Influenza B virus (IBV) is one of the main pathogens of the annual influenza epidemic, and the disease burden is significant, especially among children and young teenagers. In this study, the antiviral and anti-inflammatory effects of a traditional Chinese medicine prescription, the Lianhua-Qingwen capsule, were evaluated. Our results showed that Lianhua-Qingwen capsule can inhibit both Victoria and Yamagata lineages, and the 50% inhibitive concentrations ranged from 0.228 ± 0.150 to 0.754 ± 0.161 mg/mL. The time course results demonstrated that IBV yields were reduced with treatment at 0-4 h after infection, and the mechanistic research verified that Lianhua-Qingwen capsule has hemagglutination inhibition activity against B/Guangzhou/0215/2012 but not A/California/04/2009. In addition to antiviral activity, Lianhua-Qingwen capsule can also inhibit excessive expression of RANTES, IL-6, IL-8, IP-10, TNF-α, MCP-1, MIP-1β, and IFN-λ at the mRNA level and prevent a severe inflammatory response. The in vivo results confirmed that orally administered Lianhua-Qingwen capsule (100-400 mg/kg/day) does not reduce IBV-induced lung viral load and mortality in mice. However, the pathological change in lungs was alleviated, and there were fewer inflammatory cells in the lungs of Lianhua-Qingwen capsule treated mice than those in controls. Further research confirmed that the combination treatment of 200 mg/kg/day of Lianhua-Qingwen capsule with 2 mg/kg/day of oseltamivir significantly reduced IBV infection over the individual administration of either alone in vivo. Our findings prove that Lianhua-Qingwen capsule could be used as an assistant medicine to enhance the effect of oseltamivir against influenza B virus infection.

Low-grade gliomas (LGG), approximately constitute one-third of all types of gliomas, are prone to relapse and metastasis. MicroRNA-138 (miR-138) is reported to be dysregulated in diverse human tumors and mainly function as a tumor suppressor. In this study, we analyzed the expression profile and function of miR-138 in LGG.

The coronavirus disease 2019 (COVID-19) outbreak is evolving rapidly worldwide.

Electromechanical coupling in piezoelectric materials allows direct conversion of electrical energy into mechanical energy and vice versa. Here, we demonstrate lead-free (K x Na1-x )NbO3 single crystals with an ultrahigh large-signal piezoelectric coefficient d 33* of 9000 pm V-1, which is superior to the highest value reported in state-of-the-art lead-based single crystals (~2500 pm V-1). The enhanced electromechanical properties in our crystals are realized by an engineered compositional gradient in the as-grown crystal, allowing notable reversible non-180° domain wall motion. Moreover, our crystals exhibit temperature-insensitive strain performance within the temperature range of 25°C to 125°C. The enhanced temperature stability of the response also allows the materials to be used in a wider range of applications that exceed the temperature limits of current lead-based piezoelectric crystals.

Healing of the chronic diabetic ulceration and large burns remains a clinical challenge. Therapeutic fasting has been shown to improve health. Our study tested whether fasting facilitates diabetic and burn wound healing and explored the underlying mechanism. Methods: The effects of fasting on diabetic and burn wound healing were evaluated by analyzing the rates of wound closure, re-epithelialization, scar formation, collagen deposition, skin cell proliferation and neovascularization using histological analyses and immunostaining. In vitro functional assays were conducted to assess fasting and refeeding on the angiogenic activities of endothelial cells. Transcriptome sequencing was employed to identify the differentially expressed genes in endothelial cells after fasting treatment and the role of the candidate genes in the fasting-induced promotion of angiogenesis was demonstrated. Results: Two times of 24-h fasting in a week after but especially before wound injury efficiently induced faster wound closure, better epidermal and dermal regeneration, less scar formation and higher level of angiogenesis in mice with diabetic or burn wounds. In vitro, fasting alone by serum deprivation did not increase, but rather reduced the abilities of endothelial cell to proliferate, migrate and form vessel-like tubes. However, subsequent refeeding did not merely rescue, but further augmented the angiogenic activities of endothelial cells. Transcriptome sequencing revealed that fasting itself, but not the following refeeding, induced a prominent upregulation of a variety of pro-angiogenic genes, including SMOC1 (SPARC related modular calcium binding 1) and SCG2 (secretogranin II). Immunofluorescent staining confirmed the increase of SMOC1 and SCG2 expression in both diabetic and burn wounds after fasting treatment. When the expression of SMOC1 or SCG2 was down-regulated, the fasting/refeeding-induced pro-angiogenic effects were markedly attenuated. Conclusion: This study suggests that fasting combined with refeeding, but not fasting solely, enhance endothelial angiogenesis through the activation of SMOC1 and SCG2, thus facilitating neovascularization and rapid wound healing.

