Analysis of genetic diversity in Phytophthora colocasiae causing leaf blight of taro (Colocasia esculenta) using AFLP and RAPD markers

The oomycetous fungus Phytophthora colocasiae that causes taro leaf blight is one of the most devastating diseases of taro and is widely distributed in India. Molecular and cultural techniques were employed for assessing and exploiting the genetic variability among isolates of P. colocasiae obtained from different geographical regions of India. Analysis of the 5.8-ITS region revealed detectable intraspecific variation among isolates. Ten random amplified polymorphic DNA (RAPD) and eight amplified fragment length polymorphism (AFLP) primers produced 198 and 510 reproducible fragments, respectively. AFLP produced 100 % polymorphism, whereas RAPD showed 93.5 % polymorphism. The average value of the number of observed alleles, the number of effective alleles, mean Nei’s genetic diversity, and Shannon’s information index were 2.00–1.94, 1.53–1.36, 0.31–0.24, and 0.47–0.40, respectively, for two DNA markers used. Analysis of molecular variance (AMOVA) for both markers produced similar results with the majority (85 %, AFLP; 89 %, RAPD) of the diversity present within population of P. colocasiae. Dendrograms based on two molecular data using the unweighted pair group method with arithmetic mean (UPGMA) was incongruent and classified the P. colocasiae isolates into one and two major clusters. Cophenetic correlation coefficient between dendrogram and original similarity matrix were significant for RAPD (r?=?0.904) and AFLP (r?=?0.825). The results of this study displayed a high level of genetic variation among the isolates irrespective of the geographical origin. The possible mechanisms and implications of this genetic variation are discussed.     

Streptomyces sp. LK3 mediated synthesis of silver nanoparticles and its biomedical application

In the present study, the marine actinobacteria mediated biosynthesis of silver nanoparticles (AgNps) was achieved using Streptomyces sp LK3. The synthesized AgNps showed the characteristic absorption spectra in UV–vis at 420 nm, which confirmed the presence of nanoparticles. XRD analysis showed intense peaks at 2θ values of 27.51°, 31.87°, 45.57°, 56.56°, 66.26°, and 75.25° corresponding to (210), (113), (124), (240), (226), and (300) Bragg’s reflection based on the fcc structure of AgNps. The FTIR spectra exhibited prominent peaks at 3,417 cm^?1 (OH stretching due to alcoholic group) and 1,578 cm^?1 (C=C ring stretching). TEM micrograph showed that the synthesized AgNps were spherical in shape with an average size of 5 nm. Surface morphology and topographical structure of the synthesized AgNps were dignified by AFM. The synthesized AgNps showed significant acaricidal activity against Rhipicephalus microplus and Haemaphysalis bispinosa with LC₅₀ values of 16.10 and 16.45 mg/L, respectively. Our results clearly indicate that AgNps could provide a safer alternative to conventional acaricidal agents in the form of a topical antiparasitic formulation. The present study aimed to develop a novel, cost-effective, eco-friendly actinobacteria mediated synthesis of AgNps and its antiparasitic activity.     

Development and In Vivo Floating Behavior of Verapamil HCl Intragastric Floating Tablets

A novel gastro retentive controlled release drug delivery system of verapamil HCl was formulated in an effort to increase the gastric retention time of the dosage form and to control drug release. Hydroxypropylmethylcellulose (HPMC), carbopol, and xanthan gum were incorporated for gel-forming properties. Buoyancy was achieved by adding an effervescent mixture of sodium bicarbonate and anhydrous citric acid. In vitro drug release studies were performed, and drug release kinetics was evaluated using the linear regression method. The optimized intragastric floating tablet composed of 3:2 of HPMC K4M to xanthan gum exhibited 95.39% drug release in 24 h in vitro, while the buoyancy lag time was 36.2 s, and the intragastric floating tablet remained buoyant for >24 h. Zero-order and non-Fickian release transport was confirmed as the drug release mechanism from the optimized formulation (F7). X-ray studies showed that total buoyancy time was able to delay the gastric emptying of verapamil HCl intragastric floating tablet in mongrel dogs for more than 4 h. Optimized intragastric floating tablet showed no significant change in physical appearance, drug content, total buoyancy time, or in vitro dissolution pattern after storage at 40°C/75% relative humidity for 3 months.     

