Platelets orchestrate remote tissue damage after mesenteric ischemia-reperfusion

Ischemia-reperfusion (I/R) injury is a leading cause of morbidity and mortality. A functional role for platelets in tissue damage after mesenteric I/R is largely unknown. The hypothesis that mesenteric I/R local and remote injury are platelet dependent was tested. Using a murine mesenteric I/R model, we demonstrate that platelets orchestrate remote lung tissue damage that follows mesenteric I/R injury and also contribute, albeit to a lesser degree, to local villi damage. While lung damage is delayed compared with villi damage, it increased over time and was characterized by accumulation of platelets in the pulmonary vasculature early, followed by alveolar capillaries and extravasation into the pulmonary space. Both villi and lung tissues displayed complement deposition. We demonstrate that villi and lung damage are reduced in mice made platelet deficient before I/R injury and that platelet transfusion into previously platelet-depleted mice before I/R increased both villi and lung tissue damage. Increased C3 deposition accompanied platelet sequestration in the lung, which was mostly absent in platelet-depleted mice. In contrast, C3 deposition was only minimally reduced on villi of platelet-depleted mice. Our findings position platelets alongside complement as a significant early upstream component that orchestrates remote lung tissue damage after mesenteric I/R and strongly suggest that reperfusion injury mitigating modalities should consider the contribution of platelets.     

Copper deprivation modulates CTR1 and CUP1 expression and enhances cisplatin cytotoxicity in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been established as a model system for cancer studies, due to the widely conserved family of genes involved in cell cycle progression, proliferation and apoptosis. In the current study, we sought to determine whether copper deprivation modulates sensitivity of yeast to cisplatin. Yeast cultures grown in low copper medium and exposed to bathocuproiene disulfate (BCS) resulted in significant reduction of intracellular copper. We report here that low copper medium rendered BY4741 hypersensitive to cisplatin (CDDP). Yeast grown in low copper medium exhibited ～2.0 fold enhanced cytotoxicity in survival and colony-forming ability, compared to copper adequate control cells grown in YPD. The effect of copper restriction on CDDP sensitivity appeared to be associated with the up regulation of CTR1, facilitating enhanced uptake and accumulation of CDDP. Also, CDDP further lowered copper deprivation-induced changes in CUP1 metallothionein levels, SOD activity and GSH levels. These changes were associated with increased protein oxidation and lipid peroxidation induced by CDDP. These results thus suggest that cisplatin cytotoxicity is potentiated under low copper conditions due to enhanced uptake and accumulation of cisplatin and also in part due to lowered antioxidant defense and increased oxidative stress imposed by copper deprivation.     

Pd-mediated functionalization of polysubstituted pyrroles: Their evaluation as potential inhibitors of PDE4

Novel polysubstituted pyrroles have been designed and accessed via a one-pot multicomponent reaction followed by Pd-mediated C-C bond forming reactions. All the compounds synthesized were tested for their PDE4B inhibitory properties in vitro and two of them obtained via Heck reaction showed significant inhibition. The docking results suggested that these alkenyl derivatives containing ester moiety interact well with the PDE4B protein in silico where the ester carbonyl oxygen played a key role. The pyrrole framework presented here could be a new template for the identification of small molecule based novel inhibitors of PDE4. The single crystal X-ray data of a representative compound is presented.     

Vapor-Induced Phase Transformations in Docetaxel

Vapor-induced transformations of docetaxel anhydrous (form D(A)) under ambient conditions have been studied using methanol, ethanol, and water as the solvent media. The online vapor-induced transformations were monitored by powder X-ray diffractometry. New solid forms (solvates/hydrates/anhydrous) of docetaxel anhydrous were obtained in stoichiometric ratios which were characterized completely using powder X-ray diffraction, differential scanning calorimeter, thermogravimetric analysis, and spectroscopic ((13)C solid-state nuclear magnetic spectroscopy, solution (1)H NMR, and Fourier transform infrared) techniques. The new forms namely methanol solvate (D(M)), ethanol solvate (D(E)), monohydrate (D(MH)), trihydrate (D(TH)), and anhydrous (D(AN-I) and D(AN-II)) were identified through structural analysis.     

Role of Direct Repeat and Stem-Loop Motifs in mtDNA Deletions: Cause or Coincidence?