MBTD1, a H4K20me reader, has recently been identified as a component of the NuA4/TIP60 acetyltransferase complex, regulating gene expression and DNA repair. NuA4/TIP60 inhibits 53BP1 binding to chromatin through recognition of the H4K20me mark by MBTD1 and acetylation of H2AK15, blocking the ubiquitination mark required for 53BP1 localization at DNA breaks. The NuA4/TIP60 non-catalytic subunit EPC1 enlists MBTD1 into the complex, but the detailed molecular mechanism remains incompletely explored. Here, we present the crystal structure of the MBTD1-EPC1 complex, revealing a hydrophobic C-terminal fragment of EPC1 engaging the MBT repeats of MBTD1 in a site distinct from the H4K20me binding site. Different cellular assays validate the physiological significance of the key residues involved in the MBTD1-EPC1 interaction. Our study provides a structural framework for understanding the mechanism by which MBTD1 recruits the NuA4/TIP60 acetyltransferase complex to influence transcription and DNA repair pathway choice.

USP32, a member of the ubiquitin-specific proteases family, has been implicated in the development of breast cancer and small lung cancer. However, its biological functions and clinical significance in gastric cancer (GC) remain unclear. In the present study, we reported that knockdown or depletion of USP32 significantly inhibited GC cell proliferation and migration in vitro and in vivo, indicating that USP32 functions as an oncogene in GC. Importantly, results from immunohistochemical staining in a tissue microarray revealed that USP32 was upregulated in GC tissues compared with paracancerous tissues. Further analyses showed that high expression of USP32 was closely related with high T-staging and poor outcomes of GC patients. Mechanistically, USP32 silencing caused a decrease in the expression of SMAD2, which resulted in the inhibitory effects of GC cells on growth, motility, and chemoresistance to cisplatin. Therefore, our findings strongly suggest the involvement of USP32 in GC progression and provide a potential target for future therapy of GC.

The long non-coding RNA (lncRNA) H19 has been demonstrated to play a crucial role in carcinogenesis, including renal cell carcinoma (RCC). However, the impact of genetic variations in H19 gene on RCC has not been investigated before. In the present study, we sought to evaluate whether genetic polymorphisms in H19 are related to the susceptibility and mortality of RCC. We genotyped four widely studied polymorphisms in H19 and assessed their relationship with susceptibility and prognosis of RCC in a case-control study compromising 1,027 cases and 1,094 controls. The functionality of the important polymorphism was further investigated by real-time polymerase chain reaction and luciferase reporter assay. We found that H19 rs2839698 was significantly associated with risk and prognosis of RCC. Compared with the H19 rs2839698 CC genotype, the variant genotypes (CT/TT) were significantly associated with increased risk of RCC (P = 0.023, OR = 1.21; 95% CI = 1.03-1.45). Besides, patients with variant genotypes (CT/TT) were more likely to develop large tumor (P = 0.003, OR = 1.47; 95% CI = 1.16-1.85) and advanced disease (P = 0.010, OR = 1.59; 95% CI = 1.12-2.26); and had a significantly unfavorable overall survival than those with the rs2839698 CC genotype (CT/TT vs. CC: Log-rank P = 0.026, HR = 2.25, 95%CI = 1.07-4.75). Furthermore, the CT/TT genotypes were associated with significantly increased expression of H19 in renal tissue. The luciferase reporter assays revealed the potential effect of rs2839698 variant on the binding of microRNAs to H19. Our results suggest that the H19 rs2839698 variant may be a genetic predictor of susceptibility and mortality of RCC. The risk effects and the functional impact of the variant on H19 still need further validation.

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.

To study the clinical features of HPV58-positive cervical cancer.

