USP25 ameliorates diabetic nephropathy by inhibiting TRAF6-mediated inflammatory responses.

Diabetic kidney disease (DKD) is a common diabetic vascular complication affecting nearly 40% of patients with diabetes. The lack of efficacious therapy for DKD necessitates the in-depth investigation of the molecular mechanisms underlying the pathogenesis and progression of DKD, which remain incompletely understood. Here, we discovered that the expression of USP25, a deubiquitinating enzyme, was significantly upregulated in the kidney of diabetic mice. Ablation of USP25 had no influence on glycemic control in type 1 diabetes but significantly aggravated diabetes-induced renal dysfunction and fibrosis by exacerbating inflammation in the kidney. In DKD, USP25 was mainly expressed in glomerular mesangial cells and kidney-infiltrating macrophages. Upon stimulation with advanced glycation end-products (AGEs), USP25 markedly inhibited the production of proinflammatory cytokines in these two cell populations by downregulating AGEs-induced activation of NF-κB and MAPK pathways. Mechanistically, USP25 interacted with TRAF6 and inhibited its K63 polyubiquitination induced by AGEs. Collectively, these findings identify USP25 as a novel regulator of DKD.

Expression profiling and antibacterial analysis of cd36 in mandarin fish, Siniperca chuatsi.

Cd36 is classified as a class B scavenger receptor and has also been identified as a pattern recognition receptor. In this study, we investigated the genomic structure and molecular characteristics of cd36 in mandarin fish (Siniperca chuatsi), examined its tissue distribution, and evaluated its antibacterial activity. Genomic structure analysis showed that Sccd36 consists of 12 exons and 11 introns. Sequencing analysis confirmed that the open reading frame of Sccd36 contains 1410 bp, encoding 469 amino acids. Sccd36 is deeply conserved with other vertebrates in terms of genomic structure, gene loci and molecular evolution, and the feature of two transmembrane was observed in ScCd36 through structural prediction. Sccd36 was constitutively expressed in all tissues tested, with the strongest expression in the intestine, followed by the heart and the kidney. Dramatic changes of Sccd36 mRNA were detected in mucosal tissues, including the intestine, gill and skin, when stimulated by the microbial ligands lipopolysaccharide and lipoteichoic acid. In addition, ScCd36 was identified as having strong binding ability to microbial ligands and antibacterial activity against the gram-negative bacteria Aeromonas hydrophila and the gram-positive bacteria Streptococcus lactis. Furthermore, we verified that the genetic ablation of cd36 impaired the resistance of fish to bacterial challenge by using zebrafish cd36 knockout line. In conclusion, our findings suggest that ScCd36 plays a crucial role in the innate immune response of mandarin fish against bacterial infections. This also sets the stage for further exploration into the antibacterial function of Cd36 in lower vertebrate species.

Delayed First Feeding Chronically Impairs Larval Fish Growth Performance, Hepatic Lipid Metabolism, and Visceral Lipid Deposition at the Mouth-Opening Stage.

During the mouth-opening stage, fish larvae are susceptible to delayed first feeding (DFF). In this study, we explored the effects of DFF for two days on later growth and energy metabolism in larval fish. Results showed that DFF chronically impaired larval growth performance, thereby reducing the efficiency of feed utilization by larvae. In DFF larvae, the mRNA levels of growth inhibitors (i.e., igfbp1a and igfbp1b) were significantly upregulated and consistently maintained at high expression levels, which may be an important attribution of larval growth retardation. Concomitantly, DFF retarded the growth of adipose tissue and reduced lipid deposition in larval viscera, suggesting lipid metabolism is disordered in DFF larvae and generates inefficient lipid reserves. In the liver, we observed that DFF resulted in a significant accumulation of neutral lipids, and this phenotype did not disappear rapidly after DFF larvae received exogenous nutrition. As to the transcript analyses, we found that the expression of genes related to hepatic lipid synthesis (e.g., srebf1, srebf2, dgat1a, dgat1b, fasn, and scdb) in DFF larvae was consistently upregulated, while the expression of genes involved in lipid transport (e.g., apoa2, apoa4b.1, and apoa4b.3) was downregulated. Therefore, it appears that the inefficient lipid reserves in DFF larvae are associated with their hepatic lipid transport dysfunction. Taken together, our findings contribute to understanding the impairments to fish larvae caused by delayed first feeding during the mouth-opening stage and to aiding larval management in the aquaculture industry.

