Epidemiology and Genetic Diversity of Rotavirus Strains in Children with Acute Gastroenteritis in Lahore,Pakistan

Pakistan harbors high disease burden of gastro-enteric infections with majority of these caused by rotavirus. Unfortunately, lack of proper surveillance programs and laboratory facilities have resulted in scarcity of available data on rotavirus associated disease burden and epidemiological information in the country. We investigated 1306 stool samples collected over two years (2008–2009) from hospitalized children under 5 years of age for the presence of rotavirus strains and its genotypic diversity in Lahore. The prevalence rate during 2008 and 2009 was found to be 34% (n = 447 out of 1306). No significant difference was found between different age groups positive for rotavirus (p>0.05). A subset of EIA positive samples was further screened for rotavirus RNA through RT-PCR and 44 (49.43%) samples, out of total 89 EIA positive samples, were found positive. G and P type prevalence was found as follows: G1P [4] = 3(6.81%); G1P [6] = 9(20.45%); G1P [8] = 1(2.27%); G2P [4] = 21(47.72%); G2P [8] = 1(2.27%); G9P [4] = 1(2.27%); G9P [6] = 1(2.27%) and G9P [8] = 7(15.90%). Phylogenetic analysis revealed that the VP7 and VP4 sequences clustered closely with the previously detected strains in the country as well as Belgian rotaviruses. Antigenic characterization was performed by analyzing major epitopes in the immunodominant VP7 and VP4 gene segments. Although the neutralization conferring motifs were found variable between the Pakistani strains and the two recommended vaccines strains (Rotarix™ and RotaTeq™), we validate the use of rotavirus vaccine in Pakistan based on the proven and recognized vaccine efficacy across the globe. Our findings constitute the first report on rotavirus’ genotype diversity, their phylogenetic relatedness and epidemiology during the pre-vaccination era in Lahore, Pakistan and support the immediate introduction of rotavirus vaccine in the routine immunization program of the country.     

Effect of constant and fluctuating temperature on the circadian foraging rhythm of the red imported fire ant,Solenopsis invicta Buren (Hymenoptera: Formicidae)

Understanding circadian foraging rhythms activity of the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae) foragers at different temperatures is an important step towards developing control measures in Integrated Pest Management (IPM) programs. In this study, the circadian foraging rhythm activities of S. invicta foragersat different temperature were investigated under laboratory and field conditions. Results indicated that the foraging activity increased after sunrise, and maximum foraging occurred at 14:00 (foraging rate was 69.22 ± 0.57 and 72.58 ± 1.15 foragers/min in the first and second year, respectively) in the tea fields of Guangzhou during autumn. Furthermore, foragers demonstrated circadian rhythms and exhibited a unimodal after 24 h. A significant correlation was found between foraging activity and temperature. S. invicta colonies were active at moderate soil temperatures (approximately 26.65 °C to 29.24 °C). The preferred temperature of the colonies was 26 °C, followed by 22 °C and 18 °C in the laboratory. The individual S. invicta activity was maximum at 17:00 (18.67 ± 1.66 times /10 min) and minimum at 5:00 (8.33 ± 2.51 times/10 min) at 26 °C. The fluctuating temperature had a significant impact on individual locomotor activity (r = 0.8979, P < 0.01) but did not alter the rhythm activity. Our results demonstrated that temperature might play an important role in circadian foraging rhythms activity of S. invicta. These results may have implications for the development of more effective fire ant management strategies.     

Genetic Testing of Non-familial Deaf Patients for <Emphasis Type="Italic">CIB2</Emphasis> and <Emphasis Type="Italic">GJB2</Emphasis> Mutations: Phenotype and Genetic Counselling

