Structural understanding of stabilization patterns in engineered bispecific Ig‐like antibody molecules

Bispecific immunoglobulin‐like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin‐beta receptor (LTβR) binding Fv domain of an anti‐LTβR/anti‐TNF‐related apoptosis inducing ligand receptor‐2 (TRAIL‐R2) bispecific immunoglobulin‐like antibody. We further describe a new hierarchical structure‐guided approach toward engineering of antibody‐like molecules to enhance their thermal and chemical stability. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Diversity of an ectomycorrhizal fungal community studied by a root tip and total soil DNA approach

Molecular methods based on soil DNA extracts are increasingly being used to study the fungal diversity of ectomycorrhizal (EM) fungal communities in soil. Contrary to EM root tip identification, the use of molecular methods enables identification of extramatrical mycelia in soil. To compare fungal diversity as determined by root tip identification and mycelial identification, six soil samples were analysed. Root tips were extracted from the six samples and after amplification, the basidiomycete diversity on the root tips was analysed by denaturing gradient gel electrophoresis (DGGE). The soil from the six samples was sieved, total soil DNA was extracted and after amplification, the basidiomycete diversity in the soil fractions was analysed by DGGE. Fourteen different bands were excised from the DGGE gel and sequenced; fungal taxon names could be assigned to eight bands. Out of a total of 14 fungal taxa detected in soil, 11 fungal taxa were found on root tips, of which seven were EM fungal taxa. To examine whether the sieving treatment would affect EM species diversity, two different sieve mesh sizes were used and in addition, the organic soil fraction was analysed separately. DGGE analysis showed no differences in banding pattern for the different soil fractions. The organic fraction gave the highest DGGE band intensities. This work demonstrates that there is a high correspondence between basidiomycete diversity detected by molecular analysis of root tips and soil samples, irrespective of the soil fraction being analysed.     

Specific Chaperones for the Type VII Protein Secretion Pathway

Mycobacteria use the dedicated type VII protein secretion systems ESX-1 and ESX-5 to secrete virulence factors across their highly hydrophobic cell envelope. The substrates of these systems include the large mycobacterial PE and PPE protein families, which are named after their characteristic Pro-Glu and Pro-Pro-Glu motifs. Pathogenic mycobacteria secrete large numbers of PE/PPE proteins via the major export pathway, ESX-5. In addition, a few PE/PPE proteins have been shown to be exported by ESX-1. It is not known how ESX-1 and ESX-5 recognize their cognate PE/PPE substrates. In this work, we investigated the function of the cytosolic protein EspG(5), which is essential for ESX-5-mediated secretion in Mycobacterium marinum, but for which the role in secretion is not known. By performing protein co-purifications, we show that EspG(5) interacts with several PPE proteins and a PE/PPE complex that is secreted by ESX-5, but not with the unrelated ESX-5 substrate EsxN or with PE/PPE proteins secreted by ESX-1. Conversely, the ESX-1 paralogue EspG(1) interacted with a PE/PPE couple secreted by ESX-1, but not with PE/PPE substrates of ESX-5. Furthermore, structural analysis of the complex formed by EspG(5) and PE/PPE indicates that these proteins interact in a 1:1:1 ratio. In conclusion, our study shows that EspG(5) and EspG(1) interact specifically with PE/PPE proteins that are secreted via their own ESX systems and suggests that EspG proteins are specific chaperones for the type VII pathway.     

One-year survey of a single Micronesian reef reveals extraordinarily rich diversity of <Emphasis Type="Italic">Symbiodinium</Emphasis> types in soritid foraminifera

