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1.  Crosstalk between Edc4 and Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling in mRNA Decapping 
The mammalian target of rapamycin complex 1 (mTORC1) is involved in the cellular transcription and translation processes. The undertaken study characterized the enhancer of mRNA decapping protein 4 (Edc4) as mTORC1 interacting protein. Human T lymphoblast (CCRF-CEM) cells were used for mTORC1 purification. Co-immunoprecipitation coupled with immunoblotting analysis was used to confirm the interaction of Edc4 in mTORC1 specific purifications. Further assays were incorporated to conclude the role of mTORC1 in mRNA decapping via Edc4. Edc4 was identified as a new interacting protein with mTORC1 in both the endogenous and myc-tag raptor component mTORC1 specific purifications. Quantitative co-localization using confocal microscopy demonstrated that raptor component of mTORC1 coexists with Edc4 in processing (P) bodies, a site for mRNA degradation. Incubation of cells with rapamycin, a known inhibitor of mTOR kinase activity, increased the total Edc4 protein expression but at the same time decreased the Edc4 interaction with mTORC1. Moreover, rapamycin treatment resulted in a significant decrease in total serine phosphorylated Edc4 protein signal and the total 5'-capped mRNA. These findings provide the first evidence for the pivotal role of mTORC1 in Edc4 regulation. Further in-depth studies are required to get a complete understanding of molecular crosstalk between mTORC1 signaling and mRNA decapping pathway.
doi:10.3390/ijms151223179
PMCID: PMC4284759  PMID: 25514416
mammalian target of rapamycin complex 1 (mTORC1) purification; mTORC1 interacting proteins; enhancer of mRNA decapping protein 4 (Edc4); mRNA decapping
2.  Identification of the Novel Interacting Partners of the Mammalian Target of Rapamycin Complex 1 in Human CCRF-CEM and HEK293 Cells 
The present study was undertaken to identify proteins that interact with the mammalian target of rapamycin complex 1 (mTORC1) to enable it to carry out its crucial cell signaling functions. Endogenous and myc-tag mTORC1 was purified, in-gel tryptic digested and then identified by nano-LC ESI Q-TOF MS/MS analysis. A total of nine novel interacting proteins were identified in both endogenous and myc-tag mTORC1 purifications. These new mTORC1 interacting partners include heterogeneous nuclear ribonucleoproteins A2/B1, enhancer of mRNA decapping protein 4, 60S acidic ribosomal protein, P0, nucleolin, dynamin 2, glyceraldehyde 3 phosphate dehydrogenase, 2-oxoglutarate dehydrogenase, glycosyl transferase 25 family member 1 and prohibitin 2. Furthermore hnRNP A2/B1 and dynamin 2 interaction with mTORC1 was confirmed on immunoblotting. The present study has for the first time identified novel interacting partners of mTORC1 in human T lymphoblasts (CCRF-CEM) and human embryonic kidney (HEK293) cells. These new interacting proteins may offer new targets for therapeutic interventions in human diseases caused by perturbed mTORC1 signaling.
doi:10.3390/ijms15034823
PMCID: PMC3975426  PMID: 24646917
mTORC1 purification; mTORC1 interacting proteins; hnRNP A2/B1 and dynamin 2
3.  MHD Boundary Layer Slip Flow and Heat Transfer of Ferrofluid along a Stretching Cylinder with Prescribed Heat Flux 
PLoS ONE  2014;9(1):e83930.
This study investigates the magnetohydrodynamic (MHD) flow of ferrofluid along a stretching cylinder. The velocity slip and prescribed surface heat flux boundary conditions are employed on the cylinder surface. Water as conventional base fluid containing nanoparticles of magnetite (Fe3O4) is used. Comparison between magnetic (Fe3O4) and non-magnetic (Al2O3) nanoparticles is also made. The governing non-linear partial differential equations are reduced to non-linear ordinary differential equations and then solved numerically using shooting method. Present results are compared with the available data in the limiting cases. The present results are found to be in an excellent agreement. It is observed that with an increase in the magnetic field strength, the percent difference in the heat transfer rate of magnetic nanoparticles with Al2O3 decreases. Surface shear stress and the heat transfer rate at the surface increase as the curvature parameter increases, i.e curvature helps to enhance the heat transfer.
