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Background Stroke is a disease characterized by sudden cerebral ischemia and is the second leading cause of death worldwide. We aimed to develop and validate a nomogram model to predict mortality in intensive care unit patients with stroke. Methods All data involved in this study were extracted from the Medical Information Mart for Intensive Care III database (MIMIC-III). The data were analyzed using multivariate Cox regression, and the performance of the novel nomogram, which assessed the patient’s overall survival at 30, 180, and 360 days after stroke, was evaluated using Harrell’s concordance index (C-index) and the area under the receiver operating characteristic curve. A calibration curve and decision curve were introduced to test the clinical value and effectiveness of our prediction model. Results A total of 767 patients with stroke were randomly divided into derivation (n = 536) and validation (n = 231) cohorts at a 7:3 ratio. Multivariate Cox regression showed that 12 independent predictors, including age, weight, ventilation, cardiac arrhythmia, metastatic cancer, explicit sepsis, Oxford Acute Severity of Illness Score or OASIS score, diastolic blood pressure, bicarbonate, chloride, red blood cell and white blood cell counts, played a significant role in the survival of individuals with stroke. The nomogram model was validated based on the C-indices, calibration plots, and decision curve analysis results. Conclusions The plotted nomogram accurately predicted stroke outcomes and, thus may contribute to clinical decision-making and treatment as well as consultation services for patients.

A dual-receptor interaction with a polysialoganglioside and synaptic vesicle glycoprotein 2 (SV2) is required for botulinum neurotoxin A (BoNT) toxicity. Here, we review what is currently known about the BoNT/A-SV2 interaction based on structural studies. Currently, five crystal structures of the receptor-binding domain (Hc) of BoNT subtypes A1 and A2 complexed to the large luminal domain (LD4) of SV2C have been determined. On the basis of the available structures, we will discuss the importance of protein-protein and protein-carbohydrate interactions for BoNT/A toxicity as well as the high plasticity of BoNT/A for receptor recognition by tolerating a variety of side-chain interactions at the interface. A plausible explanation how receptor-binding specificity of BoNT/A may be achieved without an extensive and conserved side chain-side chain interaction network will be provided.

Sustained expression of B-cell receptor (BCR) critically contributes to the development of diffuse large B-cell lymphoma (DLBCL). However, little is known on the mechanism regulating BCR expression. In the present study, we explored the biological significance of functional intergenic repeating RNA element (FIRRE) in DLBCL and its regulation on BCR. Functional impacts of FIRRE on cell viability, transformation, and apoptosis were examined by MTT, colony formation, and flow cytometry, respectively. The interaction between FIRRE and polypyrimidine tract binding protein 1 (PTBP1) was identified by RNA pull-down and verified using RNA immunoprecipitation (RIP) assays. The effects of FIRRE and PTBP1 on Smurf2 mRNA were examined by RIP, RNA pull-down, and mRNA stability assays. Smurf2-mediated BCR ubiquitination was investigated using co-immunoprecipitation, ubiquitination, and protein stability assays. In vivo, xenograft models were used to assess the impacts of targeting FIRRE on DLBCL growth. FIRRE was specifically up-regulated in and essentially maintained multiple malignant behaviors of BCR-dependent DLBCL cells. Through the interaction with PTBP1, FIRRE promoted the mRNA decay of Smurf2, a ubiquitin ligase for the degradation BCR protein. Targeting FIRRE was sufficient to regulat Smurf2 and BCR expressions and inhibit DLBCL malignancy both in vivo and in vitro. FIRRE-PTBP1 interaction, by simulating Smurf2 mRNA decay and stabilizing BCR, promotes the development of DLBCL. Consequently, targeting this signaling mechanism may provide therapeutic benefits for DLBCL.

Background. Femoral nerve block (FNB) and fascia iliac compartment block (FICB) are alternative methods of pain relief during hip surgery. Nevertheless, the effectiveness and safety of FNB compared with FICB are yet to be fully determined. Methods. Electronic databases were systematically searched. Only randomized controlled trials (RCTs) on hip surgery were included. Postoperatively, the pain scores at different time points, narcotic requirements in 24 h, mean arterial pressure, spinal anesthesia (SA) time, patient satisfaction, and adverse effect rates between the two groups were extracted throughout the study. Results. Fourteen RCTs including 1179 patients were included. Compared to the FICB, FNB decreased the VAS scores postoperatively at 24 h at rest ( P < 0.05 ) and the incidence rate of some side effects (nausea, vomiting, and sedation) ( P < 0.05 ). However, compared to the FICB, no significant difference was found in the FNB regarding the VAS scores postoperatively at any of the other time points (2 min, 20 min, 2 h, 24 h at movement, 48 h at rest, and 48 h at movement). Patients in both groups had similar narcotic needs after 24 h, mean arterial pressure, SA time, and patient satisfaction ( P > 0.05 ). Conclusions. FNB has more advantages in reducing VAS scores postoperatively at 24 h at rest and the odds of some adverse effects. A better quality RCT is needed to properly compare FNB with FICB.

