Genome-wide identification and expression profiling of photosystem II(PsbX)gene family in upland cotton(Gossypium hirsutum L.)

Background Photosystem II(PSII)constitutes an intricate assembly of protein pigments,featuring extrinsic and intrinsic polypeptides within the photosynthetic membrane.The low-molecular-weight transmembrane protein PsbX has been identified in PSII,which is associated with the oxygen-evolving complex.The expression of PsbX gene protein is regulated by light.PsbX’s central role involves the regulation of PSII,facilitating the binding of quinone molecules to the Qb(PsbA)site,and it additionally plays a crucial role in optimizing the efficiency of photosynthesis.Despite these insights,a comprehensive understanding of the PsbX gene’s functions has remained elusive.Results In this study,we identified ten PsbX genes in Gossypium hirsutum L.The phylogenetic analysis results showed that 40 genes from nine species were classified into one clade.The resulting sequence logos exhibited substantial conservation across the N and C terminals at multiple sites among all Gossypium species.Furthermore,the ortholo-gous/paralogous,Ka/Ks ratio revealed that cotton PsbX genes subjected to positive as well as purifying selection pressure might lead to limited divergence,which resulted in the whole genome and segmental duplication.The expression patterns of GhPsbX genes exhibited variations across specific tissues,as indicated by the analysis.Moreover,the expression of GhPsbX genes could potentially be regulated in response to salt,intense light,and drought stresses.Therefore,GhPsbX genes may play a significant role in the modulation of photosynthesis under adverse abiotic conditions.Conclusion We examined the structure and function of PsbX gene family very first by using comparative genom-ics and systems biology approaches in cotton.It seems that PsbX gene family plays a vital role during the growth and development of cotton under stress conditions.Collectively,the results of this study provide basic information to unveil the molecular and physiological function of PsbX genes of cotton plants.

Assessment of Physico-Chemical and Microbial Quality of Sugarcane Juice Sold by Street Vendors in Three Regions of Bangladesh

Sugar cane juice is a popular refreshing drink in most part of Bangladesh. It has great taste and health benefits;also it is available most of the public places at reasonable prices which consumed by road side customers including general public, shopping personals, tourists, students. In our country, street vendors crushing sugarcane between roller crusher and sold without any heat treatment or preservative, also served with or without added ice and lemon juice. The study aimed to identify and compare the physico-chemical and microbial quality of sugarcane juice. The chemical qualities of juices including moisture, PH, ash, total soluble solid, total sugar, reducing sugar and titrable acidity were found slightly different in Mymensingh, Gazipur and Narayangonj areas. The highest and lowest value of moisture, ash, PH, total soluble solids, total sugar, reducing sugar and titrable acidity were found in 84.33% - 79.26% (Gazipur-Mymensingh), 0.57% - 0.04% (Mymensingh-Gazipur), 5.9 - 2.9 (Gazipur-Narayangonj), 17.48% - 4.98% (Mymensingh-Narayangonj), 21.9% - 6.56% (Mymensingh-Gazipur), 3.7 - 2.1 (Gazipur-Mymensingh) and 0.523% - 0.007% (Narayangonj-Gazipur) respectively. For microbiological analysis, the total viable count of sugar cane juice in Mymensingh, Gazipur and Narayangonj were ranged from 0.6 × 105 - 43.6 × 105 cfu/ml, 4.6 × 105 - 21.6 × 105 cfu/ml and 3.6 × 105 - 36.6 × 105 cfu/ml respectively, where the permitted value is 1.0 × 104 cfu/ml, whereas the total coliform count was ranged from 0.4 × 105 - 6.4 × 105 cfu/ml, 0.6 × 105 - 8.4 × 105 cfu/ml and 0.00 - 8.4 × 105 cfu/ml, where the permitted value is 100 cfu/ml and total fungal count was ranged from 5.5 × 105 - 56.5 × 105 cfu/ml, 21.5 × 105 - 54.5 × 105 cfu/ml and 32.5 × 105 - 68.5 × 105 cfu/ml, where the permitted value is 1000 cfu/ml. According to the Gulf standard, the microbiological parameters of all the collected sugar cane juice were out of the permitted standards, so that a serious health outbreak can be caused anytime.