The adaptor protein X11Lalpha/Dmint1 interacts with the PDZ-binding domain of the cell recognition protein Rst in Drosophila

The Drosophila cell adhesion molecule Rst plays key roles during the development of the embryonic musculature, spacing of ommatidia in the compound eye and of sensory organs on the antenna, as well as in the neuronal wiring of the optic lobe. In rst(CT) mutants lacking the cytoplasmic domain of the Rst protein, cell sorting and apoptosis in the eye are affected, suggesting a requirement of this domain for Rst function. To identify potential interacting proteins, yeast two-hybrid screens were performed using the cytoplasmic domains of Rst and its paralogue Kirre as baits. Among several putative interactors, two paralogous Drosophila PDZ motif proteins related to X11/Mint were identified. X11/Mint family members in C. elegans (LIN-10) and vertebrates are believed to function as adaptor proteins and to regulate the assembly of multi-subunit complexes at the synapse, thereby linking the vesicle cycle to cell adhesion. Using genetic, cell biological, and biochemical approaches, we show that the interaction of Rst with X11Lalpha is of biological significance. The proteins interact, for example, in the context of cell sorting in the pupal retina.     

Antioxidant effects of Emblica officinalis and Zingiber officinalis on arsenic and lead induced toxicity on Albino rats

It is of interest to document the effect of Emblica officinalis (E. officinalis) and Zingiber officinalae (Z. officinalae) leaf extract on reactive oxygen species, antioxidant potential changes in arsenic and lead-induced toxicity in male rats. We used 8 groups of adult male Wistar rats with 1 control group for this study. The animals were divided into Group I: Control and Group II: Lead and sodium arsenite induced rats (animals were induced for metal toxicity by the combined administration of arsenic (13.8 mg/ kg body weight) and lead (116.4 mg/kg body weight). These doses were administered by gastric intubation during 14 consecutive days using known standard procedures. Arsenic and lead induced rats treated with ethanolic extract of Emblica officinalis (60 mg/kg body weight/day, orally for 45 days) are group III rats. Group IV animals are arsenic and lead induced rats treated orally with ethanolic extracts of E. officinalis (120 mg/kg body weight/day for 45 days). Group V animals are arsenic and lead induced rats treated orally with ethanolic extracts of Z. officinalae (60 mg/kg body weight/day for 45 days). Group VI animals are arsenic and lead induced rats orally treated with ethanolic extracts of Zingiber officinalis (120 mg/kg body weight/day for 45 days). Group VII animals are arsenic and lead induced rats treated orally with ethanolic extracts of E. officinalis and Z. officinalae (60 + 60 mg/kg body weight/day for 45 days). Group VIII animals are arsenic and lead induced rats treated orally with ethanolic extracts of E. officinalis and Z. officinalae (120 + 120 mg/kg body weight/day, orally for 45 days). Normal Control animals were treated orally with ethanolic extracts of E. officinalis (120mg/kg body weight) + Z. officinalae (120mg/kg body weight) for 45 days. The control and experimental animals were then subjected to analysis for oxidative stress markers such as H2O2, *OH, and lipid peroxidation (LPO), antioxidant enzymes in addition to liver and kidney function markers. Results: Arsenic and lead induced rats showed a significant increase in the levels of reactive oxygen species (H2O2, OH* and LPO) with concomitant alterations in the renal and liver tissues. However, enzymic and non-enzymic antioxidant levels were decreased. Nevertheless, an oral effective dose of E. officinalis and Z. officinalae (120 + 120 mg/kg body weight/day increased the antioxidant enzymes and retrieved the altered levels of ROS and LPO that were induced by arsenic and lead. Thus, we show that E. officinalis and Z. officinalae leaf extract exhibits nephroprotective and hepatoprotective role through the restoration of reactive oxygen species and antioxidant enzymes in the kidney and liver tissue of Arsenic and Lead-induced nephrotoxicity and hepatotoxicity in rats. Hence, E. officinalis and Z. officinalae leaf extract are potential therapeutic options for the treatment of metal toxicity-induced kidney and liver diseases.     

