Isolation and Screening of Zn (Zn) Solubilizing Rhizosphere Bacteria from Different Vegetations for Their Ability to Improve Growth, Zn Uptake, and Expression of Zn Transporter Genes in Tomato.

Zinc-solubilizing bacteria (ZSB) can convert insoluble zinc to an accessible form and increase Zn bioavailability in soil, which helps mitigate Zn deficiency in crops. In this study, different bacterial strains were screened for different Zn solubilization and plant growth promotion traits. Two bacterial strains, Acinetobacter pittii DJ55 and Stenotrophomonas maltophilia DJ24, were tested for their Zn-solubilizing potential on plate media, and both showed variable levels of Zn solubilization. The results showed that the bacterial strains applied to the plants in the pot experiment caused improvements in growth parameters compared to control conditions. DJ55, when applied with an insoluble source, enhanced plant height, leaf number, and leaf area compared to DJ24 and control conditions, while the maximum fruit weight was noticed in plants treated with ZnSO4. An increase in chlorophyll contents was noted in plants treated with ZnSO4, while maximum carotenoid contents were observed in plants treated with DJ55 + ZnO when compared with their controls. Plants supplemented with ZnO and DJ55 showed higher zinc content and iron content as compared to their respective controls. The expression patterns of the SLZIP5 and SLZIP4 genes were changed in the root and shoot. Application of ZnO stimulates both gene expression and protein synthesis in tomato roots and shoots. Inoculation of tomato plants with ZSB and insoluble ZnO reduced the expression of the SLZIP5 and SLZIP4 genes in the root and shoot. In conclusion, both strains can be considered as potential zinc-solubilizing bioinoculants to promote the growth and production yield of tomato.

Estrogen receptor alpha (ESR1) gene polymorphism (rs2234693 and rs2046210) with breast cancer risk in pashtun population of Khyber Pakhtunkhwa.

BACKGROUND: Breast cancer susceptibility is greatly influenced by single nucleotide polymorphisms (SNPs) both in penetrance and non-penetrance genes. The Estrogen Receptor Alfa (ESR1- rs2234693 and rs2046210) have been reported as risk factor of breast cancer in different ethnic groups with inconsistent results. In this study the association of ESR1 (rs2234693 and rs2046210) with breast cancer risk was investigated in patients of Khyber Pakhtunkhwa. METHODS: A total of 312 females including 162 breast cancer patients and 150 healthy controls were enrolled in this study. The polymorphism was confirmed using T-ARMS-PCR. RESULTS: Our results revealed that ESR1-rs2234693 risk allele (C) (P = 0.21, OR = 1.27, CI = 0.87 to 1.87) and containing genotypes CC (P = 0.68, OR = 1.24, CI = 0.42 to 3.68) and TC (P = 0.23, OR = 1.32, CI = 0.83 to 2.13) were not associated with the risk of breast cancer. In case of rs2046210, the risk allele A (P < 0.0001, OR = 2.42, CI = 1.74 to 3.38) and corresponding genotypes GA (P = 0.0001, OR = 2.55, CI = 1.62 to 4.03) and AA (P = 0.02, OR = 2.20, CI = 1.12 to 4.34) were significantly associated with higher risk of breast cancer. Moreover, ESR1-rs2234693 was significantly (P < 0.05) associated with family history, stages, PR status, ER status and luminal B. The ESR1-rs2046210 showed significant (P ≤ 0.05) association with menstrual status, tumor grade and TNBC. Both the SNPs showed non-significant (P > 0.05) association with nulliparity, nodal status, HER2 status, metastasis, HER2 enriched subtype and luminal A. CONCLUSION: It is concluded that ESR1-rs2234693 is not associated with breast cancer, while rs2046210 is significantly associated with the risk of breast cancer in Khyber Pakhtunkhwa population. Further, to confirm the exact situation of ESR1 polymorphism, ESR1 existing and other SNPs need to be investigated in diverse data sets.