CIB2 and GJB2 genes variants contribute significantly in familial cases of prelingual recessive hearing loss (HL). This study was aimed to determine the CIB2 and GJB2 variants and associated phenotype in 150 non-familial individuals with HL. After getting informed consent, 150 non-familial deaf patients were enrolled and blood samples were obtained for DNA extraction. Pure tone air conduction audiometry was performed. Coding exons of CIB2 and GJB2 genes were Sanger sequenced. A tetra primer ARMS assay was developed for recurrent CIB2 variant. Four bi-allelic GJB2 variants, c.71G>A p.(Trp24*), c.231G>A p.(Trp77*), c.235delC p.(Leu79Cysfs3*) and c.35delG p.(Gly11Leufs24*), were found in nine hearing impaired individuals. We also found four homozygotes and five carriers of c.380G>A p. (Arg127His) variant of controversial clinical significance. CIB2 sequencing revealed single recurrent variant c.272T>C p. (Phe91Ser) segregating with HL in ten individuals. Among our patients, c.71G>A (p.Trp24*) was the most common variant, accounted for 45% of GJB2 variants. Two known GJB2 variants, c.235delC p. (Leu79Cysfs3*) and c.310del14 p. (Lys105Argfs2*), are reported here for the first time in Pakistani population. Our data further support the benign nature of c.380G>A p. (Arg127His) variant. For CIB2, c.272T>C p. (Phe91Ser) is the second common cause of HL among our sporadic cases. Phenotypically, in our patients, individuals homozygous for GJB2 variants had profound HL, whereas CIB2 homozygotes had severe to profound prelingual HL. Our results suggest that GJB2 and CIB2 are common cause of HL in different Pakistani ethnicities.     

Synthesis,structure-activity relationship and molecular docking studies of 3-O-flavonol glycosides as cholinesterase inhibitors

The prime objective of this research work is to prepare readily soluble synthetic analogues of naturally occurring 3-O-flavonol glycosides and then investigate the influence of various substituents on biological properties of synthetic compounds. In this context, a series of varyingly substituted 3-O-flavonol glycosides have been designed, synthesized and characterized efficiently. The structures of synthetic molecules were unambiguously corroborated by IR, ¹H, ¹³C NMR and ESI-MS spectroscopic techniques. The structure of compound 22 was also analyzed by X-ray diffraction analysis. All the synthetic compounds (21–30) were evaluated for in vitro inhibitory potential against cholinesterase enzymes. The results displayed that most of the derivatives were potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with varying degree of IC₅₀ values. The experimental results were further encouraged by molecular docking studies in order to explore their binding behavior with the active pocket of AChE and BChE enzymes. The experimental and theoretical results are in parallel with one another.     

High frequency conversion of non-embryogenic synseeds and assessment of genetic stability through ISSR markers in <Emphasis Type="Italic">Gymnema sylvestre</Emphasis>

The present study describes the first attempt of exploiting encapsulation technology for high plantlet recovery, short-term storage and conservation of Gymnema sylvestre—an antidiabetic liana. Indole-3-butyric acid (IBA) pretreated nodal segments (NS) were encapsulated in sodium alginate (Na₂-alginate) matrix and the optimal culture conditions were evaluated in terms of maximum conversion capacity of synseeds into complete plantlets. Highest conversion frequency of synseeds was obtained on Murashige and Skoog’s (MS) medium supplemented with 5.0 µM 6-benzyladenine (BA). Augmentation of Na₂-alginate matrix with plant growth regulators (PGRs) and additive further improved in vitro conversion rates and the synthetic endosperm composed of 3% Na₂-alginate in MS?+?2.5 µM BA?+?2.5 µM gibberellic acid (GA₃)?+?50 µM adenine sulphate (AdS) stimulated maximum recovery (88.2?±?0.48%) of complete plantlets from synseeds. Studies on short term cold storage of synseeds showed that nutrient encapsulation maintains the viability of NS for a storage period of 8 weeks. Ex-vitro conversion of synseeds was also carried out on soilrite and vermicompost (3:1) mixture under culture room conditions. Monomorphic DNA profiles produced through Inter-Simple Sequence Repeat (ISSR) markers confirmed the genetic uniformity between synseed derived and mother plantlets.     