Rice molecular markers and genetic mapping:Current status and prospects

Dramatic changes in climatic conditions that supplement the biotic and abiotic stresses pose severe threat to the sustainable rice production and have made it a difficult task for rice molecular breeders to enhance production and productivity under these stress factors. The main focus of rice molecular breeders is to understand the fundamentals of molecular pathways involved in complex agronomic traits to increase the yield. The availability of complete rice genome sequence and recent improvements in rice genomics research has made it possible to detect and map accurately a large number of genes by using linkage to DNA markers. Linkage mapping is an effective approach to identify the genetic markers which are co-segregating with target traits within the family. The ideas of genetic diversity, quantitative trait locus（QTL） mapping, and marker-assisted selection（MAS） are evolving into more efficient concepts of linkage disequilibrium（LD） also called association mapping and genomic selection（GS）, respectively. The use of cost-effective DNA markers derived from the fine mapped position of the genes for important agronomic traits will provide opportunities for breeders to develop high-yielding, stress-resistant, and better quality rice cultivars. Here we focus on the progress of molecular marker technologies, their application in genetic mapping and evolution of association mapping techniques in rice.

Genetic Variations and Cultivar Purity within the G. <i>hirsutum</i>Cultivars via RAPD Marker

To assess genetic variation within a cultivar, four commercial varieties with five individual G. hirsutum genotypes and their bulk were assayed with selected forty 10-mer RAPDs per cultivar via PCR. CIM-473 cultivar showed amplification of 321 bands in total per primer with an average of 8 bands. A range of similarity i.e.;80% - 100% within the cultivar has been observed for minimum and maximum respectively. CIM-448 showed amplification of 300 bands in total per primer with an average of 7.5 bands. Maximum similarity of 95% and minimum similarity of 84% was observed within the cultivar. In CIM-1100 the total numbers of fragments amplified are 304, with the average of 7.6 bands per primer and are 12% - 20% polymorphic. Similarly, in NIAB-78 the total numbers of fragments amplified were 310, with the average of 7.7 bands per primer. The reliability of RAPD technique for finding genetic resemblances is indicated by the mentioned results. Numerous groups within 5 individual genotypes bulk among 4 cultivars of G. hirsutum L. have been through cluster analysis which was by the unweighted pair group method of arithmetic means (UPGMA). Dendrograms have been used for showing cultivar relationship.

Salicylic Acid Application Mitigates Oxidative Damage and Improves the Growth Performance of Barley under Drought Stress

Drought is a severe environmental constraint,causing a significant reduction in crop productivity across the world.Salicylic acid(SA)is an important plant growth regulator that helps plants cope with the adverse effects induced by various abiotic stresses.The current study investigated the potential effects of SA on drought tolerance efficacy in two barley(Hordeum vulgare)genotypes,namely BARI barley 5 and BARI barley 7.Ten-day-old barley seedlings were exposed to drought stress by maintaining 7.5%soil moisture content in the absence or presence of 0.5,1.0 and 1.5 mM SA.Drought exposure led to severe damage to both genotypes,as indicated by phenotypic aberrations and reduction of dry biomass.On the other hand,the application of SA to drought-stressed plants protected both barley genotypes from the adverse effects of drought,which was reflected in the improvement of phenotypes and biomass production.SA supplementation improved relative water content and proline levels in drought-stressed barley genotypes,indicating the osmotic adjustment functions of SA under water-deficit conditions.Drought stress induced the accumulation of reactive oxygen species(ROS),such as hydrogen peroxide(H2O2)and superoxide(O_(2)•^(−)),and the lipid peroxidation product malondialdehyde(MDA)in the leaves of barley plants.Exogenous supply of SA reduced oxidative damage by restricting the accumulation of ROS through the stimulation of the activities of key antioxidant enzymes,including superoxide dismutase(SOD),peroxidase(POD),catalase(CAT),ascorbate peroxidase(APX)and glutathione peroxidase(GPX).Among the three-applied concentrations of SA,0.5 mM SA exhibited better mitigating effects against drought stress considering the phenotypic performance and biochemical data.Furthermore,BARI barley 5 showed better performance under drought stress than BARI barley 7 in the presence of SA application.Collectively,our results suggest that SA played a crucial role in improving water status and antioxidant defense strategy to protect barley plants from the deleterious effects of water deficiency.