Heart-on-a-chip using human iPSC-derived cardiomyocytes with an integrated vascular endothelial layer based on a culture patch as a potential platform for drug evaluation.

Many strategies have been adopted to constructin vitromyocardium models, which are of great value to both drug cardiotoxicity evaluation and cardiovascular drug development. In particular, the recent rapid development of human-induced pluripotent stem cell (hiPSC) technology and the rise of the organ-on-a-chip technique have provided great potential to achieve more physiologically relevantin vitromodels. However, recapitulating the key role of the vasculature endothelial layer in drug action on myocardium in the models is still challenging. In this work, we developed an openable heart-on-a-chip system using highly purified functional hiPSC-derived cardiomyocytes (hiPSC-CMs) with an integrated vascular endothelial layer based on our previously proposed culture-patch method. The purity and functionality of the differentiated hiPSC-CMs were characterized, which were applied into the lower chamber of the sandwich-structured device to form the CM layer. The integrity and cell morphology of the endothelial layer on the culture patch as well as the influence of fluid shear force were studied, which was integrated in between the upper and lower chambers. The constructed heart-on-a-chip was finally applied for drug testing. The effect of two cardiac targeting drugs (isoproterenol and E-4031) directly on the hiPSC-CMs or after penetrating through the endothelial layer under static or dynamic conditions was evaluated. The results demonstrated the significance of a vascular layer inin vitromyocardium models for drug testing, as well as the advantage and potential of the proposed platform for cardiovascular drug evaluation with more human physiological relevance.

Transcriptome analysis reveals the early resistance of zebrafish larvae to oxidative stress.

Oxidative stress is one of most common environmental stresses encountered by fish, especially during their fragile larval stage. More and more studies are aimed at understanding the antioxidant defense mechanism of fish larvae. Herein we characterized the early resistance of zebrafish larvae to oxidative stress and investigated the underlying transcriptional regulations using RNA-seq. We found that pre-exposure of zebrafish larvae to 2 mM H2O2 for 1 or 3 h significantly improved their survival under higher doses of H2O2 (3 mM), suggesting the antioxidant defenses of zebrafish larvae were rapidly built under pre-exposure of H2O2. Comparative transcriptome analysis showed that 310 (185 up and 125 down) and 512 (331 up and 181 down) differentially expressed genes were generated after 1 and 3 h of pre-exposure, respectively. KEGG enrichment analysis revealed that protein processing in endoplasmic reticulum is a highly enriched pathway; multiple genes (e.g., hsp70.1, hsp70.2, and hsp90aa1.2) encoding heat shock proteins in this pathway were sharply upregulated presumably to correct protein misfolding and maintaining the cellular normal functions during oxidative stress. More importantly, the Keap1/Nrf2 system-mediated detoxification enzyme system was significantly activated, which regulates the upregulation of target genes (e.g., gstp1, gsr, and prdx1) to scavenger reactive oxygen species, thereby defending against apoptosis. In addition, the MAPK, as a transmitter of stress signals, was activated, which may play an important role in activating antioxidant system in the early stages of oxidative stress. Altogether, these findings demonstrate that zebrafish larvae rapidly establish resistance to oxidative stress, and this involves changes in protein processing, stress signal transmission, and the activation of detoxification pathways.

In vitro 3D cocultured tumor-vascular barrier model based on alginate hydrogel and Transwell system for anti-cancer drug evaluation.

