P2-P1' macrocyclization of P2 phenylglycine based HCV NS3 protease inhibitors using ring-closing metathesis

Macrocyclization is a commonly used strategy to preorganize HCV NS3 protease inhibitors in their bioactive conformation. Moreover, macrocyclization generally leads to greater stability and improved pharmacokinetic properties. In HCV NS3 protease inhibitors, it has been shown to be beneficial to include a vinylated phenylglycine in the P2 position in combination with alkenylic P1' substituents. A series of 14-, 15- and 16-membered macrocyclic HCV NS3 protease inhibitors with the linker connecting the P2 phenylglycine and the alkenylic P1' were synthesized by ring-closing metathesis, using both microwave and conventional heating. Besides formation of the expected macrocycles in cis and trans configuration as major products, both ring-contracted and double-bond migrated isomers were obtained, in particular during formation of the smaller rings (14- and 15-membered rings). All inhibitors had K(i)-values in the nanomolar range, but only one inhibitor type was improved by rigidification. The loss in inhibitory effect can be attributed to a disruption of the beneficial π-π interaction between the P2 fragment and H57, which proved to be especially deleterious for the d-phenylglycine epimers.     

Direct Evidence of an Elongation Factor-Tu/Ts·GTP·Aminoacyl-tRNA Quaternary Complex

During protein synthesis, elongation factor-Tu (EF-Tu) bound to GTP chaperones the entry of aminoacyl-tRNA (aa-tRNA) into actively translating ribosomes. In so doing, EF-Tu increases the rate and fidelity of the translation mechanism. Recent evidence suggests that EF-Ts, the guanosine nucleotide exchange factor for EF-Tu, directly accelerates both the formation and dissociation of the EF-Tu-GTP-Phe-tRNA^Phe ternary complex (Burnett, B. J., Altman, R. B., Ferrao, R., Alejo, J. L., Kaur, N., Kanji, J., and Blanchard, S. C. (2013) J. Biol. Chem. 288, 13917–13928). A central feature of this model is the existence of a quaternary complex of EF-Tu/Ts·GTP·aa-tRNA^aa. Here, through comparative investigations of phenylalanyl, methionyl, and arginyl ternary complexes, and the development of a strategy to monitor their formation and decay using fluorescence resonance energy transfer, we reveal the generality of this newly described EF-Ts function and the first direct evidence of the transient quaternary complex species. These findings suggest that EF-Ts may regulate ternary complex abundance in the cell through mechanisms that are distinct from its guanosine nucleotide exchange factor functions.     

Isolation and characterisation of nuclear mutants with enhanced mitochondrial mutability in the fission yeast Schizosaccharomyces pombe

In this paper we report the isolation and preliminary characterisation of nuclear mutants with increased mitochondrial mutability in fission yeast. Screening of about 2000 clones after nitrosoguanidine mutagenesis led to the isolation of ten mutator mutants. For one of them (mut-1) we show that the mutation is chromosomally encoded. The activity of the mutator is restricted to the mitochondrial genome, since it increases the mutation rate to mitochondrially encoded drug resistance considerably, whereas the mutability of nuclear genes is not altered.     

In vivo and in silico anti-inflammatory mechanism of action of the semisynthetic (−)-cubebin derivatives (−)-hinokinin and (−)-O-benzylcubebin

(?)-Cubebin (CUB), isolated from seeds of Piper cubeba, was used as starting material to obtain the derivatives (?)-hinokinin (HK) and (?)-O-benzyl cubebin (OBZ). Using paw edema as the experimental model and different chemical mediators (prostaglandin and dextran), it was observed that both derivatives were active in comparison with both negative (5% Tween® 80 in saline) and positive (indomethacin) controls. The highest reduction in the prostaglandin-induced edema was achieved by OBZ (66.0%), while HK caused a 59.2% reduction. Nonetheless, the dextran-induced paw edema was not significantly reduced by either of the derivatives (HK or OBZ), which inhibited edema formation by 18.3% and 3.5%, respectively, in contrast with the positive control, cyproheptadine, which reduced the edema by 56.0%. The docking analysis showed that OBZ presented the most stable ligand-receptor (COX-2 – cyclooxygenase-2) interaction in comparison with CUB and HK.     

Toward a resolution of a taxonomic enigma: first genetic analyses of Paradoxornis webbianus and Paradoxornis alphonsianus (Aves: Paradoxornithidae) from China and Italy

Paradoxornis webbianus and Paradoxornis alphonsianus naturally occur in South-East Asia. Due to a recent introduction, a mixed population currently occurs in northern Italy. A preliminary phylogeographic analysis using samples from Italy and China found little genetic differentiation between the two taxa and revealed the existence of two molecular lineages, sympatric in some part of China, that do not correspond to the morphological classification. Possible taxonomic changes and preliminary inferences on the relationships between Chinese and the Italian populations and on the likely provenance of the founders introduced in Italy are also discussed.     

The Role of Lipotoxicity in Smoke Cardiomyopathy

Background/Aims

Experimental and clinical studies have shown the direct toxic effects of cigarette smoke (CS) on the myocardium, independent of vascular effects. However, the underlying mechanisms are not well known.

Methods

Wistar rats were allocated to control (C) and cigarette smoke (CS) groups. CS rats were exposed to cigarette smoke for 2 months.

