Relationship between hemagglutinin stability and influenza virus persistence after exposure to low pH or supraphysiological heating.

The hemagglutinin (HA) surface glycoprotein is triggered by endosomal low pH to cause membrane fusion during influenza A virus (IAV) entry yet must remain sufficiently stable to avoid premature activation during virion transit between cells and hosts. HA activation pH and/or virion inactivation pH values less than pH 5.6 are thought to be required for IAV airborne transmissibility and human pandemic potential. To enable higher-throughput screening of emerging IAV strains for "humanized" stability, we developed a luciferase reporter assay that measures the threshold pH at which IAVs are inactivated. The reporter assay yielded results similar to TCID50 assay yet required one-fourth the time and one-tenth the virus. For four A/TN/09 (H1N1) HA mutants and 73 IAVs of varying subtype, virion inactivation pH was compared to HA activation pH and the rate of inactivation during 55°C heating. HA stability values correlated highly with virion acid and thermal stability values for isogenic viruses containing HA point mutations. HA stability also correlated with virion acid stability for human isolates but did not correlate with thermal stability at 55°C, raising doubt in the use of supraphysiological heating assays. Some animal isolates had virion inactivation pH values lower than HA activation pH, suggesting factors beyond HA stability can modulate virion stability. The coupling of HA activation pH and virion inactivation pH, and at a value below 5.6, was associated with human adaptation. This suggests that both virologic properties should be considered in risk assessment algorithms for pandemic potential.

There is no fusion fascia in the abdomen and extraperitoneal fascia always surrounds the mesentery.

Toldt's fascia has always been described as a fusion fascia formed by two layers of visceral peritoneum when the mesentery attaches to the posterior abdominal wall. However, there is still no consensus about the mesentery and its surrounding fascia based on the current anatomic theories. This study aimed to determine the anatomical structures of the abdomen and provide a correct surgical plane for mesenteric-based surgery. Surgical videos of 121 patients who underwent laparoscopic operations of the digestive tract were reviewed to identify and compare the anatomical structures of the mesentery and associated fascia. Twenty-one postoperative specimens were stained with hematoxylin and eosin to indicate the histological appearance of the mesentery and its surrounding fascia. Furthermore, dynamic models had been established to explain the formation mechanism of the associated histological structures in different regions during the progression of mesenteric attachment. The fasciae surrounding the mesentery, including the submesothelial connective tissue, the subserosal connective tissue, Toldt's fascia, and "angel hair," have the same histological characteristic to extraperitoneal fascia. The general anatomical structure of the abdomen can be divided into three layers (abdominal wall, urogenital system, and digestive system) and two interlayers (transversalis fascia and extraperitoneal fascia). The extraperitoneal fascia surrounds the entire digestive system and is the natural layer separating adjacent structures from each other. Typical histological structures in the regions of posterior attachment include the fascia propria of the mesentery, mesofascial plane, extraperitoneal fascia, retrofascial plane, and anterior renal fascia. The urogenital system is surrounded by similar histological structures. There is no fusion fascia in the abdomen due to retreat of the visceral peritoneum, and all of the fasciae surrounding the mesentery are extraperitoneal fascia. This study demonstrates that the typical histological structures in the regions of attachment and mesofascial plane are the correct anatomic interface for mesenteric-based surgery.

Catalytic-CO2-Desorption Studies of BZA-AEP Mixed Absorbent by the Lewis Acid Catalyst CeO2-γ-Al2O3.

Traditional organic amines exhibit inferior desorption performance and high regeneration energy consumption. The implementation of solid acid catalysts presents an efficacious approach to mitigate regeneration energy consumption. Thus, investigating high-performance solid acid catalysts holds paramount importance for the advancement and implementation of carbon capture technology. This study synthesized two Lewis acid catalysts via an ultrasonic-assisted precipitation method. A comparative analysis of the catalytic desorption properties was conducted, encompassing these two Lewis acid catalysts and three precursor catalysts. The results demonstrated that the CeO2-γ-Al2O3 catalyst demonstrated superior catalytic desorption performance. Within the desorption temperature range of 90 to 110 °C, the average desorption rate of BZA-AEP catalyzed by the CeO2-γ-Al2O3 catalyst was 87 to 354% greater compared to the desorption rate in the absence of the catalyst, and the desorption temperature can be reduced by approximately 10 °C. A comprehensive analysis of the catalytic desorption mechanism of the CeO2-γ-Al2O3 catalyst was conducted, and indicated that the synergistic effect of CeO2-γ-Al2O3 conferred a potent catalytic influence throughout the entire desorption process, spanning from the rich solution to the lean solution.

