Influenza virus evolves constantly in an unpredictable fashion, making it necessary to vaccinate people annually for effective prevention and control of influenza. In general, however, during the first wave of an influenza outbreak caused by a newly emerging virus strain, influenza morbidity and mortality have been observed to rise sharply due to the lack of a matching vaccine. This necessitates the exploration of novel intervention approaches, particularly those prophylactic or therapeutic agents that have a broad range of antiviral activities and are also proven to be non-toxic. Here, we reported that stimulation of the innate immune system by intranasal administration of chitosan as a single agent was sufficient to completely protect BALB/c mice from lethal infection by H7N9 virus, a newly emerged viral strain that is highly pathogenic to humans. Remarkably, animals could still be protected against lethal challenge by H7N9 (10×LD50), even ten days after the intranasal chitosan administration. The significantly enhanced infiltration of leukocytes in the bronchoalveolar lavage and elevated levels of proinflammatory cytokines in the bronchia/lung tissues revealed the potent activation of mucosal immune responses by intranasally delivered chitosan. We also observed that chitosan can protect mice from three other virus strains. The marked breadth and magnitude of protection against diverse viral strains makes chitosan an attractive candidate as a universal anti-influenza agent.

Naringin maintains bone mass in various osteoporosis models, while its effect on bone in disuse osteoporosis has not been reported. The present study explores whether naringin can prevent disuse osteoporosis induced by unilateral sciatic neurectomy (USN) and whether the Semaphorin 3A-induced Wnt/β-catenin signalling pathway is involved in the osteoprotection of naringin. Naringin dose-dependently prevented the deterioration of bone mineral density (BMD), trabecular structure and biomechanical strength in femur due to USN. Naringin increased bone formation but inhibited resorption, as indicated by bone-turnover markers in blood and urine and the histological staining of Osteocalcin (OCN) and tartrate-resistant acid phosphatase (TRAP) in femur. Semaphorin 3A (Sema3A) and active β-catenin protein decreased after USN and could be restored by naringin to the levels of the sham-operated rats. In addition, naringin in vitro promoted the differentiation of osteoblasts and inhibited osteoclastic differentiation. Our studies suggest that the down-regulation of Sema3A and the subsequent inactivation of Wnt/β-catenin signalling may be some of the mechanisms involved in USN-induced osteoporosis. Naringin could increase the expression of Sema3A and the activation of Wnt/β-catenin signalling to prevent disuse osteoporosis induced by denervation. Thus, naringin functions in bone maintenance and could be a promising therapeutic alternative in preventing disuse osteoporosis.

The conventional chemotherapeutics could not be traced in vivo and provide timely feedback on the clinical effectiveness of drugs. In this study, poly(L-γ-glutamyl-glutamine)-paclitaxel (PGG-PTX), as a model polymer, was chemically conjugated with Gd-DTPA (Gd-diethylenetriaminepentaacetic acid), a T1-contrast agent of MRI, to prepare a Gd-DTPA-conjugated PGG-PTX (PGG-PTX-DTPA-Gd) delivery system used for tumor theranostics. PGG-PTX-DTPA-Gd can be self-assembled to NPs in water with a z-average hydrodynamic diameter about 35.9 nm. The 3 T MRI results confirmed that the relaxivity of PGG-PTX-DTPA-Gd NPs (r1 = 18.98 mM-1S-1) was increased nearly 4.9 times compared with that of free Gd-DTPA (r1 = 3.87 mM-1S-1). The in vivo fluorescence imaging results showed that PGG-PTX-DTPA-Gd NPs could be accumulated in the tumor tissue of NCI-H460 lung cancer animal model by EPR effect, which was similar to PGG-PTX NPs. The MRI results showed that compared with free Gd-DTPA, PGG-PTX-DTPA-Gd NPs showed significantly enhanced and prolonged signal intensity in tumor tissue, which should be attributed to the increased relaxivity and tumor accumulation. PGG-PTX-DTPA-Gd NPs also showed effective antitumor effect in vivo. These results indicated that PGG-PTX-DTPA-Gd NPs are an effective delivery system for tumor theranostics, and should have a potential value in personalized treatment of tumor.

