Sofosbuvir Selects for Drug-Resistant Amino Acid Variants in the Zika Virus RNA-Dependent RNA-Polymerase Complex In Vitro

The nucleotide analog sofosbuvir, licensed for the treatment of hepatitis C, recently revealed activity against the Zika virus (ZIKV) in vitro and in animal models. However, the ZIKV genetic barrier to sofosbuvir has not yet been characterized. In this study, in vitro selection experiments were performed in infected human hepatoma cell lines. Increasing drug pressure significantly delayed viral breakthrough (p = 0.029). A double mutant in the NS5 gene (V360L/V607I) emerged in 3 independent experiments at 40–80 µM sofosbuvir resulting in a 3.9 ± 0.9-fold half- maximal inhibitory concentration (IC₅₀) shift with respect to the wild type (WT) virus. A triple mutant (C269Y/V360L/V607I), detected in one experiment at 80 µM, conferred a 6.8-fold IC₅₀ shift with respect to the WT. Molecular dynamics simulations confirmed that the double mutant V360L/V607I impacts the binding mode of sofosbuvir, supporting its role in sofosbuvir resistance. Due to the distance from the catalytic site and to the lack of reliable structural data, the contribution of C269Y was not investigated in silico. By a combination of sequence analysis, phenotypic susceptibility testing, and molecular modeling, we characterized a double ZIKV NS5 mutant with decreased sofosbuvir susceptibility. These data add important information to the profile of sofosbuvir as a possible lead for anti-ZIKV drug development.     

Role of Metabolism in Bone Development and Homeostasis

Carbohydrates, fats, and proteins are the underlying energy sources for animals and are catabolized through specific biochemical cascades involving numerous enzymes. The catabolites and metabolites in these metabolic pathways are crucial for many cellular functions; therefore, an imbalance and/or dysregulation of these pathways causes cellular dysfunction, resulting in various metabolic diseases. Bone, a highly mineralized organ that serves as a skeleton of the body, undergoes continuous active turnover, which is required for the maintenance of healthy bony components through the deposition and resorption of bone matrix and minerals. This highly coordinated event is regulated throughout life by bone cells such as osteoblasts, osteoclasts, and osteocytes, and requires synchronized activities from different metabolic pathways. Here, we aim to provide a comprehensive review of the cellular metabolism involved in bone development and homeostasis, as revealed by mouse genetic studies.     

A CMOS multichannel 10-Gb/s transceiver

We describe a CMOS multichannel transceiver that transmits and receives 10 Gb/s per channel over balanced copper media. The transceiver consists of two identical 10-Gb/s modules. Each module operates off a single 1.2-V supply and has a single 5-GHz phase-locked loop to supply a reference clock to two transmitter (Tx) channels and two receiver (Rx) channels. To track the input-signal phase, the Rx channel has a clock recovery unit (CRU), which uses a phase-interpolator-based timing generator and digital loop filter. The CRU can adjust the recovered clock phase with a resolution of 1.56 ps. Two sets of two-channel transceiver units were fabricated in 0.11-/spl mu/m CMOS on a single test chip. The transceiver unit size was 1.6 mm /spl times/ 2.6 mm. The Rx sensitivity was 120-mVp-p differential with a 70-ps phase margin for a common-mode voltage ranging from 0.6 to 1.0 V. The evaluated jitter tolerance curve met the OC-192 specification.     

Visualization of mitochondria with green fluorescent protein in cultured fibroblasts from patients with mitochondrial diseases

cDNAs for green fluorescent protein (GFP) and for a GFP fusion protein containing the presequence of human ornithine transcarbamylase (pOTC-GFP) were transfected into cultured human fibroblasts. GFP cDNA gave diffuse fluorescence throughout the cytoplasm and the nucleus, whereas pOTC-GFP cDNA gave mitochondria-associated fluorescence. Fluorescent mitochondrial structures could be classified into five patterns: thread-like mitochondria, fine thread-like ones, rod-like ones, granular ones, and granular ones with weak cytosolic fluorescence. pOTC-GFP mutants resulted in a loss of mitochondrial fluorescence and an appearance of weak fluorescence throughout the cytoplasm. pOTC-GFP cDNA was transfected into fibroblasts from patients with various mitochondrial diseases. Higher ratios of fibroblasts with granular mitochondria and those with fine thread-like ones were observed in a patient with Reye's syndrome and a patient with Kearns-Sayre syndrome. Weak cytosolic fluorescence was sometimes observed in fibroblasts from these patients. This method will be useful to analyze mitochondrial structural alterations and disorders of mitochondrial protein import.     

