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Promiscuity, or selectivity on a spectrum, is an encoded feature in biomolecular anion recognition. To unravel the molecular drivers of promiscuous anion recognition, we have employed a comprehensive approach – spanning experiment and theory – with the Staphylococcus carnosus nitrate regulatory element A (ScNreA) as a model. Thermodynamic analysis reveals that ScNreA complexation with native nitrate and nitrite or non-native iodide is an exothermic process. Further deconvolution of the association and dissociation kinetics for each anion reveals that the release event can be limiting, in turn, giving rise to the observed selectivity: nitrate > iodide > nitrite. These conclusions are supplemented with molecular dynamics simulations that capture an entry and exit pathway coupled to subtle global protein motions unique to each anion. Taken together, our data point to how structural plasticity of the binding pocket controls the relative promiscuity of ScNreA to guarantee physiological nitrate sensing. Proteins can exhibit a significant degree of anion selectivity, but the factors underlying promiscuous anion coordination remain underexplored. Here, the authors study the Staphylococcus carnosus nitrate regulatory element A (ScNreA) as a model system and show that ScNreA complexation to nitrate, nitrite or iodide is an exothermic process controlled by the structural plasticity of the binding pocket.

Success in today's global and entrepreneurial economy increasing through measurement and evaluation. It highlighting as a target to all the world. It comes by term implications of an inadequate education have social and economic consequences for individuals, the communities in which they live, and the nation as a whole. As a result, the country is beginning to embrace a new goal for the public education system: graduate every child ready for college and careers in the twenty- 1st- century global economy. (Meaningful Measurement, June 2009) The assessment landscape is broad and complex. educators at the school, district, and state levels are using innovative tools such as performance assessments that engage students in their learning and give educators valuable information that can be immediately used to improve instruction. (Meaningful Measurement, June 2009) Measurement helps you decide how to interpret the data from that variable. When you know that a measure is nominal (like the one just described), then you know that the numerical values are just short codes for the longer names. Second, knowing the level of measurement helps you decide what statistical analysis is appropriate on the values that were assigned. If a measure is nominal, then you know that you would never average the data values or do a t-test on the data. \"The process of obtaining numerical description of the degree to which an individual possesses a particular characteristic. (Measurement and Evaluation, March 2015) \"The systematic process of collecting, (Classroom) analyzing and interpreting information to determine the extent to which pupils are achieving instructional objectives. (Measurement and Evaluation, March 2015) \"Evaluation involves judging the value or worth of a pupil of an instructional method or of an educational program. Such judgements may or may not be based on information obtained from tests\". (Measurement and Evaluation, March 2015) The use of assessment procedure implies that that some useful purpose is being served and that the user is clearly aware of his purpose. The blindly gather data about students and then file the information away is a waste of time and effort. Assessment is best viewed as a process of obtaining information on which to base educational decisions. (Measurement and Evaluation, March 2015).

Background Hepatitis C can lead to liver fibrosis and cirrhosis. We compared readily available non-invasive fibrosis indexes for the fibrosis progression discrimination to find a better combination of existing non-invasive markers. Methods We studied 157 HCV infected patients who underwent liver biopsy. In order to differentiate HCV fibrosis progression, readily available AAR, APRI, FI and FIB-4 serum indexes were tested in the patients. We derived a new fibrosis-cirrhosis index (FCI) comprised of ALP, bilirubin, serum albumin and platelet count. FCI = [(ALP × Bilirubin) / (Albumin × Platelet count)]. Results Already established serum indexes AAR, APRI, FI and FIB-4 were able to stage liver fibrosis with correlation coefficient indexes 0.130, 0.444, 0.578 and 0.494, respectively. Our new fibrosis cirrhosis index FCI significantly correlated with the histological fibrosis stages F0-F1, F2-F3 and F4 (r = 0.818, p < 0.05) with AUROCs 0.932 and 0.996, respectively. The sensitivity and PPV of FCI at a cutoff value < 0.130 for predicting fibrosis stage F0-F1 was 81% and 82%, respectively with AUROC 0.932. Corresponding value of FCI at a cutoff value ≥1.25 for the prediction of cirrhosis was 86% and 100%. Conclusions The fibrosis-cirrhosis index (FCI) accurately predicted fibrosis stages in HCV infected patients and seems more efficient than frequently used serum indexes.

HCV is a leading cause of hepatocellular carcinoma and cirrhosis all over the world. Claudins belong to family of tight junction's proteins that are responsible for establishing barriers for controlling the flow of molecules around cells. For therapeutic strategies, regulation of viral entry into the host cells holds a lot of promise. During HCV infection claudin-1 is highly expressed in liver and believed to be associated with HCV virus entry after HCV binding with or without co-receptor CD81. The claudin-1 assembly with tight junctions is regulated by post translational modifications. During claudins assembly and disassembly with tight junctions, phosphorylation is required at C-terminal tail. In cellular proteins, interplay between phosphorylation and O- β -GlcNAc modification is believed to be functional switch, but it is very difficult to monitor these functional and vibrant changes in vivo . Netphos 2.0 and Disphos 1.3 programs were used for potential phosphorylation; NetPhosK 1.0 and KinasePhos for kinase prediction; and YinOYang 1.2 and OGPET to predict possible O -glycosylation sites. We also identified Yin Yang sites that may have potential for O- β -GlcNAc and phosphorylation interplay at same Ser/Thr residues. We for the first time proposed that alternate phosphorylation and O- β -GlcNAc modification on Ser 192, Ser 205, Ser 206; and Thr 191 may provide an on/off switch to regulate assembly of claudin-1 at tight junctions. In addition these phosphorylation sites may be targeted by novel chemotherapeutic agents to prevent phosphorylation lead by HCV viral entry complex.

