Biochemical and Hematologic Profiles in B-Cell Acute Lymphoblastic Leukemia Children.

B-cell acute lymphoblastic leukemia is the most common type of leukemia found in children. Timely diagnosis, white blood cell count, age of onset, and sex are considered the most important prognostic factors in childhood leukemia. Hematological and biochemical profiles are crucially important to infer the health of leukemia patient pre-chemotherapy and post-chemotherapy treatment. In the current study 200 cases were taken and evaluated for hematological (complete blood count and white blood differential count) and biochemical parameters (renal function tests, liver function tests, serum electrolytes and serum proteins) by comparison with normal reference values. Most of the cases were male under 5 years of age. Hematology parameters including red blood cells, hemoglobin and platelet levels were relatively low whereas white blood cells level was high in cases as compared with normal reference value. Sex-wise and age-wise comparison of biochemical profile showed significant difference among B-cell acute lymphoblastic leukemia cases whereas hematological profile did not show any visible difference.

Fatty acid based antimicrobials from Streptomyces sp. SORS-24, an endophyte isolated from Sonchus oleraceus.

Due to the rise in bacterial resistance towards various therapeutic agents, interest is now developing towards fatty acid based antimicrobials because of their non-specific mode of action. A strain SORS 24 isolated from Sonchus oleraceus (Sow thistle) showed significant activity against Escherichia coli ATCC 25922 (25 mm), Chlorella vulgaris (20 mm), Bacillus subtilis DSM 10 (ATCC 6051) and Pseudomonas sp. (15 mm). It displayed an LC50 value of 10 µg/ml against Artemia salina (Brine shrimp) nauplii and an EC50 value of 0.8 µg/ml in the (DPPH) diphenylpicrylhydrazyl antioxidant assay. The strain also displayed genotoxicity against a PolA deficient strain, E. coli K-12 AB 3027 (15 mm). Mass spectrometry (HPLC-MS) showed that the strain produced oleamide (9-Octadecenamide) and erucamide (13-Docosenamide). Both of the purified fatty acid amides showed prominent activity against B. subtilis DSM 10 (ATCC 6051) (20 mm) and E. coli ATCC 25922 (15 mm). Significant genotoxicity was observed against E. coli K-12 AB 3027 (15 mm). The 16S gene sequencing revealed that the strain belonged to species, Streptomyces tanashiensis. As far as our understanding, this is the first report of this species producing these fatty acid based antimicrobials.

Genetic studies in the Pakistani population reveal novel associations with ventricular septal defects (VSDs).

BACKGROUND: With prevalence up to 4%, Ventricular Septal Defect (VSD) is one of the leading causes of neonatal deaths. VSD is a common complex genetic disorder that has been associated with many genetic determinants. Variants from genes for the transcription factors including T-Box TBX5 and NFATc1 (nuclear factor of activated T cells, cytoplasmic 1), Vascular endothelial growth factor (VEGF), ISLET1 (encoded by the ISL1 gene) and enzyme MTHFR, a methylene tetrahydrofolate reductase were selected. Genetic risk score (GRS) is a widely accepted approach used to convert the genetic data into prediction and assessment tool for disease susceptibility. METHODS: A total of 200 participants were recruited for the current study, 100 VSD patients and 100 controls. Genotyping of the ISL1: rs1017, NFATc1: rs7240256, VEGF: rs36208048, TBX5: rs11067075, and MTHFR: rs1801133 variants was performed using tetra primer ARMS PCR and PCR-RFLP. For the statistical analysis, the software SPSS version 23 was used. Genotypic frequencies of cases and controls were calculated using chi-square (χ²) whereas allelic frequencies were calculated by using the SNPStats tool. The association of GRS quartiles with VSD was examined using binary logistic regression. Adjusted p-value 0.01 was used as significance threshold for all analyses. RESULTS: The ISL1 (OD: 0.242, CI: 0.158-0.37, p-value: 2.15 × 10- 4 :), NFATc1 (OD: 2.53, CI: 1.64-3.89, p-value: 2.11 × 10- 5), TBX5 (OD: 2.24, CI: 1.47-3.41, p-value:1.6 × 10- 4) and MTHFR (OD: 10.46, CI: 5.68-19.26, p-value: 2.09 × 10- 9:) variants were found to be in association with VSD. In contrast, the VEGF (OD: 0.952, CI: 0.56-1.62, p-value: 0.8921) variant did not show significance association with the VSD. For cases, the mean GRS score was 3.78 ± 1.285 while in controls it was 2.95 ± 1.290 (p-value: 0.479, CI: 0.474-1.190). Comparison of GRS between cases and control showed that mean GRS of cases was 1.90 ± 0.480 while in controls it was 1.68 ± 0.490 (p-value: 0.001, CI: 0.086-0.354). Higher quartiles were more prevalent in cases whereas lower quartiles were more prevalent in controls. CONCLUSION: GRS of these five loci was strongly associated with VSD. Moreover, genetic risk score can provide better information for the association between variants and disease as compared to a single SNP. We also illustrated that the cumulative power of GRS is greater over the single SNP effect. This is a pilot scale study with a relatively small sample size whose findings should be replicated in a larger sample size for the unique local Pakistani population.

