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Recently, Deferoxamine (DFO) and magnesium (Mg) have been identified as critical factors for angiogenesis and bone formation. However, in current research studies, there is a lack of focus on whether DFO plus Mg can affect the regeneration of β-tricalcium phosphate (β-TCP) in osteoporosis and through what biological mechanisms. Therefore, the present work was aimed to preparation and evaluate the effect of Deferoxamine/magnesium modified β-tricalcium phosphate promotes (DFO/Mg-TCP) in ovariectomized rats model and preliminary exploration of possible mechanisms. The MC3T3-E1 cells were co-cultured with the exudate of DFO/Mg-TCP and induced to osteogenesis, and the cell viability, osteogenic activity were observed by Cell Counting Kit-8(CCK-8), Alkaline Phosphatase (ALP) staining, Alizarin Red Staining (RES) and Western Blot. In vitro experiments, CCK-8, ALP and ARS staining results show that the mineralization and osteogenic activity of MC3T3-E1increased significantly after intervention by DFO/Mg-TCP, as well as a higher levels of protein expressions including VEGF, OC, Runx-2 and HIF-1α. In vivo experiment, Micro-CT and Histological analysis evaluation show that DFO/Mg-TCP treatment presented the stronger effect on bone regeneration, bone mineralization and biomaterial degradation, when compared with OVX+Mg-TCP group and OVX+TCP group, as well as a higher VEGF, OC, Runx-2 and HIF-1α gene expression. The present study indicates that treatment with DFO/Mg-TCP was associated with increased regeneration by enhancing the function of osteoblasts in an OVX rat.

Hepatic sinusoidal obstruction syndrome (HSOS) via exposure to pyrrolizidine alkaloids (PAs) is with high mortality and there is no effective treatment in clinics. Bear bile powder (BBP) is a famous traditional animal drug for curing a variety of hepatobiliary diseases such as cholestasis, inflammation, and fibrosis. Here, we aim to evaluate the protective effect of BBP against HSOS induced by senecionine, a highly hepatotoxic PA compound. Our results showed that BBP treatment protected mice from senecionine-induced HSOS dose-dependently, which was evident by improved liver histology including reduced infiltration of inflammatory cells and collagen positive cells, alleviated intrahepatic hemorrhage and hepatic sinusoidal endothelial cells, as well as decreased conventional serum liver function indicators. In addition, BBP treatment lowered matrix metalloproteinase 9 and pyrrole-protein adducts, two well-known markers positively associated with the severity of PA-induced HSOS. Further investigation showed that BBP treatment prevents the development of liver fibrosis by decreasing transforming growth factor beta and downstream fibrotic molecules. BBP treatment also alleviated senecionine-induced liver inflammation and lowered the pro-inflammatory cytokines, in which tauroursodeoxycholic acid played an important role. What's more, BBP treatment also decreased the accumulation of hydrophobic bile acids, such as cholic acid, taurocholic acid, glycocholic acid, as well. We concluded that BBP attenuates senecionine-induced HSOS in mice by repairing the bile acids homeostasis, preventing liver fibrosis, and alleviating liver inflammation. Our present study helps to pave the way to therapeutic approaches of the treatment of PA-induced liver injury in clinics.

Diapause is a form of dormancy used widely by insects to survive adverse seasons. Previous studies have demonstrated that forkhead box O (FoxO) is activated during pupal diapause initiation in the moth Helicoverpa armigera . However, it is unclear how FoxO induces diapause. Here, we show that knockout of FoxO causes H. armigera diapause-destined pupae to channel into nondiapause, indicating that FoxO is a master regulator that induces insect diapause. FoxO activates the ubiquitin–proteasome system (UPS) by promoting ubiquitin c (Ubc) expression via directly binding to the Ubc promoter. Activated UPS decreases transforming growth factor beta (TGFβ) receptor signaling via ubiquitination to block developmental signaling to induce diapause. This study significantly advances the understanding of insect diapause by uncovering the detailed molecular mechanism of FoxO.

4-Hydroxyphenylpyruvate dioxygenase (HPPD), playing a critical role in vitamin E and plastoquinone biosynthesis in plants, has been recognized as one of the most important targets for herbicide discovery for over 30 years. Structure-based rational design of HPPD inhibitors has received more and more research interest. However, a critical challenge in the discovery of new HPPD inhibitors is the common inconsistency between molecular-level HPPD-based bioevaluation and the weed control efficiency in fields, due to the unpredictable biological processes of absorption, distribution, metabolism, and excretion.In this study, we developed a fluorescent-sensing platform of efficient in vivo screening for HPPD-targeted herbicide discovery. The refined sensor has good capability of in situ real-time fluorescence imaging of HPPD in living cells and zebrafish. More importantly, it enabled the direct visible monitoring of HPPD inhibition in plants in a real-time manner.We developed a highly efficient in vivo fluorescent screening method for HPPD-targeted herbicide discovery. This discovery not only offers a promising tool to advance HPPD-targeted herbicide discovery, but it also demonstrates a general path to develop the highly efficient, target-based, in vivo screening for pesticide discovery. © 2022 Society of Chemical Industry.

The common cutworm, Spodoptera litura, is a polyandrous moth with high reproductive ability. Sexual reproduction is a unique strategy for survival and reproduction of population in this species. However, to date available information about its reproductive genes is rare. Here, we combined transcriptomics, genomics and proteomics approaches to characterize reproductive-related proteins in S. litura. Illumina sequencing in parallel with the reference genome led to the yields of 12,161 reproductive genes, representing 47.83% of genes annotated in the genome. Further, 524 genes of 19 specific gene families annotated in the genome were detected in reproductive tissues of both sexes, some of which exhibited sex-biased and/or tissue-enriched expression. Of these, manual efforts together with the transcriptome analyses re-annotated 54 odorant binding proteins (OBPs) and 23 chemosensory proteins (CSPs) with an increase of 18 OBPs and one CSP compared to those previously annotated in the genome. Interestingly, at least 35 OBPs and 22 CSPs were transcribed in at least one reproductive tissue, suggestive of their involvement in reproduction. Further proteomic analysis revealed 2381 common proteins between virgin and mated female reproductive systems, 79 of which were differentially expressed. More importantly, 74 proteins exclusive to mated females were identified as transferred relatives, coupled with their specific or high expression in male reproductive systems. Of the transferred proteins, several conserved protein classes across insects were observed including OBPs, serpins, trypsins and juvenile hormone-binding proteins. Our current study has extensively surveyed reproductive genes in S. litura with an emphasis on the roles of OBPs and CSPs in reproduction, and identifies potentially transferred proteins serving as modulators of female post-mating behaviors.

Osteoporosis is one of the most common metabolic bone diseases affecting millions of people. We previously found that harmine prevents bone loss in ovariectomized mice via increasing preosteoclast platelet‐derived growth factor‐BB (PDGF‐BB) production and type H vessel formation. However, the molecular mechanisms by which harmine promotes preosteoclast PDGF‐BB generation are still unclear. In this study, we revealed that inhibitor of DNA binding‐2 (Id2) and activator protein‐1 (AP‐1) were important factors implicated in harmine‐enhanced preosteoclast PDGF‐BB production. Exposure of RANKL‐induced Primary bone marrow macrophages (BMMs), isolated from tibiae and femora of mice, to harmine increased the protein levels of Id2 and AP‐1. Knockdown of Id2 by Id2‐siRNA reduced the number of preosteoclasts as well as secretion of PDGF‐BB in RANKL‐stimulated BMMs administrated with harmine. Inhibition of c‐Fos or c‐Jun (components of AP‐1) both reversed the stimulatory effect of harmine on preosteoclast PDGF‐BB production. Dual‐luciferase reporter assay analyses determined that PDGF‐BB was the direct target of AP‐1 which was up‐regulated by harmine treatment. In conclusion, our data demonstrated a novel mechanism involving in the production of PDGF‐BB increased by harmine, which may provide potential therapeutic targets for bone loss diseases.

