Identification of the HAK gene family reveals their critical response to potassium regulation during adventitious root formation in apple rootstock

Adventitious root formation is a bottleneck during vegetative proliferation.Potassium(K^(+))is an essential macronutrient for plants.K^(+)accumulation from the soil and its distribution to the different plant organs is mediated by K^(+)transporters named K^(+)transporter(KT),K^(+)uptake(KUP),or high-affinity K^(+)(HAK).This study aimed to identify members of the HAK gene family in apples and to characterize the effects of K^(+)supply on adventitious root formation and on the expression of HAK genes and the genes that putatively control auxin transport,signaling,and cell fate during adventitious root formation.In this study,34 HAK genes(MdHAKs)were identified in the apple(Malus×domestica‘Golden Delicious’)genome.A phylogenetic analysis divided MdHAKs into four clusters(Ⅰ,Ⅱ,Ⅲ,andⅣ),comprising 16,1,4,and 13 genes,respectively.The syntenic relationships revealed that 62.5%of the total MdHAK genes arise from genomic duplication events.Chromosome location,domain structure,motif analysis,and physico-chemical characteristics were subsequently investigated.Furthermore,the application of K^(+)indicated the emergence of adventitious roots at 8 d and produced more adventitious roots at 16 d than the K^(+)-free control(CK)treatment.In addition,various MdHAKs showed root-specific expression in B9 apple rootstock stem cuttings and enhanced expression during the initiation and emergence stages of adventitious root formation in response to K^(+)treatment.Additionally,K^(+)treatment enhanced the expression levels of MdPIN1,MdPIN2,and MdAUX1.Further data indicated that a higher expression of MdWOX11,MdLBD16,and MdLBD29 and of cell cycle-related genes contributed to the auxin-stimulated adventitious root formation in response to K^(+).

Barley chitinase genes expression revamp resistance against whitefly (Bemisia Tabaci) in transgenic cotton (Gossypium hirsutum L.)

Background Chitinase is an enzyme that hydrolyzes chitin,a major component of the exoskeleton of insects,including plant pests like whiteflies.The present study aimed to investigate the expression of chemically synthesized barley ch1 and chi2 genes in cotton(Gossypium hirsutum)through Agrobacterium-mediated transformation.Fifty-five putative transgenic cotton plants were obtained,out of which fifteen plants successfully survived and were shifted to the field.Using gene-specific primers,amplification of 447 bp and 401 bp fragments confirmed the presence of the ch1 and chi2 genes in five transgenic cotton plants of the T0 generation.These five plants were further evalu-ated for their mRNA expression levels.The T0 transgenic cotton plants with the highest mRNA expression level and better yield performance in field,were selected to raise their subsequent progenies.Results The T1 cotton plants showed the highest mRNA expression levels of 3.5-fold in P10(2)for the ch1 gene and 3.7-fold in P2(1)for the chi2 gene.Fluorescent in situ hybridization(FISH)confirmed a single copy number of ch1 and chi2(hemizygous)on chromosome no.6.Furthermore,the efficacy of transgenes on whitefly was evaluated through an insect bioassay,where after 96 h of infestation,mortality rates of whitefly were calculated to be 78%–80%in transgenic cotton plants.The number of eggs on transgenic cotton plants were calculated to be 0.1%–0.12 per plant compared with the non-transgenic plants where egg number was calculated to be 0.90–1.00 per plant.Conclusion Based on these findings,it can be concluded that the chemically synthesized barley chitinase genes(ch1 and chi2)have the potential to be effective against insects with chitin exoskeletons,including whiteflies.The transgenic cotton plants expressing these genes showed increased resistance to whiteflies,resulting in reduced egg numbers and higher mortality rates.

Somatic embryogenesis in wild relatives of cotton (Gossypium Spp.)