Background Chronic HCV is one of the major causes of morbidity and mortality in the present day world. The assessment of disease progression not only provides useful information for diagnosis and therapeutic supervision judgment but also for monitoring disease. Different invasive and non invasive methods are applied to diagnose the disease from initial to end stage (mild fibrosis to cirrhosis). Although, liver biopsy is still considered as gold standard to identify liver histological stages, an assessment of the disease development based on non-invasive clinical findings is also emerging and this may replace the need of biopsy in near future. This review gives brief insight on non-invasive methods currently available for predicting liver fibrosis in HCV with their current pros and cons to make easier for a clinician to choose better marker to assess liver fibrosis in HCV infected patients. Methods More than 200 studies regarding invasive and noninvasive markers available for HCV liver disease diagnosis were thoroughly reviewed. We examined year wise results of these markers based on their sensitivity, specificity, PPV, NPV and AUROCs. Results We found that in all non-invasive serum markers for HCV, FibroTest, Forn's Index, Fibrometer and HepaScore have high five-year predictive value but with low AUROCs (0.60~0.85) and are not comparable to liver biopsy (AUROC = 0.97). Even though from its beginning, Fibroscan is proved to be best with high AUROCs (> 0.90) in all studies, no single noninvasive marker is able to differentiate all fibrosis stages from end stage cirrhosis. Meanwhile, specific genetic markers may not only discriminate fibrotic and cirrhotic liver but also differentiate individual fibrosis stages. Conclusions There is a need of marker which accurately determines the stage based on simplest routine laboratory test. Genetic marker in combination of imaging technique may be the better non invasive diagnostic method in future.

Background Hepatitis C virus (HCV) is one of the major health concerns globally, with genotype 3a as the most prevalent in Pakistan. Lack of efficient HCV genotype 3a small animal models as well as genomic replicons has hampered the complete understanding of its life cycle, pathogenesis and therapeutic options. In this study we aimed to develop a persistent HCV genotype 3a infectious cell culture model. Methods We inoculated Huh-7 cells with HCV genotype 3a serum. Cells and media supernatant were collected at different time periods up to 40 th day post infection. Culture media supernatant was also collected to find out its ability to infect naive Huh-7 cells. Results HCV replication was confirmed at both RNA and protein level through Real Time RCR and western blot using HCV core as marker. In order to validate the persistence of our model for HCV genotype 3a replication we inhibited the HCV replication through core specific siRNAs. The HCV RNA was detected intracellularly from the day one post infection up till 40 th day, while HCV core protein was detected from the second day up to 40 th day consistently. In culture media supernatant HCV RNA was also actively detected conferring its ability to infect the naive Huh-7 cells. Furthermore, core specific siRNA showed significant inhibition at 24 th hour post transfection both at RNA and protein level with progressive increase in the expression of core gene after 3 rd day. It clearly depicts that the Huh-7 successfully retained the HCV replication after degradation of siRNA. Conclusion Finally, we report that our persistent infection cell culture model consistently replicate HCV genotype 3a for more than 1 month.

Background and Aims HCV infection may lead to hepatic fibrosis. In this study, we tried to determine whether there is any correlation of HCV genotypes and viral load to the clinical parameters such as ALT, AST, ALP, bilirubin, Hb level, patient's age and gender; and then correlated this association with disease progression in liver biopsy samples. Methods In cross-sectional and observational study, 6048 serum HCV RNA positive patients were chosen. The study consists of 53 months from March 2006 to September 2010. Patients were divided into three cohorts to validate our data. Statistical analysis and correlation of lab parameters with viral factors was determined by using SPSS version 16. Results The most prevalent genotype was 3 (70.9%) followed by 1 (13.3%) and 4 (7.4%), collectively. During Univariate analysis, in all cohorts; serum bilirubin, ALP, ALT and AAR showed significant correlation with genotypes, however multivariate analysis showed that all genotypes except 4a have no association with host biochemical markers. Disease progression was also independent of all genotypes. Serum ALP, ALT, bilirubin and viremea levels were significantly elevated in patients with genotype 4a. Viral load showed negative association with serum bilirubin ( r = -0.112, P = 0.000) and ALP levels ( r = -0.098, P = 0.000). We observed positive correlation of ALP and bilirubin levels, while negative associations of viral load with HCV liver disease progression. Conclusion Disease progression seems independent of the genotypes. Relationship between ALP and bilirubin with viral load may be an attractive marker to guess disease progression in patients with hepatitis C.

