Mash1 efficiently reprograms rat astrocytes into neurons简

To date, it remains poorly understood whether astrocytes can be easily reprogrammed into neurons. Mashl and Brn2 have been previously shown to cooperate to reprogram fibroblasts into neurons. In this study, we examined astrocytes from 2-month-old Sprague-Dawley rats, and found that Brn2 was expressed, but Mashl was not detectable. Thus, we hypothesized that Mashl alone could be used to reprogram astrocytes into neurons. We transfected a recombinant MSCV-MASH1 plasmid into astrocytes for 72 hours, and saw that all cells expressed Mashl. One week later, we observed the changes in morphology of astrocytes, which showed typical neuro- nal characteristics. Moreover, β-tubulin expression levels were significantly higher in astrocytes expressing Mashl than in control cells. These results indicate that Mashl alone can reprogram astrocytes into neurons.     

Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells

Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid–bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.Plasmids serve as extrachromosomal DNA platforms for the reassortment, mobilization, and maintenance of antibiotic resistance genes in bacteria, enabling host cells to colonize environments flooded with antimicrobials and to take advantage of resources freed by the extinction of nonresistant competitors. Fueled by these selective forces and aided by their itinerant nature, plasmids disseminate resistance genes worldwide shortly after new antibiotics are developed, which is a major clinical concern (1–3). However, in antibiotic-free environments, such genes are dispensable. There, the cost that plasmid carriage imposes on cells constitutes a disadvantage in the face of competition from other cells and, because plasmids depend on their hosts to survive, also a threat to their own existence.Many plasmids keep low copy numbers (CNs) to minimize the problem above, because it reduces burdens to host cells. However, this also decreases their chances to fix in descendant cells, a new survival challenge (4). To counteract this, plasmids have evolved stability functions. Partition systems pull replicated plasmid copies to opposite poles in host cells, facilitating their inheritance by daughter cells (5). Plasmids also bear postsegregational killing (PSK) systems, which encode a stable toxin and a labile antitoxin (TA) pair that eliminates plasmid-free cells produced by occasional replication or partition failures. Regular production of the labile antitoxin protects plasmid-containing cells from the toxin. However, antitoxin replenishment is not possible in cells losing the plasmid, and this triggers their elimination (5).TA pairs are common in plasmids disseminating antibiotic resistance in bacterial pathogens worldwide (2, 6–10). The link of these systems to PSK and the exiguous list of alternatives in the pipeline have led some to propose that chemicals activating these TA pairs may constitute a powerful antibiotic approach against these organisms (5, 11–13). However, the appropriateness of these TA pairs as therapeutic targets requires unequivocal understanding of their function in plasmids. Although PSK systems encode TA pairs, not all TA pairs might function as PSK systems, as suggested by their abundance in bacterial chromosomes, where PSK seems unnecessary (14–16). Moreover, the observation that many plasmids bear several TA pairs (6–10) raises the intriguing question of why they would need more than one PSK system, particularly when they increase the metabolic burden that plasmids impose on host cells (17). Because PSK functions are not infallible, their gathering may provide a mechanism for reciprocal failure compensation, minimizing the number of cells that escape killing upon plasmid loss (5). Alternatively, some TA pairs may stabilize plasmids by mechanisms different from PSK, and their grouping might not necessarily reflect functional redundancy (18).This may be the case in plasmid R1, which encodes TA pairs hok-sok (host killing-suppressor of killing) and kis(pemI)-kid(pemK) (19–23). Inconsistent with PSK, we had noticed that activation of toxin Kid occurred in cells that still contained R1, and that this happened when CNs were insufficient to ensure plasmid transmission to descendant cells. We also found that Kid cleaved mRNA at UUACU sites, which appeared well suited to trigger a response that prevented plasmid loss and increased R1 CNs without killing cells, as suggested by our results. In view of all this, we argued that Kid and Kis functioned as a rescue system for plasmid R1, and not as a PSK system (24). This proposal cannot be supported by results elsewhere, suggesting that Kid may cleave mRNA at simpler UAH sites (with H being A, C, or U) (25, 26), a view that has prevailed in the literature (14, 16, 27–29). Moreover, other observations indicate that our past experiments may have been inappropriate to conclude that Kid does not kill Escherichia coli cells (30, 31). Importantly, Kid, Kis, and other elements that we found essential for R1 rescue are conserved in plasmids conferring resistance to extended-spectrum β-lactamases, a worrying threat to human health (1, 6–10, 32). Therapeutic options to fight pathogens carrying these plasmids are limited, and activation of Kid may be perceived as a good antibiotic alternative. Because the potential involvement of this toxin in plasmid rescue advises against such approach, we aimed to ascertain here the mode of action; the effects on cells; and, ultimately, the function of Kid (and Kis) in R1.     

