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The brain of the domestic pig (Sus scrofa domesticus) has drawn considerable attention due to its high similarities to that of humans. However, the cellular compositions of the pig brain (PB) remain elusive. Here we investigated the single-nucleus transcriptomic profiles of five regions of the PB (frontal lobe, parietal lobe, temporal lobe, occipital lobe, and hypothalamus) and identified 21 cell subpopulations. The cross-species comparison of mouse and pig hypothalamus revealed the shared and specific gene expression patterns at the single-cell resolution. Furthermore, we identified cell types and molecular pathways closely associated with neurological disorders, bridging the gap between gene mutations and pathogenesis. We reported, to our knowledge, the first single-cell atlas of domestic pig cerebral cortex and hypothalamus combined with a comprehensive analysis across species, providing extensive resources for future research regarding neural science, evolutionary developmental biology, and regenerative medicine.

Olfaction, the sense of smell, is a fundamental trait crucial to many species. The olfactory bulb (OB) plays pivotal roles in processing and transmitting odor information from the environment to the brain. The cellular heterogeneity of the mouse OB has been studied using single-cell RNA sequencing. However, the epigenetic landscape of the mOB remains mostly unexplored. Herein, we apply single-cell assay for transposase-accessible chromatin sequencing to profile the genome-wide chromatin accessibility of 9,549 single cells from the mOB. Based on single-cell epigenetic signatures, mOB cells are classified into 21 clusters corresponding to 11 cell types. We identify distinct sets of putative regulatory elements specific to each cell cluster from which putative target genes and enriched potential functions are inferred. In addition, the transcription factor motifs enriched in each cell cluster are determined to indicate the developmental fate of each cell lineage. Our study provides a valuable epigenetic data set for the mOB at single-cell resolution, and the results can enhance our understanding of regulatory circuits and the therapeutic capacity of the OB at the single-cell level.

Alternative splicing (AS) presents a key posttranscriptional regulatory mechanism associated with numerous physiological processes. However, little is known about its role in skeletal muscle atrophy. In this study, we used a rat model of denervated skeletal muscle atrophy and performed RNA-sequencing to analyze transcriptome profiling of tibialis anterior muscle at multiple time points following denervation. We found that AS is a novel mechanism involving muscle atrophy, which is independent changes at the transcript level. Bioinformatics analysis further revealed that AS transitions are associated with the appearance of the atrophic phenotype. Moreover, we found that the inclusion of multiple highly conserved exons of Obscn markedly increased at 3 days after denervation. In addition, we confirmed that this newly transcript inhibited C2C12 cell proliferation and exacerbated myotube atrophy. Finally, our study revealed that a large number of RNA-binding proteins were upregulated when the atrophy phenotype appeared. Our data emphasize the importance of AS in this process.

Vertebrate evolution was accompanied by two rounds of whole-genome duplication followed by functional divergence in terms of regulatory circuits and gene expression patterns. As a basal and slow-evolving chordate species, amphioxus is an ideal paradigm for exploring the origin and evolution of vertebrates. Single-cell sequencing has been widely used to construct the developmental cell atlas of several representative species of vertebrates (human, mouse, zebrafish, and frog) and tunicates (sea squirts). Here, we perform single-nucleus RNA sequencing (snRNA-seq) and single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) for different stages of amphioxus (covering embryogenesis and adult tissues). With the datasets generated, we constructed a developmental tree for amphioxus cell fate commitment and lineage specification and characterize the underlying key regulators and genetic regulatory networks. The data are publicly available on the online platform AmphioxusAtlas.

Heterogeneous nuclear ribonucleoprotein A1 and A2 (hnRNP A1/2) is a ubiquitously expressed RNA binding protein known to bind intronic or exonic splicing silencer. Binding of hnRNP A1/2 to survival of motor neuron gene (SMN1/2) exon 7 and flanking sequences strongly inhibits the inclusion of exon 7, which causes spinal muscular atrophy, a common genetic disorder. However, the role of hnRNP A1/2 on the side away from exon 7 is unclear. Here using antisense oligonucleotides, we fished an intronic splicing enhancer (ISE) near the 3'-splice site (SS) of intron 7 of SMN1/2. Mutagenesis identified the efficient motif of the ISE as "UAGUAGG", coupled with RNA pull down and protein overexpression, we proved that hnRNP A1/2 binding to the ISE promotes the inclusion of SMN1/2 exon 7. Using MS2-tethering array and "UAGGGU" motif walking, we further uncovered that effects of hnRNP A1/2 on SMN1/2 exon 7 splicing are position-dependent: exon 7 inclusion is inhibited when hnRNP A1/2 binds proximal to the 5'SS of intron 7, promoted when its binds proximal to the 3'SS. These data provide new insights into the splicing regulatory mechanism of SMN1/2.