Deletion mutations within mitochondrial DNA (mtDNA) have been implicated in degenerative and aging related conditions, such as sarcopenia and neuro-degeneration. While the precise molecular mechanism of deletion formation in mtDNA is still not completely understood, genome motifs such as direct repeat (DR) and stem-loop (SL) have been observed in the neighborhood of deletion breakpoints and thus have been postulated to take part in mutagenesis. In this study, we have analyzed the mitochondrial genomes from four different mammals: human, rhesus monkey, mouse and rat, and compared them to randomly generated sequences to further elucidate the role of direct repeat and stem-loop motifs in aging associated mtDNA deletions. Our analysis revealed that in the four species, DR and SL structures are abundant and that their distributions in mtDNA are not statistically different from randomized sequences. However, the average distance between the reported age associated mtDNA breakpoints and their respective nearest DR motifs is significantly shorter than what is expected of random chance in human (p<10(-4)) and rhesus monkey (p = 0.0034), but not in mouse (p = 0.0719) and rat (p = 0.0437), indicating the existence of species specific difference in the relationship between DR motifs and deletion breakpoints. In addition, the frequencies of large DRs (>10 bp) tend to decrease with increasing lifespan among the four mammals studied here, further suggesting an evolutionary selection against stable mtDNA misalignments associated with long DRs in long-living animals. In contrast to the results on DR, the probability of finding SL motifs near a deletion breakpoint does not differ from random in any of the four mtDNA sequences considered. Taken together, the findings in this study give support for the importance of stable mtDNA misalignments, aided by long DRs, as a major mechanism of deletion formation in long-living, but not in short-living mammals.     

Platelet-associated CD40/CD154 mediates remote tissue damage after mesenteric ischemia/reperfusion injury

Several innate and adaptive immune cell types participate in ischemia/reperfusion induced tissue injury. Amongst them, platelets have received little attention as contributors in the process of tissue damage after ischemia reperfusion (I/R) injury. It is currently unknown whether platelets participate through the immunologically important molecules including, CD40 and when activated, CD154 (CD40L), in the pathogenesis of I/R injury. We hypothesized that constitutive expression of CD40 and activation-induced expression of CD154 on platelets mediate local mesenteric and remote lung tissue damage after I/R injury. Wild type (WT; C57BL/6J), CD40 and CD154 deficient mice underwent mesenteric ischemia for 30 minutes followed by reperfusion for 3 hours. WT mice subjected to mesenteric I/R injury displayed both local intestinal and remote lung damage. In contrast, there was significantly less intestinal damage and no remote lung injury in CD40 and CD154 deficient mice when compared to WT mice. Platelet-depleted WT mice transfused with platelets from CD40 or CD154 deficient mice failed to reconstitute remote lung damage. In contrast, when CD40 or CD154 deficient mice were transfused with WT platelets lung tissue damage was re-established. Together, these findings suggest that multiple mechanisms are involved in local and remote tissue injury and also identify platelet-expressed CD40 and/or CD154 as mediators of remote tissue damage.     

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.^1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.^4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.^1,2The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically¹ or analytically,^2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,^1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.^1,2,7In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N₃) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein – strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.     

CYP1A1 genotypes and haplotypes and risk of oral cancer: A case-control study in South Indians

The CYP1A1 gene encodes for the enzyme, aryl hydrocarbon hydroxylase, which is involved in the biotransformation of various aromatic tobacco precarcinogens. In the present study, the association between CYP1A1 gene polymorphisms (IVS1-728G > A, Thr461Asn and Ile462Val), and the risk of oral cancer, was examined among 157 patients with oral cancer and 132 age-matched controls, in a south Indian population. The strength of the association between CYP1A1 variants and oral cancer was estimated by logistic regression. It was found that Thr461Asn was not polymorphic. Both IVS1-728G > A and Ile462Val frequencies were consistent with Hardy-Weinberg equilibrium in the control group. There were no significant differences in genotype or haplotype frequencies between controls and cases with oral cancer. Hence, CYP1A1 SNPs can be considered as not being associated with oral cancer at either the genotype or haplotype levels in the population studied.     

Biosynthesis of xylobiose: a strategic way to enrich the value of oil palm empty fruit bunch fiber

Xylooligosaccharides are functional foods mainly produced during the hydrolysis of xylan by physical, chemical, or enzymatic methods. In this study, production of xylobiose was investigated using oil palm empty fruit bunch fiber (OPEFB) as a source material, by chemical and enzymatic methods. Xylanase-specific xylan hydrolysis followed by xylobiose production was observed. Among different xylanases, xylanase from FXY-1 released maximum xylobiose from pretreated OPEFB fiber, and this fungal strain was identified as Aspergillus terreus and subsequently deposited under the accession Number MTCC- 8661. The imperative role of lignin on xylooligosaccharides enzymatic synthesis was exemplified with the notice of xylobiose production only with delignified material. A maximum 262 mg of xylobiose was produced from 1.0 g of pretreated OPEFB fiber using FXY-1 xylanase (6,200 U/ml) at pH 6.0 and 45° C. At optimized environment, the yield of xylobiose was improved to 78.67 g/100 g (based on xylan in the pretreated OPEFB fiber).