Growing evidence suggests that hypoxia-inducible factor-α (HIF-1α) plays an important role in the progression of allergic airway inflammation and remodeling. However, the biochemical mechanisms leading to the activation of HIF-1α and the effects of HIF-1α on the expression of growth factors, including vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), and fibroblast growth factor-2 (FGF-2), in allergic nasal inflammation are not clear. We examined the relationship between HIF-1α activation and production of VEGF, TGF-β1, and FGF-2 in primary cultured nasal epithelial cells (NECs) after stimulation with house dust mite (HDM) extract. Moreover, we evaluated the importance of phosphoinositide3-kinase(PI3K)/Akt signaling in HDM-induced production of these growth factors in vitro and in the nasal mucosa of a murine model of allergic rhinitis (AR). Our results indicate HDM extract induced the expression of VEGF, TGF-β1, and FGF-2 by activating the PI3K/Akt/HIF-1α pathway in human primary cultured NECs and in the nasal mucosa of a murine model. HIF-1α regulated the expression of VEGF, TGF-β1, and FGF-2 in the nasal mucosa through direct and indirect pathways, which suggested that targeting the HIF-1α pathway could be a novel therapeutic approach for reducing nasal airway inflammation and remodeling in AR.

Colorectal cancer (CRC) is one of the most common types of malignant tumor worldwide. Currently, although many researchers have been devoting themselves in CRC studies, the process of locating biomarkers for CRC early diagnosis and prognostic is still very slow. Using a centrifugal proteomic reactor-based proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling, 2620 protein groups were quantified between cancer mucosa and adjacent normal colorectal mucosa. Of which, 403 protein groups were differentially expressed with statistic significance between cancer and normal tissues, including 195 up-regulated and 208 down-regulated proteins in cancer tissues. Three proteins (SOD3, PRELP and NGAL) were selected for further Western blot validation. And the resulting Western blot experimental results were consistent with the quantitative proteomic data. SOD3 and PRELP are down-regulated in CRC mucosa comparing to adjacent normal tissue, while NGAL is up-regulated in CRC mucosa. In conclusion, the centrifugal proteomic reactor-based label-free quantitative proteomic approach provides a highly sensitive and powerful tool for analyzing minute protein sample from tiny colorectal biopsies, which may facilitate CRC biomarkers discovery for diagnoses and prognoses.

Abnormal neurogenesis occurs during embryonic development in human diabetic pregnancies and in animal models of diabetic embryopathy. Our previous studies in a mouse model of diabetic embryopathy have implicated that high glucose of maternal diabetes delays neurogenesis in the developing neuroepithelium leading to neural tube defects. However, the underlying process in high glucose-impaired neurogenesis is uncharacterized. Neurogenesis from embryonic stem (ES) cells provides a valuable model for understanding the abnormal neural lineage development under high glucose conditions. ES cells are commonly generated and maintained in high glucose (approximately 25 mM glucose). Here, the mouse ES cell line, E14, was gradually adapted to and maintained in low glucose (5 mM), and became a glucose responsive E14 (GR-E14) line. High glucose induced the endoplasmic reticulum stress marker, CHOP, in GR-E14 cells. Under low glucose conditions, the GR-E14 cells retained their pluripotency and capability to differentiate into neural lineage cells. GR-E14 cell differentiation into neural stem cells (Sox1 and nestin positive cells) was inhibited by high glucose. Neuron (Tuj1 positive cells) and glia (GFAP positive cells) differentiation from GR-E14 cells was also suppressed by high glucose. In addition, high glucose delayed GR-E14 differentiation into neural crest cells by decreasing neural crest markers, paired box 3 (Pax3) and paired box 7 (Pax7). Thus, high glucose impairs ES cell differentiation into neural lineage cells. The low glucose adapted and high glucose responsive GR-E14 cell line is a useful in vitro model for assessing the adverse effect of high glucose on the development of the central nervous system.

