Differential susceptibility to hydrogen sulfide-induced apoptosis between PHLDA1-overexpressing oral cancer cell lines and oral keratinocytes: Role of PHLDA1 as an apoptosis suppressor

Hydrogen sulfide (H₂S) is a novel gasotransmitter that plays multiple biological roles in various body systems. In addition to its endogenous production, H₂S is produced by bacteria colonizing digestive organs, including the oral cavity. H₂S was previously shown to enhance pro-apoptotic effects in cancer cell lines, although the mechanisms involved remain unclear. To properly assess the anti-cancer effects of H₂S, however, investigations of apoptotic effects in normal cells are also necessary. The aims of this study were (1) to compare the susceptibility to H₂S-induced apoptosis between the oral cancer cell line Ca9-22 and oral keratinocytes that were derived from healthy gingiva, and (2) to identify candidate genes involved in the induction of apoptosis by H₂S. The susceptibility to H₂S-induced apoptosis in Ca9-22 cells was significantly higher than that in keratinocytes. H₂S exposure in Ca9-22 cells, but not keratinocytes, enhanced the expression of pleckstrin homology-like domain, family A, member 1 (PHLDA1), which was identified through a differential display method. In addition, PHLDA1 expression increased during actinomycin D-induced apoptosis in Ca9-22 cells. Knockdown of PHLDA1 expression by small interfering RNA in Ca9-22 cells led to expression of active caspase 3, thus indicating apoptosis induction. The tongue cancer cell line SCC-25, which expresses PHLDA1 at a high level, showed similar effects. Our data indicate that H₂S is an anti-cancer compound that may contribute to the low incidence of oral cancer. Furthermore, we demonstrated the role of PHLDA1 as an apoptosis suppressor.     

Efficiency in cloning and sequencing using the single-stranded bacteriophage M13

A number of approaches to sequence DNA by the chain termination method are based on cloning into M13 phage vectors and the use of a universal primer. In this paper we investigate some of the factors which influence the speed and efficiency of these approaches. A modification of the template preparation, sequencing reaction, and gel system was used to obtain more reliable and clearer ladder gels. Redundancy and deficiency of shotgun DNA sequencing were reduced by a mapping technique and the use of synthetic primers. The mapping and cloning technique was used to organize the data entry so that the computer time necessary to reconstruct the sequence out of overlaps was reduced.     

Low genetic variation in muskoxen (Ovibos moschatus) from western Greenland using microsatellites

Muskoxen are large herbivores living in Arctic environments. Lack of genetic variation in allozymes has made it difficult to study the social and genetic structure of this species. In this study, we have tried to find polymorphic microsatellite loci using both cattle-derived microsatellite primers and primers developed from a genomic plasmid library of muskoxen. Only limited variation was found for both sets of microsatellite loci. We conclude that this consistent low genetic variation is probably due to demographic features of the muskoxen populations rather than to methodological constraints caused by the transfer of microsatellites between species.     

Multiplex PCR system applied for analysing microsatellite loci of Schlegel's black rockfish,Sebastes schlegeli

Two multiplex PCR amplifications were performed to analyse six microsatellite loci of Schlegel's black rockfish, Sebastes schlegeli, an important commercial fish in the northern part of Japan and an important species for the stock enhancement program in this area. We analysed 67 wild samples from Yamada Bay, Iwate Prefecture, Japan. The observed genotype frequencies agreed with the Hardy–Weinberg expectations at all loci, and the observed heterozygosities ranged from 0.072 to 0.897.     

The Effect of Divalent Cations and Core Polymerase in the Abortive Initiation by Escherichia Coli Dna-Dependent Rna Polymerase Using Phosphonate Dinucleotide Primers

The effect of divalent Mg²⁺ and Mn²⁺ cations on the elongation of ApU, UpA and their 3'-O- and 5'-O-phosphonylmethyl analogues by RNA polymerase holoenzyme to the corresponding trinucleo-tides on a poly(dA-dT) template was investigated. In contrast to Mg^z+ ions, Mn²⁺ ions enhance abortive trinucleotide synthesis. This effect is more pronounced with phosphonylmethyl analogues. The core enzyme cannot catalyze the elongation of either (2'-5') UpA or phosphonylmethyl analogues. The localization of the divalent cation activator, as well as the role of the σ subunit at the catalytic centre of the holoenzyme, is discussed.     

pr2-primers: An 18S rRNA primer database for protists

Metabarcoding of microbial eukaryotes (collectively known as protists) has developed tremendously in the last decade, almost solely relying on the 18S rRNA gene. As microbial eukaryotes are extremely diverse, many primers and primer pairs have been developed. To cover a relevant and representative fraction of the protist community in a given study system, an informed primer choice is necessary, as no primer pair can target all protists equally well. As such, a smart primer choice is very difficult even for experts and there are very few online resources available to list existing primers. We built a database listing 285 primers and 83 unique primer pairs that have been used for eukaryotic 18S rRNA gene metabarcoding. In silico performance of primer pairs was tested against two sequence databases: PR² version 4.12.0 for eukaryotes and a subset of silva version 132 for bacteria and archaea. We developed an R -based web application enabling browsing of the database, visualization of the taxonomic distribution of the amplified sequences with the number of mismatches, and testing any user-defined primer or primer set ( https://app.pr2-primers.org ). Taxonomic specificity of primer pairs, amplicon size and location of mismatches can also be determined. We identified universal primer sets that matched the largest number of sequences and analysed the specificity of some primer sets designed to target certain groups. This tool enables guided primer choices that will help a wide range of researchers to include protists as part of their investigations.     

