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Background: Plasmodium vivax is one of the widespread human malarial parasites accounting for 75% of malaria epidemics. However, there is no baseline information about the status and nature of genetic variation of Plasmodium species circulating in various parts of Pakistan. The present study was aimed at observing the molecular epidemiology and genetic variation of Plasmodium vivax by analysing its merozoite surface protein-3α (msp-3α) and merozoite surface protein-3β (msp-3β) genes, by using suballele, species-specific, combined nested PCR/RFLP detection techniques. Methods: A total of 230 blood samples from suspected subjects tested slide positive for vivax malaria were collected from Punjab, Sindh, Khyber Pakhtunkhwa, and Balochistan during the period May 2012 to December 2013. Combined nested PCR/RFLP technique was conducted using Pvmsp-3α and Pvmsp-3β genetic markers to detect extent of genetic variation in clinical isolates of P. vivax in the studied areas of Pakistan. Results: By PCR, P. vivax, 202/230 (87.82%), was found to be widely distributed in the studied areas. PCR/RFLP analysis showed a high range of allelic variations for both msp-3α and msp-3β genetic markers of P. vivax, i.e., 21 alleles for msp-3α and 19 for msp-3β. Statistically a significant difference (p ≤0.05) was observed in the genetic diversity of the suballelic variants of msp-3α and msp-3β genes of P. vivax. Conclusion: It is concluded that P. vivax populations are highly polymorphic and diverse allelic variants of Pvmsp-3α and Pvmsp-3β are present in Pakistan.

Determination of an individual’s hepatitis C virus (HCV) genotypes prior to antiviral therapy has become increasingly important for the clinical management and prognosis of HCV infection. Therefore, this study was conducted to investigate the prevalence of HCV genotypes in HCV infected patients of district Bannu in Khyber Pakhtunkhwa region of Pakistan. Serum samples of 117 seropositive patients were screened for HCV-RNA by using reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) and then PCR positive samples were subjected to HCV genotyping. Out of 117 seropositive samples, 110 samples were found positive by PCR analysis. Genotype 3a was the most prevalent one detected in 38% of patients, followed by genotype 3b in 21% of patients, and then genotype 2a in 12% of patients. However 21% of HCV-PCR positive samples could not be genotyped by method used in this study. Genotype 3a was the most prevalent genotype in patients of all age groups and its prevalence was found high among patients with increasing age (>34 years). Moreover, genotypes 3a and 3b were found to be the most prevalent genotypes in patients with history of shaving by barbers, receiving multiple injections, and dental procedures. In conclusion there is need of further investigation of genotypes of HCV by using more sensitive assays and considering large sample size in district Bannu.

The clinical outcomes of patients infected with hepatitis C virus (HCV) range from acute resolving hepatitis to chronic liver diseases such as liver cirrhosis or hepatocellular carcinoma. Identification of the infecting virus genotype is indispensable for the exploration of many aspects of HCV infection, including epidemiology, pathogenesis, and response to antiviral therapy. 1419 individuals were screened for anti-HCV in this study, of which 166 (11.7%) were found reactive by ICT (Immunochromatographic test). These 166 anti-HCV positive and 26 normal individuals were further analyzed. RNA was extracted from serum and reverse-transcribed to cDNA and the core region of HCV genome was targeted and amplified by multiplex PCR. HCV RNA was detected in 121 individuals, of which 87 were male and 34 were female. Genotype 3a was the most prevalent among all the genotypes observed followed by 3b. Genotypes 1a, 2a, and 2b were found in 10.89%, 13.22%, and 6.61% patients, respectively. 25.41% of the HCV RNA positive samples were not typed. 6.05% of patients were found having mixed genotypes. These findings will not only help the physicians to prescribe more appropriate treatment for the HCV infection but will also draw the attention of health-related policy makers to devise strategies to curb the disease more effectively.

The decomposition of both α- and β-anomers of ifetroban 1-O-acyl glucuronide in an aqueous medium was studied at ambient temperature (23 °C). An HPLC/MS technique was used to investigate the decomposition of these anomers via hydrolysis and acyl migration over a pH range of 1.0−13.0. It was found that while no acyl migration occurred at pH ≤4.0, hydrolysis occurred at both acidic and basic pH. The hydrolysis rate was the slowest within the pH range of 3.0−4.0. Outside this range, the rate of hydrolysis increased as the pH was increased or decreased. The acyl migration at pH 5.0 was very slow, but the rate increased as the pH was increased. First-order rate constants (in h-1), as a function of pH, were estimated according to a simplified scheme for both hydrolysis and acyl migration processes. Methanolysis of the β-anomer was studied in 80% or 100% methanol in water and also in 20% methanol in aqueous buffer solutions with apparent pH values of 3.0, 6.0 and 9.0. A systematic study of varying the pH and ion...