Wild cotton species can contribute a valuable gene pool for agronomically desirable cultivated tetraploid cultivars. In order to exploit diploid cotton a regeneration system is required to achieve transformation based goals. The present studies aimed at optimizing the conditions for regeneration of local varieties as well as wild species of cotton. Different callus induction media were tested with varying concentrations of hormones in which sucrose was used as nutritional source. Different explants (hypo-cotyls, cotyledon, root) were used to check the regeneration of both local cotton plants and wild relatives using T & G medium, BAP medium, CIM medium, EMMS medium, and cell suspension medium. Different stages of embryogenicity such as early torpedo stage, late torpedo stage, heart stage, globular stage and cotyledonary stage were observed in wild relatives of cotton. The results of this study pave the way for establishing future transformation methods.

The current status of anti-GPCR drugs against different cancers

G protein coupled receptors(GPCRs)have emerged as the most potential target for a number of drug discovery programs ranging from control of blood pressure,diabetes,cure for genetic diseases to treatment of cancer.A panel of different ligands including hormones,peptides,ions and small molecules is responsible for activation of these receptors.Molecular genetics has identified key GPCRs,whose mutations or altered expressions are linked with tumorgenicity.In this review,we discussed recent advances regarding the involvement of GPCRs in the development of cancers and approaches to manipulating the mechanism behind GPCRs involved tumor growth and metastasis to treat different types of human cancer.This review provides an insight into the current scenario of GPCR-targeted therapy,progress to date and the challenges in the development of anticancer drugs.

Phytochemical analysis of Berberis lyceum methanolic extract and its antiviral activity through the restoration of MAPK signaling pathway modulated by HCV NS5A

Objective:To evaluate the antiviral activity and phytochemicals of selected plant extracts and their effect on the mitogen-activated protein kinase(MAPK)signaling pathway modulated by hepatitis C virus(HCV)nonstructural protein 5 A(NS5A).Methods:A total of ten plant extracts were initially screened for their toxicities against Hep G2 cells.The non-toxic plants were tested for their inhibitory effect on the expression of HCV NS5A at both m RNA and protein levels using real-time PCR and Western blotting assays,respectively.The differential expression of the genes associated with MAPK pathway in the presence of NS5A gene and plant extract was measured through real-time PCR.Subsequently,the identification of secondary metabolites was carried out by phytochemical and HPLC analysis.Results:The phytochemical profiling of Berberis lyceum revealed the presence of alkaloids,phenols,saponins,tannins,flavonoids,carbohydrates,terpenoids,steroids,and glycosides.Similarly,quercetin,myricetin,gallic acid,caffeic acid,and ferulic acid were identified through HPLC analysis.The methanolic extract of Berberis lyceum strongly inhibited HCV RNA replication with an IC50 of 11.44μg/m L.RT-PCR and Western blotting assays showed that the extract reduced the expression of HCV NS5A in a dosedependent manner.Berberis lyceum extract also attenuated NS5A-induced dysregulation of the MAPK signaling pathway.Conclusions:Our findings suggest that Berberis lyceum extract strongly inhibits HCV propagation by reducing HCV NS5A-induced perturbation of MAPK signaling.

Development of 19-plex Y STR system and polymorphism studies in Pakistani population

For the development of 19-plex Y STR system and polymorphism studies in local ethnic populations sixteen markers of non-recombining regions (NRY) of Y chromosome, which show high power of discrimination among individuals, were selected in this study. Blood samples (600) were collected from the males of three most common castes of Pakistani population (Arain, Awan and Rajput) with different parent lineages. Three markers (DYS385a/b, DYS389I/II and YCAIIa/b) among 16 Y STRs are double-targeted regions of the Y chromosome and thus provide two polymorphic peaks for each respective primer set. These 16 Y-STRs were developed into Megaplex system for simultaneous amplification of all markers within the population. The overall power of discrimination observed in focused populations was 60.5%, 66.5% and 55% in Rajput, Awan and Arain casts respectively. This discrimination power will be helpful in human identification for forensic casework studies including sexual assaults and paternity testing.