doi:10.1371/journal.pone.0083930
PMCID: PMC3898924  PMID: 24465388
4.  Bilateral asymmetrical duplicated origin of vertebral arteries: Multidetector row CT angiographic study 
Bilateral duplicated origin of V-1 segment of vertebral arteries is an extremely rare vascular variant and only two such cases have been reported so far. Presence of this vascular abnormality was observed incidentally in a 36-year-old male patient, with a complaint of dizziness, evaluated by multidetector row computed tomography (CT) angiography. Two limbs of the right vertebral artery arose from the right subclavian artery and fused to form a single vessel at the interval between fourth and fifth cervical vertebrae, which entered the foramen transversarium of fourth cervical vertebra. On the left side, the medial limb originated directly from the arch of aorta and the lateral limb from the left subclavian artery, and both united at the interval between fifth and sixth cervical vertebrae to form a single vessel which entered the foramen transversarium of fifth cervical vertebra. No other cerebrovascular pathology like aneurysm, fenestration, dissection, and stenosis was detected, which could be correlated with the symptoms of the patient. This rare congenital vascular anomaly has diagnostic and therapeutic implications in any intervention involving the vertebral artery.
doi:10.4103/0971-3026.130699
PMCID: PMC4028918  PMID: 24851007
Anomalous origin; bilateral duplication; dual origin; extracranial part; vertebral artery
5.  Design of New and Potent Diethyl Thiobarbiturates as Urease Inhibitors: A Computational Approach 
Bioinformation  2014;10(5):299-307.
Urease is an important enzyme both in agriculture and medicine research. Strategies based on urease inhibition is critically considered as the first line treatment of infections caused by urease producing bacteria. Since, urease possess agro-chemical and medicinal importance, thus, it is necessary to search for the novel compounds capable of inhibiting this enzyme. Several computational methods were employed to design novel and potent urease inhibitors in this work. First docking simulations of known compounds consists of a set of arylidine barbiturates (termed as reference) were performed on the Bacillus pasteurii (BP) urease. Subsequently, two fold strategies were used to design new compounds against urease. Stage 1 comprised of the energy minimization of enzyme-ligand complexes of reference compounds and the accurate prediction of the molecular mechanics generalized born (MMGB) interaction energies. In the second stage, new urease inhibitors were then designed by the substitution of different groups consecutively in the aryl ring of the thiobarbiturates and N, N-diethyl thiobarbiturates of the reference ligands.. The enzyme-ligand complexes with lowest interaction energies or energies close to the calculated interaction energies of the reference molecules, were selected for the consequent chemical manipulation. This was followed by the substitution of different groups on the 2 and 5 positions of the aryl ring. As a result, several new and potent diethyl thiobarbiturates were predicted as urease inhibitors. This approach reflects a logical progression for early stage drug discovery that can be exploited to successfully identify potential drug candidates.
doi:10.6026/97320630010299
PMCID: PMC4070040  PMID: 24966538
Urease Inhibitor; Molecular docking; Interaction energy; H. pylori; MOE
6.  Mutational spectrum of the CYP1B1 gene in Pakistani patients with primary congenital glaucoma: Novel variants and genotype-phenotype correlations 
Molecular Vision  2014;20:991-1001.
Purpose
This study aimed to investigate the role of CYP1B1 mutations in primary congenital glaucoma (PCG) in Pakistani patients.
Methods
After consent was received, 20 families with at least more than one member affected with primary congenital glaucoma were enrolled in the study. The disease was confirmed with standard ophthalmological investigations. Genomic DNA was extracted from whole blood for localization of linkage and sequencing. Bioinformatics tools were used to assess the predicted pathological role of novel variants.
Results
Ten out of 20 families (50%, 10/20) showed homozygosity with CYP1B1-linked short tandem repeat (STR) markers. On direct sequencing of the CYP1B1 gene in the linked families, six mutations, including two novel pathogenic variants, were identified. p. R390H was the most frequently found mutation in five families (50%, 5/10), whereas c.868_869insC, p.E229K, and p.A115P were found once in three families. Two novel mutations, a missense mutation (p.G36D) and an in-frame deletion mutation (p.G67-A70del), were segregated with disease phenotype in two families. Age of disease onset was congenital in all mutations; however, disease severity and response to clinical interventions varied among the mutations and families. Haplotype analysis using five polymorphisms revealed a distinct haplotype for a common mutation.
