Inhibition of hepatitis B virus DNA replicative intermediate forms by recombinant interferon-γ

AIM: To evaluate the in vitro anti-HBV activity of recombinant human IFN-γ, alone and in combination with lamivudine. METHODS: A recombinant baculovirus-HBV/HepG2 culture system was developed which could support productive HBV infection in vitro. Expression of HBsAg and HBeAg in infected HepG2 culture medium was detected by commercial enzyme immunoassays. HBV DNA replication intermediates were detected in infected cells by Southern hybridization and viral DNA load was determined by dot hybridization. RESULTS: IFN-γat 0.1 to 5μg/L efficiently down regulated HBsAg expression in transduced HepG2 cells. At 5μg/L, IFN-γalso suppressed HBV DNA replication in these cells. While treatment with a combination of lamivudine and IFN-γshowed no additive effect, sequential treatment first with lamivudine and then IFN-γwas found to be promising. In this culture system the best HBV suppression was observed with a pulse of 2μmol/L lamivudine for two days, followed by 1μg/L IFN-γfor another four days. Compared to treatment with lamivudine alone, the sequential use of 0.2μmol/L lamivudine for two days, followed by 5μg/L IFN-γfor six days showed a 72% reduction in HBV cccDNA pool. CONCLUSION: This in vitro study warrants further evaluation of a combination of IFN-γand lamivudine, especially in IFN-αnon-responder chronic hepatitis B patients. A reduced duration of lamivudine treatment would also restrict the emergence of drug-resistant HBV mutants.

Anticancer and cytotoxic properties of the latex of Calotropis procera in a transgenic mouse model of hepatocellular carcinoma

AIM： To evaluate the anticancer property of the dried latex （DL） of Calotropis procera, a tropical medicinal plant, in the X15-myc transgenic mouse model of hepatocellular carcinoma and to elucidate its mechanism of action in cell culture. METHODS： The young transgenic mice were orally fed with the aqueous suspension of DL （400 mg/kg for 5 d/wk） for 15 wk and their liver was examined for histopathological changes at 20 wk. Serum levels of vascu- lar endothelial growth factor （VEGF） were also measured in these animals. To characterize the active fraction, DL was extracted with petroleum ether followed by methanol. The methanolic extract was sub-fractionated on a silica gel G column using a combination of non-polar and polar solvents and eleven fractions were obtained. Each fraction was analysed for cytotoxic effect on hepatoma （Huh7） and non-hepatoma （COS-1） cell lines and nontransformed hepatocytes （AML12） using tetrazolium （MTT） assay. Finally, the mechanism of cell death was investigated by measuring the levels of Bcl2, caspase 3 and DNA fragmentation. RESULTS： DL treatment of mice showed a complete protection against hepatocarcinogenesis. No adverse effect was observed in these animals. The serum VEGF level was significantly lowered in the treated mice as compared to control animals. Cell culture studies revealed that the methanolic extract of DL as well as its fraction 8 induced extensive cell death in both Huh-7 and COS-1 cells while AML12 cells were spared. This was accompanied by extensive fragmentation of DNA in Huh-7 and COS-1 cells. No change in the levels of canonical markers of apoptosis such as Bcl2 and caspase 3 was observed. CONCLUSION： DL of C. procera has the potential for anti-cancer therapy due to its differentJable targets and non-interference with regular pathway of apoptosis.

Proteomic analysis of gastric cancer and immunoblot validation of potential biomarkers

AIM： To search for and validate differentially expressed proteins in patients with gastric adenocarcinoma.METHODS： We used two-dimensional gel electrophoresis and mass spectrometry to search for differentially expressed proteins in patients with gastric adenocarcinoma. A set of proteins was validated with immunoblotting.RESULTS： We identified 30 different proteins involved in various biological processes： metabolism, development, death, response to stress, cell cycle, cell communication, transport, and cell motility. Eight proteinswere chosen for further validation by immunoblotting. Our results show that gastrokine-1, 39S ribosomal protein L12 （mitochondrial precursor）, plasma cell-induced resident endoplasmic reticulum protein, and glutathione S-transferase mu 3 were significantly underexpressed in gastric adenocarcinoma relative to adjacent non-tumor tissue samples. On the other hand, sep- tin-2, ubiquitin-conjugating enzyme E2 N, and transaldolase were significantly overexpressed. Translationally controlled tumor protein was shown to be differentially expressed only in patients with cancer of the gastric cardia/esophageal border.CONCLUSION： This work presents a set of possible diagnostic biomarkers, validated for the first time. It might contribute to the efforts of understanding gastric cancer carcinogenesis,