链霉亲和素-自噬相关基因融合蛋白的原核表达及纯化

目的 构建链霉亲和素（streptavidin,SA）和自噬相关基因（Beclin 1）重组质粒,并对融合蛋白SA-Beclin 1进行表达和纯化.方法 利用基因重组技术将核心SA与Beclin 1序列连接,克隆人pQE80形成pQE80-SA-Beclin 1重组载体,IPTG诱导融合蛋白原核表达,镍亲和凝胶层析柱纯化融合蛋白,Western blot鉴定.结果 PCR成功扩增核心SA活性中心,酶切鉴定和测序均证实重组载体pQE80-SA-Beclin 1构建成功;IPTG诱导后SA-Beclin 1融合蛋白（含His标签）在大肠杆菌中高效表达,SDS-PAGE分析表达的融合蛋白以包涵体为主,经镍亲和凝胶层析柱纯化得到融合蛋白,Western blot鉴定其相对分子量约为72000kD,与预期相符.结论 本文成功构建了重组质粒并表达纯化了SA-Beclin 1融合蛋白,为进一步研究SA-Beclin 1的功能及临床应用奠定了基础.     

Trends and molecular characteristics of carbapenemase-producing Enterobacteriaceae in Japanese hospital from 2006 to 2015

BackgroundThe increasing number of carbapenemase-producing Enterobacteriaceae (CPE) has become a global problem. Most carbapenemases detected in Japan are imipenemase, which is an imipenem-degrading enzyme with low ability; thus, CPE could have been overlooked. Therefore, this study aimed to detect and analyze CPE, without overlooking CPE showing the low minimum inhibitory concentration phenotype.MethodsCPE screening was conducted on 531 ceftazidime-resistant Enterobacteriaceae isolated from Kitasato University Hospital during 2006–2015. We confirmed the presence of the carbapenemase genes (bla_IMP, bla_VIM, bla_KPC, bla_NDM, and bla_OXA-48) by multiplex polymerase chain reaction. The detected CPE strains were analyzed by antimicrobial susceptibility testing, multilocus sequence typing, conjugal experiments, replicon typing, and plasmid profiling by restriction enzyme treatment.ResultsThe CPE detection rate in Kitasato University Hospital within the past 10 years was 0.0003% (nine CPE strains). These nine CPE strains were identified to harbor 8 bla_IMP-1 or 1 bla_NDM-5. The CPE strains consisted of five species including Klebsiella pneumoniae and Citrobacter freundii. Six of eight bla_IMP-1 were coded by IncHI2 plasmid, and the other two were coded by IncA/C plasmid. Plasmid profiling revealed that K. pneumoniae and C. freundii isolated from the same patient harbored the same plasmid.ConclusionThe CPE detection rate in this study was significantly lower than those previously reported in Japan. In one case, IncA/C plasmid transmission through different bacterial species within the body was speculated. Although the number of CPE detected was low, these results indicated that the resistance plasmid could spread to other bacterial species.     

肠毒素质粒和耐药性质粒在动物体内接合传递试验

本试验用人源ETEC 7910、7922、7926、83067菌株作为供体菌,与受体菌K_(12)1485菌株,在乳鼠、小白鼠、兔肠段、地鼠和豚鼠体内进行肠毒素质粒和耐药质粒传递。结果证实在乳鼠、小白鼠和兔肠段内二种质粒可以共同传递给受体菌,受体菌获得质粒后,具有产肠毒素和耐药性表型。动物体内接合传递需要一定菌量和一定时间,根据动物大小而定,乳鼠用供、受体菌培养液各0.1ml,小白鼠各0.5～1.0ml较适宜,接合时间以24小时比2小时好。细菌质粒能在动物体内正常的条件下接合传递,将使非致病菌成为致病菌,致泻机制更复杂,使腹泻病的防治更加困难。     

High expression of human augmenter of liver regeneration in E. coli

Heat-stable hepatocyte stimulatory activity has been described in the liver of weanling rats and pigs. This growth factor is called hepatocyte stimulator substance (HSS)[1]. Hagiya et al[2]reported the complete amino acid sequence of a 30KDa band from their purified product of rat liver and the cloning and sequence analysis of its cDNA. They called it augmenter of liver regeneration (ALR). Our previous study[3]demonstrated that human ALR had been cloned and sequenced. To further study the bioactivity of human ALR (hALR), we constructed a highly expressed vector pBV-hALR in E. coli and about 20% of somatic protein of rhALR was expressed.     

VEGF-C基因靶向RNA干扰重组表达载体的构建和表达

目的:利用质粒pSilencer3.1-H_1构建针对人血管内皮生长因子-C(VEGF-c)基因的表达载体,测序鉴定并观察在胃癌细胞中的表达.方法:根据质粒pSilencer3.1-H_1要求设计两对小干扰RNA靶序列,退火形成互补的双链,通过与线性化的pSilencer3.0-H_1相应位点连接、转化大肠杆菌,扩增、纯化得到所需质粒,酶切电泳及测序鉴定后转染胃癌细胞株SGC-7901,Western blot检测转染前后VEGF-C基因的蛋白表达.结果:经酶切和测序鉴定,针对VEGF-C基因的siRNA表达载体构建成功.转染胃癌细胞株SGC-7901后,Western blot检测显示VEGF-C基因蛋白表达明显降低,pSilencer3.1-VEGF-C1组抑制效果明显,其抑制率为81.2%,与阴性对照组相比差异具有显著性(P<0.05).结论:成功构建了针对人VEGF-C基因的siRNA表达载体和稳定转染的胃癌细胞株SGC-7901.     