Conclusions
This is the largest cohort of Pakistani patients with PCG to be genetically screened for CYP1B1 mutations. Identifying common mutation and genotype-phenotype correlations may help in genetic testing and better prognosis for the disease. Novel mutations identified in the study may help in better understanding the pathophysiology of CYP1B1-associated glaucoma.
PMCID: PMC4087121  PMID: 25018621
7.  Modelling and simulation of mutant alleles of breast cancer metastasis suppressor 1 (BRMS1) gene 
Bioinformation  2014;10(7):454-459.
Computational tools occupy the prime position in the analysis of large volume of post-genomic data. These tools have advantage over the wet lab experiments in terms of high coverage, cost and time. Breast cancer is the most common cancer in females worldwide. It is a genetically heterogeneous disorder and many genes are involved in the pathway of the disease. Mutations in metastasis suppressor gene are the major cause of the disease. In this study, the effects of mutations in breast cancer metastasis suppressor 1gene upon protein structure and function were examined by means of computational tools and information from databases.This study can be useful to predict the potential effect of every allelic variant, devise new biological experiments and to interpret and predict the patho-physiological impact of new mutations or non-synonymous polymorphisms.
doi:10.6026/97320630010454
PMCID: PMC4135295  PMID: 25187687
Breast cancer; BRMS1; Mutation analysis; Homology modeling
8.  Antibiotic resistance of E. coli isolates from urine samples of Urinary Tract Infection (UTI) patients in Pakistan 
Bioinformation  2014;10(7):419-422.
Drug resistance is becoming alarming with the passage of time worldwide in general and in third world countries in particular. Human urine specimens of patients of urinary tract infection at Sheikh Zayed hospital, Lahore, Pakistan were analyzed for drug resistance in Escherichia coli. A total of 69 Escherichia coli isolates from human urine specimens were obtained and screened for their antibiograms. A total of seven antibiotic resistance profiles were obtained with over 65% of the isolates showing multi-drug resistance. Very high resistance levels were detected against augmentin and gentamicin (87.5 &77.5 % respectively) while imipenem and tazocin recorded the least resistance levels (32.5% and 12.5% respectively) among the isolates.
doi:10.6026/97320630010419
PMCID: PMC4135289  PMID: 25187681
E.coli; Isolate; susceptibility; Antibiotic resistance; Urinart Tract infection
9.  Domain analyses of Usher syndrome causing Clarin-1 and GPR98 protein models 
Bioinformation  2014;10(8):491-495.
Usher syndrome is an autosomal recessive disorder that causes hearing loss, Retinitis Pigmentosa (RP) and vestibular dysfunction. It is clinically and genetically heterogeneous disorder which is clinically divided into three types i.e. type I, type II and type III. To date, there are about twelve loci and ten identified genes which are associated with Usher syndrome. A mutation in any of these genes e.g. CDH23, CLRN1, GPR98, MYO7A, PCDH15, USH1C, USH1G, USH2A and DFNB31 can result in Usher syndrome or non-syndromic deafness. These genes provide instructions for making proteins that play important roles in normal hearing, balance and vision. Studies have shown that protein structures of only seven genes have been determined experimentally and there are still three genes whose structures are unavailable. These genes are Clarin-1, GPR98 and Usherin. In the absence of an experimentally determined structure, homology modeling and threading often provide a useful 3D model of a protein. Therefore in the current study Clarin-1 and GPR98 proteins have been analyzed for signal peptide, domains and motifs. Clarin-1 protein was found to be without any signal peptide and consists of prokar lipoprotein domain. Clarin-1 is classified within claudin 2 super family and consists of twelve motifs. Whereas, GPR98 has a 29 amino acids long signal peptide and classified within GPCR family 2 having Concanavalin A-like lectin/glucanase superfamily. It was found to be consists of GPS and G protein receptor F2 domains and twenty nine motifs. Their 3D structures have been predicted using I-TASSER server. The model of Clarin-1 showed only α-helix but no beta sheets while model of GPR98 showed both α-helix and β sheets. The predicted structures were then evaluated and validated by MolProbity and Ramachandran plot. The evaluation of the predicted structures showed 78.9% residues of Clarin-1 and 78.9% residues of GPR98 within favored regions. The findings of present study has resulted in the three dimensional structure prediction and conserved domain analysis which will be quite beneficial in better understanding of molecular components, protein-protein interaction, clinical heterogeneity and pathophysiology of Usher syndrome.
