Search

• A rapid Elispot-based neutralization assay against Coxsackievirus B1 (CVB1) was successfully developed. • The CVB1 Nt-Elispot is objective, highly sensitive, efficient, and high-throughput. • The CVB1 Nt-Elispot could be applied in the screening of anti-CVB1 neutralizing monoclonal antibodies. • The CVB1 Nt-Elispot could be used to detect NAb levels against CVB1 in large cohorts. Coxsackievirus B1 (CVB1) is a major pathogen that causes viral myocarditis and aseptic meningitis and is implicated as a cause of type 1 diabetes mellitus. The rapid detection of neutralizing antibodies can help in the prevention and diagnosis of viral infection. The traditional cytopathic effect (CPE)-based neutralization assay (Nt-CPE) is time-consuming and labor-intensive. In this study, an efficient neutralization test based on an enzyme-linked immunospot assay and a monoclonal antibody 2E6 against CVB1 (Nt-Elispot) was developed. In this optimal Nt-Elispot, a multiplicity of infection (MOI) of 1 per well was set as the infection dose, and an incubation time of 18 hours was selected as the checkpoint. Compared with Nt-CPE, Nt-Elispot significantly shortened the detection period and displayed a good correlation with it. This established CVB1 Nt-Elispot could be applied to efficiently screen neutralizing antibodies and evaluate the level of NAb against CVB1 in large cohorts. [ABSTRACT FROM AUTHOR]

Production of normal gametes is necessary for flowering plant reproduction, which involves the transition from vegetative to reproductive stage and floral organ development. Such transitions and floral development are modulated by various environmental and endogenous stimuli and controlled by sophisticated regulatory networks. FLOWERING LOCUS T (FT) and LEAFY (LFY) are two key genes that integrate signals from multiple genetic pathways in Arabidopsis. However, the comprehensive functions and relationship between these two genes in trees are poorly understood. In this study, we found that JcFT played a vital role in regulating the flowering transition in the perennial woody species Jatropha curcas. JcLFY also involved in regulating this transition and controlled floral organ development. The non–flowering phenotype of JcFT-RNAi was rescued successfully by overexpression of JcLFY , while the abnormal flowers produced by JcLFY silencing were not recovered by JcFT overexpression via hybridization. These results indicate that JcFT , in which a mutation leads to a nonflowering phenotype, is the central gene of the floral meristem transition and that JcLFY , in which a mutation leads to striking changes in flowering and often sterility, is the central floral and inflorescence development gene. Moreover, our hybridization results suggest that JcLFY acts downstream of JcFT in Jatropha. JcLFY -silenced plants showed abnormal flowers, this picture exhibited the extremely overgrowing of floral organ in JcLFY -silenced plants. [Display omitted] • The long generation time of woody species is the key limiting factor for genetic improvement and breeding. • The non-flowering phenotype of JcFT-RNAi could be rescued by JcLFY -overexpression, while the abnormal flowers produced by JcLFY silencing could not rescued by JcFT -overexpression. • The hybridization results suggest that JcLFY acts downstream of JcFT in Jatropha. These two genes may help to guide molecular breeding of perennial plants. [ABSTRACT FROM AUTHOR]

Echovirus 25 (E-25) is a member of the enterovirus family and a common pathogen that induces hand, foot, and mouth disease (HFMD), meningitis, skin rash, and respiratory illnesses. In this study, we constructed and characterized an infectious full-length E-25 cDNA clone derived from the XM0297 strain, which was the first subgenotype D6 strain isolated in Xiamen, China. The 5′-Untranslated Regions (5′-UTR), P3 (3A-3B, 3D) and P3 (3C) regions of this E-25 (XM0297) strain were highly similar to EV-B77, E-16 and E-13, respectively. Our data demonstrate that the rescued E-25 viruses exhibited similar growth kinetics to the prototype virus strain XM0297. We observed the rescued viral particles using transmission electron microscope (TEM) and found them to possess an icosahedral structure, with a diameter of approximately 30 nm. The cross neutralization test demonstrated that the E-25 (XM0297) strain immune serum could not neutralize EV-A71, CV-A16 or CV-B3; likewise, the EV-A71 and CV-A16 immune serum could not neutralize E-25 (XM0297). The availability of this infectious clone will greatly enhance future virological investigations and possible vaccine development against E-25. [ABSTRACT FROM AUTHOR]