Complement factor H (CFH) is the major regulator of the central complement protein C3b in the alternative pathway of complement activation. A molecular view of the CFH interaction with native heparan sulfate (HS) is central for understanding the mechanism of how surface-bound CFH interacts with C3b bound to host cell surfaces. HS is composed of sulfated heparin-like S-regions that alternate with desulfated NA-regions. Solution structural studies of heparin (equivalent to the S-regions) and desulfated HS (the NA-regions) by scattering and ultracentrifugation showed that each structure was mostly extended and partially bent, but with greater bending and flexibility in the NA-regions compared to the S-regions. Their solution structures have been deposited in the Protein Data Bank. The largest HS oligosaccharides showed more bent and flexible structures than those for heparin. A folded-back domain structure for the solution structure of the 20 domains in CFH was determined likewise. CFH binds to the S-regions but less so to the NA-regions of HS. The bivalent interaction of CFH-heparin was observed by ultracentrifugation, and binding studies of CFH fragments with heparin-coated sensor chips. In common with other CFH interactions with its physiological and pathophysiological ligands, the CFH-heparin and CFH-C3b interactions have moderate micromolar dissociation constants K D, meaning that these complexes do not fully form in vivo. The combination of the solution structures and binding studies indicated a two-site interaction model of CFH with heparin at cell surfaces. By this, the bivalent binding of CFH to a cell surface is co-operative. Defective interactions at either of the two independent CFH-heparin sites reduce the CFH interaction with surface-bound C3b and lead to immune disorders.

A total of 339 patients with clinically suspected cutaneous leishmaniasis (CL) were studied from March to April, 2010 in three villages of Karak, Khyber Pakhtunkhwa, Pakistan where an epidemic of the disease was in question. Using polymerase chain reaction (PCR), 78.17% (265/339) were observed having CL. Microscopically, however, only 43.06% (146/339) were diagnosed with the disease. This study reports and confirms epidemic of CL in both gender of all ages in the area. Females (70.94%) were noted to be predominantly affected as compared to males (29%). Clinically, 12.38% of patients had more than three lesions, 29.20% had two lesions, while 58.40% had only single lesion. Most lesions were found on exposed surfaces of the body (predominantly hands, face and feet). The present study confirms that PCR was more sensitive than microscopic examination. Key words: Epidemic, cutaneous leishmaniasis, polymerase chain reaction (PCR), microscopy, Pakistan.

Toxoplasmosis is spread through contamination of water sources and results in morbidity globally. In the current study 300 water samples were processed by polymerase chain reaction (PCR) for detection of Toxoplasma gondii. The overall prevalence in different water sources was 6.6% (17/300). Among different water sources the highest prevalence was recorded in drain water at 7% (7/100), followed by tube well water at 7.5% (3/40) and open well water at 5% (5/100) ,and the lowest was recorded in tap water at 3.33% (2/60). The highest prevalence was recorded in summer. Evidence indicates that cleaning and filtration need to be adopted to avoid the health hazards of waterborne zoonotic parasites.

Background: The polysaccharide heparan sulfate (HS) exhibits key physiological roles. Results: Analytical ultracentrifugation and x-ray scattering revealed extended but bent HS solution structures. Conclusion: Scattering fits resulted in molecular models for HS in solution with glycosidic angles in good accord with a HS crystal structure. Significance: These bent HS models clarify how HS interacts with its ligands., The highly sulfated polysaccharides heparin and heparan sulfate (HS) play key roles in the regulation of physiological and pathophysiological processes. Despite its importance, no molecular structures of free HS have been reported up to now. By combining analytical ultracentrifugation, small angle x-ray scattering, and constrained scattering modeling recently used for heparin, we have analyzed the solution structures for eight purified HS fragments dp6–dp24 corresponding to the predominantly unsulfated GlcA-GlcNAc domains of heparan sulfate. Unlike heparin, the sedimentation coefficient s20,w of HS dp6–dp24 showed a small rotor speed dependence, where similar s20,w values of 0.82–1.26 S (absorbance optics) and 1.05–1.34 S (interference optics) were determined. The corresponding x-ray scattering measurements of HS dp6–dp24 gave radii of gyration RG values from 1.03 to 2.82 nm, cross-sectional radii of gyration RXS values from 0.31 to 0.65 nm, and maximum lengths L from 3.0 to 10.0 nm. These data showed that HS has a longer and more bent structure than heparin. Constrained scattering modeling starting from 5,000 to 12,000 conformationally randomized HS structures gave best fit dp6–dp24 molecular structures that were longer and more bent than their equivalents in heparin. Alternative fits were obtained for HS dp18 and dp24, indicating their higher bending and flexibility. We conclude that HS displays bent conformations that are significantly distinct from that for heparin. The difference is attributed to the different predominant monosaccharide sequence and reduced sulfation of HS, indicating that HS may interact differently with proteins compared with heparin.

Fecal samples of 720 cattle were collected from four different areas of Lahore, that is,Military dairy farm, Government dairy farm, Gawala dairy colonies and House hold dairy cows. All samples were purified and cysts were quantified microscopically. DNA was extracted from the purified cysts and was amplified through polymerase chain reaction. The overall prevalence was 31.11% (224/720), where high prevalence was recorded in Government dairy farm (46.11%), followed by Gawala dairy colonies (36.11%), Military dairy farm (25.55%) and lowest was recorded in House hold dairy animals (16.66%).Overall cattle 2-3 years were observed to have higher Giardia prevalence compared to cattle of 3-7 years. High prevalence was observed in cows (31.71%, n=640) as compared to bulls (26.25%, n=80). Giardia prevalence was higher in autumn (36.66%) and lowest in winter (25%). There was a significant difference (P