Study of variants associated with ventricular septal defects (VSDs) highlights the unique genetic structure of the Pakistani population.

BACKGROUND: Ventricular septal defects (VSDs) are one of the leading causes of death due to cardiac anomalies during the first months of life. The prevalence of VSD in neonates is reported up to 4%. Despite the remarkable progress in medication, treatment and surgical procedure for VSDs, the genetic etiology of VSDs is still in infancy because of the complex genetic and environmental interactions. METHODS: Three hundred fifty subjects (200 VSD children and 150 healthy controls) were recruited from different pediatric cardiac units. Pediatric clinical and demographic data were collected. A total of six variants, rs1017 (ISL1), rs7240256 (NFATc1), rs36208048 (VEGF), variant of HEY2, rs11067075 (TBX5) and rs1801133 (MTHFR) genes were genotyped by tetra-ARMS PCR and PCR-RFLP methods. RESULTS: The results showed that in cases, the rs1017 (g.16138A > T) variant in the ISL1 gene has an allele frequency of 0.42 and 0.58 respectively for the T and A alleles, and 0.75 and 0.25 respectively in the controls. The frequencies of the AA, TA and TT genotypes were, 52%, 11% and 37% in cases versus 21%, 8% and 71% respectively in the controls. For the NFATc1 variant rs7240256, minor allele frequency (MAF) was 0.43 in cases while 0.23 in controls. For the variant in the VEGF gene, genotype frequencies were 0% (A), 32% (CA) and 68% (CC) in cases and 0.0%, 33% and 67% respectively in controls. The allele frequency of C and A were 0.84 and 0.16 in cases and 0.83 and 0.17 respectively in controls. The TBX5 polymorphism rs11067075 (g.51682G > T) had an allelic frequency of 0.44 and 0.56 respectively for T and G alleles in cases, versus 0.26 and 0.74 in the controls. We did not detect the presence of the HEY2 gene variant (g.126117350A > C) in our pediatric cohort. For the rs1801133 (g.14783C > T) variant in the MTHFR gene, the genotype frequencies were 25% (CC), 62% (CT) and 13% (TT) in cases, versus 88%, 10% and 2% in controls. The ISL1, NFATc1, TBX5 and MTHFR variants were found to be in association with VSD in the Pakistani pediatric cohort whilst the VEGF and HEY2 variants were completely absent in our cohort. CONCLUSION: We propose that a wider programme of genetic screening of the Pakistani population for genetic markers in heart development genes would be helpful in reducing the risk of VSDs.

Drought tolerance induction and growth promotion by indole acetic acid producing Pseudomonas aeruginosa in Vigna radiata.