Biomolecular basis of the role of diabetes mellitus in osteoporosis and bone fractures

Osteoporosis has become a serious health problem throughout the world which is associated with an increased risk of bone fractures and mortality among the people of middle to old ages.Diabetes is also a major health problem among the people of all age ranges and the sufferers due to this abnormality increasing day by day.The aim of this review is to summarize the possible mechanisms through which diabetes may induce osteoporosis.Diabetes mellitus generally exerts its effect on different parts of the body including bone cells specially the osteoblast and osteoclast,muscles,retina of the eyes,adipose tissue,endocrine system specially parathyroid hormone(PTH) and estrogen,cytokines,nervous system and digestive system.Diabetes negatively regulates osteoblast differentiation and function while positively regulates osteoclast differentiation and function through the regulation of different intermediate factors and thereby decreases bone formation while increases bone resorption.Some factors such as diabetic neuropathy,reactive oxygen species,Vitamin D,PTH have their effects on muscle cells.Diabetes decreases the muscle strength through regulating these factors in various ways and ultimately increases the risk of fall that may cause bone fractures.

TGF-β/BMP signaling and other molecular events: regulation of osteoblastogenesis and bone formation

Transforming growth factor-beta（TGF-β）/bone morphogenetic protein（BMP） plays a fundamental role in the regulation of bone organogenesis through the activation of receptor serine/threonine kinases. Perturbations of TGF-β/BMP activity are almost invariably linked to a wide variety of clinical outcomes, i.e., skeletal, extra skeletal anomalies, autoimmune, cancer, and cardiovascular diseases. Phosphorylation of TGF-β（I/II） or BMP receptors activates intracellular downstream Smads, the transducer of TGF-β/BMP signals. This signaling is modulated by various factors and pathways, including transcription factor Runx2. The signaling network in skeletal development and bone formation is overwhelmingly complex and highly time and space specific.Additive, positive, negative, or synergistic effects are observed when TGF-β/BMP interacts with the pathways of MAPK, Wnt, Hedgehog（Hh）, Notch, Akt/m TOR, and mi RNA to regulate the effects of BMP-induced signaling in bone dynamics. Accumulating evidence indicates that Runx2 is the key integrator, whereas Hh is a possible modulator, mi RNAs are regulators, and b-catenin is a mediator/regulator within the extensive intracellular network. This review focuses on the activation of BMP signaling and interaction with other regulatory components and pathways highlighting the molecular mechanisms regarding TGF-β/BMP function and regulation that could allow understanding the complexity of bone tissue dynamics.

Effect of quercetin against lindane induced alterations in the serum and hepatic tissue lipids in wistar rats

Objective:To assess the effect of quercetin(flavonoid) against lindane induced alterations in lipid profile of wistar rats.Methods:Rats were administered orally with lindane(100 mg/kg body weight) and quercetin(10 mg/kg body weight) for 30 days.After the end of treatment period lipid profile was estimated in serum and tissue.Results:Elevated levels of serum cholesterol, triglycerides,low density lipoprotein(LDL),very Low Density Lipoprotein(VLDL) and tissue triglycerides,cholesterol with concomitant decrease in serum HDL and tissue phospholipids were decreased in lindane treated rats were found to be significantly decreased in the quercetin and lindane co-treated rats.Conclusions:Our study suggests that quercetin has hypolipidemic effect and offers protection against lindane induced toxicity in liver by restoring the altered levels of lipids.The quercetin cotreatment along with lindane for 30 days reversed these biochemical alterations in lipids induced by lindane.