In this study, we investigated the beneficial effects of high endogenous levels of n-3 polyunsaturated fatty acids (PUFAs) on skeletal muscle repair and regeneration using a mouse cardiotoxin (CTX, 20 μM/200 μL) -induced gastrocnemius muscle injury model. Transgenic fat-1 mice expressing the Caenorhabditis elegans fat-1 gene, encoding n-3 fatty acid desaturase, showed higher n-3 PUFA levels and lower n-6/n-3 PUFA ratios in gastrocnemius muscle tissues. Hematoxylin and eosin and Masson’s trichrome staining of gastrocnemius sections revealed increased muscle fiber size and reduced fibrosis in fat-1 mice on days 7 and 14 after CTX injections. Gastrocnemius muscle tissues from fat-1 mice showed reduced inflammatory responses and increased muscle fiber regeneration reflecting enhanced activation of satellite cells on day 3 after cardiotoxin injections. Gastrocnemius muscle tissues from cardiotoxin-treated fat-1 mice showed reduced levels of pro-apoptotic proteins (Caspase 3 and Bax) and increased levels of anti-apoptotic proteins (Bcl-2 and Survivin). Moreover, eicosapentaenoic acid (EPA) reduced the incidence of apoptosis among cardiotoxin-treated C2C12 mouse myoblasts. These findings demonstrate that higher endogenous n-3 PUFA levels in fat-1 mice enhances skeletal muscle repair and regeneration following cardiotoxin-induced injury.

Corpus luteum,an important endocrine tissue in mammalian ovary,plays a role in the regulation of reproductive cycle and the establishment of early pregnancy.While studying the luteal development and its molecular regulation,we have discovered a variety of immune cells,such as T lymphocytes,macrophages,neutrophils,and eosinophils,in the corpus luteum.These immune cells accumulate and support luteal angiogenesis and progesterone production during the luteal development,thus participating in the regulation of luteal functions.In luteal regression,prostaglandin F2 can stimulate the production of inflammatory cytokines and chemokines,which help immune cells enter the corpus luteum and enhance the decomposition of corpus luteum through inflammatory reactions.According to our research achievements,we reviewed the roles of different types of immune cells in the development and degradation of mammalian luteal functions,aiming to further understand the biology of corpus luteum and provide a reference for the clinical manipulation of luteal functions.

Emerging studies have demonstrated that long non‑coding RNAs (lncRNAs) play essential roles in tumorigenesis. However, the role and function of lncRNAs in hypopharyngeal squamous cell carcinoma (HSCC) have not been completely elucidated. The present study explored the function of a novel lncRNA, RP11‑156L14.1, in HSCC. RP11‑156L14.1 was revealed to be highly expressed in HSCC tissues and cell lines. Knockdown of RP11‑156L14.1 inhibited proliferation, migration, and invasion in HSCC cells. Furthermore, RP11‑156L14.1 regulated epithelial‑mesenchymal transition (EMT) by controlling EMT‑related protein expression. Mechanistically, RP11‑156L14.1 exerted its function as a competing endogenous RNA (ceRNA) and directly interacted with miR‑548ao‑3p. The present study also demonstrated that miR‑548ao‑3p regulated signal sequence receptor subunit 1 (SSR1) expression by targeting SSR1 3'‑UTR. Moreover, the xenograft HSCC tumor model revealed that knockdown of RP11‑156L14.1 markedly suppressed HSCC tumor growth in vivo. In summary, these findings indicated that the lncRNA RP11‑156L14.1 functions as an oncogene in HSCC by competing with miR‑548ao‑3p in regulating SSR1 expression. The RP11‑156L14.1/miR‑548ao‑3p/SSR1 axis could be utilized as a potential novel biomarker and therapeutic target for HSCC.

Background::Atrial natriuretic peptide (ANP) and its natriuretic peptide receptors A (NPR-A) and C (NPR-C) are involved in the regulation of physiological and pathophysiological process of blood pressure. The present study aimed to determine the role of NPR-C in the development of salt-sensitive hypertension.Methods::The Dahl salt-sensitive (DS) and salt-resistant (DR) rats were used in this study. Animals were matched according to their age and weight, and then placed on either a high-salt (HS, 8%) or a normal-salt (NS, 0.4%) diet for 6 weeks randomly using random number table. The systolic blood pressure (SBP), plasmatic sodium concentration (PL Na), urinary sodium excretion (UV Na), and serum creatinine concentration (Scr) were measured. The concentration of ANP in blood and tissues (heart and kidney) was detected by enzyme-linked immunosorbent assay. The expression of ANP, NPR-A, and NPR-C in kidney was evaluated with western blot analysis. Regarding renal redox state, the concentration changes in malondialdehyde (MDA), lipofuscin, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox), and nitric oxide synthase (NOS) in kidney were detected by a spectrophotometric method. The kidney damage was evaluated using pathological techniques and the succinodehydrogenase (SDHase) examination. Furthermore, after an intra-peritoneal injection of C-atrial natriuretic peptide (ANP) 4-23 (C-ANP 4-23), an NPR-C receptor agonist, the SBP, biochemical values in blood and urine, and renal redox state were evaluated. The paired Student’s t test and analysis of variance followed by the Bonferroni test were performed for statistical analyses of the comparisons between two groups and multiple groups, respectively. Results::The baseline SBP in all groups was within the normal range. At the end of the 6-week experiment, HS diet significantly increased the SBP in DS rats from 116.63 ± 2.90 mmHg to 162.25 ± 2.15 mmHg ( t = -10.213, P < 0.001). The changes of SBP were not significant in DS rats on an NS diet and DR rats on an NS diet or on an HS diet (all P > 0.05). The significant increase of PL Na, UV Na, and Scr related to an HS diet was found in both DS and DR rats (all P < 0.05). However, significant changes in the concentration ( t= -21.915, P < 0.001) and expression of renal ANP ( t= -3.566, P = 0.016) and the expression of renal NPR-C ( t = 5.864, P = 0.002) were only observed in DS hypertensive rats. The significantly higher desmin immunochemical staining score ( t = -5.715, P = 0.005) and mitochondrial injury score ( t = -6.325, P = 0.003) accompanied by the lower SDHase concentration ( t= 3.972, P = 0.017) revealed mitochondrial pathologic abnormalities in podocytes in DS rats with an HS diet. The distinct increases of MDA ( t= -4.685, P= 0.009), lipofuscin ( t= -8.195, P= 0.001), and Nox ( t= -12.733, P < 0.001) but not NOS ( t = -0.328, P = 0.764) in kidneys were also found in DS hypertensive rats. C-ANP 4-23 treatment significantly decreased the SBP induced by HS in DS rats ( P < 0.05), which was still higher than NS groups with the vehicle or C-ANP 4-23 treatment ( P < 0.05). Moreover, the HS-induced increase of MDA, lipofuscin, Nox concentrations, and Nox4 expression in DS rats was significantly attenuated by C-ANP 4-23 treatment as compared with those with HS diet and vehicle injection (all P < 0.05). Conclusions::The results indicated that the renal NPR-C might be involved in the salt-sensitive hypertension through the damage of mitochondria in podocytes and the reduction of the anti-oxidative function. Hence, C-ANP 4-23 might serve as a therapeutic agent in treating salt-sensitive hypertension.

To observe the effects of transcutaneous auricular vagus nerve stimulation (taVNS) on the motor function and the expression of glial fibrillary acidic protein (GFAP) and microtubule associated protein 2 (MAP2) in cerebral ischemic penumbra of rats with middle cerebral artery occlusion (MCAO) and explore the mechanism of taVNS in the improvement of motor function in MCAO rats.A total of 48 male SD rats were randomized into a sham-operation group, a model group, a transcutaneous auricular non-vagus nerve stimulation (tnVNS) group and a taVNS group, with 12 rats in each group. The suture-occluded method was adopted to prepare MCAO rat model. The auricular rim was stimulated in the tnVNS group and the concha stimulated in the taVNS group, 2 mA in intensity, 10 Hz in frequency, 30 min each time, once a day, for 14 days consecutively. The nerve functional assessment was recorded in each group. The expressions of nicotinic acetylcholine receptor (α7nAchR) in the cerebral ischemic penumbra and the spleen were detected by using Western blot. With the immunofluorescence, the expressions of GFAP and MAP2 were detected.After modeling, compared with the sham-operation group, the nerve functional score was increased in the model group, the tnVNS group and the taVNS group (TaVNS is effective on neuroprotection in MCAO rats, which may be related to its function of inhibition of GFAP expression and promotion of MAP2 expression in the ischemic penumbra.

