Metabolic engineering of Corynebacterium glutamicum for increasing the production of l-ornithine by increasing NADPH availability

The experiments presented here were based on the conclusions of our previous proteomic analysis. Increasing the availability of glutamate by overexpression of the genes encoding enzymes in the l-ornithine biosynthesis pathway upstream of glutamate and disruption of speE, which encodes spermidine synthase, improved l-ornithine production by Corynebacterium glutamicum. Production of l-ornithine requires 2 moles of NADPH per mole of l-ornithine. Thus, the effect of NADPH availability on l-ornithine production was also investigated. Expression of Clostridium acetobutylicum gapC, which encodes NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and Bacillus subtilis rocG, which encodes NAD-dependent glutamate dehydrogenase, led to an increase of l-ornithine concentration caused by greater availability of NADPH. Quantitative real-time PCR analysis demonstrates that the increased levels of NADPH resulted from the expression of the gapC or rocG gene rather than that of genes (gnd, icd, and ppnK) involved in NADPH biosynthesis. The resulting strain, C. glutamicum ΔAPRE::rocG, produced 14.84 g l^?1 of l-ornithine. This strategy of overexpression of gapC and rocG will be useful for improving production of target compounds using NADPH as reducing equivalent within their synthetic pathways.     

蜡状芽孢杆菌S458-1对水产养殖系统中水质调节的作用

【目的】水产养殖过程中蜡状芽孢杆菌(Bacillus cereus)作为益生菌被广泛应用。本研究旨在深入了解分离于养殖水体中的一株蜡状芽孢杆菌S458-1对养殖水体以及养殖对象的影响,以期为菌株S458-1在水产养殖生产上的应用提供理论依据。【方法】根据控制变量法,各试验组鲫鱼养殖模式设置相同温度、盐度、溶氧和pH,利用分光光度法测定水体的水化学指标;利用试剂盒法测定鲫鱼的血清生理生化指标。【结果】添加菌株S458-1处理组(终浓度分别为10~6、10~7、10~8CFU/mL)与对照组(未加S458-1菌)相比:水体活性磷浓度显著性增加(P0.05);同时具有把NH_4~+-N和NO_2~–-N转化为NO_3~–-N的趋势,但无显著性差异(P0.05);养殖鲫鱼血清检测结果显示谷草转氨酶(GOT)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)以及丙二醛(MDA)含量均显著降低(P0.05)。【结论】蜡状芽孢杆菌S458-1具有脱氮解磷、调节水质的作用,并可增强养殖对象的生理健康状态,可作为益生菌应用于水产养殖中,具有较高的应用价值。     

Characterisation of the complete mitochondrial genome of the black-footed abalone Haliotis iris

The complete mitogenome of Haliotis iris, an economically important shellfish endemic to New Zealand, was sequenced for the first time. The mitogenome was 17,131?base pairs (bp) in length and contained 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes and a control region. All 13 genes were initiated by the start codon ATG, except for nad5 (ATA). Two typical stop codons, TAA and TAG, were present. All of the tRNAs could be folded into typical cloverleaf secondary structures except tRNA^Ser1 and tRNA^Lys, which lacked a DHU stem and complete amino acid acceptor stem, respectively. The control region was 1132?bp in length and contained six AT tandem repeats. According to the gene order of the mitogenome, the 30 analysed Vetigastropoda species could be classified into three types—type I: over half of the studied species were very similar to the gastropod ancestral gene order, and the rearrangements occurred in five tRNAs; type II: eight species were found to be missing several tRNA genes; type III: Fissurellidae, Lepetodrilidae showed a large inverted fragment.     

Ionizing-radiation-induced damage in the DNA of cultured human cells. Identification of 8,5-cyclo-2-deoxyguanosine.

Epstein-Barr-virus-transformed peripheral-blood B-lymphocytes were gamma-irradiated at 0 degree C at doses from 10 to 100 Gy. The cells were immediately lysed and the DNA was isolated. Subsequently, the DNA was hydrolysed to 2'-deoxyribonucleosides with a mixture of DNAase I, venom and spleen exonucleases and alkaline phosphatase. The hydrolysate was dried, trimethylsilylated and analysed by capillary gas chromatography-mass spectrometry with selected-ion monitoring. The (5'R)- and (5'S)-diastereomers of 8,5'-cyclo-2'-deoxyguanosine were observed in a ratio of 1:3, and their formation was dose-dependent. It was possible to detect and characterize one such lesion in approx. 4 X 10(4) guanine nucleotide subunits of DNA.     

Pathway phase waveform characteristics correlated with length and rate of stylet advancement and partial stylet withdrawal in AC electrical penetration graphs of adult whiteflies

A technique was developed for measuring the length of stylet insertion during adult whitefly probing. The distance that the labium shortens during a probe was shown to be equal to the length of stylets that were inserted into the plant tissue. The length of labial shortening then was measured in high-magnification video recordings of adult female silverleaf whitefly, Bemisia argentifolii, in conjunction with recording electrical penetration graphs (EPGs – AC method). Using a split-screen device, video images of the whitefly's labium during a probe and the EPG waveforms produced during the probe were recorded simultaneously on the same video tape. On playback, changes in labial length could be measured during specific EPG waveforms to determine the length of stylet insertion that occurred during the waveforms. The focus of the study was on two characteristics of the pathway phase sawtooth waveform: the frequency of voltage peaks and the increase in voltage level that occurs over time during sawtooth waveforms. The rate of stylet penetration was significantly and positively correlated with frequency of sawtooth waveform voltage peaks (r ²=0.33) and the length of stylet penetration was significantly and positively correlated (second-order polynomial) with the relative difference in voltage level between the beginning and end of the sawtooth waveform (r ²=0.43). Stylet advancement did not appear to occur during the few low-flat waveforms (unknown behavioral correlation) and high-flat waveforms (phloem phase) that were observed. Voltage drops occur sporadically during sawtooth waveforms, and these were associated with partial stylet withdrawal (indicated when the labium increased in length, but the probe was not terminated) with an accuracy of 99%.     