Advances in single-cell sequencing technology provide a unique approach to characterize the heterogeneity and distinctive functional states at single-cell resolution, leading to rapid accumulation of large-scale single-cell datasets. A big challenge undertaken by research community especially bench scientists is how to simplify the way of retrieving, processing and analyzing the huge number of datasets. Towards this end, we developed Cell-omics Data Coordinate Platform (CDCP), a platform that aims to share and integrate comprehensive single-cell datasets, and to provide a network analysis toolkit for personalized analysis. CDCP contains single-cell RNA-seq and ATAC-seq datasets of 474 572 cells from 6 459 samples in species covering humans, non-human primate models and other animals. It allows querying and visualization of interested datasets and the expression profile of distinct genes in different cell clusters and cell types. Besides, this platform provides an analysis pipeline for non-bioinformatician experimental scientists to address questions not focused by the submitters of the datasets. In summary, CDCP provides a user-friendly interface for researchers to explore, visualize, analyze, download and submit published single-cell datasets and it will be a valuable resource for investigators to explore the global transcriptome profiling at single-cell level.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) issued a significant and urgent threat to global health. The exact animal origin of SARS-CoV-2 remains obscure and understanding its host range is vital for preventing interspecies transmission. Previously, we have assessed the target cell profiles of SARS-CoV-2 in pets, livestock, poultry and wild animals. Herein, we expand this investigation to a wider range of animal species and viruses to provide a comprehensive source for large-scale screening of potential virus hosts. Single cell atlas for several mammalian species (alpaca, hamster, hedgehog, chinchilla etc.), as well as comparative atlas for lung, brain and peripheral blood mononuclear cells (PBMC) for various lineages of animals were constructed, from which we systemically analyzed the virus entry factors for 113 viruses over 20 species from mammalians, birds, reptiles, amphibians and invertebrates. Conserved cellular connectomes and regulomes were also identified, revealing the fundamental cell-cell and gene-gene cross-talks between these species. Overall, our study could help identify the potential host range and tissue tropism of SARS-CoV-2 and a diverse set of viruses and reveal the host-virus co-evolution footprints.

Glial cells, including astrocytes, oligodendrocytes, and microglia, are the major components in the central nervous system (CNS). Studies have revealed the heterogeneity of each glial cell type and that they each may play distinct roles in physiological processes and/or neurological diseases. Single-cell sequencing (scRNA-seq) technology developed in recent years has extended our understanding of glial cell heterogeneity from the perspective of transcriptome profiling. This review summarizes the marker genes of major glial cells in the CNS and reveals their heterogeneity in different species, CNS regions, developmental stages, and pathological states (Alzheimer’s disease and spinal cord injury), expanding our knowledge of glial cell heterogeneity on both molecular and functional levels.

Vertebrate evolution was accompanied with two rounds of whole genome duplication followed by functional divergence in terms of regulatory circuits and gene expression patterns. As a basal and slow-evolving chordate species, amphioxus is an ideal paradigm for exploring the origin and evolution of vertebrates. Single cell sequencing has been widely employed to construct the developmental cell atlas of several key species of vertebrates (human, mouse, zebrafish and frog) and tunicate (sea squirts). Here, we performed single-nucleus RNA sequencing (snRNA-seq) and single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) for different stages of amphioxus (covering embryogenesis and adult tissues). With the datasets generated we constructed the developmental tree for amphioxus cell fate commitment and lineage specification, and revealed the underlying key regulators and genetic regulatory networks. The generated data were integrated into an online platform, AmphioxusAtlas, for public access at http://120.79.46.200:81/AmphioxusAtlas.

