Site-selective lateral multilayer assembly of bienzyme with polyelectrolyte on ITO electrode based on electric field-induced directly layer-by-layer deposition

We describe here a new approach to construct a multilayer enzyme/polyelectrolyte film on a structured transparent indium-tin oxide (ITO) covered glass electrode surface as micropattern, on which two different types of enzyme distributed laterally on one common substrate without interference. The multilayer film was prepared by alternate electric field directed layer-by-layer assembly deposition and alternate deposition of different redox enzymes and polyelectrolyte poly(diallyldimethylammonium chloride) (PDDA) onto the site-selective ITO glass electrode surface. The cyclic voltammogram, obtained from the ITO glass electrode modified with the glucose oxidase (GO(X))/PDDA and catalase (CA(T))/PDDA multilayers, revealed that the bioelectrocatalytic response is directly correlated to the number of deposition bilayers. From the analysis of cyclic voltammetric characterization, the coverage of catalytically active enzymes per enzyme/PDDA bilayer during the multilayer formation was homogeneous, which demonstrates that the multilayer is constructed in a spatially ordered manner. Also, from the atomic force microscopy and Brewster angle microscopy measurements, more information of the multilayer constructed by different methods on the modified electrode surface is obtained and compared. This fabrication technique is simple and would be applicable to the construction of a thickness- and area-controlled biopattern composed of multi-enzymes as well as multiple biomaterials.     

海洋芽孢杆菌（Bacillus marinus）B-9987菌株抑制病原真菌机理的研究

摘要：【目的】：探讨海洋芽孢杆菌（Bacillus marinus）B-9987菌株的代谢产物BMME-1,对植物病原真菌茄链格孢菌的抑菌作用机理。【方法】分别使用分光光法、气相色谱-质谱GC-MS联用技术、红外光谱法等,检测了BMME-1处理病原真菌后,菌体渗透性、细胞壁及细胞膜成份的变化。【结果】BMME-1对茄链格孢菌的抑菌中浓度（MIC50）为6.2 mg/L,最小杀菌浓度（MFC）为50 mg/L,在MIC50浓度或高于此浓度处理靶标菌,将导致菌体蛋白质、核酸等大分子物质的外流;处理菌株葡聚糖结     

我国分离的肠道病毒71型(SHZH03病毒株)全基因组核苷酸序列分析

对肠道病毒71型(enterovirus 71,EV71)中国(深圳)分离株SHZH03进行了全基因组(未包括多聚腺苷尾)7406个碱基的核苷酸序列测定.结果表明,SHZH03株与其它肠道病毒71型毒株相比,在编码区没有核苷酸的缺失和插入,其5′UTR和3′UTR区的长度和序列有一定的差异.核苷酸同源性比较结果表明,在P1区SHZH03株与SHZH98株、中国台湾流行株(TW2086、TW2272)的同源性较高(分别为92.5%,90.1%和87.9%),与新加坡流行株SIN5666、SIN5865及标准株MS、BrCr的同源性则在81%左右,而与Coxsackievirus A16(Cox.A16)的同源性最低(63.6%).氨基酸同源性比较结果表明,在P1区SHZH03株与Cox. A16的同源性最低,但在P2和P3区SHZH03株与Cox.A16的同源性最高.P1区的遗传进化分析表明,SHZH03株和中国台湾1998年流行的EV71毒株的亲缘关系较近,属于同一型(genogroup),而与标准株BrCr和MS的亲缘关系较远.上述结果有助于肠道病毒71型的基础研究和中国对于EV71所致疾病的预防.     