Transient receptor potential channel M2 (TRPM2) is a Ca2+-permeable channel that is activated by reactive oxygen species (ROS). In many cell types, ROS activate TRPM2 to induce excessive Ca2+ influx, resulting in Ca2+ overload and consequent cell death. Recent studies suggest that TRPM2 may also regulate autophagy in pericytes and cancer cells by acting on the early step of autophagy, i.e. autophagic induction. However, there is no report on the role of TRPM2 in autophagic degradation, which is the late stage of autophagy. In the present study, we found abundant TRPM2 expression in lysosomes/autolysosomes in the primary cultured mouse aortic smooth muscle cells (mASMCs). Nutrient starvation stimulated autophagic flux in mASMCs mainly by promoting autophagic degradation. This starvation-induced autophagic degradation was reduced by TRPM2 knockout. Importantly, starvation-induced lysosomal/autolysosomal acidification and cell death were also substantially reduced by TRPM2 knockout. Taken together, the present study uncovered a novel mechanism that lysosomal TRPM2 facilitates lysosomal acidification to stimulate excessive autolysosome degradation and consequent cell death.

The activation/inactivation of HIF1α is precisely regulated in an oxygen-dependent manner. HIF1α is essential for hypoxia induced apoptosis and cell cycle arrest. Several recent studies indicated that the expression of miRNAs can be modulated by hypoxia. However, the involvement of miRNAs in the regulation of HIF1α induction remains elusive. In present study, we demonstrated that miR-101 was rapidly and transiently induced after hypoxia in breast cancer cells. Over-expression of miR-101 significantly inhibited cell proliferation in breast cancer cells through increased apoptosis and cell cycle arrest in normoxia condition. This inhibitory phenomenon seems due to miR-101-mediated induction of HIF1α, because we identified that VHL, a negative regulator of HIF1α, is a novel target of miR-101 and over-expression of miR-101 decreased VHL levels and subsequently stabilized HIF1α and induced its downstream target VEGFA. Furthermore, we demonstrated that siRNA-mediated knockdown of VHL or HIF1α overexpression could also induce apoptosis and cell cycle arrest whereas enforced expression of VHL, administration of anti-miR-101 oligos or treatment of 2-MeOE2, an inhibitor of HIF1α, could rescue cells from such inhibition. These results reveal a novel regulatory mechanism of HIF1α induction in normoxia and suggest that miR-101 mediated proliferation inhibition may through HIF1α mediated apoptosis and cell cycle arrest.

Long noncoding RNA(lncRNA)s are new regulators governing the metabolism in adipose tissue. In this study, we aimed to understand how lncRNAs respond to insulin signalling and explore whether lncRNAs have a functional role in insulin signalling pathway. We treated primary adipocyte cultures with insulin and collected RNA for RNA-sequencing to profile the non-coding transcriptome changes, through which we identified a top Adipose Specific Insulin Responsive LncRNA (LncASIR). To determine its biological function, we knocked down LncASIR using dcas9-KRAB, followed by RNA-seq to examine the effect on insulin-induced gene expression program. We identified a set of lncRNAs regulated by insulin signalling pathway. LncASIR is transcribed from a super enhancer region and responds robustly to insulin treatment. Silencing LncASIR resulted in an impaired global insulin-responsive gene program. LncASIR is a novel and integral component in the insulin signalling pathway in adipocytes.

Circulating long non-coding RNAs (lncRNAs) serve as valuable biomarkers in a number of human diseases. However, lncRNA biomarkers have yet to be identified in obesity. We aim to characterize circulating lncRNA expression in obese and non-obese human subjects. First, we assessed the genome-wide circulating lncRNA expression profiles in blood from 3 obese and 3 non-obese human subjects. We found a significant decrease in circulating levels of three lncRNAs (lncRNA-p5549, lncRNA-p21015 and lncRNA-p19461) in obese human subjects only. Next, using RT-PCR we measured the expression levels of these three lncRNAs in 33 obese and 33 non-obese human subjects and found similar differences. Moreover, we found a negative correlation between circulating levels of these three lncRNAs and body mass index (BMI), waist circumference, waist to hip ratio and fasting insulin. There was also a significant negative correlation between expression of lncRNA-p19461 and homeostasis model assessment-estimated insulin resistance. Finally, we tested the circulating levels of these three lncRNAs in 8 obese human subjects after a 12-week diet-induced weight loss program. We found that only lncRNA-p19461 expression level significantly increased. In summary, circulating lncRNAs are deregulated in obesity. Weight loss-induced changes in this profile support this observation and suggest a potential mechanistic relevance.