SummaryGrain size is determined by the number of cells and cell size of the grain. Regulation of grain size is crucial for improving crop yield. However, the genes and underlying molecular mechanisms controlling grain size remain elusive. Here we report a member of Detoxification efflux carrier (DTX)/Multidrug and Toxic Compound Extrusion (MATE) family transporter, BIG RICE GRAIN 1 (BIRG1), negatively regulates the grain size in rice. BIRG1 is highly expressed in reproductive organs and roots. In birg1 grain, the size of the outer parenchyma layer cells of spikelet hulls is noticeably larger but the cell number is not altered compared with that in the wild-type (WT) grain. When expressed in Xenopus oocytes, BIRG1 exhibits chloride efflux activity. In line with the role of BIRG1 in mediating chloride efflux, the birg1 mutant shows reduced tolerance to salt stress under which the chloride level is toxic. Moreover, the birg1 grains contain higher level of chloride compared to WT grains when grown under normal paddy field. The birg1 roots accumulate more chloride than those of WT under saline condition. Collectively, our findings suggest that BIRG1 functions as a chloride efflux transporter regulating grain size and salt tolerance via controlling chloride homeostasis in rice.

Antisense nucleic acids are single-stranded oligonucleotides that have been specially chemically modified, which can bind to RNA expressed by target genes through base complementary pairing and affect protein synthesis at the level of posttranscriptional processing or protein translation. In recent years, the application of antisense nucleic acid technology in the treatment of neuromuscular diseases has made remarkable progress. In 2016, the US FDA approved two antisense nucleic acid drugs for the treatment of Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA), and the development to treat other neurodegenerative diseases has also entered the clinical stage. Therefore, ASO represents a treatment with great potential. The article will summarize ASO therapies in terms of mechanism of action, chemical modification, and administration methods and analyze their role in several common neurodegenerative diseases, such as SMA, DMD, and amyotrophic lateral sclerosis (ALS). This article systematically summarizes the great potential of antisense nucleic acid technology in the treatment of hereditary neurodegenerative diseases.

Heterogeneous nuclear ribonucleoprotein (HNRNP) A1 and A2 are the most abundant HNRNPs with nearly identical functions, and play important roles in regulating gene expression at multiple levels (i.e. transcription, posttranscription, and translation). However, the expression and regulation mechanism of HNRNPA1 and A2 themselves remain unclear. In this study, the amino acid sequences of HNRNPA1 and HNRNPA2 were compared and found to have 78% and 86% homology in key functional domains. Transfection of HEK293 cells with small interfering RNA and overexpression vectors of HNRNPA1 and HNRNPA2 demonstrated that HNRNPA1 and HNRNPA2 paralogs regulate each other’s expression in a compensatory manner at both the RNA and protein levels. Multiprimer reverse transcription-polymerase chain reaction showed that HNRNPA1 and HNRNPA2 did not affect splicing of the HNRNPA2 and HNRNPA1 gene. Using luciferase reporting system, we found that compensatory degradation was mediated by the 3′UTR of the two genes rather than by the promoter. Moreover, treatment with cycloheximide inhibited the compensatory regulation. Our results indicate a novel regulation mechanism of HNRNPA1 and A2 expression. Through compensatory regulation, the expression levels of HNRNPA1 and HNRNPA2 are strictly controlled within a certain range to maintain normal cellular activities under different physiological conditions.

SummarySystem-wide molecular characteristics of COVID-19, especially in those patients without comorbidities, have not been fully investigated. We compared extensive molecular profiles of blood samples from 231 COVID-19 patients, ranging from asymptomatic to critically ill, importantly excluding those with any comorbidities. Amongst the major findings, asymptomatic patients were characterized by highly activated anti-virus interferon, T/natural killer (NK) cell activation, and transcriptional upregulation of inflammatory cytokine mRNAs. However, given very abundant RNA binding proteins (RBPs), these cytokine mRNAs could be effectively destabilized hence preserving normal cytokine levels. In contrast, in critically ill patients, cytokine storm due to RBPs inhibition and tryptophan metabolites accumulation contributed to T/NK cell dysfunction. A machine-learning model was constructed which accurately stratified the COVID-19 severities based on their multi-omics features. Overall, our analysis provides insights into COVID-19 pathogenesis and identifies targets for intervening in treatment.

