Characterization Ofp53 Gene Sequence in Exons 5-8 of the Western Mosquito Fish, Gambusia Affinis.

Cancer is a multistage disease that involves both genetic and epigenetic factors. The number of physical and chemical agents with which human beings come in contact on a regular basis is increasing everyday. Somatic mutations can result due to such exposures and trigger uncontrolled cell division resulting in cancer. The p53 tumor suppressor gene has been identified in several vertebrate species ranging from man to fish. In addition, inactivation of p53 has been observed in a wide variety of tumors. Fish models are becoming increasingly popular for assessing environmental exposure. Low background incidence of mutations, relatively low cost tumor studies, and the ability to extrapolate the results to humans makes these models viable alternatives. The Western mosquito fish (Gambusia affinis) a fresh water species (order Atheriniformes; family Poeciliidae) was chosen as a model organism for this study. Polymerase chain reaction with rainbow trout genomic DNA as a positive control was conducted. Fragments encompassing exons 5-6 and 7-8 were isolated and sequenced. Alignment of the sequences (exons 5-8) of the mosquito fish with that of rainbow trout in the similar regions revealed high homology between the species. Southern transfer of restricted genomic DNA of Gambusia affinis was conducted (target DNA). A PCR product (exons 5-6) of 450 base pairs was digoxigenin (DIG) labeled (probe DNA). In a separate set of experiments, PCR product from Gambusia affinis exons 7-8 (target DNA) was probed with another DIG-labeled PCR product (350 base pairs) from similar regions of rainbow trout genomic DNA. Hybridization of the probe and target DNA followed by chemiluminiscent detection resulted in visualization as bands on X-ray film indicating high homology between mosquito fish and rainbow trout p53 gene. While rainbow trout can function only in cold-temperatures, medaka, being exotic, is restricted to use only in the laboratory. In the present work the mosquito fish can withstand temperature ranging from \rm 40\sp\circ F{-}110\sp\circ F. In addition, this study can help promote the concept of the mosquito fish as a sentinel species for environmental monitoring and replace rainbow trout and medaka for direct validation in field

Role of Host Proteins in HIV-1 Early Replication

After 33 years of the identification of HIV-1 infection, very little is known about the role of host cellular proteins. Till now considerable work has been done in the area of host- pathogen interactions facilitated by the viral proteins and host receptors. The role of the main receptor CD4 and co-receptors like CCR5, CXCR4 and their alternative receptors were well studied in disease progression. But the intracellular events during the host- pathogen interactions were poorly understood. Much data is available based on the global analysis of genome-wide RNA interference screens, yeast two-hybrid system and co-immunoprecipitation studies but their exact roles are not yet characterized. There are very few host proteins like APOBEC3G, LEDGF/p75, INI1, HMG I(Y), BAF which are well studied and characterized. Majority of the reported proteins are attributed to multiple functions. It will be useful to study such proteins to develop as future candidates in HIV-1 therapeutics

Evaluation of Abelmoschus moschatus extracts for antioxidant, free radical scavenging, antimicrobial and antiproliferative activities using in vitro assays