Cardiovascular complications account for a substantial proportion of morbidity and mortality in diabetic patients. Abnormalities of cardiac microvascular endothelial cells (CMECs) lead to impaired cardiac microvascular vessel integrity and subsequent cardiac dysfunction, underlining the importance of coronary microvascular dysfunction. In this study, experimental diabetes models were constructed using Mst1 transgenic, Mst1 knockout and sirt1 knockout mice. Diabetic Mst1 transgenic mice exhibited impaired cardiac microvessel integrity and decreased cardiac function. Mst1 overexpression deceased CMECs autophagy as evidenced by decreased LC3 expression and enhanced protein aggregation when subjected to high glucose culture. Mst1 knockout improved cardiac microvessel integrity and enhanced cardiac functions in diabetic mice. Mst1 knockdown up-regulated autophagy as indicated by more typical autophagosomes and increased LC3 expression in CMECs subjected to high glucose cultures. Mst1 knockdown also promoted autophagic flux in the presence of bafilomycin A1. Mst1 overexpression increased CMECs apoptosis, whereas Mst1 knockout decreased CMECs apoptosis. Sirt1 knockout abolished the effects of Mst1 overexpression in cardiac microvascular injury and cardiac dysfunction. In conclusion, Mst1 knockout preserved cardiac microvessel integrity and improved cardiac functions in diabetic mice. Mst1 decreased sirt1 activity, inhibited autophagy and enhanced apoptosis in CMECs, thus participating in the pathogenesis of diabetic coronary microvascular dysfunction.

Thyroid hormones (TH) control brown adipose tissue (BAT) activation and differentiation, but their subsequent homeostatic response following BAT activation remains obscure. This study aimed to investigate the relationship between cold- and capsinoids-induced BAT activation and TH changes between baseline and 2 hours post-intervention. Nineteen healthy subjects underwent 18F-fluorodeoxyglucose positron-emission tomography (18F-FDG PET) and whole-body calorimetry (WBC) after 2 hours of cold exposure (~14.5 °C) or capsinoids ingestion (12 mg) in a crossover design. Standardized uptake values (SUV-mean) of the region of interest and energy expenditure (EE) were measured. Plasma free triiodothyronine (FT3), free thyroxine (FT4) and thyroid stimulating hormone (TSH) were measured before and 2 hours after each intervention. Subjects were divided into groups based on the presence (n = 12) or absence (n = 7) of BAT after cold exposure. 12 of 19 subjects were classified as BAT-positive. Subjects with BAT had higher baseline FT3 concentration, baseline FT3/FT4 ratio compared with subjects without BAT. Controlling for body fat percentage, FT3 concentration at baseline was associated with EE change from baseline after cold exposure (P = 0.037) and capsinoids (P = 0.047). Plasma FT4 level significantly increased associated with reciprocal decline in TSH after acute cold exposure and capsinoids independently of subject and treatment status. Circulating FT3 was higher in BAT-positive subjects and was a stronger predictor of EE changes after cold exposure and capsinoids in healthy humans. BAT activation elevates plasma FT4 acutely and may contribute towards augmentation of thermogenesis via a positive feedback response.

Bailinggu (Pleurotus tuoliensis) is a major, commercially cultivated mushroom and widely used for nutritional, medicinal, and industrial applications. Yet, the mushroom's genetic architecture and the molecular mechanisms underlying its formation are largely unknown. Here we performed comparative transcriptomic analysis during Bailinggu's mycelia, primordia, and fruiting body stages to identify genes regulating fruiting body development and develop EST-SSR markers assessing the genetic value of breeding materials. The stage-specific and differentially expressed unigenes (DEGs) involved in morphogenesis, primary carbohydrate metabolism, cold stimulation and blue-light response were identified using GO and KEGG databases. These unigenes might help Bailinggu adapt to genetic and environmental factors that influence fructification. The most pronounced change in gene expression occurred during the vegetative-to-reproductive transition, suggesting that is most active and key for Bailinggu development. We then developed 26 polymorphic and informative EST-SSR markers to assess the genetic diversity in 82 strains of Bailinggu breeding materials. These EST-SSRs exhibited high transferability in closely related species P. eryngii var. ferulae and var. eryngii. Genetic population structure analysis indicated that China's Bailinggu has low introgression with these two varieties and likely evolved independently. These findings provide new genes, SSR markers, and germplasm to enhance the breeding of commercially cultivated Bailinggu.