A few animals have been suspected to be intermediate hosts of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, a large-scale single-cell screening of SARS-CoV-2 target cells on a wide variety of animals is missing. Here, we constructed the single-cell atlas for 11 representative species in pets, livestock, poultry, and wildlife. Notably, the proportion of SARS-CoV-2 target cells in cat was found considerably higher than other species we investigated and SARS-CoV-2 target cells were detected in multiple cell types of domestic pig, implying the necessity to carefully evaluate the risk of cats during the current COVID-19 pandemic and keep pigs under surveillance for the possibility of becoming intermediate hosts in future coronavirus outbreak. Furthermore, we screened the expression patterns of receptors for 144 viruses, resulting in a comprehensive atlas of virus target cells. Taken together, our work provides a novel and fundamental strategy to screen virus target cells and susceptible species, based on single-cell transcriptomes we generated for domesticated animals and wildlife, which could function as a valuable resource for controlling current pandemics and serve as an early warning system for coping with future infectious disease threats.

Background: Glucocorticoids, including dexamethasone (Dex), are corticosteroids secreted by the adrenal gland, which are used as potent anti-inflammatory, anti-shock, and immunosuppressive agents. Dex is commonly used in patients with malignant tumors, such lung cancer. However, administration of high-dose Dex induces severe atrophy of the skeletal muscle, and the underlying mechanisms of this skeletal muscle atrophy remain unclear. Abundant miRNAs of skeletal muscle, such as miR-351, play an important role in the regulation of extenuating the process of muscle atrophy. Methods: The mRNA and protein expression of TRAF6, MuRF1, MAFbx was determined by real-time PCR and western blot, while the expression of miR-351 was detected by real-time PCR. The myotubes were transfected with miR-351 mimic, negative control, or miR-351 inhibitor. The C2C12 myotubes diameter was measured. Results: MicroRNA351 (miR-351) level was markedly reduced and the mRNA and protein levels of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) were increased in Dex-induced C2C12 myotube atrophy. miR-351 directly interacted with the 3'-untranslated region (3'UTR) of TRAF6. Interestingly, miR-351 administration notably inhibited the reduction of the C2C12 myotube diameter induced by Dex treatment and reduced the levels of TRAF6, muscle-RING-finger protein-1 (MuRF1), and muscle atrophy F-box (MAFbx). Conclusions: miR-351 counteracts Dex-induced C2C12 myotube atrophy by repressing the TRAF6 expression as well as E3 ubiquitin ligase MuRF1 and MAFbx. miR-351 maybe a potential target for development of a new strategy for skeletal muscle atrophy.

Domestic pig (Sus scrofa domesticus) has drawn much attention from researchers worldwide due to its implications in evolutionary biology, regenerative medicine and agriculture. The brain atlas of Homo sapiens (primate), Mus musculus (rodent), Danio rerio (fish) and Drosophila melanogaster (insect) have been constructed at single cell resolution, however, the cellular compositions of pig brain remain largely unexplored. In this study, we investigated the single-cell transcriptomic profiles of five distinct regions of domestic pig brain, from which we identified 21 clusters corresponding to six major cell types, characterized by unique spectrum of gene expression. By spatial comparison, we identified cell types enriched or depleted in certain brain regions. Inter-species comparison revealed cell-type similarities and divergences in hypothalamus between mouse and pig, providing invaluable resources for the evolutionary exploration of brain functions at single cell level. Besides, our study revealed cell types and molecular pathways closely associated with several diseases (obesity, anorexia, bulimia, epilepsy, intellectual disability, and autism spectrum disorder), bridging the gap between gene mutations and pathological phenotypes, which might be of great use to the development precise therapies against neural system disorders. Taken together, we reported, so far as we know, the first single cell brain atlas of Sus scrofa domesticus, followed by comprehensive comparisons across brain region and species, which could throw light upon future evo-devo, regenerative medicine, and agricultural studies.