Background: Areal bone mineral density (aBMD) applied for osteoporosis diagnosis unavoidably results in the missingdiagnosis in patients with large bones and misdiagnosis in those with small bones. Therefore, we try to find a new adjusted index of bone mineral content (BMC) to make up shortcomings of aBMD in osteoporosis diagnosis. Methods: In this multi-center epidemiological study, BMC and aBMD of lumbar spines (n = 5510) and proximal femurs (n = 4710) were measured with dual energy X-ray absorptiometry (DXA). We analyzed the correlation between the bone mass and body weight in all subjects including four age groups ( 50 years). And then the body weight was used for standardizing BMC (named wBMC) and applied for the epidemiological analysis of osteoporosis. Results: The correlation of body weight and BMC is 0.839 to 0.931 of lumbar vertebra 1–4 (L1–4), and 0.71 to 0.95 of femoral neck in different age groups. When aBMD was applied for diagnosing osteoporosis, the prevalence was 7.55%, 16.39%, and 25.83% in patients with a high, intermediate, and low body weight respectively. However, the prevalence was 21.8%, 18.03%, and 11.64% by wBMC applied for diagnosing osteoporosis. Moreover, the prevalence of osteoporosis increased by 3.76% by wBMC with the body weight increased by 5 kg. The prevalence decreased by 1.94% when the body weight decreased by 5 kg. Conclusions: wBMC can reduce the missed diagnosis in patients with large body weight and reduce misdiagnosis in those with small body weight. Including children, wBMC may be feasible for osteoporosis diagnosis individuals at any age. Key words: Bone mineral content; Weight-standardized bone mineral content; Bone density; Dual-energy X-ray absorptiometry; Osteoporosis

Background: This study investigated the scientific research capacity and related factors of nurses with Master of Science in Nursing (MSN) degrees to provide information on hierarchical management and continuing education of nurses in China. Method: This multi-center cross-sectional study included 221 MSN hospital nurses in China. Results: Ninety-one percent of MSN nurses had good or excellent research capacity. Continuing education requirements existed regarding research practice and design. Research time, teamwork, leadership support, and retraining opportunities influenced research capacity, which decreased with increasing age and years of work, especially at 3 to 5 years after initial employment. Conclusion: Clinical managers should pay attention to factors that influence nurses' research capacity and continuing education requirements. Multiple incentives should be used for MSN nurses, especially for those with 3 to 5 years of employment, to provide more chances for self-actualization, to take advantage of their talents, and to promote the development of clinical nursing. [ J Contin Educ Nurs. 2019;50(2):61–68.]

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, which is highly pathogenic and classified as a biosafety level 3 (BSL-3) agent, has greatly threatened global health and efficacious antivirals are urgently needed. The high requirement of facilities to manipulate the live virus has limited the development of antiviral study. Here, we constructed a reporter replicon of SARS-CoV-2, which can be handled in a BSL-2 laboratory. The Renilla luciferase activity effectively reflected the transcription and replication levels of the replicon genome. We identified the suitability of the replicon in antiviral screening using the known inhibitors, and thus established the replicon-based high-throughput screening (HTS) assay for SARS-CoV-2. The application of the HTS assay was further validated using a few hit natural compounds, which were screened out in a SARS-CoV-2 induced cytopathic-effect-based HTS assay in our previous study. This replicon-based HTS assay will be a safe platform for SARS-CoV-2 antiviral screening in a BSL-2 laboratory without the live virus.

COVID-19 caused by SARS-CoV-2 has posed a significant threat to global public health since its outbreak in late 2019. Although there are a few drugs approved for clinical treatment to combat SARS-CoV-2 infection currently, the severity of the ongoing global pandemic still urges the efforts to discover new antiviral compounds. As the viral spike (S) protein plays a key role in mediating virus entry, it becomes a potential target for the design of antiviral drugs against COVID-19. Here, we tested the antiviral activity of berbamine hydrochloride, a bis-benzylisoquinoline alkaloid, against SARS-CoV-2 infection. We found that berbamine hydrochloride could efficiently inhibit SARS-CoV-2 infection in different cell lines. Further experiments showed berbamine hydrochloride inhibits SARS-CoV-2 infection by targeting the viral entry into host cells. Moreover, berbamine hydrochloride and other bis-benzylisoquinoline alkaloids could potently inhibit S-mediated cell-cell fusion. Furthermore, molecular docking results implied that the berbamine hydrochloride could bind to the post fusion core of SARS-CoV-2 S2 subunit. Therefore, berbamine hydrochloride may represent a potential efficient antiviral agent against SARS-CoV-2 infection.

Asthma is an inflammatory syndrome characterized by airway hyperresponsiveness, bronchial inflammation, and airway remodeling. The hypertrophy and hyperplasia of airway smooth muscle cells (ASMCs) are hallmarks of bronchial remodeling in asthma. In this study, the regulatory effects of microRNA‐620 (miR‐620) on ASMC proliferation and apoptosis in response to transforming growth factor β1 (TGF‐β1) stimulation was investigated. The expression of miR‐620 was significantly upregulated in TGF‐β1‐treated ASMCs compared with vehicle‐treated cells. Downregulation of miR‐620 suppressed the proliferation and increased apoptosis in TGF‐β1‐stimulated ASMCs. Phosphatase and tensin homolog (PTEN) was predicted and confirmed as a downstream target of miR‐620. PTEN was upregulated in miR‐620‐inhibitor transfected ASMCs, but decreased in cells delivered with miR‐620 mimics. Moreover, knocking down miR‐620 alone efficiently reduced the phosphorylation of protein kinase B (AKT), decreased TGF‐β1‐induced proliferation and promoted apoptosis in ASMCs, whereas downregulation of PTEN in miR‐620 inhibitor‐transfected cells restored the activation of AKT, increased TGF‐β1‐triggered proliferation, and partially inhibited ASMC apoptosis. Taken together, the present study provided evidence that miR‐620 increased TGF‐β1‐mediated proliferation and suppressed apoptosis in ASMCs via the regulation of PTEN and AKT expression. These findings suggest that miR‐620/PTEN/AKT axis may be considered as a therapeutic target for asthma treatment.