Synthesis of new sulfonamides as lipoxygenase inhibitors

The present study describes a convenient method for the synthesis of new lipoxygenase inhibitors, 4-(toluene-4-sulfonylamino)-benzoic acids from p-amino benzoic acid. Reaction of p-amino benzoic acid with p-toluenesulfonyl chloride provided thirteen N- and O-alkylation products 4a-4m in moderate to good yields. Lipoxygenase inhibition of newly formed sulfonamide derivatives was investigated and some of these compounds 4m, 4g, 4e, 4f and 4j showed good lipoxygenase inhibitory activities with IC(50) values ranged between 15.8 ± 0.57 and 91.7 ± 0.61 μmol whilst all other compounds exhibited mild anti-lipoxygenase activities with IC(50) values ranged between 139.2 ± 0.75 and 232.1 ± 0.78 μmol. N-alkylated products were more active against the enzyme than O-alkylated or both N- and O-alkylated ones. All synthesized sulfonamides were recrystallized in chloroform to give these title compounds which were characterized using FTIR, (1)H NMR, (13)C NMR, elemental analysis and single crystal X-ray diffraction techniques.     

DNA mismatch repair MSH2 gene-based SNP associated with different populations

We screened for the major essential single-nucleotide polymorphism (SNP) variant that might be associated with the MSH2 gene based on the data available from three types of human tissue samples [156 lymphoblastoid cell variations (LCL), 160 epidermis, 166 fat]. An association analysis confirmed that the KCNK12 SNP variant (rs748780) was highly associated (p value 9 × 10^?4) with the MSH2 gene for all three samples. Using SNP identification, we further found that the recognized SNP was also relevant among Hapmap populations. Techniques that display specific SNPs associated with the gene of interest or nearby genes provide more reliable genetic associations than techniques that rely on data from individual SNPs. We investigated the MSH2 gene regional linkage association with the determined SNP (rs748780), KCNK12 variant (Allele T>C) in the intronic region, in HapMap3 full dataset populations, Yoruba in Ibadan, Nigeria (YRI), Utah residents with ancestry from northern Europe (CEU), Han Chinese in Beijing, China (CHB), and a population of Mexican ancestry in Los Angeles, California (MEX). A gene-based SNP association analysis analyzes the combined impact of every variant within the gene while creating referrals to linkage disequilibrium or connections between markers. Our results indicated that among the four populations studied, this association was highest in the MEX population based on the r ² value; a similar pattern was also observed in the other three populations. The relevant SNP rs748780 in KCNK12 is related to a superfamily of potassium channel pore-forming P-domain proteins as well as to other non-pore-forming proteins and has been shown to be relevant to neurological disorder predisposition in MEX as well as in other populations.     

Effectiveness of phytase pre-treatment on growth performance,nutrient digestibility and mineral status of common carp (Cyprinus carpio) juveniles fed Moringa by-product based diet

Several anti-nutritional substances are found in plant derivatives for example phytate, that make the nutrients and minerals unavailable to fish, hence leading to poor growth performance. Presence of the anti-nutrient factor such as phytate is a chelated compound and need enzyme for its breakdown and availability of nutrients to improve fish growth. This research work was performed to check the improvement of overall performance of Cyprinus carpio fingerlings by the help of phytase addition in Moringa oleifera by- products based diet. Combination of Moringa seed meal and Moringa leaf meal was utilized as test ingredient to formulate seven test feeds, containing graded levels of phytase (0, 500, 650, 800, 950, 1100 and 1250 FTU kg⁻¹). In feeding trial of 70 days, fingerlings were given feed two times in a day at the rate of 4% of wet weight of their bodies and faeces were collected. According to current results, it was found that growth performance parameters i.e. weight gain; 25 g, specific growth rate; 1.67 and feed conversion ratio; 1.10 were improved to maximum at 950 FTU kg⁻¹. Digestibility of nutrients (crude protein; 73%, crude fat; 71% and gross energy; 67%) and minerals absorption was also maximum (Ca; 70%, Zn; 66%, K; 74%, Mn; 66% and P; 71%) at 950 FTU kg⁻¹. Lowest growth efficiency, nutrient digestibility and mineral absorption were observed in fingerlings fed at control diet (0 FTU kg⁻¹). Results of the current study, proved that 950 FTU kg⁻¹ is the most optimum level of phytase to formulate economical and ecofriendly feed for improved growth of C. carpio fingerlings as it decreases the discharge of minerals and nutrients in water bodies.