Butein imparts free radical scavenging, anti-oxidative and proapoptotic properties in the flower extracts of Butea monosperma

The flower of Butea monosperma(Lam.)(Fabaceae)has been used in traditional Indian medicine in the treatment of many ailments including liver disorders.To understand the pharmacological basis of its beneficial effects,the extracts of dried flowers in water,methanol,butanol,ethyl acetate and acetone were evaluated for free radical scavenging and pro-apoptotic activities in cell cultures(human hepatoma Huh-7 cell line and immortalized AML-12 mouse hepatocytes).Butrin and butein-the active constituents of flower extracts-were used as reference molecules.The levels of cell injury markers like lactate dehydrogenase,glutathione and lipid peroxidation and primary antioxidant enzymes glutathione S-transferase and catalase were also measured.The aqueous and butanolic extracts exhibited better 2,2-diphenyl-1-picrylhydrazyl scavenging and cytotoxic activities in hepatoma cells than in immortalized hepatocytes.Interestingly,butein inhibited 2,2-diphenyl-1-picrylhydrazyl radical better than butrin.The aqueous and butanolic extracts were further investigated for hepatoprotection against carbon tertrachloride-induced biochemical changes and cell death.Both extracts,just as butrin and butein,significantly reversed the cellular glutathione levels and lipid peroxidation,and glutathione–S-transferase activity.Lactate dehydrogenase leakage and cell death were also prevented.However,only butein revived the catalase activity.Thus,the butein content of Butea monosperma flower extracts is important for free radical scavenging activity,apoptotic cell death and protection against oxidative injury in hepatic cells.

In vivo modulation of androgen receptor by androgens

Aim: To study the effect of androgen and antiandrogen on the level of androgen receptor (AR) mRNA. Methods: The total RNA was extracted from the prostate and analyzed by slot blot analysis. The blots were hybridized with AR cDNA probe and 1A probe (internal control) and autoradiography was performed. The intensity of signal was measured with a densitometer and the ratio of AR RNA and 1A RNA was calculated. Results: Androgenic deprivation produced by castration decreased the weight of the prostate and increased the levels of AR mRNA. Treatment of the castrated rats with testostrone increased the weight of prostate and decreased the levels of AR mRNA. Treatment of normal rats with flutamide decreased the weight of the gland and increased the levels Of AR mRNA. Conclusion: Androgens produce proliferative effect on the prostate and negatively regulate the AR transcription.

Phytochemical profiling and biological activity of Plectranthus amboinicus(Lour.) mediated by various solvent extracts against Aedes aegypti larvae and toxicity evaluation

Objective:To analyze the phytochemical compounds and to investigate the bio-toxic efficacy of various solvent extracts of Plectranthus amboinicus against mosquito larvae activity and lethality on non-targeting organisms.Methods:The methanol,ethyl acetate,hexane,and aqueous extracts of Plectranthus amboinicus were subjected to analyze the mosquitocidal activity against the dengue vector,Aedes aegypti and toxicity assays on zebra fish and brine shrimp.Three replications were performed,and negative control was also maintained.Amongst,ethyl acetate extract of Plectranthus amboinicus was chosen for the determination of bio-active compounds.Results:The mosquitocidal assays of methanol and ethyl acetate extracts of Plectranthus amboinicus showed the maximal activity with minimal concentration against the 4 th instar mosquito-larvae of Aedes aegypti through the following lethal concentration(LC50 and LC90)values:53.36&92.51μg/m L and 13.64&86.09μg/mL,respectively.In addition,the plant extracts showed no toxicity on zebra fish embryo and brine shrimp assays.The gas-chromatography analysis of the ethyl acetate extract of Plectranthus amboinicus revealed the presence of seven different compounds.Among them,PAEA-fraction 60 contained a major active bioactive compound,hexadecanoic acid,methyl ester(270.0).Conclusions:Plectranthus amboinicus possesses mosquitocidal properties and could be used as a potential alternative source for preparing the mosquitocidal agents.