doi:10.6026/97320630010491
PMCID: PMC4166767  PMID: 25258483
10.  Prevalence of plasmid-mediated AmpC β-lactamases in Escherichia coli and Klebsiella pneumonia at tertiary care hospital of Islamabad, Pakistan 
Enterobacteriaceae produces AmpC β-lactamases that make them resistant to commonly used antibiotics. AmpC β-lactamases can be chromosomal-mediated or plasmid-mediated AmpC β-lactamases (PABLs). The present study was undertaken to determine the occurrence of PABLs production in clinical isolates in Escherichia coli and Klebsiella pneumoniae.
Among 1328 culture positive samples, 511 isolates were identified as E. coli (81.02%, n = 414) and K. pneumonia (18.98%, n = 97). Cefoxitin resistance was observed in E. coli (19.57%, n = 81) and K. pneumoniae (22.68%, n = 22). Out of these cefoxitin resistant isolates, 40.74% (n = 33) E. coli and 54.55% (n = 12) K. pneumoniae came out to be PABL producers. Prevalence of both PABLs and ESBLs in E. coli and K. pneumoniae was 29.24% (n = 8) and 47% (n = 5), respectively. Isolates coproducing PABLs and ESBL exhibited increased minimum inhibitory concentrations (MICs) for selected cephalosporins. This study documented a high frequency of PABLs producing isolates from hospital which may lead to serious therapeutic problem.
doi:10.1556/EuJMI.3.2013.4.5
PMCID: PMC3838542  PMID: 24294496
antibiogram; E. coli; ESBLs; K. pneumoniae; PABLs
11.  Antibacterial activity of some medicinal plants against selected human pathogenic bacteria 
Medicinal plants are traditionally used for the treatment of human infections. The present study was undertaken to investigate Bergenia ciliata, Jasminum officinale, and Santalum album for their potential activity against human bacterial pathogens.
B. ciliata, J. officinale, and S. album extracts were prepared in cold and hot water. The activity of plant extracts and selected antibiotics was evaluated against five bacterial pathogens including Staphylococcus aureus, Bacillus subtilis, Proteus vulgaris, Pseudomonas aeruginosa, and Escherichia coli using agar well diffusion method.
Among the three medicinal plants, B. ciliata extracts displayed potential activity against bacterial pathogens. Cold water extract of Bergenia ciliate showed the highest activity against B. subtilis, which is comparable with a zone of inhibition exhibited by ceftriaxone and erythromycin. J. officinale and S. album extracts demonstrated variable antibacterial activity. Further studies are needed to explore the novel antibacterial bioactive molecules.
doi:10.1556/EuJMI.3.2013.4.6
PMCID: PMC3838543  PMID: 24294497
antibacterial activity; Bergenia ciliata; Jasminum officinale; plant extracts; Santalum album
12.  Initiation and progression of mechanical damage in the intervertebral disc under cyclic loading using continuum damage mechanics methodology: A finite element study 
Journal of biomechanics  2012;45(11):1934-1940.
It is difficult to study the breakdown of disc tissue over several years of exposure to bending and lifting by experimental methods. There is also no finite element model that elucidates the failure mechanism due to repetitive loading of the lumbar motion segment. The aim of this study was to refine an already validated poro-elastic finite element model of lumbar motion segment to investigate the initiation and progression of mechanical damage in the disc under simple and complex cyclic loading conditions. Continuum damage mechanics methodology was incorporated into the finite element model to track the damage accumulation in the annulus in response to the repetitive loading. The analyses showed that the damage initiated at the posterior inner annulus adjacent to the endplates and propagated outwards towards its periphery under all loading conditions simulated. The damage accumulated preferentially in the posterior region of the annulus. The analyses also showed that the disc failure is unlikely to happen with repetitive bending in the absence of compressive load. Compressive cyclic loading with low peak load magnitude also did not create the failure of the disc. The finite element model results were consistent with the experimental and clinical observations in terms of the region of failure, magnitude of applied loads and the number of load cycles survived.
