Quantitative neuroproteomics of an in vivo rodent model of focal cerebral ischemia/reperfusion injury reveals a temporal regulation of novel pathophysiological molecular markers

Cerebral ischemia or stroke, an acute neurological injury lacking an effective therapy, is the second leading cause of death globally. The unmet need in stroke research is to identify viable targets and to understand their interplay during the temporal evolution of ischemia/reperfusion (I/R) injury. Here we report a temporal signature of the ischemic hemisphere revealed by the isobaric tag for relative and absolute quantification (iTRAQ)-based 2D-LC-MS/MS strategy in an in vivo middle cerebral artery occlusion (MCAO) model of focal cerebral I/R injury. To recapitulate clinical stroke, two hours of MCAO was followed by 0, 4, and 24 h of reperfusion to capture ischemia with an acute and subacute durations of reperfusion injury. The subsequent iTRAQ experiment identified 2242 proteins from the ischemic hemisphere with <1.0% false discovery rate. Data mining revealed that (1) about 2.7% of detected proteins were temporally perturbed having an involvement in the energy metabolism (Pygb, Atp5b), glutamate excitotoxicity (Slc1a3, Glud1), neuro-inflammation (Tf, C3, Alb), and cerebral plasticity (Gfap, Vim, Gap43); (2) astrocytes participated actively in the neurometabolic coupling underlining the importance of a cerebro-protective rather than a neuro-protective approach; and (3) hyper-acute yet progressive opening of the blood brain barrier (BBB), accompanied by stimulation of an innate immune response and late activation of a regenerative response, which provides an extended therapeutic window for intervention. Several regulated proteins (Caskin1, Shank3, Kpnb1, Uchl1, Mtap6, Epb4.1l1, Apba1, and Ube1x) novel in the context of stroke were also discovered. In conclusion, our result supports a dynamic multitarget therapy rather than the traditional approach of a unilateral and sustained modulation of a single target to address the phasic regulation of an ischemic proteome.     

Epithelial‐mesenchymal transition softens head and neck cancer cells to facilitate migration in 3D environments

The biological impact and signalling of epithelial‐mesenchymal transition (EMT) during cancer metastasis has been established. However, the changes in biophysical properties of cancer cells undergoing EMT remain elusive. Here, we measured, via video particle tracking microrheology, the intracellular stiffness of head and neck cancer cell lines with distinct EMT phenotypes. We also examined cells migration and invasiveness in different extracellular matrix architectures and EMT‐related signalling in these cell lines. Our results show that when cells were cultivated in three‐dimensional (3D) environments, the differences in cell morphology, migration speed, invasion capability and intracellular stiffness were more pronounced among different head and neck cancer cell lines with distinct EMT phenotypes than those cultivated in traditional plastic dishes and/or seated on top of a thick layer of collagen. An inverse correlation between intracellular stiffness and invasiveness in 3D culture was revealed. Knock‐down of the EMT regulator Twist1 or Snail or inhibition of Rac1 which is a downstream GTPase of Twist1 increased intracellular stiffness. These results indicate that the EMT regulators, Twist1 and Snail and the mediated signals play a critical role in reducing intracellular stiffness and enhancing cell migration in EMT to promote cancer cells invasion.     

Characterization of N-glycosylated type I collagen in streptozotocin-induced diabetes.

The N epsilon-glycosylation of lysine and hydroxylysine residues in collagen from streptozotocin-induced-diabetic rats was confirmed and the stability of the complex shown to be due to an Amadori rearrangement. The studies also demonstrate the relative specificities of glucose, galactose and mannose in their reaction with collagen. The glycosylation of lysine in vitro occurs with glucose and galactose, but not with mannose, whereas only gucose reacts with hydroxylysine to any significant extent. Glycosylation of collagen occurs slowly during normal aging, but in contrast with reports suggesting accelerated aging of collagen in diabetic animals, we clearly demonstrated that the apparent increased stability is not due to an acceleration of the normal maturation process involving the reducible cross-links.     

The sequence and duration of mitotic cycles in the apical root meristem ofVicia faba l

A study was made on the mitotic cycle times in meristematic cells of Vicia faba root tips and on the relationship between their duration and the position of a certain cell in the column of proliferating cells. For the demonstration of the sequence and duration of mitotic oycles a single-column model was used. The results of experiments show that the great variability in the duration of mitotic cycles (from 12 to l20 h)is the result of a different program of the apical meristem cells. The long duration of the cycle of initials corresponds to the sum of cycle times of their descendants.     

Effective High-Throughput Blood Pooling Strategy before DNA Extraction for Detection of Malaria in Low-Transmission Settings

In the era of (pre) elimination setting, the prevalence of malaria has been decreasing in most of the previously endemic areas. Therefore, effective cost- and time-saving validated pooling strategy is needed for detection of malaria in low transmission settings. In this study, optimal pooling numbers and lowest detection limit were assessed using known density samples prepared systematically, followed by genomic DNA extraction and nested PCR. Pooling strategy that composed of 10 samples in 1 pool, 20 µl in 1 sample, was optimal, and the parasite density as low as 2 p/µl for both falciparum and vivax infection was enough for detection of malaria. This pooling method showed effectiveness for handling of a huge number of samples in low transmission settings (<9% positive rate). The results indicated that pooling of the blood samples before DNA extraction followed by usual nested PCR is useful and effective for detection of malaria in screening of hidden cases in low-transmission settings.     