Analgesic,anti-inflammatory and anti-diarrheal activities of ethanolic leaf extract of Typhonium trilobatum L.Schott

Objective:To explore the efficacy of ethanolic leaf extract of Typhonium trilobatum L.Schott in treating diarrhea,pain and inflammation using experimental models.Methods:In the present study,acetic acid-induced writhing,xylene-induced ear edema and castor oil-imluced diarrlieal model were used to evaluate the analgesic,anti-inflammatory and anti-diarrheal activities,respectively.Acute toxicity test was carried out to fix the safe doses of the plant extract.Results:The plant extract demonstrated a significant inhibition of writhing(P<0.01)compared with the control group in acetic acid-induced writhing test in mice.The extract also significantly inhibited the xylene induced ear edema formation(P<0.05).In anti-diarrheal test,the extract significantly decreased the frequency of defecation and increased the mean latent period(P<0.0l)in castor oil-induced diarrheal model mice at the doses of 250 and 500 mg/kg body weight.Conclusions:These results suggest that the extract possesses significant analgesic,anti-inflammatory and anti-diarrheal activities that support to the ethnopharmacologicl uses of this plant.

In vitro callus induction and plantlet regeneration of Achyranthes aspera L.,a high value medicinal plant

Objective:To study callus induction from different explants(internode,leaf,root)and in vitro plantlets propagation from medicinally important plant Achyranthes aspera L.Methods:Sterilized explants were prepared by uning 0.1%HgCl_2 and 0.5%Bavistin and callus was obtained when cultured onto Murashige Skoog's(MS)medium by using different concentrations and combination of 2,4-D.NAA.BAP,IAA,IBA with 3%sucrose and 0.8%agar.Induced callus was immediately transferred to MS medium containing at different concentrations of phytohormones for shootlets and rootlets induction respectively.Results:Sterilization treatment of 0.1%HgCl_2.for 2-3 min and Bavistin 0.5%for 10-12 min showed the highest percentage of asepsis and survival rate.Maximum induction of callus was obtained from a combination of 2.0 mg/L 2,4-D and 0.5 mg/L NAA from leaf.Highest shootlets number(4.83±0.l7)and length(3.8±0.16)cm were observed on full strength MS medium when fortified with BAP 4.0 mg/L and KIN 0.5 mg/L.Concerted efforts of BAP 10 mg/L and NAA 0.5 mg/L on full strength MS medium showed highest leaf number(6.77±0.94).In vitro raised shoots were allowed to root on different strengths of MS medium fortified with IAA and IBA at different concentrations.Experimentally,3.0 mg/L IBA was enabled to induce maximum rootlets number(10.0±9.82)on full strength MS medium.Afterwards,regenerated shoots with well developed roots were successfully subjected to hardening process and were acclimatized.The survived plantlets showed 66.67%survival frequency without any morphological abnormality.Conclusions:The results demonstrated that different explants were good source of callus induction,morphology analysis as well as indirect plantlets regeneration.

Hepatitis C virus prevalence and genotype distribution inPakistan:Comprehensive review of recent data

Hepatitis C virus(HCV) is endemic in Pakistan and its burden is expected to increase in coming decades owing mainly to widespread use of unsafe medical procedures. The prevalence of HCV in Pakistan has previously been reviewed. However, the literature search conducted here revealed that at least 86 relevant studies have been produced since the publication of these systematic reviews. A revised updated analysis was therefore needed in order to integrate the fresh data. A systematic review of data published between 2010 and 2015 showed that HCV seroprevalence among the general adult Pakistani population is 6.8%, while active HCV infection was found in approximately 6% of the population. Studies included in this review have also shown extremely high HCV prevalence in rural and underdeveloped peri-urban areas(up to 25%), highlighting the need for an increased focus on this previously neglected socioeconomic stratum of the population. While a 2.45% seroprevalence among blood donors demands immediate measures to curtail the risk of transfusion transmitted HCV, a very high prevalence in patients attending hospitals with various non-liver disease related complaints(up to 30%) suggests a rise in the incidence of nosocomial HCV spread. HCV genotype 3a continues to be the most prevalent subtype infecting people in Pakistan(61.3%). However, recent years have witnessed an increase in the frequency of subtype 2a in certain geographical sub-regions within Pakistan. In Khyber Pakhtunkhwa and Sindh provinces, 2a was the second most prevalent genotype(17.3% and 11.3% respectively). While the changing frequency distribution of various genotypes demands an increased emphasis on research for novel therapeutic regimens, evidence of high nosocomial transmission calls for immediate measures aimed at ensuring safe medical practices.