Huangqin Decoction(HQD) is a classic prescription for treating dysentery in the Treatise on Cold Damage and now is mainly used for the treatment of ulcerative colitis(UC). Since there are no requirements on specific Paeonia species, both Paeoniae Radix Alba(white peony root, WPR) and Paeoniae Radix Rubra(red peony root, RPR) are clinically used in HQD now. Although the two types of peony roots are close in origin and similar in primary components, the medicinal properties and efficacies are different. Furthermore, the systematic comparative analysis on the efficacy differences in treating UC of HQD with the roots of multi-originated peony has been seldom reported. This study compared and evaluated the pharmacological effects of HQD prepared from the roots of multi-originated peony, including WPR, RPR-l(derived from P. lactiflora), and RPR-v(derived from P. veitchii) based on the mouse model of UC induced by dextran sodium sulfate(DSS) by animal behaviors, pathological section(colon), and cytokine expression(IL-1β and IL-6), aiming to provide evidence for the identification of the original resource of peony root in HQD. The results indicated that all HQD samples prepared from WPR, RPR-l, and RPR-v could improve the symptoms of UC. Compared with the HQD-WPR, HQD-RPR-l and HQD-RPR-v were significantly different in weight loss, colon length, and disease activity index(DAI) score, but there was no significant difference between HQD-RPR-l and HQD-RPR-v. Moreover, HQD-RPR-v exhibited the most significant improvement in the pathological morphology of colonic tissue and mucosal defects. According to the previous comparative analysis of chemical profiling and content distribution of HQD prepared from the roots of multi-originated peony, RPR-v in HQD was potent in protecting against UC, which was presumedly attributed to a large number of monoterpene glycosides and galloyl glucoses. This study provided a scientific basis for the determination of peony root in HQD and its clinical medication.

Honeybees (Apis mellifera) are important economic insects and play important roles in pollination and maintenance of ecological balance. However, the use of pesticides has posed a substantial threat to bees in recent years, with the more widely used deltamethrin being the most harmful. In this study, we found that deltamethrin exposure significantly reduced bee survival in a dose-dependent manner (p = 0.025). In addition, metagenomic sequencing further revealed that DM exposure significantly reduced the diversity of the bee gut microbiota (Chao1, p 0.0001; Shannon, p 0.0001; Simpson, p 0.0001) and decreased the relative abundance of core species of the gut microbiota. Importantly, in studies of GF-bees, we found that the colonization of important gut bacteria such as Gilliamella apicola and Lactobacillus kunkeei significantly increased bee resistance to DM (survival rate increased from 16.7 to 66.7%). Interestingly, we found that the immunity-genes Defensin-2 and Toll were significantly upregulated in bees after the colonization of gut bacteria. These results suggest that gut bacteria may protect against DM stress by improving host immunity. Our findings provide an important rationale for protecting honeybees from pollutants from the perspective of gut microbes.

Autoinducer-2 (AI-2) is believed to be a bacterial interspecies signaling molecule that plays an important role in the regulation of the physiological behaviors of bacteria. The effect of AI-2 on the process of necrotizing enterocolitis (NEC) is unknown, and the aim of this study was to study the effect of AI-2 in a mouse NEC model. C57BL/6 mouse pups were randomly divided into three groups: the control group, the NEC group, and the NEC+AI-2 (NA) group. Exogenous AI-2 (500 nM) was added to the formula milk of the NA group. The concentrations of fecal AI-2 and flora were tested. The expression of cytokines, TLR4 and NF-κB in intestinal tissue was detected. The AI-2 level was significantly decreased in the NEC group (PPPPP < 0.05). Exogenous AI-2 partially reverses flora disorder and decreases inflammation in an NEC mouse model.

The microbiome associated with brown planthopper (BPH) plays an important role in mediating host health and fitness. Characterization of the microbial community and its structure is prerequisite for understanding the intricate symbiotic relationships between microbes and host insect. Here, we investigated the bacterial and fungal communities of BPH at different developmental stages using high-throughput amplicon sequencing. Our results revealed that both the bacterial and fungal communities were diverse and dynamic during BPH development. The bacterial communities were generally richer than fungi in each developmental stage. At 97% similarly, 19 phyla and 278 genera of bacteria were annotated, while five fungal phyla comprising 80 genera were assigned. The highest species richness for the bacterial communities was detected in the nymphal stage. The taxonomic diversity of the fungal communities in female adults was generally at a relatively higher level when compared to other developmental stages. The most dominant phylum of bacteria and fungi at each developmental stage all belonged to Proteobacteria and Ascomycota, respectively. A significantly lower abundance of bacterial genus Acinetobacter was recorded in the egg stage when compared to other developmental stages, while the dominant fungal genus Wallemia was more abundant in the nymph and adult stages than in the egg stage. Additionally, the microbial composition differed between male and female adults, suggesting that the microbial communities in BPH were gender-dependent. Overall, our study enriches our knowledge on the microbial communities associated with BPH and will provide clues to develop potential biocontrol techniques against this rice pest.

Osteoarthritis (OA) is the most common degenerative joint disease and involves the loss of articular cartilage integrity, formation of articular osteophytes, remodeling of subchondral bone, and synovitis. Knockdown of receptor interacting serine/threonine kinase (RIPK) 1 leads to anti-inflammatory and anti-apoptotic effects. However, the involvement of RIPK1 in the pathogenesis of OA is unclear. Here, we evaluated the effect of RIPK1 on chondrocytes and elaborated the underlying molecular mechanism. Knockdown of RIPK1 protected chondrocytes against inflammation and apoptosis induced by interleukin (IL)-1β in vitro and in vivo. RIPK1 was required for myeloid differentiation primary response 88 (MyD88)- and TIR-domain-containing adapter-inducing interferon b (TRIF)-mediated production of matrix metalloproteinases (MMPs) in OA. Moreover, overexpression of RIPK1 promoted the expression of tumor necrosis factor receptor-associated factor 2 (TRAF2), which blocked the expression and phosphorylation of RIPK1. Upregulation of TRAF2 decreased the expression of TRIF, MyD88, and MMPs in chondrocytes. Furthermore, knockdown of RIPK1 blocked activation of the nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) signaling pathways. In summary, knockdown of RIPK1 alleviated OA in a manner mediated by the TRIF/MyD88-RIPK1-TRAF2 negative feedback loop and activation of the NF-κB and JNK signaling pathways.

Background Highly sensitized patients are difficult to match with suitable renal allograft donors and may benefit from xenotransplant trials. We evaluate antibody binding from sensitized patients to pig cells and engineered single allele cells to identify anti-human leukocyte antigen (HLA) antibody cross-species reactivity with swine leukocyte antigen (SLA). These novel testing strategies assess HLA/SLA epitopes and antibody-binding patterns and introduce genetic engineering of SLA epitopes. Methods Sensitized patient sera were grouped by calculated panel reactive antibody and luminex single antigen reactivity profile and were tested with cloned GGTA1/CMAH/B4GalNT2 glycan knockout porcine cells. Pig reactivity was assessed by direct flow cytometric crossmatch and studied following elution from pig cells. To study the antigenicity of individual class I HLA and SLA alleles in cells, irrelevant sera binding to lymphoblastoid cells were minimized by CRISPR/Cas9 elimination of endogenous class I and class II HLA, B-cell receptor, and Fc receptor genes. Native HLA, SLA, and mutants of these proteins after mutating 144K to Q were assessed for antibody binding. Results Those with predominately anti-HLA-B&C antibodies, including Bw6 and Bw4 sensitization, frequently have low pig reactivity. Conversely, antibodies eluted from porcine cells are more commonly anti-HLA-A. Single HLA/SLA expressing engineered cells shows variable antigenicity and mutation of 144K to Q reduces antibody binding for some sensitized patients. Conclusions Anti-HLA antibodies cross-react with SLA class I in predictable patterns, which can be identified with histocompatibility strategies, and SLA class I is a possible target of genetic engineering.

Jinzhen oral liquid (JZ) is a classical traditional Chinese medicine formula used for the treatment of children lung disease. However, the effective substance of JZ is still unclear. In this study, we used lung injury rat model to study the protective effect of JZ, through UPLC-Q-TOF/MS detection coupled with metabolic research and network pharmacology analysis. Fortunately, 31 absorbed prototype constituents and 41 metabolites were identified or tentatively characterized based on UPLC-Q-TOF/MS analysis, and the possible metabolic pathways were hydroxylation, sulfation and glucuronidation. We optimized the data screening in the early stage of network pharmacology by collecting targets based on adsorbed constituents, and further analyzed the main biological processes and pathways. 24 selected core targets were frequently involved in reactive oxygen species metabolic process, dopaminergic synapse pathway and so on, which might play important roles in the mechanisms of JZ for the treatment of lung injury. Overall, the absorbed constituents and their possible metabolic pathways, as well as the absorbed constituent-target-disease network provided insights into the mechanisms of JZ for the treatment of lung injury. Further studies are needed to validate the biological processes and effect pathways of JZ. Keywords: Metabolic research, TCM, Network pharmacology

Circular RNAs have been found to be aberrantly expressed in tumors and their significance in tumorigenesis has been focused on. The role of circDYNC1H1 in hepatocellular carcinoma (HCC) pathogenesis and its relationship with miR-140-5p were explored. The expression of circDYNC1H1, miR-140-5p, and SULT2B1 in HCC tissues and cells was measured, and Pearson's analysis was used to analyze their expression correlation. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Transwell assays were performed to determine cell proliferation and migration. Binding between circDYNC1H1 and miR-140-5p was evaluated with RNA pull-down assay. A luciferase reporter assay was conducted to assess the interaction between circDYNC1H1 and miR-140-5p and between miR-140-5p and SULT2B1. circDYNC1H1 was highly expressed in HCC tissues (n = 20), and it was negatively associated with the expression of miR-140-5p but positively correlated with SULT2B1 messenger RNA expression. circDYNC1H1 was upregulated in cell lines of HCC. Interference of circDYNC1H1 suppressed cell proliferation and migration of HCC. circDYNC1H1 acted as a sponge of miR-140-5p. miR-140-5p controlled SULT2B1 expression by targeting its 3'-untranslated region. circDYNC1H1 enhanced SULT2B1 expression via sponging miR-140-5p. Downregulation of circDYNC1H1 disturbed cell proliferation and migration of HCC through miR-140-5p/SULT2B1 pathway. Silencing of circDYNC1H1 delayed tumor growth in HCC mouse model. Acting like a sponge of miR-140-5p, silenced circDYNC1H1 downregulated SULT2B1 to restrain HCC cell proliferation and migration, which is adverse to HCC growth and progression.

