T-Cell Infiltration Correlates with CXCL10 Expression in Ganglia of Cynomolgus Macaques with Reactivated Simian Varicella Virus

Ganglia of monkeys with reactivated simian varicella virus (SVV) contained more CD8 than CD4 T cells around neurons. The abundance of CD8 T cells was greater less than 2 months after reactivation than that at later times and correlated with that of CXCL10 RNA but not with those of SVV protein or open reading frame 61 (ORF61) antisense RNA. CXCL10 RNA colocalized with T-cell clusters. After SVV reactivation, transient T-cell infiltration, possibly mediated by CXCL10, parallels varicella zoster virus (VZV) reactivation in humans.     

Development and integration of EST–SSR markers into an established linkage map in switchgrass

Switchgrass (Panicum virgatum L.) is a model cellulosic biofuel crop in the United States. Simple sequence repeat (SSR) markers are valuable resources for genetic mapping and molecular breeding. A large number of expressed sequence tags (ESTs) of switchgrass are recently available in our sequencing project. The objectives of this study were to develop new SSR markers from the switchgrass EST sequences and to integrate them into an existing linkage map. More than 750 unique primer pairs (PPs) were designed from 243,600 EST contigs and tested for PCR amplifications, resulting in 538 PPs effectively producing amplicons of expected sizes. Of the effective PPs, 481 amplifying informative bands in NL94 were screened for polymorphisms in a panel consisting of NL94 and its seven first-generation selfed (S1) progeny. This led to the selection of 117 polymorphic EST–SSRs to genotype a mapping population encompassing 139 S1 individuals of NL94. Of 83 markers demonstrating clearly scorable alleles in the mapping population, 79 were integrated into a published linkage map, with three linked to accessory loci and one unlinked. The newly identified EST–SSR loci were distributed in 17 of 18 linkage groups with 27 (32.5 %) exhibiting distorted segregations. The integration of EST–SSRs aided in reducing the average marker interval (cM) to 3.7 from 4.2, and reduced the number of gaps (each >15 cM) to 10 from 23. Developing new EST–SSRs and constructing a higher density linkage map will facilitate quantitative trait locus mapping and provide a firm footing for marker-assisted breeding in switchgrass.     

Lucanthone is a novel inhibitor of autophagy that induces cathepsin D-mediated apoptosis

Cellular stress induced by nutrient deprivation, hypoxia, and exposure to many chemotherapeutic agents activates an evolutionarily conserved cell survival pathway termed autophagy. This pathway enables cancer cells to undergo self-digestion to generate ATP and other essential biosynthetic molecules to temporarily avoid cell death. Therefore, disruption of autophagy may sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis. Chloroquine and its analog hydroxychloroquine are the only clinically relevant autophagy inhibitors. Because both of these agents induce ocular toxicity, novel inhibitors of autophagy with a better therapeutic index are needed. Here we demonstrate that the small molecule lucanthone inhibits autophagy, induces lysosomal membrane permeabilization, and possesses significantly more potent activity in breast cancer models compared with chloroquine. Exposure to lucanthone resulted in processing and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, but impaired autophagic degradation as revealed by transmission electron microscopy and the accumulation of p62/SQSTM1. Microarray analysis, qRT-PCR, and immunoblotting determined that lucanthone stimulated a large induction in cathepsin D, which correlated with cell death. Accordingly, knockdown of cathepsin D reduced lucanthone-mediated apoptosis. Subsequent studies using p53(+/+) and p53(-/-) HCT116 cells established that lucanthone induced cathepsin D expression and reduced cancer cell viability independently of p53 status. In addition, lucanthone enhanced the anticancer activity of the histone deacetylase inhibitor vorinostat. Collectively, our results demonstrate that lucanthone is a novel autophagic inhibitor that induces apoptosis via cathepsin D accumulation and enhances vorinostat-mediated cell death in breast cancer models.     

A New Ibuprofen Derivative Inhibits Platelet Aggregation and ROS Mediated Platelet Apoptosis

Thrombocytopenia is a serious issue connected with the pathogenesis of several human diseases including chronic inflammation, arthritis, Alzheimer''s disease, cardiovascular diseases (CVDs) and other oxidative stress-associated pathologies. The indiscriminate use of antibiotics and other biological drugs are reported to result in thrombocytopenia, which is often neglected during the treatment regime. In addition, augmented oxidative stress induced by drugs and pathological conditions has also been shown to induce thrombocytopenia, which seems to be the most obvious consequence of elevated rate of platelet apoptosis. Thus, blocking oxidative stress-induced platelet apoptosis would be of prime importance in order to negotiate thrombocytopenia and associated human pathologies. The current study presents the synthesis and platelet protective nature of novel ibuprofen derivatives. The potent anti-oxidant ibuprofen derivative 4f was selected for the study and the platelet protective efficacy and platelet aggregation inhibitory property has been demonstrated. The compound 4f dose dependently mitigates the oxidative stress-induced platelet apoptosis in both platelet rich plasma and washed platelets. The platelet protective nature of compound 4f was determined by assessing various apoptotic markers such as ROS generation, cytosolic Ca²⁺ levels, PS externalization, cytochrome C translocation, Caspase activation, mitochondrial membrane depolarization, cytotoxicity, LDH leakage and tyrosine phosphorylation of cytosolic proteins. Furthermore, compound 4f dose dependently ameliorated agonist induced platelet aggregation. Therefore, compound 4f can be estimated as a potential candidate in the treatment regime of pathological disorders associated with platelet activation and apoptosis. In addition, compound 4f can be used as an auxiliary therapeutic agent in pathologies associated with thrombocytopenia.     