Mosquito-borne flaviviruses consist of a positive-sense genome RNA flanked by the untranslated regions (UTRs). There is a panel of highly complex RNA structures in the UTRs with critical functions. For instance, Xrn1-resistant RNAs (xrRNAs) halt Xrn1 digestion, leading to the production of subgenomic flaviviral RNA (sfRNA). Conserved short direct repeats (DRs), also known as conserved sequences (CS) and repeated conserved sequences (RCS), have been identified as being among the RNA elements locating downstream of xrRNAs, but their biological function remains unknown. In this study, we revealed that the specific DRs are involved in the production of specific sfRNAs in both mammalian and mosquito cells. Biochemical assays and structural remodeling demonstrate that the base pairings in the stem of these DRs control sfRNA formation by maintaining the binding affinity of the corresponding xrRNAs to Xrn1. On the basis of these findings, we propose that DRs functions like a bracket holding the Xrn1-xrRNA complex for sfRNA formation. IMPORTANCE Flaviviruses include many important human pathogens. The production of subgenomic flaviviral RNAs (sfRNAs) is important for viral pathogenicity as a common feature of flaviviruses. sfRNAs are formed through the incomplete degradation of viral genomic RNA by the cytoplasmic 5ʹ–3ʹ exoribonuclease Xrn1 halted at the Xrn1-resistant RNA (xrRNA) structures within the 3ʹ-UTR. The 3ʹ-UTRs of the flavivirus genome also contain distinct short direct repeats (DRs), such as RCS3, CS3, RCS2, and CS2. However, the biological functions of these ancient primary DR sequences remain largely unknown. Here, we found that DR sequences are involved in sfRNA formation and viral virulence and provide novel targets for the rational design of live attenuated flavivirus vaccine.

West Nile virus (WNV) is a neurotropic human pathogen that has caused increasing infected cases over recent years. There is currently no licensed vaccine or effective drug for prevention and treatment of WNV infection in humans. To facilitate antiviral drug discovery and neutralizing antibody detection, a WNV cDNA clone containing a luciferase reporter gene was constructed through incorporating Gaussia luciferase (Gluc) gene within the capsid-coding region of WNV genome. Transfection of BHK-21 cells with the cDNA clone-derived RNA generated luciferase reporter WNV (WNV-Gluc) and the stable WNV-Gluc with high titers (>10(7)PFU/ml) was obtained through plaque purification. Luciferase activity was used to effectively quantify the viral production of WNV-Gluc. Using the reporter virus WNV-Gluc, we developed a luciferase based assay in a 12-well format for evaluating neutralizing antibodies. The reporter virus could be a powerful tool for epidemiological investigation of WNV, vaccine evaluation, antiviral drug screening, and the study of WNV replication and pathogenesis.

Japanese encephalitis virus (JEV), a major cause of Japanese encephalitisis, is an arbovirus that belongs to the genus Flavivirus of the family Flaviviridae. Currently, there is no effective drugs available for the treatment of JEV infection. Therefore, it is important to establish efficient antiviral screening system for the development of antiviral drugs. In this study, we constructed a full-length infectious clone of eGFP-JEV reporter virus by inserting the eGFP gene into the capsid-coding region of the viral genome. The reporter virus RNA transfected-BHK-21 cells generated robust eGFP fluorescence signals that were correlated well with viral replication. The reporter virus displayed growth kinetics similar to wild type (WT) virus although replicated a little slower. Using a known JEV inhibitor, NITD008, we demonstrated that the reporter virus could be used to identify inhibitors against JEV. Furthermore, an eGFP-JEV-based high throughput screening (HTS) assay was established in a 96-well format and used for screening of 1,443 FDA-approved drugs. Sixteen hit drugs were identified to be active against JEV. Among them, five compounds which are lonafarnib, cetylpyridinium chlorid, cetrimonium bromide, nitroxoline and hexachlorophene, are newly discovered inhibitors of JEV, providing potential new therapies for treatment of JEV infection., Highlights • The eGFP-JEV reporter virus was established. • The eGFP-JEV -based high throughput screening (HTS) assay was developed with a calculated Z′ factor of >0.5. • A library of 1,443 FDA-approved drugs was screened to determine their ability to inhibit JEV infection using this HTS assay. • 5 novel hit drugs were identified to be active against JEV infection.

International audience; There is growing interest in the use of mammalian protein expression systems, and in the use of antibody-derived chaperones, for structural studies. Here, we describe protocols ranging from the production of recombinant membrane proteins in stable inducible cell lines to biophysical characterization of purified membrane proteins in complex with llama antibody domains. These protocols were used to solve the structure of the mouse 5-HT3 serotonin receptor but are of broad applicability for crystallization or cryo-electron microscopy projects.

We report a stable Gaussia luciferase enterovirus 71 (Gluc-EV71) reporter virus to facilitate drug discovery. The Gluc-EV71 reporter virus was generated by engineering the Gaussia luciferase (Gluc) gene between the 5' untranslated region and VP4 gene of the EV71 genome. We could recover Gluc-EV71 after transfection of Vero cells with the cDNA clone-derived RNA. The reporter virus efficiently infects and replicates in various cell types (Vero, human rhabdomyosarcoma, and HeLa cells), producing robust luciferase activity. The Gluc-EV71 virus replicates slower than the wild-type virus in cell culture. The reporter virus is stable in maintaining the Gluc gene after five rounds of continuous passaging in Vero cells. Using known EV71 inhibitors, we demonstrate that the reporter virus can be used for antiviral testing. However, the Gluc-EV71 infection assay cannot be adapted to a homogenous format for high throughput screen, mainly due to the secreted nature of the Gluc protein and the short half-life of the Gluc luminescence signal. The Gluc-EV71 and its infection assay could be useful for antiviral drug discovery as well as for studying EV71 replication and pathogenesis.