Varicella-zoster virus (VZV) is a highly infectious agent that causes varicella and herpes zoster (HZ), which may be associated with severe neuralgia. Vaccination is the most effective way to reduce the burden of the diseases. VZV glycoprotein E (gE) is the major and most immunogenic membrane protein that plays important roles in vaccine efficacy. A quantitative assay for gE content is desirable for the VZV vaccine process monitoring and product analysis. In this study, 70 monoclonal antibodies (mAbs) were obtained after immunizing mice with purified recombinant gE (rgE). The collection of mAbs was well-characterized, and a pair of high-affinity neutralization antibodies (capture mAb 4A2 and detection mAb 4H10) was selected to establish a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the native and recombinant gE. The detection limit of this assay was found to be 1.95 ng/mL. Furthermore, a reasonably good correlation between the gE content (as measured by the mAb-based quantitative ELISA) and the virus titer (as measured by the 'gold standard' plaque assay) was observed when both assays were performed for tracking the kinetics of virus growth during cell culture. A total of 16 batches of lyophilized VZV vaccine were tested using the newly developed quantitative ELISA and classical plaque assay, demonstrating reasonably good correlation between gE content and virus titer. Therefore, this mAb-based gE quantitative assay serves as a rapid, stable, and sensitive method for monitoring viral antigen content, one additional quantitative method for VZV vaccine process and product characterization. This quantitative ELISA may also serve as a complementary method for virus titering. [ABSTRACT FROM AUTHOR]

Mechanisms governing a neuron's regenerative ability are important but not well understood. We identify Rtca (RNA 3′-terminal phosphate cyclase) as an inhibitor of axon regeneration. Removal of Rtca cell-autonomously enhanced axon regrowth in the Drosophila CNS, whereas its overexpression reduced axon regeneration in the periphery. Rtca along with the RNA ligase Rtcb and its catalyst Archease operate in the RNA repair and splicing pathway important for stress-induced mRNA splicing, including that of Xbp1, a cellular stress sensor. Drosophila Rtca and Archease had opposing effects on Xbp1 splicing, and deficiency of Archease or Xbp1 impeded axon regeneration in Drosophila. Moreover, overexpressing mammalian Rtca in cultured rodent neurons reduced axonal complexity in vitro, whereas reducing its function promoted retinal ganglion cell axon regeneration after optic nerve crush in mice. Our study thus links axon regeneration to cellular stress and RNA metabolism, revealing new potential therapeutic targets for treating nervous system trauma. [ABSTRACT FROM AUTHOR]

Coxsackievirus A16 (CA16) is one of the major pathogens responsible for hand, foot and mouth disease (HFMD). The assessment of the humoral immunity response is indispensable in the development of vaccines against enteroviruses. The neutralization test based on the inhibition of cytopathic effects (Nt-CPE) is a common method for measuring neutralizing antibodies against CA16. However, an efficient neutralization test needs to be developed for seroepidemiological surveys and clinical trials of CA16 vaccines because Nt-CPE is time-consuming and labor-intensive. In this study, a high-throughput neutralization test for CA16 based on the enzyme-linked immunospot assay (Nt-ELISPOT) was developed. The monoclonal antibody 7D10, which reacted with the viral protein VP1, was used to detect the cells infected with CA16. The neutralizing titers of sera were proven to be unchanged over an infectious dose range from 10 to 10,000 TCID 50 per well. The Nt-ELISPOT results correlated well with the Nt-CPE results ( R 2 = 0.9250), and the detection period was shortened from five days to approximately 30 h. Overall, the Nt-ELISPOT is a reliable and efficient method for measuring neutralizing antibodies against CA16. [ABSTRACT FROM AUTHOR]

Enterovirus 71 (EV71) is a neurotropic virus capable of inducing severe neurological symptoms and death. No direct targeting antivirals are useful in the treatment of severe EV71 infection. Because of low toxicity and good specificity, monoclonal antibodies (MAb) are a potential candidate for the treatment of viral infections. Therefore, we developed an EV71-specific conformational MAb with high in vitro cross-neutralization activity to heterologous EV71 subgenotypes. The in vivo treatment experiment at different days post-infection indicated that a single treatment of MAb CT11F9 within day 3 post-infection fully protected mice from morbidity and mortality (0% PBS vs. 100% at 10 µg/g per body weight ***P<0.0001). Immunohistochemical and histological analysis confirmed that CT11F9 significantly prohibited EV71 VP1 expression in various tissues and prevented EV71-induced myonecrosis. Moreover, thrice-treatment at day 4, 5, 6 post-infection was associated with an increased survival rate (18.2% single vs. 50% thrice at 20 µg/g per body weight), and the mice recovered from limb paralysis. Competitive ELISA also confirmed that CT11F9-recognized epitopes were immunodominant in humans. In conclusion, MAb CT11F9 is an ideal candidate to be humanized and used in severe EV71 infection. [ABSTRACT FROM AUTHOR]