Study on Benzylamine(BZA) and Aminoethylpiperazine(AEP) Mixed Absorbent on Ship-Based Carbon Capture.

To find suitable absorbents for ship-based carbon capture, the absorption and desorption properties of four mixed aqueous amines based on BZA were investigated, and the results indicated that BZA-AEP had the best absorption and desorption performance. Then, the absorption and desorption properties of different mole ratios of BZA-AEP were tested. The results showed that the average CO2 absorption rate had the highest value at the mole ratio of BZA to AEP of three. The average CO2 desorption rate had the maximum value at the mole ratio of BZA to AEP of one. Three fitted models of the absorption and desorption performance of BZA-AEP based on the test data were obtained. The p-values of all three models were less than 0.0001. Considering the performance and material cost, the BZA-AEP mole ratio of 1.5 is more appropriate for ship carbon capture. Compared with MEA, the average CO2 absorption rate increased by 48%, the CO2 desorption capacity increased by 120%, and the average CO2 desorption rate increased by 161%.

1T-MoS2 Nanosheets Coupled with CoS2 Nanoparticles: Electronic Modulation for Efficient Electrochemical Nitrogen Fixation.

Electrocatalytic nitrogen reduction reaction (eNRR), a substitute process for the conventional Haber-Bosch for NH3 production, has drawn tremendous attention due to its merits in mild conditions, abundant reactant sources, low energy consumption, and environmental protection. However, electrocatalysts for eNRR are still subjected to low catalytic activity and selectivity. Herein, we constructed a CoS2/1T-MoS2 heterostructure with CoS2 nanoparticles uniformly loaded on 1T-MoS2 nanosheets and applied it as an eNRR electrocatalyst for the first time. Theoretical calculation suggests that electron transfer from CoS2 to 1T-MoS2 across their contact interface optimizes the local electronic structure of 1T-MoS2, where the electron-depletion region near CoS2 is in favor of accepting lone-pair electrons from N2 to enable N2 absorption, and the electron-accumulation region near 1T-MoS2 is conductive to break inert N≡N triple bonds. Unlike pure 1T-MoS2, the potential-determining step (PDS) demonstrates a significantly lower energy barrier. In addition, the weak interaction between CoS2/1T-MoS2 and hydrogen discourages competitive hydrogen evolution reaction. As a result, CoS2/1T-MoS2 exhibited noticeably improved eNRR activity and selectivity, with an NH3 yield of 59.3 µg h-1 mg-1 and a high Faradaic efficiency of 26.6%.

Hemagglutinin Stability Regulates H1N1 Influenza Virus Replication and Pathogenicity in Mice by Modulating Type I Interferon Responses in Dendritic Cells.

