Rational Design of Bipolar Host and Thermally Activated Delayed Fluorescence Materials in Donor-Acceptor Molecular Architecture: A Theoretical Study.

Donor-acceptor (D-A) molecules have drawn massive attention recently in the design of high-performance materials, but the underlying reasons for the magic abilities of D-A architecture in building very different organic semiconductors are still unclear. Here, based on a series of experimentally bipolar host and thermally activated delayed fluorescence (TADF) molecules with the same donor but different acceptor units, it was found that TADF emitters have more effective charge transfer between donor and acceptor units than bipolar host molecules. More efficient conjugation effects between the donor and acceptor units of host materials were identified from the lower dihedral angles of the D-A structure, smaller and even negative charge transfer amount, shorter charge-transfer length, and larger hole-electron overlap extent. These findings with in-depth insights into different interaction models of donor and acceptor units shed important light on the molecular design of TADF emitters and bipolar materials in a D-A architecture.

Long noncoding RNA plasmacytoma variant translocation 1 is overexpressed in cutaneous squamous cell carcinoma and exon 2 is critical for its oncogenicity.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) is one of the most common and fastest increasing forms of cancer worldwide with metastatic potential. Long noncoding RNAs (lncRNAs) are a group of RNA molecules with essential regulatory functions in both physiological and pathological processes. OBJECTIVES: To investigate the function and mode of action of lncRNA plasmacytoma variant translocation 1 (PVT1) in cSCC. METHODS: Quantitative reverse transcriptase polymerase chain reaction and single-molecule in situ hybridization were used to quantify the expression level of PVT1 in normal skin, premalignant skin lesions, actinic keratosis (AK) and primary and metastatic cSCCs. The function of PVT1 in cSCC was investigated both in vivo (tumour xenografts) and in vitro (competitive cell growth assay, 5-ethynyl-2'-deoxyuridine incorporation assay, colony formation assay and tumour spheroid formation assay) upon CRISPR-Cas9-mediated knockout of the entire PVT1 locus, the knockout of exon 2 of PVT1, and locked nucleic acid (LNA) gapmer-mediated PVT1 knockdown. RNA sequencing analysis was conducted to identify genes and processes regulated by PVT1. RESULTS: We identified PVT1 as a lncRNA upregulated in cSCC in situ and cSCC, associated with the malignant phenotype of cSCC. We showed that the expression of PVT1 in cSCC was regulated by MYC. Both CRISPR-Cas9 deletion of the entire PVT1 locus and LNA gapmer-mediated knockdown of PVT1 transcript impaired the malignant behaviour of cSCC cells, suggesting that PVT1 is an oncogenic transcript in cSCC. Furthermore, knockout of PVT1 exon 2 inhibited cSCC tumour growth both in vivo and in vitro, demonstrating that exon 2 is a critical element for the oncogenic role of PVT1. Mechanistically, we showed that PVT1 was localized in the cell nucleus and its deletion resulted in cellular senescence, increased cyclin-dependent kinase inhibitor 1 (p21/CDKN1A) expression and cell cycle arrest. CONCLUSIONS: Our study revealed a previously unrecognized role for exon 2 of PVT1 in its oncogenic role and that PVT1 suppresses cellular senescence in cSCC. PVT1 may be a potential biomarker and therapeutic target in cSCC.

Serum Exosomal Long Noncoding RNA Growth Arrest-Specific 5 Predicts 3-Month Mortality in Acute-on-Chronic Hepatitis B Liver Failure.

Background: Acute-on-chronic hepatitis B liver failure (ACHBLF) is a clinical syndrome with an extremely high mortality. In this study, we aim to evaluate the potential role of serum exosomal long noncoding RNA (lncRNA) growth arrest-specific 5 (GAS5) in ACHBLF and its predictive value for 3-month mortality. Methods: From December 2017 to June 2022, we enrolled 110 patients with ACHBLF and 42 healthy controls (HCs). Exosomes were isolated from the serum of the participants. Serum exosomal lncRNA GAS5 was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The functional role of lncRNA GAS5 on hepatocyte phenotypes was investigated through loss-of-function and gain-of-function assays. Exosomal labeling and cell uptake assay were used to determine the exosomes-mediated transmission of lncRNA GAS5 in hepatocytes in vitro. Results: The serum exosomal lncRNA GAS5 was identified to be an independent predictor for 3-month mortality of ACHBLF. It yielded an area under the receiver operating characteristic curve (AUC) of 0.88, which was significantly higher than MELD score (AUC 0.73; P < 0.01). Further study found that lncRNA GAS5 could inhibit hepatocytes proliferation and increase hepatocytes apoptosis. Exosomes-mediated lncRNA GAS5 transfer promoted hepatocytes injury. The knocked down of lncRNA GAS5 weakened H2O2-induced hepatocytes injury. Conclusion: We revealed that serum exosomal lncRNA GAS5 might promote hepatocytes injury and showed high predictive value for 3-month mortality in ACHBLF.