Caffeine is a substance that is consumed worldwide, and it may exert neuroprotective effects against various cerebral insults, including neurotrauma, which is the most prevalent injury among military personnel. To investigate the effects of caffeine on high-intensity blast wave-induced severe blast injury in mice, three different paradigms of caffeine were applied to male C57BL/6 mice with severe whole body blast injury (WBBI). The results demonstrated that chronic caffeine treatment alleviated blast-induced traumatic brain injury (bTBI); however, both chronic and acute caffeine treatments exacerbated blast-induced lung injuries and, more importantly, increased both the cumulative and time-segmented mortalities postinjury. Interestingly, withdrawing caffeine intake preinjury resulted in favorable outcomes in mortality and lung injury, similar to the findings in water-treated mice, and had the trend to attenuate brain injury. These findings demonstrated that although drinking coffee or caffeine preparations attenuated blast-induced brain trauma, these beverages may place personnel in the battlefield at high risk of casualties, which will help us re-evaluate the therapeutic strategy of caffeine application, particularly in multiple-organ-trauma settings. Furthermore, these findings provided possible strategies for reducing the risk of casualties with caffeine consumption, which may help to change the coffee-drinking habits of military personnel.

The current evidence is that sensitization to a pig xenograft does not result in the development of antibodies that cross-react with alloantigens, and therefore, sensitization to a pig xenograft would not be detrimental to the outcome of a subsequent allograft. This evidence relates almost entirely to the transplantation of cells or organs from wild-type or α1,3-galactosyltransferase gene-knockout (GTKO) pigs. However, it is not known whether recipients of triple-knockout (TKO) pig grafts who become sensitized to TKO pig antigens develop antibodies that cross-react with alloantigens and thus be detrimental to a subsequent organ allotransplant. We identified a single baboon (B1317) in which no (or minimal) serum anti-TKO pig antibodies could be measured-in our experience unique among baboons. We sensitized it by repeated subcutaneous injections of TKO pig peripheral blood mononuclear cells (PBMCs) in the absence of any immunosuppressive therapy. After TKO pig PBMC injection, there was a transient increase in anti-TKO pig IgM, followed by a sustained increase in IgG binding to TKO cells. In contrast, there was no serum IgM or IgG binding to PBMCs from any of a panel of baboon PBMCs (n = 8). We conclude that sensitization to TKO pig PBMCs in the baboon did not result in the development of antibodies that also bound to baboon cells, suggesting that there would be no detrimental effect of sensitization on a subsequent organ allotransplant.

Background The brown planthopper (BPH) is one of the most destructive pests of rice, causing tremendous yield and economic losses every year. The fungal entomopathogen Metarhizium anisopliae was previously proved to have great potential for BPH biocontrol. Genome-wide insight into the insect-fungus interaction is crucial for genetic improvement of M. anisopliae to enhance its virulence to BPH but still has been poorly explored. Results Using dual RNA-seq approach, we present here a global view of host and fungal gene expressions in BPH adults during the fungal infection. The results revealed that BPH could initiate strong defense responses against the fungal attack by upregulating the expressions of a large number of genes, including genes involved in cuticle formation, immune response, cell detoxification and biomacromolecule metabolism. Correspondingly, the fungal entomopathogen could induce a series of genes to infect and modulate BPH, including genes involved in fungal penetration, invasive growth, stress resistance and virulence. Three host defense-related genes (NlPCE4, NlPOD1 and NlCYP4DE1) were chose for further function analysis. RNAi-mediated knockdown of NlPCE4 caused a significant decrease in BPH survival, but no obvious effects on the survival rates were detected by the suppression of NlPOD1 and NlCYP4DE1. Combination of dsRNA injection and fungal infection could significantly enhance the BPH-killing speed, as synergistic mortalities were observed in co-treatments of RNAi and M. anisopliae infection. Conclusion Our study provides a comprehensive insight into molecular mechanisms of host-pathogen interaction between BPH and M. anisopliae and contributes to future development of new efficient biocontrol strategies for BPH biocontrol.

Simulated hypobaric hypoxia (SHH) training has been used to enhance running performance. However, no studies have evaluated the effects of a single SHH exposure on healthy mice performance and analyzed the changes of mitochondria-related genes in the central nervous system. The current study used a mouse decompression chamber to simulate mild hypobaric hypoxia at the high altitude of 5000 m or severe hypobaric hypoxia at 8000 m for 16 h (SHH5000 & SHH8000, respectively). Then, the mouse behavioral tests were recorded by a modified Noldus video tracking. Third, the effects of SHH on 8 mitochondria-related genes of Drp1, Mfn1, Mfn2, Opa1, TFAM, SGK1, UCP2 and UCP4, were assessed in cerebellum, hippocampus and gastrocnemius muscles. The results have shown that a single mild or severe HH improves healthy mice performance. In cerebellum, 6 of all 8 detected genes (except Mfn2 and UCP4) did not change after SHH. In hippocampus, all detected genes did not change after SHH. In muscles, 7 of all 8 detected genes (except Opa1) did not change after SHH. The present study has indicated the benefit of a single SHH in healthy mice performance, which would due to the stabilized mitochondria against a mild stress state.

Gene editing is a genetic engineering technology that can modify, delete, or insert a small piece of DNA at a specific point in the genome of cells and organisms. Gene editing technology holds great promises in the fields of disease treatment, gene function regulation, gene detection, drug research and development, and crop breeding. However, side effects, such as off-target editing, genotoxicity and other issues, have gradually emerged in the application. In the CRISPR (clustered regularly interspaced short palindromic repeats) system, the Cas9 nuclease can specifically recognize the target DNA by the base pairing of a guide RNA (gRNA) with the target DNA. Upon target recognition, the two DNA strands are cleaved by distinct domains of the Cas9 nuclease. Since both Cas9 nuclease and gRNA possess different characteristics in their own activities, recognition sites and binding ability to specific target, it is essential to precisely regulate the activity of Cas9 nuclease and gRNA in both time and space manners, thus preventing the risk of side effects and enhancing the precise regulation of the CRISPR/Cas9 gene editing technology. In this review, we summarize the advances in the precise control of gene editing, especially CRISPR/cas9 over several dimensions using fusion Cas9 proteins regulated by light, temperature and drugs, exploiting and screening anti-CRISPRs proteins, synthesizing and identifying small molecules- inhibitors, and developing other therapeutic agents, thereby providing a reference and research ideas for human disease treatment, crop and livestock improvement and prevention of biotechnology misuse.基因编辑(gene editing)是一种能对细胞和生物体基因组一小段DNA进行定点修饰或者删除、插入的基因工程技术。基因编辑技术在疾病治疗、基因功能调控、基因检测、药物研发和作物育种等方面具有广阔的应用前景,但在应用中也逐渐显现出脱靶、基因毒性等副作用问题。CRISPR (clustered regularly interspaced short palindromic repeats)系统中核酸酶Cas9蛋白能通过与gRNA (guide RNA)结合特异性识别靶DNA并进行酶切反应,由于Cas9蛋白和gRNA在其自身活性、识别位点及结合能力等方面具有不同的特性,因此在应用中可以通过对Cas9蛋白酶的活性以及与靶DNA在时间和空间上的结合进行精准调控,主要调控方法包括使用光、温度和药物等调节Cas9融合蛋白、抗CRISPR蛋白、核酸类和小分子类化合物抑制剂的使用等,从而能有效地防范基因编辑技术的风险和增强精准调控基因编辑技术的实际应用性。本文就目前如何精准调控基因编辑技术,尤其是精准调控CRISPR/Cas9基因编辑技术的方法进行了综述,以期为人类疾病治疗、作物育种、家畜遗传改良和防范生物技术缪用等提供借鉴和研究思路。.