Identification,Isolation and Characterization of GaCyPI Gene in Gossypium arboreum under Cotton Leaf Curl Virus Disease Stress

Pakistan is facing the threat of Cotton Leaf Curl Virus(CLCuV)which is transmitted through whitefly to cotton crop.Molecular mechanism of leaf epicuticular wax protects the plants from different pathogens including insect attack and disease transmission.Objective of current study is the isolation and characterization of a wax related gene GaCyPI from Gossypium arboreum under CLCuV infection.A fragment of 475 bp was isolated from the total RNA and 3’and 5’RACE-PCR products were arranged by overlapping the extended sequences at both the ends.Deduced protein sequence of GaCyPI showed homology with Cyclophilin cis-trans isomerase gene of Gossypium ramondii and Gossypium barbadanse.Multiple sequence alignment also revealed homology among the coding sequences of same gene.GaCyPI protein comprised of 173 amino acids and ORF finder revealed the 69 bases upstream at 5’while 350 bp at 3’UTR.InterProScan revealed that it belongs to Cyclophilin-type peptidyl prolyl cis/trans isomerase(PPIase)family.Active sites are visible at specific amino acid positions and 3D structure was stable in Ramachandran plot.Prosa server showed protein residues have average 3D-1D score>=0.2 and Z-Score was−6.74.Phylogenetic analysis revealed that G.raimondii is the closest species that shares the same sequence.Hence,GaCyPI has strong role in plants’epicuticular wax and its genetic transformation may protect the cotton from whitefly which transmits CLCuV.

Analysis of Bifenthrin Degrading Bacteria from Rhizosphere of Plants Growing at Tannery Solid Waste

Bifenthrin is an insecticide which is used to control insects, mites, and ticks. It poses a solemn en-vironmental threat and health risk to living organisms. It may be bioaccumulated or biomagnified at different trophic levels in the food chain by biota. Microbes are hidden creature of earth’s biodiversity. For isolation of bifenthrin degrading bacteria, rhizospheric soil samples of plants like Pisum sativum, Triticun aestvum, Chenopodium album were taken from tannery solid waste, Kasur, Pakistan. Enrichment culture techniques were used for the isolation of bacterial strains that showed luxurious growth on minimal growth media with bifenthrin dose was selected for biodegradation study. Bacteria were further screened out based on their morphological, biochemical parameters and degradation efficiency. Furthermore the effect of different growth factors like temperature, pH, inoculum concencentration, minimal inhibitory concentration of heavy metals and antibiotics were also studied. Bacterial strains of Xanthomonas and Bacillus sp. were identified as efficient degrading microbes. Maximum bifenthrin utilization were observed at 25°C (pH 7), with 500 μL inoculum of Bacillus sp., while Xanthomonas sp. gave optimm utilization at 30°C (pH 7) at the same inoculum volume of bacteria. The Rf values of Bacillus sp. and Xanthomonas sp. were 0.91 and 0.90 respectively, which indicated their potential to metabolize bifenthrin into nontoxic forms. These strains can be used to clean up the sites polluted with pesticides and tannery wastes when present in rhizosphere of plants.

Evidence-based consensus on the diagnosis, prevention and management of hepatitis C virus disease

Hepatitis C virus(HCV) is a potent human pathogen and is one of the main causes of chronic hepatitis round the world. The present review describes the evidencebased consensus on the diagnosis, prevention and management of HCV disease. Various techniques, for the detection of anti-HCV immunoglobulin G immunoassays, detection of HCV RNA by identifying virus-specific molecules nucleic acid testings, recognition of core antigen for diagnosis of HCV, quantitative antigenassay, have been used to detect HCV RNA and core antigen. Advanced technologies such as nanoparticlebased diagnostic assays, loop-mediated isothermal amplification and aptamers and Ortho trak-C assay have also come to the front that provides best detection results with greater ease and specificity for detection of HCV. It is of immense importance to prevent this infection especially among the sexual partners, injecting drug users, mother-to-infant transmission of HCV, household contact, healthcare workers and people who get tattoos and piercing on their skin. Management of this infection is intended to eradicate it out of the body of patients. Management includes examining the treatment(efficacy and protection), assessment of hepatic condition before commencing therapy, controlling the parameters upon which dual and triple therapies work, monitoring the body after treatment and adjusting the co-factors. Examining the treatment in some special groups of people(HIV/HCV co-infected, hemodialysis patients, renal transplanted patients).