Manipulating Multiple Resonance-Charge Transfer Hybrid Proportion for Developing Red Narrowband Thermally Activated Delayed Fluorescence Materials.

Multiple resonance thermally activated delayed fluorescence (MR-TADF) materials have attracted increasing attention because of their 100% exciton utilization capability and narrowband emissions. However, it remains a formidable challenge to develop such red materials. Herein, we perform a theoretical investigation on the design of red narrowband TADF materials via manipulating the MR-charge transfer (CT) hybrid proportion by regulating the types of MR cores and peripheral electron-donating units. The results indicate that the MR-CT proportion in the excited states is closely relevant to the frontier molecular orbital (FMO)/hole-electron overlap, which is mainly determined by the dihedral angle between the MR cores and the peripheral units for the MR donor-acceptor molecules. The electron-donating ability of the peripheral substituents has little influence on the FMO/hole-electron overlap. Finally, c1-a and c2-a with red narrowband emissions were revealed. These findings with rich physical insights into the structure-property relationship should provide important clues for designing red narrowband optoelectronic materials.

Cutaneous squamous cell carcinoma-derived extracellular vesicles exert an oncogenic role by activating cancer-associated fibroblasts.

Cutaneous squamous cell carcinoma (cSCC) is a fast-increasing cancer with metastatic potential. Extracellular vesicles (EVs) are small membrane-bound vesicles that play important roles in intercellular communication, particularly in the tumor microenvironment (TME). Here we report that cSCC cells secrete an increased number of EVs relative to normal human epidermal keratinocytes (NHEKs) and that interfering with the capacity of cSCC to secrete EVs inhibits tumor growth in vivo in a xenograft model of human cSCC. Transcriptome analysis of tumor xenografts by RNA-sequencing enabling the simultaneous quantification of both the human and the mouse transcripts revealed that impaired EV-production of cSCC cells prominently altered the phenotype of stromal cells, in particular genes related to extracellular matrix (ECM)-formation and epithelial-mesenchymal transition (EMT). In line with these results, co-culturing of human dermal fibroblasts (HDFs) with cSCC cells, but not with normal keratinocytes in vitro resulted in acquisition of cancer-associated fibroblast (CAF) phenotype. Interestingly, EVs derived from metastatic cSCC cells, but not primary cSCCs or NHEKs, were efficient in converting HDFs to CAFs. Multiplex bead-based flow cytometry assay and mass-spectrometry (MS)-based proteomic analyses revealed the heterogenous cargo of cSCC-derived EVs and that especially EVs derived from metastatic cSCCs carry proteins associated with EV-biogenesis, EMT, and cell migration. Mechanistically, EVs from metastatic cSCC cells result in the activation of TGFß signaling in HDFs. Altogether, our study suggests that cSCC-derived EVs mediate cancer-stroma communication, in particular the conversion of fibroblasts to CAFs, which eventually contribute to cSCC progression.

MicroRNA-23b Plays a Tumor-Suppressive Role in Cutaneous Squamous Cell Carcinoma and Targets Ras-Related Protein RRAS2.

Cutaneous squamous cell carcinoma (cSCC) is one of the most common types of cancer with metastatic potential. MicroRNAs regulate gene expression at the post-transcriptional level. In this study, we report that miR-23b is downregulated in cSCCs and in actinic keratosis and that its expression is regulated by the MAPK signaling pathway. We show that miR-23b suppresses the expression of a gene network associated with key oncogenic pathways and that the miR-23b-gene signature is enriched in human cSCCs. miR-23b decreased the expression of FGF2 both at mRNA and protein levels and impaired the angiogenesis-inducing ability of cSCC cells. miR23b overexpression suppressed the capacity of cSCC cells to form colonies and spheroids, whereas the CRISPR/Cas9-mediated deletion of MIR23B resulted in increased colony and tumor sphere formation in vitro. In accordance with this, miR-23b-overexpressing cSCC cells formed significantly smaller tumors upon injection into immunocompromised mice with decreased cell proliferation and angiogenesis. Mechanistically, we verify RRAS2 as a direct target of miR-23b in cSCC. We show that RRAS2 is overexpressed in cSCC and that interference with its expression impairs angiogenesis and colony and tumorsphere formation. Taken together, our results suggest that miR-23b acts in a tumor-suppressive manner in cSCC, and its expression is decreased during squamous carcinogenesis.