Putrescine, spermidine, and spermine play distinct roles in rice salt tolerance

Polyamines(PAs) play diverse roles in plant growth and development,as well as responses to environmental stimuli.In this study,the effects of PAs on rice salt tolerance were investigated.Salt stress resulted in the alteration of endogenous PAs levels in rice roots and leaves,where spermidine(Spd) and spermine(Spm) contents were increased,and putrescine(Put) content was decreased.RT-qPCR analysis revealed that PAs biosynthesis-related genes ADC1,ODC,and Arginase were significantly downregulated by salt treatment;however,SAMDC transcription was significantly upregulated.Exogenous Spm enhanced rice salt tolerance remarkably;however,exogenous Put and Spd undermined rice salt tolerance.Transgenic rice plants overexpressing SAMDC display a higher ratio of Spm/(Put+Spd) and enhanced salt tolerance.Salt stress also increased polyamine oxidase activities in rice,resulting in elevated reactive oxygen species(ROS) production.Our findings revealed that accumulation of Put and Spd substantially reduced salt tolerance in rice,likely by facilitating ROS production;whereas,conversion of Put and Spd to Spm contributes to rice salt tolerance.

Emblica officinalis improves glycemic status and oxidative stress in STZ induced type 2 diabetic model rats

Objective:To evaluate the antidiabetic and antioxidant potential of Emblica officinalis(E.officinalis)fruit on normal and type 2 diabetic rats.Methods:Type 2 diabetes was induced into the male Long-Evans rats.The rats were divided into nine groups including control groups receiving water,type 2 diabetic controls,type 2 diabetic rats treated with glibenclamide(T2GT)and type 2diabetic rats treated with aqueous extract of fruit pulp of E.officinalis.They were fed orally for8 weeks with a single feeding.Blood was collected by cutting the tail tip on 0 and 28 days and by decapitation on 56 day.Packed red blood cells and serum were used for evaluating different biochemical parameters.Results:Four weeks administration of aqueous extract of E.officinalis improved oral glucose tolerance in type 2 rats and after 8 weeks it caused significant(P<0.007)reduction in fasting serum glucose level compared to 0 day.Triglycerides decreased by 14%but there was no significant change in serum ALT,creatinine,cholesterol and insulin level in any group.Furthermore,reduced erythrocyte malondialdehyde level showed no significant change(P<0.07)but reduced glutathione content was found to be increased significantly(P<0.05).Conclusions:The aqueous extract of E.officinalis has a promising antidiabetic and antioxidant properties and may be considered for further clinical studies in drug development.

Rapid Detection of Wheat Blast Pathogen Magnaporthe oryzae Triticum Pathotype Using Genome-Specific Primers and Cas12a-mediated Technology

Wheat blast,caused by the fungus Magnaporthe oryzae Triticum(MoT)pathotype,is a devastating disease persistent in South America and Bangladesh.Since MoT generally fails to cause visual symptoms in wheat until the heading stage when the infection would have advanced,disease control by fungicide application solely based on the detection of visual symptoms is ineffective.To develop an accurate and sensitive method to detect MoT at the seedling and vegetative stages for disease control,we sequenced the genomes of two MoT isolates from Brazil and identified two DNA fragments,MoT-6098 and MoT-6099,that are present in the MoT genome but not in the genome of the rice-infecting Magnaporthe oryzae Oryzae(MoO)pathotype.Using polymerase chain reaction(PCR),we confirmed the specificity of the two markers in 53 MoT and MoO isolates from South America and Bangladesh.To test the efficiency of the two markers,we first established a loop-mediated isothermal amplification(LAMP)method to detect MoT at isothermal conditions,without the use of a PCR machine.Following this,we used the Cas12a protein and guide RNAs(gRNAs)to target the MoT-6098 and MoT-6099 sequences.The activated Cas12a showed indiscriminate single-stranded deoxyribonuclease(ssDNase)activity.We then combined targetdependent Cas12a ssDNase activation with recombinase polymerase amplification(RPA)and nucleic acid lateral flow immunoassay(NALFIA)to develop a method that accurately,sensitively,and cost-effectively detects MoT-specific DNA sequences in infected wheat plants.This novel technique can be easily adapted for the rapid detection of wheat blast and other important plant diseases in the field.