The epidemic of obesity and diabetes has markedly spurred the research interest in adipocyte biology. Brown adipocytes are specialized for energy expenditure and of therapeutic interest for treatment of metabolic diseases, but how brown adipocytes are distinguished from white adipocytes at the level of translational regulation remains poorly understood. To systemically determine the translational control of gene expression in adipose tissue, we performed ribosome profiling and RNA-seq in parallel to depict the translatome and transcriptome changes during primary brown and white adipogenesis, and between brown and white adipose tissue. The most prominent layer of translational regulation was the increased translation efficiency of genes encoding mitochondria components in brown adipocytes relative to white. Systemic analysis of the regulatory interactions between microRNAs and their targets revealed that microRNAs were more active in repressing targets' mRNA abundance and translation in brown fat. Together, our data comprehensively delineated a landscape integrating transcriptome and translatome in adipose tissue.

To investigate the mitigation effect and mechanism of oestrogen and PTH on disc degeneration in rats after ovariectomy, as well as on Wnt/β-catenin pathway activity, thirty 3-month-old rats were ovariectomized and divided into three groups. Ten additional rats were used as controls. Eight weeks later, the rats were administered oestrogen or PTH for 12 weeks, and then discs were collected for tests. Results showed that nucleus pulposus cells in the Sham group were mostly notochord cells, while in the OVX group, cells gradually developed into chondrocyte-like cells. Oestrogen or PTH could partly recover the notochord cell number. After ovariectomy, the endplate roughened and endplate porosity decreased. After oestrogen or PTH treatment, the smoothness and porosity of endplate recovered. Compared with the Sham group, Aggrecan, Col2a and Wnt/β-catenin pathway expression in OVX group decreased, and either oestrogen or PTH treatment improved their expression. The biomechanical properties of intervertebral disc significantly changed after ovariectomy, and oestrogen or PTH treatment partly recovered them. Disc degeneration occurred with low oestrogen, and the underlying mechanisms involve nutrition supply disorders, cell type changes and decreased Wnt/β-catenin pathway activity. Oestrogen and PTH can retard disc degeneration in OVX rats and enhance Wnt/β-catenin pathway activity in nucleus pulposus.

Obesity is associated with an increasing prevalence of cardiovascular diseases and metabolic syndrome. It is of paramount importance to reduce obesity-associated cardiac dysfunction and impaired energy metabolism. In this study, the activation of the AMP-activated protein kinase (AMPK) pathway by punicalagin (PU), a major ellagitannin in pomegranate was investigated in the heart of a rat obesity model. In male SD rats, eight-week administration of 150 mg/kg pomegranate extract (PE) containing 40% punicalagin sufficiently prevented high-fat diet (HFD)-induced obesity associated accumulation of cardiac triglyceride and cholesterol as well as myocardial damage. Concomitantly, the AMPK pathway was activated, which may account for prevention of mitochondrial loss via upregulating mitochondrial biogenesis and amelioration of oxidative stress via enhancing phase II enzymes in the hearts of HFD rats. Together with the normalized expression of uncoupling proteins and mitochondrial dynamic regulators, PE significantly prevented HFD-induced cardiac ATP loss. Through in vitro cultures, we showed that punicalagin was the predominant component that activated AMPK by quickly decreasing the cellular ATP/ADP ratio specifically in cardiomyocytes. Our findings demonstrated that punicalagin, the major active component in PE, could modulate mitochondria and phase II enzymes through AMPK pathway to prevent HFD-induced cardiac metabolic disorders.