Lipid rafts play an important role in the life cycle of many viruses. Cholesterol is a critical structural component of lipid rafts. Although the porcine reproductive and respiratory syndrome virus (PRRSV) has restricted cell tropism for cells of the monocyte/macrophage lineage, a non-macrophage cell MARC-145 was susceptible to PRRSV because of the expression of virus receptor CD163 on the cell surface, therefore MARC-145 cells is used as model cell for PRRSV studies. In order to determine if cholesterol is involved in PRRSV infection in MARC-145 cells, we used three pharmacological agents: methyl-β cyclodextrin (MβCD), mevinolin, and filipin complex to deplete cholesterol in MARC-145. Although these agents act by different mechanisms, they all significantly inhibited PRRSV infection. The inhibition could be prevented by addition of exogenous cholesterol. Cell membrane cholesterol depletion after virus infection had no effect on PRRSV production and cholesterol depletion pre-infection did not reduce the virus attachment, suggesting cholesterol is involved in virus entry. Further results showed that cholesterol depletion did not change expression levels of the PRRSV receptor CD163 in MARC-145, had no effect on clathrin-mediated endocytosis, but disturbed lipid-raft-dependent endocytosis. Collectively, these studies suggest that cholesterol is critical for PRRSV entry, which is likely to be mediated by a lipid-raft-dependent pathway.

Treatment of cells with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial proton gradient uncoupler, can result in mitochondrial damage and autophagy activation, which in turn eliminates the injured mitochondria in a Parkin-dependent way. How CCCP mobilizes the autophagy machinery is not fully understood. By analyzing a key autophagy step, LC3 lipidation, we examined the roles of two kinase complexes typically involved in the initiation and nucleation phases of autophagy, namely the ULK kinase complex (UKC) and the Beclin 1/Atg14 complex. We found that CCCP-induced LC3 lipidation could be independent of Beclin 1 and Atg14. In addition, deletion or knockdown of the UKC component FIP200 or Atg13 only led to a partial reduction in LC3 lipidation, indicating that UKC could be also dispensable for this step during CCCP treatment. In contrast, Atg9, which is important for transporting vesicles to early autophagosomal structure, was required for CCCP-induced LC3 lipidation. Taken together, these data suggest that CCCP-induced autophagy and mitophagy depends more critically on Atg9 vesicles than on UKC and Beclin 1/Atg14 complex.

Several studies have demonstrated that allergic asthma can induce atherosclerosis formation in mice. Moreover, allergic asthma and atherosclerosis have been shown to be strongly associated with dyslipidemia. In this study, we investigated the underlying mechanism of allergic asthma-aggravated atherosclerosis-induced cholesterol metabolism disorder in asthmatic apoE-/- mice. We found that allergic asthma increased the expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) in the liver and CD36 in the aorta during the acute and advanced stages of atherosclerosis, respectively. These results indicate that cholesterol biosynthesis is increased during acute atherosclerosis and cholesterol uptake and foam cell formation is increased during advanced atherosclerosis. Simvastatin administration significantly ameliorated the aortic root lesion size of asthmatic mice and significantly decreased HMGCR and CD36 expression. However, the expression of the low-density lipoprotein receptor and ATP-binding cassette transporter A1 was markedly increased, indicating that the beneficial effect of statins in allergic asthma and coronary artery disease was mediated, at least in part, by decreasing cholesterol biosynthesis and foam cell formation. In conclusion, allergic asthma aggravates atherosclerosis by regulating cholesterol metabolism in apoE-/- mice. Allergic asthma selectively promotes cholesterol biosynthesis in acute atherosclerosis and increases foam cell formation in advanced atherosclerosis.

Accumulating studies have appreciated circular RNAs (circRNAs) as novel prognostic biomarkers and therapeutic targets in malignant carcinomas. Here, we aim to investigate the expression of a novel circRNA, circ_0070934. The biological roles and mechanisms of circ_0070934 in cutaneous squamous cell carcinoma (CSCC) were explored. The expression of circ_0070934 in CSCC tissues and cell lines was evaluated by qRT-PCR. Loss-of-function and gain-of-function assays were performed to detect cell proliferation, apoptosis, migration and invasion in vitro. Moreover, the underlying molecular mechanism of circ_0070934 was predicted by online database and luciferase reporter assay. Abnormally overexpression of circ_0070934 was detected in CSCC samples and cell lines. Inhibition of cell proliferation, invasion, migration, and increased apoptosis were observed upon circ_0070934 knockdown. The opposite effect was observed in the circ_0070934 overexpression cells. Circ_0070934 expression was negatively correlated with miR-1238 and miR-1247-5p expression in CSCC and luciferase reporter experiment verified the binding ability between circ_0070934 and miR-1238/miR-1247-5p. Rescue experiments further identified that the oncogenic role of circ_0070934 is attributed to its suppression of miR-1238 and miR-1247-5p. Taken together, our results implicate that circ_0070934 is correlated with tumor aggressiveness by serving as an oncogenic circRNA in CSCC.