doi:10.1016/j.jbiomech.2012.05.022
PMCID: PMC3787695  PMID: 22682891
Lumbar spine; Disc degeneration; Fatigue failure; Finite element modeling; Continuum damage mechanics
13.  A Biomechanical Comparison of Intralaminar C7 Screw Constructs with and without Offset Connector Used for C6-7 Cervical Spine Immobilization : A Finite Element Study 
Objective
The offset connector can allow medial and lateral variability and facilitate intralaminar screw incorporation into the construct. The aim of this study was to compare the biomechanical characteristics of C7 intralaminar screw constructs with and without offset connector using a three dimensional finite element model of a C6-7 cervical spine segment.
Methods
Finite element models representing C7 intralaminar screw constructs with and without the offset connector were developed. Range of motion (ROM) and maximum von Mises stresses in the vertebra for the two techniques were compared under pure moments in flexion, extension, lateral bending and axial rotation.
Results
ROM for intralaminar screw construct with offset connector was less than the construct without the offset connector in the three principal directions. The maximum von Misses stress was observed in the C7 vertebra around the pedicle in both constructs. Maximum von Mises stress in the construct without offset connector was found to be 12-30% higher than the corresponding stresses in the construct with offset connector in the three principal directions.
Conclusion
This study demonstrated that the intralaminar screw fixation with offset connector is better than the construct without offset connector in terms of biomechanical stability. Construct with the offset connector reduces the ROM of C6-7 segment more significantly compared to the construct without the offset connector and causes lower stresses around the C7 pedicle-vertebral body complex.
doi:10.3340/jkns.2013.53.6.331
PMCID: PMC3756124  PMID: 24003366
Cervical spine; Intralaminar screw; Finite element method; Biomechanics
14.  Heat Transfer in a Micropolar Fluid over a Stretching Sheet with Newtonian Heating 
PLoS ONE  2013;8(4):e59393.
This article looks at the steady flow of Micropolar fluid over a stretching surface with heat transfer in the presence of Newtonian heating. The relevant partial differential equations have been reduced to ordinary differential equations. The reduced ordinary differential equation system has been numerically solved by Runge-Kutta-Fehlberg fourth-fifth order method. Influence of different involved parameters on dimensionless velocity, microrotation and temperature is examined. An excellent agreement is found between the present and previous limiting results.
doi:10.1371/journal.pone.0059393
PMCID: PMC3614560  PMID: 23565151
15.  Prevalence of HCV among the young male blood donors of Quetta region of Balochistan, Pakistan 
Virology Journal  2013;10:83.
Background
Hepatitis C, caused by hepatitis C virus (HCV) is a contagious disease of the liver which infects more than 170 million people world-wide and around 16 million in Pakistan. HCV associated infection spreads mainly by blood-to-blood contact. In recent years, many studies have been conducted to determine the prevalence of HCV infection in Pakistan; however, no data is available on HCV infection from the largest province of Pakistan. Therefore, the present study focuses on the prevalence of HCV infection in the young male blood donor population of Quetta region of Balochistan, Pakistan.
Methods
A total of 356 blood samples were collected from blood donors (age range 17–25 years) at Combined Military Hospital (CMH), Quetta, Balochistan, Pakistan. Blood samples were screened for HCV positivity by Immunochromatographic test (ICT) and Enzyme Linked Immunosorbant Assay (ELISA).
Results
Out of 356 blood samples, the overall HCV prevalence was 20.8%. Among the HCV positive cases, the age group with 25 years was more frequently infected with a prevalence of 26.3%.
Conclusions
The present study provides the preliminary information about high HCV prevalence among the young male donor population in Balochistan province. This data may be helpful in formulating public health strategy for the prevention of risk factors associated with spreading of the disease. Furthermore, we recommend that in public sector hospitals and health care units ELISA should be preferred for anti-HCV detection over ICT.
doi:10.1186/1743-422X-10-83
PMCID: PMC3600024  PMID: 23497435
Enzyme linked immunosorbant assay; Hepatitis C virus; Immunochromatography; Quetta; Pakistan
16.  Computer aided screening of Accacia nilotica phytochemicals against HCV NS3/4a 
Bioinformation  2013;9(14):710-714.