In this experiment, the effects of a sudden drop of salinity on the immune response mechanisms of the ark shell Anadara kagoshimensis were examined by simulating the sudden drop of salinity that occurs in seawater after a rainstorm. Additionally, the differentially expressed genes (DEGs) were identified using transcriptome sequencing. When the salinity dropped from 30‱ (S30) to 14‱ (S14), the phagocytic activity of blood lymphocytes, the O2- levels produced from respiratory burst, the content of reactive oxygen species, and the activities of lysozymes and acid phosphatases increased significantly, whereas the total count of blood lymphocytes did not increase. Total count of blood lymphocytes in 22‱ salinity (S22) was significantly higher than that in any other group. The raw data obtained from sequencing were processed with Trimmomatic (Version 0.36). The expression levels of unigenes were calculated using transcripts per million (TPM) based on the effects of sequencing depth, gene length, and sample on reads. Differential expression analysis was performed using DESeq (Version 1.12.4). Transcriptome sequencing revealed 269 (101 up-regulated, 168 down-regulated), 326 (246 up-regulated, 80 down-regulated), and 185 (132 up-regulated, 53 down-regulated) significant DEGs from comparison of the S14 vs. S22, S22 vs. S30, and S14 vs. S30 groups, respectively. Gene Ontology enrichment analysis of the DEGs in these salinity comparison groups revealed that the cellular amino acid metabolic process, the regulation of protein processing, the regulation of response to stress, and other terms were significantly enriched. Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that nucleotide-binding, oligomerization domain (NOD)-like receptor signaling pathway (ko04621), apoptosis-multiple species (ko04215), Toll and Imd signaling pathway (ko04624), NF-κB signaling pathway (ko04064), apoptosis (ko04210), and focal adhesion (ko04510) were significantly enriched in all salinity comparison groups. qRT-PCR verification of 12 DEGs in the above six pathways was conducted, and the results were consistent with the transcriptome sequencing results in terms of up-regulation and down-regulation, which illustrates that the transcriptome sequencing data are credible. These results were used to preliminarily explore the effects of a sudden drop of salinity on blood physiological and biochemical indexes and immunoregulatory mechanisms of A. kagoshimensis. They also provide a theoretical basis for the selection of bottom areas optimal for release and proliferation of A. kagoshimensis required to restore the declining populations of this species.

One of the key events during the development of alcoholic liver disease (ALD) is that alcohol inhibits the insulin signaling pathway in liver and leads to disorders of glucose and lipid metabolism. Methyl ferulic acid (MFA) is a biologically active monomer isolated from the root of Securidaca inappendiculata Hasskarl. It has been reported that MFA has a hepatoprotective effect against alcohol-induced liver injury in vivo and in vitro. However, the effect of MFA on ethanol-induced insulin resistance in ALD remains unclear. In this study, we investigated whether MFA could exert protective effects against hepatic insulin resistance in ethanol-induced L-02 cells and ALD rats. ALD was induced in vivo by feeding Lieber-DeCarli diet containing 5% (w/v) alcohol for 16 weeks to Sprague-Dawley rats. Insulin resistance was induced in vitro in human hepatocyte L-02 cells with 200 mM ethanol for 24 h followed by 10-7 nM insulin for 30 min. MFA exhibited the effects of inhibited insulin resistance, reduced enzymatic capacity for hepatic gluconeogenesis, and increased hepatic glycogen synthesis both in vivo and in vitro. In addition, the results of transcriptome sequencing of liver tissues in the ethanol- and MFA-treated groups indicated that "pyruvate metabolism," "glycolysis/gluconeogenesis," and "fatty acid metabolism" were significantly different between ethanol- and MFA-treated groups. Further studies suggested that MFA activated the hepatic phosphatidylinositol 3-kinase (PI3K)/AKT pathway in vivo and in vitro. Taken together, these findings suggested that MFA effectively ameliorated hepatic insulin resistance in ALD at least partially by acting on the PI3K/AKT pathway.

The immune system is involved in the development of diabetes complications and IgG Fc gamma receptors (FcgRs) are key immune receptors responsible for the effective control of both humoral and innate immunity. We investigated the effects of members of the FcgR superfamily into both the streptozotocin plus high fat-induced type 2 diabetes and high fat diet (HFD) models. FcgRIII-/- diabetic mice and FcgRIIb-/- diabetic mice had elevated levels of serum creatinine compared with wildtype (WT) diabetic mice. Renal histology of diabetic FcgRIII knockout and FcgRIIb knockout mice showed mesangial expansion and GBM thickening; the mechanistic study indicated a higher expression of TGF-β1, TNF-α, and p-NFκB-p65 compared with wild type mouse. The HFD mouse with FcgRIII knockout or FcgRIIb knockout had increased biochemical and renal injury factors, but oxLDL deposition was higher than in FcgRIII-/- diabetic mice and FcgRIIb-/- diabetic mice. In vitro we further examined the mechanism by which the Fc gamma receptor promoted renal injury and transfected glomerular mesangial cells (GMCs) with FcgRI siRNA attenuated the level of TGF-β1, TNF-α expression. In summary, FcgRI knockdown downregulated kidney inflammation and fibrosis and FcgRIIb knockout accelerated inflammation, fibrosis, and the anomalous deposition of oxLDL whereas FcgRIII deficiency failed to protect kidney from diabetic renal injury. These observations suggested that FcgRs might represent a novel target for the therapeutic intervention of diabetic nephropathy.