Hemagglutinin (HA) stability, or the pH at which HA is activated to cause membrane fusion, has been associated with the replication, pathogenicity, transmissibility, and interspecies adaptation of influenza A viruses. Here, we investigated the mechanisms by which a destabilizing HA mutation, Y17H (activation pH, 6.0), attenuates virus replication and pathogenicity in DBA/2 mice compared to wild-type (WT) virus (activation pH, 5.5). The extracellular lung pH was measured to be near neutral (pH 6.9 to 7.5). WT and Y17H viruses had similar environmental stability at pH 7.0; thus, extracellular inactivation was unlikely to attenuate the Y17H virus. The Y17H virus had accelerated replication kinetics in MDCK, A549, and RAW 264.7 cells when inoculated at a multiplicity of infection (MOI) of 3 PFU/cell. The destabilizing mutation also increased early infectivity and type I interferon (IFN) responses in mouse bone marrow-derived dendritic cells (DCs). In contrast, the HA-Y17H mutation reduced virus replication in murine airway murine nasal epithelial cell and murine tracheal epithelial cell cultures and attenuated virus replication, virus spread, the severity of infection, and cellular infiltration in the lungs of mice. Normalizing virus infection and weight loss in mice by inoculating them with Y17H virus at a dose 500-fold higher than that of WT virus revealed that the destabilized mutant virus triggered the upregulation of more host genes and increased type I IFN responses and cytokine expression in DBA/2 mouse lungs. Overall, HA destabilization decreased virulence in mice by boosting early infection in DCs, resulting in the greater activation of antiviral responses, including the type I IFN response. These studies reveal that HA stability may regulate pathogenicity by modulating IFN responses.IMPORTANCE Diverse influenza A viruses circulate in wild aquatic birds, occasionally infecting farm animals. Rarely, an avian- or swine-origin influenza virus adapts to humans and starts a pandemic. Seasonal and many universal influenza vaccines target the HA surface protein, which is a key component of pandemic influenza viruses. Understanding the HA properties needed for replication and pathogenicity in mammals may guide response efforts to control influenza. Some antiviral drugs and broadly reactive influenza vaccines that target the HA protein have suffered resistance due to destabilizing HA mutations that do not compromise replicative fitness in cell culture. Here, we show that despite not compromising fitness in standard cell cultures, a destabilizing H1N1 HA stalk mutation greatly diminishes viral replication and pathogenicity in vivo by modulating type I IFN responses. This encourages targeting the HA stalk with antiviral drugs and vaccines as well as reevaluating previous candidates that were susceptible to destabilizing resistance mutations.

Analytical and Clinical Performance of a Liquid Chromatography-Tandem Mass Spectrometry Method for Measuring Gastrin Subtypes G34 and G17 in Serum.

BACKGROUND: Two major forms of gastrin, gastrin-17 (G17) and gastrin-34 (G34), exist in blood. However, conventional immunoassay methods can only quantify total gastrin or G17 alone. Here, we aimed to establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify G17 and G34 simultaneously. METHODS: Serum samples were prepared by anion-exchange solid-phase extraction. The analytical performance of the LC-MS/MS method was validated and the method was compared to chemiluminescence immunoassay (CLIA) and radioimmunoassay (RIA). The G17 and G34 concentrations in 245 serum samples from healthy controls, individuals with gastrinoma, and individuals with other diseases were analyzed. RESULTS: The total runtime of the LC-MS/MS method was 6 min. No substantial matrix effect was observed with internal standard correction. The intraassay coefficients of variation (CVs) for G17 and G34 were 4.0%-14.2% and 4.4%-10.4%, respectively, and total CVs were 5.2%-14.1% and 4.6%-12.4%, respectively. The correlation coefficient between LC-MS/MS and CLIA was 0.87, and between LC-MS/MS and RIA was 0.84. The G17+G34 concentrations for 87.5% of individuals with gastrinoma were higher than the 95th percentile of healthy controls (18.1 pg/mL), whereas the concentrations for individuals with other diseases and gastrinoma overlapped. Based on the Youden indices calculated for G17+G34, G34, and G17, the most specific biomarker was G17 (96.9% clinical specificity at 209.8 pg/mL) for gastrinoma. CONCLUSIONS: This method should aid in the diagnosis of diseases associated with increased gastrin concentrations.

Thioredoxin-interacting protein promotes activation and inflammation of monocytes with DNA demethylation in coronary artery disease.

Numerous studies have demonstrated that thioredoxin-interacting protein (TXNIP) expression of peripheral blood leucocytes is increased in coronary artery disease (CAD). However, the molecular mechanism of this phenomenon remained unclear. DNA methylation plays important roles in the regulation of gene expression. Therefore, we speculated there might be a close association between the expression of TXNIP and methylation. In this study, we found that compared with controls, DNA methylation at cg19693031 was decreased in CAD, while mRNA expressions of TXNIP and inflammatory factors, NLRP3, IL-1ß, IL-18, were increased. Methylation at cg19693031 was negatively associated with TXNIP expression in the cohort, THP-1 and macrophages/foam cells. Furthermore, Transwell assay and co-cultured adhesion assay were performed to investigate functions of TXNIP on the migration of THP-1 or the adhesion of THP-1 on the surface of endothelial cells, respectively. Notably, overexpressed TXNIP promoted the migration and adhesion of THP-1 cells and expressions of NLRP3, IL-18 and IL-1ß. Oppositely, knock-down TXNIP inhibited the migration and adhesion of THP-1 and expressions of NLRP3, IL-18. In conclusion, increased TXNIP expression, related to cg19693031 demethylation orientates monocytes towards an inflammatory status through the NLRP3 inflammasome pathway involved in the development of CAD.