Drought accompanied with reduced precipitation is one of the key manacles to global agricultural throughput and is expected to escalate further hence posing major challenges to future food safety. For a sustainable agricultural environment, drought resistant plant growth promoting rhizobacteria (PGPR) are new encouraging prospect, which are inexpensive and have no side effects, as those of synthetic fertilizers. In the present study, five strains of Pseudomonas aeruginosa, the strain MK513745, strain MK513746, strain MK513747, strain MK513748, and strain MK513749 were used as drought tolerant PGPR with multiple traits of IAA production, N fixation, P solubilization, siderophore producing capabilities. The strain MK513745 and strain MK513749 produced higher quantities of indole acetic acid (116±0.13 and 108±0.26 µg ml-1). MK513749 yielded 12 different indole compounds in GCMS analysis. The strain MK513748 yielded maximum S.I. (3.33mm) for phosphate solubilizing test. Maximum nitrogen concentration was produced (0.18 µg ml-1) by strain MK513746. Percent siderophore units ranged from 2.65% to 2.83% as all five pseudomonas strains were siderophore positive. In all growth experiments of plant microbe interaction two varieties of Vigna radiata (AZRI-06, NM-11) plants inoculated with P. aeruginosa strains under drought stress responded significantly (P<0.05) better than control stressed plants. Maximum shoot length was enhanced up-to 125%, pod/plant 172%, number of grains 65%, 100 seed weight 95%, 100 seed straw weight 124% and total yield 293% were recorded in plants inoculated with drought stress tolerant PGPR in both varieties as compared to respective stressed control plants. Photosynthetic activity, membrane stability (45%), water content (68%) and antioxidant efficacy (19%) were improved with PGPR inoculations. The variety NM-11 (V2) was more tolerant to drought stress with inoculations of Pseudomonas strains than AZRI-06 (V1). Inoculations with these indole acetic acid producing strains would be suitable for plant growth promotion in areas facing water deficiency.

Dengue and Chikungunya virus co-infection in major metropolitan cities of provinces of Punjab and Khyber Pakhtunkhwa: A multi-center study.

Dengue has become endemic in Pakistan with annual recurrence. A sudden increase in the dengue cases was reported from Rawalpindi in 2016, while an outbreak occurred for the first time in Peshawar in 2017. Therefore, a multi-center study was carried out to determine the circulating dengue virus (DENV) serotypes and Chikungunya virus (CHIKV) co-infection in Lahore, Rawalpindi, and Peshawar cities in 2016-18. A hospital-based cross-sectional study was carried out in Lahore and Rawalpindi in 2016-18, while a community-based study was carried out in Peshawar in 2017. The study participants were tested for dengue NS1 antigen using an immunochromatographic device while anti-dengue IgM/IgG antibodies were detected by indirect ELISA. All NS1 positive samples were used for DENV serotyping using multiplex real-time PCR assay. Additionally, dengue samples were tested for CHIKV co-infection using IgM/IgG ELISA. A total of 6291 samples were collected among which 8.11% were NS1 positive while 2.5% were PCR positive. DENV-2 was the most common serotype (75.5%) detected, followed by DENV-1 in 16.1%, DENV-3 in 3.9% and DENV-4 in 0.7% while DENV-1 and DENV-4 concurrent infections were detected in 3.9% samples. DENV-1 was the predominant serotype (62.5%) detected from Lahore and Rawalpindi, while DENV-2 was the only serotype detected from Peshawar. Comorbidities resulted in a significant increase (p-value<0.001) in the duration of hospital stay of the patients. Type 2 diabetes mellitus substantially (p-value = 0.004) contributed to the severity of the disease. Among a total of 590 dengue positive samples, 11.8% were also positive for CHIKV co-infection. Co-circulation of multiple DENV serotypes and CHIKV infection in Pakistan is a worrisome situation demanding the urgent attention of the public health experts to strengthen vector surveillance.

Association of variable number of tandem repeats (VNTR) and T941G polymorphism of monoamine oxidase (MAO-A) gene with aggression in Pakistani subjects.

BACKGROUND: Human behavioral traits are known to be significantly heritable. Certain individuals have a greater tendency of negative behavioral aspects including aggression. The quest to identify tunderlying genetic causes has led to identification of a number of genetic markers, one of them is the monoamine oxidase-A (MAO-A) gene. OBJECTIVE: We aimed to genotype a variable number of tandem repeats (VNTRs) in the promoter region and a functional SNP within this gene (T941G, dbSNP ID: rs6323) in the recruited cohort of 482 subjects. METHODS: After DNA isolation, genotyping was done by PCR-RFLP and the results were confirmed by sequencing. RESULTS: For VNTRs, the results showed, highest frequency of 3.5 repeats in males and 4 repeats in females in the promoter region. The genotype frequencies for the SNP in cases were GG=16.3%, TG=20.6% and TT=63.1%, while in controls, the frequencies were GG=12.7%, TG=6.3%, and TT=81.0%. The allele frequencies were significantly different between cases and controls (p=0.015; OR=1.51; CI=1.085-2.102). CONCLUSION: The selected VNTR and SNP appeared to be significantly associated with aggression. These VNTRs and SNP have not been studied previously in the Pakistani population, hence they represent a unique ethnic group. These results, however, would have to be replicated in larger cohorts.