High-sensitive electrochemical detection of point mutation based on polymerization-induced enzymatic amplification

Here a highly sensitive electrochemical method is described for the detection of point mutation in DNA. Polymerization extension reaction is applied to specifically initiate enzymatic electrochemical amplification to improve the sensitivity and enhance the performance of point mutation detection. In this work, 5'-thiolated DNA probe sequences complementary to the wild target DNA are assembled on the gold electrode. In the presence of wild target DNA, the probe is extended by DNA polymerase over the free segment of target as the template. After washing with NaOH solution, the target DNA is removed while the elongated probe sequence remains on the sensing surface. Via hybridizing to the designed biotin-labeled detection probe, the extended sequence is capable of capturing detection probe. After introducing streptavidin-conjugated alkaline phosphatase (SA-ALP), the specific binding between streptavidin and biotin mediates a catalytic reaction of ascorbic acid 2-phosphate (AA-P) substrate to produce a reducing agent ascorbic acid (AA). Then the silver ions in solution are reduced by AA, leading to the deposition of silver metal onto the electrode surface. The amount of deposited silver which is determined by the amount of wild target can be quantified by the linear sweep voltammetry (LSV). The present approach proved to be capable of detecting the wild target DNA down to a detection limit of 1.0×10(-14) M in a wide target concentration range and identifying -28 site (A to G) of the β-thalassemia gene, demonstrating that this scheme offers a highly sensitive and specific approach for point mutation detection.     

Short exposure to paclitaxel induces multipolar spindle formation and aneuploidy through promotion of acentrosomal pole assembly

Paclitaxel is a widely used microtubule drug and cancer medicine. Here we report that by short exposure to paclitaxel at a low dose, multipolar spindles were induced in mitotic cells without centrosome amplification. Both TPX2 depletion and Aurora-A overexpression antagonized the multipolarity. Live cell imaging showed that some paclitaxel-treated cells accomplished multipolar cell division and a portion of the daughter cells went on to the next round of mitosis. The surviving cells grew into clones with varied genome content. The results indicated that an aneuploidy population could be induced by short exposure to paclitaxel at a low dose, implicating potential side effects of paclitaxel.     

Discovery of benzimidazole derivatives as novel multi-target EGFR, VEGFR-2 and PDGFR kinase inhibitors

Multi-target EGFR, VEGFR-2 and PDGFR inhibitors are highly useful anticancer agents with improved therapeutic efficacies. In this work, we used two virtual screening methods, support vector machines (SVM) and molecular docking, to identify a novel series of benzimidazole derivatives, 2-aryl benzimidazole compounds, as multi-target EGFR, VEGFR-2 and PDGFR inhibitors. 2-Aryl benzimidazole compounds were synthesized and their biological activities against a tumor cell line HepG-2 and specific kinases were evaluated. Among these compounds, compounds 5a and 5e exhibited high cytotoxicity against HepG-2 cells with IC?? values at ～2 μM. Further kinase assay study showed that compound 5a have good EGFR inhibitory activity and moderate VEGFR-2 and PDGFR inhibitory activities, while 5e have moderate EGFR inhibitory activity and slightly weaker VEGFR-2 and PDGFR inhibitory activities. Molecular docking analysis suggested that compound 5a more tightly interacts with EGFR and PDGFR than compound 5e. Our study discovered a novel series of benzimidazole derivatives as multi-target EGFR, VEGFR-2 and PDGFR kinases inhibitors.     

<Emphasis Type="Italic">Rubellimicrobium roseum</Emphasis> sp. nov., a Gram-negative bacterium isolated from the forest soil sample

A novel pink-coloured, non-spore-forming, non-motile, Gram-negative bacterium, designated YIM 48858^T, is described by using a polyphasic approach. The strain can grow at pH 6.5–9 (optimum at pH 7) and 25–30°C (optimum at 28°C). NaCl is not required for its growth. Positive for oxidase and catalase. Urease activity, nitrate reduction, starch and Tween 80 tests are negative reaction. 16S rRNA gene sequence similarity studies showed that strain YIM 48858^T is a member of the genus Rubellimicrobium, with similarities of 96.3, 95.7 and 95.5% to Rubellimicrobium mesophilum MSL-20^T, Rubellimicrobium aerolatum 5715S-9^T and Rubellimicrobium thermophilum DSM 16684^T, respectively. Q-10 was the predominant respiratory ubiquinone as in the other members of the genus Rubellimicrobium. The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphoglycolipid, glycolipid and the major fatty acids were C18:1 ω7c, C16:0 and C10:0 3-OH, which are very different from the valid published species. The DNA G + C content was 67.7 mol%. Both phylogenetic and chemotaxonomic evidence supports that YIM 48858^T is a novel species of the genus Rubellimicrobium, for which the name Rubellimicrobium roseum sp. nov. is proposed. The type strain is YIM 48858^T (=CCTCC AA 208029^T =KCTC 23202^T).