Long‐term stability of entomopathogenic nematode spatial patterns in soil as measured by sentinel insects and real‐time PCR assays

Quantitative real‐time PCR (qPCR) techniques are being increasingly used to provide accurate and reliable methods to identify and quantify cryptic organisms in soil ecology. Entomopathogenic nematode (EPN) diversity in Florida is known to be extensive and our phylogenetic studies of the D2D3 and ITS regions showed the occurrence of an additional species‐complex in the Steinernema glaseri‐ group in widely separated locations of the peninsula. To address ecological studies, we developed and used qPCR assays to detect and quantify six species of EPN that are naturally distributed in Florida citrus orchards (Steinernema diaprepesi, Steinernema riobrave, Heterorhabditis indica, Heterorhabditis zealandica, Heterorhabditis floridensis and an undescribed species in the S. glaseri group) and an exotic species, S. glaseri. Species‐specific primers and TaqMan® probes were designed from the ITS rDNA region. No nonspecific amplification was observed in conventional or qPCR when the primers and probes were tested using several populations of each of the Florida species and other exotic EPN species. Standard curves were established using DNA from pure cultures. We optimised a protocol for extracting nematodes and DNA from soil samples that can detect one EPN added to nematode communities recovered by conventional extraction protocols. A survey of an 8‐ha orchard in April 2009 compared the EPN spatial patterns derived from qPCR to that obtained by baiting soil samples with Galleria mellonella larvae. The patterns were also compared to those derived from the same site in 2000–01 by repeatedly (12 sampling events) baiting soil in situ with caged larvae of the root weevil Diaprepes abbreviatus. The qPCR assay was more efficient than the Galleria baiting method for detecting the EPN species composition in population mixtures. Moreover, the spatial patterns of EPN in this orchard were remarkably stable over the course of nearly a decade. The pattern of H. zealandica detected at the site 8 years earlier was related to those derived by qPCR (P = 0.002) and from sample baiting (P = 0.02). The spatial pattern of H. indica derived from qPCR, but not that from sample baiting, was also related to the earlier pattern (P = 0.01). The qPCR assay developed here is a fast, affordable and accurate method to detect and quantify these EPN species in soil and offers great potential for studying the ecology of EPN.     

小桐子甘油-3-磷酸酰基转移酶（JcGPAT）cDNA的克隆与序列分析

甘油-3-磷酸酰基转移酶是植物生物合成储存油脂过程中的关键酶,对油料作物种子含油量具有重要的限制作用。本研究以植物甘油-3-磷酸酰基转移酶同源基因的保守区域序列为基础,设计简并引物,结合RACE技术,从能源植物小桐子种子中克隆获得JcGPAT基因的cDNA全长序列（GenBank登录号HQ395225）。JcGPAT cDNA核苷酸序列长度为1672bp,开放阅读框为1125bp,编码375个氨基酸。该基因具有明显的GPAT基因结构域,其编码的氨基酸序列与油桐、蓖麻等植物具有很高的同源性。RT-PCR表达分析表明,该基因在小桐子发育的种子、叶、根尖等多个组织表达。     

Microeukaryotes in animal and plant microbiomes: Ecologies of disease?

Studies of animal and plant microbiomes are burgeoning, but the majority of these focus on bacteria and rarely include microeukaryotes other than fungi. However, there is growing evidence that microeukaryotes living on and in larger organisms (e.g. plants, animals, macroalgae) are diverse and in many cases abundant. We present here a new combination of ‘anti-metazoan’ primers: 574*f–UNonMet_DB that amplify a wide diversity of microeukaryotes including some groups that are difficult to amplify using other primer combinations. While many groups of microeukaryotic parasites are recognised, myriad other microeukaryotes are associated with hosts as previously unknown parasites (often genetically divergent so difficult to amplify using standard PCR primers), opportunistic parasites, commensals, and other ecto- and endo-symbionts, across the ‘symbiotic continuum'. These fulfil a wide range of roles from pathogenesis to mutually beneficial symbioses, but mostly their roles are unknown and likely fall somewhere along this spectrum, with the potential to switch the nature of their interactions with the host under different conditions. The composition and dynamics of host-associated microbial communities are also increasingly recognised as important moderators of host health. This ‘pathobiome’ approach to understanding disease is beginning to supercede a one-pathogen-one-disease paradigm, which cannot sufficiently explain many disease scenarios.