Hepatitis B virus (HBV) infection is a major health problem in the developing countries including Pakistan. This study aimed to investigate various risk factors and prevalence of HBV in different areas of Khyber Pakhtunkhwa province, Pakistan. A total of 1439 individuals (1021 males and 418 females) suspected for hepatitis B infection were screened for HBsAg. All the samples were blindly analyzed for HBV DNA by nested polymerase chain reaction (PCR). Of the total, 49.5% were found positive for HBsAg. Of these HBsAg positive patients, 83.03% were confirmed for HBV DNA. Of the 726 HBsAg negative individuals, 37 (24 males and 13 females) were found positive for HBV DNA. 629 HBV DNA positive individuals include 70.43% male and 29.57% female. Higher prevalence rate (16.53%) was observed in Malakand and lowest (13.35%) in Mardan. Mostly young people with age 16 to 30 years were infected as compared to other age group. Risk factors observed in HBV positive individuals were unhygienic barber practice, blood transfusion, general and dental surgery, unsafe injection and sharing personal items. Trend of sharing personal items was common (20.19%). Extensive vaccination and other preventive measures should be taken to stop the spread of this dreadful disease in the study area. Key words: Hepatitis B Virus, prevalence, polymerase chain reaction (PCR), risk factor

Retraction The editors regretfully retract the article [1] by Jabbar Khan, Sanaullah Khan, Sobia Attaullah, Ijaz Ali, and Shahid Khan (BMC Cell Biology 2012, 13:8) due to significant overlap with previously published article “Overexpression of Active Aurora-C Kinase Results in Cell Transformation and Tumour Formation” by Jabbar Khan, Frederic Ezan, Jean-Yves Cremet, Alain Fautre, David Gilot, Marine Lambert, Christelle Benaud, MarieBerengere Troadec, and Claude Prigent (PLoS ONE 6 (10): e26512. doi:10.1371/journal.pone.0026512). We apologise to all affected parties for the inconvenience caused.

Modern world is under tremendous pressure for alternative sources of energy and the trends are towards sustainable energy. Present power systems are facing heavy economic losses for poor power qualities. Efficiency of sustainable energy depends on the power quality of the supply. Presently, Power Quality issues are major problems for utilities, customers and end users. The cost of poor PQ is high and gradually rising. The paper gives insights on global economical losses due to poor PQ. The business risk posed by PQ problems is a real one with even low tech industries exposed to serious financial losses. It has been observed that the consequences of poor PQ would have large financial impacts on a country's economy, and more initiatives are required from the concerned parties and regulating bodies to take corrective measures for maintaining better power quality. Especially for successful sustainable energy programme, Power Quality Monitoring can help identify the cause of power system disturbances and the problem conditions on a system before they cause interruptions or disturbances. In an environment of sustainable energy and modern grid, intelligent PQ monitoring is essentially required to solve different PQ related problems. Authors proposed an intelligent power quality monitoring system using virtual instrumentation and appropriate data acquisition system to detect different PQ disturbances. The system is user friendly due to its graphical user interface. The tests were done in the Energy Laboratory of NIT, Silchar (India) and Research Lab, Electrical Engineering Department, AMU, Aligarh (India). The available energy in both the labs was solar energy. The qualities monitored include voltage fluctuations, sag, swell, interruptions, harmonics etc. The huge amount of acquired data has been analyzed using quadratic discriminant analysis technique to determine the condition of the waveforms. The system shows fast response with accuracy in monitoring desired power qualities.

Background Aurora kinases (Aurora-A, B and C) belong to a family of conserved serine/threonine kinases which are key regulators of cell cycle progression. Aurora-A and Aurora-B are expressed in somatic cells and involved in cell cycle regulation while aurora-C is meiotic chromosome passenger protein. As Aurora kinase C is rarely expressed in normal somatic cells and has been found over expressed in many cancer lines. It is suggested that Aurora-C-T191D is not hyperactive mutant. Result Aurora-C-T191D variant form was investigated and compared with wild type. The overexpression of Aurora-C-T191D was observed that it behaves like Aurora-C wild type (aurC-WT). Both Aurora-C-T191D and aurC-WT induce abnormal cell division resulting in centrosome amplification and multinucleation in transiently transfected cells as well as in stable cell lines. Similarly, Aurora-C-T191D and aurC-WT formed foci of colonies when grown on soft agar, indicating that a gain of Aurora-C activity is sufficient to transform cells. Furthermore, we reported that NIH-3 T3 stable cell lines overexpressing Aurora-C-T191D and its wild type partner induced tumour formation when injected into nude mice, demonstrating the oncogenic activity of enzymatically active Aurora kinase C. Interestingly enough tumour aggressiveness was positively correlated with the rate of kinase activity, making Aurora-C a potential anti-cancer therapeutic target. Conclusion These findings proved that Aurora C-T191D is not hyperactive but is constitutively active mutant.

Rabies is an avertable viral disease caused by the rabid animal to the warm blooded animals (zoonotic) especially human. Rabies occurs in more than 150 countries and territories. According to an estimation by WHO, almost 55,000 people die because of rabies every year. The Dogs are the major reason behind this, approximately 99% human deaths caused by dog's bites. Developing and under developing countries, both are the victims of rabies. With the post-exposure preventive regimes, 327,000 people can prevent this disease annually. The current article mainly covers the genome, virology, symptoms, epidemiology, diagnostic methods, and the high risk countries around the globe.