Background: Skeletal muscle atrophy, characterized by accelerated protein degradation, occurs in such conditions as unloading, immobilization, fasting, and denervation. Effective treatments for skeletal muscle atrophy are not yet available. Considering that microRNAs (miRs) may play an important role in the regulation of muscle atrophy, in the present study, we aimed to examine the effect of miR-125b-5p-based therapeutic strategies on skeletal muscle atrophy, and to explore the underlying mechanisms. Methods: Fasting-induced atrophic mouse C2C12 myotubes and denervated rat tibialis anterior (TA) muscles were used as in vitro and in vivo models of skeletal muscle atrophy, respectively. The morphological parameters of skeletal muscle were measured by immunostaining-based quantification. The interaction between miR-125b-5p and TRAF6 3'-UTR was detected by luciferase reporter analysis. The mRNA and protein expressions were determined by real-time qPCR and Western blot analysis respectively. The miR mimics/agomir and miR inhibitor/antagomir were transfected into C2C12 myotubes and TA muscles respectively to alter the expression of miR-125b-5p. Results: The expression of miR-125b-5p was down-regulated in both atrophic C2C12 myotubes and denervated TA muscles. The interaction between miR-125b-5p and TRAF6 3'-UTR was identified. Overexpression of miR-125b-5p protected skeletal muscle samples from atrophy in vitro and in vivo by targeting TRAF6 through inactivation of several ubiquitin-proteasome system (UPS)- and autophagy-lysosome system (ALS)-related proteins. Conclusions: Overexpression of miR-125b-5p may provide a promising therapeutic approach to treat muscle atrophy.

Glucocorticoids, including dexamethasone (Dex), are corticosteroids secreted by the adrenal gland, which are used as potent anti-inflammatory, anti-shock, and immunosuppressive agents. Dex is commonly used in patients with malignant tumors, such lung cancer. However, administration of high-dose Dex induces severe atrophy of the skeletal muscle, and the underlying mechanisms of this skeletal muscle atrophy remain unclear. Abundant miRNAs of skeletal muscle, such as miR-351, play an important role in the regulation of extenuating the process of muscle atrophy.The mRNA and protein expression of TRAF6, MuRF1, MAFbx was determined by real-time PCR and western blot, while the expression of miR-351 was detected by real-time PCR. The myotubes were transfected with miR-351 mimic, negative control, or miR-351 inhibitor. The C2C12 myotubes diameter was measured.MicroRNA351 (miR-351) level was markedly reduced and the mRNA and protein levels of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) were increased in Dex-induced C2C12 myotube atrophy. miR-351 directly interacted with the 3'-untranslated region (3'UTR) of TRAF6. Interestingly, miR-351 administration notably inhibited the reduction of the C2C12 myotube diameter induced by Dex treatment and reduced the levels of TRAF6, muscle-RING-finger protein-1 (MuRF1), and muscle atrophy F-box (MAFbx).miR-351 counteracts Dex-induced C2C12 myotube atrophy by repressing the TRAF6 expression as well as E3 ubiquitin ligase MuRF1 and MAFbx. miR-351 maybe a potential target for development of a new strategy for skeletal muscle atrophy.

MicroRNAs (miRs) have been observed to be involved in the modulation of various physiopathological processes. However, the impacts of miRNAs on muscle atrophy have not been fully investigated. In the present study, the results demonstrated that miR-351 was differentially expressed in the tibialis anterior (TA) muscle at various times following sciatic nerve transection, and the time-dependent expression profile of miR-351 was inversely correlated with that of tumor necrosis factor receptor-associated factor 6 (TRAF6) at the mRNA and protein levels. The dual luciferase reporter assay indicated that miR-351 was able to significantly downregulate the expression levels of TRAF6 by directly targeting the 3′-untranslated region of TRAF6. Overexpression of miR-351 inhibited a significant decrease in the wet weight ratio or cross-sectional area of the TA muscle following sciatic nerve transection. Western blot analysis indicated that the protein expression levels of TRAF6, muscle ring-finger protein 1 (MuRF1) and muscle atrophy F-box (MAFBx) in denervated TA muscles were suppressed by overexpression of miR-351. These results demonstrate that miR-351 inhibits denervation-induced atrophy of TA muscles following sciatic nerve transection at least partially through negative regulation of TRAF6 as well as MuRF1 and MAFBx, the two downstream signaling molecules of TRAF6.