The development of three-dimensional (3D) in vitro model to recapitulate the in vivo tumor tissue is essential for studying tumor biology, discovering anti-cancer drugs, and evaluating anti-cancer drug efficacy. However, most of the previous models lack the involvement of vascular barrier. Here, we proposed an in vitro 3D cocultured tumor-vascular barrier model by the combination of alginate hydrogels beads and Transwell system. PC-3 cells and NIH/3T3 cells were encapsulated in alginate hydrogel beads, which were cultured in the bottom chamber of Transwell, while human umbilical vein endothelial cells (HUVECs) were cultured on the porous membrane in the upper chamber to form vascular barrier. The effect of the concentration of alginate sodium on the morphology, diameter and swelling ratio of the beads was studied. The alginate sodium content and cell seeding density were further optimized according to cell proliferation ability. The formation of endothelial barrier was verified by immunostaining with tight junction protein VE-cadherin and transendothelial electrical resistance (TEER) monitoring. Finally, the drug response of 3D cocultured tumor-vascular barrier model to curcumin was evaluated. Compared with two-dimensional (2D) coculture model and 3D coculture spheroid model, 3D tumor-vascular barrier model showed the highest activity of cancer cells and the strongest drug resistance. The developed 3D cocultured tumor-vascular barrier model possesses great potential to be applied for in vitro evaluation of anti-tumor drugs.

Flavokawain B alleviates LPS-induced acute lung injury via targeting myeloid differentiation factor 2.

Acute lung injury (ALI) is a sudden onset systemic inflammatory response. ALI causes severe morbidity and death and currently no effective pharmacological therapies exist. Natural products represent an excellent resource for discovering new drugs. Screening anti-inflammatory compounds from the natural product bank may offer viable candidates for molecular-based therapies for ALI. In this study, 165 natural compounds were screened for anti-inflammatory activity in lipopolysaccharide (LPS)-challenged macrophages. Among the screened compounds, flavokawain B (FKB) significantly reduced LPS-induced pro-inflammatory IL-6 secretion in macrophages. FKB also reduced the formation of LPS/TLR4/MD2 complex by competitively binding to MD2, suppressing downstream MAPK and NF-κB signaling activation. Finally, FKB treatment of mice reduced LPS-induced lung injury, systemic and local inflammatory cytokine production, and macrophage infiltration in lungs. These protective activities manifested as increased survival in the ALI model, and reduced mortality upon bacterial infection. In summary, we demonstrate that the natural product FKB protects against LPS-induced lung injury and sepsis by interacting with MD2 and inhibiting inflammatory responses. FKB may potentially serve as a therapeutic option for the treatment of ALI.

Fabrication and in vitro evaluation of PCL/gelatin hierarchical scaffolds based on melt electrospinning writing and solution electrospinning for bone regeneration.

As an emerging 3D printing technique, melt electrospinning writing (MEW) has been used to fabricate scaffolds with controllable structure and good mechanical strength for bone regeneration. However, how to further improve MEW scaffolds with nanoscale extracellular matrix (ECM) mimic structure and bioactivity is still challenging. In this study, we proposed a simple composite process by combining MEW and solution electrospinning (SE) to fabricate a micro/nano hierarchical scaffold for bone tissue engineering. The morphological results confirmed the hierarchical structure with both well-defined MEW microfibrous grid structure and SE random nanofiber morphology. The addition of gelatin nanofibers turned the scaffolds to be hydrophilic, and led to a slight enhancement of mechanical strength. Compared with PCL MEW scaffolds, higher cell adhesion efficiency, improved cell proliferation and higher osteoinductive ability were achieved for the MEW/SE composite scaffolds. Finally, multilayer composite scaffolds were fabricated by alternately stacking of MEW layer and SE layer and used to assess the effect on cell ingrowth in the scaffolds. The results showed that gelatin nanofibers did not inhibit cell penetration, but promoted the three-dimensional growth of bone cells. Thus, the strategy of the combined use of MEW and SE is a potential method to fabricate micro/nano hierarchical scaffolds to improve bone regeneration.