Hepatitis C virus genotype 3a infection and hepatocellular carcinoma: Pakistan experience

AIM: To assess the association between chronic hepatitis C virus (HCV) infection and hepatocellular carcinoma (HCC) in Pakistan, and the genotype distribution among these HCC patients.METHODS: One hundred and sixty-one subjects with HCC were included in this study. Liver biopsy was performed on 145 of the patients; sixteen were excluded because they failed to fulfill the inclusion criteria. Qualitative polymerase chain reaction (PCR) was performed for hepatitis B virus and HCV. Samples positive for HCV RNA were genotyped using genotype-specif ic PCR and conf irmed by HCV 5' noncoding region sequencing analysis. RESULTS: Chronic HCV infection was identified a major risk factor (63.44% of tested HCC patients) forthe development of HCC. The time from HCV infection to appearance of cancer was 10-50 years. In the HCC patient population, broader distributions of genotypes were present with genotype 3a as the predominant genotype. Using the type-specific genotyping method, we found HCV genotype 3a in 40.96%, 3b in 15.66%, 1a in 9.63%, and 1b in 2.40% of HCC tissue samples. About 28% of cases were found with mixed genotypes. Two cases were unable to be genotyped because of low viral load. Sixty-six percent of treated patients with cirrhosis had an end of treatment response, but unfortunately they relapsed quickly when the treatment was discontin-ued, and HCC developed during a median 3.8 years. CONCLUSION: There was a strong association between chronic HCV infection and HCC in Pakistan, and between HCV genotype 3a and HCC.

HCV genotype distribution and possible transmission risks in Lahore, Pakistan

AIM: To investigate the prevalence of hepatitis C virus (HCV) genotypes and their association with possible transmission routes in the general population of Lahore, as the data exclusively related to this city is limited. METHODS: Complete data regarding patient's history, possible route of infection and biochemical tests was collected from the public hospital for 1364 patients. SPSS version 16 windows software was used for data analysis by univariate and multivariate techniques. RESULTS: Age range ≤ 40 years showed high prevalence of HCV infection. HCV genotype 3a was dominant (55.9%), followed by 1a (23.6%), 4a (12.5%), 3b (3.2%), untypable (2.5%), 4b (1.2%) and mixed type (1.2%). Blood transfusion, dental surgery and barber shops were the main risk factors for HCV transmission. Genotype prevalence was independent of age (P = 0.971) and gender (P = 0.122) while risk factors showed a significant association with age (P = 0.000) and genotypes (P = 0.000). We observed an independent association of risk factors and genotype 3a, while patients with genotype 1 and 4 were mostly infected due to dental surgery blood transfusion and barber shops. Risk factors of intravenous drug use and sexual exposure were exclusively found in ≤ 40 years age group. CONCLUSION: An increase in genotypes 1a and 4a suggest migration of people, possibly from Balochistan and the northern war-zone area. Government should focus on public education regarding infection routes.

Potato Virus Y mRNA Expression Knockdown Mediated by siRNAs in Cultured Mammalian Cell Line

RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the capsid protein gene of potato virus Y(CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.

Genes and Transcriptional Factors in Chili Plant with Aspect to Metabolism and Resistance against Virus, Bacteria and Fungi： A Review

These days, there is a lot of discussion about genetically modified plants. There are different schools of thoughts in public, and some people adjusted while others are reluctant to accept genetically modified organism foods. Many vegetables are transformed and are used in daily life. Chili is one of those which is genetically modified and used in our food. Race specific genes can be used more efficiently for disease resistance and improving metabolic pathways. Different genes and transcriptional factors are available in Capsicum for this purpose. We can optimize and use the better expressed genes while engineering the chili plants, Genetic modifications causing significant changes are related with metabolism, which cause disease resistance.