<p>Abstract</p> <p>Background</p> <p><it>Abelmoschus moschatus </it>Medik. leaves and seeds are considered as valuable traditional medicine. The aromatic seeds of this plant are aphrodisiac, ophthalmic, cardio tonic, antispasmodic and used in the treatment of intestinal complaints and check queasiness. To give a scientific basis for traditional usage of this medicinal plant, the seed and leaf extracts were evaluated for their antioxidant, free radical scavenging, antimicrobial and antiproliferative activities.</p> <p>Methods</p> <p>In this study, antioxidant, antimicrobial and antiproliferative activities of <it>A. moschatus </it>extracts were evaluated in a series of <it>in vitro </it>assay involving free radicals, reactive oxygen species and their IC₅₀values were also determined. The antioxidant activities of the seed and leaf extracts of <it>A. moschatus </it>were determined by total antioxidant, DPPH, and ferrous reducing antioxidant property (FRAP) methods. In addition, the antiproliferative activity was also evaluated using colorectal adenocarcinoma and retinoblastoma human cancer cell lines. Moreover, six bacterial reference strains, two gram-positive (<it>Bacillus subtilis </it>and <it>Staphylococcus aureus</it>), four gram-negative (<it>Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris </it>and <it>Salmonella enterica paratyphi</it>) and one fungal strain (<it>Candida albicans</it>) were used to evaluate its antimicrobial activity.</p> <p>Results</p> <p>The results from this study showed that the antioxidant activities of <it>A. moschatus </it>as determined by the total phenol, flavonoids, total antioxidant and FRAP methods were higher in leaf than that of the seed extracts. On the other hand, the aqueous overnight seed extract (AMS-I) has shown significant radical scavenging activity as in 1, 1- Diphenyl-2-picrylhydrazyl (DPPH), hydrogen peroxide, hydroxyl radical, superoxide and lipid peroxidation as compared to other seed and leaf extracts. The AMS-I and AML-IV have shown activity against six and seven microorganisms respectively. Simulteneously, AMS-IV and AML-IV have demonstrated potential antiproliferative activity against two human cell lines - Colorectal adenocarcinoma (COLO-205) and retinoblastoma (Y79).</p> <p>Conclusion</p> <p>The seed and leaf extracts of <it>A. moschatus </it>possess significant antioxidant activity and could serve as free radical inhibitors or scavenger, or substitute, probably as primary antioxidants. The plant possesses moderate antibacterial activity against bacterial strains used in this study. Hydroalcoholic seed and leaf extracts also exhibited antiproliferative activity against two human cancer cell lines. <it>A. moschatus </it>may therefore, be a good candidate for functional foods as well as pharmaceutics.</p

An Efficient Targeted Drug Delivery through Apotransferrin Loaded Nanoparticles

BACKGROUND: Cancerous state is a highly stimulated environment of metabolically active cells. The cells under these conditions over express selective receptors for assimilation of factors essential for growth and transformation. Such receptors would serve as potential targets for the specific ligand mediated transport of pharmaceutically active molecules. The present study demonstrates the specificity and efficacy of protein nanoparticle of apotransferrin for targeted delivery of doxorubicin. METHODOLOGY/PRINCIPAL FINDINGS: Apotransferrin nanoparticles were developed by sol-oil chemistry. A comparative analysis of efficiency of drug delivery in conjugated and non-conjugated forms of doxorubicin to apotransferrin nanoparticle is presented. The spherical shaped apotransferrin nanoparticles (nano) have diameters of 25-50 etam, which increase to 60-80 etam upon direct loading of drug (direct-nano), and showed further increase in dimension (75-95 etam) in conjugated nanoparticles (conj-nano). The competitive experiments with the transferrin receptor specific antibody showed the entry of both conj-nano and direct-nano into the cells through transferrin receptor mediated endocytosis. Results of various studies conducted clearly establish the superiority of the direct-nano over conj-nano viz. (a) localization studies showed complete release of drug very early, even as early as 30 min after treatment, with the drug localizing in the target organelle (nucleus) (b) pharmacokinetic studies showed enhanced drug concentrations, in circulation with sustainable half-life (c) the studies also demonstrated efficient drug delivery, and an enhanced inhibition of proliferation in cancer cells. Tissue distribution analysis showed intravenous administration of direct nano lead to higher drug localization in liver, and blood as compared to relatively lesser localization in heart, kidney and spleen. Experiments using rat cancer model confirmed the efficacy of the formulation in regression of hepatocellular carcinoma with negligible toxicity to kidney and liver. CONCLUSIONS: The present study thus demonstrates that the direct-nano is highly efficacious in delivery of drug in a target specific manner with lower toxicity to heart, liver and kidney

Curcumin-Loaded Apotransferrin Nanoparticles Provide Efficient Cellular Uptake and Effectively Inhibit HIV-1 Replication In Vitro