Congenital Asplenia Interrupts Immune Homeostasis and Leads to Excessive Systemic Inflammation in Zebrafish.

Splenectomy or congenital asplenia in humans increases susceptibility to infections. We have previously reported that congenital asplenia in zebrafish reduces resistance to Aeromonas hydrophila infection. However, the molecular mechanism of systemic immune response in congenitally asplenic individuals is largely unexplored. In this study, we found that pro-inflammatory cytokines were more highly induced in congenitally asplenic zebrafish than wild-type after pathogenic A. hydrophila infection and lipopolysaccharide exposure. In addition, a higher aggregation of apoptotic cells was observed in congenitally asplenic zebrafish than that in wild-type. Next, we examined the transcriptome profiles of whole kidneys from wild-type and congenitally asplenic zebrafish to investigate the effects of congenital asplenia on innate and adaptive immune responses induced by the inactivated A. hydrophila. Congenital asplenia inactivated the splenic anti-inflammatory reflex, disrupted immune homeostasis, and induced excessive inflammation as evidenced by the highly induced stress response-related biological processes, inflammatory and apoptosis-associated pathways, and pro-inflammatory cytokines/chemokines in congenitally asplenic zebrafish compared with wild-type after vaccination. In addition, complement component genes (c3a.1, c3a.6, c4, c6, and c9) and several important immune-related genes (tabp.1, tap1, hamp, prg4b, nfil3, defbl1, psmb9a, tfr1a, and sae1) were downregulated in congenitally asplenic zebrafish. Furthermore, congenital asplenia impaired adaptive immunity as demonstrated by downregulation of biological processes and signaling pathways involved in adaptive immune response after vaccination in congenitally asplenic zebrafish. The expression of MHCII/IgM was also significantly reduced in the congenitally asplenic zebrafish when compared with wild-type. Together, our study provides an in-depth understanding of spleen function in controlling immune homeostasis and may offer insight into the pathological response in splenectomized or congenitally asplenic patients after infections.

Transcriptome analysis reveals the importance of exogenous nutrition in regulating antioxidant defenses during the mouth-opening stage in oviparous fish.

Antioxidant system is crucial for protecting against environmental oxidative stress in fish life cycle. Although the effects of starvation on the antioxidant defenses in several adult fish have been defined, no relevant researches have been reported in the larval stage, particularly during the transition from endogenous to exogenous feeding. To clarify the molecular response of antioxidant system that occurs during the mouth-opening stage under starvation stress and explore its association with energy metabolism, we employed RNA-seq to analyze the gene expression profiles in zebrafish larvae that received a delayed first feeding for 3 days. Our data showed that delayed feeding resulted in downregulation of 7078 genes and upregulation of 497 genes. These differentially expressed genes are mainly involved in growth regulation (i.e., DNA replication and cell cycle), energy metabolism (i.e., glycolysis/gluconeogenesis and fatty acid metabolism), and antioxidant defenses. We demonstrated that the starved larvae are in an extremely malnourished state in the absence of exogenous nutrition, and the consequence is that numerous antioxidant genes are downregulated. Meanwhile, the antioxidant defenses also respond negatively to oxidative stress. After nutritional supply, the expression of these inhibited antioxidant genes was restored. These results suggest that the establishment of antioxidant defenses during the mouth-opening stage depends highly on exogenous nutrition. Our findings would contribute to comprehending the nutritional stress and metabolic switches during the mouth-opening stage and are essential for reducing high mortality in commercial fish farming.

Extracellular vesicles in neuroinflammation: Pathogenesis, diagnosis, and therapy.