In the present study, liquid chromatography-mass spectrometer (LC-MS) was used to perform untargeted metabolomics analysis of white adipose tissue (WAT) and brown adipose tissue (BAT) in Tupaia belangeri during cold acclimation. Differences in biochemical composition between WAT and BAT were compared. Clarifying how the two adipose tissues respond to the lower temperature in terms of metabolomics, which elucidate the metabolic process and energy homeostasis regulation mechanism in T. belangeri. The results showed that there were 34, 59 and 20 differential metabolites in the WAT, BAT and WAT compared with BAT, respectively. WAT and BAT had significant differences in various metabolic pathways such as sugar metabolism, amino acid metabolism, lipid metabolism, and nucleotide metabolism, which were closely related to the different biological roles of the two tissues. Increasing the concentrations of intermediate products of tricarboxylic acid (TCA) cycle, pyruvic acid, and phosphoenolpyruvic acid (PEP) in WAT and increasing the metabolites in TCA cycle, glyoxylate and dicarboxylate metabolism pathways in BAT, likely to increase the thermogenic capacity in T. belangeri in response to cold stress. There were more differential metabolic pathways in BAT during cold acclimation than that of in WAT. Moreover, compared to WAT, BAT responds to cold stress by adjusting the concentration of nucleotide metabolites.

To investigate adaptive strategies of Tupaia belangeri to environmental factors in different populations, 12 locations were selected, including higher and lower altitude areas. Total of 96 and 90 metabolites were annotated in serum and liver respectively, which were mainly concentrated in primary metabolites. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) showed that the locations were divided into two groups in serum metabolites, but each group had a few samples overlap. The samples of each group overlap to some degree in the liver metabolites. The tricarboxylic acid (TCA) cycle occupies a central position in metabolism. The concentrations of TCA intermediates, lipid metabolites and amino acid metabolites were higher in higher altitude areas, and the concentrations of carbohydrate and glycolysis intermediates were higher in lower altitude areas. Different areas adapted to the changes of environmental and altitude by regulating the concentration of metabolites in serum and liver, and revealed the adaptive mechanism of T. belangeri in different living environments.

RNAi is an essential technology for studying gene function in eukaryotes, and is also considered to be a potential strategy for pest control. However, the mechanism behind the cellular uptake of dsRNA in aphids, a group of important agricultural sucking pests, remains unknown. Here, using the pea aphid Acyrthosiphon pisum as model for aphids, we identified two core genes of clathrin-dependent endocytosis (CDE), Apchc and Apvha16. We confirmed that expression of Apchc, Apvha16 and RNAi core component genes (ApAgo2, ApDcr2 and ApR2d2) were simultaneously induced at 12 h after feeding dsRNA. By using an RNAi-of-RNAi approach, we demonstrated that suppression of Apchc and Apvha16 transcripts by RNAi significantly impaired RNAi efficiency of selected reporter genes (RGs), including ApGNBP1, Apmts and Aphb, suggesting the involvement of CDE in cellular dsRNA uptake in aphids. Further confirmation was also provided using two inhibitors, chlorpromazine (CPZ) and bafilomycin A1 (BafA1). Administration of CPZ and of BafA1 both led to an impaired silencing efficiency of the RGs in the pea aphid. Finally, these RNAi-of-RNAi results were reconfirmed in the peach aphid Myzus persicae. Taking these findings together, we conclude that CDE is involved in cellular dsRNA uptake in aphids.

The underlying mechanisms of wound healing are complex but inflammation is one of the determining factors. Besides its traditional role in combating against infection upon injury, the characteristics and magnitude of inflammation have dramatic impacts on the pathogenesis of scar. Keloids and hypertrophic scars are pathological scars that result from aberrant wound healing. They are characterized by continuous local inflammation and excessive collagen deposition. In this review, we aim at discussing how dysregulated inflammation contributes to the pathogenesis of scar formation. Immune cells, soluble inflammatory mediators, and the related intracellular signal transduction pathways are our three subtopics encompassing the events occurring in inflammation associated with scar formation. In the end, we enumerate the current and potential medicines and therapeutics for suppressing inflammation and limiting progression to scar. Understanding the initiation, progression, and resolution of inflammation will provide insights into the mechanisms of scar formation and is useful for developing effective treatments.

Fracture healing is a complicated process affected by many factors, such as inflammatory responses and angiogenesis. Omentin-1 is an adipokine with anti-inflammatory properties, but whether omentin-1 affects the fracture healing process is still unknown. Here, by using global omentin-1 knockout (omentin-1

Pig organ xenotransplantation offers a solution to the shortage of deceased human organs for transplantation. The pathobiological response to a pig xenograft is complex, involving antibody, complement, coagulation, inflammatory, and cellular responses. To overcome these barriers, genetic manipulation of the organ-source pigs has largely been directed to two major aims-(a) deletion of expression of the known carbohydrate xenoantigens against which humans have natural (preformed) antibodies, and (b) transgenic expression of human protective proteins, for example, complement- and coagulation-regulatory proteins. Conventional (FDA-approved) immunosuppressive therapy is unsuccessful in preventing an adaptive immune response to pig cells, but blockade of the CD40:CD154 costimulation pathway is successful. Survival of genetically engineered pig kidneys in immunosuppressed nonhuman primates can now be measured in months. Non-immunological aspects, for example, pig renal function, a hypovolemia syndrome, and rapid growth of the pig kidney after transplantation, are briefly discussed. We suggest that patients on the wait-list for a deceased human kidney graft who are unlikely to receive one due to long waiting times are those for whom kidney xenotransplantation might first be considered. The potential risk of infection, public attitudes to xenotransplantation, and ethical, regulatory, and financial aspects are briefly addressed.

Aberrant scar formation, which includes keloid and hypertrophic scars, is associated with a pathological disorganized wound healing process with chronic inflammation. The TGF-β/Smad signaling pathway is the most canonical pathway through which the formation of collagen in the fibroblasts and myofibroblasts is regulated. Sustained activation of the TGF-β/Smad signaling pathway results in the long-term overactivation of fibroblasts and myofibroblasts, which is necessary for the excessive collagen formation in aberrant scars. There are two categories of therapeutic strategies that aim to target the TGF-β/Smad signaling pathway in fibroblasts and myofibroblasts to interfere with their cellular functions and reduce cell proliferation. The first therapeutic strategy includes medications, and the second strategy is composed of genetic and cellular therapeutics. Therefore, the focus of this review is to critically evaluate these two main therapeutic strategies that target the TGF-β/Smad pathway to attenuate abnormal skin scar formation.

Progress has been made in overcoming antibody-mediated rejection of porcine xenografts by deleting pig genes that produce unique carbohydrate epitopes. Pigs deficient in galactose α-1,3 galactose (gene modified: GGTA1) and neu5Gc (gene modified: CMAH) have reduced levels of human antibody binding. Previously we identified α-fucose as a glycan that was expressed in high levels on cells of GGTA1/CMAH KO pigs. To validate the α-fucose phenotype observed previously we compared lectin affinity toward human and pig serum glycoproteins by dot blot analysis and confocal microscopy. Human anti-fucose antibody isolated by affinity chromatography was tested for specificity to L-fucose by custom macroarray. The affinity and cytotoxicity of the isolated human anti-fucose antibody toward human and GGTA1/CMAH KO pig PBMCs was determined by flow cytometry. Dot blot and confocal analysis support out previous findings that α-fucose is more highly expressed in pigs than humans. Pig kidney glomeruli and tubules contain abundant α-fucose and may represent focal sites for anti-α-fucose antibody binding. The Isolated human anti-fucose IgA, IgG and IgM bound to GGTA1/CMAH KO pig PBMC and were cytotoxic. Interestingly, the isolated human IgG cross reacted with the methyl pentose, L-rhamnose. Human anti-fucose antibody bound and was cytotoxic to GGTA1/CMAH KO pig peripheral blood monocytes. We have shown that α-fucose is an abundant target for cytotoxic human antibody in the organs of genetically modified pigs important to xenotransplantation.