Significant association of BRCA1 (rs1799950), BRCA2 (rs144848) and TP53 (rs1042522) polymorphism with breast cancer risk in Pashtun population of Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: Single nucleotide polymorphism (SNPs) in BRCA1, BRCA2 and TP53 has been widely associated with breast cancer risk in different ethnicities with inconsistent results. There is no such study conducted so far in the Pashtun population of Khyber Pakhtunkhwa, Pakistan. Therefore, this study was conducted to check BRCA1 (rs1799950), BRCA2 (rs144848) and TP53 (rs1042522) polymorphism with breast cancer risk in Pashtun population of Khyber Pakhtunkhwa, Pakistan. METHODS: This study, consisting 140 breast cancer patients and 80 gender and age matched healthy controls were subjected to confirm BRCA1, BRCA2 and TP53 polymorphism. Clinicopathological data and blood samples were taken from all the participants. DNA was extracted and SNPs were confirmed using T-ARMS-PCR protocol. RESULTS: Our data indicated that BRCA1, BRCA2, and TP53 selected SNPs risk allele and risk allele containing genotypes displayed significant association (p < 0.05) with breast cancer risk in the Pashtun population of Khyber Pakhtunkhwa, Pakistan. CONCLUSION: All the three selected SNPs of BRCA1, BRCA2 and TP53 showed significant association with breast cancer risk in the Pashtun population of Khyber Pakhtunkhwa, Pakistan. However, more investigation will be required on large data sets to confirm the selected SNPs and other SNPs in the selected and other related genes with the risk of breast cancer.

Prevalence of dengue virus serotypes in the 2017 outbreak in Peshawar, KP, Pakistan.

BACKGROUND: Dengue is a viral disease, transmitted by infected Aedes aegypti and Aedes albopictus female mosquitoes. Worldwide, 96 million infections were estimated in 2010. The dengue virus comprises four distinct serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) which belong to the genus Flavivirus. Determining the serotypes during dengue outbreaks is crucial for its effective management in terms of diagnostics improvement and polyvalent vaccine development. The aim of the present study is to determine the prevalence rate of dengue virus serotypes in the samples collected from patients during the 2017 outbreak in Khyber Pakhtunkhwa, Pakistan. METHODS: A total of 800 ELISA-positive samples were collected, of which 513 (290 males, 223 females) samples were confirmed positive by PCR. RESULTS: Out of 513, 25 were found serotype 1 (5%), 196 were serotype 2 (38%), 192 were serotype 3 (37%), 56 were serotype 4 (11%), and 44 (8%) were found to have mix serotypes. CONCLUSION: We can conclude that serotypes 2 and 3 of dengue virus were the predominated serotypes of dengue virus in the 2017 outbreak in Peshawar, capital city of Khyber Pakhtunkhwa, Pakistan.

Molecular charactarization of wheat advanced lines for leaf rust resistant genes using SSR markers.

A total of fifty seven wheat advanced lines were screened to detect the existence of leaf rust resistant genes (Lr9, Lr13, Lr19, Lr24, Lr26, Lr28, Lr32, Lr34, Lr35, Lr36, Lr37, Lr39 and Lr46) using thirteen SSR markers. Only four markers for Lr13, Lr32, Lr34 and Lr35 produced separate, reproducible bands which indicated the positive linkage of leaf rust resistance with these SSR markers. The highest frequency was observed for Lr32 (100%), as it was detected in all fifty seven lines, followed by Lr34 (89.4%) in 51 lines, Lr35 (87.7%) in 50 lines and Lr13 (31.5%) in 18 lines respectively. All the four resistant genes were identified in fifteen lines which is only 26% of the studied population. These results indicate that there are limited number of variant genes for leaf rust resistance in the studied wheat advanced lines. Therefore, strategies for arraying these genes to lengthen infection resistance are advised to eliminate wheat rust diseases. In addition, more reliable and capable markers are essential to be settled for marker assisted selection of these and other genes.

Genetic Polymorphism and Bottleneck Analysis of Balkhi, Hashtnagri, and Michni Sheep Populations Using Microsatellite Markers.

Pakistan is rich in sheep genetic resources. Balkhi, Hashtnagri, and Michni are neighboring sheep populations found in Khyber Pakhtunkhwa province of Pakistan. In this study, we analyzed the genetic structures and bottleneck incidents within these sheep populations using 31 microsatellite DNA markers. Total numbers of 116, 100, and 95 alleles, with average numbers of 3.20, 3.26, and 3.74 alleles per locus were observed, respectively, in Balkhi, Hashtnagri, and Michni population. Mean observed heterozygosity was 0.402 in Balkhi, 0.416 in Hashtnagri, and 0.522 in Michni population. All the three sheep populations showed significantly high inbreeding. Michni population was found to be in mutation drift equilibrium, showing the absence of genetic bottleneck. The data of Balkhi and Hashtnagri indicated the presence of genetic bottleneck in these populations. These results suggest a moderate level of genetic diversity within Michni population that may be useful for breed improvement programs. Hashtnagri and Balkhi populations having low within breed genetic variability may contain some valuable characteristics that need to be conserved.