Genome-wide characterization of lncRNAs in acute myeloid leukemia.

Long noncoding RNAs (lncRNAs) are a large family of noncoding RNAs that play a critical role in various normal bioprocesses as well as tumorigenesis. However, the expression patterns and biological functions of lncRNAs in acute leukemia have not been well studied. Here, we performed transcriptome-wide lncRNA expression profiling of acute myeloid leukemia (AML) patient samples, along with non-leukemia control hematopoietic samples. We found that lncRNAs were differentially expressed in AML samples relative to control samples. Notably, we identified that lncRNAs upregulated in AML (relative to the control samples) are associated with a lower degree of DNA methylation and a higher ratio of being bound by transcription factors such as SP1, STAT4, ATF-2 and ELK-1 compared with those downregulated in AML. Moreover, an enrichment of H3K4me3 and a depletion of H3K27me3 were observed in upregulated lncRNAs in AML. Expression patterns of three types of lncRNAs (antisense, enhancer and intergenic lncRNAs) have previously been characterized. Of the identified lncRNAs, we found that high expression level lncRNA LOC285758 is associated with the poor prognosis in AML patients. Furthermore, we found that LOC285758 regulates proliferation of AML cell lines by enhancing the expression of HDAC2, a key factor in carcinogenesis. Collectively, our study depicts a landscape of important lncRNAs in AML and provides novel potential therapeutic targets and prognostic markers for AML treatment.

MiR-199a-3p/5p participated in TGF-ß and EGF induced EMT by targeting DUSP5/MAP3K11 in pterygium.

BACKGROUND: Recently, it has been reported that miRNA is involved in pterygium, however the exact underlying mechanism in pterygium is unrevealed and require further investigation. METHODS: The differential expression of miRNA in pterygium was profiled using microarray and validated with quantitative real-time polymerase chain reaction (qRT-PCR). Human conjunctival epithelial cells (HCEs) were cultured and treated with transforming growth factor ß (TGF-ß) and epidermal growth factor (EGF) and transfected with miR-199a-3p/5p mimic and inhibitor. Markers of epithelial-mesenchymal transition (EMT) in HCEs were detected using western blot and immunohistochemistry. Cell migration ability was determined using wound healing and transwell assay, while apoptosis was determined by flow cytometry. The target genes of miR-199a were confirmed by the dual-luciferase reporter assay. RESULTS: TGF-ß and EGF could induced EMT in HCEs and increase miR-199a-3p/5p but suppress target genes, DUSP5 and MAP3K11. With the occurrence of EMT, cell migration ability was enhanced, and apoptosis was impeded. Promoting miR-199a-3p/5p expression could induce EMT in HCEs without TGF-ß and EGF, while suppressing miR-199a-3p/5p could inhibit EMT in TGF-ß and EGF induced HCEs. In a word, TGF-ß and EGF induced EMT could be regulated with miR-199a-3p/5p-DUSP5/MAP3K11 axes. The validated results in tissues showed that, compared with control conjunctival tissues, miR-199a-3p/5p were more overexpressed in pterygium, while DUSP5/MAP3K11 were lower expressed. In addition, bioinformatics analysis indicated the miR-199a-3p/5p-DUSP5/MAP3K11 was belong to MAPK signalling pathway. CONCLUSIONS: TGF-ß and EGF induce EMT of HCEs through miR-199a-3p/5p-DUSP5/MAP3K11 axes, which explains the pathogenesis of EMT in pterygium and may provide new targets for pterygium prevention and therapy.

Immunohistochemical study of STAT3, HIF-1α and VEGF in pterygium and normal conjunctiva: Experimental research and literature review.