Genetically engineered pigs are now available for xenotransplantation in which all three known carbohydrate xenoantigens, against which humans have natural antibodies, have been deleted (triple-knockout [TKO] pigs). Furthermore, multiple human transgenes have been expressed in the TKO pigs, all of which are aimed at protecting the cells from the human immune response. Many human sera demonstrate no or minimal antibody binding to, and little or no cytotoxicity of, cells from these pigs, and this is associated with a relatively low T-cell proliferative response. Unfortunately, baboons and other Old World NHPs have antibodies against TKO pig cells, apparently directed to a fourth xenoantigen that appears to be exposed after TKO. In our experience, most, if not all, humans do not have natural antibodies against this fourth xenoantigen. This discrepancy between NHPs and humans is providing a hurdle to successful translation of pig organ transplantation into the clinic, and making it difficult to provide pre-clinical data that support initiation of a clinical trial. The potential methods by which this obstacle might be overcome are discussed. We conclude that, whatever currently available genetically engineered pig is selected for the final pre-clinical studies, this may not be the optimal pig for clinical trials.

Autoinducer-2 (AI-2) has a widely accepted role in bacterial intra- and interspecies communication. Little is known about the relationships between AI-2 and NEC. This study found that AI-2 levels in patients and in a NEC mouse model were detected using the Vibrio harveyi BB170 assay system. Bacterial communities of the newborns' stool microbiota (NEC acute group, NEC recovery group, control group, and antibiotics-free group) and of the NEC mouse model (NEC group and control group) were detected by high-throughput sequencing. Intestinal histopathological changes were observed after HE staining. The AI-2 level in the NEC acute group (44.75 [40.17~65.52]) was significantly lower than that in the control group, NEC recovery group and antibiotics-free group. The overall microbiota compositions of each group at the phylum level were not significantly different. The proportions of Enterococcus, Clostridium_sensu_stricto_1, Peptoclostridium, and Veillonella had significant differences among the 4 groups at the genus level. In animal experiments, the AI-2 level in feces of NEC mice (56.89 ± 11.87) was significantly lower than that in the feces of control group mice (102.70 ± 22.97). The microbiota compositions of NEC and control group mice at the phylum level were not significantly different. At the genus level, Klebsiella, Clostridium_sensu_stricto_1, and Peptoclostridium abundances in the NEC group increased significantly compared with those in the control group (P < 0.05). In addition, Lactobacillus, Pasteurella, and Parabacteroides abundances in the NEC group decreased significantly compared with those in the normal control group (P < 0.05), while Lactobacillus, Pasteurella, and Parabacteroides abundances had the opposite trend. The AI-2 concentration decreased significantly in the acute phase of NEC and increased gradually in the convalescent phase. We conclude that the concentration of AI-2 was correlated with intestinal flora disorder and different stages of disease. AI-2 may be a new biomarker for the diagnosis and monitoring of NEC.Trial Registry: ClinicalTrials.gov; ChiCTR-ROC-17013746; URL: www.clinicaltrials.gov

Objectives Drp1 is widely expressed in the mouse central nervous system and plays a role in inducing the mitochondrial fission process. Many diseases are associated with Drp1 and mitochondria. However, since the exact distribution of Drp1 has not been specifically observed, it is difficult to determine the impact of anti-Drp1 molecules on the human body. Clarifying the specific Drp1 distribution could be a good approach to targeted treatment or prognosis. Methods We visualized the distribution of Drp1 in different brain regions and explicated the relationship between Drp1 and mitochondria. GAD67-GFP knock-in mice were utilized to detect the expression patterns of Drp1 in GABAergic neurons. We also further analyzed Drp1 expression in human malignant glioma tissue. Results Drp1 was widely but heterogeneously distributed in the central nervous system. Further observation indicated that Drp1 was highly and heterogeneously expressed in inhibitory neurons. Under transmission electron microscopy, the distribution of Drp1 was higher in dendrites than other areas in neurons, and only a small amount of Drp1 was localized in mitochondria. In human malignant glioma, the fluorescence intensity of Drp1 increased from grade I-III, while grade IV showed a declining trend. Conclusion In this study, we observed a wide heterogeneous distribution of Drp1 in the central nervous system, which might be related to the occurrence and development of neurologic disease. We hope that the relationship between Drp1 and mitochondria may will to therapeutic guidance in the clinic.

Wilms' tumour 1 gene (WT1) is essential for the development of mammalian urogenital system. However, the expression pattern of WT1 in the development of porcine urogenital organs is still unclear. Here, we examined the expression of WT1 mRNA and protein in porcine kidneys, ovaries and testes from embryonic days 35 and 60 (E35d, E60d, n = 3) to the newborn (0d, n = 4) and adult (210d, n = 3) stages, using real-time PCR and immunofluorescent staining. Real-time PCR analysis showed that porcine kidneys, ovaries and testes all expressed high level of WT1 mRNAs, especially in adult testes (p < 0.05 or 0.01 vs. kidney and ovary, respectively). Morphologically, characteristic microstructures of the kidneys, ovaries and testes were observed and discerned at all four stages. Immunofluorescently, WT1 expression was detected in a dynamic and context-specific pattern during the development of these organs. Taken together, porcine urogenital organs express relatively high levels of WT1 mRNA. Dynamical and context-specific expression profile of WT1 in these organs occurs during their development, implying its close association with the development and function of porcine kidney, ovary and testis.

Background Tools for genome editing in pigs are improving rapidly so that making precise cuts in DNA for the purposes of deleting genes is straightforward. Development of means to replace pig genes with human genes with precision is very desirable for the future development of donor pigs for xenotransplantation. Materials and methods We used Cas9 to cut pig thrombomodulin (pTHBD) and replace it with a plasmid containing a promoterless antibiotic selection marker and the exon for human thrombomodulin. PhiC31 recombinase was used to remove the antibiotic selection marker to create porcine aortic endothelial cells expressing human instead of pTHBD, driven by the endogenous pig promoter. Results The promoterless selection cassette permitted efficient enrichment of cells containing correctly inserted transgene. Recombinase treatment of selected cells excised the resistance marker permitting expression of the human transgene by the endogenous pTHBD promoter. Gene regulation was maintained after gene replacement because pig endogenous promoter was kept intact in the correct position. Conclusions Cas9 and recombinase technology make orthotopic human for pig gene exchange feasible and pave the way for creation of pigs with human genes that can be expressed in the appropriate tissues preserving gene regulation.

Background Over 130 000 patients in the United States alone need a lifesaving organ transplant. Genetically modified porcine organs could resolve the donor organ shortage, but human xenoreactive antibodies destroy pig cells and are the major barrier to clinical application of xenotransplantation. The objective of this study was to determine whether waitlisted patients possess preformed antibodies to swine leukocyte antigen (SLA) class II, homologs of the class II HLA. Methods Sera from people currently awaiting solid organ transplant were tested for IgG binding to class II SLA proteins when expressed on mammalian cells. Pig fibroblasts were made positive by transfection with the class II transactivator. As a second expression system, transgenes encoding the alpha and beta chains of class II SLA were transfected into human embryonic kidney cells. Results Human sera containing IgG specific for class II HLA molecules exhibited greater binding to class II SLA positive cells than to SLA negative cells. Sera lacking antibodies against class II HLA showed no change in binding regardless of the presence of class II SLA. These antibodies could recognize either SLA-DR or SLA-DQ complexes. Conclusions Class II SLA proteins may behave as xenoantigens for people with humoral immunity toward class II HLA molecules.

During the past decade, antennal transcriptome sequencing has been applied to at least 50 species from 16 families of the Lepidoptera order of insects, emphasizing the identification and characterization of chemosensory-related genes. However, little is known about the chemosensory genes in the Zygaenidae family of Lepidoptera. Herein, we report the transmembrane protein gene repertoires involved in chemoreception from Achelura yunnanensis (Lepidoptera: Zygaenidae) through transcriptome sequencing, bioinformatics, phylogenetics and polymerase chain reaction (PCR) approaches. Transcriptome analysis led to the generation of 555.47 million clean reads and accumulation of 83.30 gigabases of data. From this transcriptome, 132 transcripts encoding 69 odorant receptors (ORs), 33 gustatory receptors (GRs), 26 ionotropic receptors (IRs), and four sensory neuron membrane proteins (SNMPs) were identified, 69 of which were full-length sequences. Notably, the number of SNMPs in A. yunnanensis was the largest set in Lepidoptera to date. Phylogenetic analysis combined with sequence homology highlighted several conserved groups of chemoreceptors, including pheromone receptors (a so-called pheromone receptor (PR) clade: AyunOR50 and novel PR members: AyunOR39 and OR40), a phenylacetaldehyde-sensing OR (AyunOR28), carbon dioxide receptors (AyunGR1-3), and antennal IRs (13 A-IRs). In addition, a Zygaenidae-specific OR expansion was observed, including 15 A. yunnanensis members. Expression profiles revealed 99 detectable chemosensory genes in the antennae and 20 in the reproductive tissues, some of which displayed a sex-biased expression. This study identifies potential olfactory molecular candidates for sensing sex pheromones, phenylacetaldehyde or other odorants, and provides preliminary evidence for the putative reproductive function of chemosensory membrane protein genes in A. yunnanensis.

