The ORF3 protein of hepatitis E virus interacts with hemopexin by means of its 26 amino acid N-terminal hydrophobic domain II

Hepatitis E virus (HEV) is a nonenveloped plus-stranded RNA virus that is a major cause of acute hepatitis in many developing countries. Recent work has shown HEV may be endemic in developed countries also. The 5' two-thirds of the 7.2 kb single-stranded RNA genome of HEV encodes ORF1, and the 3' end encodes the structural proteins ORF2 and ORF3. ORF1 is the nonstructural protein involved in viral RNA synthesis, and ORF2 is the major capsid protein, whereas ORF3 is a very small protein of only 123 amino acids. The precise cellular functions of ORF3 protein remain obscure, although it has been postulated to be a viral regulatory protein. To elucidate the role of ORF3 in viral pathogenesis, the yeast two-hybrid system was used to screen a human liver cDNA library for proteins interacting with ORF3. One of the ORF3-interacting partners thus isolated and identified was hemopexin, a 60 kDa acute-phase plasma glycoprotein with a high binding affinity to heme. The two-hybrid result was validated by in vitro pull-down and co-immunoprecipitation assays and finally by intracellular fluorescence resonance energy transfer. Using a deletion mapping approach, the hydrophobic domain II of ORF3 (spanning amino acids 37 to 62) was found to be responsible for binding to Hpx, with amino acids 63 to 77 possibly contributing to the strength of the interaction. The biological significance of this interaction in the virus life cycle has been discussed.     

Divanadium(V) complexes with 4-R-benzoic acid (1-methyl-3-oxo-butylidene)-hydrazides: Syntheses, structures and properties

In acetonitrile, reactions of bis(acetylacetonato)oxidovanadium(IV) ([VO(acac)₂]) with 4-R-benzoylhydrazine in 1:1 mole ratio provide coordinatively symmetrical complexes (1-5) of the {OV(μ-O)VO}⁴⁺ motif in 40-47% yields. On the other hand, in methanol the same reactants provide complexes (6-10) containing the {OV(μ-OMe)₂VO}⁴⁺ core in 37-50% yields. In both series of complexes, the ligand is the O,N,O-donor deprotonated Schiff base system 4-R-benzoic acid (1-methyl-3-oxo-butylidene)-hydrazide formed by template condensation of acac⁻ with 4-R-benzoylhydrazine (R = H, Cl, OMe, NO₂ and NMe₂). All the complexes have been characterized by elemental analysis, magnetic and spectroscopic (IR, UV-Vis and NMR) measurements. Molecular structures of three representative complexes (4, 6 and 7) have been determined by X-ray crystallography. In each complex, the dianionic planar ligand is coordinated to the metal centre via the enolate-O, the imine-N and the O-atom of the deprotonated amide functionality. Cyclic voltammetric measurements in dichloromethane revealed that complexes 1-5 are redox inactive, while complexes 6-10 display a metal centred reduction in the potential range −0.06 to 0.0.32 V (versus Ag/AgCl).     

Studies on Stibanate unresponsive isolates of<Emphasis Type="Italic">Leishmania donovani</Emphasis>

Visceral leishmaniasis, also known as kala-azar (KA) is generally caused byLeishmania donovani. Organic pentavalent antimonials (SbV) is the first line of treatment for KA. However, the number of KA patients unresponsive to treatment with Sb(V) is steadily increasing in India and elsewhere. The primary objective of this work is to determine the factor(s) associated with the rise of unresponsiveness. Analysis of the clonal population of parasites clearly indicated that wild type parasites isolated from KA patients who were clinically cured after treatment with Sb(V), were a mixture of resistant and sensitive cells. The resistant promastigotes were also resistant as amastigotesin vivo. It was further observed that Stibanate sensitive parasites can be made resistant to the drug by repeated passages in experimental animals followed by incomplete treatment with suboptimal doses of the drug. These results suggest that the steady rise in Sb(V) unresponsiveness of KA patients in India is due to infection with resistant parasites, generated as a result of irregular and often incomplete treatment of the patients.     

A375 melanoma cells are sensitized to cisplatin-induced toxicity by a synthetic nitro-flavone derivative 2-(4-Nitrophenyl)-4H-chromen-4-one through inhibition of PARP1

Background

Cisplatin has been extensively used in therapeutics for its broad-spectrum anticancer activity and frequently used for the treatment of solid tumors. However, it presents several side-effects and several cancers develop resistance. Combination therapy of cisplatin with poly (ADP-ribose) polymerase 1 (PARP1) inhibitors has been effective in increasing its efficacy at lower doses.