Purpose: Signal transducer and activator of transcription 3 (STAT3) is a DNA-binding protein that regulates various biologic processes, including cell growth, apoptosis, and malignant transformation. Abnormal activation of STAT3 is associated with many diseases, and there is currently no relevant study on the pathogenesis of pterygium. The purpose of this study was to investigate the expression and clinical significance of STAT3, HIF-1α, and VEGF in pterygium at different stages. Methods: Immunohistochemistry was used to study the expression levels of STAT3, HIF-1α, and VEGF in 50 cases of pterygium and 20 cases of control conjunctival tissue. The expression intensity of the three proteins was evaluated with Image-Pro Plus 6.0 image analysis software. Results: In the pterygium group, the positive rates for STAT3, HIF-1α, and VEGF were 82.0%, 86.0%, and 84.0%, respectively, while those in the normal conjunctiva group were 40.0%, 25.0%, and 15.0%. The expression of STAT3, HIF-1α, and VEGF in pterygium was higher than that in control conjunctiva, and the expression in advanced pterygium was statistically significantly higher than that in stationary pterygium (p < 0.01). The expression levels of STAT3 and HIF-1α in pterygium were related to the length and depth of the corneal invasion of pterygium. The expression level of VEGF in pterygium was related to the length of pterygium, but not to the depth. In addition, there was a significant positive correlation between the expression of STAT3, HIF-1α, and VEGF (p < 0.01). Conclusions: For the first time, the expression levels of the STAT3, HIF-1α, and VEGF proteins were detected simultaneously in pterygium tissue. Compared with normal conjunctiva, STAT3, HIF-1α, and VEGF were highly expressed in pterygium, and the expression in advanced pterygium tissue was more significant than in the stationary pterygium tissue. It is suggested that STAT3 may directly or through HIF-1α promote VEGF expression and participate in the growth and angiogenesis of pterygium. Targeting STAT3 may provide a new direction for the treatment of pterygium.

tRFs as Potential Exosome tRNA-Derived Fragment Biomarkers for Gastric Carcinoma.

BACKGROUND: Gastric Carcinoma (GC) is one of the common diseases induced by the interaction of genes and environment. Exosomes are potential markers for several health problems, which contain lipids, proteins, long non-coding RNAs, microRNAs (miRNAs), and tRNA-derived fragments (tRFs). The roles of mRNAs and miRNAs in GC have been studied comprehensively; however, little research was focused on the function of plasma exosomal tRFs. METHODS: We collected plasma samples from fifty healthy controls and fifty GC patients, and all exosomes were isolated with a combined centrifugation and characterized by electron microscopy, western blot, and flow cytometry. The small RNA sequence was performed to detect the plasma exosomal tRFs, and tRFs markers were validated by real-time quantitative PCR. Three exosomal diagnostic tRFs were confirmed by receiver operating characteristic analyses. RESULTS: In this study, we found higher plasma exosomal tRF-25, tRF-38, tRF-18 expression in GC than in controls. Plasma exosomal tRF-25, tRF-38, and tRF-18 showed better accuracy for GC diagnosis. CONCLUSIONS: Our results suggest that plasma exosomal tRF-25, tRF-38, and tRF-18 were biomarkers for GC detection; tRF-25, tRF-38 and tRF-18 might be predictive of GC prognosis.

Physicochemical properties and microbial community dynamics during Chinese horse bean-chili-paste fermentation, revealed by culture-dependent and culture-independent approaches.

The production of Chinese horse bean-chili-paste (CHCP) involves three fermentation phases: chili-to-moromi fermentation (CF) phase, horse bean-to-meju fermentation (HF) phase and moromi-meju mixed fermentation (MF) phase. To understand the microbial dynamics among these three phases and the potential roles of viable microbes for fermentation, microbial community dynamics was investigated by using culture-dependent and culture-independent methods. Furthermore, the capacities of enzyme-producing of the isolates were determined. During the CF phase, reducing sugar content increased from 3.1% to 3.49%, while pH declined from 4.85 to 4.5. The protein content in the HF phase and MF phase reduced sharply from 22.23% to 10.29% and 4.39%-1.19%, respectively. Bacillus sp., Staphylococcus sp., Oceanobacillus sp., Candida sp., Zygosaccharomyces sp. and Aspergillus sp. dominated the CF phase, while Bacillus sp., Candida sp. and Zygosaccharomyces sp. were the dominant microorganisms in both the HF and MF phases. B. amyloliquefaciens, B. methylotrophicus, B. subtilis, B. licheniformis and A. oryzae possessed strong capacities of producing enzymes, i.e. α-amylase, cellulase and xylanase, acid protease and leucine aminopeptidase, and could make a great contribution to CHCP fermentation.