Ethnopharmacological relevance The twigs and bark of Cinnamomum cassia Presl (Lauraceae) are widely used in traditional Chinese medicine in the treatment of tumor, abdominal pain, dysmenorrhea, digestive system disease and inflammatory diseases. The aim of this study was to determine the inhibitory effect of the essential oil from the twigs of Cinnamomum cassia Presl (EOCC) on uterine contraction in vitro and in vivo. Materials and methods The Institute of Cancer Research (ICR) mouse uterine contraction was induced by oxytocin (OT) exposure following estradiol benzoate pretreatment. Mice were given the EOCC (60, 30, and 15 mg/kg) by gavage. The level of prostaglandin F2α (PGF2α) in uterine tissue were determined according to specification of enzyme linked immunosorbent assay (ELISA) kit. Uterine tissue was collected for histopathological analysis (H&E). Myosin light chain 20 (MLC20), phosphorylation of myosin light chain 20 (p-MLC20) and cyclooxygenase-2 (COX-2) proteins in uterine tissue were assessed by Western Blot. Mouse isolated uterus strips were mounted in tissue organ baths containing Locke's solution. The contractile responses were recorded with Power Lab recording system. The effect of the EOCC on uterine contraction induced by OT, PGF2α, and acetylcholine (Ach) was observed. Myometrial cells were exposed to OT (7 μM) to induce Ca2+ release, and the effect of the EOCC (100, 50, and 25 μg/ml) on intracellular Ca2+ was analysed with fluorometry imaging. Results In vivo study demonstrated that the EOCC significantly reduced OT-induced writhing responses with a maximal inhibition of 66.5%. It also decreased the level of PGF2α in OT-induced mice uterine tissue. Moreover, Western blot analysis showed that COX-2 and p-MLC20 expressions in uterine tissue of dysmenorrhea mice were significantly reduced. EOCC inhibited spontaneous uterus contractions in a dose-dependent manner, and the concentration of the EOCC giving 50% of maximal contraction (IC50) value was 61.3 μg/ml. The IC50 values of the EOCC on OT, PGF2α, and Ach-induced contractions were 113.0 μg/ml, 94.7 μg/ml, and 61.5 μg/ml, respectively. Further in vitro studies indicated that the EOCC could restrain intracellular Ca2+ levels in favour of uterine relaxation. Conclusion Both in vivo and in vitro results suggest that the EOCC possesses significant spasmolytic effect on uterine contraction. Thus, the EOCC yields a possible therapeutic choice for the prevention and treatment of primary dysmenorrhea.

Background: The autologous saphenous vein is the most common conduit for coronary artery bypass grafting, but the vein graft disease will occur. This study used Matrigel basement membrane matrix with many different growth factors to promote vasa vasorum neovascularization and extenuate the hypoxia to improve remodeling. Methods: This study observed the hypoxia and thickness of the vein grafts at different times. Normal veins and vein grafts with 15 min of ischemia one day postoperatively were harvested in the neck of rabbits. Paired vein grafts with 15 min ischemia bilaterally (control vs. Matrigel basement membrane matrix) were performed and harvested at 2, 6, and 12 weeks postoperatively. The rabbits were randomly divided into four postoperative groups (six rabbits in each group): Group 1, one day postoperatively; Group 2, 2 weeks postoperatively; Group 3, 6 weeks postoperatively; and Group 4, 12 weeks postoperatively. The dimensions of vessel wall were captured, and the mean thicknesses of intima, media, and adventitia were measured. The hypoxia-inducible factor (HIF)-1α and HIF-2α labeling indices of intima, media, and adventitia were also measured. Results: In Group 1, the labeling index of HIF-1α was high in the normal vein and decreased significantly in the vein graft one day postoperatively (intima: 80 ± 3% vs. 12 ± 1%, P = 0.01; media: 67 ± 5% vs. 11 ± 1%, P = 0.01; adventitia: 40 ± 10% vs. 7 ± 2%, P = 0.03). The labeling index of HIF-2α had similar trend as HIF-1α (intima: 80 ± 10% vs. 10 ± 5%, P = 0.02; media: 60 ± 14% vs. 12 ± 2%, P = 0.01; adventitia: 45 ± 20% vs. 10 ± 4%, P = 0.03). Compared with the control vein grafts, vein grafts with Matrigel basement membrane matrix had lower labeling indices of HIF-1α and HIF-2α in media and adventitia at Group 2 (HIF-1α: 34 ± 5% vs. 20 ± 4%, P = 0.04 for media; 23 ± 3% vs. 11 ± 2%, P = 0.03 for adventitia; HIF-2α: 37 ± 6% vs. 21 ± 4%, P = 0.03 for media; 24 ± 4% vs. 13 ± 2%, P = 0.04 for adventitia) and Group 3 (HIF-1α: 33 ± 4% vs. 7 ± 2%, P = 0.04 for media; 13 ± 3% vs. 3 ± 1%, P = 0.02 for adventitia; HIF-2α: 27 ± 4% vs. 12 ± 3%, P = 0.02 for media; 19 ± 2% vs. 6 ± 1%, P = 0.02 for adventitia). There were no differences in mean thickness of intima, media, and adventitia between bilateral vein grafts at 2, 6, and 12 weeks postoperatively. Conclusions: This study indicated that promoting vasa vasorum neovascularization of vein grafts extenuated hypoxia, but did not influence the intimal hyperplasia of the wall. Key words: Hypoxia; Hypoxia-inducible Factor; Intimal Hyperplasia; Vein Graft

Background Nuclease-based genome editing has rapidly sped the creation of new models of human disease. These techniques also hold great promise for the future of clinical xenotransplantation and cell-based therapies for cancer or immunodeficient pathology. However, to fully realize the potential of nuclease editing tools, the efficiency and precision of their application must be optimized. The object of this study was to use nonintegrating selection and nuclease-directed homologous recombination to efficiently control the genetic modification of the porcine genome. Methods Clustered randomly integrating spaced palindromic repeats and associated Cas9 protein (CRISPR/Cas9)-directed mutagenesis with a single-guide RNA target was designed to target the alpha-1,3-galactosyltransferase locus (GGTA1) of the porcine genome. A vector expressing a single-guide RNA, Cas9 protein, and green fluorescent protein was used to increase plasmid-delivered mutational efficiency when coupled with fluorescence sorting. Single and double-strand DNA oligonucleotides with a restriction site replacing the start codon were created with variable homology lengths surrounding the mutational event site. Finally, a transgene construct was flanked with 50 base pairs of homology directed immediately 5′ to a nuclease cut site. These products were introduced to cells with a constant concentration of CRISPR/cas9 vector. Phenotype-specific mutational efficiency was measured by flow cytometer. Controlled homologous insertion was measured by Sanger sequence, restriction enzyme digest and flow cytometry. Results Expression of a fluorescence protein on the Cas9 vector functioned as a nonintegrating selection marker. Selection by this marker increased phenotype-silencing mutation rates from 3.5% to 82% (P = 0.0002). Insertion or deletion mutation increased from 11% to 96% (P = 0.0007). Co-transfection with homologous DNA oligonucleotides increased the aggregate phenotype-silencing mutation rates up to 22% and increased biallelic events. Single-strand DNA was twice as efficient as double-strand DNA. Furthermore, nuclease-mediated insertion by homology-directed repair successfully drove locus-specific transgene expression in the porcine genome. Conclusions A nonintegrating selection strategy based on fluorescence expression can increase the mutational efficiency of the CRISPR/Cas9 system. The precision of this system can be increased by the addition of a very short homologous template sequence and can serve as a method for locus-specific transgene delivery. Together these strategies may be used to efficiently control mutational events. This system may be used to better use the potential of nuclease-mediated genomic editing.

ObjectiveTo investigate the cellular mechanisms of action of tanshinone IIA on the fatty liver disease induced by a high-fat diet in an animal model of non-alcoholic fatty liver disease (NAFLD).MethodsAdult male Sprague Dawley rats were randomized into one of three groups: regular rat diet (CON group) for 4 months; high-fat diet (HFD group) for 4 months; HFD for 2 months followed by tanshinone IIA treatment plus HFD (TAN group) for a further 2 months. A range of physical and biochemical markers of lipid accumulation and fatty liver disease were measured and compared between the groups.ResultsTanshinone IIA treatment significantly reduced fat accumulation in the liver and plasma lipid levels that had been increased by HFD. The toll-like receptor (TLR4)/nuclear factor kappa B (NF-κB) signalling pathway was silenced by tanshinone IIA treatment. Tumour necrosis factor-α and interleukin-6 were reduced by tanshinone IIA. Hepatocyte apoptosis was inhibited by tanshinone IIA. Tanshinone IIA upregulated peroxisome proliferator-activated receptor gamma (PPAR-γ), which resulted in an improvement in the oxidative status.ConclusionTanshinone IIA ameliorates NAFLD by targeting PPAR-γ and TLR4, resulting in decreased plasma lipids and oxidative stress, suggesting this strategy may form the basis of novel NAFLD therapies.