The role of Mesenchymal-endothelial transition (MEndoT) in cardiac hypertrophy is unclear. To determine the difference between MEndoT-derived and coronary endothelial cells is essential for understanding the revascularizing strategy in cardiac repair. Using lineage tracing we demonstrated that MEndoT-derived cells exhibit highly heterogeneous which were characterized with highly expression of endothelial markers such as vascular endothelial cadherin(VECAD) and occludin but low expression of Tek receptor tyrosine kinase(Tek), isolectin B4, endothelial nitric oxide synthase(eNOS), von Willebrand factor(vWF), and CD31 after cardiac hypertrophy. RNA-sequencing showed altered expression of fibroblast lineage commitment genes in fibroblasts undergoing MEndoT. Compared with fibroblasts, the expression of p53 and most endothelial lineage commitment genes were upregulated in MEndoT-derived cells; however, the further analysis indicated that MEndoT-derived cells may represent an endothelial-like cell sub-population. Loss and gain function study demonstrated that MEndoT-derived cells are substantial sources of neovascularization, which can be manipulated to attenuate cardiac hypertrophy and preserve cardiac function by improving the expression of endothelial markers in MEndoT-derived cells. Moreover, fibroblasts undergoing MEndoT showed significantly upregulated anti-hypertrophic factors and downregulated pro-hypertrophic factors. Therefore MEndoT-derived cells are an endothelial-like cell population that can be regulated to treat cardiac hypertrophy by improving neovascularization and altering the paracrine effect of fibroblasts.

Developing an effective universal influenza vaccine against influenza virus with highly conserved antigenic epitopes could induce a broad-spectrum immune response to prevent infection. The soluble protein M1 that can induce the M1 specific immune response was first confirmed in our previous study. In this study, we characterized the immune response induced by DNA prime-subunit protein boost strategy based on the relatively conserved matrix protein 1 (M1) in the BALB/c mouse model, and evaluated its protection ability against a lethal challenge of homologous H9N2 avian influenza virus (A/Chicken/Jiangsu/11/2002). The results showed that 100 μg DNA prime + 100 μg M1 subunit protein boost-strategy significantly increased antibody levels more than vaccination with M1 DNA or M1 subunit protein alone, and induced a more balanced Th1 / Th2 immune response, which not only can provide protection against the homologous virus but also can provide part of the cross-protection against the heterosubtypic PR8 H1N1 strain. In addition, we used an Elispot assay to preliminary screen the T cell epitope in M1 protein, and identified that p22 (M111-25 VLSIIPSGPLKAEIA) epitope was the only immunodominant M1-specific CD4+ T cell epitopes, which could be helpful in understanding the function of influenza virus T cell epitopes.

We demonstrate the use of cryogenic super-resolution correlative light and electron microscopy (csCLEM) to precisely determine the spatial relationship between proteins and their native cellular structures. Several fluorescent proteins (FPs) were found to be photoswitchable and emitted far more photons under our cryogenic imaging condition, resulting in higher localization precision which is comparable to ambient super-resolution imaging. Vitrified specimens were prepared by high pressure freezing and cryo-sectioning to maintain a near-native state with better fluorescence preservation. A 2-3-fold improvement of resolution over the recent reports was achieved due to the photon budget performance of screening out Dronpa and optimized imaging conditions, even with thin sections which is at a disadvantage when calculate the structure resolution from label density. We extended csCLEM to mammalian cells by introducing cryo-sectioning and observed good correlation of a mitochondrial protein with the mitochondrial outer membrane at nanometer resolution in three dimensions.

Our previous studies indicate that Cyclophilin A (CypA) impairs the replication of influenza A virus in vitro. To further evaluate the antiviral functions of CypA and explore its mechanism, transgenic mice with overexpression of CypA by two specific promoters with SPC (CypA-SPC) or CMV (CypA-CMV) were developed. After challenge with the A/WSN/33(H1N1) influenza virus, CypA-SPC and CypA-CMV transgenic mice displayed nearly 2.5- and 3.8-fold stronger disease resistance to virus infection, respectively, compared to wild-type animals. Virus replication, pathological lesions and inflammatory cytokines were substantially reduced in both lines of transgenic mice. In addition, after infection there was an upregulation of genes associated with cell migration, immune function, and organ development; and a downregulation of genes associated with the positive regulation of immune cells and apoptosis in the peritoneal macrophages of CypA-overexpressing transgenic mice (CypA+). These results indicate that CypA is a key modulator of influenza virus resistance in mice, and that CypA+ mice constitutes an important model to study the roles of CypA in the regulation of immune responses and infections.