Genome Sequence of Hybrid Vibrio cholerae O1 MJ-1236, B-33, and CIRS101 and Comparative Genomics with V. cholerae

The genomes of Vibrio cholerae O1 Matlab variant MJ-1236, Mozambique O1 El Tor variant B33, and altered O1 El Tor CIRS101 were sequenced. All three strains were found to belong to the phylocore group 1 clade of V. cholerae, which includes the 7th-pandemic O1 El Tor and serogroup O139 isolates, despite displaying certain characteristics of the classical biotype. All three strains were found to harbor a hybrid variant of CTXΦ and an integrative conjugative element (ICE), leading to their establishment as successful clinical clones and the displacement of prototypical O1 El Tor. The absence of strain- and group-specific genomic islands, some of which appear to be prophages and phage-like elements, seems to be the most likely factor in the recent establishment of dominance of V. cholerae CIRS101 over the other two hybrid strains.Vibrio cholerae, a bacterium autochthonous to the aquatic environment, is the causative agent of cholera, a life-threatening disease that causes severe, watery diarrhea. Cholera bacteria are serogrouped based on their somatic O antigens, with more than 200 serogroups identified to date (6). Only toxigenic strains of serogroups O1 and O139 have been identified as agents of cholera epidemics and pandemics; serogroups other than O1 and O139 have the potential to cause mild gastroenteritis or, rarely, local outbreaks. Genes coding for cholera toxin (CTX), ctxAB, and other virulence factors have been shown to reside in bacteriophages and various mobile genetic elements. In addition, V. cholerae serogroup O1 is differentiated into two biotypes, classical and El Tor, by a combination of biochemical traits, by sensitivity to biotype-specific bacteriophages, and more recently by nucleotide sequencing of specific genes and by molecular typing (5, 17, 19).There have been seven pandemics of cholera recorded throughout human history. The seventh and current pandemic began in 1961 in the Indonesian island of Sulawesi and subsequently spread to Asia, Africa, and Latin America; the six previous pandemics are believed to have originated in the Indian subcontinent. Isolates of the sixth pandemic were almost exclusively of the O1 classical biotype, whereas the current (seventh) pandemic is dominated by the V. cholerae O1 El Tor biotype as the causative agent, a transition occurring between 1923 and 1961. Today, the disease continues to remain a scourge in developing countries, confounded by the fact that V. cholerae is native to estuaries and river systems throughout the world (8).Over the past 20 years, several new epidemic lineages of V. cholerae O1 El Tor have emerged (or reemerged). For example, in 1992, a new serogroup, namely, O139 of V. cholerae, was identified as the cause of epidemic cholera in India and Bangladesh (25). The initial concern was that a new pandemic was beginning; however, the geographic range of V. cholerae O139 is currently restricted to Asia. Additionally, V. cholerae O1 hybrids and altered El Tor variants have been isolated repeatedly in Bangladesh (Matlab) (23, 24) and Mozambique (1). Altered V. cholerae O1 El Tor isolates produce cholera toxin of the classical biotype but can be biotyped as El Tor by conventional phenotypic assays, whereas V. cholerae O1 hybrid variants cannot be biotyped based on phenotypic tests and can produce cholera toxin of either biotype. These new variants have subsequently replaced the prototype seventh-pandemic V. cholerae O1 El Tor strains in Asia and Africa, with respect to frequency of isolation from clinical cases of cholera (27).Here, we report the genome sequence of three V. cholerae O1 variants, MJ-1236, a Matlab type I hybrid variant from Bangladesh that cannot be biotyped by conventional methods, CIRS101, an altered O1 El Tor isolate from Bangladesh which harbors ctxB of classical origin, and B33, an altered O1 El Tor isolate from Mozambique which harbors classical CTXΦ, and we compare their genomes with prototype El Tor and classical genomes. From an epidemiological viewpoint, among the three variants characterized in this study, V. cholerae CIRS101 is currently the most “successful” in that strains belonging to this type have virtually replaced the prototype El Tor in Asia and many parts of Africa, notably East Africa. This study, therefore, gives us a unique opportunity to understand why V. cholerae CIRS101 is currently the most successful El Tor variant.     

Rainfall manipulation experiments as simulated by terrestrial biosphere models: Where do we stand?