Immunoinformatics design of multivalent epitope vaccine against monkeypox virus and its variants using membrane-bound, enveloped, and extracellular proteins as targets.

Introduction: The current monkeypox (MPX) outbreak, caused by the monkeypox virus (MPXV), has turned into a global concern, with over 59,000 infection cases and 23 deaths worldwide. Objectives: Herein, we aimed to exploit robust immunoinformatics approach, targeting membrane-bound, enveloped, and extracellular proteins of MPXV to formulate a chimeric antigen. Such a strategy could similarly be applied for identifying immunodominant epitopes and designing multi-epitope vaccine ensembles in other pathogens responsible for chronic pathologies that are difficult to intervene against. Methods: A reverse vaccinology pipeline was used to select 11 potential vaccine candidates, which were screened and mapped to predict immunodominant B-cell and T-cell epitopes. The finalized epitopes were merged with the aid of suitable linkers, an adjuvant (Resuscitation-promoting factor), a PADRE sequence (13 aa), and an HIV TAT sequence (11 aa) to formulate a multivalent epitope vaccine. Bioinformatics tools were employed to carry out codon adaptation and computational cloning. The tertiary structure of the chimeric vaccine construct was modeled via I-TASSER, and its interaction with Toll-like receptor 4 (TLR4) was evaluated using molecular docking and molecular dynamics simulation. C-ImmSim server was implemented to examine the immune response against the designed multi-epitope antigen. Results and discussion: The designed chimeric vaccine construct included 21 immunodominant epitopes (six B-cell, eight cytotoxic T lymphocyte, and seven helper T-lymphocyte) and is predicted non-allergen, antigenic, soluble, with suitable physicochemical features, that can promote cross-protection among the MPXV strains. The selected epitopes indicated a wide global population coverage (93.62%). Most finalized epitopes have 70%-100% sequence similarity with the experimentally validated immune epitopes of the vaccinia virus, which can be helpful in the speedy progression of vaccine design. Lastly, molecular docking and molecular dynamics simulation computed stable and energetically favourable interaction between the putative antigen and TLR4. Conclusion: Our results show that the multi-epitope vaccine might elicit cellular and humoral immune responses and could be a potential vaccine candidate against the MPXV infection. Further experimental testing of the proposed vaccine is warranted to validate its safety and efficacy profile.

Structure-based identification of potential substrate antagonists for isethionate sulfite-lyase enzyme of Bilophila Wadsworthia: Towards novel therapeutic intervention to curb gut-associated illness.

Bilophila wadsworthia is one of the prominent sources of hydrogen sulfide (H2S) production in appendices, excessive levels of which can result in a weaker colonic mucus barrier, inflammatory bowel disease, and colorectal cancer. Isethionate sulfite-lyase (IslA) enzyme catalyzes H2S production by cleaving CS bond in isethionate, producing acetaldehyde and sulfite. In this study, we aimed to identify potential substrate antagonists for IsIA using a structure-based drug design. Initially, pharmacophore-based computational screening of the ZINC20 database yielded 66 hits that were subjected to molecular docking targeting the isethionate binding site of IsIA. Based on striking docking scores, nine compounds showed strong interaction with critical IsIA residues (Arg189, Gln193, Glu470, Cys468, and Arg678), drug-like features, appropriate adsorption, metabolism, excretion, and excretion profile with non-toxicity. Molecular dynamics simulations uncovered the significant impact of binding the compounds on protein conformational dynamics. Finally, binding free energies revealed substantial binding affinity (ranging from -35.23 to -53.88 kcal/mol) of compounds (ZINC913876497, ZINC913856647, ZINC914263733, ZINC914137795, ZINC915757996, ZINC914357083, ZINC913934833, ZINC9143362047, and ZINC913854740) for IsIA. The compounds proposed herein through a multi-faceted computational strategy can be experimentally validated as potential substrate antagonists of B. wadsworthia's IsIA for developing new medications to curb gut-associated illness in the future.

Detrimental effects of Cd and temperature on rice and functions of microbial community in paddy soils.