Screening of sunflower associated bacteria as biocontrol agents for plant growth promotion.

Deleterious effects of artificially applied chemicals have highlighted the significance of biocontrol agents as suitable substitute for sustainable agriculture. In present study, three endophytic bacterial strains SV7, SV10 and LV19 showed extensive range of antifungal as well as plant growth promoting activities signifying potential to accomplish the requirement. Phylogenetic analysis revealed 100% similarity of three strains with taxon Firmicute. However, there was division among these isolates on basis of subgroups as SV7 belonged to Exiguobacterium auranticum, SV10 belonged to Paenibacillus sp. and LV19 was best fit in subgroup Priestia koreensis. All strains showed antifungal activity against Fusarium oxysporum on three different media (PDA, NA, LA) with maximum activity (53%) of LV19 strain on NA and least activity (13%) on PDA medium as recorded by zones of inhibition. In growth promotion experiments, combination of LV19 with Fusarium significantly suppressed chances of Fusarium wilt which is commonly caused by Fusarium oxysporum in sunflower plants. Diverse growth parameters (seed germination percentage, lengths and fresh weights of root and shoot) were significantly increased from 34 to 909% over pathogen infected plants only which was further proved by their root colonization analysis. Based on most efficient growth promotion by LV19 strain, expression of five plant defense related genes (SOD, PAL, NPR1, PR5, Chitinase) was evaluated revealing enhanced expression by 1.7-270-folds in consortium of LV19 and Fusarium. Thus, current study provided a scientific justification that bacterial strains in specific LV19 (Priestia koreensis) could be further developed as biocontrol agent with potential of plant growth promotion.

First report of polymorphisms in MTRR, GATA4, VEGF, and ISL1 genes in Pakistani children with isolated ventricular septal defects (VSD).

BACKGROUND: Ventricular septal defects (VSDs) are malformations in the septum separating the heart's ventricles. VSDs may present as a single anomaly (isolated/nonsyndromic VSD) or as part of a group of phenotypes (syndromic VSD). The exact location of the defect is crucial in linking the defect to the underlying genetic cause. The number of children visiting cardiac surgery units is constantly increasing. However, there are no representative data available on the genetics of VSDs in Pakistani children. METHODS: Two hundred forty-two subjects (121 VSD children and 121 healthy controls) were recruited from pediatric cardiac units of Lahore. The clinical and demographic data of the subjects were collected. A total of four SNPs, one each from MTRR, GATA4, VEGF, and ISL1 genes were genotyped by PCR-RFLP. RESULTS: The results showed that the minor allele (T) frequency (MAFs) for the MTRR gene variant rs1532268 (c.524C > T) was 0.20 and 0.41 in the controls and the cases, respectively, with the genotype frequencies 3, 35, 62% in the controls and 12, 59 and 29% in the cases for TT, CT, CC genotypes, respectively (allelic OR: 5.73, CI: 3.82-8.61, p-value: 5.11 × 10- 7). For the GATA4 variant rs104894073 (c.886G > A), the MAF for the controls and the cases was 0.16 and 0.37, respectively, the frequencies of AA, GA and GG genotypes were 2, 28, and 70% in the controls and 5, 64 and 31% of the cases (allelic OR: 3.08, CI: 2.00-4.74, p-value: 8.36 × 10- 8). The rs699947 (c.-2578C > A) of VEGF gene showed MAF 0.36 and 0.53 for the controls and cases, respectively, with the genotype frequencies 13, 42, and 45% in the controls and 22, 15, and 63% in the cases for the AA, CA, CC (allelic OR: 2.03, CI: 1.41-2.92, p-value: 0.0001). The ISL1 gene variant rs6867206 (g.51356860 T > C), the MAFs were 0.26 and 0.31 in the controls and cases, respectively. The genotype frequencies were 48, 52, 0% in the controls and 39, 61, 0% in the cases for TT, TC, CC genotypes (allelic OR: 0.27, CI: 0.85-1.89, p-value: 0.227). The MTRR, GATA4 and VEGF variants showed association while ISL1 variant did not appear to be associated with the VSD in the recruited cohort. CONCLUSION: This first report in Pakistani children demonstrates that single nucleotide polymorphisms in genes encoding transcription factors, signaling molecules and structural heart genes involved in fetal heart development are associated with developmental heart defects., however further work is needed to validate the results of the current investigation.