Background and aim Phylogenetic analysis has led to the classification of hepatitis C virus (HCV) into 1-6 major genotypes. HCV genotypes have different biological properties, clinical outcome and response to antiviral treatment and provide important clues for studying the epidemiology, transmission and pathogenesis. This article deepens the current molecular information about the geographical distribution of HCV genotypes and subgenotypes in population of four provinces of Pakistan. 34 published papers (1996-2011) related to prevalence of HCV genotypes/serotypes and subgenotypes in Pakistan were searched. Result HCV genotype/s distribution from all 34 studies was observed in 28,400 HCV infected individuals in the following pattern: 1,999 (7.03%) cases of genotype 1; 1,085 (3.81%) cases of genotype 2; 22,429 (78.96%) cases of genotype 3; 453 (1.59%) cases of genotype 4; 29 (0.10%) cases of genotype 5; 37 (0.13%) cases of genotype 6; 1,429 (5.03%) cases of mixed genotypes, and 939 (3.30%) cases of untypeable genotypes. Overall, genotype 3a was the predominant genotype with a rate of 55.10%, followed by genotype 1a, 3b and mixed genotype with a rate of 10.25%, 8.20%, and 5.08%, respectively; and genotypes 4, 5 and 6 were rare. Genotype 3 occurred predominately in all the provinces of Pakistan. Second more frequently genotype was genotype 1 in Punjab province and untypeable genotypes in Sindh, Khyber Pakhtunkhwa and Balochistan provinces.

Background The structural and functional differences between hepatitis B virus (HBV) genotypes are the mainstay to severity, complications, treatment and possibly vaccination against the virus. This study was conducted to determine the HBV genotypes in HBsAg positive patients of Afghanistan as no such large scale data available previously. Methods Two hundred and fourteen HBsAg-positive patients were included in this study. All patients were anti-HCV and anti-HIV negative. All the samples were confirmed for HBV DNA with nested PCR while HBV DNA positive samples were subjected to type specific PCR for HBV genotyping (A-F). Results Of the total samples, 168 (78.5%) were males and 46 (21.49%) females, aged ranged between 18 to 71 years. This study demonstrated that genotype D (35.67%) is the predominant genotype circulating in Afghani's population. Genotype C was observed in 32.16% followed by genotype A (19.30%), and genotype B (7.02%) while 6.07% of the individuals were not typed. Conclusion This study has shown a heterogeneous distribution of HBV genotypes. Further more, extensive studies are required to investigate genetic and geographical divergence and characteristics of the virus in the country, as no such large sample sized study has been carried out so far in this country.

Injection drug users (IDUs) are considered as a high risk group to develop hepatitis C due to needle sharing. In this study we have examined 200 injection drug users from various regions of the Khyber Pakhtunkhwa province for the prevalence of active HCV infection and HCV genotypes by Immunochromatographic assays, RT-PCR and Type-specific PCR. Our results indicated that 24% of the IDUs were actively infected with HCV while anti HCV was detected among 31.5% cases. Prevalent HCV genotypes were HCV 2a, 3a, 4 and 1a. Majority of the IDUs were married and had attained primary or middle school education. 95% of the IDUs had a previous history of needle sharing. Our study indicates that the rate of active HCV infection among the IDUs is higher with comparatively more prevalence of the rarely found HCV types in KPK. The predominant mode of HCV transmission turned out to be needle sharing among the IDUs.

Background Hepatitis C virus (HCV) genotype 3a is known to show comparatively better response to combination therapy than genotype 1 and 4. Mutations within NS5A gene of HCV have earlier been implicated with response to interferon (IFN) therapies in chronic HCV patients among various populations. As response to therapy are available in different populations because of the ethnic and viral factors and there was no study available on the phenomenon of resistivity to IFN. Results Chronic HCV 3a infected Pakistani patients were kept on IFN-α and ribavirin therapy for six months. NS5A gene of HCV was amplified and sequenced in the case of all the patients prior to therapy and the sequences were analysed for mutations. Out of the total 27 patients, 20 (74.07%) were observed with sustained virological response (SVR), 4 (14.81%) patients were non responder (NR) while 3 (11.11%) patients exhibited in end of treatment response (ETR). Three (3/20) (15%) SVR patients and two (2/3) ETR patients had mutations (ranging from I-V amino acids) within the NS5A ISDR regions. While the rest of the SVR patients (85%) and the NR had no mutations at ISDR region when compared with HCV K3a ISDR. Conclusions Mutations within the NS5A gene of HCV 3a genotype may not influence the outcome of combination therapy in Pakistani populations.

The highly sulfated polysaccharides heparin and heparan sulfate (HS) play key roles in the regulation of physiological and pathophysiological processes. Despite its importance, no molecular structures of free HS have been reported up to now. By combining analytical ultracentrifugation, small angle x-ray scattering, and constrained scattering modeling recently used for heparin, we have analyzed the solution structures for eight purified HS fragments degree of polymerization 6-18 (dp6-dp18) and dp24, corresponding to the predominantly unsulfated GlcA-GlcNAc domains of heparan sulfate. Unlike heparin, the sedimentation coefficient s(20,)(w) of HS dp6-dp24 showed a small rotor speed dependence, where similar s(20,)(w) values of 0.82-1.26 S (absorbance optics) and 1.05-1.34 S (interference optics) were determined. The corresponding x-ray scattering measurements of HS dp6-dp24 gave radius of gyration (R(G)) values from 1.03 to 2.82 nm, cross-sectional radius of gyration (R(XS)) values from 0.31 to 0.65 nm, and maximum lengths (L) from 3.0 to 10.0 nm. These data showed that HS has a longer and more bent structure than heparin. Constrained scattering modeling starting from 5000-8000 conformationally randomized HS structures gave best fit dp6-dp16 molecular structures that were longer and more bent than their equivalents in heparin. No fits were obtained for HS dp18 or dp24, indicating their higher flexibility. We conclude that HS displays an extended bent conformation that is significantly distinct from that for heparin. The difference is attributed to the different predominant monosaccharide sequence and reduced sulfation of HS, indicating that HS may interact differently with proteins compared with heparin.