Increasing life expectancy is causing the prevalence of age-related diseases to rise, and there is an urgent need for new strategies to improve health at older ages. Reduced activity of insulin/insulin-like growth factor signaling (IIS) and mechanistic target of rapamycin (mTOR) nutrient-sensing signaling network can extend lifespan and improve health during aging in diverse organisms. However, the extensive feedback in this network and adverse side effects of inhibition imply that simultaneous targeting of specific effectors in the network may most effectively combat the effects of aging. We show that the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib, the mTOR complex 1 (mTORC1) inhibitor rapamycin, and the glycogen synthase kinase-3 (GSK-3) inhibitor lithium act additively to increase longevity in Drosophila Remarkably, the triple drug combination increased lifespan by 48%. Furthermore, the combination of lithium with rapamycin cancelled the latter's effects on lipid metabolism. In conclusion, a polypharmacology approach of combining established, prolongevity drug inhibitors of specific nodes may be the most effective way to target the nutrient-sensing network to improve late-life health.

Overexpressing Tau counteracts apoptosis and increases dephosphorylated β-catenin levels, but the underlying mechanisms are elusive. Here, we show that Tau can directly and robustly acetylate β-catenin at K49 in a concentration-, time-, and pH-dependent manner. β-catenin K49 acetylation inhibits its phosphorylation and its ubiquitination-associated proteolysis, thus increasing β-catenin protein levels. K49 acetylation further promotes nuclear translocation and the transcriptional activity of β-catenin, and increases the expression of survival-promoting genes (bcl2 and survivin), counteracting apoptosis. Mutation of Tau's acetyltransferase domain or co-expressing non-acetylatable β-catenin-K49R prevents increased β-catenin signaling and abolishes the anti-apoptotic function of Tau. Our data reveal that Tau preserves β-catenin by acetylating K49, and upregulated β-catenin/survival signaling in turn mediates the anti-apoptotic effect of Tau.

Aspergillus ochraceus is reported to be the major contributor of ochratoxin A (OTA), classified as one of the possible human carcinogen (group 2B) by the International Agency for Research on Cancer. The heterotrimeric velvet complex proteins, LaeA/VeA/VelB, have been most studied in fungi to clarify the relation between light-dependent morphology and secondary metabolism. To explore possible genetic targets to control OTA contamination, we have identified laeA, veA, and velB in A. ochraceus. The loss of laeA, veA, and velB yielded mutants with differences in vegetative growth and conidial production. Especially, ΔlaeA almost lost the ability to generate conidiaphore under dark condition. The deletion of laeA, veA, and velB drastically reduced the production of OTA. The wild-type A. ochraceus produced about 1 and 7 μg/cm2 OTA under light and dark conditions on media, whereas the three gene deletion mutants produced less than 20 ng/cm2 OTA, which was correlated with a down regulation of OTA biosynthetic genes. Pathogenicity studies of ΔlaeA, ΔveA, and ΔvelB showed their reduction in disease severity in pears. Furthermore, 66.1% of the backbone genes in secondary metabolite gene cluster were significantly regulated, among which 81.6% were downregulated. Taking together, these results revealed that velvet complex proteins played crucial roles in asexual development, secondary metabolism, and fungal virulence in A. ochraceus.

Mast cells (MCs) are known to participate in a variety of patho-physiological processes depending largely on the intragranular mediators and the production of cytokines and chemokines during degranulation. Recently, extracellular vesicles (EVs) have been implicated important functions for MCs, but the components of MC-derived EVs have not yet been well-characterized. In this study, we aimed to identify signatures of proteins, long non-coding RNAs (lncRNAs), and microRNAs (miRNAs) in EVs derived from resting (Rest-EV) and degranulated (Sti-EV) MCs by differential ultracentrifugation. Using tandem mass tag (TMT)-based quantitative proteomics technology and RNA sequencing, we identified a total of 1988 proteins, 397 lncRNAs, and 272 miRNAs in Rest-EV and Sti-EV. The proteins include common EVs markers (cytoskeletal proteins), MCs markers (FcεRI and tryptase), and some preformed MCs mediators (lysosomal enzymes) as well. The global expression profiles of lncRNAs and miRNAs identified, for the first time, from Rest-EV and Sti-EV, strongly suggest a potential regulatory function of MC-derived EVs. We have also performed Western blotting and qRT-PCR analysis to further verify some of the proteins, lncRNAs, and miRNAs identified from Rest-EV and Sti-EV. Our findings will help to elucidate the functions of MC-derived EVs, and provide a reference dataset for future translational studies involving MC-derived EVs.