Anti-Cholinesterase Combination Drug Therapy as a Potential Treatment for Alzheimer's Disease.

Alzheimer's disease (AD) is a burgeoning social and healthcare problem. Cholinesterase inhibitors (ChEIs) are employed for symptomatic treatment of AD, but often elicit adverse drug reactions (ADRs). Herein, the potency of the ChEIs, donepezil, tacrine, berberine, and galantamine to inhibit human or Torpedo californica acetylcholinesterase (tcAChE) proteins were evaluated. The efficacy of dual-drug combinations to inhibit human AChE directly and within differentiated neurons was also quantified. ChEI potency was in the order: donepezil > tacrine > berberine > galantamine for both AChEs. Dual-drug combinations of berberine and tacrine (BerTac), berberine and galantamine (BerGal), and tacrine and donepezil (TacDon) all produced synergistic outcomes for AChE inhibition. Donepezil and berberine (DonBer) and tacrine and galantamine (TacGal) elicited antagonistic responses. Donepezil and galantamine (DonGal) was synergistic for human AChE but antagonistic for tcAChE. After application of dual-drug combinations to neuronal cells, BerTac, BerGal, DonGal, and DonBer all showed synergistic inhibition of AChE, TacDon additive, and TacGal antagonistic effects. BerGal produced the most potent synergism and reduced total drug dose by 72%. Individual ChEIs or dual-drug combinations were relatively non-toxic to neuronal cells, and only reduced cell viability at concentrations two-three orders of magnitude greater than that required to inhibit AChE. In summary, dual-drug combinations of ChEIs potentially represent a novel means of AD patient treatment, with reduced and more cost-effective drug dosing, and lowered likelihood of ADRs.

Association of serotonin system-related genes with homicidal behavior and criminal aggression in a prison population of Pakistani Origin.

The serotonin transporter (SLC6A4), 5-HT2A (HTR2A) and 5-HT2B (HTR2B) recepter genes, express proteins that are important regulators of serotonin reuptake and signaling, and thereby may contribute to the pathogenesis of aggressive criminal behavior. 370 sentenced murderers in Pakistani prisons and 359 men without any history of violence or criminal delinquency were genotyped for six candidate polymorphisms in SLC6A4, HTR2A and HTR2B genes. An association of higher expressing L/L and LA/LA variants of the 5-HTTLPR polymorphism was observed with homicidal behavior (bi-allelic: OR = 1.29, p = 0.016, tri-allelic: OR = 1.32, p = 0.015) and in the murderer group only with response to verbal abuse (OR = 2.11, p = 0.015), but not with other measures of self-reported aggression. L/L and LA/LA genotypes of the 5-HTTLPR polymorphism were associated with higher aggression scores on STAX1 scale of aggression compared to lower expressing genotypes (S/S, S/LG, LG/LG) in prison inmates. No associations were apparent for other serotonergic gene polymorphisms analyzed. Using the Braineac and GTEx databases, we demonstrated significant eQTL based functional effects for rs25531 in HTTLPR and other serotonergic polymorphisms analyzed in different brain regions and peripheral tissues. In conclusion, these findings implicate SLC6A4* HTTLPR as a major genetic determinant associated with criminal aggression. Future studies are needed to replicate this finding and establish the biologic intermediate phenotypes mediating this relationship.

In Silico Design of Dual-Binding Site Anti-Cholinesterase Phytochemical Heterodimers as Treatment Options for Alzheimer's Disease.