Background: HCV has become a leading cause of liver cirrhosis and hepatocellular carcinoma and is a major health concern worldwide. To date, there is no vaccine available in the market to tackle this disease, therefore there is a strong need to develop antiviral compounds that can target all genotypes of HCV with the same efficiency. Medicinal plants have low cost and are less toxic therefore, extracts of medicinal plants can serve as important antiviral agents against HCV. This study was designed to screen phytochemicals of Accacia nilotica to find a potent drug candidate that can inhibit HCV infection effectively.
Results: Docking of NS3/4A protease and Flavonoids of Accacia nilotica revealed that most of the flavonoids bound deeply with the active site of NS3/4A protease. Compound 01 showed a high ranking on docking score. All other compounds also showed reliable docking scores and had interactions with the binding cavity of NS3/4A protease, suggesting them as a potent drug candidate to block HCV replication.
Conclusion: To recognize binding interactions of Accacia nilotica phytochemicals with NS3/4A protease, molecular docking was performed to find potential inhibitor against NS3/4A protease of HCV. After post docking analysis, important interactions were found between active compounds and active site of NS3/4A protease. It can be concluded from the study that phytochemicals of Accacia nilotica may serve as a potential drug candidate with relatively simple structural changes against HCV NS3/4A protease.
doi:10.6026/97320630009710
PMCID: PMC3746092  PMID: 23976825
17.  Molecular screening of phytochemicals from Amelanchier Alnifolia against HCV NS3 protease/helicase using computational docking techniques 
Bioinformation  2013;9(19):978-982.
Hepatitis C is serious health concern worldwide caused by HCV. It causes liver cirrhosis and hepato-cellular carcinoma. Development of prevention solutions is under progress. Meanwhile, the treatment of the viral disease using compounds isolated from natural medicinal plants is promising. The traditional use of photo-chemicals from medicinal plants like Amelanchier alnifolia for viral treatment is hopeful. Therefore, it is of interest to screen for flavonoids from Amelanchier alnifolia against protein targets of HCV. Hence, we assessed the binding of flavonoids to HCV NS3/4A protease and helicase proteins. Results show that Quercitin 3- galactoside and 3-glucosideshowed good binding score with protease and helicase respectively. Their interaction/binding sites are documented in this report. This data provide insights for the consideration of flavonoids as potential inhibitors of HCV/NS3/4A protease and helicase.
doi:10.6026/97320630009978
PMCID: PMC3867651  PMID: 24391361
18.  Rabies molecular virology, diagnosis, prevention and treatment 
Virology Journal  2012;9:50.
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.
doi:10.1186/1743-422X-9-50
PMCID: PMC3307483  PMID: 22348291
Rabies; Zoonosis; Vaccine; Prevention
19.  Hepatitis C virus to hepatocellular carcinoma 
Hepatitis C virus causes acute and chronic hepatitis and can lead to permanent liver damage and hepatocellular carcinoma (HCC) in a significant number of patients via oxidative stress, insulin resistance (IR), fibrosis, liver cirrhosis and HCV induced steatosis. HCV induced steatosis and oxidative stress causes steato-hepatitis and these pathways lead to liver injury or HCC in chronic HCV infection. Steatosis and oxidative stress crosstalk play an important role in liver damage in HCV infection. This Review illustrates viral and host factors which induce Oxidative stress, steatosis and leads toward HCC. It also expresses Molecular cascade which leads oxidative stress and steatosis to HCC.
doi:10.1186/1750-9378-7-2
PMCID: PMC3293064  PMID: 22289144
20.  Popliteal Pterygium Syndrome: A Rare Entity 
The popliteal pterygium syndrome is a congenital malformation that includes orofacial, musculoskeletal and genitourinary anomalies. It is a rare autosomal dominant disorder. We report one family with popliteal pterygium syndrome affecting father and his two daughters, who underwent surgical corrections for multiple congenital malformations.