Changes in rainfall amounts and patterns have been observed and are expected to continue in the near future with potentially significant ecological and societal consequences. Modelling vegetation responses to changes in rainfall is thus crucial to project water and carbon cycles in the future. In this study, we present the results of a new model‐data intercomparison project, where we tested the ability of 10 terrestrial biosphere models to reproduce the observed sensitivity of ecosystem productivity to rainfall changes at 10 sites across the globe, in nine of which, rainfall exclusion and/or irrigation experiments had been performed. The key results are as follows: (a) Inter‐model variation is generally large and model agreement varies with timescales. In severely water‐limited sites, models only agree on the interannual variability of evapotranspiration and to a smaller extent on gross primary productivity. In more mesic sites, model agreement for both water and carbon fluxes is typically higher on fine (daily–monthly) timescales and reduces on longer (seasonal–annual) scales. (b) Models on average overestimate the relationship between ecosystem productivity and mean rainfall amounts across sites (in space) and have a low capacity in reproducing the temporal (interannual) sensitivity of vegetation productivity to annual rainfall at a given site, even though observation uncertainty is comparable to inter‐model variability. (c) Most models reproduced the sign of the observed patterns in productivity changes in rainfall manipulation experiments but had a low capacity in reproducing the observed magnitude of productivity changes. Models better reproduced the observed productivity responses due to rainfall exclusion than addition. (d) All models attribute ecosystem productivity changes to the intensity of vegetation stress and peak leaf area, whereas the impact of the change in growing season length is negligible. The relative contribution of the peak leaf area and vegetation stress intensity was highly variable among models.     

A highly selective and colorimetric assay of lysine by molecular-driven gold nanorods assembly

In this contribution, a simple, rapid, colorimeteric and selective assay for lysine was achieved by a controllable end-to-end assembly of gold nanorods (AuNRs) in the presence of Eu(3+) and lysine. This one-pot end-to-end assembly of 11-mercaptoundecanoic acid (MUA) modified AuNRs was occurred in Britton-Robinson buffer of pH 6.0, which involves the coordination binding between Eu(3+) and COO(-) groups as well as the electrostatic interaction of the COO(-) groups of MUA with the -NH(3)(+) group of lysine. As monitored by absorption spectra, scanning electron microscopic (SEM) images and dynamic light scattering (DLS) measurement, the end-to-end chain assembly results in large red-shift in the longitudinal plasmon resonance absorption (LPRA), giving red-to-blue color change of AuNRs. Importantly, it was found that the red-shift of LPRA is linearly proportional to the concentrations of lysine in the range of 5.0×10(-6)-1.0×10(-3)M with the limit of detection (LOD) being 1.6×10(-6)M (3σ/k). This red-shift of LPRA is highly selective, making it possible to develop a rapid, selective and visual assay for lysine in food samples.     

Studies on Some Properties of the Apoplastic β-N-Acetyl-D-Hexosaminidase from Tomato Leaves

Some properties of the β-N-acetyl-D-hexosaminidase purified from intercellular fluid of tomato leaves after the plant was systematically infected by TMV (tobacco mosaic virus) were studied. When pNP β-D-GlcNAc (p nitrophenyl-N-aeetyl β-D-glucosaminide) or pNP β-D- GalNAc (p-nitrophenyl-N-acetyl-β-D galactosaminide) was used as the substrate, it showed the optical pH between 4. 8--5.0 and optical temperature between 44— 47℃. Studies of thermostabillty indicated that the enzyme had a biphasic denaturation curve. Using pNP-β-D-GIcNAc or pNP-β-D GalNAc as the substrate, the Km value of the enzyme was 0. 36 and 0. 67 mmol/L respectively. N acetyi-D glucosamine and N acetyl-D-galactosamine were competitive inhibitors of the enzyme activities. Ag+ and Hg2+ were sensitive inhibitors and Fe2+ . Fe3+ and Cu2+ were also inhibitors enzyme activities.     

Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation

Background

Apolipoprotein E (apoE) is a major carrier of cholesterol and essential for synaptic plasticity. In brain, it’s expressed by many cells but highly expressed by the choroid plexus and the predominant apolipoprotein in cerebrospinal fluid (CSF). The role of apoE in the CSF is unclear. Recently, the glymphatic system was described as a clearance system whereby CSF and ISF (interstitial fluid) is exchanged via the peri-arterial space and convective flow of ISF clearance is mediated by aquaporin 4 (AQP4), a water channel. We reasoned that this system also serves to distribute essential molecules in CSF into brain. The aim was to establish whether apoE in CSF, secreted by the choroid plexus, is distributed into brain, and whether this distribution pattern was altered by sleep deprivation.

Methods

We used fluorescently labeled lipidated apoE isoforms, lenti-apoE3 delivered to the choroid plexus, immunohistochemistry to map apoE brain distribution, immunolabeled cells and proteins in brain, Western blot analysis and ELISA to determine apoE levels and radiolabeled molecules to quantify CSF inflow into brain and brain clearance in mice. Data were statistically analyzed using ANOVA or Student’s t- test.

Results

We show that the glymphatic fluid transporting system contributes to the delivery of choroid plexus/CSF-derived human apoE to neurons. CSF-delivered human apoE entered brain via the perivascular space of penetrating arteries and flows radially around arteries, but not veins, in an isoform specific manner (apoE2?>?apoE3?>?apoE4). Flow of apoE around arteries was facilitated by AQP4, a characteristic feature of the glymphatic system. ApoE3, delivered by lentivirus to the choroid plexus and ependymal layer but not to the parenchymal cells, was present in the CSF, penetrating arteries and neurons. The inflow of CSF, which contains apoE, into brain and its clearance from the interstitium were severely suppressed by sleep deprivation compared to the sleep state.

Conclusions

Thus, choroid plexus/CSF provides an additional source of apoE and the glymphatic fluid transporting system delivers it to brain via the periarterial space. By implication, failure in this essential physiological role of the glymphatic fluid flow and ISF clearance may also contribute to apoE isoform-specific disorders in the long term.

     

Treatment of donor cells with trichostatin A improves in vitro development and reprogramming of buffalo (Bubalus bubalis) nucleus transfer embryos

It has been reported that buffalo (Bubalus bubalis) embryos reconstructed by somatic cell nucleus transfer (SCNT) can develop to the full term of gestation and result in newborn calves. However, the developmental competence of reconstructed embryos is still low. Recently, it has been reported that treating donor cells or embryos with trichostatin A (TSA) can increase the cloning efficiency in some species. Thus, the present study was undertaken to improve the development of buffalo SCNT embryos by treatment of donor cells (buffalo fetal fibroblasts) with TSA and explore the relation between histone acetylation status of donor cells and developmental competence of SCNT embryos. Treatment of donor cells with either 0.15 or 0.3 μM TSA for 48 hours resulted in a significant increase in the cleavage rate and blastocyst yield of SCNT embryos (P < 0.05). Meanwhile, the expression level of HDAC1 in donor cells was also decreased (0.4–0.6 fold, P < 0.05) by TSA treatment, although the expression level of HAT1 was not affected. Further measurement of the epigenetic maker AcH4K8 in buffalo IVF and SCNT embryos at the eight-cell stage revealed that the spatial distribution of acH4K8 staining in SCNT embryos was different from the IVF embryos. Treatment of donor cells with TSA resulted in an increase in the AcH4K8 level of SCNT embryos and similar to fertilized counterparts. These results suggest that treatment of donor cells with TSA can facilitate their nucleus reprogramming by affecting the acetylated status of H4K8 and improving the in vitro development of buffalo SCNT embryos. The AcH4K8 status at the eight-cell stage can be used as an epigenetic marker for predicting the SCNT efficiency in buffalos.