Recent molecular studies of symbiotic dinoflagellates (genus Symbiodinium) from a wide array of invertebrate hosts have revealed exceptional fine-scale symbiont diversity whose distribution among hosts, regions and environments exhibits significant biogeographic, ecological and evolutionary patterns. Here, similar molecular approaches using the internal transcribed spacer-2 (ITS-2) region were applied to investigate cryptic diversity in Symbiodinium inhabiting soritid foraminifera. Approximately 1,000 soritid specimens were collected and examined during a 12-month period over a 40 m depth gradient from a single reef in Guam, Micronesia. Out of 61 ITS-2 types distinguished, 46 were novel. Most types found are specific for soritid hosts, except for three types (C1, C15 and C19) that are common in metazoan hosts. The distribution of these symbionts was compared with the phylotype of their foraminiferal hosts, based on soritid small subunit ribosomal DNA sequences, and three new phylotypes of soritid hosts were identified based on these sequences. Phylogenetic analyses of 645 host-symbiont pairings revealed that most Symbiodinium types associated specifically with a particular foraminiferal host genus or species, and that the genetic diversity of these symbiont types was positively correlated with the genetic diversity found within each of the three host genera. Compared to previous molecular studies of Symbiodinium from other locations worldwide, the diversity reported here is exceptional and suggests that Micronesian coral reefs are home to a remarkably large Symbiodinium assemblage.     

利诺吡啶对豚鼠耳蜗外毛细胞和Deiters细胞钾电流的抑制

为探讨KCNQ家族钾通道在耳蜗外毛细胞和Deiters细胞的功能性表达,我们观察并记录了KCNQ家族钾通道阻滞剂利诺吡啶对豚鼠耳蜗单离外毛细胞(outer hair cells,OHCs)和Deiters细胞总钾电流的影响。采用酶孵育加机械分离法分离豚鼠耳蜗单个OHCs和Deiters细胞：运用膜片钳技术,在全细胞模式下记录正常细胞外液中8个外毛细胞和5个Deiters细胞的总钾电流,并观察100μmol／L和200μmol／L利诺吡啶对外毛细胞和Deiters细胞总钾电流的影响。结果观察到,在正常细胞外液中的单离外毛细胞,可记录到四乙基二乙胺敏感的外向性钾电流和静息膜电位附近激活的内向性钾电流(the K^ current activated at negative potential,IKa)两种钾电流,而在单离Deiters细胞中只记录到外向整流性钾电流。在细胞外液中,加入100μmol／L利诺吡啶后,OHCs中的四乙基二乙胺敏感的钾电流峰电流成分被抑制,稳态电流幅值减小,且电流的失活时问常数明显延长;在细胞外液中加入100μmol／L和200μmol／L利诺吡啶后,OHCs的内向性钾电流IKa被完全抑制;而细胞外液中利诺吡啶终浓度为200μmol／L时,Deiters细胞的外向整流性钾电流幅值无明显变化。由此我们推测,KCNQ家族钾通道存在于豚鼠耳蜗外毛细胞,其介导的钾电流是四乙基二乙胺敏感的钾电流的组成部分,并构成全部的IKn,其功能是介导细胞内K^ 外流和防止细胞过度去极化;KCNQ家族钾通道不存在于豚鼠耳蜗Dciters细胞。     

高血压病患者肠系膜动脉平滑肌细胞钙激活钾通道研究

目的:研究高血压病患者肠系膜动脉平滑肌细胞钙激活钾通道(KCa)的功能活动。方法:应用膜片钳制技术内面向外式单通道记录方法。结果:①人肠系膜动脉平滑肌细胞KCa开放具有电压依赖性。KCa通道电导在高血压组、正常组分别为191.4pS、197.7pS。胞内侧应用TEA可阻断通道。②增加浴液中Ca2 浓度(从0增至10-8、10-7、5×10-7、10-6mol/L),各组KCa开放概率(Po)均呈浓度依赖性增加,高血压组Po从0.016增至0.023、0.031、0.053、0.094,正常组Po从0.004增至0.023、0.041、0.072、0.184。通道平均开放时间延长,平均关闭时间缩短。③Ca2 浓度为0时,高血压组KCa开放概率明显高于正常组,在其它Ca2 浓度下高血压组KCa开放概率等于或低于正常组。结论:高血压病患者肠系膜动脉平滑肌细胞KCa的Ca2 敏感性较低,可能促进高血压的发生。     

Ancient Mitochondrial DNA Reveals the Origin of Sus scrofa from Rebun Island, Japan