Methods and results

In this work, we have shown that the nitro-flavone derivative, 2-(4-Nitrophenyl)-4H-chromen-4-one (4NCO), can improve the sensitivity of cancer cells to cisplatin through inhibition of PARP1. The effect of 4NCO on cisplatin toxicity was studied through combination therapy in both exponential and density inhibited A375 melanoma cells. Combination index (CI) was determined from isobologram analysis. The mechanism of cell killing was assessed by lactate dehydrogenase (LDH) assay. Temporal nicotinamide adenine dinucleotide (NAD⁺) assay was done to show the inhibition of PARP1. We also performed in silico molecular modeling studies to know the binding mode of 4NCO to a modeled PARP1-DNA complex containing cisplatin-crosslinked adduct. The results from both in silico and in cellulo studies confirmed that PARP1 inhibition by 4NCO was most effective in sensitizing A375 melanoma cells to cisplatin. Isobologram analysis revealed that 4NCO reduced cell viability both in exponential and density inhibited A375 cells synergistically. The combination led to cell death through apoptosis.

Conclusion

The synthetic nitro-flavone derivative 4NCO effectively inhibited the important nuclear DNA repair enzyme PARP1 and therefore, could complement the DNA-damaging anticancer drug cisplatin in A375 cells and thus, could act as a potential adjuvant to cisplatin in melanoma therapy.

     

Scale up, optimization and stability analysis of Curcumin C3 complex-loaded nanoparticles for cancer therapy

ABSTRACT: BACKGROUND: Nanoparticle based delivery of anticancer drugs have been widely investigated. However, a very important process for Research & Development in any pharmaceutical industry is scaling nanoparticle formulation techniques so as to produce large batches for preclinical and clinical trials. This process is not only critical but also difficult as it involves various formulation parameters to be modulated all in the same process. METHODS: In our present study, we formulated curcumin loaded poly (lactic acid-co-glycolic acid) nanoparticles (PLGA-CURC). This improved the bioavailability of curcumin, a potent natural anticancer drug, making it suitable for cancer therapy. Post formulation, we optimized our process by Reponse Surface Methodology (RSM) using Central Composite Design (CCD) and scaled up the formulation process in four stages with final scale-up process yielding 5 g of curcumin loaded nanoparticles within the laboratory setup. The nanoparticles formed after scale-up process were characterized for particle size, drug loading and encapsulation efficiency, surface morphology, in vitro release kinetics and pharmacokinetics. Stability analysis and gamma sterilization were also carried out. RESULTS: Results revealed that that process scale-up is being mastered for elaboration to 5 g level. The mean nanoparticle size of the scaled up batch was found to be 158.5 [PLUS-MINUS SIGN] 9.8 nm and the drug loading was determined to be 10.32 [PLUS-MINUS SIGN] 1.4 %. The in vitro release study illustrated a slow sustained release corresponding to 75 % drug over a period of 10 days. The pharmacokinetic profile of PLGA-CURC in rats following i.v. administration showed two compartmental model with the area under the curve (AUC0-[INFINITY]) being 6.139 mg/L h. Gamma sterilization showed no significant change in the particle size or drug loading of the nanoparticles. Stability analysis revealed long term physiochemical stability of the PLGA-CURC formulation. CONCLUSIONS: A successful effort towards formulating, optimizing and scaling up PLGA-CURC by using Solid-Oil/Water emulsion technique was demonstrated. The process used CCD-RSM for optimization and further scaled up to produce 5 g of PLGA-CURC with almost similar physicochemical characteristics as that of the primary formulated batch.     

Variations in the Rhythms of Respiration and Nitrogen Fixation in Members of the Unicellular Diazotrophic Cyanobacterial Genus Cyanothece