Background and Aims Erythropoietin (EPO) is a glycoprotein hormone which is required to regulate the production of red blood cells. Deficiency of EPO is known to cause anemia in chronically infected renal patients and they require regular blood transfusion. Availability of recombinant EPO has eliminated the need for blood transfusion and now it is extensively used for the treatment of anemia. Glycosylation of erythropoietin is essential for its secretion, stability, protein conformation and biological activity. However, maintenance of human like glycosylation pattern during manufacturing of EPO is a major challenge in biotechnology. Currently, Chinese hamster ovary (CHO) cell line is used for the commercial production of erythropoietin but this cell line does not maintain glycosylation resembling human system. With the trend to eliminate non-human constituent from biopharmaceutical products, as a preliminary approach, we have investigated the potential of human hepatoma cell line (Huh-7) to produce recombinant EPO. Materials and methods Initially, the secretory signal and Kozak sequences was added before the EPO mature protein sequence using overlap extension PCR technique. PCR-amplified cDNA fragments of EPO was inserted into mammalian expression vector under the control of the cytomegalovirus (CMV) promoter and transiently expressed in CHO and Huh-7 cell lines. After RT-PCR analysis, ELISA and Western blotting was performed to verify the immunochemical properties of secreted EPO. Results Addition of secretory signal and Kozak sequence facilitated the extra-cellular secretion and enhanced the expression of EPO protein. Significant expression ( P < 0.05) of EPO was observed in the medium from Huh-7 cell line. Conclusion Huh-7 cell line has a great potential to produce glycosylated EPO, suggesting the use of this cell line to produce glycoproteins of the therapeutic importance resembling to the natural human system.

Background Several factors have been proposed to assess the clinical outcome of HCV infection. The correlation of HCV genotypes to possible serum markers in clinical prediction is still controversial. The main objective of this study was to determine the existence of any correlation between HCV genotypes to viral load and different clinical serum markers. Methods We performed a prospective cross-sectional and observational study. About 3160 serum HCV RNA positive patients were chosen from 4020 randomly selected anti-HCV positive patients. Statistical analysis was performed using the SPSS 16 software package. ROC (receiver operating characteristics) curves were used to compare diagnostic values of serum markers to predict genotypes. Results The most prevalent genotype was 3a (73.9%) followed by 1a (10.7%), 4a (6.4%) and 3b (6.1%) in Pakistani population. No correlation was found between viral load and serum markers for genotype 3a in a large no. of sample (n = 2336). While significant correlation was observed between viral load and AST in genotype 3b, ALP with viral load and ALT for genotype 1a. Patients with genotype 4a showed a significant inverse correlation with viral load and Hb level and AST with ALP. For genotype 4a, AUC (area under the curve) of ALT, ALP, AST, bilirubin, Hb level and viral load was 0.790, 0.763, 0.454, 0.664, 0.458 and 0.872 respectively. Conclusions In conclusion, there was a significant variable response of HCV genotypes with serum markers. Severity of disease is independent of serum marker level in genotype 3a, while the liver damage in genotype 4a may associate with viral cytopathic effect as well as the immune-mediated process. An index using six serum markers may correctly predict genotype 4a in patients with ≥75% accuracy.

Background The 9.6 kb long RNA genome of Hepatitis C virus (HCV) is under the control of RNA dependent RNA polymerase, an error-prone enzyme, for its transcription and replication. A high rate of mutation has been found to be associated with RNA viruses like HCV. Based on genetic variability, HCV has been classified into 6 different major genotypes and 11 different subtypes. However this classification system does not provide significant information about the origin of the virus, primarily due to high mutation rate at nucleotide level. HCV genome codes for a single polyprotein of about 3011 amino acids which is processed into structural and non-structural proteins inside host cell by viral and cellular proteases. Results We have identified a conserved NS4A protein sequence for HCV genotype 3a reported from four different continents of the world i.e. Europe, America, Australia and Asia. We investigated 346 sequences and compared amino acid composition of NS4A protein of different HCV genotypes through Multiple Sequence Alignment and observed amino acid substitutions C 22 , V 29 , V 30 , V 38 , Q 46 and Q 47 in NS4A protein of genotype 1b. Furthermore, we observed C 22 and V 30 as more consistent members of NS4A protein of genotype 1a. Similarly Q 46 and Q 47 in genotype 5, V 29 , V 30 , Q 46 and Q 47 in genotype 4, C 22 , Q 46 and Q 47 in genotype 6, C 22 , V 38 , Q 46 and Q 47 in genotype 3 and C 22 in genotype 2 as more consistent members of NS4A protein of these genotypes. So the different amino acids that were introduced as substitutions in NS4A protein of genotype 1 subtype 1b have been retained as consistent members of the NS4A protein of other known genotypes. Conclusion These observations indicate that NS4A protein of different HCV genotypes originally evolved from NS4A protein of genotype 1 subtype 1b, which in turn indicate that HCV genotype 1 subtype 1b established itself earlier in human population and all other known genotypes evolved later as a result of mutations in HCV genotype 1b. These results were further confirmed through phylogenetic analysis by constructing phylogenetic tree using NS4A protein as a phylogenetic marker.