Overexpression of the phytochrome B gene from Arabidopsis thaliana increases plant growth and yield of cotton (Gossypium hirsutum)

The phytochrome B (PHYB) gene of Arabidopsis thaliana was introduced into cotton through Agrobacterium tumefaciens.Integration and expression of PHYB gene in cotton plants were confirmed by molecular evidence.Messenger RNA (mRNA) expression in one of the transgenic lines,QCC11,was much higher than those of control and other transgenic lines.Transgenic cotton plants showed more than a two-fold increase in photosynthetic rate and more than a four-fold increase in transpiration rate and stomatal conductance.The increase in photosynthetic rate led to a 46% increase in relative growth rate and an 18% increase in net assimilation rate.Data recorded up to two generations,both in the greenhouse and in the field,revealed that overexpression of Arabidopsis thaliana PHYB gene in transgenic cotton plants resulted in an increase in the production of cotton by improving the cotton plant growth,with 35% more yield.Moreover,the presence of the Arabidopsis thaliana PHYB gene caused pleiotropic effects like semi-dwarfism,decrease in apical dominance,and increase in boll size.

Curcumin preconditioning enhances the efficacy of adipose-derived mesenchymal stem cells to accelerate healing of burn wounds

Background:Following recent findings from our group that curcumin preconditioning augments the therapeutic efficacy of adipose-derived stem cells in the healing of diabetic wounds in rats,we aimed to investigate the regenerative effects of curcumin preconditioned adipose-derived mesenchymal stem cells(ASCs)for better recovery of acid inflicted burns in this study.Methods:ASCs were preconditioned with 5μM curcumin for 24 hours and assessed for proliferation,migration,paracrine release potential and gene expression comparative to naive ASCs.Subsequently,the healing capacity of curcumin preconditioned ASCs(Cur-ASCs)versus naive ASCs was examined using acidic wounds in rats.For this,acid inflicted burns of 20 mm in diameter were made on the back of male Wistar rats.Then,2×10^(6) cells of Cur-ASCs and naive ASCs were intradermally injected in the wound periphery(n=6)for comparison with an untreated saline control.Post-transplantation,wounds were macroscopically analysed and photographed to evaluate the percentage of wound closure and period of re-epithelization.Healed wound biopsies were excised and used for histological evaluation and expression analysis of wound healing markers at molecular level by quantitative PCR and western blotting.Results:We found that Cur-ASCs exhibited greater proliferation,migration and paracrine potential in vitro.Further,Cur-ASCs showed more effective recovery than naive ASCs as exhibited by gross morphology,faster wound closure and earlier re-epithelialization.Masson’s trichrome and hematoxylin and eosin staining demonstrated the improved architecture of the healing burns,as evidenced by reduced infiltration of inflammatory cells,compact collagen and marked granulation in Cur-ASC treated rats.Corroborating these findings,molecular assessment showed significantly reduced expressions of pro-inflammatory factors(interleukin-1 beta,interleukin-6,tumor necrosis factor alpha)a with striking upsurge of an oxidative marker(superoxide dismutase 1),proangiogenic factors(vascular endothelial growth factor,hepatocyte growth factor,hypoxia-inducible factor-1 alpha)and collagen markers(transforming growth factor beta 1,fibroblast growth factor-2,collagen type 1 alpha 1),verifying that Cur-ASCs modulate the regulation of pro-inflammatory and healing markers at burn sites.Conclusions:Treatment with Cur-ASCs resulted in faster re-epithelization of acid inflicted burns compared to the treatment with naive ASCs.Based on observed findings,we suggest the transplantation of Cur-ASCs is a valuable therapy for the potent clinical management of acidic burns.