The nucleocapsid protein is significant in the formation of viral RNA of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), accounting for the largest proportion of viral structural proteins. Here, we report for the first time that the 11S proteasomal activator PA28γ regulates the intracellular abundance of the SARS-CoV-2 N protein (nCoV N). Furthermore, we have identified proteasome activator PA28γ as a nCoV N binding protein by co-immunoprecipitation assay. As a result of their interaction, nCoV N could be degraded by PA28γ-20S in vitro degradation assay. This was also demonstrated by blocking de novo protein synthesis with cycloheximide. The stability of nCoV N in PA28γ-knockout cells was greater than in PA28γ-wildtype cells. Notably, immunofluorescence staining revealed that knockout of the PA28γ gene in cells led to the transport of nCoV N from the nucleus to the cytoplasm. Overexpression of PA28γ enhanced proteolysis of nCoV N compared to that in PA28γ-N151Y cells containing a dominant-negative PA28γ mutation, which reduced this process. These results suggest that PA28γ binding is important in regulating 20S proteasome activity, which in turn regulates levels of the critical nCoV N nucleocapsid protein of SARS-CoV-2, furthering our understanding of the pathogenesis of COVID-19.

Ischemia-reperfusion injury (IR) is the leading cause of acute kidney injury (AKI). No effective drugs to treat IR-related AKI are currently available. Recent pre-clinical trials have evaluated the therapeutic potential of extracellular vesicles-exosomes to chronic kidney disease. Here, we found exosomes derived from the tubular epithelial cell in IR condition (ExoIR) enriched CD26, compared with control (ExoNormal). Tracking exosomes in vivo certified tubular epithelial cell uptake exosomes. We have isolated exosomes with overexpression of CD26 (ExoCD26+) from culture media from tubular epithelial cell line transferred by adenovirus vectors. After administration of exosomes (100 mg) or bovine serum albumin (BSA, equivalent protein control) in IR or sham operation mice after 72 h via tail vein injection, the renal function impairment and histology injury were relived in mice receiving ExoCD26+. Immunofluorescence staining with proliferating cell nuclear antigen revealed ExoCD26+ recovered proliferation of cells partly after IR injury. Cell cycle modulator, p53 and p21 were upregulated in IR mice receiving BSA control, ExoNormal, and ExoIR. ExoCD26+ significantly blunt this protein upregulation. Inflammatory cell infiltration and chemokine receptor (CXCR4) were dissipated in IR mice receiving ExoCD26+. Downstream chemokine of CXCR4, stromal derived factor-1 (SDF1) also decreased after administration of ExoCD26+ in IR mice. Finally, ExoCD26+ suppressed inundant collagenⅠ expression in IR kidney. In conclusion, Tubular epithelial cells derived-exosomes containing CD26 might be one of the therapy modes for IR-AKI by maintaining proliferation and dissipating inflammation.

Genistein, a naturally occurring phytoestrogen and a member of the large class of compounds known as isoflavones, exerts protective effects in several diseases. Recent studies indicate that genistein plays a critical role in controlling body weight, obesity-associated insulin resistance, and metabolic disorders, but its target organs in reversing obesity and related pathological conditions remain unclear. In this study, we showed that mice supplemented with 0.2% genistein in a high-fat diet for 12 weeks showed enhanced metabolic homeostasis, including reduced obesity, improved glucose uptake and insulin sensitivity, and alleviated hepatic steatosis. We also observed a beiging phenomenon in the white adipose tissue and reversal of brown adipose tissue whitening in these mice. These changes led to enhanced resistance to cold stress. Altogether, our data suggest that the improved metabolic profile in mice treated with genistein is likely a result of enhanced adipose tissue function.

Understanding the molecular mechanisms of lipid synthesis in the mammary gland is crucial for regulating the level and composition of lipids in milk. This study aimed to investigate the functional and molecular mechanisms of miR-204-5p in mammary epithelial cells to provide a theoretical basis for milk lipid synthesis.