Second to Fourth Digit Ratio(2D:4D) as a Predictor of Adult Circulating Sex Hormones and Overweight/Obesity in Ado-Ekiti Nigeria

The ratio of the second-to-fourth digit length（2D：4D）has been shown to be sexually dimorphic,with males having lower mean than females and the ratio relates to masculinity and femininity in adults.2D：4D is influenced by digit growth pattern which is related to the ratio of prenatal androgen to estrogen levels in the later part of the first trimester.

Differential Expression of miRNAs in Sorghum bicolor under Drought and Salt Stress

The regulatory mechanisms of drought and salt-associated miRNAs have not been fully understood in Sorghum bicolor. In this study, we investigated the effect of salinity stress (200 and 300 mM NaCl) and drought stress at pre- and post-flowering stages on the expression pattern of small regulatory RNAs in six Sorghum genotypes using semi-quantitative reverse transcriptase PCR (RT-qPCR). The results indicated that both drought and salt stresses altered the expression pattern of miRNAs in a dose-dependent manner. However, each miRNA responded to drought and salt stress in a different pattern among the six sorghum genotypes. miR156, miR167, miR168 and miR399 give different expressions levels compared to other studied miRNAs which may attribute to the adaption of sorghum to drought and salt stress and are good candidates for improving sorghum by transgenic technology.

Soybean Improvement for Lipoxygenase-free by Simple Sequence Repeat(SSR)Markers Selection

Beany flavor of soybean(Glycine max(L.)Merr.)is caused by oxidation of polyunsaturated fatty acids by the action of three lipoxygenases(LOX1,LOX2 and LOX3)present in mature seeds.The unpleasant flavor restricts human consumption of soybean products.This problem could be solved through genetic elimination of alleles that code these enzymes.Parental cultivars and two hybrid population were selected and analyzed using genetic markers for alleles locus,encoding L_(ox1),L_(ox2)and L_(ox3)free.The SSR marker Satt 212 confirmed the presence of the homozygous null-allele L_(x3)in the cultivar BRS 213,which were used for hybridization with BR 36.Heterozygote F1 hybrid plants and homozygous L_(x3)lines in F_(2)segregating populations were successfully identified.The SSR markers Sat090 and Sat417 were the most effective diagnostic markers among all SSR markers tested.Satt090 and Satt417 confirmed the presence of the homozygous L_(x2)null-allele in the parental cultivar BRS 213 by flanking L_(x2)loci at 3,00 and 2,77 cM,respectively.The presence of L_(x2)null allele in the F_(2)segregating populations between BRS 213 and BRS 155 was successfully identified with a selection efficiency of 98%and have great potential for further application in the Brazilian breeding program aimed at improving soybean seed quality.

Identification,Structure Analyses and Expression Pattern of the ERF Transcription Factor Family in Coffea arabica

Members of the ERF Family of Transcription Factors play an important role in plant development and gene expression that regulates responses to biotic and abiotic stress.This work identified 36 ERF family genes in Coffea arabica within the AP2/ERF full domain,using the EST-based genomic resource of the Brazilian Coffee Genome Project.The ERF family genes were classified into nine of the ten existing groups through phylogenetic analysis of the deduced amino acid sequences and comparison with the sequences of the ERF family genes in Arabidopsis.In addition to the AP2 domain,other conserved domains were identified,typical of members of each group.The in silico analysis and expression profiling showed high levels of expression for libraries derived from tissues of fruits,leaves and flowers as well as for libraries subjected to water stress.These results suggest the participation of the ERF family genes of C.arabica in distinct biological functions,such as control of development,maturation,and responses to water stress.The results of this work imply in the selection of promising genes for further functional characterizations that will provide a better understanding of the complex regulatory networks related to plant development and responses to stress,opening up opportunities for coffee breeding programs.

