on siRNA inhibition prediction

MotivationThe use of exogenous small interfering RNAs (siRNAs) for gene silencing has become a widespread molecular tool for gene function study and new drug identification. Although the pathway of RNAi to mediate gene expression has been widely investigated, the selection of hyperfunctional siRNA with high inhibition remains challenging. ResultsIn this study, we build a deep-learning-based approach on siRNA inhibition prediction, named DeepSipred. It combines features from sequence context, thermodynamic property, and other expert knowledge together to predict the inhibition more accurately than existing methods. The sequence features from siRNA and local target mRNA are generated via one-hot encoding and pretrained RNA-FM encoding. The convolution layers with multiple kernels in DeepSipred can detect various decisive motifs, which will determine the actual inhibition of siRNA. The thermodynamic features are calculated from Gibbs Free Energy. In addition, the expert knowledge includes those design criteria from previous studies. Benchmarked on large available public datasets, the 10-fold cross-validation results indicate that our predictor achieving the state-of-the-art performance. Contactyuanye_auto@sjtu.edu.cn or chengjin520@sjtu.edu.cn.

bioinformatics↗

bioRxiv2023

Tumor- and Nerve-Derived Axon Guidance Molecule Promotes Pancreatic Ductal Adenocarcinoma Progression and Metastasis through Macrophage Reprogramming

Axon guidance molecules were found to be the gene family most frequently altered in pancreatic ductal adenocarcinoma (PDA) through mutations and copy number changes. However, the exact molecular mechanism regarding PDA development remained unclear. Using genetically engineered mouse models to examine one of the axon guidance molecules, semaphorin 3D (SEMA3D), we found a dual role for tumor-derived SEMA3D in malignant transformation of pancreatic epithelial cells and a role for nerve-derived SEMA3D in PDA development. This was demonstrated by the pancreatic-specific knockout of the SEMA3D gene from the KRASG12D and TP53R172H mutation knock-in, PDX1-Cre (KPC) mouse model which demonstrated a delayed tumor initiation and growth comparing to the original KPC mouse model. Our results showed that SEMA3D knockout skews the macrophages in the pancreas away from M2 polarization, providing a potential mechanistic role of tumor-derived SEMA3D in PDA development. The KPC mice with the SEMA3D knockout remained metastasis-free, however, died from primary tumor growth. We then tested the hypothesis that a potential compensation mechanism could result from SEMA3D which is naturally expressed by the intratumoral nerves. Our study further revealed that nerve-derived SEMA3D does not reprogram macrophages directly, but reprograms macrophages indirectly through ARF6 signaling and lactate production in PDA tumor cells. SEMA3D increases tumor-secreted lactate which is sensed by GPCR132 on macrophages and subsequently stimulates pro-tumorigenic M2 polarization in vivo. Tumor intrinsic- and extrinsic-SEMA3D induced ARF6 signaling through its receptor Plexin D1 in a mutant KRAS-dependent manner. Consistently, RNA sequencing database analysis revealed an association of higher KRASMUT expression with an increase in SEMA3D and ARF6 expression in human PDAs. Moreover, multiplex immunohistochemistry analysis showed an increased number of M2-polarized macrophages proximal to nerves in human PDA tissue expressing SEMA3D. Thus, this study suggests altered expression of SEMA3D in tumor cells lead to acquisition of cancer-promoting functions and the axon guidance signaling originating from nerves is "hijacked" by tumor cells to support their growth. Other axon guidance and neuronal development molecules may play a similar dual role which is worth further investigation. One sentence summaryTumor- and nerve-derived SEMA3D promotes tumor progression and metastasis through macrophage reprogramming in the tumor microenvironment. STATEMENT OF SIGNIFICANCEThis study established the dual role of axon guidance molecule, SEMA3D, in the malignant transformation of pancreatic epithelial cells and of nerve-derived SEMA3D in PDA progression and metastasis. It revealed macrophage reprogramming as the mechanism underlying bothroles. Together, this research elucidated how inflammatory responses promote invasive PDA progression and metastasis through an oncogenic process.

cancer biology↗

bioRxiv2023

Integrating large-scale meta-GWAS and PigGTEx resources to decipher the genetic basis of complex traits in pig.

