Role of the varicella-zoster virus gene product encoded by open reading frame 35 in viral replication in vitro and in differentiated human skin and T cells in vivo

Although genes related to varicella-zoster virus (VZV) open reading frame 35 (ORF35) are conserved in the herpesviruses, information about their contributions to viral replication and pathogenesis is limited. Using a VZV cosmid system, we deleted ORF35 to produce two null mutants, designated rOkaDelta35(#1) and rOkaDelta35(#2), and replaced ORF35 at a nonnative site, generating two rOkaDelta35/35@Avr mutants. ORF35 Flag-tagged recombinants were made by inserting ORF35-Flag at the nonnative Avr site as the only copy of ORF35, yielding rOkaDelta35/35Flag@Avr, or as a second copy, yielding rOka35Flag@Avr. Replication of rOkaDelta35 viruses was diminished in melanoma and Vero cells in a 6-day analysis of growth kinetics. Plaque sizes of rOkaDelta35 mutants were significantly smaller than those of rOka in melanoma cells. Infection of melanoma cells with rOkaDelta35 mutants was associated with disrupted cell fusion and polykaryocyte formation. The small plaque phenotype was not corrected by growth of rOkaDelta35 mutants in melanoma cells expressing the major VZV glycoprotein E, gE. The rOkaDelta35/35@Avr viruses displayed growth kinetics and plaque morphologies that were indistinguishable from those of rOka. Analysis with ORF35-Flag recombinants showed that the ORF35 gene product localized predominantly to the nuclei of infected cells. Evaluations in the SCIDhu mouse model demonstrated that ORF35 was required for efficient VZV infection of skin and T-cell xenografts, although the decrease in infectivity was most significant in skin. These mutagenesis experiments indicated that ORF35 was dispensable for VZV replication, but deleting ORF35 diminished growth in cultured cells and was associated with attenuated VZV infection of differentiated human skin and T cells in vivo.     

Double-edge sword roles of iron in driving energy production versus instigating ferroptosis

Iron is vital for many physiological functions, including energy production, and dysregulated iron homeostasis underlies a number of pathologies. Ferroptosis is a recently recognized form of regulated cell death that is characterized by iron dependency and lipid peroxidation, and this process has been reported to be involved in multiple diseases. The mechanisms underlying ferroptosis are complex, and involve both well-described pathways (including the iron-induced Fenton reaction, impaired antioxidant capacity, and mitochondrial dysfunction) and novel interactions linked to cellular energy production. In this review, we examine the contribution of iron to diverse metabolic activities and their relationship to ferroptosis. There is an emphasis on the role of iron in driving energy production and its link to ferroptosis under both physiological and pathological conditions. In conclusion, excess reactive oxygen species production driven by disordered iron metabolism, which induces Fenton reaction and/or impairs mitochondrial function and energy metabolism, is a key inducer of ferroptosis.Subject terms: Cell biology, Biochemistry     

The outbreak of rabbit hemorrhagic virus type 2 in the interior of China may be related to imported semen

Highlights:
1. We identified one RHD case caused by a new RHDV variant (GI.2) in China through HA, TEM, and genome sequencing.
2. This is the first study to demonstrate that GI.2 can replicate efficiently in the reproductive system.
3. Our evidence suggests that GI.2 might be introduced into China by contaminated rabbit semen.     

A novel system of galangin–potassium permanganate–polyphosphoric acid for the determination of tryptophan and its chemiluminescence mechanism

A novel galangin–potassium permanganate (KMnO₄)–polyphosphoric acid (PPA) system was found to have an outstanding response to tryptophan (Trp). Trp determination using this KMnO₄–PPA system was enhanced significantly in the presence of galangin. A highly sensitive flow‐injection chemiluminescence (CL) method to determine Trp was developed based on the CL reaction of galangin–KMnO₄–Trp in PPA media. The presence of galangin, a member of the flavonol class of flavonoid complexes, greatly increased the luminous intensity of Trp in KMnO₄–PPA systems. Under optimized conditions, Trp was determined in the 0.05–10 µg/mL range, with a detection limit (3σ) of 5.0 × 10^?3 µg/mL. The relative standard deviation (RSD) was 1.0% for 11 replicate determinations of 1.0 µg/mL Trp. Two synthetic samples were determined selectively with recoveries of 98.4–100.1% in the presence of other amino acids. The possible mechanism is summarized as follows: excited states of Mn(II)^* and Mn(III ^* types are the main means of generating chemical luminescent species, and Trp concentration and luminescence intensity have a linear relationship, which enables quantitative analysis. Copyright © 2014 John Wiley & Sons, Ltd.     

miRNA-192 and -215 activate Wnt/β-catenin signaling pathway in gastric cancer via APC

Although great progress has been made in surgical techniques, traditional radiotherapy, and chemotherapy, gastric cancer (GC) is still the most common malignant tumor and has a high mortality, which highlights the importance of novel diagnostic markers. Emerging studies suggest that different microRNAs (miRNAs) are involved in tumorigenesis of GC. In this study, we found that miRNA-192 and -215 are significantly upregulated in GC and promote cell proliferation and migration. Adenomatous polyposis coli (APC), a well-known negative regulator in Wnt signaling, has been proved to be a target of miRNA-192 and -215. Inhibition of miRNA-192 or -215 reduced the Topflash activities and repressed the expression of Wnt signaling pathway proteins, while APC small interfering RNAs reversed the inhibitory effects, suggesting that miRNA-192 and -215 activate Wnt signaling via APC. In addition, APC mediates the cell proliferation and migration regulated by miRNA-192 and -215. Furthermore, APC is downregulated in GC tissues and negatively correlated with the expression of miRNA-192 and -215. In summary, miRNA-192 and -215 target APC and function as oncogenic miRNAs by activating Wnt signaling in GC, revealing to be potential therapeutic targets.     