West Nile virus (WNV) is a mosquito-borne flavivirus that causes epidemics of encephalitis and viscerotropic disease worldwide. This virus has spread rapidly and has posed a significant public health threat since the outbreak in New York City in 1999. The interferon (IFN)-mediated antiviral response represents an important component of virus-host interactions and plays an essential role in regulating viral replication. Previous studies have suggested that multifunctional nonstructural proteins encoded by flaviviruses antagonize the host IFN response via various means in order to establish efficient viral replication. In this study, we demonstrated that the nonstructural protein 1 (NS1) of WNV antagonizes IFN-β production, most likely through suppression of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) activation. In a dual-luciferase reporter assay, WNV NS1 significantly inhibited the activation of the IFN-β promoter after Sendai virus infection or poly(I·C) treatment. NS1 also suppressed the activation of the IFN-β promoter when it was stimulated by interferon regulatory factor 3 (IRF3)/5D or its upstream molecules in the RLR signaling pathway. Furthermore, NS1 blocked the phosphorylation and nuclear translocation of IRF3 upon stimulation by various inducers. Mechanistically, WNV NS1 targets RIG-I and melanoma differentiation-associated gene 5 (MDA5) by interacting with them and subsequently causing their degradation by the proteasome. Furthermore, WNV NS1 inhibits the K63-linked polyubiquitination of RIG-I, thereby inhibiting the activation of downstream sensors in the RLR signaling pathway. Taken together, our results reveal a novel mechanism by which WNV NS1 interferes with the host antiviral response. IMPORTANCE WNV Nile virus (WNV) has received increased attention since its introduction to the United States. However, the pathogenesis of this virus is poorly understood. This study demonstrated that the nonstructural protein 1 (NS1) of WNV antagonizes the induction of interferon beta (IFN-β) by interacting with and degrading retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), which are crucial viral sensors in the host innate immune system. Further experiments suggested that NS1-mediated inhibition of the RIG-I-like receptor (RLR) signaling pathway involves inhibition of RIG-I K63-linked polyubiquitination and that the proteasome plays a role in RIG-I degradation. This study provides new insights into the regulation of WNV NS1 in the RLR signaling pathway and reveals a novel mechanism by which WNV evades the host innate immune response. The novel findings may guide us to discover new therapeutic targets and develop effective vaccines for WNV infections.

Background Pain management after total hip arthroplasty (THA) varies and has been widely studied in recent years. Gabapentin as a third-generation antiepileptic drug that selectively affects the nociceptive process has been used for pain relief after THA. This meta-analysis was conducted to examine the efficacy of gabapentin in THA. Methods An electronic-based search was conducted using the following databases: PubMed, EMBASE, Ovid MEDLINE, ClinicalTrials.gov, and Cochrane Central Register of Controlled Trials (CENTRAL). Randomised controlled trials (RCTs) involving gabapentin and a placebo for THA were included. The meta-analysis was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Results Five trials met the inclusion criteria. The cumulative narcotic consumption and the visual analogue scale (VAS) scores at 24 and 48 h postoperatively were used for postoperative pain assessment. There was a significant decrease in morphine consumption at 24 h (P = 0.00). Compared with the control group, the VAS score (at rest) at 48 h was less in the gabapentin group (P = 0.00). Conclusion The administration of gabapentin is effective in decreasing postoperative narcotic consumption and the VAS score.

The present study aimed to re-evaluate the incidence of early dysphonia after anterior cervical spine surgery (ACSS) and to determine the related risk factors.Patients underwent ACSS between January 2011 and December 2013 at two sites were identified retrospectively from hospital's patient databases. A total of 233 cases were included in this study. Dysphonia developed 1 month postoperatively was recorded. Follow-up was conducted in all positive-response patients. Those reporting severe or persistent voice symptoms were referred to otolaryngologists for further assessments and (or) treatments. Pre and intraoperative factors were collected to determine their relationships with dysphonia one month postoperatively.45 patients developed dysphonia at one month, including 23 males and 22 females, yielding to an incidence of 19.3%. 34 cases resolved themselves in 3 months, leaving the remaining 11 patients considered to be severe or persistent cases. However, 10 of them recovered spontaneously in the next 9 months, while the last case received vocal cord medialization and returned to almost normal speech function at 18 months. In univariate analysis, only approaching level involving C6-C7 or (and) C7-T1 was significantly associated with postoperative dysphonia (P0.001). This association was not weakened in multiple logistic regression analysis (OR 2.348, 95% CI 1.467-3.659, P0.001).The incidence of early dysphonia following ACSS was relatively high and approaching at lower cervical levels was an independent predictive factor.

Flavivirus nonstructural protein 2B (NS2B) is a transmembrane protein that functions as a cofactor for viral NS3 protease. The cytoplasmic region (amino acids 51 to 95) alone of NS2B is sufficient for NS3 protease activity, whereas the role of transmembrane domains (TMDs) remains obscure. Here, we demonstrate for the first time that flavivirus NS2B plays a critical role in virion assembly. Using Japanese encephalitis virus (JEV) as a model, we performed a systematic mutagenesis at the flavivirus conserved residues within the TMDs of NS2B. As expected, some mutations severely attenuated (L38A and R101A) or completely destroyed (G12L) viral RNA synthesis. Interestingly, two mutations (G37L and P112A) reduced viral RNA synthesis and blocked virion assembly. None of the mutations affected NS2B-NS3 protease activity. Because mutations G37L and P112A affected virion assembly, we selected revertant viruses for these two mutants. For mutant G37L, replacement with G37F, G37H, G37T, or G37S restored virion assembly. For mutant P112A, insertion of K at position K127 (leading to K127KK) of NS2B rescued virion assembly. A biomolecular fluorescent complementation (BiFC) analysis demonstrated that (i) mutation P112A selectively weakened NS2B-NS2A interaction and (ii) the adaptive mutation K127KK restored NS2B-NS2A interaction. Collectively, our results demonstrate that, in addition to being a cofactor for NS3 protease, flavivirus NS2B also functions in viral RNA replication, as well as virion assembly. IMPORTANCE Many flaviviruses are important human pathogens. Understanding the molecular mechanisms of the viral infection cycle is essential for vaccine and antiviral development. In this study, we demonstrate that the TMDs of JEV NS2B participate in both viral RNA replication and virion assembly. A viral genetic study and a BiFC assay demonstrated that interaction between NS2B and NS2A may participate in modulating viral assembly in the flavivirus life cycle. Compensatory-mutation analysis confirmed that there was a correlation between viral assembly and NS2B-NS2A interaction. TMDs of NS2B may serve as novel antiviral targets to prevent flavivirus infection, and the structure determination of NS2B will help us to understand the functional mechanism of NS2B in viral RNA replication and assembly. The results have uncovered a new function of flavivirus NS2B in virion assembly, possibly through interaction with the NS2A protein.