Understanding the molecular and cellular mechanisms that underlie complex traits in pigs is crucial for enhancing their genetic improvement program and unleashing their substantial potentials in human biomedicine research. Here, we conducted a meta-GWAS analysis for 232 complex traits with 28.3 million imputed whole-genome sequence variants in 70,328 individuals from 14 pig breeds. We identified a total of 6,878 genomic regions associated with 139 complex traits. By integrating with the Pig Genotype-Tissue Expression (PigGTEx) resource, we systemically explored the biological context and regulatory circuits through which these trait-associated variants act and finally prioritized 16,664 variant-gene-tissue-trait circuits. For instance, rs344053754 regulates the expression of UGT2B31 in the liver by affecting the activity of regulatory elements and ultimately influences litter weight at weaning. Furthermore, we investigated the conservation of genetic and regulatory mechanisms underlying 136 human traits and 232 pig traits. Overall, our multi-breed meta-GWAS in pigs provides invaluable resources and novel insights for understanding the regulatory and evolutionary mechanisms of complex traits in both pigs and humans.

genetics↗

bioRxiv2023

A muscle hypertrophy-derived myokine reprograms the stromal vascular fractions differentiation towards thermogenic adipocytes in subcutaneous adipose tissue

Skeletal muscle plays a significant role in both local and systemic energy metabolism; however, the cross-talk between skeletal muscle and adipose tissue remains largely unexplored. In this study, we identify the HIF2-Bambi-Thbs4 axis as a critical driver of muscle hypertrophy and metabolic improvement. Deletion of Bambi induces muscle hypertrophy and oxidative switching, thereby enhancing local and systemic metabolism under conditions of rodent chow and high-fat diets (HFD). Other than the metabolic regulation of skeletal muscle hypertrophy, we hypothesize that a portion of this improvement may be attributed to the metabolic reprogramming of the stromal vascular fraction (SVFs) of iWAT, promoting the development of beige adipocytes. Leveraging multi-omics approaches, we establish Thbs4 as a newly identified, muscle hypertrophy-derived myokine that serves as an essential regulator of this metabolic reprogramming. Thbs4 is upregulated in skeletal muscle during hypertrophy and aerobic exercise, localizes to the iWAT membrane, and facilitates the transformation of white adipocytes into beige adipocytes under conditions such as cold exposure, muscle hypertrophy, or aerobic exercise. Moreover, overexpression of Thbs4 elevates its plasma levels and leads to the transformation of white to beige adipocytes, therefore offering long-term protection against HFD-induced metabolic disorders. Conversely, Thbs4 knockout (Thbs4-KO) disrupts the metabolic reprogramming of SVFs and exacerbates metabolic syndromes induced by an HFD, mirroring the observed decline in Thbs4 levels in aged mice. Therefore, our findings underscore Thbs4s potential as both a long-term metabolic protective factor and a therapeutic target for the treatment of metabolic disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/558627v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@1178e6org.highwire.dtl.DTLVardef@1184d4eorg.highwire.dtl.DTLVardef@7b592aorg.highwire.dtl.DTLVardef@1b0b03_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBambi, a direct target gene of HIF2 in skeletal muscle, when deleted, triggers muscle hypertrophy and the transformation of white into beige adipocytes, improving systemic energy metabolism. C_LIO_LIThe metabolic reprogramming of stromal vascular fractions (SVFs) contributes to the observed improvements in energy metabolism and adipocyte hyperplasia. C_LIO_LIThbs4, a novel myokine produced in skeletal muscle during hypertrophy and aerobic exercise, plays a key role in the transformation of white into beige adipocytes. C_LIO_LIOverexpression of Thbs4 provides enduring protection against high-fat diet (HFD)-induced metabolic disorders, demonstrating its potential as a therapeutic target. C_LIO_LIThbs4 deletion abrogates the beneficial metabolic reprogramming of SVFs and exacerbates HFD-induced metabolic syndromes, further underlining its crucial role in metabolic regulation. C_LI