Background: Cachexia, a wasting syndrome, is most commonly observed in individuals with advanced cancer including lung cancer, esophageal cancer, liver cancer, etc. The characteristic sign of cachexia is muscle atrophy. To date, effective countermeasures have been still deficiency to alleviate muscle atrophy. Reactive oxygen species (ROS) are important regulators of muscle atrophy. Therefore, the effects of a naturally antioxidant, pyrroloquinoline quinone (PQQ), were explored on muscle atrophy induced by cachexia in the present study. Methods: Tumor necrosis factor-α (TNF-α) induced C2C12 myotubes atrophy model was constructed. The atrophied C2C12 myotubes were dealt with the presence or absence of N-acetyl-L-cysteine (NAC, an antioxidant for ROS abolition) (5 mM) or PQQ (80 µM) for 24 hours. ROS content was determined by dichlorodihydrofluorescein diacetate (DCFH-DA) staining. The diameter of myotubes was analyzed by myosin heavy chain (MHC) staining. The protein levels of MHC, muscle atrophy F-box (MAFbx) and muscle RING finger-1 (MuRF-1) in each group were observed by Western blotting. Results: First, ROS generation was enhanced in C2C12 myotubes treated with TNF-α. NAC treatments significantly avoided the reduction in the diameter of C2C12 myotubes, and concomitantly increased MHC levels, and decreased ROS contents, MuRF1 and MAFbx levels. These data suggested that the increased ROS induced by TNF-α might play a central role in muscle wasting. PQQ (a naturally occurring antioxidant) administration inhibited C2C12 myotubes atrophy induced by TNF-α, as evidenced by the increase of the diameter of C2C12 myotubes, together with increased MHC levels and decreased ROS, MAFbx and MuRF-1 levels. Conclusions: PQQ resists atrophic effect dependent on, at least in part, decreased ROS in skeletal muscle treated with TNF-α.

Skeletal muscle atrophy occurs under various conditions, such as disuse, denervation, fasting, aging, and various diseases. Although the underlying molecular mechanisms are still not fully understood, skeletal muscle atrophy is closely associated with reactive oxygen species (ROS) overproduction. In this study, we aimed to investigate the involvement of ROS in skeletal muscle atrophy from the perspective of gene regulation, and further examine therapeutic effects of antioxidants on skeletal muscle atrophy. Microarray data showed that the gene expression of many positive regulators for ROS production were up-regulated and the gene expression of many negative regulators for ROS production were down-regulated in mouse soleus muscle atrophied by denervation (sciatic nerve injury). The ROS level was significantly increased in denervated mouse soleus muscle or fasted C2C12 myotubes that had suffered from fasting (nutrient deprivation). These two muscle samples were then treated with N-acetyl-L-cysteine (NAC, a clinically used antioxidant) or pyrroloquinoline quinone (PQQ, a naturally occurring antioxidant), respectively. As compared to non-treatment, both NAC and PQQ treatment (1) reversed the increase in the ROS level in two muscle samples; (2) attenuated the reduction in the cross-sectional area (CSA) of denervated mouse muscle or in the diameter of fasted C2C12 myotube; (3) increased the myosin heavy chain (MHC) level and decreased the muscle atrophy F-box (MAFbx) and muscle-specific RING finger-1 (MuRF-1) levels in two muscle samples. Collectively, these results suggested that an increased ROS level was, at least partly, responsible for denervation- or fasting-induced skeletal muscle atrophy, and antioxidants might resist the atrophic effect via ROS-related mechanisms.