Successful ear reconstruction depends on two factors: an ear framework and the skin covering the framework. However, the relative deficiency of skin for coverage of the cartilage framework remains an issue. This new method for total auricular reconstruction is a three-stage operation and involves the use of two tissue expanders. First, two skin expanders are implanted, one underneath the scalp and the other sited behind the microtic ear. At the second stage, after lobule transposition, the two expanded skin flaps (upper and lower) and mastoid fascial flap are raised. At the same time, the autogenous rib cartilage is harvested and the framework constructed. The cartilage framework is then anchored between the upper expanded skin flap and the fascial flap with its inferior pole inserted into the rotated earlobe. The upper expanded skin flap covers the whole anterior surface of the framework and drapes over the margins of the fascial flap, which wrap the framework from beneath. The raw surface of fascial flap is covered with the lower expanded skin flap. The formation of a pseudomeatus and tragus is performed at the third stage. Deficiency of skin is the major problem encountered with the other conventional methods. Our innovations using two tissue expanders in combination with an autogenous rib cartilage framework eliminate this problem completely.

Protein-reversible ADP-ribosylation is emerging as an important post-translational modification used to control enzymatic and protein activity in different biological systems. This modification regulates nitrogenase activity in several nitrogen-fixing bacterial species. ADP-ribosylation is catalyzed by ADP-ribosyltransferases and is reversed by ADP-ribosylhydrolases. The structure of the ADP-ribosylhydrolase that acts on Azospirillum brasilense nitrogenase (dinitrogenase reductase-activating glycohydrolase, DraG) has been solved at a resolution of 2.5 A. This bacterial member of the ADP-ribosylhydrolase family acts specifically towards a mono-ADP-ribosylated substrate. The protein shows an all-alpha-helix structure with two magnesium ions located in the active site. Comparison of the DraG structure with orthologues deposited in the Protein Data Bank from Archaea and mammals indicates that the ADP-ribosylhydrolase fold is conserved in all domains of life. Modeling of the binding of the substrate ADP-ribosyl moiety to DraG is in excellent agreement with biochemical data.

MicroRNA-18a (miR-18a) has been reported to be upregulated in gastric cancer (GC) tissues compared with normal gastric tissues. However, little is known about its prognostic value and biological roles.In this study, miR-18a expression in gastric adenocarcinoma (GAC) tissues and adjacent non-tumor tissues was validated by in situ hybridization, and the predictive values of miR-18a were explored. The biological roles of miR-18a and the underlying signal pathway were investigated in GC cell lines.Overexpressed intra-tumoral miR-18a was associated with poor survival rate and was an independent prognostic factor for overall survival rate (P0.001) in GC patients. Forced expression of miR-18a remarkably enhanced cell proliferation, migration, and invasion in GC cells, while inhibition of miR-18a caused the opposite effects. Further study showed that miR-18a suppressed the expression of interferon regulatory factor 2 (IRF2) by directly binding to its 3'-untranslated region. Moreover, miR-18a expression levels are inversely correlated with IRF2 in human GC tissues. Western blot showed that forced expression of miR-18a could not only downregulate the expression of IRF2, but also inhibit the expression of P53, suggesting that IRF2 might play as a tumor suppressor by regulating P53 signaling in GC.miR-18a modulated P53 expression by directly targeting IRF2 and had a high predictive value for prognosis of GAC patients. These results may lead to identification of therapeutic candidates of GC.

X-ray crystallography revealed a similar architecture of the ammonium transport protein AmtB from Escherichia coli and the homologous protein Amt-1 from Archaeoglobus fulgidus. Furthermore, the atomic structures suggest that the proteins conduct ammonia (NH3) rather than ammonium ions (NH4+). These findings indicate that the more than 350 members of the ammonium transporter/methylamine permease/Rhesus (Amt/Mep/Rh) protein family found in archaea, bacteria, fungi, plants and animals are ammonia-conducting channels rather than ammonium ion transporters. The essential part of these proteins is the narrow hydrophobic ammonia-conducting pore with two highly conserved histidine residues located in the middle of the pore. A specific ammonium ion binding site is found at the extracellular entry site of E. coli AmtB. E. coli AmtB and its regulator GlnK form an effective ammonium sensory system that couples intracellular gene regulation by the nitrogen control system to external changes in ammonium availability. Based on structural and functional analysis of various mutants, two conserved histidine residues were found to be essential for substrate conductance also in the functional eukaryotic ammonium transporters. The next big challenge in the field surely is to determine the atomic structure of Rh proteins.

Posttranslational modification of proteins plays a key role in the regulation of a plethora of metabolic functions. Protein modification by mono-ADP-ribosylation was first described as a mechanism of action of bacterial toxins. Since these pioneering studies, the number of pathways regulated by ADP-ribosylation in organisms from all domains of life expanded significantly. However, in only a few cases the full regulatory ADP-ribosylation circuit is known. Here, we review the system where mono-ADP-ribosylation regulates the activity of an enzyme: the regulation of nitrogenase in bacteria. When the nitrogenase product, ammonium, becomes available, the ADP-ribosyltransferase (DraT) covalently links an ADP-ribose moiety to a specific arginine residue on nitrogenase switching-off nitrogenase activity. After ammonium exhaustion, the ADP-ribosylhydrolase (DraG) removes the modifying group, restoring nitrogenase activity. DraT and DraG activities are reversibly regulated through interaction with PII signaling proteins . Bioinformatics analysis showed that DraT homologs are restricted to a few nitrogen-fixing bacteria while DraG homologs are widespread in Nature. Structural comparisons indicated that bacterial DraG is closely related to Archaea and mammalian ADP-ribosylhydrolases (ARH). In all available structures, the ARH active site consists of a hydrophilic cleft carrying a binuclear Mg(2+) or Mn(2+) cluster, which is critical for catalysis.