Effects of intramyocardial injection of phVEGF-A165 as sole therapy in patients with refractory coronary artery disease--12-month follow-up: angiogenic gene therapy

OBJECTIVE: To test the safety and bioactivity of phVEGF-A165 after intramyocardial injection during 12-month follow-up. DESIGN: Open-labelled study. SUBJECTS: Inclusion criteria were angina pectoris, Canadian Cardiovascular Society (CCS) class III-IV, unamenable to further revascularization, ejection fraction (EF) >30%, perfusion defects extending over >10% of the anterolateral left ventricle wall detectable with adenosine single photon emission computerized tomography (SPECT) and at least one patent vessel visible by coronary angiography. Seven of 39 patients referred for gene therapy were included. INTERVENTION: Via a mini-thoracotomy under general anaesthesia. phVEGF-A165 was injected directly into the myocardium at four sites in the anterolateral region of the left ventricle. RESULTS: Operative procedures were uneventful. Perioperative release of myocardial markers and electrocardiogram (ECG) changes were detected in two patients. There were no perioperative deaths but one patient died 7 months postoperatively because of myocardial infarction. Plasma vascular endothelial growth factor (VEGF)-A levels increased two to threefold peaking 6 days postoperatively (P < 0.004) and returning to baseline by day 30. A significant reduction in angina pectoris was reported. The CCS class improved from 3.3+/-0.2 to 1.9+/-0.3 (P < 0.01) and nitroglycerine intake decreased from 39+/-15 to 12+/-5 tablets week(-1) (P < 0.001) 2 months after gene transfer. Improvements remained after 12 months when nitroglycerine consumption approached zero. Improved myocardial function in the phVEGF-A165 injection region was documented in all patients (P < 0.016) by tissue velocity imaging (TVI). Reduced reversible ischaemia was detected by adenosine SPECT in four patients. Improved collateralization was detected in four patients with coronary angiography. CONCLUSION: Intramyocardial injection of phVEGF-A165 is safe and may lead to improved myocardial perfusion and function with longstanding symptomatic relief in end-stage angina pectoris. Based on these results this therapeutic potential is being tested in a double-blind placebo controlled multicentre trial.     

弓形虫表面抗原P30、P22与霍乱毒素A2／B复合基因真核表达质粒的构建

目的选择弓形虫 RH 株主要表面抗原 P30、P22的有效基因片段和霍乱毒素 A_2/B 亚基共同构建在同一真核表达载体 pcDNA3.1(-)上,并保证其连接方向和开放读码框的正确。方法用PCR 技术分别从弓形虫 RH 株基因组 DNA 和 pUAB024-CTXA_2/B 质粒中扩增编码 P30、P22基因片段和 CTXA_2/B,定向重组入 pUC18克隆载体,然后酶切释放 P30-P22-CTXA_2/B 复合基因片段,亚克隆入pcDNA3.1(-)真核表达载体,再经含氨苄青霉素的 LB 培养基筛选、酶切及测序鉴定。结果酶切产物经电泳显示条带清晰,P30、P22和 CTXA_2/B 基因片段的泳动位置分别在786bp、492bp、512bp 的位置,与预计结果一致;测序结果表明插入的基因片段方向及序列均正确。结论成功地构建了真核表达重组质粒 pcDNA3.1-P30-P22-CTXA_2/B,保证了三个基因连接方向,序列及开放读码框的正确,为下一步融合蛋白 P30-P22-CTXA_2/B 在哺乳动物细胞中的表达及动物实验奠定了基础。     

细粒棘球蚴2HSP70重组质粒的构建、原核表达、纯化和初步鉴定

目的构建细粒棘球蚴（Echinococcus granulosus，EG）2热休克蛋白70（heat shock protein，2HSPT0）表达重组质粒，原核表达及纯化重组蛋白，并对重组蛋白进行免疫学特性的初步鉴定。方法从EG2HSP70／pGEM-T／JM109质粒中分离EG2HSP70目的基因，将此片段重组入表达载体pGEX-6P-1后转化入大肠杆菌BL21，鉴定后以IPTG诱导表达，通过SDS-PAGE检测其表达水平，用亲和层析纯化并分离重组蛋白，利用Western blot来鉴定重组蛋白的免疫学特性。结果酶切鉴定及测序分析表明，成功构建细粒棘球蚴pGEX-6P-1／EG2HSP70重组质粒；IPTG诱导表达后，融合蛋白占菌体总蛋白的39％，占裂解物上清总蛋白的70．4%，表明重组的EG2HSP70基因能在BL21中高效表达，表达的蛋白大部分以可溶性状态存在。亲和层析法纯化重组蛋白EG2HSP70，通过Western blot证实该重组蛋白能被细粒棘球蚴囊壁免疫兔血清识别。结论成功构建细粒棘球蚴中国大陆株pGEX-6P-1／EG2HSP70重组质粒，重组的EG2HSP70能够高效表达，表达的EG2HSP70具有免疫学活性。