Hand, foot and mouth disease, associated with enterovirus 71 (EV71) infections, has recently become an important public health issue throughout the world. Serum neutralizing antibodies are major indicators of EV71 infection and protective immunity. However, the potential for cross-reactivity of neutralizing antibodies for different EV71 genotypes and subgenotypes is unclear. Here we measured the cross-reactive neutralizing antibody titers against EV71 of different genotypes or subgenotypes in sera collected from EV71-infected children and vaccine-inoculated children in a phase III clinical trial (ClinicalTrials.gov Identifier: NCT01636245) using a new pseudovirus-based neutralization assay. Antibodies induced by EV71-C4a were cross-reactive for different EV71 genotypes, demonstrating that C4a is a good candidate strain for an EV71 vaccine. Our study also demonstrated that this new assay is practical for analyses of clinical samples from epidemiological and vaccine studies. [ABSTRACT FROM AUTHOR]

Highlights: [•] Similar to the ELISPOT method, we developed a VZV neutralization enzyme-linked immunosorbent spot (N-ELISPOT) system. [•] We applied it to quantify virus, optimizing the test antibody dilution. [•] Compared with the plaque assay, the VZV N-ELISPOT was accurate and reliable for the detection of neutralizing serum samples. [ABSTRACT FROM AUTHOR]

Abstract: In recent year, Enterovirus 71 (EV71)-associated hand, foot and mouth disease (HFMD) has become an important public health issue in China. EV71 has been classified into genotypes A, B1–B5 and C1–C5. With such genetic diversity, whether the convalescent or recovery antibody responses can cross-protect infections from other genotypes remains a question. Understanding of the antigenicity of such diverse genetic EV71 isolates is crucial for the EV71 vaccine development. Here, a total of 186 clones anti-EV71 MAbs was generated and characterized with Western blot and cell-based neutralization assay. Forty neutralizing anti-EV71 MAbs were further used to analyze the antigenic properties of 18 recent EV71 isolates representing seven genotypes in neutralization assay. We found that most neutralizing anti-EV71 MAbs are specific to conformational epitopes. We also classified the 40 neutralizing anti-EV71 MAbs into two classes according to their reactivity patterns with 18 EV71 isolates. Class I MAb can neutralize all isolates, suggesting conserved epitopes are present among EV71. Class II MAb includes four subclasses (IIa–IId) and neutralizes only subgroups of EV71 strains. Conversely, 18 EV71 strains were grouped into antigenic types 1 and four antigenic subtypes (2.1–2.4). These results suggest that the current genotyping of EV71 does not reflect their antigenicity which may be important in the selection of EV71 vaccine strains. This panel of neutralizing anti-EV71 MAbs may be useful for the recognition of emerging antigenic variants of EV71 and vaccine development. [Copyright &y& Elsevier]

Summary: Collapse of membrane lipid asymmetry is a hallmark of blood coagulation. TMEM16F of the TMEM16 family that includes TMEM16A/B Ca2+-activated Cl− channels (CaCCs) is linked to Scott syndrome with deficient Ca2+-dependent lipid scrambling. We generated TMEM16F knockout mice that exhibit bleeding defects and protection in an arterial thrombosis model associated with platelet deficiency in Ca2+-dependent phosphatidylserine exposure and procoagulant activity and lack a Ca2+-activated cation current in the platelet precursor megakaryocytes. Heterologous expression of TMEM16F generates a small-conductance Ca2+-activated nonselective cation (SCAN) current with subpicosiemens single-channel conductance rather than a CaCC. TMEM16F-SCAN channels permeate both monovalent and divalent cations, including Ca2+, and exhibit synergistic gating by Ca2+ and voltage. We further pinpointed a residue in the putative pore region important for the cation versus anion selectivity of TMEM16F-SCAN and TMEM16A-CaCC channels. This study thus identifies a Ca2+-activated channel permeable to Ca2+ and critical for Ca2+-dependent scramblase activity during blood coagulation. PaperFlick: Display Omitted [ABSTRACT FROM AUTHOR]