To investigate the expression and significance of beta-receptor ( p-R) in infantile hemangioma.The expression of beta-R was detected by immunohistochemistry in infantile hemangioma (40 cases), venous malformation (20 cases) and normal skin (10 cases).The positive expression rate of beta2-R was 70% (28/40) in infantile hemangioma, while no expression of beta2-R in venous malformation and normal skin. Among 28 cases with positive expression of beta2-R, 24 cases were in proliferative phase. The positive expression of beta2-R in infantile hemangioma was significantly higher than that in venous malformation and normal skin tissue. The positive expression of beta2-R in the proliferative hemangioma and non-proliferative hemangioma was also significantly different (P0.05).beta2-R is specifically expressed in infantile hemangioma, especially in proliferative hemangioma.

To reduce the negative intervention for the transplant kidney and explore the optimal operational method for treating urinary tumor in patients after renal transplantation.From March 2006 to March 2013, a total of 12 post renal transplantation patients with renal pelvis tumor/ureter tumor underwent operations. After the general anesthesia, the patient was placed in lithotomy position. The 2 micron microwave resection was performed, with the ureteral orifice as the center. After the ureteral wall section was resected, the resection was forwarded 6 cm. Then the patient was placed in lateral position. The laparoscopic radical nephrectomy was performed, and when this procedure was completed, the laparoscopy was converted to the other side to observe to the bladder side. Meanwhile, the operator pulled the ureter and maintained some tension, using the ultrasound knife to dissect the adjacent tissue and avoiding injuring the transplant kidney. With this method, the resected distal ureter and partial bladder were resected en bloc. In some condition, the uerteral tumor was tightly connected to the adjacent tissue, and the upper ureter failed to be removed completely, a short incision could be performed besides the musculus rectus abdominis. The Foley catheter had remained for 2 weeks after the operation.The 12 operations were successfully performed. The operation duration ranged from 110 to 290 minutes, and the bleeding volume was from 50 to 200 mL. Only one female patient developed secondary bleeding 18 hours after the operation, however, the secondary operation successfully stopped the bleeding. The mean hospital stay was 10.5 days.The combination use of 2 micron microwave resection and laparoscopy in the treatment of urinary tumor in patients after renal transplantation is effective, and has its advantages: lowering the risk of damaging the transplant kidney and easing the operation. Furthermore, this procedure is minimally invasive and safe. The patient could recover soon.

To investigate a method of auricular reconstruction by soft tissue expansion techniques without skin grafting.Two tissue expanders (50 ml and 70 ml) were implanted under the skin of mastoid in 15 patients with grade II or III microtia. One big expander (100 ml) were implanted under the skin of mastoid in 13 patients with grade I microtia and 3 patients with grade II or III microtia. After skin expansion, the expanders were taken out. The autologous rib cartilage or Medpor scaffolds were implanted. The superior expanded skin flap was used to cover the frontal surface and the upper part of the back surface of the framework. The inferior expanded skin flap was transplanted to cover the lower part of the back surface of the framework. The remained expanded skin flap was transplanted to cover the wound in the lateral of head.No skin graft was needed in all the patients. Epidermis blister occurred at the distal part of flap in one case. No other complication was happened. A follow-up of 6 to 12 months (mean 10.9 months) was carried out in all patients with good cosmetic result when the reconstructed ear underwent second-stage operation. The scar size on the dornor site was (5.2 +/- 0.6) cm2 The satisfactory rate was 90% (28/31).The expanded skin with this new method is enough for auricular reconstruction without skin grafting, leaving less complication and less scar at donor site.

Analyze the proliferation of different host H1N1 subtype influenza viruses in A549 and BEAS-2B cells.Human, avain and swine three hosts of the H1N1 influenza viruses infected A549 and BEAS-2B cells and analyze the characteristics of different periods after inocubation. Determine the receptor binding specificity of influenza virus by hemagglutination (HA) test with RBCs with two types of receptor. And the receptors on surfaces of A549 and BEAS-2B cells were tested by flow cytometry.The Cell Pathologic Effect (CPE) is obvious after 24 h inoculation in A549 cells by all the H1N1 influenza viruses, moreover, the peak hemagglutinin (HA) and 50% tissue culture cell infected dose (TCID50) titers was observed after 36 h of culturing in A549 cells. Otherwise, the CPE is not typical from 24 h-120 h inoculated by the same viruses and the HA, TCID50 titers were keep low all the periods in the BEAS-2B cell after inoculation. The receptor-binding preference of H1N1 viruses used in the study was screened by HA assay and some were found with 2-6-receptor binding affinity. Both SA a-2, 3Gal and SA a-2, 6Gal receptors were detected on A549 and BEAS-2B, furthermore, receptor density on A549 cells was significantly higher than that of BEAS-2B cells.A549 cells were susceptible to human, avian and swine H1N1 influenza viruses infection and permissively for viral replication. However, BEASE-2B cells with similar receptor pattern and epithelium-derived propriety as A549 cells were unsusceptible to their infection and replication. Possible host factors involved in effective viral infection and replication were needed further study.