HCV infection causes cirrhosis in human by step-wise regulation of host genes involved in cellular functioning and defense during fibrosis:Identification of bio-markers

Chronic Hepatitis C Viral(HCV)infection is a leading health problem worldwide and resulted in fibrotic scar formation,and finally liver-cirrhosis.Although contemporary therapies can partially reverse this destructive process,the rehabilitation is too slow and unsuitable for all chronic infections.The current study elucidates the mechanism of disease progression from early(F1)to moderate(F2,F3),and to severe fibrosis(F4)/cirrhosis in HCV genotype 3a infected patients to find out new candidates as potential disease progression markers and antiviral therapeutic agents.A total of 550 genes were found differentially regulated in the four fibrosis stages and grouped in 22 classes according to their biological functions.Gene set enrichment(GSEA)and Ingenuity pathway analysis(IPA)were used to identify the regulation of crucial biological functions and pathways involved in HCV progression.HCV differentially regulated the expression of genes involved in apoptosis,cell structure,signal transduction,proliferation,metabolism,cytokine signaling,immune response,cell adhesion and maintenance,and post translational modifications by pathway analysis.There was an increasing trend of proliferative and cell growth related genes and shutting down of immune response as the disease progress mild to moderate to advanced stage cirrhosis.The myriad of changes in gene expression showed more chances of developing liver cancer in patients infected with HCV genotype 3a in a systematic manner.The identified gene set can act as disease markers for prediction,whether the fibrosis lead to cirrhosis and its association with end stage liver disease development.

Antimicrobial potential of Pakistani medicinal plants against multi-drug resistance Staphylococcus aureus

Objective:To determine resistance patterns of Staphylococcus aureus(S.aureus)isolated from different areas of Pakistan and to identify antimicrobial agents against multi-drug resistant S.aureus strains.Methods:A total of 67 samples(sewerage,nasal and milk)were collected from different farm areas of Pakistan to identify local strains of S.aureus.Sixteen out of 67 samples were positive for S.aureus.Only 6 out of 16 S.aureus strains showed resistance to antibiotics.Then the antibacterial effect of 29 medicinal plants was evaluated on these S.aureus isolates and a standard S.aureus strain ATCC 25923.The solvents used for the extraction of plants were acetone,dimethyl sulfoxide and methanol.The in vitro antibacterial activity was performed using agar disc diffusion method.Moreover,minimum inhibitory concentration of effective medicinal plant extracts was identified through micro-dilution method to find out their 50%inhibitory concentration.Results:Plant extracts of 5 medicinal plants(Psidium guajava,Nigella sativa,Piper nigrum,Valeriana jatamansi,and Cucurbita pepo)exhibited antibacterial activity against locally isolated multidrug resistant strains of S.aureus.The minimum inhibitory concentration of these extracts was ranged from 0.328 to 5.000 mg/mL.Conclusions:Plant extracts of Psidium guajava,Piper nigrum seed,Valeriana jatamansi,Cucurbita pepo and Nigella sativa showed significant in vitro antibacterial activity and thus,such findings may serve as valuable contribution in the treatment of infection and may contribute to the development of potential antimicrobial agents against multi drug resistant strains of S.aureus.

In vitro Selection for Fusarium Wilt Resistance in Gladiolus

Cormels pieces of four Fusarium susceptible Gladiolus cultivars （Friendship, Peter Pears, Victor Borge and Novalux） formed friable calli when cultured in vitro on Murashige and Skoog basal medium containing various concentrations of auxin and cytokinin. The friable calli established cell suspensions. Plantlet regeneration was obtained from the control callus, control cell suspension derived callus and in vitro selected Fusarium oxysporum Schlecht. resistant cell-lines of Friendship. The in vitro cormlets showed 85-95% germination after breaking dormancy of 8 weeks at 4℃. Cell suspensions of all four Gladiolus cultivars were found to be highly sensitive to fusaric acid. Gradual increase in fusaric acid concentrations to the cell-suspension cultures decreased cell growth considerably. One albino plant was found from the second generation of the in vitro selected cell line of Friendship. The albino plant was found to be highly susceptible to F. oxysporum. The cormlets of all in vitro selected cell lines of Friendship were inoculated with a conidial suspension of the F. oxysporum before planting and were also sprayed with the same spore suspension for further characterization when the height of plants was about 6cm. The four selected cell lines showed the same response whether or not they were inoculated with conidia of the F. oxysporum. Plantlets of all of the selected cell lines exhibited significant growth as compared with the control after application of conidia of the F. oxysporum.