PMCID: PMC3418038  PMID: 22953299
Popliteal pterygium syndrome;  Autosomal dominant disorder;  Familial
21.  Fetal calf serum heat inactivation and lipopolysaccharide contamination influence the human T lymphoblast proteome and phosphoproteome 
Proteome Science  2011;9:71.
Background
The effects of fetal calf serum (FCS) heat inactivation and bacterial lipopolysaccharide (LPS) contamination on cell physiology have been studied, but their effect on the proteome of cultured cells has yet to be described. This study was undertaken to investigate the effects of heat inactivation of FCS and LPS contamination on the human T lymphoblast proteome. Human T lymphoblastic leukaemia (CCRF-CEM) cells were grown in FCS, either non-heated, or heat inactivated, having low (< 1 EU/mL) or regular (< 30 EU/mL) LPS concentrations. Protein lysates were resolved by 2-DE followed by phospho-specific and silver nitrate staining. Differentially regulated spots were identified by nano LC ESI Q-TOF MS/MS analysis.
Results
A total of four proteins (EIF3M, PRS7, PSB4, and SNAPA) were up-regulated when CCRF-CEM cells were grown in media supplemented with heat inactivated FCS (HE) as compared to cells grown in media with non-heated FCS (NHE). Six proteins (TCPD, ACTA, NACA, TCTP, ACTB, and ICLN) displayed a differential phosphorylation pattern between the NHE and HE groups. Compared to the low concentration LPS group, regular levels of LPS resulted in the up-regulation of three proteins (SYBF, QCR1, and SUCB1).
Conclusion
The present study provides new information regarding the effect of FCS heat inactivation and change in FCS-LPS concentration on cellular protein expression, and post-translational modification in human T lymphoblasts. Both heat inactivation and LPS contamination of FCS were shown to modulate the expression and phosphorylation of proteins involved in basic cellular functions, such as protein synthesis, cytoskeleton stability, oxidative stress regulation and apoptosis. Hence, the study emphasizes the need to consider both heat inactivation and LPS contamination of FCS as factors that can influence the T lymphoblast proteome.
doi:10.1186/1477-5956-9-71
PMCID: PMC3280938  PMID: 22085958
CCRF-CEM cells; FCS heat inactivation; LPS; proteome; phosphoproteome
22.  Inhibition of HCV 3a genotype entry through Host CD81 and HCV E2 antibodies 
Background
HCV causes acute and chronic hepatitis which can eventually lead to permanent liver damage hepatocellular carcinoma and death. HCV glycoproteins play an important role in HCV entry by binding with CD81 receptors. Hence inhibition of virus at entry step is an important target to identify antiviral drugs against HCV.
Methods and result
The present study elaborated the role of CD81 and HCV glycoprotein E2 in HCV entry using retroviral pseudo-particles of 3a local genotype. Our results demonstrated that HCV specific antibody E2 and host antibody CD81 showed dose- dependent inhibition of HCV entry. HCV E2 antibody showed 50% reduction at a concentration of 1.5 ± 1 μg while CD81 exhibited 50% reduction at a concentration of 0.8 ± 1 μg. In addition, data obtained with HCVpp were also confirmed with the infection of whole virus of HCV genotype 3a in liver cells.
Conclusion
Our data suggest that HCV specific E2 and host CD81 antibodies reduce HCVpp entry and full length viral particle and combination of host and HCV specific antibodies showed synergistic effect in reducing the viral titer.
doi:10.1186/1479-5876-9-194
PMCID: PMC3228851  PMID: 22074322
23.  Differential proteome analysis of human embryonic kidney cell line (HEK-293) following mycophenolic acid treatment 
Proteome Science  2011;9:57.
Background
Mycophenolic acid (MPA) is widely used as a post transplantation medicine to prevent acute organ rejection. In the present study we used proteomics approach to identify proteome alterations in human embryonic kidney cells (HEK-293) after treatment with therapeutic dose of MPA. Following 72 hours MPA treatment, total protein lysates were prepared, resolved by two dimensional gel electrophoresis and differentially expressed proteins were identified by QTOF-MS/MS analysis. Expressional regulations of selected proteins were further validated by real time PCR and Western blotting.