A bead-based approach for large-scale identification of in vitro kinase substrates

Deciphering the kinase-substrate relationship is vital for the study of phosphorylation network. The use of immobilized proteins on protein chip as the library for screening of potential kinase substrates is a tried-and-tested method. However, information on phosphorylation sites is lacking and the creation of the library with proteins of whole proteome by recombinant expression is costly and difficult. In this study, a new solid-phase approach by immobilization of proteins from cell lysate onto beads as a protein library for kinase substrate screening was developed. It was found that consensus phosphorylation sites motif for kinase substrates could be accurately determined and hundreds of in vitro kinase substrates and their phosphorylation sites could be identified by using this method.     

Chromosome‐level genome assembly of Triplophysa tibetana,a fish adapted to the harsh high‐altitude environment of the Tibetan Plateau

Triplophysa is an endemic fish genus of the Tibetan Plateau in China. Triplophysa tibetana, which lives at a recorded altitude of ~4,000 m and plays an important role in the highland aquatic ecosystem, serves as an excellent model for investigating high‐altitude environmental adaptation. However, evolutionary and conservation studies of T. tibetana have been limited by scarce genomic resources for the genus Triplophysa. In the present study, we applied PacBio sequencing and the Hi‐C technique to assemble the T. tibetana genome. A 652‐Mb genome with 1,325 contigs with an N50 length of 3.1 Mb was obtained. The 1,137 contigs were further assembled into 25 chromosomes, representing 98.7% and 80.47% of all contigs at the base and sequence number level, respectively. Approximately 260 Mb of sequence, accounting for ~39.8% of the genome, was identified as repetitive elements. DNA transposons (16.3%), long interspersed nuclear elements (12.4%) and long terminal repeats (11.0%) were the most repetitive types. In total, 24,372 protein‐coding genes were predicted in the genome, and ~95% of the genes were functionally annotated via a search in public databases. Using whole genome sequence information, we found that T. tibetana diverged from its common ancestor with Danio rerio ~121.4 million years ago. The high‐quality genome assembled in this work not only provides a valuable genomic resource for future population and conservation studies of T. tibetana, but it also lays a solid foundation for further investigation into the mechanisms of environmental adaptation of endemic fishes in the Tibetan Plateau.     

Detecting structural variations in the human genome using next generation sequencing

Structural variations are widespread in the human genome and can serve as genetic markers in clinical and evolutionary studies. With the advances in the next-generation sequencing technology, recent methods allow for identification of structural variations with unprecedented resolution and accuracy. They also provide opportunities to discover variants that could not be detected on conventional microarray-based platforms, such as dosage-invariant chromosomal translocations and inversions. In this review, we will describe some of the sequencing-based algorithms for detection of structural variations and discuss the key issues in future development.     

Purification and characterization of a β-1,3-glucanase from the novel mycoparasite <Emphasis Type="Italic">Periconia byssoides</Emphasis>

An extracellular β-1,3-glucanase with antifungal properties was secreted by the novel mycoparasite, Periconia byssoides. The glucanase has a molecular mass of 35 kDa estimated by SDS-PAGE. Its optimum activity was at pH 6.0 and 50°C (over 2 h). The purified β-1,3-glucanase was capable of degrading cell walls, and inhibiting mycelia growth and spore germination of plant pathogenic fungi including Fulvia fulva, Fusarium sp. and Rhizoctonia solani. The N-terminal amino acid residues of the purified β-1,3-glucanase are LKNGGPSFGA, which do not have any homology with previously described glucanases, suggesting it may be a novel member of the fungal β-1,3-glucanases. Chao Lin and Jinkui Yang contributed equally to this work.     

PQ-69, a novel and selective adenosine A1 receptor antagonist with inverse agonist activity

Potent and selective adenosine A₁ receptor (A₁AR) antagonists with favourable pharmacokinetic properties used as novel diuretics and antihypertensives are desirable. Thus, we designed and synthesized a series of novel 4-alkylamino substitution-2-arylpyrazolo[4,3-c]quinolin-3-one derivatives. The aim of the present study is to characterize the biological profiles of the optimized compound, PQ-69. In vitro binding assay revealed a K_i value of 0.96 nM for PQ-69 in cloned hA₁ receptor, which was 217-fold more selective compared with hA_2A receptors and >1,000-fold selectivity for hA₁ over hA₃ receptor. The results obtained from [³⁵S]-GTPγS binding and cAMP concentration assays indicated that PQ-69 might be an A₁AR antagonist with inverse agonist activity. In addition, PQ-69 displayed highly inhibitory activities on isolated guinea pig contraction (pA2 value of 8.99) induced by an A₁AR agonist, 2-chloro-N6-cyclopentyl adenosine. Systemic administration of PQ-69 (0.03, 0.3, 3 mg/kg) increased urine flow and sodium excretion in normal rats. Furthermore, PQ-69 displayed better metabolic stability in vitro and longer terminal elimination half-life (t_1/2) in vivo compared with 1,3-dipropyl-8-cyclopentylxanthine. These findings suggest that PQ-69 exhibits potent antagonist effects on A₁AR in vitro, ex vivo and in vivo, it might be a useful research tool for investigating A₁AR function, and it could be developed as a potential therapeutic agent.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9424-5) contains supplementary material, which is available to authorized users.