Residues in the HIV-1 capsid assembly inhibitor binding site are essential for maintaining the assembly-competent quaternary structure of the capsid protein

Morphogenesis of infectious HIV-1 involves budding of immature virions followed by proteolytic disassembly of the Gag protein shell and subsequent assembly of processed capsid proteins (CA) into the mature HIV-1 core. The dimeric interface between C-terminal domains of CA (C-CA) has been shown to be important for both immature and mature assemblies. We previously reported a CA-binding peptide (CAI) that blocks both assembly steps in vitro. The three-dimensional structure of the C-CA/CAI complex revealed an allosteric effect of CAI that alters the C-CA dimer interface. Based on this structure, we now investigated the phenotypes of mutations in the binding pocket. CA variants carrying mutations Y169A, L211A, or L211S had a reduced affinity for CAI and were unable to form mature-like particles in vitro. These mutations also blocked morphological conversion to mature virions in tissue culture and abolished infectivity. X-ray crystallographic analyses of the variant C-CA domains revealed that these alterations induced the same allosteric change at the dimer interface observed in the C-CA/CAI complex. These results point to a role of key interactions between conserved amino acids in the CAI binding pocket of C-CA in maintaining the correct conformation necessary for mature core assembly.     

敦煌西湖湿地鸟类栖息地重要性模糊综合评判

根据2007～2008年在甘肃敦煌西湖国家级自然保护区进行的湿地鸟类调查种类和数量数据,运用模糊综合评判法对保护区内的8块湿地进行了湿地鸟类栖息地重要性评价. 评判中隶属度的确定采用最佳因子值法,并分春、秋两种最佳因子值进行评判.结果表明:(1)春季各湿地均比秋季的重要性程度高,春季是管理的重点;(2)春秋两季南湖湿地重要性(0.938和0.966)都要远大于其它的湿地.春季盐池湾、羊水海子、南大湖的结果较相近,重要性程度次之,可持有相同程度的管理水平.党河水库、墩子湾、马圈湾、南园湖的重要性程度较低,只需一般水平的管理;(3)秋季各湿地水平除了南湖外普遍较低,羊水海子和盐池湾的重要性(0.340和0.269)相对较高些,但也只需一般水平的管理.     

Sensitive Detection of Colorectal Cancer in Peripheral Blood by Septin 9 DNA Methylation Assay

Background

Colorectal cancer (CRC) is the second leading cause of cancer deaths despite the fact that detection of this cancer in early stages results in over 90% survival rate. Currently less than 45% of at-risk individuals in the US are screened regularly, exposing a need for better screening tests. We performed two case-control studies to validate a blood-based test that identifies methylated DNA in plasma from all stages of CRC.

Methodology/Principal Findings

Using a PCR assay for analysis of Septin 9 (SEPT9) hypermethylation in DNA extracted from plasma, clinical performance was optimized on 354 samples (252 CRC, 102 controls) and validated in a blinded, independent study of 309 samples (126 CRC, 183 controls). 168 polyps and 411 additional disease controls were also evaluated. Based on the training study SEPT9-based classification detected 120/252 CRCs (48%) and 7/102 controls (7%). In the test study 73/126 CRCs (58%) and 18/183 control samples (10%) were positive for SEPT9 validating the training set results. Inclusion of an additional measurement replicate increased the sensitivity of the assay in the testing set to 72% (90/125 CRCs detected) while maintaining 90% specificity (19/183 for controls). Positive rates for plasmas from the other cancers (11/96) and non-cancerous conditions (41/315) were low. The rate of polyp detection (>1 cm) was ∼20%.

Conclusions/Significance

Analysis of SEPT9 DNA methylation in plasma represents a straightforward, minimally invasive method to detect all stages of CRC with potential to satisfy unmet needs for increased compliance in the screening population. Further clinical testing is warranted.     

A simpler way of comparing the labelling densities of cellular compartments illustrated using data from VPARP and LAMP-1 immunogold labelling experiments

Quantitative immunoelectron microscopy of gold label in intracellular compartments often involves calculating labelling densities (LDs). These are related to antigen concentrations and usually refer gold particle counts to the sizes of compartments on sections (for example, golds per microm(2) of organelle profile area or per microm of membrane trace length). Here, we show how LD values can be estimated more simply (without estimating areas or lengths) and also how observed and expected LD values can be used to calculate a relative labelling index (RLI) for each compartment and then test statistically for preferential (non-random) labelling. For random labelling, RLI=1. Compartment size is estimated stereologically by superimposing random test points (which hit organelle profiles in proportion to their area) or test lines (which intersect membrane traces in proportion to their length). By this means, the observed LD of a compartment (LD(obs)) can be expressed simply as golds per test point (organelles) or per intersection (membranes). Furthermore, the LD obtained by dividing total golds (on all compartments) by total points or intersections (on all compartments) is the value to be expected (LD(exp)) when compartments label randomly. For each compartment, RLI=LD(obs)/LD(exp). Statistical analysis is undertaken by comparing observed distributions of golds with predicted random distributions (calculated from point or intersection counts). A compartment is preferentially labelled if two criteria are met: (1) its RLI>1 (i.e. LD(obs) is greater than LD(exp)) and (2) its partial chi-squared value makes a substantial contribution to total chi-squared value. This approach provides a simple and efficient way of comparing LDs in different compartments. Its utility is illustrated using data from VPARP and LAMP-1 labelling experiments.     

Cell vacuolization induced by Helicobacter pylori VacA cytotoxin does not depend on late endosomal SNAREs

Cellular vacuoles induced by the Helicobacter pylori vacuolating cytotoxin VacA originate from late endosomal compartments. Their biogenesis requires the activity of both rab7 GTPase and the ATPase proton pump. The toxin has been suggested to cause an increased luminal osmotic pressure via its anion-specific channel activity localized on late endosomal compartments after endocytosis. Here, we show that the extensive membrane fusion that takes place in the transition from the small late endosomal compartments to the large vacuoles does not depend on soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins. The process of vacuolization leads to disappearance of the large array of internal membranes of late endosomes. We suggest that most of the vacuole-limiting membrane derives from internal membranes.     

Homology-based functional proteomics by mass spectrometry: application to the Xenopus microtubule-associated proteome

The application of functional proteomics to important model organisms with unsequenced genomes is restricted because of the limited ability to identify proteins by conventional mass spectrometry (MS) methods. Here we applied MS and sequence-similarity database searching strategies to characterize the Xenopus laevis microtubule-associated proteome. We identified over 40 unique, and many novel, microtubule-bound proteins, as well as two macromolecular protein complexes involved in protein translation. This finding was corroborated by electron microscopy showing the presence of ribosomes on spindles assembled from frog egg extracts. Taken together, these results suggest that protein translation occurs on the spindle during meiosis in the Xenopus oocyte. These findings were made possible due to the application of sequence-similarity methods, which extended mass spectrometric protein identification capabilities by 2-fold compared to conventional methods.