Hepatitis C is a fatal liver disease caused by the hepatitis C virus. In this study, blood donors, from various districts of the KPK province and the federally administered tribal area (FATA) of Pakistan were tested for anti-HCV antibodies and HCV RNA by ICT (Immuno-chromatographic test), ELISA and RT-PCR. Out of the 7148 blood donors, 224 (3.13%) were positive for anti-HCV antibodies by ICT, 135 (1.89%) by ELISA while 118 (1.65%) blood donors had active HCV infection as detected by RT-PCR. We suggest that ELISA should be used for anti-HCV screening in public sector hospitals and health care units.

Background Studies of the molecular epidemiology and risk factors for hepatitis C virus (HCV) in health care workers (HCWs) of Peshawar, Khyber Pakhtunkhwa region are scarce. Lack of awareness about the transmission of HCV and regular blood screening is contributing a great deal towards the spread of hepatitis C. This study is an attempt to investigate the prevalence of HCV and its possible association with both occupational and non-occupational risk factors among the HCWs of Peshawar. Results Blood samples of 824 HCWs, aged between 20-59 years were analysed for anti-HCV antibodies, HCV RNA and HCV genotypes by Immunochromatographic tests and PCR. All relevant information was obtained from the HCWs with the help of a questionnaire. The study revealed that 4.13% of the HCWs were positive for HCV antibodies, while HCV RNA was detected in 2.79% of the individuals. The most predominant HCV genotype was 3a and 2a. Conclusion A program for education about occupational risk factors and regular blood screening must be implemented in all healthcare setups of Khyber Pakhtunkhwa province in order to help reduce the burden of HCV infection.

Several types of whole pulses (green lentils, red lentils, yellow lentils, chickpeas, green peas, cowpeas and yellow peas) and grams (black grams, red grams and white grams) have been investigated for the identification of radiation treatment using microgel electrophoresis of single cells (DNA comet assay). Pulses and grams were exposed to the radiation doses of 0.5, 1.0 and 5 kGy covering the legalized commercial dose range for protection from insect/pest infestations. All irradiated samples showed comet like stretching of fragmented DNA toward anode, which is expected for irradiated samples. Unirradiated samples showed many intact cells/nuclei in form of round stains or with short faint tails, which is typical for unirradiated food samples. The study shows that DNA comet assay can be used as a rapid, inexpensive and highly effective screening test for the detection of radiation treatment of foods, like pulses and grams.

Analytical ultracentrifugation and solution scattering provide different multi-parameter structural and compositional information on proteins. The joint application of the two methods supplements high resolution structural studies by crystallography and NMR. We summarise the procedures required to obtain equivalent ultracentrifugation and X-ray and neutron scattering data. The constrained modelling of ultracentrifugation and scattering data is important to confirm the experimental data analysis and yields families of best-fit molecular models for comparison with crystallography and NMR structures. This modelling of ultracentrifugation and scattering data is described in terms of starting models, their conformational randomisation in trial-and-error fits, and the identification of the final best-fit models. Seven applications of these methods are described to illustrate the current state-of-the-art. These include the determination of antibody solution structures (the human IgG4 subclass, and oligomeric forms of human IgA and its secretory component), the solution structures of the complement proteins of innate immunity (Factor H and C3/C3u) and their interactions with macromolecular ligands (C-reactive protein), and anionic polysaccharides (heparin). Complementary features of joint ultracentrifugation and scattering experiments facilitate an improved understanding of crystal structures (illustrated for C3/C3u, C-reactive protein and heparin). If a large protein or its complex cannot be crystallised, the joint ultracentrifugation-scattering approach provides a means to obtain an overall macromolecular structure.

Factor H (FH) is the major regulator of the central complement protein C3b in the alternative pathway of complement activation, and is comprised of 20 SCR domains. A FH Tyr402His polymorphism in SCR-7 is associated with age-related macular degeneration (AMD) and leads to deposition of complement in drusen. The unravelling of how FH interacts with five major physiological and patho-physiological ligands is complicated by the weak nature of these interactions, coupled with the multivalency of FH. Using multiple biophysical methods, we summarise our recent results for these five FH ligands: (1) FH by itself shows a folded-back SCR domain structure in solution, and self-associates in a manner dependent on electrostatic forces. (2) FH activity is inhibited by zinc, which causes FH to aggregate. The onset of FH-zinc aggregation for zinc concentrations above 20 muM appears to be enhanced with the His402 allotype, and may be relevant to AMD. (3) The FH and C-reactive protein (CRP) interaction has been controversial; however our new work resolves earlier discrepancies. The FH-CRP interaction is only observed when native CRP is at high acute-phase concentration levels, and CRP binds weakly to the His402 FH allotype to suggest a molecular mechanism that leads to AMD. (4) Heparin is an analogue of the polyanionic host cell surface, and FH forms higher oligomers with larger heparin fragments, suggesting a mechanism for more effective FH regulation. (5) The interaction of C3b with FH also depends on buffer, and FH forms multimers with the C3d fragment of C3b. This FH-C3d interaction at high FH concentration may also facilitate complement regulation. Overall, our results to date suggest that the FH interactions involving zinc and native CRP have the closest relevance for explaining the onset of AMD.