Effects of selenite on the responses of lettuce (Lactuca sativa L.) to polystyrene nano-plastic stress.

Nowadays, nano-plastics are widespread in agricultural soils and could be uptaken by crops to cause an increased risk for food safety. As a beneficial element for plants, selenium (Se) can alleviate oxidative damages under various environmental stresses (eg. heavy metals, heat, cold). This study investigated the single and co-applications of nano-size polystyrene (PS) (80 nm and 200 nm) and selenite (0.8 ppm and 5 ppm) in lettuce (Lactuca sativa L.). Results showed nano-PS significantly decreased the root-shoot fresh biomass ratios, inhibited physiological functions in roots and leaves (e.g. root length, chlorophyll content and net photosynthetic rate), as well as stimulated the activities of the antioxidant enzymes in roots and shoots with greater toxicity at the smaller particle size (80 nm). However, both exogenous selenite applications significantly alleviated the above toxic effects of nano-PS in lettuces, especially at a high Se level of 5 ppm. Regression Path Analysis (RPA) revealed that regulation of chlorophyll levels by Se might be a key mechanism for counteracting PS stress in lettuces, which led to the increase in indigenous defense capacity. The present findings provide a novel but safer and cleaner agricultural strategy to alleviate or minimize nano-plastics toxicity in agricultural soils for staple crops and vegetables via application of Se.

Identification and validation of volatile organic compounds in bile for differential diagnosis of perihilar cholangiocarcinoma.

Early and differential diagnosis of perihilar cholangiocarcinoma (PHCCA) is highly challenging. This study aimed to evaluate whether volatile organic compounds (VOCs) in bile samples could be emerging diagnostic biomarkers for PHCCA. We collected 200 bile samples from patients with PHCCA and benign biliary diseases (BBD), including a 140-patient training cohort and an 60-patient test cohort. Gas chromatography-ion mobility spectrometry (GC-IMS) was used for VOCs detection. The predictive models were constructed using machine learning algorithms. Our analysis detected 19 VOC substances using GC-IMS in the bile samples and resulted in the identification of three new VOCs, 2-methoxyfuran, propyl isovalerate, and diethyl malonate that were found in bile. Unsupervised hierarchical clustering analysis supported that VOCs detected in the bile could distinguish PHCCA from BBD. Twelve VOCs defined according to 32 signal peaks had significant statistical significance between BBD and PHCCA, including four up-regulated VOCs in PHCCA, such as 2-ethyl-1-hexanol, propyl isovalerate, cyclohexanone, and acetophenone, while the rest eight VOCs were down-regulated. ROC curve analysis revealed that machine learning models based on VOCs could help diagnosing PHCCA. Among them, SVM provided the highest AUC of 0·966, with a sensitivity and specificity of 93·1% and 100%, respectively. The diagnostic model based on different VOC spectra could be a feasible method for the differential diagnosis of PHCCA.

Generation of Endogenous Promoter-Driven Luciferase Reporter System Using CRISPR/Cas9 for Investigating Transcriptional Regulation of the Core Clock Gene BMAL1.

Humans and other organisms are continuously exposed to thousands of chemicals through the atmosphere, drinking water, food, or direct contact. A large proportion of such chemicals are present in very low concentrations and may have synergistic effects, even at their no-observed-adverse-effect level (NOAEL). Complex mixtures of contaminants are very difficult to assess by traditional toxicological methods. There is increasing attention on how different pollutants induce adverse physiological functions in the human body through effects on the circadian rhythm. However, it is very difficult to screen for compounds with circadian-rhythm-disrupting effects from a large number of chemicals or their complex mixtures. We established a stable firefly luciferase reporter gene knock-in U2-OS cell line by CRISPR/Cas9 to screen circadian-rhythm-disrupting pollutants. The luciferase gene was inserted downstream of the core clock gene BMAL1 and controlled by an endogenous promoter. Compared to detection systems using exogenous promoters, these cells enable the detection of compounds that interfere with the circadian rhythm system mediated by BMAL1 gene expression. The U2-OS knock-in cells showed BMAL1 and luciferase activity had parallel changes when treated with BMAL1 inhibitor and activator. Furthermore, the luciferase reporter gene has high sensitivity and is faster and more cost-effective than classic toxicology methods. The knock-in cell line can be used for high-throughput and efficient screening of circadian-rhythm-disrupting chemicals such as drugs and pollutants.