H1N1 influenza viruses varying widely in hemagglutinin stability transmit efficiently from swine to swine and to ferrets.

A pandemic-capable influenza virus requires a hemagglutinin (HA) surface glycoprotein that is immunologically unseen by most people and is capable of supporting replication and transmission in humans. HA stabilization has been linked to 2009 pH1N1 pandemic potential in humans and H5N1 airborne transmissibility in the ferret model. Swine have served as an intermediate host for zoonotic influenza viruses, yet the evolutionary pressure exerted by this host on HA stability was unknown. For over 70 contemporary swine H1 and H3 isolates, we measured HA activation pH to range from pH 5.1 to 5.9 for H1 viruses and pH 5.3 to 5.8 for H3 viruses. Thus, contemporary swine isolates vary widely in HA stability, having values favored by both avian (pH >5.5) and human and ferret (pH ≤5.5) species. Using an early 2009 pandemic H1N1 (pH1N1) virus backbone, we generated three viruses differing by one HA residue that only altered HA stability: WT (pH 5.5), HA1-Y17H (pH 6.0), and HA2-R106K (pH 5.3). All three replicated in pigs and transmitted from pig-to-pig and pig-to-ferret. WT and R106 viruses maintained HA genotype and phenotype after transmission. Y17H (pH 6.0) acquired HA mutations that stabilized the HA protein to pH 5.8 after transmission to pigs and 5.5 after transmission to ferrets. Overall, we found swine support a broad range of HA activation pH for contact transmission and many recent swine H1N1 and H3N2 isolates have stabilized (human-like) HA proteins. This constitutes a heightened pandemic risk and underscores the importance of ongoing surveillance and control efforts for swine viruses.

Reference intervals for thyroid-stimulating hormone, free thyroxine, and free triiodothyronine in elderly Chinese persons.

Background Thyroid hormone levels are essential for diagnosing and monitoring thyroid diseases. However, their reference intervals (RIs) in elderly Chinese individuals remain unclear. We aimed to identify factors affecting thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroxine (FT4) levels using clinical "big data" to establish hormone level RIs for elderly Chinese individuals. Methods We examined 6781, 6772, and 6524 subjects aged ≥65 years who underwent FT3, FT4, and TSH tests, respectively, at the Peking Union Medical College Hospital between September 1, 2013, and August 31, 2016. Hormones were measured using an automated immunoassay analyzer (ADVIA Centaur XP). RIs were established using the Clinical Laboratory Standards Institute document C28-A3 guidelines. Results The median TSH was significantly higher in women than in men; the opposite was true for median FT3 and FT4 levels. No differences were observed in TSH or FT4 by age in either sex or overall; FT3 levels significantly decreased with age. Seasonal differences were observed in TSH and FT3 levels but not FT4 levels; the median TSH was the highest in winter and lowest in summer, whereas the median FT3 was the lowest in summer (albeit not significantly). RIs for TSH were 0.53-5.24 and 0.335-5.73 mIU/L for men and women, respectively; those for FT3 were 3.76-5.71, 3.60-5.42, and 3.36-5.27 pmol/L in 64- to 74-, 75- to 84-, and 85- to 96-year-old subjects, respectively. The RI for FT4 was 11.70-20.28 pmol/L. Conclusions RIs for TSH in elderly individuals were sex specific, whereas those for FT3 were age specific.

The TET2/E-cadherin/ß-catenin regulatory loop confers growth and invasion in hepatocellular carcinoma cells.