Factor H (FH) is the major regulator of the central complement protein C3b in the alternative pathway of complement activation, and is comprised of 20 SCR domains. A FH Tyr402His polymorphism in SCR-7 is associated with age-related macular degeneration (AMD) and leads to deposition of complement in drusen. The unravelling of how FH interacts with five major physiological and patho-physiological ligands is complicated by the weak nature of these interactions, coupled with the multivalency of FH. Using multiple biophysical methods, we summarise our recent results for these five FH ligands: (1) FH by itself shows a folded-back SCR domain structure in solution, and self-associates in a manner dependent on electrostatic forces. (2) FH activity is inhibited by zinc, which causes FH to aggregate. The onset of FH-zinc aggregation for zinc concentrations above 20 μM appears to be enhanced with the His402 allotype, and may be relevant to AMD. (3) The FH and C-reactive protein (CRP) interaction has been controversial; however our new work resolves earlier discrepancies. The FH-CRP interaction is only observed when native CRP is at high acute-phase concentration levels, and CRP binds weakly to the His402 FH allotype to suggest a molecular mechanism that leads to AMD. (4) Heparin is an analogue of the polyanionic host cell surface, and FH forms higher oligomers with larger heparin fragments, suggesting a mechanism for more effective FH regulation. (5) The interaction of C3b with FH also depends on buffer, and FH forms multimers with the C3d fragment of C3b. This FH-C3d interaction at high FH concentration may also facilitate complement regulation. Overall, our results to date suggest that the FH interactions involving zinc and native CRP have the closest relevance for explaining the onset of AMD.

All computer software systems, whether online or offline, require a help system. Help texts are traditionally written by software development companies and answer targeted questions in the form of how-tos and troubleshooting procedures. However, when the popularity of an application grows, users of the application themselves start adding to the corpus of help for the system in the form of online tutorials. There is, however, one problem with such tutorials. They have no direct link with the software for which they are written. Users have to search the Internet for different tutorials that are usually hosted on dispersed locations, and there is no ideal way of finding the relevant information without ending up with lots of noise in the search results. In this chapter, we describe a model for a help system which enhances this concept using collaborative tagging for categorization of “helplets.” For the knowledge retrieval part of the system, we utilize a previously developed technique based on common sense and user personalization. We use a freely available common sense reasoning toolkit for knowledge retrieval. Our architecture can be implemented in Web-based systems as well as in stand-alone desktop applications.

Modern mobile devices serve as platforms that consume services from multiple service providers. It is vital for such an open cell phone environment to secure the information flows of the stakeholders on the platform. Recent emergence of trusted computing technologies provides a root of trust in hardware, which can be used to construct a chain of trust. This chain of trust can be used to remotely verify that the platform is capable to manage information flows in a trusted manner. This work highlights how trusted computing technologies can be complemented with existing Mandatory Access Control mechanisms to verify the runtime and dynamic behaviors of a platform by using a high level, managerial policy - hence enabling a trustworthy platform with dynamic behavior management.

The anionic polysaccharides heparin and heparan sulphate play essential roles in the regulation of many physiological processes. Heparin is often used as an analogue for heparan sulphate. Despite knowledge of an NMR solution structure and 19 crystal structures of heparin-protein complexes for short heparin fragments, no structures for larger heparin fragments have been reported up to now. Here, we show that solution structures for six purified heparin fragments dp6-dp36 (where dp stands for degree of polymerisation) can be determined by a combination of analytical ultracentrifugation, synchrotron X-ray scattering, and constrained modelling. Analytical ultracentrifugation velocity data for dp6-dp36 showed sedimentation coefficients that increased linearly from 1.09 S to 1.84 S with size. X-ray scattering of dp6-dp36 gave radii of gyration R(G) that ranged from 1.33 nm to 3.12 nm and maximum lengths that ranged from 3.0 nm to 12.3 nm. The higher resolution of X-ray scattering revealed an increased bending of heparin with increased size. Constrained molecular modelling of 5000 randomised heparin conformers resulted in 9-15 best-fit structures for each of dp18, dp24, dp30, and dp36 that indicated flexibility and the presence of short linear segments in mildly bent structures. Comparisons of these solution structures with crystal structures of heparin-protein complexes revealed similar ranges of phi (phi) and psi (psi) angles between iduronate and glucosamine rings. We conclude that heparin in solution has a semi-rigid and extended conformation that is preformed for its optimal binding to protein targets without major conformational changes.

This study describes the pharmacokinetic parameters of gemopatrilat, a potent vasopeptidase inhibitor, in humans and the comparative biotransformation of the compound in rats, dogs, and humans after administration of a single oral dose of [14C]gemopatrilat. Gemopatrilat was rapidly absorbed in humans with an oral bioavailability of 49%. Within 5 h after dose, the mean concentrations of gemopatrilat were less than 1% of the mean Cmax values. The total area under the first-moment time curve extrapolated to infinity [AUC(INF)] value for gemopatrilat was only 2% of the AUC(INF) of radioactivity in plasma. Gemopatrilat showed a large apparent steady-state volume of distribution (2500 liters) and a prolonged terminal-phase decline in plasma concentration. These results are consistent with the idea that the free sulfhydryl group of gemopatrilat forms reversible disulfide linkages with plasma and tissue proteins and is thus eliminated from the body at a very slow rate. Approximately half of the drug-related radioactivity in 1-h plasma samples from rat, dog, and human was reduced chemically with dithiothreitol to gemopatrilat, suggesting that disulfide linkage occurred in all species. In addition, metabolites formed through S-methylation and amide hydrolysis were also detected in rat, dog, and human plasma. No gemopatrilat was detected in urine and fecal samples from all three species, indicating that the compound is extensively metabolized in vivo. The major metabolites identified in human urine and feces were also present in rat and dog. These data suggest that the metabolism of gemopatrilat in all three species were qualitatively very similar.