Extracellular vesicles (EVs) are bilayer membrane vesicles and act as key messengers in intercellular communication. EVs can be secreted by both neurons and glial cells in the central nervous system (CNS). Under physiological conditions, EVs contribute to CNS homeostasis by facilitating omnidirectional communication among CNS cell populations. In response to CNS injury, EVs mediate neuroinflammatory responses and regulate tissue damage and repair, thereby influencing the pathogenesis, development, and/or recovery of neuroinflammatory diseases, including CNS autoimmune diseases, neurodegenerative diseases, stroke, CNS traumatic injury, and CNS infectious diseases. The unique ability of EVs to pass through the blood-brain barrier further confers them an important role in the bidirectional communication between the CNS and periphery, and application of EVs enables the diagnosis, prognosis, and therapy of neuroinflammatory diseases in a minimally invasive manner.

Coaxial electrospun PVA/PCL nanofibers with dual release of tea polyphenols and ε-poly (L-lysine) as antioxidant and antibacterial wound dressing materials.

Preparing wound dressing with dual-delivery of antioxidant and antibacterial agents is highly desirable in clinical wound treatment. Herein, a series of coaxial nanofiber membranes loaded with antioxidant tea polyphenols (TP) in the core and antibacterial ε-poly (L-lysine) (ε-PL) in the shell layer were successfully fabricated by coaxial electrospinning. The physicochemical characterizations by transmission electron microscopy, inverted fluorescence microscopy and fourier transform infrared spectroscopy confirmed the formation of core-shell structure. The results of in vitro drug release indicated that ε-PL exhibited a fast release profile while TP released in a sustained manner, which is favorable to the achievement of quick bacteria inhibition in the initial phase as well as long-term antioxidant activity during wound healing. The antioxidant activity of coaxial nanofibers was found to be increased with the increment of TP content and incubation time. The antibacterial assays against Escherichia coli and Staphylococcus aureus demonstrated that the incorporation of ε-PL in the coaxial nanofibers led to strong antibacterial activity. Additionally, all the coaxial nanofibers possessed good cytocompatibility. Therefore, the prepared coaxial nanofibers simultaneously incorporated with ε-PL and TP are promising as potential wound dressing materials.

A Model Construction of Starvation Induces Hepatic Steatosis and Transcriptome Analysis in Zebrafish Larvae.

Hepatic steatosis caused by starvation, resulting in non-alcoholic fatty liver disease (NAFLD), has been a research topic of human clinical and animal experiments. To understand the molecular mechanisms underlying the triggering of abnormal liver metabolism by starvation, thus inducing hepatic lipid accumulation, we used zebrafish larvae to establish a starvation-induced hepatic steatosis model and conducted comparative transcriptome analysis by RNA-seq. We demonstrated that the incidence of larvae steatosis is positively correlated with starvation time. Under starvation conditions, the fatty acid transporter (slc27a2a and slc27a6-like) and fatty acid translocase (cd36) were up-regulated significantly to promote extrahepatic fatty acid uptake. Meanwhile, starvation inhibits the hepatic fatty acid metabolism pathway but activates the de novo lipogenesis pathway to a certain extent. More importantly, we detected that the expression of numerous apolipoprotein genes was downregulated and the secretion of very low density lipoprotein (VLDL) was inhibited significantly. These data suggest that starvation induces hepatic steatosis by promoting extrahepatic fatty acid uptake and lipogenesis, and inhibits hepatic fatty acid metabolism and lipid transport. Furthermore, we found that starvation-induced hepatic steatosis in zebrafish larvae can be rescued by targeting the knockout cd36 gene. In summary, these findings will help us understand the pathogenesis of starvation-induced NAFLD and provide important theoretical evidence that cd36 could serve as a potential target for the treatment of NAFLD.

Multidrug-loaded electrospun micro/nanofibrous membranes: Fabrication strategies, release behaviors and applications in regenerative medicine.