The poor outcome of hepatocellular carcinoma (HCC) is mainly due to the development of fast growth, invasion and metastasis. The role of TET2 has been implicated in some cancer types, but its role and mechanisms in HCC remains elusive. In this study, our findings indicated that TET2 expression frequently increased in HCC and that TET2 expressional upregulation correlated with HCC progression. TET2 knockdown inhibited HCC cells proliferation in vitro and growth in vivo, and inhibited the invasion potential of HCC cells. Mechanically, TET2 knockdown upregulated E-cadherin expression and then attenuated ß-catenin transactivation in HCC cells. TET2 repressed E-cadherin expression via recruited HDAC1 to E-cadherin promoter to reduce the H3K9Ac and H4K16Ac levels. Moreover, ß-catenin signaling transcriptionally regulated TET2 expression to form a positive feedback in HCC cells. These findings indicate that the dysregulation of TET2/E-cadherin/ß-catenin regulatory loop is a critical oncogenic event in HCC progression.

Influences of sterol regulatory element binding protein-1c silencing on glucose production in HepG2 cells treated with free fatty acid.

BACKGROUND: Elevation of exogenous free fatty acid (FFA) level leads to insulin resistance (IR) in liver, IR is manifested by elevated hepatic glucose production. We aim to study whether inhibition of endogenous fatty acid synthesis could decrease hepatic glucose production. METHODS: Low-passage HepG2 cells derived from human liver tissue were cultured in medium supplemented with FFA to induce IR, the influences of sterol regulatory element binding protein-1c (SREBP-1c) silencing on glucose production of HepG2 cells were investigated, and genes responsible for fatty acid and glucose metabolism were detected by real-time PCR. RESULTS: Compared with HepG2 cells cultured in normal growth medium, glucose production of HepG2 cells treated by FFA was significantly increased {[(0.28 ± 0.01) vs (0.83 ± 0.02)] umol.ug- 1 protein, n = 6 wells, P < 0.01}; the mRNA expression of phosphoenolpyruvate carboxylase kinase (PEPCK) and glucose-6-phosphatase (G6PC) in HepG2 cells increased by more than 5-fold and 3-fold, respectively; the mRNA expression of fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD1) increased by approximately 4-fold and 1.1-fold, respectively; the mRNA expression of carnitine palmitoyltransferase-1 (CPT-1) changed slightly. Compared with the scrambled siRNA control, glucose production of HepG2 cells treated by FFA significantly increased after SREBP-1c silencing {[(0.018 ± 0.001) vs (0.028 ± 0.002)] umol.ug- 1 protein, n = 6 wells, P < 0.01}; the mRNA expression of PEPCK and G6PC increased by approximately 1.5-fold and 5-fold, respectively, but the mRNA expression of FAS, SCD1 and CPT-1 changed slightly. CONCLUSIONS: SREBP-1c silencing further augmented glucose production of HepG2 cells treated by FFA significantly, genes responsible for fatty acid synthesis and gluconeogenesis played an important role in this process. SREBP-1c functions not only as a lipid regulator but also plays an important role in regulation of glucose metabolism.

C-terminal domain (CTD) phosphatase links Rho GTPase signaling to Pol II CTD phosphorylation in Arabidopsis and yeast.

Rho GTPases, including the Rho, Cdc42, Rac, and ROP subfamilies, act as pivotal signaling switches in various growth and developmental processes. Compared with the well-defined role of cytoskeletal organization in Rho signaling, much less is known regarding transcriptional regulation. In a mutant screen for phenotypic enhancers of transgenic Arabidopsis plants expressing a constitutively active form of ROP2 (designated CA1-1), we identified RNA polymerase II (Pol II) C-terminal domain (CTD) phosphatase-like 1 (CPL1) as a transcriptional regulator of ROP2 signaling. We show that ROP2 activation inhibits CPL1 activity by promoting its degradation, leading to an increase in CTD Ser5 and Ser2 phosphorylation. We also observed similar modulation of CTD phosphorylation by yeast Cdc42 GTPase and enhanced degradation of the yeast CTD phosphatase Fcp1 by activated ROP2 signaling. Taken together, our results suggest that modulation of the Pol II CTD code by Rho GTPase signaling represents an evolutionarily conserved mechanism in both unicellular and multicellular eukaryotes.