Mechanisms and molecular approaches for heat tolerance in rice (Oryza sativa L.) under climate change scenario

Rice, a staple cereal crop in many parts of the world, has been confronted with multiple environmental stresses including high temperature, negatively impacts the booting as well as anthesis growth stages. The situation is further complicated by the changing climatic conditions, resulting in gradual escalation of temperature as well as changing the rainfall pattern and frequency, thus raising a concern of food security worldwide. The situation can be combat by developing rice varieties with excellent genetics with improved morpho-physiological, biochemical, and molecular mechanisms, together can minimize the adverse effects of heat stress. Here, several strategies(encompassing genetic and genomic, and mechanisms involved) for mitigating the impact of high temperature on rice have been discussed. Finally, the utilization of genomic knowledge in augmenting the conventional breeding approaches have been comprehensively elaborated to develop heat tolerant germplasm.

Antioxidant,antimicrobial,cytotoxic and analgesic activities of ethanolic extract of Mentha arvensis L.

Objective:To investigate potential antioxidant,antimicrobial,cytotoxic and analgesic activities of ethanolic extract of Mentha arrensis l.,in different in vivo and in vitro experimental models.Methods:In vitro DPPH radical scavenging assay was used to evaluate the antioxidant activity of the plant extract.In vivo analgesic activity was carried out by acetic acid—induced writhing test in Swiss albino mice.All studies in mice were undertaken at the doses of 250 and 500 mg/kg body weight.Antibacterial activilv was studied by disk diffusion assay against some Gram—positive and Gram—negative bacterial strains.Brine shrimp lethality assay was used to investigate cyloloxicity effects of the plant extract.Results:The extract showed free radical scavenging activity in the DPPH assay(IC5041 μg/mL)compared to the standard antioxidant ascorbic acid(IC5019 μg/mL).The extract also produced prominent antimicrobial activity against Salmonella typhi.Salmonella paratyphi.Shigella boydii,Streptococcus pyogenes and Streptococcus aureus compared to standard drug kanamycin at the dose of 30 μg/disc.The extract exhibited lethality against the brine shrimp nauplii with the LC50,values of 40 μg/mL.and also 90%mortality(LC90) value was found to be 160 μg/mL.In analgesic test.the extract demonstrated statistically significant（P【0.01） analgesic effect in acetic acid induced writhing in white albino mice al both dose levels.Conclusions:These results suggest that the ethanolic extract of Mentha arvenns L.has potential antioxidant,antibacterial,cytotoxic and analgesic activities that support the ethnopharmacological uses of this plant.

Role of the CXCR4-SDF1-HMGB1 pathway in the directional migration of cells and regeneration of affected organs

In recent years,several studies have reported positive outcomes of cell-based therapies despite insufficient engraftment of transplanted cells.These findings have created a huge interest in the regenerative potential of paracrine factors released from transplanted stem or progenitor cells.Interestingly,this notion has also led scientists to question the role of proteins in the secretome produced by cells,tissues or organisms under certain conditions or at a particular time of regenerative therapy.Further studies have revealed that the secretomes derived from different cell types contain paracrine factors that could help to prevent apoptosis and induce proliferation of cells residing within the tissues of affected organs.This could also facilitate the migration of immune,progenitor and stem cells within the body to the site of inflammation.Of these different paracrine factors present within the secretome,researchers have given proper consideration to stromal cell-derived factor-1(SDF1)that plays a vital role in tissue-specific migration of the cells needed for regeneration.Recently researchers recognized that SDF1 could facilitate site-specific migration of cells by regulating SDF1-CXCR4 and/or HMGB1-SDF1-CXCR4 pathways which is vital for tissue regeneration.Hence in this study,we have attempted to describe the role of different types of cells within the body in facilitating regeneration while emphasizing the HMGB1-SDF1-CXCR4 pathway that orchestrates the migration of cells to the site where regeneration is needed.

