Mitochondrial DNA diversity patterns in Pakistani buffalo

The Indian subcontinent is considered to be the likely centre of river buffalo domestication, based on population dynamics, archaeological evidence and genetic diversity. Recent studies on mitochondrial DNA diversity have drawn useful conclusions about the domestication history of Bubalus bubalis. The conclusions of these studies are, however, incomplete, unless samples can also be analysed from Pakistan, which contains the second largest buffalo population of the world. Here, we report the results of the first study on mitochondrial D-loop sequence diversity in five breeds of Pakistani buffalo. Analysis of sequence variations in 503-bp of the D-loop region of 123 animals revealed 52 haplotypes, including 40 singletons. Multidimensional display of breed pairwise F(ST) values revealed no strong clustering of breeds. Bayesian, maximum parsimony, neighbour joining and UPGMA trees revealed a topology consistent with domestication as well as subsequent introgression of multiple maternal lineages from the wild stocks. Reduced median network analysis provided evidence of population expansion from more than one set of haplotypes. The study also confirmed that Pakistani buffalo are of the river type. The observed mitochondrial D-loop sequence diversity suggests that Pakistani areas bordering India might have contributed to the initiation of domestication of the present-day river buffalo.     

A spectrum of novel NPHS1 and NPHS2 gene mutations in pediatric nephrotic syndrome patients from Pakistan

Background

Mutations in the NPHS1 and NPHS2 genes are among the main causes of early-onset and familial steroid resistant nephrotic syndrome respectively. This study was carried out to assess the frequencies of mutations in these two genes in a cohort of Pakistani pediatric NS patients.

Methods

Mutation analysis was carried out by direct sequencing of the NPHS1 and NPHS2 genes in 145 nephrotic syndrome (NS) patients. This cohort included 36 samples of congenital or infantile onset NS cases and 39 samples of familial cases obtained from 30 families.

Results

A total of 7 homozygous (6 novel) mutations were found in the NPHS1 gene and 4 homozygous mutations in the NPHS2 gene. All mutations in the NPHS1 gene were found in the early onset cases. Of these, one patient has a family history of NS. Homozygous p.R229Q mutation in the NPHS2 gene was found in two children with childhood-onset NS.

Conclusions

Our results show a low prevalence of disease causing mutations in the NPHS1 (22% early onset, 5.5% overall) and NPHS2 (3.3% early onset and 3.4% overall) genes in the Pakistani NS children as compared to the European populations. In contrast to the high frequency of the NPHS2 gene mutations reported for familial SRNS in Europe, no mutation was found in the familial Pakistani cases. To our knowledge, this is the first comprehensive screening of the NPHS1 and NPHS2 gene mutations in sporadic and familial NS cases from South Asia.     

Association of the ACE-II genotype with the risk of nephrotic syndrome in Pakistani children

Nephrotic syndrome is a common pediatric glomerular disease associated with heavy proteinuria. Since, the angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism is a putative genetic risk factor for NS, in this study, ACE (I/D) polymorphism was analyzed in 268 NS and 223 control samples by a PCR-based method. The genotypic and allelic frequencies were determined and the association between ACE I/D polymorphism and NS was evaluated. The frequency distribution of the II, ID and DD genotypes was 82 (30.6%), 128 (47.8%) and 58 (21.6%) in the NS patients and 9 (4.0%), 171 (76.7%) and 43 (19.3%) in the control samples respectively. In the Pakistani pediatric NS population, the II genotypic and allelic frequencies were found to be significantly associated with the disease (OR = 6.755; C.I = 3-14.9). No significant association was found between this polymorphism and the response to standard steroid therapy. Thus, in contrast to reports from other parts of the world, the II genotype was found to be significantly associated with NS in the Indian and Malay populations and in the Pakistani population described here. To our knowledge, this is the first report from Pakistan describing the association of the ACE I/D polymorphism with pediatric NS. On the basis of these results, it is suggested that analysis of the ACE (I/D) polymorphism should be performed for the early diagnosis in the high risk NS patients in South Asia.     