Molecular requirements for a pandemic influenza virus: An acid-stable hemagglutinin protein.

Influenza pandemics require that a virus containing a hemagglutinin (HA) surface antigen previously unseen by a majority of the population becomes airborne-transmissible between humans. Although the HA protein is central to the emergence of a pandemic influenza virus, its required molecular properties for sustained transmission between humans are poorly defined. During virus entry, the HA protein binds receptors and is triggered by low pH in the endosome to cause membrane fusion; during egress, HA contributes to virus assembly and morphology. In 2009, a swine influenza virus (pH1N1) jumped to humans and spread globally. Here we link the pandemic potential of pH1N1 to its HA acid stability, or the pH at which this one-time-use nanomachine is either triggered to cause fusion or becomes inactivated in the absence of a target membrane. In surveillance isolates, our data show HA activation pH values decreased during the evolution of H1N1 from precursors in swine (pH 5.5-6.0), to early 2009 human cases (pH 5.5), and then to later human isolates (pH 5.2-5.4). A loss-of-function pH1N1 virus with a destabilizing HA1-Y17H mutation (pH 6.0) was less pathogenic in mice and ferrets, less transmissible by contact, and no longer airborne-transmissible. A ferret-adapted revertant (HA1-H17Y/HA2-R106K) regained airborne transmissibility by stabilizing HA to an activation pH of 5.3, similar to that of human-adapted isolates from late 2009-2014. Overall, these studies reveal that a stable HA (activation pH ≤ 5.5) is necessary for pH1N1 influenza virus pathogenicity and airborne transmissibility in ferrets and is associated with pandemic potential in humans.

Zoonotic Risk, Pathogenesis, and Transmission of Avian-Origin H3N2 Canine Influenza Virus.

Two subtypes of influenza A virus (IAV), avian-origin canine influenza virus (CIV) H3N2 (CIV-H3N2) and equine-origin CIV H3N8 (CIV-H3N8), are enzootic in the canine population. Dogs have been demonstrated to seroconvert in response to diverse IAVs, and naturally occurring reassortants of CIV-H3N2 and the 2009 H1N1 pandemic virus (pdmH1N1) have been isolated. We conducted a thorough phenotypic evaluation of CIV-H3N2 in order to assess its threat to human health. Using ferret-generated antiserum, we determined that CIV-H3N2 is antigenically distinct from contemporary human H3N2 IAVs, suggesting that there may be minimal herd immunity in humans. We assessed the public health risk of CIV-H3N2 × pandemic H1N1 (pdmH1N1) reassortants by characterizing their in vitro genetic compatibility and in vivo pathogenicity and transmissibility. Using a luciferase minigenome assay, we quantified the polymerase activity of all possible 16 ribonucleoprotein (RNP) complexes (PB2, PB1, PA, NP) between CIV-H3N2 and pdmH1N1, identifying some combinations that were more active than either parental virus complex. Using reverse genetics and fixing the CIV-H3N2 hemagglutinin (HA), we found that 51 of the 127 possible reassortant viruses were viable and able to be rescued. Nineteen of these reassortant viruses had high-growth phenotypes in vitro, and 13 of these replicated in mouse lungs. A single reassortant with the NP and HA gene segments from CIV-H3N2 was selected for characterization in ferrets. The reassortant was efficiently transmitted by contact but not by the airborne route and was pathogenic in ferrets. Our results suggest that CIV-H3N2 reassortants may pose a moderate risk to public health and that the canine host should be monitored for emerging IAVs.IMPORTANCE IAV pandemics are caused by the introduction of novel viruses that are capable of efficient and sustained transmission into a human population with limited herd immunity. Dogs are a a potential mixing vessel for avian and mammalian IAVs and represent a human health concern due to their susceptibility to infection, large global population, and close physical contact with humans. Our results suggest that humans are likely to have limited preexisting immunity to CIV-H3N2 and that CIV-H3N2 × pdmH1N1 reassortants have moderate genetic compatibility and are transmissible by direct contact in ferrets. Our study contributes to the increasing evidence that surveillance of the canine population for IAVs is an important component of pandemic preparedness.