Abstract: Diagnosis of Coxsackievirus A16 (CA16) infection in China relies mainly on reverse transcription-polymerase chain reaction (RT-PCR) that require expensive equipment and special trained personnel, thus making its wide application in health care settings unlikely. In this study, a novel IgM anti-CA16 assay was developed for the detection of IgM antibodies to CA16 in serum. The responses and diagnostic value of IgM for the CA16 infection were assessed by testing 1970 serum samples. The results showed that sensitivity of IgM test was 84.6% (259/306, 95% CI: 80.1–88.5), and specificity in control subjects and patients with CA16 HFMD was 99.2% (1508/1520, 95% CI: 98.6–99.6) and 90.3% (14/144, 95% CI: 84.2–94.6), respectively. The IgM positive rate reached 56.3% in the sera collected within the first day after onset, increased continuously to 95.3% at day 5 to day 7 after onset, and then reached 100% after more than 8 days. The cross-reaction rate in patients infected with other non-CA16 enteroviruses was 9.7% (14/144). These results suggest that the IgM anti-CA16 assay offers a rapid, convenient, and reliable method to detect acute CA16 infections. [ABSTRACT FROM AUTHOR]

A robust adaptive tracking control approach is presented for a class of strict-feedback single-input-single-output nonlinear systems. By employing radial-basis-function neural networks to account for system uncertainties, the proposed scheme is developed by combining "dynamic surface control" and "minimal learning parameter" techniques. The key features of the algorithm are that, first, the problem of "explosion of complexity" inherent in the conventional backstepping method is avoided, second, the number of parameters updated online for each subsystem is reduced to 2, and, third, the possible controller singularity problem in the approximation-based adaptive control schemes with feedback linearization technique is removed. These features result in a much simpler adaptive control algorithm, which is convenient to implement in applications. In addition, it is shown via input-to-state stability theory and small gain approach that all signals in the closed-loop system are semiglobal uniformly ultimately bounded. Finally, three simulation examples are used to demonstrate the effectiveness of the proposed scheme. [ABSTRACT FROM AUTHOR]

Genetic variation in the human angiotensinogen gene (AGT) influences plasma AGT concentration and susceptibility to essential hypertension by a mechanism that remains to be clarified. When one or two additional copies of the gene were inserted by gene titration (by homologous recombination with gap-repair at the AGT locus), both plasma AGT and arterial pressure were elevated in the physiological range in the mouse. The causal dependency between plasma AGT and blood pressure and the relative contribution of the various tissues that express AGT to these two phenotypic parameters remained to be determined. To address these issues, we generated a transgenic mouse with overexpression of the mouse AGT gene restricted to the liver. The transgene was examined in two contrasted genetic backgrounds, the sodium-sensitive C57BL/6J and the sodium-resistant A/J. Transgenic and control male animals underwent continuous cardiovascular monitoring by telemetry for 14 days while under a standard sodium diet (0.2%). Moderate but significant increases in plasma AGT (40%, p = 0.01) and systolic blood pressure (4–6 mmHg, p ranging from 0.01 to <0.001) were observed in the sodium-sensitive background, but not in the sodium-resistant animals. Statistical analysis of a large number of consecutive, repeated measurements of blood pressure afforded power to detect small effects in the physiological range by use of advanced mixed models of analysis of variances and covariances. Although plasma renin activity was increased in the sodium-sensitive background, it did not reach statistical significance. These observations underline a potential contribution of systemic AGT to the mechanism of AGT-mediated hypertension, but the significance of sodium sensitivity in the genetic background suggests participation of the kidney in expression of the elevated blood pressure phenotype, a matter that will warrant further studies. They also highlight the challenge of identifying the contribution of individual genes in complex inheritance, as their effects are modulated by other genetic and environmental determinants. [ABSTRACT FROM AUTHOR]

Abstract: The candidate particulate hepatitis E vaccine, HEV 239, has been shown to be an efficacious vaccine in primates, and clinical study to date shows it to be safe and immunogenic for humans. The antigenicity of HEV 239 is virtually identical to its N-terminal 26 amino acids truncated protein, E2, which is not particulate but soluble. However, HEV 239 is over 200 times more immunogenic than E2. In present study, several events underlying this dramatic immunogenicity difference have been addressed. (1) HEV 239 can efficiently evoke a vigorous and predominant T cell response while E2 cannot induce detectable T cell response; (2) the dominant T cell epitopes in HEV 239 are identified, and both are also contained integrally in E2; (3) priming mice with Th epitope peptide can partially rescue the weak immunogenicity of E2 in alum adjuvant and (4) HEV 239 but not E2 can induce significant antibody response in athymic mice, which indicates that HEV 239 can directly activate B cell more efficiently. These results contribute to a better understanding of the mechanisms involved in the significant high immunogenicity of particulate antigen and may provide knowledge for the rational design and development of future vaccines. [Copyright &y& Elsevier]