Leonurus sibiricus L.(honeyweed):A review of its phytochemistry and pharmacology

Leonurus sibiricus is a herbaceous plant found in many countries in Asia and America.This plant is widely practiced as a remedy for the treatment of diabetes,menstrual irregularities,and bronchitis.The approval of therapeutic implications of any drugs depends on the well characterized mode of actions of the compounds.The bioactive compounds like diterpenes,triterpenes,flavonoids and phenolic acids in Leonurus sibiricus show analgesic,anti-inflammatory,anti-oxidant,anti-atherogenic and antihemorrhagic,anti-diabetic,anti-bacterial and allelopathic potency.Interestingly,the expression level of some genes is altered by the crude extract treatments,which are effective against cancers,diabetes and cardiovascular diseases where the molecular mechanisms are yet to be explored.Intriguingly,the extracts significantly induce nitric oxide production by endothelial nitric oxide synthase,a signaling molecule of vasodilation in combination with interferon-y indicating positive effect on atherosclerosis.Further investigations are required to unlock the effects of bioactive compounds found in extracts at clinical settings.

Development of Gene Expression and Gene Silencing Vectors Based onCotton Leaf Curl Virus for Functional Genomics in Cotton

Cotton leaf curl virus is the first example of acotton-infecting virus where infectious clones areavailable.Plant viruses are valuable tools inunderstanding plant biology as they can beengineered for expression of foreign genes orsilencing of genes homologous to cloned genes.

Screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonance energy transfer assay and mass spectrometry

BACKGROUND Hepatitis C virus genotype 3a(HCV G3a)is highly prevalent in Pakistan.Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV,medicinal natural products of potent antiviral activity should be screened for the cost-effective treatment of the disease.Furthermore,from natural products,active compounds against vital HCV proteins like non-structural protein 3(NS3)protease could be identified to prevent viral proliferation in the host.AIM To develop cost-effective HCV genotype 3a NS3 protease inhibitors from citrus fruit extracts.METHODS Full-length NS3 without co-factor non-structural protein 4A(NS4A)and codon optimized NS3 protease in fusion with NS4A were expressed in Escherichia coli.The expressed protein was purified by metal ion affinity chromatography and gel filtration.Citrus fruit extracts were screened using fluorescence resonance energy transfer(FRET)assay against the protease and polyphenols were identified as potential inhibitors using electrospray ionization-mass spectrometry(MS)/MS technique.Among different polyphenols,highly potent compounds were screened using molecular modeling approaches and consequently the most active compound was further evaluated against HCV NS4A-NS3 protease domain using FRET assay.RESULTS NS4A fused with NS3 protease domain gene was overexpressed and the purified protein yield was high in comparison to the lower yield of the full-length NS3 protein.Furthermore,in enzyme kinetic studies,NS4A fused with NS3 protease proved to be functionally active compared to full-length NS3.So it was concluded that co-factor NS4A fusion is essential for the purification of functionally active protease.FRET assay was developed and validated by the half maximal inhibitory concentration(IC50)values of commercially available inhibitors.Screening of citrus fruit extracts against the native purified fused NS4A-NS3 protease domain showed that the grapefruit mesocarp extract exhibits the highest percentage inhibition 91%of protease activity.Among the compounds identified by LCMS analysis,hesperidin showed strong binding affinity with the protease catalytic triad having S-score value of-10.98.CONCLUSION Fused NS4A-NS3 protease is functionally more active,which is effectively inhibited by hesperidin from the grapefruit mesocarp extract with an IC50 value of 23.32μmol/L.