Expression of HIV-1 env Glycoprotein gp120 and gp160 in Vaccinia Virus System and Their Antigenicity Analysis

Human immunodeficiency virus type 1 (HIV-1)is the principal causative agent of AIDS which is threatening human health. The most important antigen of HIV1 is the viral envelope glycoprotein, gp160. The mature form of the HIV-1 gp160 is made up of two major regions:the extracellular (gq120) and the transmembrane (gp41) domains. The gp120 binds with high affinity to CD4. he cellular receptor for HIV-1, and antibodies that prevent the CD4-gq120 interaction are able to block HIV-1 infection. A principal neutralizing deter-

Compartmentalized Metabolic Engineering for Artemisinin Biosynthesis and Effective Malaria Treatment by Oral Delivery of Plant Cells

Artemisinin is highly effective against drug-resistant malarial parasites, which affects nearly half of the global population and kills 〉500 000 people each year. The primary cost of artemisinin is the very expensive process used to extract and purify the drug from Artemisia annua. Elimination of this apparently unnecessary step will make this potent antimalarial drug affordable to the global population living in endemic regions. Here we reported the oral delivery of a non-protein drug artemisinin biosynthesized （~0.8 mg/g dry weight） at clinically meaningful levels in tobacco by engineering two metabolic pathways targeted to three different cellular compartments （chloroplast, nucleus, and mitochondria）. The doubly transgenic lines showed a three-fold enhancement of isopentenyl pyrophosphate, and targeting AACPR, DBR2, and CYP71AV1 to chloroplasts resulted in higher expression and an efficient photo-oxidation of di- hydroartemisinic acid to artemisinin. Partially purified extracts from the leaves of transgenic tobacco plants inhibited in vitro growth progression of Plasmodium falciparum-infected red blood cells. Oral feeding of whole intact plant cells bioencapsulating the artemisinin reduced the parasitemia levels in challenged mice in comparison with commercial drug. Such novel synergistic approaches should facilitate low-cost production and delivery of artemisinin and other drugs through metabolic engineering of edible plants.

Changes in gene expression in liver tissue from patients with fulminant hepatitis E

AIM: To study host gene expression and numberof immune cells in liver tissues from patients with fulminant hepatitis E(FH-E).METHODS: Microarray-based expression profiling was done using Illumina Human WG-6_v3_Bead Chip arrays on post-mortem liver tissue from 5 patients with FH-E,and compared with similar tissue from 6 patients with fulminant hepatitis B(FH-B; disease controls) and normal liver tissue from 6 persons.Differential expression was defined as ≥ 2.0-fold change with Benjamini-Hochberg false discovery rate below 0.05 using t-test in liver tissue from FH-B and FH-E,than healthy liver tissue.For some genes that showed differential expression in FH-E,microarray data were validated using quantitative reverse transcription PCR.Differentially expressed gene lists were then subjected to "Gene Ontology" analysis for biological processes,and pathway analysis using Bio Carta database on the DAVID server.In addition,tissue sections were stained for CD4+,CD8+ and CD56+ cells using indirect immunohistochemistry; cells staining positive for each of these markers were counted and compared between groups.RESULTS: Compared to normal livers,those from patients with FH-E and FH-B showed differential expression of 3377 entities(up-regulated 1703,downregulated 1674) and 2572 entities(up 1164,down 1408),respectively.This included 2142(up 896,down 1246) entities that were common between the two sets; most of these belonged to metabolic,hemostatic and complement pathways,which are active in normal livers.Gene expression data from livers of patients with FH-E but not those of FH-B showed activation of several immune response pathways,particularly those involving cytotoxic T cells.The fold-change values of m RNA for selected genes in livers from FH-E than in normal liver tissue determined using quantitative reverse transcription PCR showed excellent concordance with microarray analysis.At immunohistochemistry,CD8+ T cells showed an increase in liver biopsies from both FH-E [median 53.4 per arbitrary unit area(range 31.2-99.9)] and FH-B [median 49.3(19.3-51.0); P = 0.005] compared to control liver tissue [median 6.9(3.1-14.9)].CONCLUSION: FH-E patients show CD8+ T cell infiltration and increased gene expression of cytotoxic T cell pathways in liver,suggesting a possible pathogenetic role for these cells.