Cauliflower Orange (Or) mutant is characterized by high level of β-carotene in its curd. Or mutation affects the OR protein that was shown to be involved in the posttranslational control of phytoene synthase (PSY), a major rate-limiting enzyme of carotenoid biosynthesis, and in maintaining PSY proteostasis with the plastid Clp protease system. A transposon integration into the cauliflower wild-type Or gene (BoOR-wt) results in the formation of three differently spliced transcripts. One of them is characterized by insertion (BoOR-Ins), while the other two have exon-skipping deletions (BoOR-Del and BoOR-LD). We investigated the properties of individual BoOR variants and examined their effects on carotenoid accumulation. Using the yeast split-ubiquitin system, we showed that all variants were able to form OR dimers except BoOR-LD. The deletion in BoOR-LD eliminated the first of two adjacent transmembrane domains and was predicted to result in a misplacement of the C-terminal zinc finger domain to the opposite side of membrane, thus preventing OR dimerization. As interaction with PSY is mediated by the N-terminus of BoOR, which remains unaffected after splicing, all BoOR variants including BoOR-LD maintained interactions with PSY. Expression of individual BoOR mutant variants in Arabidopsis revealed that their protein stability varied greatly. While expression of BoOR-Del and BoOR-Ins resulted in increased BoOR protein levels as BoOR-wt, minimal amounts of BoOR-LD protein accumulated. Carotenoid accumulation showed correlated changes in calli of Arabidopsis expressing these variants. Furthermore, we found that OR also functions in E. coli to increase the proportion of native, enzymatically active PSY from plants upon co-expression, but not of bacterial phytoene synthase CrtB. Taken together, these results suggest that OR dimerization is required for OR stability in planta and that the simultaneous presence of PSY interaction-domains in both OR and PSY proteins is required for the holdase function of OR. The more pronounced effect of simultaneous expression of all BoOR variants in cauliflower Or mutant compared with individual overexpression on carotenoid accumulation suggests an enhanced activity with possible formation of various BoOR heterodimers.

BACKGROUND Osteosarcoma is a common malignant tumor of musculoskeletal stromal cells. Osteosarcoma clinical behavior depends mostly on the histologic grade, the site of primary tumor, the response to chemotherapy, and the presence of pulmonary metastases. The aim of this study was to knockout SHOX CNE9/10 in U2OS osteosarcoma cells and to analyze the effects on cell growth and apoptosis. MATERIAL AND METHODS U2OS cells with CNE9 knockout and U2OS cells with CNE10 knockout were established via the CRISPR/Cas9 system. Sanger sequencing was used to detect the success of the knockdown experiment. Western blotting and quantitative polymerase chain reaction were used to detect the expression levels of short stature homeobox-containing gene (SHOX) protein and messenger RNA (mRNA) after knockdown of CNE9 and CNE10. The cell viability and apoptotic rate were detected by the Cell Counting Kit-8 method and by flow cytometry. RESULTS The Sanger sequencing results showed that the knockdown experiment was successful. The levels of SHOX mRNA and protein were significantly reduced after knocking down CNE9 and CNE10. Knockdown of CNE9 and CNE10 significantly increased the growth and inhibited the apoptosis of U2OS osteosarcoma cells. CNE9/CNE10 knockdown U2OS cells were successfully constructed. CONCLUSIONS Knockdown of CNE9 and CNE10 promoted U2OS cell growth and inhibited apoptosis by decreasing SHOX expression. This CNE9/CNE10 knockout U2OS cell model could provide a bridge for the research on SHOX and CNEs in osteosarcoma.

Irinotecan-induced diarrhea (IID) results from intestinal damages by its active metabolite SN-38. Alleviation of these damages has focused on lowering luminal SN-38 concentrations. However, it is unclear if the enteric bioavailability of SN-38 is mostly dependent on luminal SN-38 concentrations.