The number of patients with neurodegenerative diseases, particularly Alzheimer's disease (AD), continues to grow yearly. Cholinesterase inhibitors (ChEIs) represent the first-line symptomatic drug treatment for mild-to-moderate AD; however, there is an unmet need to produce ChEIs with improved efficacy and reduced side effects. Herein, phytochemicals with reported anti-acetylcholinesterase (AChE) activity were ranked in silico for their anti-AChE potential. Ligands with a similar or higher binding affinity to AChE than galantamine were then selected for the design of novel dual-binding site heterodimeric drugs. In silico molecular docking of heterodimers with the target enzymes, AChE and butyrylcholinesterase (BuChE), were performed, and anti-cholinesterase binding affinities were compared with donepezil. Drug-likeliness properties and toxicity of the heterodimers were assessed using the SwissADME and ProTox-II webservers. Nine phytochemicals displayed similar or higher binding affinities to AChE than galantamine: sanguinarine > huperzine A > chelerythrine > yohimbine > berberine > berberastine > naringenin > akuammicine > carvone. Eleven heterodimeric ligands were designed with phytochemicals separated by four- or five-carbon alkyl-linkers. All heterodimers were theoretically potent AChE and BuChE dual-binding site inhibitors, with the highest affinity achieved with huperzine-4C-naringenin, which displayed 34% and 26% improved affinity to AChE and BuChE, respectively, then the potent ChEI drug, donepezil. Computational pharmacokinetic and pharmacodynamic screening suggested that phytochemical heterodimers would display useful gastrointestinal absorption and with relatively low predicted toxicity. Collectively, the present study suggests that phytochemicals could be garnered for the provision of novel ChEIs with enhanced drug efficacy and low toxicity.

Potential Nutraceutical Properties of Leaves from Several Commonly Cultivated Plants.

Chronic dietary ingestion of suitable phytochemicals may assist with limiting or negating neurodegenerative decline. Current therapeutics used to treat Alzheimer disease elicit broad adverse drug reactions, and alternative sources of cholinesterase inhibitors (ChEIs) are required. Herein, we screened methanolic extracts from seven commonly cultivated plants for their nutraceutical potential; ability to inhibit acetylcholinesterase (AChE) and butyryl-cholinesterase (BuChE), and provision of antioxidant activity through their 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical scavenging capabilities. Potential neurotoxicity of plant extracts was examined via application to SHSY-5Y neuroblastoma cells and quantitation of cell viability. Methanolic extracts of Citrus limon (Lemon), Bombax ceiba (Red silk-cotton), Lawsonia inermis (Henna), Eucalyptus globulus (Eucalyptus), Ocimum basilicum (Basil), Citrus reticulata (Mandarin orange), and Mentha spicata (Spearmint) all displayed concentration-dependent inhibition of AChE and BuChE. The majority of extracts inhibited AChE and BuChE to near equipotency, with Henna and Eucalyptus extracts the two most potent ChEIs. All plant extracts were able to scavenge free radicals in a concentration-dependent manner, with Eucalyptus the most potent antioxidant. Toxicity of plant extracts to neuronal cells was concentration dependent, with Eucalyptus also the most toxic extract. Fractionation of plant extracts and analysis by mass spectrometry identified a number of plant polyphenols that might have contributed to the cholinesterase inhibition: 3-caffeoylquinic acid, methyl 4-caffeoylquinate, kaempferol-acetyl-glycoside, quercetin 3-rutinoside, quercetin-acetyl-glycoside, kaempferol 3-O-glucoside, and quercetin 3-O-glucoside. In silico molecular modeling of these polyphenols demonstrated their improved AChE and BuChE binding affinities compared to the current FDA-approved dual ChEI, galantamine. Collectively, all the plant extracts contained nutraceutical agents as antioxidants and ChEIs and, therefore, their chronic consumption may prove beneficial to combat the pathological deficits that accrue in Alzheimer disease.

Correction To: Obesity, More than a 'Cosmetic' Problem. Current Knowledge and Future Prospects of Human Obesity Genetics.

The authors regret that the reference provided to Fig. 1 of their publication was incorrect in the original publication. The authors obtained permission to reprint Fig. 1 retrospectively. The correct reference is: Nat Rev Genet. 2005 Mar;6(3):221-34. https://doi.org/10.1038/nrg1556 .

PCR-based Screening Approach: A Rapid Method to Detect the Biosynthetic Potential of Antimicrobials in Actinobacterial Strains.