The effect of endosulfan, an Organochlorine pesticide, on bluegill testes was studied. Endosulfan is aqua-toxic and has an immediate effect on fish and other aquatic life. In this experiment, we exposed the fish for 24-, 48-, 72-, 96-h, and 1- and 2-week periods. A second group of fish without exposure to endosulfan served as the control. The control testis appeared structurally normal. The seminiferous tubules were of round or oval shape and contained primary spermatogonia, primary spermatocytes, secondary spermatocytes, spermatozoa, spermatids, Sertoli cells, and interstitial cells of Leydig. Within the connective tissue that connected the seminiferous tubules were Leydig cells. After 24 h of exposure, there was evidence of slight signs of connective tissue splintering. The 48-h exposure resulted in breakage of primary spermatocyte walls and separation from the seminiferous tubules. The 72-h testis showed further connective tissue damage and migration of primary spermatogonia into the lumen. After 96 h, there was significant damage to connective tissue and the seminiferous tubules were less pronounced. After 1 and 2 weeks, the seminiferous tubule walls were disrupted and missing in places and the structure of the testis was very disorganized compared to the control testis. Biometric analysis indicated that the diameter of the primary spermatogonia decreased from 24 h to two weeks. There also appeared to be fewer Leydig cells, responsible for testosterone production, over the exposure period and damaged Sertoli cells, which support, protect, and nourish the spermatogonic cells, synthesize ABP, and assist in maintaining the necessary concentration of testosterone in the seminiferous tubules so that spermatogenesis can progress. These kinds of damage could affect the spermatids and spermatozoa and possibly have a negative impact on spermatogenesis and male fertility, affecting bluegill fish population.

To find out the frequency of common genotypes of hepatitis-B virus (HBV).An analytical study.The present study was carried out at Division of Infectious Diseases and Molecular Diagnostics, Centre for Applied Molecular Biology, Ministry of Science and Technology, Lahore, Pakistan from May 2002 to February 2004.HBV genotypes were determined in 112 HBV DNA positive sera by a simple and precise molecular genotyping system based on PCR using type-specific primers for the determination of genotypes of HBV A through H.Four genotypes (A, B, C and D) out of total eight reported genotypes so far were identified. Genotypes A, B and C were predominant. HBV genotype C was the most predominant in this collection, appearing in 46 samples (41.07%). However, the genotypes of a total of 5 (4.46%) samples could not be determined with the present genotyping system. Mixed genotypes were seen in 8 (7.14%) HBV isolates. Five of these were infected with genotypes A/D whereas two were with genotypes C/D. One patient was infected with 4 genotypes (A/B/C/D). Genotype A (68%) was predominant in Sindh; genotype C was most predominant in North West Frontier Province (N.W.F.P.) (68.96) whereas genotypes C and B were dominant in Punjab (39.65% and 25.86% respectively).All the four common genotypes of HBV found worldwide (A, B, C and D) were isolated. Genotype C is the predominant. Genotypes B and C are predominant in Punjab and N.W.F.P whereas genotype A is predominant in Sindh.

Omapatrilat, a novel vasopeptidase inhibitor, is under development for the treatment of hypertension and congestive heart failure. This study describes the comparative biotransformation of radiolabeled [(14)C]- and stable-labeled [(13)C(2)]omapatrilat after administration of single oral doses to rats, dogs, and humans. The metabolites were identified by a combination of methods including reduction, hydrolysis, and comparison of high performance liquid chromatography retention times with those of the synthetic standards. Urinary metabolites were further characterized by liquid chromatography tandem mass spectrometry analysis. Prominent metabolites identified in human plasma, which were also present in rat and dog plasma, were S-methyl omapatrilat and S-2-thiomethyl-3-phenylpropionic acid. Omapatrilat accounted for only a small portion of the extractable radioactivity in plasma in all three species. A portion of the plasma radioactivity was unextractable in all three species (27-53%). The majority of unextractable radioactivity in plasma was characterized after dithiothreitol reduction to be omapatrilat and (S)-2-thio-3-phenylpropionic acid, both apparently bound to plasma proteins by reversible disulfide bonds. The major human urinary metabolites were the amine hydrolysis product, diasteromeric sulfoxide of (S)-2-thiomethyl-3-phenylpropionic acid, acyl glucuronide of S-methyl omapatrilat, and S-methyl omapatrilat. The minor metabolites were acyl glucuronide of (S)-2-thiomethyl-3-phenylpropionic acid, L-cysteine mixed disulfide of omapatrilat, diastereomers of S-methyl sulfoxide of omapatrilat, and S-methyl omapatrilat ring sulfoxide. The metabolic profiles of dog and human urine were qualitatively similar whereas rat urine showed only metabolites arising from hydrolysis of omapatrilat. Unchanged omapatrilat was not found in rat, dog, or human urine samples indicating extensive metabolism in vivo.