Novel Benzoxazine-Based Aglycones Block Glucose Uptake In Vivo by Inhibiting Glycosidases

Glycoside hydrolases catalyze the selective hydrolysis of glycosidic bonds in oligosaccharides, polysaccharides, and their conjugates. β-glucosidases occur in all domains of living organisms and constitute a major group among glycoside hydrolases. On the other hand, the benzoxazinoids occur in living systems and act as stable β-glucosides, such as 2-(2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one)-β-D-gluco-pyranose, which hydrolyse to an aglycone DIMBOA. Here, we synthesized the library of novel 1,3-benzoxazine scaffold based aglycones by using 2-aminobenzyl alcohols and aldehydes from one-pot reaction in a chloroacetic acid catalytic system via aerobic oxidative synthesis. Among the synthesized benzoxazines, 4-(7-chloro-2,4-dihydro-1H-benzo[d][1,3]oxazin-2-yl)phenol (compound 7) exhibit significant inhibition towards glucosidase compared to acarbose, with a IC₅₀ value of 11.5 µM. Based upon results generated by in silico target prediction algorithms (Naïve Bayesian classifier), these aglycones potentially target the additional sodium/glucose cotransporter 1 (where a log likelihood score of 2.70 was observed). Furthermore, the in vitro glucosidase activity was correlated with the in silico docking results, with a high docking score for the aglycones towards the substrate binding site of glycosidase. Evidently, the in vitro and in vivo experiments clearly suggest an anti-hyperglycemic effect via glucose uptake inhibition by 4-(7-chloro-2,4-dihydro-1H-benzo[d][1,3]oxazin-2-yl)phenol in the starved rat model. These synthetic aglycones could constitute a novel pharmacological approach for the treatment, or re-enforcement of existing treatments, of type 2 diabetes and associated secondary complications.     

Effect of Experimental Temperature on the Permeation of Model Diffusants Across Porcine Buccal Mucosa

The influence of experimental temperature on the permeability of model diffusants across porcine buccal mucosa was investigated in vitro. The permeability increased significantly as the experimental temperature was increased in increments of approximately 7°C. It was observed that the apparent permeability and temperature were related by an exponential relationship that conformed to the Arrhenius equation. Diffusants with higher lipophilicities—buspirone and bupivacaine—had lower activation energies for diffusion when compared to hydrophilic diffusants—antipyrine and caffeine. The activation energy for diffusion of the model diffusants decreased linearly with increasing distribution coefficients across porcine buccal mucosa. The results suggested that the buccal mucosa acts as a stronger barrier to the diffusion of hydrophilic diffusants than the lipophilic ones. The log-linear relationship between permeability and temperature indicates that temperature should be carefully controlled in diffusion experiments. These results also point to the possibility of developing heat-generating buccal delivery devices, especially for hydrophobic diffusants.     

Atomic Basis for the Species-specific Inhibition of αV Integrins by Monoclonal Antibody 17E6 Is Revealed by the Crystal Structure of αVβ3 Ectodomain-17E6 Fab Complex

The function-blocking, non-RGD-containing, and primate-specific mouse monoclonal antibody 17E6 binds the αV subfamily of integrins. 17E6 is currently in phase II clinical trials for treating cancer. To elucidate the structural basis of recognition and the molecular mechanism of inhibition, we crystallized αVβ3 ectodomain in complex with the Fab fragment of 17E6. Protein crystals grew in presence of the activating cation Mn²⁺. The integrin in the complex and in solution assumed the genuflected conformation. 17E6 Fab bound exclusively to the Propeller domain of the αV subunit. At the core of αV-Fab interface were interactions involving Propeller residues Lys-203 and Gln-145, with the latter accounting for primate specificity. The Propeller residue Asp-150, which normally coordinates Arg of the ligand Arg-Gly-Asp motif, formed contacts with Arg-54 of the Fab that were expected to reduce soluble FN10 binding to cellular αVβ3 complexed with 17E6. This was confirmed in direct binding studies, suggesting that 17E6 is an allosteric inhibitor of αV integrins.