Significant Upregulation of HERV-K (HML-2) Transcription Levels in Human Lung Cancer and Cancer Cells.

Lung cancer is the second most common cancer worldwide and the leading cause of cancer death in the world. Therefore, there is an urgent need to develop new and effective biomarkers for diagnosis and treatment. Under this circumstance, human endogenous retroviruses (HERVs) were recently introduced as novel biomarkers for cancer diagnosis. This study focused on the correlation between lung cancer and HERV-K (HML-2) transcription levels. At the cellular level, different types of lung cancer cells and human normal lung epithelial cells were used to analyze the transcription levels of the HERV-K (HML-2) gag, pol, and env genes by RT-qPCR. At the level of lung cancer patients, blood samples with background information from 734 lung cancer patients and 96 healthy persons were collected to analyze the transcription levels of HERV-K (HML-2) gag, pol, and env genes. The results showed that the transcriptional levels of the HERV-K (HML-2) gag, pol, and env genes in lung cancer cells and lung cancer patient blood samples were significantly higher than those in the healthy controls, which was also verified by RNAScope ISH technology. In addition, we also found that there was a correlation between the abnormal transcription levels of HERV-K (HML-2) genes in lung cancer patients and the clinicopathological parameters of lung cancer. We also identified the distribution locations of the gag, pol, and env primer sequences on each chromosome and analyzed the function of these loci. In conclusion, HERV-K (HML-2) genes may be a potential biomarker for the diagnosis of lung cancer.

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 to Close Contacts, China, January-February 2020.

We estimated the symptomatic, PCR-confirmed secondary attack rate (SAR) for 2,382 close contacts of 476 symptomatic persons with coronavirus disease in Yichang, Hubei Province, China, identified during January 23-February 25, 2020. The SAR among all close contacts was 6.5%; among close contacts who lived with an index case-patient, the SAR was 10.8%; among close-contact spouses of index case-patients, the SAR was 15.9%. The SAR varied by close contact age, from 3.0% for those <18 years of age to 12.5% for those >60 years of age. Multilevel logistic regression showed that factors significantly associated with increased SAR were living together, being a spouse, and being >60 years of age. Multilevel regression did not support SAR differing significantly by whether the most recent contact occurred before or after the index case-patient's onset of illness (p = 0.66). The relatively high SAR for coronavirus disease suggests relatively high virus transmissibility.

Roles of conserved residues in the receptor binding sites of human parainfluenza virus type 3 HN protein.

Human parainfluenza virus type 3 (hPIV-3) entry and intrahost spread through membrane fusion are initiated by two envelope glycoproteins, hemagglutinin-neuraminidase (HN) and fusion (F) protein. Binding of HN protein to the cellular receptor via its receptor-binding sites triggers conformational changes in the F protein leading to virus-cell fusion. However, little is known about the roles of individual amino acids that comprise the receptor-binding sites in the fusion process. Here, residues R192, D216, E409, R424, R502, Y530 and E549 located within the receptor-binding site Ⅰ, and residues N551 and H552 at the putative site Ⅱ were replaced by alanine with site-directed mutagenesis. All mutants except N551A displayed statistically lower hemadsorption activities ranging from 16.4% to 80.2% of the wild-type (wt) level. With standardization of the number of bound erythrocytes, similarly, other than N551A, all mutants showed reduced fusogenic activity at three successive stages: lipid mixing (hemifusion), content mixing (full fusion) and syncytium development. Kinetic measurements of the hemifusion process showed that the initial hemifusion extent for R192A, D216A, E409A, R424A, R502A, Y530A, E549A and H552A was decreased to 69.9%, 80.6%, 71.3%, 67.3%, 50.6%, 87.4%, 84.9% and 25.1%, respectively, relative to the wt, while the initial rate of hemifusion for the E409A, R424A, R502A and H552A mutants was reduced to 69.0%, 35.4%, 62.3%, 37.0%, respectively. In addition, four mutants with reduced initial hemifusion rates also showed decreased percentages of F protein cleavage from 43.4% to 56.3% of the wt. Taken together, Mutants R192A, D216A, E409A, R424A, R502A, Y530A, E549A and H552A may lead to damage on the fusion activity at initial stage of hemifusion, of which decreased extent and rate may be associated with impaired receptor binding activity resulting in the increased activation barrier of F protein and the cleavage of it, respectively.