Heavy metal (HM) contamination and high environmental temperature (HT) are caused by anthropogenic activities that negatively impact soil microbial communities and agricultural productivity. Although HM contaminations have deleterious effects on microbes and plants; there are hardly any reports on the combined effects of HM and HT. Here, we reported that HT coupled with cadmium (Cd) accumulation in soil and irrigated water could seriously affect crop growth and productivity, alternatively influencing the microbial community and nutrient cycles of paddy soils in rice fields. We analyzed different mechanisms of plants and microflora in the rhizospheric region, such as plant rhizospheric nitrification, endophytes colonization, nutrient uptake, and physiology of temperature-sensitive (IR64) and temperature-resistant Huanghuazhan (HZ) rice cultivars against different Cd levels (2, 5 and 10 mg kg-1) with rice plants grown under 25 °C and 40 °C temperatures. Consequently, an increment in Cd accumulation was observed with rising temperature leading to enhanced expression of OsNTRs. In contrast, a greater decline in the microbial community was detected in IR64 cultivar than HZ. Similarly, ammonium oxidation, root-IAA, shoot-ABA production, and 16S rRNA gene abundance in the rhizosphere and endosphere were significantly influenced by HT and Cd levels, resulting in a significant decrease in the colonization of endophytes and the surface area of roots, leading to a decreased N uptake from the soil. Overall, the outcomes of this study unveiled the novel effects of Cd, temperature, and their combined effect on rice growth and functions of the microbial community. These results provide effective strategies to overcome Cd-phytotoxicity on the health of endophytes and rhizospheric bacteria in Cd-contaminated soil by using temperature-tolerant rice cultivars.

Identifying non-nucleoside inhibitors of RNA-dependent RNA-polymerase of SARS-CoV-2 through per-residue energy decomposition-based pharmacophore modeling, molecular docking, and molecular dynamics simulation.

BACKGROUND AND OBJECTIVE: The current coronavirus disease-2019 (COVID-19) pandemic has triggered a worldwide health and economic crisis. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes the disease and completes its life cycle using the RNA-dependent RNA-polymerase (RdRp) enzyme, a prominent target for antivirals. In this study, we have computationally screened ∼690 million compounds from the ZINC20 database and 11,698 small molecule inhibitors from DrugBank to find existing and novel non-nucleoside inhibitors for SARS-CoV-2 RdRp. METHODS: Herein, a combination of the structure-based pharmacophore modeling and hybrid virtual screening methods, including per-residue energy decomposition-based pharmacophore screening, molecular docking, pharmacokinetics, and toxicity evaluation were employed to retrieve novel as well as existing RdRp non-nucleoside inhibitors from large chemical databases. Besides, molecular dynamics simulation and Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method were used to investigate the binding stability and calculate the binding free energy of RdRp-inhibitor complexes. RESULTS: Based on docking scores and significant binding interactions with crucial residues (Lys553, Arg557, Lys623, Cys815, and Ser816) in the RNA binding site of RdRp, three existing drugs, ZINC285540154, ZINC98208626, ZINC28467879, and five compounds from ZINC20 (ZINC739681614, ZINC1166211307, ZINC611516532, ZINC1602963057, and ZINC1398350200) were selected, and the conformational stability of RdRp due to their binding was confirmed through molecular dynamics simulation. The free energy calculations revealed these compounds possess strong binding affinities for RdRp. In addition, these novel inhibitors exhibited drug-like features, good absorption, distribution, metabolism, and excretion profile and were found to be non-toxic. CONCLUSION: The compounds identified in the study by multifold computational strategy can be validated in vitro as potential non-nucleoside inhibitors of SARS-CoV-2 RdRp and holds promise for the discovery of novel drugs against COVID-19 in future.

Heavy metal ATPase genes (HMAs) expression induced by endophytic bacteria, "AI001, and AI002" mediate cadmium translocation and phytoremediation.

Contamination of heavy metals is a serious threat, which causes threats to the environment. Our study aimed to determine the role of endophytic bacteria in Cd phytoremediation and heavy metal ATPase gene expression. Cadmium (Cd) resistant endophytic bacteria were isolated from Solanum nigrum on LB agar plates, contaminated with 0-30 mg/L Cd. The phosphate solubilization and indole-3-acetic acid (IAA) production of endophytes were estimated by growing them on Pikovskaya agar medium and GC-MS analysis, respectively. An experiment in a pot was performed to evaluate the effects of bacteria on rice plants contaminated with 5-25 mg/L of Cd. Expression of Cd response genes was quantified through qRT-PCR and Cd translocation from one part to another part of the plant was measured through the ICP. BLAST alignment of 16 S-rDNA gene sequences confirmed the bacterial isolates as Serratia sp. AI001 and Klebsiella sp. Strain AI002. Both strains tolerated Cd up to 25 mg/L and produced 27-30 µg/mL of IAA. Inoculation of AI001 and AI002 improved plant growth dynamics (i.e., plant length, biomass, chlorophyll contents), relieved electrolyte leakage, and improved reduced glutathione significantly (P < 0.05). The inoculation of AI001 and AI002 significantly (P < 0.05) induced the expression of heavy metal ATPase genes ie., "HMA2, HMA3, and HMA4" and Cd translocation compared to uninoculated plants. Both AI001 and AI002 exhibited very prominent plant-growth-promoting and Cd phytoremediation properties. The results revealed that isolates also contributed a lot to the expression of rice plant heavy metal ATPase genes and in the Cd translocation in the plant.