This study aimed to investigate the PCR-based screening strategy for the prediction of the antimicrobial biosynthetic potential of the selected Streptomyces strains originated from an extreme environment (Cholistan Desert, Pakistan). The biosynthetic potential was determined by using both molecular and culture-dependent screening approaches. The four biosynthetic genes clusters, including the pks-1, nrps, cyp P450 hydroxylase (cyps), and glycopeptide oxy b genes, were investigated in the selected strains by PCR amplification, sequencing, and by subsequent bioinformatics approaches. Among the 40 selected Streptomyces strains, 33 strains possessed the nrps gene, 17 strains carried the pks-1 gene, four strains were found to have the cyps gene, and none of the strain carried oxy b gene. The Streptomyces strains including NR-1, NR-10, NR-14, and NR-15 were investigated for in vitro antifungal activity against Fusarium oxysporum, Rhizoctonia solani, and Aspergillus sp. The extracts were analyzed for chemical profiling (TLC and HPLC-UV), and a unique pattern of secondary metabolites was observed. The selected strains exhibited pronounced antifungal activity against the fungal test strains with the zone of inhibition up to 17, 18, and 19 mm, respectively. The study depicts that gene-based screening can be successfully applied to identify potentially bioactive strains by usin a single screening process. This PCR-based approach is rapid and can be used for sorting out and selecting the potential candidate among actinobacterial culture collections. Such a preselection or strain prioritization consequently decreases the time and efforts required for selecting the potential bioactive strain, which then can be subjected to the detailed chemical analysis.This study aimed to investigate the PCR-based screening strategy for the prediction of the antimicrobial biosynthetic potential of the selected Streptomyces strains originated from an extreme environment (Cholistan Desert, Pakistan). The biosynthetic potential was determined by using both molecular and culture-dependent screening approaches. The four biosynthetic genes clusters, including the pks-1, nrps, cyp P450 hydroxylase (cyps), and glycopeptide oxy b genes, were investigated in the selected strains by PCR amplification, sequencing, and by subsequent bioinformatics approaches. Among the 40 selected Streptomyces strains, 33 strains possessed the nrps gene, 17 strains carried the pks-1 gene, four strains were found to have the cyps gene, and none of the strain carried oxy b gene. The Streptomyces strains including NR-1, NR-10, NR-14, and NR-15 were investigated for in vitro antifungal activity against Fusarium oxysporum, Rhizoctonia solani, and Aspergillus sp. The extracts were analyzed for chemical profiling (TLC and HPLC-UV), and a unique pattern of secondary metabolites was observed. The selected strains exhibited pronounced antifungal activity against the fungal test strains with the zone of inhibition up to 17, 18, and 19 mm, respectively. The study depicts that gene-based screening can be successfully applied to identify potentially bioactive strains by usin a single screening process. This PCR-based approach is rapid and can be used for sorting out and selecting the potential candidate among actinobacterial culture collections. Such a preselection or strain prioritization consequently decreases the time and efforts required for selecting the potential bioactive strain, which then can be subjected to the detailed chemical analysis.

Comparative analysis of endophytic bacterial diversity between two varieties of sunflower Helianthus annuus with their PGP evaluation.

Endophytic bacterial diversity shows an intricate network of interactions with host plants as they reside in various tissues and organs at certain stages or all stages of their life cycle stimulating the plant growth and fitness. Sunflower is a trendy oilfield crop and variation in its varieties is associated with the dynamics of endophytic diversity. The present study is undertaken to identify and compare the ecological niche of endophytic bacterial communities amongst different tissues of two hybrids varieties Hysun-33 and Hysun-39 of sunflower (Helianthus annuus) at three developmental stages which are vegetative stage I (after 15 days of seeds germination), vegetative stage II (after 30 days of germination) and reproductive stage (after 90 days of germination). A total of 74 endophytes from Hysun-33 and 115 endophytes from Hysun-39 have been isolated from different tissues and growth stages. Amongst plant parts, root tissues harbored higher bacterial inhabitants (44) followed by stem (33), leaf (30) and flower (7) of Hysun-39. Likewise, Hysun-33 endophytes colonized roots more abundantly followed by leaves, stem and flowers. All strains are found to be gram positive with the exception of only RA9 from Hysun-33 and RB9 from Hysun-39 that are gram negative. Among different growth stages, the maximum bacterial population (CFU of 320 × 103) was found amongst root microflora at vegetative stage II of plant in Hysun-39 variety as compared to root endophytes of Hysun-33 having (CFU of 10 × 103). The evaluation of their growth promoting features revealed that among 74 isolates of Hysun-33, 70% exhibited the ability of hydrogen cyanide production, 43% IAA production, 36% siderophore production and 4% nitrogen fixation and also phosphate solubilization. However among 115 isolates of Hysun-39, 64% appeared as hydrogen cyanide producers, 56% IAA producers, 33% siderophore producers, 2% nitrogen fixers and 4% as phosphate solubilizers. Therefore our study reveals understanding of wide-ranging diversity of endophytic bacteria and their beneficial relationship with internal tissues of host plant which may recommend their implementation to crops for better development of agricultural systems.