Abstract: Protein C, a vitamin K-dependent serine protease zymogen that circulates in plasma, is converted by limited proteolysis to activated protein C (APC) by the thrombin–thrombomodulin complex. APC exerts anticoagulant, antiinflammatory, cytoprotective, and antiapoptotic activities. Recombinant APC therapy reduces mortality in severe sepsis patients. This review summarizes data from clinical observations, from in vitro studies, and from animal models of focal ischemic injury that provide a compelling rationale for clinical trials of APC for ischemic stroke. [Copyright &y& Elsevier]

Activated protein C (APC), a serine protease with anticoagulant and anti-inflammatory activities, exerts direct cytoprotective effects on endothelium via endothelial protein C receptor-dependent activation of protease activated receptor 1 (PAR1). Here, we report that APC protects mouse cortical neurons from two divergent inducers of apoptosis, N-methyl-D-aspartate (NMDA) and staurosporine. APC blocked several steps in NMDA-induced apoptosis downstream to nitric oxide, i.e., caspase-3 activation, nuclear translocation of apoptosis-inducing factor (AIF), and induction of p53, and prevented staurosporine-induced apoptosis by blocking caspase-8 activation upstream of caspase-3 activation and AIF nuclear translocation. Intracerebral APC infusion dose dependently reduced NMDA excitotoxicity in mice. By using different anti-PARs antibodies and mice with single PAR1, PAR3, or PAR4 deletion, we demonstrated that direct neuronal protective effects of APC in vitro and in vivo require PAR1 and PAR3. Thus, PAR1 and PAR3 mediate anti-apoptotic signaling by APC in neurons, which may suggest novel treatments for neurodegenerative disorders. [Copyright &y& Elsevier]

Two previously undescribed seco-type C 19 -diterpenoid alkaloids, stylosines A and B (1 – 2), which possess an unprecedented opened D ring by the C (15) –C (16) broken bond forming a five-membered C (15) −O–C (14) lactone ring, together with 14 known diterpenoid alkaloids were isolated from the roots of Aconitum stylosum (Ranunculaceae). Compound 1 is the first reported C 19 -diterpenoid alkaloid with an α -orientation at H-13. Their structures were determined by extensive spectroscopic analyses, NMR calculations and DP4+ analysis. A plausible biosynthetic pathway of the undescribed compounds was proposed. Compounds 1 and 2 showed significant inhibitory activity against the production of inflammatory cytokines (interleukin 1 β, cyclooxygenase-2, and tumor necrosis factor α) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Compounds 1 and 2 exhibited antibacterial activity against Staphylococcus aureus with MIC values of 2.00 and 32.00 μ g/mL, respectively. Image 1 • Stylosines A and B (1 – 2), which possess an unprecedented opened D ring by the C (15) –C (16) broken bond forming a five-membered C (15) −O–C (14) lactone ring. • Compound 1 is the first reported C 19 -diterpenoid alkaloid with α -orientation at H-13. • Compounds 1 and 2 showed significant anti-inflammatory effects. • Compounds 1 and 2 exhibited antibacterial activity against Staphylococcus aureus. [ABSTRACT FROM AUTHOR]

Noroviruses are the primary cause of gastroenteritis and foodborne diseases, affecting millions of individuals annually worldwide. Oysters are frequently associated with norovirus outbreaks. Hence, inexpensive and simple norovirus detection methods are important to detect and contain foodborne illness outbreaks. The objective of this study is to develop a high-efficient concentrated pretreatment method for reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) detection. Based on the existing ISO/TS 15216-2:2013 standard (ISO/TS 15216-2., 2013), four methods are compared for recovery of norovirus from spiked digestive tissue of oysters. A method is found to be the most efficient based on protease K method of increasing buffer volume and PEG precipitation method. The recovery rate and amplification efficiency approached 11.07 ± 0.09% and 124.12 ± 5.99%, respectively, being 7-fold that of the original ISO/TS 15216-2:2013 method. This method serves as a rapid (1.5h) sample concentration tool with a limit of detection as low as 7.05 × 103 copies. Thirty-eight oyster samples from an aquatic products market in Guangzhou are tested using this method, and noroviruses are detected in 13 samples. This method is an effective extension of the existing ISO/TS 15216-2:2013 standard and it is potentially applicable for detecting norovirus contamination in oysters. • PEG precipitation with proteinase K treatment was performed to detect NoV in independent oysters. • This method had a higher recovery rate than the standard ISO/TS 15216-2:2013 method. • This method had a better amplification efficiency upon RT-qPCR-based monitoring. [ABSTRACT FROM AUTHOR]