To analyze and summarize the risk factors of malignant tumor after renal transplantation.We retrospectively analyzed 435 renal recipients who underwent renal transplantation in our institute from May 1991 to March 2011, and found 14 patients developed malignant tumor, of which 12 were from our institute, and the other 2 were referred from other institute. We analyzed the risk factors of those tumors for their incidence, classification, time of final diagnosis, immunity suppression therapy and prognosis.The incidence of malignant tumors after renal transplantation in our hospital was 2.8%.Of the 14 cases, 11 were urothelial tumor (6 cases developed both in pelvis and ureter, 2 cases of primary ureter cancer and 3 cases of primary bladder cancer), and the other 3 were thyroid carcinoma, colon carcinoma and lymphoma. We carried out radical corrections in 13 patients,of whom 10 were in good status, 2 underwent the secondary operation in 1 year due to relapse (one patient died in half a year due to tumor size expansion), and 1 died from repeated relapse in 3 years. Chemotherapy was used for the patient with lymphoma.Immunity suppression and surgery are associated with the high incidence of maglinant tumor (78% were urinary tract tumors) after renal transplantation. The risk of tumor is strongly associated with the recipient's survival. The key point of management is early diagnosis and treatment. Further attention should be emphasized to painless gross hematuria, unconfirmed back pain, hydronephrosis, hydroureter and infections.

To evaluate the effect of auricular reconstruction and correction of mild hemifacial microsomia with dermal fat graft in one stage.28 cases with microtia and grade I and II hemifacial microsomia were treated. The tissue expander was implanted under the skin of mastoid at the first stage. At the second stage, the auricular reconstruction was performed with autologous rib cartilage framework. The remained dermal fat tissue from the donor site was inserted subcutaneously to correct the asymmetric face of microsomia. 20 cases, who underwent auricular reconstruction only, were used as control group.The facial asymmetry was greatly improved after operation in the 28 cases. The complications happened in 7% (2/28) of the patients, including one case of infection and one case of framework exposure. While it was 5% (1/20) in control group, showing no significant difference between the two groups (P0.05). The satisfactory rate was 93% (26/28) in one-staged group and 80% (16/20) in control group (P0.05).The dermal fat graft which should be abandoned in the traditional auricular reconstructions could be used to correct mild hemifacial microsomia with satisfactory appearance improvement.

A new flu caused by a novel influenza A(H1N1) virus has spread over the United States, Mexico and more than 40 other countries. And because of the immediate global concern, WHO has announced that the current level of influenza pandemic alert is raised to phase 5, indicating approaching of an influenza pandemic. As patients suffering from the influenza A (H1N1) have the similar symptoms as patients with seasonal influenza, differential detection and identification of the influenza virus have to depend on specific laboratory tests. We have successfully developed a RT-PCR based method for detection of the influenza A (H1N1) virus, and had applied the method to detection of clinical samples.

To investigate the temporomandibular joint (TMJ) function of the condylar hyperplasia patients after condylectomy.Twelve patients with condylar hyperplasia were included in this study. Before and after condylectomy, they were examined and the Fricton' s Craniomandibular Index (CMI) was calculated to assess the functional disorder of TMJ.(1) The facial asymmetry was improved after condylectomy. The morphological symmetry of the bilateral condyle and ramus was displayed by X-ray examinations. There was new cortex formed on the surface of the operated condyle. (2) The TMJ function was improved in terms of Fricton's DI and CMI, which decreased from 0.131 and 0.066 to 0.042 and 0.021 respectively (P0.05).Condylectomy is an effective method to remedy facial asymmetry and, at the same time, improve the TMJ function of the condylar hyperplasia patients.

To investigate the electrosonography character of sounds emanating from anterior disc displacement with reduction of TMJ and the value of it in clinical diagnosis.The sounds from healthy TMJ, anterior disc displacement with or without reduction of TMJ, and osteoarthritis of TMJ were recorded and analyzed by K6-I system, then the data was used for diagnosis of anterior disc displacement with reduction in clinic.A special kind of waveform was found in the electrosonography of sounds from anterior disc displacement with reduction repeatedly, and seldom or not in sounds from healthy joints, anterior disc displacement without reduction or osteoarthritis of TMJ. The diagnostic sensitivity of anterior disc displacement with reduction by using electrosonography analysis was 77.2% and specificity was 93.3% when compared with the clinical diagnosis based on clinical appearance and radiography evidence.The special kind of waveform may be characteristic wave of sounds from anterior disc displacement with reduction of TMJ, which is useful for assistant diagnosis of anterior disc displacement with reduction in clinic.

To measure the activity of oxygen free radicals and the level of antioxidant enzyme superoxide dismutase (SOD) in the synovial fluid (SF) of the temporomandibular joints (TMJs) of patients with temporomandibular disorders (TMD).Thirty-two patients were divided into 3 subgroups: anterior disc displacement with reduction, anterior disc displacement without reduction, and osteoarthrosis. Six healthy volunteers served as controls. A pumping procedure was used to take SF from the superior TMJ space. The concentration of lipid peroxidation products was assessed by means of the thiobarbituric acid reaction, and the level of SOD was assessed with spectrophotometry.SF levels of lipid peroxides (LPO) in the control and patient groups were 0.010 (+/- 0.004) x 10(-3) nmol/mg protein and 0.61 (+/- 0.121) x 10(-3) nmol/mg protein, respectively. SF SOD levels were 4.61 (+/- 1.30) NU/mg protein and 9.83 (+/- 2.66) NU/mg protein, respectively. Both the concentration of LPO and the level of SOD activity were significantly higher in the TMD patients than in the normal control subjects (P.001 and P.01, respectively). There were no significant differences in the level of LPO or SOD between the 3 subgroups.These results demonstrate oxygen free radicals and antioxidant enzymes may be connected in the pathogenesis of TMD.