Detection of common mutations in the GALT gene through ARMS

Type I galactosemia is an inborn error resulting from mutations on both alleles of the GALT gene, which leads to the absence or deficiency of galactose-1-phosphate uridyltranseferase (GALT), the second of three enzymes catalyzing the conversion of galactose into glucose. On the basis of residual GALT activity, Type I galactosemia is classified into severe “Classical” and mild “Duarte” phenotypes. Classical galactosemia is frequently associated with S135L, Q188R and K285N mutations in the GALT gene. The functionally neutral N314D variation in the GALT gene is associated with Duarte galactosemia and is widespread among various worldwide populations. The present study aimed at detecting S135L, Q188R and K285N mutations and the N314D variant in the GALT gene by PCR using amplification refractory mutation system (ARMS). ARMS assays were established using standard DNA samples and were used for 8 galactosemia patients and 190 unrelated normal subjects all of Pakistani origin. S135L and K285N mutations were present neither in galactosemia patients nor in normal subjects. Only one galactosemia patient carried Q188R mutation that was in homozygous state. However, the N314D variant was frequently found both in affected (7 out of 16 alleles) and normal subjects (55 out of 380 alleles). This finding indicates that Duarte allele D314 might be far more common in Pakistani population than in European and North American ones.     

Root colonization and growth promotion of sunflower (Helianthus annuus L.) by phosphate solubilizing Enterobacter sp. Fs-11

An Enterobacter sp. Fs-11 was isolated from sunflower rhizosphere, identified on the basis of 16S rRNA gene sequence analysis (GeneBank accession no. GQ179978) and studied for its root colonization and growth promotion ability in sunflower. Morphologically, it was rod shaped Gram-negative, motile bacterium, producing 4.5 μg mL(-1) indole acetic acid in tryptophan-supplemented medium. It utilized 27 out of 95 substrates in BIOLOG GN2 micro plate system. It was able to convert insoluble tri-calcium phosphate to soluble phosphorus up to 43.5 μg mL(-1) with decrease in pH of the medium up to 4.5 after 10 days incubation at 28 ± 2 °C in the Pikovskaya's broth. High performance liquid chromatography of cell free supernatant showed that Fs-11 produced malic acid and gluconic acid (2.43 and 16.64 μg mL(-1), respectively) in Pikovskaya's broth. Analysis of 900 bp fragment of pyrroloquinoline quinine pqqE gene sequence showed 98 % homology with that of E. cloacae pqqE gene. Confocal laser scanning microscope revealed strong colonization of fluorescently labeled Fs-11 with sunflower roots. Sunflower inoculation with Fs-11 and its rifampicin resistant derivative in sterile sand and natural soil showed that Fs-11 colonized sunflower roots up to 30 days after transplanting in both sterile sand as well as natural soil. Moreover, Fs-11 inoculation resulted in increased plant height, fresh weight, dry weight and total phosphorus contents as compared to un-inoculated plants. The data showed that Enterobacter sp. Fs-11 is an efficient phosphate solubilizing and plant growth promoting rhizobacterium and has great potential to be used as bio-inoculant for sunflower under phosphorus deficient conditions.     

3D-QSAR studies of triazolopyrimidine derivatives of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> dihydroorotate dehydrogenase inhibitors using a combination of molecular dynamics,docking, and genetic algorithm-based methods

A series of 35 triazolopyrimidine analogues reported as Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors were optimized using quantum mechanics methods, and their binding conformations were studied by docking and 3D quantitative structure–activity relationship studies. Genetic algorithm-based criteria was adopted for selection of training and test sets while maintaining structural diversity of training and test sets, which is also very crucial for model development and validation. Both the comparative molecular field analyses ( q_\textLOO² = 0.841 q_{\text{LOO}}^2 = 0.{841} , r_\textncv² = 0.99 r_{\text{ncv}}^2 = 0.{99} ) and comparative molecular similarity indices analyses ( q_\textLOO² = 0.757 q_{\text{LOO}}^2 = 0.{757} , r_\textncv² = 0.943 r_{\text{ncv}}^2 = 0.{943} ) show excellent correlation and high predictive power. Furthermore, molecular dynamics simulations were performed to explore the binding mode of the two of the most active compounds of the series, 10 and 14. Harmonization in the two simulation results validate the analysis and therefore applicability of docking parameters based on crystallographic conformation of compound 14 bound to receptor molecule. This work provides useful information about the inhibition mechanism of this class of molecules and will assist in the design of more potent inhibitors of PfDHODH.     

Virtual Fossils: a New Resource for Science Communication in Paleontology

Computer-aided 3-D reconstruction of fossils, or virtual paleontology, is an increasingly common and powerful technique. It is now regularly used for research in paleontology, yet to date has impacted little on public outreach and science communication; however, it is ideally suited for these purposes, being increasingly cheap and available, dynamic and exciting, and applicable to a range of topics. Here, we provide an introduction to the field, and a case study of its use for a public engagement event. The steps involved in creating such an educational resource are outlined, and include computed tomography scanning, digital visualization, and 3-D printing of fossils. We emphasize the value of virtual fossils for science communication; they allow for diverse learning styles in a variety of topics. In the future, we hope that virtual paleontology will become a mainstay of communicating the history of life, thereby promoting accurate understanding of evolution.