• A continuous DFB-QCL is first used in TDLAS for measuring emitted NO of cigarette. • High dynamic response of system is achieved due to a low volume absorption cell. • An adaptive Kalman filter is employed to reduce the concentration measurement noise. • Emitted NO masses of cigarette smoke are determined at deferent burning behaviors. Nitric oxide (NO) is an important product released by cigarette burning with significantly negative effects to both environment and human health, but it is hard to be measured in-situ in the process of burning. In this paper, we report a tunable diode laser absorption spectroscopy (TDLAS) set-up using a quantum cascade laser emitting at 5.24 μ m to continuously determine the emitted NO mass in cigarette smoke during burning. Wavelength modulation spectroscopy was applied to improve the detection sensitivity and robustness of the measurement system. A simple single-pass gas cell with a volume of 28 cm3 was used to record the absorption spectra, and then the mass of NO was derived based on the measured line strength. Allan variance analysis demonstrated that the limit of detection of system reached 28 ppb over 180 s integration time. Due to the low effective volume, the rise time and the fall time were rapid with 3 s and 6 s, respectively. An adaptive Kalman filter was used to reduce the noise on the concentration measurement. As a result, the measurement precision is improved by a factor of 4.5. For accurate determination the total NO mass, the emissions of mainstream (MS) and sidestream (SS) of a burning cigarette were individually measured at the same continuous flow rate. The recorded results of three types of cigarettes revealed that the NO mass emitted in the MS is correlated with the sampling flow rate, while the NO mass emitted by the same cigarette in the SS is hardly influenced by at different smoke flow rates. And different cigarette types with identical physical dimensions emit approximately equivalent mass of NO. This work provides a promising method for determining the mass of emitted NO and other compositions of cigarettes smoke. [ABSTRACT FROM AUTHOR]

• The H 2 S sensor is developed for detecting domestic natural gas based on TDLAS. • A home-made embedded electronics system with an MPC fabricates a compact sensor. • The WMS-2 f /DS-sine technique is employed to reduce external interferences. • The sensor was deployed for real-time and field measurements of H 2 S of natural gas. Hydrogen sulfide (H 2 S) is a highly toxic gaseous component of natural gas that poses a significant hazard during the use of natural gas for domestic purposes; therefore, a high-sensitivity, on-line detection method is extremely important to ensure its safety for domestic use. In this work, a portable sensor was developed based on near-infrared tunable diode laser absorption spectroscopy (TDLAS). A special Herriott multipass cell (MPC) combined with a home-made embedded electronics system allowed for the fabrication of a compact sensor with a size of 50 × 20 × 10 cm3. A rugged movable cell was used for detecting contaminated gas samples. An embedded electronic system with a diameter of 8 cm was implemented to control the laser, acquire and process electronic signal. To reduce external interference, the calibration-free TDLAS (wavelength modulation frequency-2 f /direct sine signal) was employed to suppress common field measurement noise. Based on the results of Allan deviation analysis, the limit of system detection could reach 0.14 ppm using the H 2 S absorption line at 6336.61 cm−1. It is suitable for on-line measurement because of its rapid response time. Its feasibility is validated for monitoring H 2 S in a domestic natural gas of pipeline. This work demonstrates a system for real-time and field measurement of H 2 S, which can be used to address the security risks associated with using natural gas. [ABSTRACT FROM AUTHOR]

The abuse of antibiotics has led to the emergence of multidrug-resistant bacteria, which is becoming a serious worldwide problem people have to face. In our previous study, temporin-GHa (GHa) cloned from Hylarana guentheri showed antimicrobial activity against Gram-positive bacteria. In order to improve its therapeutic potential, we used a template-based and a database-assisted design to obtain three derived peptides by replacing the histidine at both ends of GHa with lysine, which exhibited faster and stronger bactericidal activity and a broader spectrum than the parent peptide. GHaK and GHa4K targeted to the bacterial membrane to exert their antibacterial activities at a faster membrane damage rate. The derived peptides inhibited the initial adhesion and the formation of Staphylococcus aureus biofilms, and eradicated the mature biofilms, which indicated that the derived peptides effectively penetrated the biofilm and killed bacteria. The therapeutic index (TI) and cell selectivity index (CSI) of the derived peptides increased significantly, which means a broader therapeutic window of the derived peptides. The derived peptides with improved activity and cell selectivity have the potential to be the promising candidates for the treatment of S. aureus infections. Our research also provides new insights into the design and development of antimicrobial peptides. [ABSTRACT FROM AUTHOR]