Demethylated miR-216a Regulates High Mobility Group Box 3 Promoting Growth of Esophageal Cancer Cells Through Wnt/ß-Catenin Pathway.

BACKGROUND: Esophageal cancer (EC) is the eighth most common cause of cancer-associated mortality in humans. Recent studies have revealed the important roles of microRNAs (miRs) in mediating tumor initiation and progression. miR-216a has been found to be involved in the progression of EC, but the underlying mechanisms remain largely unknown. The aim of this study is to explore the mechanism of miR-216a and the downstream molecules in esophageal cancer. MATERIALS AND METHODS: The degree of methylation of miR-216a promoter in EC tissues and cell lines was determined with methylation specific polymerase chain reaction (MSP). The levels of miR-216a and HMGB3 in EC cells were quantified by quantitative PCR (qPCR) and Western blot (WB). EC cell lines were treated with DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AZ), miR-216a mimics, and HMGB3 siRNA to explore the effects of miR-216a and HMGB3 on the proliferation, migration, invasion, and apoptosis of cells. Dual-luciferase reporter assay was employed to verify the binding of miR-216a to the 3'UTR of HMGB2 mRNA. RESULTS: The promoter of MiR-216a was hypermethylated and the expression of miR-216a was down-regulated in EC, while HMGB3 was up-regulated. Dual luciferase reporter assay confirmed the binding of miR-216a to the 3'UTR of HMGB3 mRNA. Demethylated miR-216a and miR-216a mimics elevated miR-216a expression and down-regulated HMGB3, as well as inhibited cell proliferation, migration, and invasion. Inhibiting the expression of HMGB3 played an important role in inducing apoptosis, suppressing cell expansion, and down-regulating the activity of Wnt/ß-catenin pathway. CONCLUSIONS: Hypermethylation in the promoter of miR-216a upregulated HMGB3 and the Wnt/ß-catenin pathway, resulting in enhanced EC progression.

Using machine learning to develop an autoverification system in a clinical biochemistry laboratory.

OBJECTIVES: Autoverification systems have greatly improved laboratory efficiency. However, the long-developed rule-based autoverfication models have limitations. The machine learning (ML) algorithm possesses unique advantages in the evaluation of large datasets. We investigated the utility of ML algorithms for developing an artificial intelligence (AI) autoverification system to support laboratory testing. The accuracy and efficiency of the algorithm model were also validated. METHODS: Testing data, including 52 testing items with demographic information, were extracted from the laboratory information system and Roche Cobas® IT 3000 from June 1, 2018 to August 30, 2019. Two rounds of modeling were conducted to train different ML algorithms and test their abilities to distinguish invalid reports. Algorithms with the top three best performances were selected to form the finalized ensemble model. Double-blind testing between experienced laboratory personnel and the AI autoverification system was conducted, and the passing rate and false-negative rate (FNR) were documented. The working efficiency and workload reduction were also analyzed. RESULTS: The final AI system showed a 89.60% passing rate and 0.95 per mille FNR, in double-blind testing. The AI system lowered the number of invalid reports by approximately 80% compared to those evaluated by a rule-based engine, and therefore enhanced the working efficiency and reduced the workload in the biochemistry laboratory. CONCLUSIONS: We confirmed the feasibility of the ML algorithm for autoverification with high accuracy and efficiency.

Molecular epidemiology of GII noroviruses in outpatients with acute gastroenteritis in Shandong Province, China.