Rewilding staple crops for the lost halophytism:Toward sustainability and profitability of agricultural production systems

Abiotic stress tolerance has been weakened during the domestication of all major staple crops.Soil salinity is a major environmental constraint that impacts over half of the world population;however,given the increasing reliance on irrigation and the lack of available freshwater,agriculture in the 21st century will increasingly become saline.Therefore,global food security is critically dependent on the ability of plant breeders to create high-yielding staple crop varieties that will incorporate salinity tolerance traits and ac-count for future climate scenarios.Previously,we have argued that the current agricultural practices and reliance on crops that exclude salt from uptake is counterproductive and environmentally unsustainable,and thus called for a need for a major shift in a breeding paradigm to incorporate some halophytic traits that were present in wild relatives but were lost in modern crops during domestication.In this review,we provide a comprehensive physiological and molecular analysis of the key traits conferring crop halophy-tism,such as vacuolar Na+sequestration,ROS desensitization,succulence,metabolic photosynthetic switch,and salt deposition in trichomes,and discuss the strategies for incorporating them into elite germ-plasm,to address a pressing issue of boosting plant salinity tolerance.

Light Regulated Osa-miR169e Is Implicated during Priming under High Temperature Stress in Rice

Plants face biotic and abiotic stresses during their entire life cycle, which leads to the loss in crop productivity. It has been shown that a relatively shorter exposure to heat stress, called priming, results in better adaptation of plants under subsequent stresses, which plants may face. While rice plants in nature often encounter high temperature stress conditions, the strategies to cope with those are poorly understood. We identified the involvement of microRNA pathways in the adaptation to heat stress (HS) at the physiological and molecular levels. It was observed that osa-miR169 levels are altered after HS and in response to light conditions. Its expression was also regulated by heat priming during anthesis and effectively responds to the successive exposure to high temperature stress during grain filling in rice. Osa-miR169 targets nuclear factor Y (NF-Y). We propose that osa-miR169: NF-Y regulatory module may be important for HS memory induced during high temperature priming and thus may serve to integrate stress responses with light regulated development. The future study in this direction will be useful to understand how plants acclimatize to the changing environment and thus help in generating stress tolerant crops.

Protecting future antimalarials from the trap of resistance:Lessons from artemisinin-based combination therapy (ACT) failures

Having faced increased clinical treatment failures with dihydroartemisinin-piperaquine(DHA-PPQ),Cambodia swapped the first line artemisinin-based combination therapy(ACT)from DHA-PPQ to artesunate-mefloquine given that parasites resistant to piperaquine are susceptible to mefloquine.However,triple mutants have now emerged,suggesting that drug rotations may not be adequate to keep resistance at bay.There is,therefore,an urgent need for alternative treatment strategies to tackle resistance and prevent its spread.A proper understanding of all contributors to artemisinin resistance may help us identify novel strategies to keep artemisinins effective until new drugs become available for their replacement.This review highlights the role of the key players in artemisinin resistance,the current strategies to deal with it and suggests ways of protecting future antimalarial drugs from bowing to resistance as their predecessors did.

Folate-producing rhizobacteria of Hippophae rhamnoides L.from Indian trans-Himalaya low atmospheric zone

Hippophae rhamnoides L.is a plant of immense ethnopharmacological importance and is a known source for various valuable biochemicals and nutraceuticals.The production of folate,a vitamin involved in several vital functions,in this plant is rather poorly understood.Herein,we investigate the hypothesis that rhizobial bacteria serve the plant in this essential vitamin’s biosynthesis.Bacterial strains of Bacillus,Azorhizobium,Frankia,Paenibacillus,Brevibacillus and Pseudomonas,were isolated from the rhizosphere of the plant.HPLC and LCMS were used to trace the production of intra and extra-cellular folate by representative rhizospheric bacterial strains in vitro.From the seventeen functionally characterized bacterial strains of the plant’s rhizosphere,thirteen produced significant amounts of folate.Azorhizobium BR5401 produced the maximum amount of folic acid(424μg/mL),and Bacillus GY779 was the only strain capable of producing both intracellular and extra-cellular folic acid.The Open Reading Frame coding for dihydroneopterin aldolase,an enzyme involved in folate biosynthesis,was found in one of the representative isolates.Our experimental findings help us to suggest that the folate synthesized by rhizobial bacteria is transported to the plant,highlighting a significant benefit of coexistence.