To evaluate the value of sialoendoscopy in the diagnosis and treatment of chronic obstructive sialadenitis (COS).25 patients (34 sides) who complained of recurrent swelling in the salivary gland region were selected for study from Jan. 2003 to Jun. 2003 at the Department of salivary gland diseases, School of Stomatology, Wuhan University, including 19 parotid cases (27 sides) and 6 submandibular gland cases (7 sides); 16 females and 9 males. And their age range was 17 - 77 years, with an average of 43.72 years. Before sialoendoscopy, 22 parotid cases underwent sialography, and 3 submandibular gland sialoliths cases received an additional standard mandibular occlusal film examination.Conventional radiographs revealed that 3 cases had submandibular gland radiopaque stones, and 22 had COS. Dilatation of main duct of parotid and submandibular gland were also found in the radiographs. Sialoendoscopy showed: proliferative fibrous materials in the duct; stenosis or dilatation of duct; vasodilatation, erythema on the wall of the duct; mucoid plugs and pyogenic plugs; irregular lumen shape; radiopaque and radiolucent stones.Sialoendoscopy may be a safe, effective, and minimal invasive method in the diagnosis and treatment of COS.

To evaluate the advantages, disadvantages and their indications of total nasal reconstruction with different techniques.A series of total nasal reconstruction were treated with four methods from 1975 to 2003. These methods were tubed flap of arm,midline forehead flap with skin graft, midline forehead flap with bilateral frontotemporal flaps for repairing the donor site, and expanded forehead flap.All of the patients were treated successfully. The shape and function of the reconstructed noses were satisfactory. However, the traditional forehead flap with skin graft may leave a unsightly big and black scar on the forehead. The technique of the tubed flap of arm could provide enough tissue without remaining forehead scar and be easily shaped, but it required long period, multiple procedures and body fixation for three weeks.Midline forehead flap with bilateral frontotemporal flaps for repairing the donor site may be good for small nose reconstruction while expanded forehead flap could reconstruct a big nose. Tubed flap of arm may be used to the patients who do not wish to leave any scar on the forehead. Forehead flap with skin graft to repair the donor sit- should generally be avoided for nose reconstruction.

To evaluate the inhibitory effect of endostatin on tumor growth of human laryngeal squamous carcinoma in nude mice and to explore the possible mechanism of the inhibition and the possible way of biological therapy.Nude mice model bearing laryngocarcinoma was established by using human laryngeal squamous carcinoma cell line ( Hep-II). The animals were given endostatin (20 mg x kg(-1) x d(-1)) or PBS, for 21 consecutive days. The volumes of the subcutaneous tumor were observed. The microstructure in which the general 2-step immuohistochemical examination was adopted and ultra-microstructural changes of carcinoma after administration of endostatin were observed under light and electron microscopes for pathology examination.The differences were statistically significant for the net mice weight, tumor weight, and tumor volume and weight/net mice weight between the treatment group and the control group. The restrained percentage of tumor was 45.9%. The necrosis and apoptosis of the tumor cell and the angiogenesis reduction were found under light and electron microscope in the treatment group. The expression of MVD, PCNA and VEGF of the treatment group is lower than that of the control group, and T test showed that P0.01, P0.05, P0.05 respectively, the differences were statistically significant.These studies showed that endostatin could significantly restrain the development of laryngocarcinoma. The mechanism may be due to the effect of antiangiogesis.

To confirm the expression and distribution of Fas and Fas-L in the nasal polyps and to illustrate the role of the Fas/Fas-L system in the pathogenesis of human nasal polyps.Investigating the transcripts of the Fas/Fas-L gene in 30 human nasal polyp tissues and 30 nasal turbinate mucosa specimens using reverse transcription-polymerase chain reaction. Localization of Fas/Fas-L was performed with immunohistochemistry.The transcripts of the Fas/Fas-L gene were detected at similar levels in both polyps and nasal mucosa. There was a significant overexpression of Fas-L protein on nasal polyps (epithelium: 25 +/- 21, glands: 19 +/- 14) compared to nasal mucosa (epithelium: 14 +/- 13, glands: 12 +/- 10), (t = 1.66, P0.01), while Fas was overexpressed on nasal mucosa (epithelium: 17 +/- 11, glands: 17 +/- 13) compared to nasal polyps (epithelium: 13 +/- 10, glands: 11 +/- 9), (t = 1.98, P0.01). Fas-L positive cells were localized on the epithelial layers of cystically dilated glands and the down-growing epithelium of nasal polyps. Fas positive cells were localized on the cilia of the epithelial of nasal mucosa and mainly on the infiltrative cells.Fas/Fas-L may play an important role on the pathogenesis of human nasal polyps, including cystic degeneration of submucosal glands, apoptosis and conferring of immune privilege to nasal polyp formation.

Membrane transporter proteins play critical physiological roles in the cell and constitute 5–10% of prokaryotic and eukaryotic genomes. High-resolution structural information is essential for understanding the functional mechanism of these proteins. A prerequisite for structural study is to overexpress such proteins in large quantities. In the last few years, over 20 bacterial membrane transporters were overexpressed at a level of 1 mg/l of culture or higher, most often in Escherichia coli. In this review, we analyzed those factors that affect the quantity and quality of the protein produced, and summarized recent progress in overexpression of membrane transporters from bacterial inner membrane. Rapid progress in genome sequencing provides opportunities for expressing several homologues and orthologues of the target protein simultaneously, while the availability of various expression vectors allows flexible experimental design. Careful optimization of cell culture conditions can drastically improve the expression level and homogeneity of the target protein. New sample preparation techniques for mass spectrometry of membrane proteins have enabled one to identity the rigid protein core, which can be subsequently overexpressed. Size-exclusion chromatography on HPLC has proven to be an efficient method in screening detergent, pH an other conditions required for maintaining the stability and monodispersity of the protein. Such high-quality preparations of membrane transporter proteins will probably lead to successful crystallization and structure determination of these proteins in the next few years.