Objectives We aimed to investigate whether skeletal-specific H-type blood vessels exist in alveolar bone and how they function in alveolar bone remodeling. Materials and methods H-type vessels with high expression of CD31 and Endomucin (CD31hi Emcnhi ) were immunostained in alveolar bone. Abundance and age-related changes in CD31hi Emcnhi endothelial cells (H-ECs) were detected by flow cytometry. Osteoprogenitors association with H-type vessels and bone mass were detected in tooth extraction model of alveolar bone remodeling by immunohistofluorescence and micro-CT, respectively. Transcription and expression of H-EC feature genes during in vitro Notch inhibition were measured by RT-qPCR and immunocytofluorescence. Results We verified that H-type vessels existed in alveolar bone, the abundance of which was highest at infancy age, then decreased but maintained a constant level during aging. In tooth extraction model, H-ECs significantly increased with concomitant perivascular accumulation of Runx2+ osteoprogenitors and gradually augmentation of bone mass. Notch inhibition of in vitro cultured H-ECs resulted in decreased expression levels of Emcn and hes1, but not Pecam1 or Kdr genes, with decreased expression levels of H-EC numbers, accordingly. Conclusions The present study suggests that H-type vessels promote osteogenesis during alveolar bone remodeling. Notch signaling pathway regulates expression of Emcn and possibly determines fate and functions of alveolar H-ECs.

Background To initiate insect infection, entomopathogenic fungi produce diverse cuticle-degrading enzymes. Of those, lipolytic enzymes participate in epicuticular lipid hydrolysis and thus facilitate fungal penetration through the outermost cuticular barrier of the insect host. The Far/CTF1-type zinc finger transcription factors play an important role in the regulation of lipolytic activity and fungal pathogenicity in plant pathogens but remain functionally unknown in fungal insect pathogens. Results Two Far/CTF1-type transcription factor Bbctf1α and Bbctf1β, which are essential for differential expression of genes involved in the fungal lipid degradation, were identified and functionally characterized in a fungal entomopathogen Beauveria bassiana. Disruption of each gene led to drastic losses of extracellular lipolytic activities under lipidic substrate-inducing conditions, followed by remarkable phenotypic defects associated with the fungal biocontrol potential. These defects mainly included severe impairments of mycelial growth and conidium formation, and drastic losses of tolerance to the stresses of oxidation and cell wall perturbation during colony growth under either normal or induction conditions. Bioassays showed that the virulence of each disruption mutant on the greater wax moth was remarkably attenuated in topical immersion. However, there was no significant difference in intrahemolymph injection when the cuticle penetration process was bypassed. Conclusions Bbctf1α and Bbctf1β are multifunctional transcription factors that play vital roles in the regulation of fungal lipid utilization and contribute to the vegetative growth, sporulation capacity, environmental fitness and pest control potential in B. bassiana. © 2020 Society of Chemical Industry.

Diabetic nephropathy (DN) is a severe end-stage kidney disease developed from diabetes mellitus. The involvement of circular RNAs (circRNAs) in modulating DN pathogenesis has been implied, but underlying mechanism is still lacking. In this study, we demonstrated that the expression of circ_0080425 correlated with the DN progression, and exerted positive effect on cell proliferation and fibrosis in mesangial cells. Further assessment suggested that circ_0080425 function as sponge harboring miR-24-3p. Moreover, miR-24-3p negatively correlated with the DN progression, and showed an antagonistic effect to circ_0080425on regulating MCs cell proliferation and fibrosis. Bioinformatics analysis predicted fibroblast growth factor 11 (FGF11) acting as direct downstream target of miR-24-3p. Indeed, the expression of FGF11 was significantly activated by circ_0080425 while suppressed by miR-24-3p. Knockdown of FGF11 resulted in a significant reduced cell proliferation rate and fibrosis. In addition, miR-24-3p inhibitor rescued the suppression of si-circ_0080425 on FGF11, suggesting that circ_0080425 competitive binding to miR-24-3p could release FGF11 from miR-24-3p suppression, which subsequently promoted DN progression.In conclusion, we have reported a novel circ_0080425-miR-24-3p-FGF11 axis, and explored the underlying mechanism in regulating DN pathogenesis.

Fertilization failure often occurs during in vitro fertilization (IVF) cycles despite apparently normal sperm and oocytes. Accumulating evidence suggests that mitochondria play crucial roles in the regulation of sperm function and male fertility. 3-Nitrophthalic acid (3-NPA) can induce oxidative stress in mitochondria, and melatonin, as an antioxidant, can improve mitochondrial function by reducing mitochondrial oxidative stress. The role of sperm mitochondrial dysfunction in fertilization failure during IVF is unclear. The present study revealed that spermatozoa with low, or poor, fertilization rates had swollen mitochondria, increased mitochondria-derived ROS, and attenuated mitochondrial respiratory capacity. 3-NPA treatment enhanced mitochondrial dysfunction in sperm. Spermatozoa with poor fertilization rates, and spermatozoa treated with 3-NPA, had reduced penetration ability. The concentration of melatonin was decreased in semen samples with low and poor fertilization rates. Melatonin, not only decreased excessive mitochondria-derived ROS, but also ‘rescued’ the reduced penetration capacity of spermatozoa treated with 3-NPA. Taken together, the study suggested that mitochondria-derived ROS and mitochondrial respiratory capacity are independent bio-markers for sperm dysfunction, and melatonin may be useful in improving sperm quality and overall male fertility.

Since the mid-1990s detailed studies and field investigations on the Tibetan Plateau have revealed human echinococcosis to be an under-reported major public health problem, particularly in the dominant pastoral communities in the eastern and central regions. Human prevalence surveys showed that cystic echinococcosis (CE, caused by Echinococcus granulosus) and alveolar echinococcosis (AE, caused by Echinococcus multilocularis) are co-endemic with higher burdens of each disease than other endemic world regions. Epidemiological investigations identified some major risk factors for human CE and AE including dog ownership, husbandry practices and landscape features. Dogs appear to be the major zoonotic reservoir for both E. granulosus and E. multilocularis, but the latter is also transmitted in complex wildlife cycles. Small mammal assemblages especially of vole and pika species thrive on the Plateau and contribute to patterns of E. multilocularis transmission which are influenced by landscape characteristics and anthropogenic factors. Tibetan foxes are a principal definitive host for both E. multilocularis and E. shiquicus. In 2006 a national echinococcosis control programme was initiated in Tibetan communities in northwest Sichuan Province and rolled out to all of western China by 2010, and included improved surveillance (and treatment access) of human disease and regular deworming of dogs with annual copro-testing. Control of echinococcosis in Tibetan pastoral communities poses a difficult challenge for delivery and sustainability.

Osteoarthritis (OA) is the most common type of degenerative joint disease and secreted inflammatory molecules serve a pivotal role in it. Peimine has been reported to have anti‑inflammatory activity. In order to investigate the potential therapeutic role of Peimine in OA, mouse articular chondrocytes were treated with IL‑1β and different doses of Peimine in vitro. The data revealed that Peimine not only suppressed IL‑1β‑induced production of nitric oxide (NO) and prostaglandin E2, but also reduced the protein levels of inducible NO synthase (iNOS) and cyclooxygenase‑2 (COX‑2). In addition, Peimine inhibited the IL‑1β‑induced mRNA expression of matrix metalloproteinase (MMP)‑1, MMP‑3, MMP‑9, MMP‑13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)‑4 and ADAMTS‑5. Furthermore, Peimine inhibited IL‑1β‑induced activation of the mitogen‑activated protein kinase (MAPK) pathway. The protective effect of Peimine on IL‑1β‑treated chondrocytes was attenuated following activation of the MAPK pathway, as demonstrated by the increased expression levels of MMP‑3, MMP‑13, ADAMTS‑5, iNOS and COX‑2 compared with the Peimine group. The in vivo data suggested that Peimine limited the development of OA in the mouse model. In general, the data indicate that Peimine suppresses IL‑1β‑induced inflammation in mouse chondrocytes by inhibiting the MAPK pathway, suggesting a promising therapeutic role for Peimine in the treatment of OA.