physiology↗

bioRxiv2023

CRISPR-Cas9 Extracellular Vesicles for Treating Hearing Loss

Clinical translation of gene therapy has been challenging, due to limitations in current delivery vehicles such as traditional viral vectors. Herein, we report the use of gRNA:Cas9 ribonucleoprotein (RNP) complexes engineered extracellular vesicles (EVs) for in vivo gene therapy. By leveraging a novel high-throughput microfluidic droplet-based electroporation system (DES), we achieved 10-fold enhancement of loading efficiency and more than 1000-fold increase in processing throughput on loading RNP complexes into EVs (RNP-EVs), compared with conventional bulk electroporation. The flow-through droplets serve as enormous bioreactors for offering millisecond pulsed, low-voltage electroporation in a continuous-flow and scalable manner, which minimizes the Joule heating influence and surface alteration to retain natural EV stability and integrity. In the Shaker-1 mouse model of dominant progressive hearing loss, we demonstrated the effective delivery of RNP-EVs into inner ear hair cells, with a clear reduction of Myo7ash1 mRNA expression compared to RNP-loaded lipid-like nanoparticles (RNP-LNPs), leading to significant hearing recovery measured by auditory brainstem responses (ABR). One sentence summaryThe scalable microfluidic electroporation system enables the loading of gRNA:Cas9 ribonucleoprotein (RNP) complexes into extracellular vesicles, which leads to clinical translation potential employed in hearing disease gene therapy.

bioengineering↗

bioRxiv2023

Convergent alterations in the tumor microenvironment of MYC-driven human and murine prostate cancer

The tissue microenvironment in prostate cancer is profoundly altered. While such alterations have been implicated in driving prostate cancer initiation and progression to aggressive disease, how prostate cancer cells and their precursors mediate those changes is unclear, in part due to the inability to longitudinally study the disease evolution in human tissues. To overcome this limitation, we performed extensive single-cell RNA-sequencing (scRNA-seq) and rigorous molecular pathology of the comparative biology between human prostate cancer and key time points in the disease evolution of a genetically engineered mouse model (GEMM) of prostate cancer. Our studies of human tissues, with validation in a large external data set, revealed that cancer cell-intrinsic activation of MYC signaling was the top up-regulated pathway in human cancers, representing a common denominator across the well-known molecular and pathological heterogeneity of human prostate cancer. Likewise, numerous non-malignant cell states in the tumor microenvironment (TME), including non-cancerous epithelial, immune, and fibroblast cell compartments, were conserved across individuals, raising the possibility that these cell types may be a sequelae of the convergent MYC activation in the cancer cells. To test this hypothesis, we employed a GEMM of prostate epithelial cell-specific MYC activation in two mouse strains. Cell communication network and pathway analyses suggested that MYC oncogene-expressing neoplastic cells, directly and indirectly, reprogrammed the TME during carcinogenesis, leading to the emergence of cascading cell state alterations in neighboring epithelial, immune, and fibroblast cell types that paralleled key findings in human prostate cancer. Importantly, among these changes, the progression from a precursor-enriched to invasive-cancer-enriched state was accompanied by a cell-intrinsic switch from pro-immunogenic to immunosuppressive transcriptional programs with coinciding enrichment of immunosuppressive myeloid and Treg cells in the immune microenvironment. These findings implicate activation of MYC signaling in reshaping convergent aspects of the TME of prostate cancer as a common denominator across the otherwise well-documented molecular heterogeneity of human prostate cancer.

cancer biology↗