Monomeric, monovalent derivative of Maackia amurensis leukoagglutinin. Preparation and application to the study of cell surface glycoconjugates by flow cytometry

A stable subunit of Maackia amurensis leukoagglutinin (MMAL) was prepared by the selective reduction of disulfide bridges between the subunits followed by alkylation with 4-vinylpyridine. MMAL failed to precipitate fetuin, whereas it retained its ability to bind to the same glycoprotein coated on a plastic plate, indicating the monovalency of this derivative. This binding to immobilized fetuin was inhibited by a haptenic sugar, Neu5Ac alpha 2-3lactose, with the same inhibitory potency as against the native M. amurensis leukoagglutinin. Microscopic observation as well as flow cytometric analyses showed that Chinese hamster ovary cells were clearly stained with fluorescein isothiocyanate-labeled MMAL without any detectable agglutination. This staining was inhibited by the addition of fetuin or by the sugar chains of fetuin. Differences in the types of sialylated glycoconjugates on the cell surface of several cell lines were detected by the combined use of fluorescein isothiocyanate-labeled MMAL and the monomeric derivative of elderberry bark lectin (specific for the Neu5Ac alpha 2-6Gal/GalNAc sequence) by flow cytometry. These results demonstrate the usefulness of these monovalent derivatives of sialylated oligosaccharide-specific lectins as probes for the analysis of cell surface glycoconjugates containing sialic acid by the technique of flow cytometry.     

Transmission study of germanium using free-electron laser

We have measured optical transmittance through germanium in the mid-infrared region at room temperature using the Osaka free-electron laser (FEL). In spite of the fact that germanium is transparent in the mid-infrared region, we observed strong suppression of optical transmission under high-intensity FEL excitation. We found that the observed suppression is due to optical absorption in germanium. To analyze the experimental results, we have calculated optical transmittance of germanium within Keldysh theory     

A pseudoaneurysm after repair of thoraco-abdominal aortic aneurysm

We report a 63-year-old female patient developing a pseudoaneurysm three years after patch-plasty of an aneurysm located at the distal thoracic aorta. Redo-operation was performed including total replacement of the descending thoracic aorta with reimplantation of distal intercostal arteries using small-caliber interposition grafts. This case presentation underlines the ineffectiveness of patch repair for the treatment of aortic aneurysms. Furthermore, the surgical technique of reimplantation of intercostal arteries using a separate graft is discussed.     

Dynamical Properties of Vortices in a Bose-Einstein Condensate in a Rotating Lattice

We present simulation results of the vortex dynamics in a trapped Bose-Einstein condensate in the presence of a rotating optical lattice. Changing the potential amplitude and the relative rotation frequency between the condensate and the optical lattice, we find a rich variety of dynamical phases of vortices. In particular, when the optical lattice rotates faster than the condensate, the competition between the pinning force and the interactions by nucleated interstitial vortices leads to the melting of vortex lattice, yielding a vortex liquid phase.     

Staurosporine and its derivatives enhance f-Met-Leu-Phe-induced superoxide production via phospholipase D activation in human polymorphonuclear leukocytes

Staurosporine (STAR) is one of the most potent inhibitors of protein kinase C (PKC). It is known that in human polymorphonuclear leukocytes (PMNs), the phorbol ester-induced generation of superoxide anion (respiratory burst) is effectively inhibited by STAR in a dose-dependent manner, whereas superoxide generation induced by chemoattractants, e.g. n-formyl-methionyl-leucyl-phenylalanine (FMLP) or PAF, is regulated biphasically by STAR. We compared the effects of STAR and K252a on FMLP-induced superoxide production from PMNs and examined the effects of propranolol, a inhibitor of phosphatidic acid (PA) phosphohydrolase, on the potentiation of the production by STAR. We also examined the effects of some derivatives of STAR and K252a on the production and the alteration of the effects induced by propranolol pretreatment. When PMNs were stimulated with FMLP, STAR potentiated superoxide production by 240.5 +/- 30.9% at a low concentration (100 nmol/l). Propranolol pretreatment specifically inhibited the potentiation. When phorbol-12-myristate-13-acetate (PMA) was used as a stimulant, STAR inhibited superoxide production dose-dependently and did not enhance the production. K252a inhibited PMA or FMLP-induced superoxide production dose-dependently and did not enhance FMLP-induced superoxide production. STAR derivatives showed potentiation of FMLP-induced superoxide production similar to that of STAR at concentrations ranging from 10-100 nmol/l, and propranolol (200 mumol/l) effectively inhibited it. K252a derivative NA332 did not show any potentiative effect on the production. PMA-induced superoxide production was inhibited by all compounds dose-dependently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficacy of interferons in treating children with chronic hepatitis C

Twenty-two children with chronic hepatitis serologically positive for hepatitis C virus (HCV) were treated with interferon-alpha (IFN-alpha). Liver biopsy showed chronic active hepatitis in 13 and chronic persistent hepatitis in 9 patients. A sustained clearance of HCV was observed in 8/22 children 12 months after the administration of IFN-alpha for 26 weeks, associated with normalization of HCV core antibody. Of these eight patients six had HCV genotype III and two HCV genotype II or IV. Hepatitis relapsed in seven other patients after completion of IFN-alpha with an increase in HCV core antibody titre, five with HCV genotype II, and two with HCV genotype III or IV. A second course of IFN-alpha suppressed the reactivation of HCV in all seven patients. Three of seven responders who relapsed after the first course remained negative for HCV RNA 12 months after their second course of IFN-alpha. However, the remaining four patients with HCV genotype II again relapsed after completing their second course of IFN-alpha. Seven children with the HCV genotype II resistant to IFN, including 8 weeks of IFN-beta administration, and showed no significant reduction in HCV core antibody titre. CONCLUSION: The genotype of HCV (III) and a reduction in the core antibody titre appear to be useful parameters for predicting the response to IFN-alpha therapy.