The Kabukai A site (5 to 8C A.D.) of the Okhotsk cultural area is on Rebun Island, a small island near the coast, north–northwest of Hokkaido, Japan. Specimens of Sus scrofa, called the Sakhalin pig, were discovered in five cultural layers at the Kabukai A site. Ancient DNA was extracted from the remains of 42 Sakhalin pig bones. Thirty-nine nucleotide sequences of the 574-bp mitochondrial DNA control region, estimated to have originated from at least 21 individuals, were amplified and analyzed phylogenetically. Nine distinct haplotypes (A1, A2, A3, B1, B2, C1, C2, D1, and D2) from this site were classified into four haplotype groups (A, B, C, and D) by parsimonious network analysis. Phylogenetic analysis of 9 ancient and 55 modern haplotypes indicated that the population of Sakhalin pigs at the Kabukai A site belonged to two distinct clusters; haplotype groups A and B formed a cluster comprised only of themselves, and haplotype groups C and D belonged to the cluster of one of the two genetic groups of Japanese wild boars uniquely distributed in the western part of Japan, including one northeast Mongolian wild boar. Analysis of the haplotype distribution among three archaeological sites and their historical transitions among the five layers reflecting the cultural periods at the Kabukai A site suggests that the Sakhalin pig populations were introduced from Sakhalin island and the Amur River basin in the northeastern Eurasian continent together with some cultural influences. Received: 18 April 2000 / Accepted: 24 November 2000     

Non-invasive Parenchymal,Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.     

Assessment of variability among morphological and molecular characters in wild populations of mint [Mentha longifolia (L.) L.] germplasm

Mentha longifolia is an important medicinal and aromatic perennial herb that exhibits wide distribution range from sub-tropical to temperate regions. In the present study, agro-morphological traits and genetic differences in 19 different populations of M. longifolia were studied to evaluate the level and extent of its diversity. Analysis of variance (ANOVA) showed that the different phenotypic characters show considerable differences among various populations and was significant at p < 0.05. Molecular diversity analysis performed by using arbitrary amplified eleven ISSR primers generated a total of 121 amplicons that range within the size of 200–2500 base pairs (bp). Each primer on average generated 11 amplicons with percentage polymorphism being 100. The analysis of molecular variance (AMOVA) showed more (64%) among population genetic diversity and less (36%) within the populations. Greater genetic differentiation (Gst = 0.6852) among these populations occurs due to low gene flow (Nm = 0.2297) and greater habitat variability. Geographic and genetic distances were positively correlated according to Mantel’s test. In order to remove any kind of biases, we used R software to perform cluster and redundancy analysis to analyse the extent of relatedness among studied populations. In terms of morphological and molecular aspects, the populations were grouped into four and five clusters respectively based on hierarchical clustering method. The results demonstrated that M. longifolia displays a great degree of morphological and genetic variation and can be utilized in breeding, genetic improvement, and gene bank conservation programmes in future.     

Integrated computational approaches to screen gene expression data to determine key genes and therapeutic targets for type-2 diabetes mellitus

There is a rapid rise in cases of Type-2-diabetes mellitus (T2DM) globally, irrespective of the geography, ethnicity or any other variable factors. The molecular mechanisms that could cause the condition of T2DM need to be more thoroughly analysed to understand the clinical manifestations and to derive better therapeutic regimes. Tools in bioinformatics are used to trace out key gene elements and to identify the key causative gene elements and their possible therapeutic agents. Microarray datasets were retrieved from the Gene expression omnibus database and studied using R to derive different expressed gene (DEG) elements. With the comparison of the expressed genes with disease specific genes in DisGeNET, the final annotated genes were taken for analysis. Gene Ontology studies, Protein–protein interaction (PPI), Co-expression analysis, Gene-drug interactions were performed to scale down the hub genes and to identify the novelty across the genes analysed so far. In vivo and invitro analysis of key genes and the trace of interaction pathway is crucial to better understand the unique outcomes from the novel genes, forming the basis to understand the pathway that ends up causing T2DM. Afterwards, docking was executed enabling recognition of interacting residues involved in inhibition. The complex CCL5-265 and CD8A-40585 thus docked showed best results as is evident from its PCA analysis and MMGBSA calculation. There is now scope for deriving candidate drugs that could possibly detect personalized therapies for T2DM.