Generation of a purely clonal defective interfering influenza virus

Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus‐based antivirals.     

Controlled release of adeno-associated virus from alginate hydrogel microbeads with enhanced sensitivity to ultrasound

Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l -lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.     

Signal transduction pathways in guinea pig sperm

Trifluoperazine (TFP), the antagonist of calmodulin (CaM). significantly stimulated the capacitation and acrosome reaction of guinea pig spermatozoa at the concentration of 10-100μmol/L, independent of the external Ca2+. Forskolin, dbcAMP and caffeine evidently promoted the occurrence of acrosome reaction of spermatozoa at early capacitation stage (5 h) in nonsynchronous system but not in synchronous system. If the spermatozoa were capacitated for 15 h in synchronous system, the above three drugs significantly stimulated acrosome reaction in a Ca2+-independent manner. Protein kinase C activators, i.e. phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDB) did not influence the occurrence of acrosome reaction of spermatozoa at early capacitation stage, but significantly increased the acrosome reaction rate in capacitated spermatozoa in a Ca2+-independent manner. In contrast. PKC inhibitor staurosporine significantly inhibited the occurrence of acrosome reaction.     

Anatomy of pharynx in patients with obstructive sleep apnea and in normal subjects

Isono, Shiroh, John E Remmers, Atsuko Tanaka, Yasuhide Sho,Jiro Sato, and Takashi Nishino. Anatomy of pharynx in patients with obstructive sleep apnea and in normal subjects.J. Appl. Physiol. 82(4):1319-1326, 1997.Anatomic abnormalities of the pharynx arethought to play a role in the pathogenesis of obstructive sleep apnea(OSA), but their contribution has never been conclusively proven. Thepresent study tested this anatomic hypothesis by comparing themechanics of the paralyzed pharynx in OSA patients and in normalsubjects. According to evaluation of sleep-disordered breathing (SDB)by nocturnal oximetry, subjects were divided into three groups: normalgroup (n = 17), SDB-1(n = 18), and SDB-2(n = 22). The static pressure-arearelationship of the passive pharynx was quantified under generalanesthesia with complete paralysis. Age and body mass index werematched among the three groups. The site of the primary closure was thevelopharynx in 49 subjects and the oropharynx in only 8 subjects.Distribution of the location of the primary closure did not differamong the groups. Closing pressure(PC) of the velopharynx forSDB-1 and SDB-2 groups (0.90 ± 1.34 and 2.78 ± 2.78 cmH₂O, respectively) wassignificantly higher than that for the normal group (3.77 ± 3.44 cmH₂O;P < 0.01). Maximal velopharyngealarea for the normal group (2.10 ± 0.85 cm²) was significantly greaterthan for SDB-1 and SDB-2 groups (1.15 ± 0.46 and 1.06 ± 0.75 cm², respectively). Theshape of the pressure-area curve for the velopharynx differed betweennormal subjects and patients with SDB, being steeper in slope nearPC in patients with SDB.Multivariate analysis of mechanical parameters and oxygen desaturationindex (ODI) revealed that velopharyngealPC was the only variable highly correlated with ODI. VelopharyngealPC was associated withoropharyngeal PC, suggestingmechanical interdependence of these segments. We conclude that thepassive pharynx is more narrow and collapsible in sleep-apneic patientsthan in matched controls and that velopharyngeal PC is the principal correlate ofthe frequency of nocturnal desaturations.

     

Combined tazemetostat and MAPKi enhances differentiation of papillary thyroid cancer cells harbouring BRAFV600E by synergistically decreasing global trimethylation of H3K27

Clinical efficacy of differentiation therapy with mitogen-activated protein kinase inhibitors (MAPKi) for lethal radioiodine-refractory papillary thyroid cancer (RR-PTC) urgently needs to be improved and the aberrant trimethylation of histone H3 lysine 27 (H3K27) plays a vital role in BRAF^V600E-MAPK-induced cancer dedifferentiation and drug resistance. Therefore, dual inhibition of MAPK and histone methyltransferase (EZH2) may produce more favourable treatment effects. In this study, BRAF^V600E-mutant (BCPAP and K1) and BRAF-wild-type (TPC-1) PTC cells were treated with MAPKi (dabrafenib or selumetinib) or EZH2 inhibitor (tazemetostat), or in combination, and the expression of iodine-metabolizing genes, radioiodine uptake, and toxicity were tested. We found that tazemetostat alone slightly increased iodine-metabolizing gene expression and promoted radioiodine uptake and toxicity, irrespective of the BRAF status. However, MAPKi induced these effects preferentially in BRAF^V600E mutant cells, which was robustly strengthened by tazemetostat incorporation. Mechanically, MAPKi-induced decrease of trimethylation of H3K27 was evidently intensified by tazemetostat in BRAF^V600E-mutant cells. In conclusion, tazemetostat combined with MAPKi enhances differentiation of PTC cells harbouring BRAF^V600E through synergistically decreasing global trimethylation of H3K27, representing a novel differentiation strategy.     

Involvement of Putrescine,Nitric Oxide,and Hydrogen Peroxide in Methyl Jasmonate-Induced Ginsenoside Synthesis in Adventitious Root Cultures of Panax ginseng C.A. Meyer

Journal of Plant Growth Regulation - The adventitious root (AR) culture of Panax ginseng C.A. Meyer is an alternative route for mass production of ginsenosides. During the AR culture of P. ginseng,...     

Integrative genomic phylogeography reveals signs of mitonuclear incompatibility in a natural hybrid goby population

Hybridization between divergent lineages generates new allelic combinations. One mechanism that can hinder the formation of hybrid populations is mitonuclear incompatibility, that is, dysfunctional interactions between proteins encoded in the nuclear and mitochondrial genomes (mitogenomes) of diverged lineages. Theoretically, selective pressure due to mitonuclear incompatibility can affect genotypes in a hybrid population in which nuclear genomes and mitogenomes from divergent lineages admix. To directly and thoroughly observe this key process, we de novo sequenced the 747‐Mb genome of the coastal goby, Chaenogobius annularis, and investigated its integrative genomic phylogeographics using RNA‐sequencing, RAD‐sequencing, genome resequencing, whole mitogenome sequencing, amplicon sequencing, and small RNA‐sequencing. Chaenogobius annularis populations have been geographically separated into Pacific Ocean (PO) and Sea of Japan (SJ) lineages by past isolation events around the Japanese archipelago. Despite the divergence history and potential mitonuclear incompatibility between these lineages, the mitogenomes of the PO and SJ lineages have coexisted for generations in a hybrid population on the Sanriku Coast. Our analyses revealed accumulation of nonsynonymous substitutions in the PO‐lineage mitogenomes, including two convergent substitutions, as well as signals of mitochondrial lineage‐specific selection on mitochondria‐related nuclear genes. Finally, our data implied that a microRNA gene was involved in resolving mitonuclear incompatibility. Our integrative genomic phylogeographic approach revealed that mitonuclear incompatibility can affect genome evolution in a natural hybrid population.