Loss of viability during freeze–thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to␣apoptosis rather than cellular necrosis

Summary A major challenge in the widespread application of human embryonic stem (hES) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of <5% as reported by previous studies. This study characterized cell death within frozen–thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (≈98%), as assessed by the trypan blue exclusion test. However, when the freshly-thawed hES colonies were incubated within a 37 °C incubator, there was observed to be a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed-down by keeping the freshly-thawed hES colonies at 4 °C, with >90% of cells remaining viable after 90 min of incubation at 4 °C. This effect was reversible upon re-exposing the cells to physiological temperature. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37 °C. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay confirmed apoptosis-induced nuclear DNA fragmentation in frozen–thawed hES cells after incubation at 37 °C for 90 min. Expression of active caspase-3 enzyme, which is another prominent marker of apoptosis, was confirmed by immunocytochemical staining, while transmission electron microscopy showed typical ultrastructural features of apoptosis such as chromatin condensation and margination to the nuclear membrane. Hence, our results demonstrated that apoptosis instead of cellular necrosis, is the major mechanism of the loss of viability of cryopreserved hES cells during freeze–thawing with conventional slow-cooling protocols.     

Genetic diversity of phenazine- and pyoluteorin-producing pseudomonads isolated from green pepper rhizosphere

The genetic diversity among indigenous phenazine-1-carboxylic acid (PCA)-producing and pyoluteorin (Plt)-producing isolates of pseudomonads screened from green pepper rhizosphere was exploited in this study. A total of 48 bacterium isolates producing one or both of these antibiotics were screened from green pepper rhizosphere in diverse regions in China. Among these isolates, 45 could produce PCA, 3 could produce both PCA and Plt, and none could produce Plt only. Based on the restriction patterns of partial 16S and 16S-23S internal transcribed spacer (ITS) PCR fragments generated by enzyme HaeIII or HinfI, these isolates fell into 19 or 17 distinct groups respectively, indicating that there was a significant diversity among them. Polygenetic analysis of the partial 16S rDNA and 16S-23S ITS sequence from the representative in each group in the context of similar sequence from previously described bacterial species indicated that most isolates were closely related to the species of Pseudomonas fluorescens, P. putida, and Stenotrophomonas maltophilia. Some of these representatives of these isolates, then, are likely to be novel strains or species in these two genera. The GenBank accession numbers for DNA sequences of the partial 16S rDNA with ITS region in each isolate determined in this study were: GP30 DQ003219; GP127 DQ003220; GP83 DQ003221; GP42, DQ003222; GP59 DQ003223; GP50 DQ003224; GP36 DQ003225; GP110 DQ003226; GP26 DQ003227; GP37 DQ003228; GP60 DQ003229; GP31 DQ003230; GP57 DQ003231; GP75 DQ003232; GP115 DQ003233; GP65 DQ003234; GP32 DQ003235; GP76 DQ003236; GP78 DQ003237.     

麻疯树脂酶全长基因克隆、表达及其蛋白质结构预测

脂酶(Lipase,EC3.1.1.3)是普遍应用于皮革、饲料及生物柴油工业的工业酶制剂,具有广泛的应用价值。目前对植物来源的脂酶研究较少。本研究用在生物柴油中具有应用前景的油料植物——麻疯树(Jatrophacurcas)作为研究对象,克隆了该物种的脂酶基因(JcLIP)。通过多序列比对并结合物种的亲缘关系设计了具有较高特异性的简并引物,通过使用RT-PCR和RACE技术,最终获得了麻疯树脂酶基因的全长序列并成功地在大肠杆菌中表达,酶活测定结果表明,麻疯树脂酶在大肠杆菌中表达在包涵体中,但是能产生具有活力的蛋白质,酶活约为0.8U.mL-1。结构预测和比较表明,JcLIP蛋白质具有脂酶的结构核心和催化活性中心,而在非核心区具有较毛霉脂酶更多的插入和随机卷曲,这可能是决定二者之间酶活差异的重要原因。     

Novel potent agonist [(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 and antagonist [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 of nociceptin/orphanin FQ receptor

Two novel ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), [(pF)Phe4,Aib7, Aib11,Arg14,Lys15]N/OFQ-NH2 (peptide-1) and [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 (peptide-2), have been generated by combining different modifications of N/OFQ sequence. In the present study, we investigated the actions of two analogues and compared them with those of N/OFQ in four assays. Peptide-1 mimicked N/OFQ effects in mouse vas deferens and mouse colon and showed similar maximal effects but higher potency relative to N/OFQ. The effects of peptide-1 were sensitive to NOP receptor selective antagonist ([Nphe1]N/OFQ(1-13)-NH2) but not to naloxone in vitro. Peptide-1 (25 pmol, i.c.v.) mimicked the pronociceptive action of N/OFQ (2.5 nmol, i.c.v.) in mouse tail withdrawal assay, displaying higher potency and longer lasting effects. In anesthetized rats, peptide-1 (1 nmol/kg, i.v.) produced a marked decrease in mean arterial pressure, which was comparable to that evoked by i.v. N/OFQ (100 nmol/kg). Peptide-2 did not produce any effect per se but antagonized N/OFQ actions in mouse vas deferens and mouse colon assays. Peptide-2 is active in vivo where it prevented the pronociceptive effect induced by 2.5 nmol N/OFQ i.c.v. in the mouse tail withdrawal assay. Furthermore, peptide-2 at 5 nmol produced alone a robust and long lasting antinociceptive effect. Moreover, peptide-2 (10 and 40 nmol/kg i.v.) didn't produce any effect per se but antagonized hypotensive actions produced by i.v. administration of N/OFQ. Collectively, these findings demonstrate that [(pF)Phe4,Aib7,Aib11, Arg14,Lys15]N/OFQ-NH2 behaves as a highly potent NOP receptor agonist which produces long lasting effects in vivo and [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 acts as a pure and competitive antagonist of the NOP receptor.     