Results

After that morphometric, functional and biochemical parameters were measured. The echocardiographic study showed enlargement of the left atria, increase in the left ventricular systolic volume and reduced systolic function. Within the cardiac metabolism, exposure to CS decreased beta hydroxy acyl coenzyme A dehydrogenases and citrate synthases and increased lactate dehydrogenases. Peroxisome proliferator-activated receptor alpha (PPARα) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) were expressed similarly in both groups. CS increased serum lipids and myocardial triacylglycerols (TGs). These data suggest that impairment in fatty acid oxidation and the accumulation of cardiac lipids characterize lipotoxicity. CS group exhibited increased oxidative stress and decreased antioxidant defense. Finally, the myocyte cross-sectional area and active Caspase 3 were increased in the CS group.

Conclusion

The cardiac remodeling that was observed in the CS exposure model may be explained by abnormalities in energy metabolism, including lipotoxicity and oxidative stress.     

Impact of Human Cytomegalovirus Infection and its Immune Response on Survival of Patients with Ovarian Cancer

Human cytomegalovirus (HCMV) has been detected in various types of tumors. We studied the prevalence of HCMV in ovarian cancer and its relation to clinical outcome. Paraffin-embedded tissues obtained prospectively from 45 patients with ovarian cancer and 30 patients with benign ovarian cystadenoma were analyzed for expression of HCMV immediate-early protein (IE) and HCMV tegument protein (pp65) by immunohistochemistry. Plasma was analyzed for HCMV serology. HCMV-IgG levels were higher in patients with ovarian cancer or benign cystadenoma than in age-matched controls (P?=?.002, P?<?.0001, respectively). HCMV IgM was detected in 12% of ovarian cancer patients and 3% of patients with benign tumors but was absent in controls. In patients with ovarian cancer, higher IgG levels were associated with better outcomes (P?=?.04). Extensive HCMV-IE protein expression was detected in 75% of ovarian cancers and 26% of benign tumors; pp65 was detected in 67% of ovarian cancers and 14% of benign tumors. A higher grade of HCMV infection was associated with higher stage of disease. Extensive HCMV-pp65 expression was associated with shorter median overall survival than focal expression (39 versus 42.5?months, P?=?.03). At study closure, 58% of ovarian cancer patients with focal pp65 expression were alive versus 27% of patients with extensive pp65 expression (P?=?.03). Thus, HCMV proteins are detected at different levels in ovarian tumors and benign cystadenomas. Ovarian cancer patients with focal HCMV-pp65 expression in their tumors and high IgG levels against HCMV lived longer, highlighting a need for in-depth studies of the oncomodulatory role of HCMV in ovarian cancer.     

Microresonator mass sensors for detection of Bacillus anthracis Sterne spores in air and water

Towards the goal of developing a real-time monitoring device for microorganisms, we demonstrate the use of microcantilevers as resonant mass sensors for detection of Bacillus anthracis Sterne spores in air and liquid. The detection scheme was based on measuring resonant frequency decrease driven by thermally induced oscillations, as a result of the added mass of the spores with the use of a laser Doppler vibrometer (LDV). Viscous effects were investigated by comparing measurements in air and deionized (DI) water along with theoretical values. Moreover, biological experiments were performed which involved suspending spores onto the cantilevers and performing mass detection in air and water. For detection of spores in water, the cantilevers were functionalized with antibodies in order to fix the spores onto the surface. We demonstrate that as few as 50 spores on the cantilever can be detected in water using the thermal noise as excitation source. Measurement sensitivity of 9.23 Hz/fg for air and 0.1 Hz/fg for water were obtained. These measurements were compared with theoretical values and sources of improvement in cantilever sensitivity in a viscous medium were also discussed. It is expected that by driving the cantilevers and using higher order modes, detection of a single spore in liquids should be achievable.     

MicroRNA-539 Is Up-regulated in Failing Heart,and Suppresses O-GlcNAcase Expression

Derangements in metabolism and related signaling pathways characterize the failing heart. One such signal, O-linked β-N-acetylglucosamine (O-GlcNAc), is an essential post-translational modification regulated by two enzymes, O-GlcNAc transferase and O-GlcNAcase (OGA), which modulate the function of many nuclear and cytoplasmic proteins. We recently reported reduced OGA expression in the failing heart, which is consistent with the pro-adaptive role of increased O-GlcNAcylation during heart failure; however, molecular mechanisms regulating these enzymes during heart failure remain unknown. Using miRNA microarray analysis, we observed acute and chronic changes in expression of several miRNAs. Here, we focused on miR-539 because it was predicted to target OGA mRNA. Indeed, co-transfection of the OGA-3′UTR containing reporter plasmid and miR-539 overexpression plasmid significantly reduced reporter activity. Overexpression of miR-539 in neonatal rat cardiomyocytes significantly suppressed OGA expression and consequently increased O-GlcNAcylation; conversely, the miR-539 inhibitor rescued OGA protein expression and restored O-GlcNAcylation. In conclusion, this work identifies the first target of miR-539 in the heart and the first miRNA that regulates OGA. Manipulation of miR-539 may represent a novel therapeutic target in the treatment of heart failure and other metabolic diseases.     

The Ultrastructure of a Cyanophage Attack on Anabaena variabilis

Cyanophages multiplying on the nitrogen fixing blue-green alga Anabaena variabilis Kütz. were revealed by electron microscopy. Severe ultrastructural changes have been observed in the vegetative cells, whereas the heterocysts appeared resistant to the cyanophage. A lytic cycle was observed from adsorption to lysis.