Results
The proliferation assay demonstrated that therapeutic MPA concentration causes a dose dependent inhibition of HEK-293 cell proliferation. A significant apoptosis was observed after MPA treatment, as revealed by caspase 3 activity. Proteome analysis showed a total of 12 protein spots exhibiting differential expression after incubation with MPA, of which 7 proteins (complement component 1 Q subcomponent-binding protein, electron transfer flavoprotein subunit beta, cytochrome b-c1 complex subunit, peroxiredoxin 1, thioredoxin domain-containing protein 12, myosin regulatory light chain 2, and profilin 1) showed significant increase in their expression. The expression of 5 proteins (protein SET, stathmin, 40S ribosomal protein S12, histone H2B type 1 A, and histone H2B type 1-C/E/F/G/I) were down-regulated. MPA mainly altered the proteins associated with the cytoskeleton (26%), chromatin structure/dynamics (17%) and energy production/conversion (17%). Both real time PCR and Western blotting confirmed the regulation of myosin regulatory light chain 2 and peroxiredoxin 1 by MPA treatment. Furthermore, HT-29 cells treated with MPA and total kidney cell lysate from MMF treated rats showed similar increased expression of myosin regulatory light chain 2.
Conclusion
The emerging use of MPA in diverse pathophysiological conditions demands in-depth studies to understand molecular basis of its therapeutic response. The present study identifies the myosin regulatory light chain 2 and peroxiredoxin 1 along with 10 other proteins showing significant regulation by MPA. Further characterization of these proteins may help to understand the diverse cellular effects of MPA in addition to its immunosuppressive activity.
doi:10.1186/1477-5956-9-57
PMCID: PMC3189873  PMID: 21933383
HEK-293 cells; proteome; mycophenolic acid; drug toxicology; differential proteomics
24.  Epigastric Heteropagus Twin 
Parasitic twining is a rare type of monozygotic monochorionic monoamniotic asymmetrical conjoined twin. We report a case of epigastric heteropagus twin. An ultrasound scan showed a defect of 1.5 cm in the epigastrium. CT showed soft tissue lobulated mass with fat and air components coming out of the epigastric defect. At operation rudimentary alimentary canal with no viscera, was found in the parasite. The parasite was easily separated from the host.
PMCID: PMC3418030  PMID: 22953291
Conjoined twin;  Monochorionic monoamniotic;  Epigastric heteropagus twin
25.  Gene structure and mutant alleles of PCDH15: nonsyndromic deafness DFNB23 and type 1 Usher syndrome 
Human genetics  2008;124(3):215-223.
Mutations of PCDH15, encoding protocadherin 15, can cause either combined hearing and vision impairment (type 1 Usher syndrome; USH1F) or nonsyndromic deafness (DFNB23). Human PCDH15 is reported to be comprised of 35 exons and encodes a variety of isoforms with 3 to 11 ectodomains (EC), a transmembrane domain and a carboxy-terminal cytoplasmic domain (CD). Building on these observations we describe an updated gene structure that has four additional exons of PCDH15 and isoforms that can be subdivided into four classes. Human PCDH15 encodes three alternative, evolutionarily conserved unique cytoplasmic domains (CD1, CD2 or CD3). Families ascertained on the basis of prelingual hearing loss were screened for linkage of this phenotype to markers for PCDH15 on chromosome 10q21.1. In seven of twelve families segregating USH1 we identified homozygous mutant alleles (1 missense, 1 splice site, 3 nonsense and 2 deletion mutations) of which six are novel. One family was segregating nonsyndromic deafness DFNB23 due to a homozygous missense mutation. To date in our cohort of 557 Pakistani families, we have found 11 different PCDH15 mutations that account for deafness in 13 families. Molecular modeling provided mechanistic insight into the phenotypic variation in severity of the PCDH15 missense mutations. We did not find pathogenic mutations in five of the twelve USH1 families linked to markers for USH1F, which suggest either the presence of mutations of yet additional undiscovered exons of PCDH15, mutations in the introns or regulatory elements of PCDH15, or an additional locus for type I USH at chromosome 10q21.1.
doi:10.1007/s00439-008-0543-3
PMCID: PMC2716558  PMID: 18719945
DFNB23; Usher syndrome; protocadherin 15; PCDH15; deafness; retinitis pigmentosa

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