Exploring whole proteome to contrive multi-epitope-based vaccine for NeoCoV: An immunoinformtics and in-silico approach.

Neo-Coronavirus (NeoCoV) is a novel Betacoronavirus (ß-CoVs or Beta-CoVs) discovered in bat specimens in South Africa during 2011. The viral sequence is highly similar to Middle East Respiratory Syndrome, particularly that of structural proteins. Thus, scientists have emphasized the threat posed by NeoCoV associated with human angiotensin-converting enzyme 2 (ACE2) usage, which could lead to a high death rate and faster transmission rate in humans. The development of a NeoCoV vaccine could provide a promising option for the future control of the virus in case of human infection. In silico predictions can decrease the number of experiments required, making the immunoinformatics approaches cost-effective and convenient. Herein, with the aid of immunoinformatics and reverse vaccinology, we aimed to formulate a multi-epitope vaccine that may be used to prevent and treat NeoCoV infection. Based on the NeoCoV proteins, B-cell, cytotoxic T lymphocyte (CTL), and helper T lymphocyte (HTL) epitopes were shortlisted. Four vaccines (Neo-1-4) were devised by fusing shortlisted epitopes with appropriate adjuvants and linkers. The secondary and three-dimensional structures of final vaccines were then predicted. The binding interactions of these potential vaccines with toll-like immune receptors (TLR-2, TLR-3, and TLR-4) and major histocompatibility complex molecules (MHC-I and II) reveal that they properly fit into the receptors' binding domains. Besides, Neo-1 and Neo-4 vaccines exhibited better docking energies of -101.08 kcal/mol and -114.47 kcal/mol, respectively, with TLR-3 as compared to other vaccine constructs. The constructed vaccines are highly antigenic, non-allergenic, soluble, non-toxic, and topologically assessable with good physiochemical characteristics. Codon optimization and in-silico cloning confirmed efficient expression of the designed vaccines in Escherichia coli strain K12. In-silico immune simulation indicated that Neo-1 and Neo-4 vaccines could induce a strong immune response against NeoCoV. Lastly, the binding stability and strong binding affinity of Neo-1 and Neo-4 with TLR-3 receptor were validated using molecular dynamics simulations and free energy calculations (Molecular Mechanics/Generalized Born Surface Area method). The final vaccines require experimental validation to establish their safety and effectiveness in preventing NeoCoV infections.

Response to combination therapy of HCV 3a infected Pakistani patients and the role of NS5A protein.

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

Prevalence of HCV among the high risk groups in Khyber Pakhtunkhwa.

Hepatitis C is an infectious disease, caused by blood borne pathogen; the Hepatitis C Virus. In this study we analyzed blood samples collected from various risk groups for the prevalence of anti-HCV and active HCV infection with the help of Immunochromtographic tests and nested PCR. The prevalence of active HCV infection among the high risk groups was 15.57% (26/167). The prevalence of HCV in individual risk groups was 15%, 28%, 8%, 14.28% and 14.28% in the case of thalassemics, dialysis, major surgery group, dental surgery group and injection drug users respectively. Our analysis reveals the fact that health care facilities in the Khyber Pakhtunkhwa province of Pakistan are contributing a great deal towards the spread of HCV infection.

Active hepatitis C infection and HCV genotypes prevalent among the IDUs of Khyber Pakhtunkhwa.

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

Prevalence of active HCV infection among the blood donors of Khyber Pakhtunkwa and FATA region of Pakistan and evaluation of the screening tests for anti-HCV.

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

Duplex PCR assay for the detection of avian adeno virus and chicken anemia virus prevalent in Pakistan.