Curcumin (diferuloylmethane) shows significant activity across a wide spectrum of conditions, but its usefulness is rather limited because of its low bioavailability. Use of nanoparticle formulations to enhance curcumin bioavailability is an emerging area of research.In the present study, curcumin-loaded apotransferrin nanoparticles (nano-curcumin) prepared by sol-oil chemistry and were characterized by electron and atomic force microscopy. Confocal studies and fluorimetric analysis revealed that these particles enter T cells through transferrin-mediated endocytosis. Nano-curcumin releases significant quantities of drug gradually over a fairly long period, ∼50% of curcumin still remaining at 6 h of time. In contrast, intracellular soluble curcumin (sol-curcumin) reaches a maximum at 2 h followed by its complete elimination by 4 h. While sol-curcumin (GI(50) = 15.6 µM) is twice more toxic than nano-curcumin (GI(50) = 32.5 µM), nano-curcumin (IC(50)<1.75 µM) shows a higher anti-HIV activity compared to sol-curcumin (IC(50) = 5.1 µM). Studies in vitro showed that nano-curcumin prominently inhibited the HIV-1 induced expression of Topo II α, IL-1β and COX-2, an effect not seen with sol-curcumin. Nano-curcumin did not affect the expression of Topoisomerase II β and TNF α. This point out that nano-curcumin affects the HIV-1 induced inflammatory responses through pathways downstream or independent of TNF α. Furthermore, nano-curcumin completely blocks the synthesis of viral cDNA in the gag region suggesting that the nano-curcumin mediated inhibition of HIV-1 replication is targeted to viral cDNA synthesis.Curcumin-loaded apotransferrin nanoparticles are highly efficacious inhibitors of HIV-1 replication in vitro and promise a high potential for clinical usefulness

CARMUSTINE LOADED LACTOFERRIN NANOPARTICLES DEMONSTRATES AN ENHANCED ANTIPROLIFERATIVE ACTIVITY AGAINST GLIOBLASTOMA IN VITRO

Objective: Despite sophisticated treatment regimens, there is no significant improvement in the mortality rates of glioblastoma due to insufficient dosage delivery, reoccurrence of tumors, higher systemic toxicity, etc. Since brain endothelial cells and glioblastoma cells express lactoferrin receptors, a target-specific drug delivery vehicle was developed using lactoferrin itself as a matrix, into which carmustine was loaded. The objective was to use carmustine loaded lactoferrin nanoparticles (CLN) to achieve higher therapeutic efficacy and target specificity compared to free carmustine.Methods: CLN were prepared using the Sol-oil method. The nanoparticles prepared were characterized for their size, shape, polydispersity, and stability using FESEM and DLS methods. Drug loading and drug releasing efficiencies were also estimated. Further, cellular uptake of nanoparticles and their antiproliferative efficacy against glioblastoma cells were evaluated.Results: Characterization of CLN showed that they were spherical with ≤ 41 nm diameter and exhibited homogeneously dispersed stable distribution. Loading efficiency of carmustine in CLN was estimated to be 43±3.7 %. Drug release from the nanoparticles was pH dependent with the maximum observed at pH 5. At physiological and gastric pH, drug release was lower, whereas maximum release was observed at endocytotic vesicular and around tumor extracellular pH. Confocal microscopic studies showed an active cellular uptake of nanoparticles. Results of antiproliferative analysis substantiated a higher antiproliferative effect for CLN compared to free carmustine.Conclusion: The results of the study demonstrated that CLN serves as a vital tool, in designing an effective treatment strategy for targeted drug delivery to glioblastoma

Fabrication of Hybrid Optical Electrical Platforms for Potential Applications in Neuromodulation

State-of-the art neuromodulators are bulky, and they are mostly fabricated on rigid substrates. Furthermore, they are not provided with a feedback control loop re- sulting in undesired off-target effects. This thesis paves the way for fabrication of lightweight neural interfaces with the capability to implement closed-loop controlled neuromodulation down to a single-cell resolution. In this work, an approach based on hybrid optical-electrical devices to implement neuromodulation is presented. The rationale of this choice, design illustration, fabrication and characterization is discussed. In addition, a proof-of-concept neural interface is illustrated. Specif- ically, hybrid optical-electrical devices are developed based on inorganic thin films, i.e., nanomembranes (NMs) formed in ordered arrays of buckled channels on com- pliant substrates. The buckled NMs include light-emitting structures in the visible range (namely Si nano-crystals) and graphene electrodes to control and record neu- ral activity, respectively. The compliant substrates of choice is polydimethylsiloxane (PDMS). Buckled NMs are obtained by guided self-assembly of the supported thin films under compressive strain. The cross-sectional size of buckled NM channels is scaled to match the dimensions of single neurons. This work primarily focuses on the fabrication and integration of an optically active NM, with a conductive film, thus obtaining a hybrid optical-electrical platform for potential neuronal in-vitro studies. A process is established based on multiple layer releases and transfers which enables graphene electrodes to be fabricated on the inner side of the buckle-delaminated channels.In addition, structural characterization of the fabricated devices is performed, along with current/voltage measurements of the graphene electrodes, and photoluminescence spectroscopy to assess the optical emission from the buckled NMs