Several novel human RNA viruses were identified in 1995-96 and were partially characterized that apparently can cause acute and chronic hepatitis both in monkeys and humans. These new viruses are related to the flavivirus hepatitis C. It is known to be distinct from other human hepatitis viruses (A, B, C, D, E). Three viruses, identified by investigators at Abbott Labs, have been termed GB-A, GB-B and GB-C. GB-A and GB-B are likely tamarin viruses whereas GB-C infects humans only. All these three viruses are the isolates of the same virus termed HGV, which is positive stranded RNA virus. The genomic sequences of these viruses have been determined by different researchers, which were found to be 1600 nucleotide long. Another group at Genelabs Technologies has identified and determined the complete genomic sequence of a virus they termed hepatitis G virus (HGV). Based on genomic sequence comparisons HGV is probably the same as GB-C. It is highly controversial virus regarding pathogenecity, mode of transmission and site of replication.

Omapatrilat, the most clinically advanced member of a new class of cardiovascular agents, vasopeptidase inhibitors, is under development at Bristol-Myers Squibb Pharmaceutical Research Institute for the treatment of hypertension and heart failure. An electrospray LC/MS/MS method has been developed and validated for the simultaneous determination of omapatrilat and its four metabolites in human plasma. Since omapatrilat and two of the metabolites are sulfhydryl-containing compounds, methyl acrylate was used to stabilize these compounds in human blood and plasma samples. Methyl acrylate reacted instantly with the sulfhydryl group to form a derivative that was stable in blood and plasma. Extraction of the analytes from plasma samples was achieved by semiautomated liquid-liquid extraction, where a robotic liquid handler performed the liquid-transferring steps. The mass spectrometer was operated in the negative ion selected-reaction-monitoring mode. The calibration curve ranges were 0.5-250 ng/mL for omapatrilat and one metabolite and 2.0-250 ng/mL for the other three metabolites.

Biological samples are normally collected and stored frozen in capped tubes until analysis. To obtain aliquots of biological samples for analysis, the sample tubes have to be thawed, uncapped, samples removed and then recapped for further storage. In this paper, we report an automated method of sample transfer devised to eliminate the uncapping and recapping process. This sampling method was incorporated into an automated liquid–liquid extraction procedure of plasma samples. Using a robotic system, the plasma samples were transferred directly from pierceable capped tubes into microtubes contained in a 96-position block. The aliquoted samples were extracted with methyl- tert -butyl ether in the same microtubes. The supernatant organic layers were transferred to a 96-well collection plate and evaporated to dryness. The dried extracts were reconstituted and injected from the same plate for analysis by liquid chromatography with tandem mass spectrometry.

Three extraction procedures were developed for the quantitative determination of a carboxylic acid containing analyte (I) in human plasma by high-performance liquid chromatography (HPLC) with negative ion electrospray tandem mass spectrometry (MS-MS). The first procedure was based on the manual liquid-liquid extraction (LLE) of the acidified plasma samples with methyl tert.-butyl ether. The second procedure was based on the automation of the manual LLE procedure using 96-well collection plates and a robotic liquid handling system. The third approach was based on automated solid-phase extraction (SPE) using 96-well SPE plates and a robotic liquid handling system. A lower limit of quantitation of 50 pg/ml was achieved using all three extraction procedures. The total time required to prepare calibration curve standards, aliquot the standards and plasma samples, and process a total of 96 standards and samples by manual LLE was three-times longer than the time required for 96-well SPE or 96-well LLE (4 h, 50 min vs. 1 h, 43 min). Even more importantly, the time the bioanalyst physically spent on the 96-well LLE or 96-well SPE procedure was only a small fraction of the time spent on the manual LLE procedure (

Background Transfusion transmitted infections create significant burden on health care system. Donor selection is of paramount importance because infected individuals serve as an asymptomatic reservoir and a potential source of transmission. Methods A retrospective study was carried out in healthy blood donors in the Lady Reading Hospital Peshawar, Pakistan over a period of three and a half years i.e., from January 2008 to June 2011, to determine the prevalence of HBV, HCV, HIV and syphilis in order to provide information for relevant polices. Results Out of 1,27,828 sample of blood donors, recorded mean prevalence for HBs Ag, anti-HCV, anti-HIV and syphilis was 2.68%, 2.46%, 0.06% and 0.43%, respectively, with an increasing trend in frequencies of transfusion transmitted infections (TTIs). Conclusions This study reflects that blood transfusion is one of the leading risk factor of spread of the TTIs, which showed the need and importance of the mandatory screening of these infectious markers in blood donations.

There is a notable dearth of data about Hepatitis B Virus (HBV) and Hepatitis C Virus(HCV) prevalence in Afghanistan. Awareness program and research capacity in the field of hepatitis are very limited in Afghanistan. Number of vulnerabilities and patterns of risk behaviors signal the need to take action now. Thirty one studies dating from October 2003 to 2011 were included, consisting the data of 1,32,981 individuals for HBV and 1,32,500 individuals for HCV. Percentage prevalence was 1.9% for HBV and 1.1% for HCV in all available Afghanistan population. Most at risk population to hepatitis include injecting drug users who share needles and female sex workers, while truck drivers, prisoners and homosexual men needs attention, as their statistical figure are missing. Data suggests that high incidence of intravenous drug use, sexual activities, unsafe blood transfusion procedures and mobility are major risk factors for hepatitis transmission. This review is based on analysis of the limited available data in Afghanistan. Although there are many underlying vulnerability factors, it appears that Afghanistan remains at an early epidemic phase. Further research is required to determine the seroprevalence and prevalent genotype(s) of HBV and HCV in all provinces in Afghanistan. This article provides some key insights into the potential and likely future transmission dynamics of Hepatitis which will serve as a guide in the identification of priority areas in term of high risk groups and risk behaviours in the country and will assist to develop urgent strategic plans to combat the future burden of Hepatitis in Afghanistan.