Background: Mutations of human androgen receptor (AR) gene are responsible for androgen insensitivity syndrome (AIS). Variable phenotypes and androgen receptor binding activity have permitted the classification of AIS into complete (CAIS), partial (PAIS), and minimal or mild (MAIS) forms. Somatic mosaicism in AIS is a rare condition which happened when de novo mutations occur after the zygotic stage. Methods: Clinical evaluation, hormone measurements, and molecular analysis were performed to diagnose the patient in the study. Results: A 46, XY girl who conceived through in vitro fertilization (IVF), presented with partial virilization of external genitalia, was found to have the p.C620R in heterozygosity. The variant p.C620R of AR has been previously reported in a patient with completely feminized external genitalia, which was inherited from the heterozygote carrier mother. Mutation analysis of the mother of our patient revealed that the variant was de novo and presented as a somatic mosaicism which indicated an insufficient amount of wild‐type AR in our patient. Conclusion: This is the first case that AIS was caused by de novo mutation of AR in a 46, XY Disorder of Sexual Development (DSD) patient by the assisted reproduction technique (ART). The phenotype of partial virilization could be explained by AR mutation in somatic mosaicism. [ABSTRACT FROM AUTHOR]

Background: Endometrial cancer (EC) is one of the most common gynecological malignancies globally. Although progress has been made in surgical and other adjuvant therapies, there is still a great need to develop new approaches to further reduce the incidence and mortality of EC. Oncolytic virotherapy offers a novel promising option of cancer treatment and has demonstrated good efficacy in preclinical models and clinical trials. However, only few oncolytic viruses have been tested for EC treatment. In this study, the potential of an oncolytic coxsackievirus B3 (CV-B3) strain 2035A (CV-B3/2035A) was investigated as a novel biotherapeutic agent against EC. Methods: Human EC cell lines (Ishikawa, HEC-1-A and HEC-1-B) were infected with CV-B3/2035A, and viral replication and cytotoxic effects were evaluated in vitro. CV-B3/2035A-induced oncolysis was also investigated in nude mice bearing EC xenografts in vivo and in patient-derived EC samples ex vivo. Results: Human EC cell lines expressing different levels of CAR and DAF were all susceptible to infection by CV-B3/2035A and supported efficient viral replication in vitro. In the EC xenograft/nude mouse model, both intratumoral and intravenous administrations of CV-B3-2035A exerted significant therapeutic effects against pre-established EC tumors without causing significant treatment-related toxicity and mortality in nude mice. Moreover, CV-B3/2035A treatment resulted in decreased viability of patient-derived EC samples ex vivo. Conclusions: CV-B3/2035A showed oncolytic activity in human EC cell lines both in vitro and in vivo as well as in patient-derived EC samples ex vivo and thus could be used as an alternative virotherapy agent for the treatment of EC. [ABSTRACT FROM AUTHOR]

Selective splicing is a feature of luteinizing hormone receptor (LHCGR). A cryptic exon (LHCGR-exon 6A) was found to be derived from alternative splicing in intron 6 of the LHCGR gene, which including two transcripts LHCGR-exon 6A-long and LHCGR-exon 6A-short. We addressed the functional consequences of SNP rs68073206, located at the +5 position of an alternative 5′ splice donor site, and observed its association with male infertility in the subjects with azoospermia, oligoasthenozoospermia and normozoospermia. The translation product of splicing variant LHCGR-exon 6A was expressed in the cytoplasm and exhibited no affinity with [125I]-hCG. No dominant negative effect was observed in cells co-expressed with LHCGR-exon 6A and wild-type LHCGR. The long transcript (LHCGR-exon 6A-long) was significantly elevated in the granulosa cells with G/G genotypes, which could be reproduced in vitro by mini-gene construct transfection. Genotyping analysis showed no association between rs68073206 and male infertility. However, this polymorphism was significantly associated with testosterone levels in normozoospermic subjects (n = 210). In conclusion, SNP rs68073206 in the splicing site of the cryptic exon 6A of the LHCGR gene affect the splicing pattern in the gene, which may play a role in the modulation of the LHCGR sensitivity in the gonads. [ABSTRACT FROM AUTHOR]