Avian Adeno viruses and Chicken Anemia Viruses cause serious economic losses to the poultry industry of Pakistan each year. Timely and efficient diagnosis of the viruses is needed in order to practice prevention and control strategies. In the first part of this study, we investigated broilers, breeder and Layer stocks for morbidity and mortality rates due to AAV and CAV infections and any co-infections by examining signs and symptoms typical of their infestation or post mortem examination. In the second part of the study, we developed a duplex PCR assay for the detection of AAV and CAV which is capable to simultaneously detect both the viral types prevalent in Pakistan with high sensitivity and 100% specificity.

Identification and characterization of thermophilic amylase producing bacterial isolates from the brick kiln soil.

The present experiment was designed to isolate bacterial strains from the brick kiln soil and to check the activity and enzyme kinetics of amylase from these isolates. The bacterial colonies were isolated from soil samples through the serial dilution method. The bacterial isolates were identified through morphological, electron microscopic and molecular analysis. The 16S ribosomal RNA sequences of the isolates IR-1, IR-2, IR-3, IR-8, and IR-9 showed high similarities with Bacillus tequilensis, Bacillus paramycoides, Proteus alimentorum, Bacillus wiedmannii, and Pseudomonas aeruginosa, respectively. All of the bacterial isolates showed a positive catalase activity except IR-9. Furthermore, the isolates showed variable antagonistic effects against different bacterial pathogens. All of the strains produced indole acetic acid (IAA), and the concentrations increased in the presence of tryptophan application. The isolates showed the amylase enzyme activity and maximum activity of isolates was achieved in 4% starch concentration. The IR-9 isolate showed the highest amylase activity of 5.9 U/ml. The V max values of the extracellular amylase from different bacterial isolates ranged between 12.90 and 50.00 IU ml-1. The lowest K m value of 6.33 mg starch was recorded for IR-8 and the maximum K cat value of 2.50 min-1 was observed for IR-3. The amylase activity of the isolates was significantly affected by a range of different incubation time, temperature, and pH values. Further tests are required before the potential utilization of these isolates for amylase production, and in the biopesticide and biofertilizer applications.

Determination of mutations in iron regulating genes of beta thalassemia major patients of Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: Hepcidin and hemochromatosis (HFE) are iron regulatory proteins that are encoded by HAMP and HFE genes. Mutation in either HAMP gene or HFE gene causes Hepcidin protein deficiency that can lead to iron overload in beta thalassemia patients. The aim of this research work was to study the presence of G71D mutation of HAMP gene and H63D mutation of HFE gene in beta thalassemia major and minor group to check the association of these mutations with serum ferritin level of beta thalassemia patients. METHODS: The study was conducted on 42 beta thalassemia major and 20 beta thalassemia minor samples along with 20 control samples. The genotyping of both mutations has done by ARM-PCR technique with specific set of primers. RESULTS: Significant effect of G71D and H63D mutations was observed on serum ferritin level of thalassemia major group. The risk allele of HAMP G71D and HFE H63D was found with high frequency (48% and 49%, respectively) in beta thalassemia major than in control group. High genotypic frequency of HAMP and HFE gene mutation gene mutation was observed in beta thalassemia major than beta thalassemia minor and control group (7% and 9%, respectively). CONCLUSION: It can be concluded that both HAMP and HFE gene mutations show high frequency in beta thalassemia major patients and mean significant association between mutations and high serum ferritin level of beta thalassemia major patients but the nonsignificant results of Odd ratios showed that both mutations do not act as major risk factor in beta thalassemia major.

Identification and characterization of a novel multi-stress responsive gene in Arabidopsis.

Abiotic stresses especially salinity, drought and high temperature result in considerable reduction of crop productivity. In this study, we identified AT4G18280 annotated as a glycine-rich cell wall protein-like (hereafter refer to as GRPL1) protein as a potential multistress-responsive gene. Analysis of public transcriptome data and GUS assay of pGRPL1::GUS showed a strong induction of GRPL1 under drought, salinity and heat stresses. Transgenic plants overexpressing GRPL1-3HA showed significantly higher germination, root elongation and survival rate under salt stress. Moreover, the 35S::GRPL1-3HA transgenic lines also showed higher survival rates under drought and heat stresses. GRPL1 showed similar expression patterns with Abscisic acid (ABA)-pathway genes under different growth and stress conditions, suggesting a possibility that GRPL1 might act in the ABA pathway that is further supported by the inability of ABA-deficient mutant (aba2-1) to induce GRPL1 under drought stress. Taken together, our data presents GRPL1 as a potential multi-stress responsive gene working downstream of ABA.