Biochemical, serological, and genetic aspects related to gene HLA-DQB1 and its association with type 1 diabetes mellitus (T1DM).

BACKGROUND: Type 1 Diabetes Mellitus (T1DM) is the autoimmune disorder of destruction of ß cells of pancreas, creating insulin deficiency condition, which leads to hyperglycemia, polyuria, polydipsia, ketoacidosis, and other metabolic disorder especially in children. Different genetic aspects and environmental factors are involved in pathophysiology of the disease. About 20 genes are associated with this disease in which the most common is the different combination of haplotype DRB1-DQA1-DQB1 present at HLA gene. At HLA-DQB1, there are some SNPs which are associated with T1DM. In T1DM, there are number of biochemical, serological parameters which show some abnormalities leading to some complications. METHODS: Samples were subjected to all biochemical and serological techniques to get the measurement of concentration of glucose, lipid profile (cholesterol, triglycerides, and HDL and LDL cholesterol), urea, creatinine, albumin, insulin, anti-insulin antibodies, C-peptides, and leptin. All these values were compared with controls values and statistical analysis was also done on these values. At molecular level, two primers set which were allele specific at HLA-DQB1, were used to amplify the SNPs, homozygous and heterozygous conditions were stated. RESULTS: PCR results for the studied population showed that most of samples have heterozygous condition for these SNPs of this allele specific region on HLA-DQB1. Very few of them have homozygous state for it. Even in the control sample have the same conditions. CONCLUSION: In Pakistan, there is dire need of studies about SNPs and haplotypes related to HLA-DQB1 which show association with T1DM.

Virtual screening of antitumour phytochemical against peroxisome proliferators activated receptor proteins PPARs.

Cancers are caused by the defects in apoptosis process which leads to uncontrolled proliferation, therefore, most attractive drug target discovery strategy is to find ligands which have the ability to activate or regulate the apoptotic machinery. Peroxisome-proliferator-activated receptors (PPARs) are nuclear hormone receptors their over expression is observed in many tumours and contributes to chemotherapy resistance. The goal of this study to scrutinized antitumor phytochemicals from Alysicarpus bupleurifolius, Piper nigrum and Plumeria obtuse and potential energy values render from interactions between active site residues and ligands. The potential phytochemicals with significant binding affinity are ursolic acid, cis-4-decenoic acid and p-coumaric acid respectively most effective compounds in high throughput virtual screening belongs to Plumeria obtuse against PPARs associated with tumour development and progression. This modern drug designing modeling in silico approach, therefore, identifies the potential leads against over expressed tumours.

Chromosomal region 1q24.1 is associated with increased risk of schizophrenia in Pakistani population.

Schizophrenia is a stressful, chronic and in many cases incorrigible psychological disorder. Till now no biomarker is available for diagnosis of this condition and diagnosis is done purely on psychiatric bases. A strong genetic association of human genome region 1q24.1 is implicated with onset of schizophrenia in many studies. Present study is first from Pakistan to report effect of this locus in transmission and liaison of schizophrenia in Pakistani population. For this analysis 300 samples were genotyped of four SNPs, rs1417584, rs1954175, rs821616 and rs113012343 that were selected on basis of minor allele frequency and effects on schizophrenia. Haplotype and transmission disequilibrium analysis was also performed on data. Association of SNPs revealed a significant relation between MAF of rs1417584 and schizophrenia in given samples (χ2 5.57; p 0.02). Haplotype association led to formation of three significant blocks TCAG (OR 20.06), TTAG (OR 4.65) and CCTG (OR 26.17) for rs1417584, rs1954175, rs821616 and rs113012343 that were expected to cause schizophrenia in said combinations. rs1417584, rs1954175 and rs821616 were found to be in a linkage block based on D' value (p < 0.0001) with 22% co inheritance alongside disease onset. This block was represented by 325 kb on chromosome 1. It is concluded from this study that this 325 Kb region can be considered prognostic marker for schizophrenia development in Pakistani population.