In order to accommodate the physiologically incompatible processes of photosynthesis and nitrogen fixation within the same cell, unicellular nitrogen-fixing cyanobacteria have to maintain a dynamic metabolic profile in the light as well as the dark phase of a diel cycle. The transition from the photosynthetic to the nitrogen-fixing phase is marked by the onset of various biochemical and regulatory responses, which prime the intracellular environment for nitrogenase activity. Cellular respiration plays an important role during this transition, quenching the oxygen generated by photosynthesis and by providing energy necessary for the process. Although the underlying principles of nitrogen fixation predict unicellular nitrogen-fixing cyanobacteria to function in a certain way, significant variations are observed in the diazotrophic behavior of these microbes. In an effort to elucidate the underlying differences and similarities that govern the nitrogen-fixing ability of unicellular diazotrophic cyanobacteria, we analyzed six members of the genus Cyanothece. Cyanothece sp. ATCC 51142, a member of this genus, has been shown to perform efficient aerobic nitrogen fixation and hydrogen production. Our study revealed significant differences in the patterns of respiration and nitrogen fixation among the Cyanothece spp. strains that were grown under identical culture conditions, suggesting that these processes are not solely controlled by cues from the diurnal cycle but that strain-specific intracellular metabolic signals play a major role. Despite these inherent differences, the ability to perform high rates of aerobic nitrogen fixation and hydrogen production appears to be a characteristic of this genus.Nitrogen fixation is an important global phenomenon by which molecular nitrogen, one of the most abundant components of the earth’s atmosphere, is converted into a more reduced form suitable for incorporation into living systems. The majority of this nitrogen fixation is achieved by biological means through the activity of microorganisms (Burris and Roberts, 1993; Raymond et al., 2004; Rubio and Ludden, 2008). This process is energy intensive, and nitrogenase, the enzyme complex involved in the biological nitrogen fixation reaction, is generally known to be extremely sensitive to oxygen (Robson and Postgate, 1980; Hill et al., 1981; Berman-Frank et al., 2005). Thus, most microbes participating in this process fix nitrogen only when suitable anaerobic or microaerobic conditions are established in an otherwise oxygen-rich environment. However, some nitrogen-fixing (diazotrophic) microbes have the advantage of being able to fix nitrogen in aerobic environments. Outstanding among these are the photosynthetic prokaryotes called cyanobacteria, an extremely successful group of microbes with plant-like traits. These microbes are considered to be the progenitors of plant chloroplasts. Cyanobacteria perform both oxygen-evolving photosynthesis and oxygen-sensitive nitrogen fixation, thereby providing a platform to power the most metabolically expensive biological process (Simpson and Burris, 1984) with solar energy.Among the nitrogen-fixing cyanobacteria, filamentous strains have been extensively studied for their contribution to the nitrogen cycle in marine and terrestrial ecosystems (Mulligan and Haselkorn, 1989; Kaneko et al., 2001; Meeks et al., 2001; Sañudo-Wilhelmy et al., 2001; Wong and Meeks, 2001; Gomez et al., 2005). Some of these filamentous strains develop specialized cells called heterocysts that allow the spatial segregation of photosynthesis and nitrogen fixation. These heterocysts also have higher rates of respiratory oxygen consumption, which results in a virtually anoxic environment conducive for the nitrogenase enzyme (Bergman et al., 1997). All heterocystous strains are known to fix nitrogen aerobically. In contrast, nonheterocystous cyanobacteria lack any specialized oxygen-free compartments and often require incubation under microoxic or anaerobic conditions for nitrogen fixation (Rippka and Waterbury, 1977; Rippka et al., 1979; Brass et al., 1992). However, some nonheterocystous cyanobacterial strains can fix nitrogen under aerobic conditions. These include some filamentous genera like Trichodesmium spp., Lyngbya spp., and Oscillatoria spp. (Jones, 1990; Janson et al., 1994; Finzi-Hart et al., 2009) as well as unicellular genera like Gloeothece spp. and Cyanothece spp. (Wyatt and Silvey, 1969; Rippka and Waterbury, 1977; Huang and Chow, 1988; Van Ni et al., 1988; Schütz et al., 2004).In comparison with filamentous cyanobacteria, which have long been recognized for their nitrogen-fixing ability, the importance of unicellular cyanobacteria as key components of the environmental nitrogen cycle has only been recently uncovered. Studies over the last decade have established unicellular strains like Crocosphaera spp., Cyanothece spp., and UCYN-A as important players in the marine nitrogen cycle (Zehr et al., 2001; Montoya et al., 2004; Zehr, 2011). Since unicellular diazotrophic cyanobacteria utilize the same cellular platform for photosynthesis and nitrogen fixation, they are required to adjust their cellular metabolism to accommodate these two antagonistic processes. Systems-level studies in the unicellular genus Cyanothece have revealed a temporal separation of the two processes, photosynthesis occurring during the day and nitrogen fixation occurring at night (Stöckel et al., 2008; Toepel et al., 2008; Welsh et al., 2008). Cellular respiration plays a critical role during the transition from one phase to the next, rapidly freeing the intracellular environment of the photosynthetically generated oxygen and rendering it conducive for the induction of nitrogenase activity. In addition, respiration also sustains the process of nitrogen fixation, not only by maintaining a low-oxygen environment required for the functioning of the nitrogenase enzyme but also by mobilizing the stored solar energy to fuel this energy-intensive process.Unicellular diazotrophs exhibit great diversity in the efficiency of nitrogen fixation as well as in the physiological regulation of the process. For instance, members of the genus Gloeothece fix nitrogen aerobically during the day, but at 0% dissolved oxygen concentration, nitrogen fixation is shifted entirely to the dark period (Ortega-Calvo and Stal, 1991; Taniuchi et al., 2008). In contrast, some Synechococcus spp. strains can fix nitrogen only when incubated under anoxic conditions (Steunou et al., 2006). Members of the genus Cyanothece have been reported to engage in both aerobic and anaerobic nitrogen fixation, with nitrogenase activity peaking during the night (Reddy et al., 1993; Bergman et al., 1997; Turner et al., 2001). This suggests that, in addition to the regulations imposed by the diurnal cycle, strain-specific intracellular cues govern the process of nitrogen fixation in unicellular cyanobacteria, which may vary according to the genotype or the ecotype of the strains.Members of the unicellular cyanobacterial genus Cyanothece are diazotrophs that thrive in marine as well as terrestrial environments. This genus was originally grouped together with Synechococcus spp. but was later separated on the basis of distinct morphological and biochemical differences between the two genera (Komárek, 1976; Rippka and Cohen-Bazire, 1983). Some of the features that define the largely heterogeneous genus Cyanothece are oval to cylindrical cells, larger than 3 µm in size (they can be as large as 24 µm in diameter), radially arranged thylakoids, and a mucilaginous layer surrounding the cells (Komárek and Cepák, 1998; Porta et al., 2000; Liberton et al., 2011).It was recently demonstrated that Cyanothece sp. ATCC 51142, a member of the genus Cyanothece, has the unique ability to produce molecular hydrogen at exceptionally high rates under aerobic conditions (Bandyopadhyay et al., 2010). This striking observation was attributed to the nitrogenase enzyme system of Cyanothece sp. ATCC 51142. Our study also indicated that high rates of respiration in this strain might contribute to its nitrogenase-mediated aerobic hydrogen production. Glycerol was found to be an efficient source of reductants and energy for this process. In an effort to investigate if this atypical cyanobacterial trait was a characteristic of the genus Cyanothece, five additional Cyanothece spp. strains from different ecological habitats were sequenced to completion. The six strains display more than 90% identity in their 16S ribosomal RNA sequence but exhibit striking variability with respect to their genome sizes (with the largest genome being 7.8 Mb and the smallest being 4.4 Mb), the number of plasmids, and the percentage of pseudogenes (Bandyopadhyay et al., 2011). In addition, two of the strains possess linear chromosomal elements, features not known to occur in any other photosynthetic bacteria sequenced to date, which may impart niche-specific advantages to these strains. Analysis of the genome sequence of the Cyanothece spp. strains showed the presence of a nitrogenase gene cluster in all five strains, and preliminary analysis showed that four of the five strains were capable of aerobic nitrogen fixation and hydrogen production (Bandyopadhyay et al., 2011). In this study, we have focused on the patterns of nitrogen fixation and respiration in six different Cyanothece spp. strains in an effort to elucidate the underlying differences and similarities in these processes in unicellular diazotrophic strains with similar genotypic but varied ecological backgrounds. Our study reveals inherent differences in the regulation of these processes, which are likely controlled by strain-specific cellular signals. However, despite the differences in the patterns of nitrogenase activity, aerobic nitrogen fixation and hydrogen production was found to be a characteristic of this genus, with most members exhibiting nitrogenase-mediated hydrogen production at rates higher than any other wild-type cyanobacterial strain.     