Comparative Analysis of RNAi Suppression Activity of Proteins from Two Disparate Viruses

RNAi is an efficient surveillance machinery that plays a robust defensive role in shielding plant and animal hosts against viral infections. In counter-defense viruses encode suppressor proteins that have the ability to restrict the RNAi machinery to ensure successful systemic invasion. The B2 protein of insect Flock House Virus (FHV-B2) and AC2 protein of Mungbean Yellow Mosaic India Virus (MYMIV-AC2) are two well-characterized suppressors of RNAi, capable of reversing reporter gene silencing. In this study, we compared the strength of the two suppressors by assaying for the degree of RNAi reversion and the duration of sustaining the reversal in planta. The suppression activity was observed by assaying for GFP fluorescence at 3 dpi, 7 dpi and 14 dpi. The phenotypic observations were corroborated with small RNA Northern Blotting and semi-quantitative RT-PCR. The results indicate that suppressor strength of FHVB2 is comparable to MYMIV-AC2, although they are encoded by virus infecting host from two different eukaryotic kingdoms. This study will provide new insights to dissect the conservation in the RNAi pathways during the host-virus interactions.

Antileishmanial Potential of <i>Piper</i><i>nigrum</i>Seed Extracts against <i>Leishmania</i><i>donovani</i>

Visceral leishmaniasis (VL) is a pestilent form of leishmaniasis that chiefly impinges the poorest sections of the society. The prototypical therapeutic interventions in vogue are handicapped due to toxicity and alarming increase in drug resistance. In the absence of vaccines, progressive emergence of HIV-VL co-infection and relapse in the form of post kala-azar dermal leishmaniasis, have fuelled the quest for alternative therapies. Herein, we report antileishmanial activity of Piper nigrum, which is endowed with multifarous medicinal properties. Hexane (PNH) and ethanolic (PNE) extracts of P. nigrum substantially inhibited the growth of Leishmania donovani promastigotes with 50% inhibitory concentration (IC50) of 31.6 and 37.8 μg&#183ml-1, respectively. Growth reversibility analysis revealed the leishmanicidal effect of PNH which caused cell shrinkage and flagellar disruption. In contrast, PNE treated promastigotes showed partial effect. PNH and PNE also abrogated the growth of intra-macrophagic Leishmania amastigotes with IC50 of 14.6 and 18.3 μg&#183ml-1, respectively. Anti-amastigote efficacy of PNH was accompanied by higher selectivity over host macrophages than PNE. Gas-Chromatography-Mass Spectrometry showed the presence of several secondary metabolites such as trans-β-caryophyllene, piperine, β-bisabolene and other sesquiterpenes in PNH and piperine, δ-(sup 9)-cis oleic acid and piperyline in PNE. Conclusively, our work revealed discernible antileishmanial activity of P. nigrum extracts.

Revisiting the role of mesenchymal stem cells in tuberculosis and other infectious diseases

Mesenchymal stem cells(MsCs)play diverse roles ranging from regeneration and wound healing to immune signaling.Recent investigations have indicated the crucial role of these multipotent stem cells in regulating various aspects of the immune system.MsCs express unique signaling molecules and secrete various soluble factors that play critical roles in modulating and shaping immune responses,and in some other cases,MSCs can also exert direct antimicrobial effects,thereby helping with the eradication of invading organisms.Recently,it has been demonstrated that MSCs are recruited at the periphery of the granuloma containing Mycobacterium tuberculosis and exert"Janus"-like functions by harboring pathogens and mediating host protective immune responses.This leads to the establishment of a dynamic balance between the host and the pathogen.MsCs function through various immunomodulatory factors such as nitric oxide(NO),IDO,and immunosuppressive cytokines.Recently,our group has shown that M.tb uses MSCs as a niche to evade host protective immune surveillance mechanisms and establish dormancy.MSCs also express a large number of ABC efflux pumps;therefore,dormant M.tb residing in MSCs are exposed to a suboptimal dose of drugs.Therefore,it is highly likely that drug resistance is coupled with dormancy and originates within MSCs.In this review,we discussed various immunomodulatory properties of MsCs,their interactions with important immune cells,and soluble factors.We also discussed the possible roles of MsCs in the outcome of multiple infections and in shaping the immune system,which may provide insight into therapeutic approaches using these cells in different infection models.