Noroviruses have been recognized as the most important causative agents of acute gastroenteritis. The present study was carried out to investigate the molecular epidemiological features of genotype II (GII) norovirus in outpatients with acute gastroenteritis in Shandong province in China from July 2017 to June 2018. In total, 151 (10.30%) samples were positive for NoV GII strains by RT-PCR. Eight genotypes were detected: GII.2, GII.3, GII.4, GII.6, GII.7, GII.12, GII.13 and GII.17. GII.4 (43.71%) was the most prevalent genotype, and the dominant strains belonged to the group of Sydney-2012 strains. GII.17 (27.15%), which has become the main cause of outbreaks of acute gastroenteritis in China, also accounted for a high proportion. Meanwhile, three recombinant types (GII.P17-GII.7, GII.P3-GII.4 and GII.P12-GII.4) were observed and authenticated using Simplot software. The results showed that GII norovirus was the main cause of acute gastroenteritis in Shandong province. GII.4 and GII.17 were the dominant genotypes. Continuous observation and identification of emerging genotypes are necessary for understanding the evolution of the virus, control of infection, and development of vaccines.

Comparative Genomics Studies on the dmrt Gene Family in Fish.

Doublesex and mab-3-related transcription factor (dmrt) genes are widely distributed across various biological groups and play critical roles in sex determination and neural development. Here, we applied bioinformatics methods to exam cross-species changes in the dmrt family members and evolutionary relationships of the dmrt genes based on genomes of 17 fish species. All the examined fish species have dmrt1-5 while only five species contained dmrt6. Most fish harbored two dmrt2 paralogs (dmrt2a and dmrt2b), with dmrt2b being unique to fish. In the phylogenetic tree, 147 DMRT are categorized into eight groups (DMRT1-DMRT8) and then clustered in three main groups. Selective evolutionary pressure analysis indicated purifying selections on dmrt1-3 genes and the dmrt1-3-2(2a) gene cluster. Similar genomic conservation patterns of the dmrt1-dmrt3-dmrt2(2a) gene cluster with 20-kb upstream/downstream regions in fish with various sex-determination systems were observed except for three regions with remarkable diversity. Synteny analysis revealed that dmrt1, dmrt2a, dmrt2b, and dmrt3-5 were relatively conserved in fish during the evolutionary process. While dmrt6 was lost in most species during evolution. The high conservation of the dmrt1-dmrt3-dmrt2(2a) gene cluster in various fish genomes suggests their crucial biological functions while various dmrt family members and sequences across fish species suggest different biological roles during evolution. This study provides a molecular basis for fish dmrt functional analysis and may serve as a reference for in-depth phylogenomics.

Genetic characterization and phylogenetic analysis of Newcastle disease virus from China.

The avian infectious disease, Newcastle disease (ND), caused by Newcastle disease virus (NDV) can cause severe economic losses to poultry whether vaccinated or not in many countries. In this study, a strain of NDV isolated from an outbreak in China was subjected to biological, phylogenetic and genetic characterization. The results showed that the mean death time (MDT) was 52.4 h and the intracerebral pathogenicity indices (ICPI) value was 1.95. In addition, amino acid sequencing result showed that it had a sequence 112R-R-Q-R-R↓F117 at fusion protein cleaving site (FPCS) indicating a velogenic strain. And its genome length is 15,192 nucleotide (nt) with the conserved complementary 3' leader and 5' trailer regions encoding six genes, 3'-NP-P-M-F-HN-L-5'. Based on phylogenetic analyses for hyper-variable region and complete genome of F gene, the strain studied here can be clustered into genotype IX, Class II, which has little evolution distance with strains of genotype III, being considered as a transitional strain in the evolution history of NDV. The rescue of infectious cDNA is proceeded in 9-day-old embryonated SPF chicken eggs. Despite the death of the first generation, the allantoic fluid harvested from the first generation lost its pathogenicity after passage. And we found the phenomenon happened due to the antibody appearing in the allantoic fluid. These findings offer our understanding of circulating strains of NDV in China and lay scientific foundations for making more efficient vaccines for Newcastle disease.

Correlation between RAGE gene promoter methylation and diabetic retinal inflammation.