Bt rice expressing Cry1Ab does not stimulate an outbreak of its non-target herbivore, Nilaparvata lugens

In this study, the non-target effects of Bt rice “KMD2” expressing a Cry1Ab protein on the performance of the brown planthopper (BPH), Nilaparvata lugens, over multiple generations were evaluated under laboratory and field conditions. In the laboratory, BPH was reared to observe the impact of the Bt rice as compared to its parental non-Bt cultivar Xiushui 11, while the population dynamics and oviposition performance of BPH were investigated in the field. The survival of BPH nymphs fed Bt and non-Bt rice did not differ significantly. The nymph developmental duration of BPH was significantly delayed by the Bt rice by comparison with the non-Bt rice for the 1st and 2nd but not the 4th generation. Most importantly, the fecundity of BPH on the Bt rice was significantly decreased in every generation when compared with the non-Bt rice. In the field investigations, the population density of BPH nymphs was significantly lower in the Bt rice field. However, the temporal pattern of population dynamics of BPH adults was similar between the Bt and non-Bt rice, presumably due to migratory interference of the adults. In the Bt rice field, the percentage of tillers with eggs and the number of eggs per tiller were also significantly lower from tillering to mature stage. Additionally, Cry1Ab protein could not be detected in guts from single BPH adults. In general, our results suggest that the Bt rice “KMD2” could not stimulate an outbreak of BPH.     

杂交试验中的最佳样本含量

杂交试验是一项费时费钱的工作,因此在进行试验之前如能进行严密的设计,给出试验所需的样本大小是十分必要的,统计学中常见的估计样本容理的公式不宜应用于杂交试验,本文分两种情况给出了杂交试验中样本容量的估计公式,据此估计出的样本容量安排杂交试验,可在满足试验者要求的条件下,使试验的总成本最低或使试畜的总头数最少。     

Non-contiguous finished genome sequence of Anoxybacillus flavithermus subsp. yunnanensis type strain (E13T), a strictly thermophilic and organic solvent-tolerant bacterium

Anoxybacillus flavithermus subsp. yunnanensis is the only strictly thermophilic bacterium that is able to tolerate a broad range of toxic solvents at its optimal temperature of 55-60°C. The type strain E13^T was isolated from water-sediment slurries collected from a hot spring. This study presents the draft genome sequence of A. flavithermus subsp. yunnanensis E13^T and its annotation. The 2,838,393bp long genome (67 contigs) contains 3,035 protein-coding genes and 85 RNA genes, including 10 rRNA genes, and no plasmids. The genome information has been used to compare with the genomes from A. flavithermus subsp. flavithermus strains.     

Comparison of the Accuracy of Two Conventional Phenotypic Methods and Two MALDI-TOF MS Systems with That of DNA Sequencing Analysis for Correctly Identifying Clinically Encountered Yeasts

We assessed the accuracy of species-level identification of two commercially available matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems (Bruker Biotyper and Vitek MS) and two conventional phenotypic methods (Phoenix 100 YBC and Vitek 2 Yeast ID) with that of rDNA gene sequencing analysis among 200 clinical isolates of commonly encountered yeasts. The correct identification rates of the 200 yeast isolates to species or complex (Candida parapsilosis complex, C. guilliermondii complex and C. rugosa complex) levels by the Bruker Biotyper, Vitek MS (using in vitro devices [IVD] database), Phoenix 100 YBC and Vitek 2 Yeast ID (Sabouraud''s dextrose agar) systems were 92.5%, 79.5%, 89%, and 74%, respectively. An additional 72 isolates of C. parapsilosis complex and 18 from the above 200 isolates (30 in each of C. parapsilosis, C. metapsilosis, and C. orthopsilosis) were also evaluated separately. Bruker Biotyper system could accurately identify all C. parapsilosis complex to species level. Using Vitek 2 MS (IVD) system, all C. parapsilosis but none of C. metapsilosis, or C. orthopsilosis could be accurately identified. Among the 89 yeasts misidentified by the Vitek 2 MS (IVD) system, 39 (43.8%), including 27 C. orthopsilosis isolates, could be correctly identified Using the Vitek MS Plus SARAMIS database for research use only. This resulted in an increase in the rate of correct identification of all yeast isolates (87.5%) by Vitek 2 MS. The two species in C. guilliermondii complex (C. guilliermondii and C. fermentati) isolates were correctly identified by cluster analysis of spectra generated by the Bruker Biotyper system. Based on the results obtained in the current study, MALDI-TOF MS systems present a promising alternative for the routine identification of yeast species, including clinically commonly and rarely encountered yeast species and several species belonging to C. parapsilosis complex, C. guilliermondii complex, and C. rugosa complex.