Implantable gastric stimulator does not prevent the increase in plasma ghrelin levels that occurs with weight loss

The SHAPE (Screened Health Assessment and Pacer Evaluation) trial was a 24 month randomized multicenter placebo-controlled study to determine the efficacy of an implantable gastric stimulator (IGS) for weight loss. This report is an investigator-initiated sub-study at one site designed to assess whether IGS affects plasma levels of ghrelin and peptide YY (PYY). The device was implanted in all subjects but was activated in the Treatment group (n = 7, BMI = 41.5 ± 2.0 kg/m2) and remained inactive in the Control (n = 6, BMI = 39.5 ± 1.7 kg/m2) during the first 12 months. IGS was activated in both groups during months 12-24. Fasting venous blood was drawn at months 0, 12, and 24 and an oral glucose tolerance test (OGTT) was performed at month 12. Although there was no difference in weight loss at 6 months (Control: -6.6 ± 1.5% vs. Treatment: -6.2 ± 1.4%), at 24 months the Control group exhibited weight gain from baseline (+2.2 ± 1.5%) that was significantly different from the weight loss in the Treatment group (-1.9 ± 1.4%; P < 0.05). At 12 months, fasting ghrelin was significantly increased (P < 0.05) in the Treatment group (285 ± 35 to 336 ± 35 pg/ml; weight change, -4.9 ± 1.4%), but not in the Control (211 ± 36 to 208 ± 35 pg/ml; weight change, -3.4 ± 1.5%). No significant change was observed in postprandial suppression of plasma ghrelin or in fasting and postprandial PYY levels. In conclusion, IGS does not prevent the increase in fasting plasma ghrelin levels associated with weight loss. Further studies are needed to determine whether changes in technology can improve weight loss and maintenance, perhaps using gut hormones as biomarkers of possible efficacy.