Tumor necrosis factor receptor-associated factor 6 (TRAF6), a unique E3 ubiquitin ligase and adaptor protein, is involved in activation of various signaling cascades. Recent studies identify TRAF6 as one of the novel regulators of skeletal muscle atrophy. The role of TRAF6 in glucocorticoid-induced muscle atrophy, however, remains to be elucidated. In this study, we show that TRAF6 and its downstream signaling molecules, muscle atrophy F-box (MAFBx) and muscle ring finger 1 (MuRF1), were all upregulated in dexamethasone-induced atrophy of mouse C2C12 myotubes or mouse tibialis anterior (TA) muscle. To further investigate the role of TRAF6 in dexamethasone-induced muscle atrophy, TRAF6-siRNA was used to transfect cultured C2C12 myotubes or was injected into the TA muscle of mice respectively, and we note that TRAF6 knockdown attenuated dexamethasone-induced muscle atrophy in vitro and in vivo, and concomitantly decreased the expression of MuRF1 and MAFBx. Our findings suggest that a decreased expression of TRAF6 could rescue dexamethasone-induced skeletal muscle atrophy through, at least in part, regulation of the expression of MAFBx and MuRF1.

Background: Oxidative stress has been recognized to play a crucial role in the pathogenesis of peripheral nerve injury. Isoquercitrin (quercetin-3-glucoside) is a flavonoid that exhibited many biological activities, including anti- oxidative effect. However, it is unclear whether isoquercitrin has protective effects on peripheral nerve injury. Methods: Mice treated by isoquercitrin were used as a case group, and mice injected with saline was the control group. Sciatic behavioral function was assessed using SFI and CMAPs were measured by electrophysiology. Schwann cells proliferation and migration were tested using EdU staining and Transwell migration chambers respectively. The expression of oxidative stress related factors were detected by qRT-PCR and Western blotting. Results: In present study, our results demonstrated that isoquercitrin (20 mg/kg/day) treatment achieved significantly higher SFI and higher amplitude of CMAP, promoted the nerve regeneration and remyelination, increased the production of GAP43, NF200, MAG and PMP22, alleviated target muscle atrophy and autophagy, and suppressed the expression of ATG7, PINK1 and Beclin1 in soleus muscles after sciatic nerve crush. In vitro studies found that isoquercitrin promoted the axonal regeneration of DRGs neurons, the proliferation and migration of Schwann cells, and the expression of proliferating cell nuclear antigen (PCNA) in Schwann cells. The administration of isoquercitrin at 40 and 320 μM showed a dose dependent, and high doses of isoquercitrin (160 and 320 μM) showed better performance in promoting axonal regeneration of DRGs neurons, and the proliferation and migration of Schwann cells than low dose of isoquercitrin (40 μM). Furthermore, isoquercitrin significantly inhibited oxidative stress through reducing the production of Nox4 and Duox1, and promoting the expression of Nrf2 and SOD2 in soleus muscles after sciatic nerve crush. Conclusions: Isoquercitrin may promote motor functional recovery and nerve regeneration following peripheral nerve injury though inhibition of oxidative stress, which highlighted the therapeutic values of isoquercitrin as a neuroprotective drug for peripheral nerve repair applications.

The aim of this study was to improve our understanding and the current treatment of denervation-induced skeletal muscle atrophy. We used isobaric tags for relative and absolute quantification (iTRAQ) coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) to identify the differentially expressed proteins in the tibialis anterior (TA) muscle of rats at 1 and 4 weeks following sciatic nerve transection. A total of 110 proteins was differentially expressed and was further classified using terms from the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to unravel their molecular functions. Among the differentially expressed metabolic enzymes involved in glycolysis, Krebs cycle and oxidative phosphorylation, α- and β-enolase displayed an increased and decreased expression, respectively, which was further validated by western blot analysis and immunohistochemistry. These findings suggest that the enolase isozymic switch during denervation-induced muscle atrophy is the reverse of that occurring during muscle maturation. Notably, protein‑protein interaction analysis using the STRING database indicated that the protein expression of tumor necrosis factor receptor-associated factor-6 (TRAF6), muscle ring-finger protein 1 (MuRF1) and muscle atrophy F-box (MAFBx) was also upregulated during denervation‑induced skeletal muscle atrophy, which was confirmed by western blot analysis. TRAF6 knockdown experiments in L6 myotubes suggested that the decreased expression of TRAF6 attenuated glucocorticoid‑induced myotube atrophy. Therefore, we hypothesized that the upregulation of TRAF6 may be involved in the development of denervation‑induced muscle atrophy, at least in part, by regulating the expression of MAFBx and MuRF1 proteins. The data from the present study provide valuable insight into the molecular mechanisms regulating denervation-induced muscle atrophy.