NAC transcription factors are plant-specific and play important roles in many processes including plant development, response to biotic and abiotic stresses and hormone signaling. So far, only a few NAC genes have been identified to mediate cell death. In this study, we identified a novel NAC gene from canola (Brassica napus L.), BnaNAC103 which induces reactive oxygen species (ROS) accumulation and cell death in Nicotianabenthamiana leaves. We found that BnaNAC103 responded to multiple signalings, including cold, salicylic acid (SA) and a fungal pathogen Sclerotinia sclerotiorum. BnaNAC103 is located in the nucleus. Expression of full-length BnaNAC103, but not either the N-terminal NAC domain or C-terminal regulatory domain, was identified to induce hypersensitive response (HR)-like cell death when expressed in N. benthamiana. The cell death triggered by BnaNAC103 is preceded by accumulation of ROS, with diaminobenzidine (DAB) staining supporting this. Moreover, quantification of ion leakage and malondialdehyde (MDA) of leaf discs indicates significant cell membrane breakage and lipid peroxidation induced by BnaNAC103 expression. Taken together, our work has identified a novel NAC transcription factor gene modulating ROS level and cell death in plants.

Senescence-associated autophagy downregulation leads to cardiomyocyte dysfunction. Coactivator-associated arginine methyltransferase 1 (CARM1) participates in many cellular processes, including autophagy in mammals. However, the effect of CARM1 in aging-related cardiac autophagy decline remains undefined. Moreover, AMP-activated protein kinase (AMPK) is a key regulator in metabolism and autophagy, however, the role of nuclear AMPK in autophagy outcome in aged hearts still unclear. Hers we identify the correlation between nuclear AMPK and CARM1 in aging heart. We found that fasting could promote autophagy in young hearts but not in aged hearts. The CARM1 stabilization is markedly decrease in aged hearts, which impaired nucleus TFEB-CARM1 complex and autophagy flux. Further, S-phase kinase-associated protein 2(SKP2), responsible for CARM1 degradation, was increased in aged hearts. We further validated that AMPK dependent FoxO3 phosphorylation was markedly reduced in nucleus, the decreased nuclear AMPK-FoxO3 activity fails to suppress SKP2-E3 ubiquitin ligase. This loss of repression leads to The CARM1 level and autophagy in aged hearts could be restored through AMPK activation. Taken together, AMPK deficiency results in nuclear CARM1 decrease mediated in part by SKP2, contributing to autophagy dysfunction in aged hearts. Our results identified nuclear AMPK controlled CARM1 stabilization as a new actor that regulates cardiac autophagy.

BNIP3 is found to eliminate cancer cells via causing mitochondrial damage and endoplasmic reticulum stress, but it remains elusive of its role in regulating DNA double strand breaks (DSBs). In this study, we find that silibinin triggers DNA DSBs, ROS accumulation and expressional upregulation of BNIP3 in glioma cells. Mitigation of ROS with antioxidant GSH significantly inhibits silibinin-induced DNA DSBs and glioma cell death. Then, we find knockdown of BNIP3 with SiRNA obviously prevents silibinin-induced DNA DSBs and ROS accumulation. Mechanistically, BNIP3 knockdown not only reverses silibinin-triggered depletion of cysteine and GSH via maintaining xCT level, but also abrogates catalase decrease. Notably, silibinin-induced dephosphorylation of mTOR is also prevented when BNIP3 is knocked down. Given that activated mTOR could promote xCT expression and inhibit autophagic degradation of catalase, our data suggest that BNIP3 contributes to silibinin-induced DNA DSBs via improving intracellular ROS by inhibition of mTOR.

Inflammation or trauma occurring on one side of the body can cause pathological pain on the contralateral noninjured side in a phenomenon called mirror-image pain (MIP). Although some potential mechanisms involved in MIP have been reported, including those involving the immune system and glial cells as well as neural mechanisms, the molecular mechanisms are not well understood. In this study, we aimed to understand the molecular mechanisms in MIP using quantitative proteomics and whole-cell patch clamp recordings. Behavioral test results showed that complete Freund's adjuvant could induce MIP in the mice. The results of isobaric tags for relative and absolute quantification (iTRAQ) quantitative proteomics showed that 108 proteins were dysregulated, and these proteins may represent potential targets. Furthermore, bioinformatics analysis was applied to explore the potential molecular mechanisms during MIP after complete Freund's adjuvant (CFA) treatment. Parallel reaction monitoring (PRM) results showed that PKCδ and seven other dysregulated proteins were related to MIP after CFA treatment. Patch clamp recording results showed that CFA treatment could increase intrinsic excitability and spontaneous firing in spinal cord neurons during MIP. In summary, we found that CFA could induce MIP. The results of proteomic research on the spinal cord after CFA treatment could provide new insight into the molecular mechanisms of MIP. Moreover, the neuronal activity of spinal cord neurons was upregulated during MIP after CFA treatment. In summary, the results of the spinal cord proteomic profile provide a potential molecular mechanism for understanding MIP.