Towards the genetic control of insect vectors: An overview

Insects are responsible for the transmission of major infectious diseases. Recent advances in insect genomics and transformation technology provide new strategies for the control of insect borne pathogen transmission and insect pest management. One such strategy is the genetic modification of insects with genes that block pathogen development. Another is to suppress insect populations by releasing either sterile males or males carrying female‐specific dominant lethal genes into the environment. Although significant progress has been made in the laboratory, further research is needed to extend these approaches to the field. These insect control strategies offer several advantages over conventional insecticide‐based strategies. However, the release of genetically modified insects into the environment should proceed with great caution, after ensuring its safety, and acceptance by the target populations.     

Autocrine/paracrine pattern of superoxide production through NAD(P)H oxidase in coronary arterial myocytes

The present study tested the hypothesis that membrane-bound NAD(P)H oxidase in coronary arterial myocytes (CAMs) is capable of producing superoxide (O(2)(*-)) toward extracellular space to exert an autocrine- or paracrine-like action in these cells. Using a high-speed wavelength-switching fluorescent microscopic imaging technique, we simultaneously monitored the binding of dihydroethidium-oxidizing product to exogenous salmon testes DNA trapped outside CAMs and to nuclear DNA as indicators of extra- and intracellular O(2)(*-) production. It was found that a muscarinic agonist oxotremorine (OXO; 80 microM) increased O(2)(*-) levels more rapidly outside than inside CAMs. In the presence of superoxide dismutase (500 U/ml) plus catalase (400 U/ml) and NAD(P)H oxidase inhibitor diphenylene iodonium (50 microM) or apocynin (100 microM), these increases in extra- and intracellular O(2)(*-) levels were substantially abolished or attenuated. The O(2)(*-) increase outside CAMs was also confirmed by detecting oxidation of nitro blue tetrazolium and confocal microscopic localization of Matrigel-trapped OxyBURST H(2)HFF Green BSA staining around these cells. By electron spin resonance spectrometry, the extracellular accumulation of O(2)(*-) was demonstrated as a superoxide dismutase-sensitive component outside CAMs. Furthermore, RNA interference of NAD(P)H oxidase subunits Nox1 or p47 markedly blocked OXO-induced increases in both extra- and intracellular O(2)(*-) levels, whereas small inhibitory RNA of Nox4 only attenuated intracellular O(2)(*-) accumulation. These results suggest that Nox1 represents a major NAD(P)H oxidase isoform responsible for extracellular O(2)(*-) production. This rapid extracellular production of O(2)(*-) seems to be unique to OXO-induced M(1)-receptor activation, since ANG II-induced intra- and extracellular O(2)(*-) increases in parallel. It is concluded that the outward production of O(2)(*-) via NAD(P)H oxidase in CAMs may represent an important producing pattern for its autocrine or paracrine actions.     

福建植物生物多样性的特点及其生物安全问题

对福建植物生物多样性从物种多样性、遗传多样性和生态系统多样性三个层面进行了评价,分析了植物生物多样性的特点。在此基础上提出了影响福建植物生物多样性的主要生物安全问题。研究表明,福建植物生物多样性具有植物种类丰富,热带和亚热带的科属种类多、起源古老,中国特有成分比例较高和国家重点保护植物较为丰富等特点。影响植物生物多样性的主要生物安全问题包括阔叶林面积不断减少,森林资源总体质量下降;濒危野生植物比重增大,名木古树破坏严重;外来物种入侵呈加重趋势。     

铜绿假单胞菌LexA蛋白的纯化及免疫活性分析

目的：对铜绿假单胞菌(Pseudomonas aeruginosa,PA)的LexA蛋白进行表达、纯化,并检测其免疫活性。方法： lexA基因片段插入表达载体pET32a(+),在E.coli BL21(DE3)中表达。包涵体经洗涤并用8M尿素溶解,镍离子亲合柱层析为第一步纯化,Superdex 75凝胶过滤层析作为第二步精细纯化,HPLC测定蛋白的浓度,将纯化的LexA蛋白经注射途径免疫家兔,制备兔抗LexA血清,采用免疫双扩、ELISA及Western Blot分析LexA的免疫活性。结果：LexA以包涵体形式表达,经镍离子亲合柱层析和凝胶过滤层析二步组合纯化目的蛋白,经HPLC测定目的蛋白的最终纯度为98.97%,表达及纯化的LexA具有良好的免疫活性。     

污染作物籽实中As的分布及其结合形态初探

对水稻和小麦籽实中Ａｓ的分布,结合形态及加工去除进行了初步研究,结果表明,Ａｓ在谷物各结构部位中的分布不均,在水稻籽实中浓度分布顺序为胚〉种皮〉颖壳〉胚乳,小麦中Ａｓ的浓度分布顺序为胚〉种皮〉胚乳;Ａｓ在各形态部位的累积量不同,主要存留在胚乳中;Ａｓ主要与蛋白质相结合的形态存在;在稻谷去颖壳、粗米糠和精米糠的过程中,可分别去除Ａｓ１６．５１％、１２．４１％和１０．２６％,小麦也随着加工深度的升级,