Resistance to anoikis and Epithelial-mesenchymal transition (EMT) are two processes critically involved in cancer metastasis. In this study, we demonstrated that after anchorage deprival, hepatocellular carcinoma (HCC) cells not only resisted anoikis, but also exhibited EMT process. Microarray expression profiling revealed that expression of miR-424-5p was significantly decreased in anoikis-resistant HCC cells. Ectopic overexpression of miR-424-5p was sufficient to reverse resistance to anoikis, block EMT process and inhibit malignant behaviors of HCC cells. Target analysis showed that a potent β-catenin inhibitor, ICAT/CTNNBIP1 was a direct target of miR-424-5p. Further study demonstrated that miR-424-5p reversed resistance to anoikis and EMT of HCCs by directly targeting ICAT and further maintaining the E-cadherin/β-catanin complex on the cellular membrance. In vivo study further demonstrated that miR-424-5p significantly inhibited the tumorigenicity of HCC cells in nude mice. Clinical investigation demonstrated that miR-424-5p was significantly downregulated in HCC tissues compared with that of the non-cancerous liver tissues, and this decreased expression of miR-424-5p was significantly correlated with higher pathological grades and more advanced TNM stages. Therefore, aberrant expression of miR-424-5p is critically involved in resistance to anoikis and EMT during the metastatic process of HCC, and its downregulation significantly contributes to liver cancer progression.

Microtus fortis exhibits natural resistance against Schistosoma japonicum, and the parasite cannot grow and develop in M. fortis. Extensive research has been carried out, however, the associated mechanism remains unclear. In the present study, we analysed the combined data obtained from a cytokine chip assay, transcriptome, and metabolome. The cytokine profile from C57BL/6 and M. fortis mice was assessed before and after infection. Several cytokines increased during the second and third week post-infection. Some transcripts related to cytokine genes and associated proteins were also highly expressed (i.e., Hgf, C3, and Lbp). The liver metabolism of M. fortis following infection with S. japonicum was assessed. We identified 25 different metabolites between the uninfected and infected M. fortis, and 22 different metabolites between infected M. fortis and C57BL/6 mice. The metabolomic pathways of these differential metabolites were then analysed with MetPA, revealing that they were involved in histidine metabolism, valine, leucine, and isoleucine biosyntheses, and lysine degradation. Thus, the elevated expression of these metabolites and pathways may promote the phagocytic function of the neutrophils and natural killer cell activity following TLR activation. These results provide novel insight into the resistance mechanism of M. fortis against S. japonicum.

Fennel contains many antioxidant and antibacterial substances, and it has very important applications in food flavoring and other fields. The kinds and contents of chemical substances in fennel vary from region to region, which can affect the taste and efficacy of the fennel and its derivatives. Therefore, it is of great significance to accurately classify the origin of the fennel. Recently, origin detection methods based on deep networks have shown promising results. However, the existing methods spend a relatively large time cost, a drawback that is fatal for large amounts of data in practical application scenarios. To overcome this limitation, we explore an origin detection method that guarantees faster detection with classification accuracy. This research is the first to use the machine learning algorithm combined with the Fourier transform-near infrared (FT-NIR) spectroscopy to realize the classification and identification of the origin of the fennel. In this experiment, we used Rubberband baseline correction on the FT-NIR spectral data of fennel (Yumen, Gansu and Turpan, Xinjiang), using principal component analysis (PCA) for data dimensionality reduction, and selecting extreme learning machine (ELM), Convolutional Neural Network (CNN), recurrent neural network (RNN), Transformer, generative adversarial networks (GAN) and back propagation neural network (BPNN) classification model of the company realizes the classification of the sample origin. The experimental results show that the classification accuracy of ELM, RNN, Transformer, GAN and BPNN models are above 96%, and the ELM model using the hardlim as the activation function has the best classification effect, with an average accuracy of 100% and a fast classification speed. The average time of 30 experiments is 0.05 s. This research shows the potential of the machine learning algorithm combined with the FT-NIR spectra in the field of food production area classification, and provides an effective means for realizing rapid detection of the food production area, so as to merchants from selling shoddy products as good ones and seeking illegal profits.