Drug-proton antiporters (DHA) play an important role in multi-drug resistance, utilizing the proton-motive force to drive the expulsion of toxic molecules, including antibiotics and drugs. DHA transporters belong to the major facilitator superfamily (MFS), members of which deliver substrates by utilizing the alternating access model of transport. However, the transport process is still elusive. Here, we report the structures of SotB, a member of DHA1 family (TCDB: 2.A.1.2) from Escherichia coli. Four crystal structures of SotB were captured in different conformations, including substrate-bound occluded, inward-facing, and inward-open states. Comparisons between the four structures reveal nonlinear rigid-body movements of alternating access during the state transition from inward-open to occluded conformation. This work not only reveals the conformational dynamics of SotB but also deepens our understanding of the alternating access mechanism of MFS transporters.

Immunocytokines (antibody-cytokine fusions) have been proved to be a promising class of therapeutic agents for tumors. Anti-PD-L1 antibodies or IL-2 have been used to treat a variety of cancers. Here, in order to remove T cell inhibition and increasing the IL-2 concentration in the tumor microenvironment, we engineered a novel anti-PD-L1 antibody based immunocytokine by fusing hIL-2 to the C-Term of atezolizumab, denoted as BIPI. Our results revealed that BIPI was effective in stimulating T cell activation in vitro and could selectively localize to the tumor. Furthermore, tumor regression and prolonged survival were also observed in the metastatic colorectal cancer mouse model. The obviously longer survival mice in BIPI treatment group turned out depending on the function of CD8+ T cells. The IFN- secreted from CD8+ T cells in the spleen also contributed to the better tumor inhibition profile in BIPI treatment group than in anti-PD-L1 or IL-2 treatment alone. Taken together, our data evidenced the enhanced antitumor potency of BIPI, suggesting its potential use for cancers with a low response to the anti-PD-L1 or IL-2 treatment.

The present study was to evaluate the prognostic value of protein expression of Pofut1 and Notch1 signaling in breast cancer.

DUX4 plays critical role in the molecular pathogenesis of the neuromuscular disorder facioscapulohumeral muscular dystrophy and acute lymphoblastic leukemia in humans. As a master transcription regulator, DUX4 can also bind the promoters and activate the transcription of hundreds ZGA-associated genes. Here we report on the structural and biochemical studies of DUX4 double homeodomains (DUX4-DH), representing the only structures contain both homeodomain 1 (HD1) and homeodomain 2 (HD2). HD1 and HD2 adopt classical homeobox fold; via the helix inserted into the major groove and the N-terminal extended loop inserted into the minor groove, HD1 and HD2 recognize the box1 (5'-TAA-3') and box2 (5'-TGA-3') nucleotides of the consensus sequence, respectively. Among the box1 and box2 linking nucleotides (CCTAA), the two adenine residues are reported to be highly conserved; however, they are not directly recognized by DUX4-DH in the structures. Besides different nucleotides, our ITC analysis indicated that DUX4-DH can also tolerate various changes in the linker length. Our studies not only revealed the basis for target DNA recognition by DUX4, but also advanced our understanding on multiple gene activation by DUX4.

Hepatocellular carcinoma (HCC) is associated with poor prognosis due to many unknowns about its inflammatory microenvironment. As a pivotal proinflammatory cytokine, IL-17A exerts a protective effect on the survival and function of HCC cells. It is widely accepted that IL-17A plays an important role in regulating autophagy. Bcl2, a key molecule promoting the survival of HCC cells, also plays an indispensable role as an autophagy regulator. The aim of this study was to investigate the role of Bcl2 in IL-17A-regulated autophagy of HCC cells. The results showed that IL-17A not only inhibited autophagic activity, but also increased Bcl2 levels in HCC cells under starvation. Besides, IL-17A could prevent the dissociation of autophagy protein Beclin1 from Bcl2-Beclin1 complex upon starvation. Overexpression of Beclin1 rescued the autophagy deficiency of HCC cells in presence of IL-17A. Moreover, RNAi-induced Bcl2 silencing impaired the function of IL-17A in inhibiting the activation of autophagy, subsequently reducing the viability and migration of HCC cells, while the inhibition of Beclin1 by spautin-1 could reduce autophagic activity to a certain degree, thus restoring the viability and migration of HCC cells. In summary, it was suggested that the inhibition of Bcl2 degradation may be an important mechanism by which IL-17A inhibits autophagy response, subsequently maintaining the survival in HCC cells.