Hepatitis D virus (HDV) is a defective RNA virus that requires the presence of hepatitis B virus (HBV) for its life cycle. The in vitro HDV infection system is widely used as a surrogate model to study cellular infection with both viruses owing to its practical feasibility. However, previous methods for running this system were less efficient for high-throughput screening and large-scale studies. Here, we developed a novel method for the production of infectious HDV by adenoviral vector (AdV)-mediated transduction. We demonstrated that the AdV-based method yields 10-fold higher viral titers than the transient-transfection approach. The HDV-containing supernatant derived from AdV-infected Huh7 cells can be used as the inoculum in infectivity assays without requiring further concentration prior to use. Furthermore, we devloped a chemiluminescent immunoassay (HDV-CLEIA) to quantitatively determine intracellular HDAg with a dynamic range of 5-11,000 pg/mL. HDV-CLEIA can be used as an alternative approach to assess HDV infection. The advantages of our updated methodology were demonstrated through in vitro HDV infection of HepaRG cells and by evaluating the neutralization activity using antibodies that target various regions of the HBV/HDV envelope proteins. Together, the methods presented here comprise a novel toolbox of in vitro assays for studying HDV infection. [ABSTRACT FROM AUTHOR]

Human enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the major etiological agents of hand, foot and mouth disease (HFMD) and are often associated with neurological complications. Currently, several vaccine types are being developed for EV71 and CA16. In this study, we constructed a bivalent chimeric virus-like particle (VLP) presenting the VP1 (aa208-222) and VP2 (aa141-155) epitopes of EV71 using hepatitis B virus core protein (HBc) as a carrier, designated HBc-E1/2. Immunization with the chimeric VLPs HBc-E1/2 induced higher IgG titers and neutralization titers against EV71 and CA16 in vitro than immunization with only one epitope incorporated into HBc. Importantly, passive immunization with the recombinant HBc-E2 particles protected neonatal mice against lethal EV71 and CA16 infections. We demonstrate that anti-VP2 (aa141-155) sera bound authentic CA16 viral particles, whereas anti-VP1 (aa208-222) sera could not. Moreover, the anti-VP2 (aa141-155) antibodies inhibited the binding of human serum to virions, which demonstrated that the VP2 epitope is immunodominant between EV71 and CA16. These results illustrated that the chimeric VLP HBc-E1/2 is a promising candidate for a broad-spectrum HFMD vaccine, and also reveals mechanisms of protection by the neighboring linear epitopes of the VP1 GH and VP2 EF loops. [ABSTRACT FROM AUTHOR]

Objective: To summarize clinical observations, animal model experimentation, and in vitro studies that advance knowledge of the protein C system, including activated protein C (APC), in the setting of ischemic stroke.Data Source: Narrative review of selected published primary basic and clinical literature from MEDLINE for 2000-2003.Data Synthesis: Low levels of plasma APC and a poor response to APC in clotting assays may be markers or risk factors for ischemic stroke. Ischemia during routine endarterectomy causes APC generation in the affected region of the human brain. The prospective epidemiologic Atherosclerosis Risk in Communities (ARIC) study reported that plasma protein C may be protective for ischemic stroke. In murine models of focal cerebral ischemia, APC provided remarkable anti-inflammatory and neuroprotective effects in vivo and increased survival at 24 hrs. Recent in vitro and in vivo studies provide remarkable insights into mechanisms of the neuroprotective activities of APC. Independent of its well-known anticoagulant activity, APC acts directly on cells and alters gene expression profiles, inhibits apoptosis, and down-regulates inflammation. These effects require protease-activated receptor-1 and the endothelial protein C receptor. In an in vitro model involving hypoxia-induced apoptosis of human brain endothelial cells, protease-activated receptor-1 and endothelial protein C receptor were required for APC to exert its anti-apoptotic effects. In these cells, APC blunts hypoxia-induced increases in p53 messenger RNA and protein, reduces pro-apoptotic Bax, and increases anti-apoptotic Bcl-2, thereby inhibiting mitochondrial-dependent apoptosis. Murine ischemic stroke model studies have provided in vivo evidence for the physiologic roles of protease-activated receptor-1 and endothelial protein C receptor in the neuroprotective activities of APC. Because the low doses required for recombinant murine APC to provide neuroprotection do not cause observable anticoagulant effects, the in vivo neuroprotective action of APC seems, at least in part, to be independent of its anticoagulant activity and is likely to involve its anti-apoptotic activity.Conclusions: There is compelling evidence that ischemic stroke is an attractive target for therapy with APC. [ABSTRACT FROM AUTHOR]