The methylation status of the receptor for advanced glycation end products (RAGE) gene promoter in peripheral blood mononuclear cells (PBMCs) of type 2 diabetic retinopathy (DR) patients was evaluated to investigate the correlation between RAGE gene promoter methylation and diabetic retinal inflammation. Eighty patients admitted and diagnosed as type 2 DR in Qilu Hospital, Shandong University during the period from October, 2013 to October, 2015 were enrolled in this study. They were the observation group and 40 healthy subjects were enrolled in the control group. PBMCs were collected from patients using density gradient centrifugation, and the methylation status of RAGE gene promoters was detected using methylation-specific PCP (MSP). Interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor-α (TNF-α) levels of in the serum were measured using enzyme-linked immunosorbent assay (ELISA). PBMCs in patients with positive RAGE gene promoter methylation were isolated and cultured and RAGE gene promoter methylation was inhibited using the demethylating agent, 5'-aza-2'-deoxycytidine (5-aza-dC). The methylation status of RAGE gene promoters in PBMCs was detected via MSP. IL-1ß, IL-6 and TNF-α levels in the supernatant of PBMC culture solution were evaluated using ELISA. MSP results showed that there were 26 cases (32.50%) of RAGE gene promoter methylation in PBMCs in DR patients. RAGE gene promoters were methylated in all normal healthy subjects. IL-1ß, IL-6 and TNF-α levels in serum for positive RAGE gene promoter methylation group were significantly lower than those in negative RAGE gene promoter methylation group (p<0.01). 5-aza-dC inhibited the RAGE gene promoter methylation of PBMCs in patients with positive RAGE gene promoter methylation. The inhibition of methylation in RAGE gene promoter increased the levels of IL-1ß, IL-6 and TNF-α in supernatant of culture solution. In conclusion, RAGE gene promoter hypomethylation was detected in DR patients, indicating that RAGE gene promoter methylation could inhibit the diabetic retinal inflammation.

Mutations in the DI-DII Linker of Human Parainfluenza Virus Type 3 Fusion Protein Result in Diminished Fusion Activity.

Human parainfluenza virus type 3 (HPIV3) can cause severe respiratory tract diseases in infants and young children, but no licensed vaccines or antiviral agents are currently available for treatment. Fusing the viral and target cell membranes is a prerequisite for its entry into host cells and is directly mediated by the fusion (F) protein. Although several domains of F are known to have important effects on regulating the membrane fusion activity, the roles of the DI-DII linker (residues 369-374) of the HPIV3 F protein in the fusogenicity still remains ill-defined. To facilitate our understanding of the role of this domain might play in F-induced cell-cell fusion, nine single mutations were engineered into this domain by site-directed mutagenesis. A vaccinia virus-T7 RNA polymerase transient expression system was employed to express the wild-type or mutated F proteins. These mutants were analyzed for membrane fusion activity, cell surface expression, and interaction between F and HN protein. Each of the mutated F proteins in this domain has a cell surface expression level similar to that of wild-type F. All of them resulted in a significant reduction in fusogenic activity in all steps of membrane fusion. Furthermore, all these fusion-deficient mutants reduced the amount of the HN-F complexes at the cell surface. Together, the results of our work suggest that this region has an important effect on the fusogenic activity of F.

Roles of the highly conserved amino acids in the globular head and stalk region of the Newcastle disease virus HN protein in the membrane fusion process.

Newcastle disease virus (NDV), an avain paramyxovirus, has been assigned to the genus Avulavirus within the family Paramyxoviridae. It causes Newcastle disease (ND) that is a highly contagious and fatal viral disease affecting poultry and most species of birds. The hemagglutinin-neuraminidase (HN) protein of NDV has multiple functions including mediating hemadsorption (HAD), neuraminidase (NA), and fusion promotion activities affecting the process of viral attachment, entry, replication and dissemination. Fusion ability of the NDV was highly correlated to its virulence. Mutations in the HN globular head and headless HN of NDV were constructed to determinate the impact of highly conserved amino acids in the globular head of paramyxovirus HN proteins and the roles of the stalk region of HN in the fusion process. It was found that the interaction between F and HN mutants E401A, G402A, G468A, V469A, Y526A, and T527A was equal to that in F and wt HN. The mutations of G402A, G468A, V469A, and T527A had various effects on cell fusion promotion, receptor binding ability, and NA activity, but the membrane merging rate was comparable to wt HN. The elimination of hemadsorption ability and NA activity of E401A and Y526A resulted in the loss of the fusion promotion function of HN. The conclusion was that receptor binding and NA had a common active site and E401 and Y526 amino acids were essential for virus attachment, entry, and dissemination. In addition, G468A mutation made different contributions to HAD and NA, which indicated that G468 was one of the potential key amino acids in switching the two functions between receptor binding and sialic acid destruction of HN. It was also proven that the headless HN of NDV could promote the fusion event mediated by F. Thus, it revealed a novel mechanism in F activation of NDV.