Rhaponticin suppresses osteosarcoma through the inhibition of PI3K-Akt-mTOR pathway

Osteosarcoma is the frequent pediatric bone cancer where pediatric osteosarcoma incidences are more than 10% within the population. Most of the patients with osteosarcoma fall within the age of 15–30 years. Therefore, in this research, we examined the anticancer effect of Rhaponticin against the human osteosarcoma (MG-63) cells. The cytotoxicity of Rhaponticin on the MC3T3-E1 and MG-63 cells was examined through the MTT assay. The intracellular ROS accumulation, cell nuclear morphological alterations, apoptotic cell death and nuclear damages, and MMP status of Rhaponticin administered MG-63 cells were inspected by fluorescent staining techniques. The cell migration was assessed through scratch assay. The mRNA expressions of PI3K-Akt-mTOR signaling proteins were studied by RT-PCR analysis. Rhaponticin showed potent cytotoxicity, substantially inhibited the MG-63 cell growth, and displayed morphological alterations. However, rhaponticin did not affect the MC3T3-E1 cell viability. Rhaponticin administered MG-63 cells demonstrated augmented intracellular ROS accretion, weakened MMP, increased nuclear damages, and increased apoptosis. Rhaponticin effectively down-regulated the PI3K-Akt-mTOR signaling cascade in the MG-63 cells. These outcomes proved that the Rhaponticin can be a hopeful chemotherapeutic agent in the future to treat human osteosarcoma.     

Designed Chemical Intervention with Thiols for Prophylactic Contraception

Unlike somatic cells, sperm have several-fold more available-thiols that are susceptible to redox-active agents. The present study explains the mechanism behind the instant sperm-immobilizing and trichomonacidal activities of pyrrolidinium pyrrolidine-1-carbodithioate (PPC), a novel thiol agent rationally created for prophylactic contraception by minor chemical modifications of some known thiol drugs. PPC, and its three derivatives (with potential active-site blocked by alkylation), were synthesized and evaluated against live human sperm and metronidazole-susceptible and resistant Trichomonas vaginalis, in vitro. Sperm hexokinase activity was evaluated by coupled enzyme assay. PPC irreversibly immobilized 100% human sperm in ∼30 seconds and totally eliminated Trichomonas vaginalis more efficiently than nonoxynol-9 and metronidazole. It significantly inhibited (P<0.001) thiol-sensitive sperm hexokinase. However, the molecule completely lost all its biological activities once its thiol group was blocked by alkylation. PPC was subsequently formulated into a mucoadhesive vaginal film using GRaS excipients and evaluated for spermicidal and microbicidal activities (in vitro), and contraceptive efficacy in rabbits. PPC remained fully active in quick-dissolving, mucoadhesive vaginal-film formulation, and these PPC-films significantly reduced pregnancy and fertility rates in rabbits. The films released ∼90% of PPC in simulated vaginal fluid (pH 4.2) at 37°C in 5 minutes, in vitro. We have thus discovered a common target (reactive thiols) on chiefly-anaerobic, redox-sensitive cells like sperm and Trichomonas, which is susceptible to designed chemical interference for prophylactic contraception. The active thiol in PPC inactivates sperm and Trichomonas via interference with crucial sulfhydryl-disulfide based reactions, e.g. hexokinase activation in human sperm. In comparison to non-specific surfactant action of OTC spermicide nonoxynol-9, the action of thiol-active PPC is apparently much more specific, potent and safe. PPC presents a proof-of-concept for prophylactic contraception via manipulation of thiols in vagina for selective targeting of sperm and Trichomonas, and qualifies as a promising lead for the development of dually protective vaginal-contraceptive.