Electrospun micro/nanofibrous membranes (EFMs) have been widely investigated as local drug delivery systems. Multiple drugs can be simultaneously incorporated into one EFM to create synergistic effects, reduce side effects, and play their respective roles in the complex physiological processes of tissue regeneration and postoperative adhesion prevention. Due to the versatile electrospinning techniques, sustained and programmed release behaviors of multiple drugs could be achieved by modulating the structure of the EFMs and the location of the drugs. In this review, various multidrug incorporation approaches based on electrospinning are overviewed. In particular, the advantages and limitations of each drug incorporation technique, the methods to control drug release and the effect of one drug release on another are discussed. Then the applications of multidrug-loaded EFMs in regenerative medicine, including wound healing, bone regeneration, vascular tissue engineering, nerve regeneration, periodontal regeneration and adhesion prevention are comprehensively reviewed. Finally, the future perspectives and challenges in the research of multidrug-loaded EFMs are discussed.

Angelica Essential Oil Loaded Electrospun Gelatin Nanofibers for Active Food Packaging Application.

The development of food packaging possessing bioactivities which could extend the shelf life of food has gained increased interest in recent years. In this study, gelatin nanofibers with encapsulated angelica essential oil (AEO) were fabricated via electrospinning. The morphology of gelatin/AEO nanofibers was examined by scanning electron microscopy (SEM) and the addition of AEO resulted in the increase of fiber diameter. The proton nuclear magnetic resonance (1H-NMR) spectra were measured to confirm the presence of AEO in nanofibers. The hydrophobic property of gelatin nanofibers was also found to be improved with the addition of AEO. The nanofibers incorporated with AEO showed significant antioxidant activity and inhibitory effect against both Gram-negative and Gram-positive bacteria in a concentration dependent manner. Furthermore, the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay demonstrated that the developed gelatin/AEO nanofibers revealed no cytotoxicity effect. Thus, gelatin nanofibers incorporated with AEO can be used as potential food packaging.

Honey loaded alginate/PVA nanofibrous membrane as potential bioactive wound dressing.

Honey is an ancient natural wound-healing agent and has been reintroduced to modern clinical wound care as it has various bioactivities. In this study, honey was incorporated into an alginate/PVA-based electrospun nanofibrous membrane to develop an efficient wound dressing material. The morphology and chemical composition of the nanofibrous membrane were observed by scanning electron microscopy and characterized via Fourier transform infrared spectroscopy, respectively, demonstrating that honey was successfully introduced to the nanofibers. The nanofibrous membranes with increasing honey content showed enhanced antioxidant activity, suggesting the ability to control the overproduction of reactive oxygen species. Disc diffusion assay and dynamic contact assay proved the antibacterial activity of the honey loaded nanofibers towards Gram-positive bacterium (Staphylococcus aureus) and Gram-negative bacterium (Escherichia coli). The cytotoxicity assay illustrated the non-cytotoxicity and biocompatibility of the nanofibrous membranes. Therefore, the developed honey/alginate/PVA nanofibrous membranes are promising for wound dressings.

Electrospun Gelatin Nanofibers Encapsulated with Peppermint and Chamomile Essential Oils as Potential Edible Packaging.

Natural and edible materials have attracted increasing attention in food packaging, which could overcome the serious environmental issues caused by conventional non-biodegradable synthetic packaging. In this work, gelatin nanofibers incorporated with two kinds of essential oil (EO), peppermint essential oil (PO) and chamomile essential oil (CO), were fabricated by electrospinning for potential edible packaging application. Electron microscopy showed that smooth and uniform morphology of the gelatin/EOs was obtained, and the diameter of nanofibers was mostly enlarged with the increase of the EO content. The proton nuclear magnetic resonance spectrum confirmed the existence of PO and CO in nanofibers after electrospinning. The addition of EOs led to an enhancement of the water contact angle of nanofibers. The antioxidant activity was significantly improved for the nanofibers loaded with CO, while the antibacteria activity against Escherichia coli and Staphylococcus aureus was better for the fibers with PO addition. The combination of half PO and half CO in nanofibers compensated for their respective limitations and exhibited optimum bioactivities. Finally, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay with NIH-3T3 fibroblasts demonstrated the absence of cytotoxicity of the gelatin/EO nanofibers. Thus, our studies suggest that the developed gelatin/PO/CO nanofiber could be a promising candidate for edible packaging.