Inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent HIV-1 entry

We previously reported that monoclonal antibodies to protein-disulfide isomerase (PDI) and other membrane-impermeant PDI inhibitors prevented HIV-1 infection. PDI is present at the surface of HIV-1 target cells and reduces disulfide bonds in a model peptide attached to the cell membrane. Here we show that soluble PDI cleaves disulfide bonds in recombinant envelope glycoprotein gp120 and that gp120 bound to the surface receptor CD4 undergoes a disulfide reduction that is prevented by PDI inhibitors. Concentrations of inhibitors that prevent this reduction and inhibit the cleavage of surface-bound disulfide conjugate prevent infection at the level of HIV-1 entry. The entry of HIV-1 strains differing in their coreceptor specificities is similarly inhibited, and so is the reduction of gp120 bound to CD4 of coreceptor-negative cells. PDI inhibitors also prevent HIV envelope-mediated cell-cell fusion but have no effect on the entry of HIV-1 pseudo-typed with murine leukemia virus envelope. Importantly, PDI coprecipitates with both soluble and cellular CD4. We propose that a PDI.CD4 association at the cell surface enables PDI to reach CD4-bound virus and to reduce disulfide bonds present in the domain of gp120 that binds to CD4. Conformational changes resulting from the opening of gp120-disulfide loops may drive the processes of virus-cell and cell-cell fusion. The biochemical events described identify new potential targets for anti-HIV agents.     

N-linked glycosylation of CXCR4 masks coreceptor function for CCR5-dependent human immunodeficiency virus type 1 isolates

The chemokine receptors CXCR4 and CCR5 are the principal coreceptors for infection of X4 and R5 human immunodeficiency virus type 1 (HIV-1) isolates, respectively. Here we report on the unexpected observation that the removal of the N-linked glycosylation sites in CXCR4 potentially allows the protein to serve as a universal coreceptor for both X4 and R5 laboratory-adapted and primary HIV-1 strains. We hypothesize that this alteration unmasks existing common extracellular structures reflecting a conserved three-dimensional similarity of important elements of CXCR4 and CCR5 that are involved in HIV envelope glycoprotein (Env) interaction. These results may have far-reaching implications for the differential recognition of cell type-dependent glycosylated CXCR4 by HIV-1 isolates and their evolution in vivo. They also suggest a possible explanation for the various observations of restricted virus entry in some cell types and further our understanding of the framework of elements that represent the Env-coreceptor contact sites.     

The Mechanism of Henipavirus Fusion： Examining the Relationships between the Attachment and Fusion Glycoproteins

The henipaviruses, represented by Nipah virus and Hendra virus, are emerging zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These viruses enter host cells via a class I viral fusion mechanism mediated by their attachment and fusion envelope glycoproteins; efficient membrane fusion requires both these glycoproteins in conjunction with specific virus receptors present on susceptible host cells. The henipavirus attachment glycoprotein interacts with a cellular B class ephrin protein receptor triggering conformational alterations leading to the activation of the viral fusion (F) glycoprotein. The analysis of monoclonal antibody (mAb) reactivity with G has revealed measurable alterations in the antigenic structure of the glycoprotein following its binding interaction with receptor. These observations only appear to occur with full-length native G glycoprotein, which is a tetrameric oligomer, and not with soluble forms of G (sG), which are disulfide-linked dimers. Single amino acid mutations in a heptad repeat-like structure within the stalk domain of G can disrupt its association with F and subsequent membrane fusion promotion activity. Notably, these mutants of G also appear to confer a postreceptor bound conformation implicating the stalk domain as an important element in the G glycoprotein's structure and functional relationship with F. Together, these observations suggest fusion is dependent on a specific interaction between the F and G glycoproteins of the henipaviruses. Further, receptor binding induces measurable changes in the G glycoprotein that appear to be greatest in respect to the interactions between the pairs of dimers comprising its native tetrameric structure. These receptor-induced conformational changes may be associated with the G glycoprotein's promotion of the fusion activity of F.     

Bias and Sampling Error of the Estimated Proportion of Genotypic Variance Explained by Quantitative Trait Loci Determined From Experimental Data in Maize Using Cross Validation and Validation With Independent Samples.

Cross validation (CV) was used to analyze the effects of different environments and different genotypic samples on estimates of the proportion of genotypic variance explained by QTL (p). Testcrosses of 344 F(3) maize lines grown in four environments were evaluated for a number of agronomic traits. In each of 200 replicated CV runs, this data set was subdivided into an estimation set (ES) and various test sets (TS). ES were used to map QTL and estimate p for each run (p(ES)) and its median (p(ES)) across all runs. The bias of these estimates was assessed by comparison with the median (p(TS.ES)) obtained from TS. We also used two independent validation samples derived from the same cross for further comparison. The median p(ES) showed a large upward bias compared to p(TS.ES). Environmental sampling generally had a smaller effect on the bias of p(ES) than genotypic sampling or both factors simultaneously. In independent validation, p(TS.ES) was on average only 50% of p(ES). A wide range among p(ES) reflected a large sampling error of these estimates. QTL frequency distributions and comparison of estimated QTL effects indicated a low precision of QTL localization and an upward bias in the absolute values of estimated QTL effects from ES. CV with data from three QTL studies reported in the literature yielded similar results as those obtained with maize testcrosses. We therefore recommend CV for obtaining asymptotically unbiased estimates of p and consequently a realistic assessment of the prospects of MAS.     

Inter-Observer Agreement in Measuring Respiratory Rate

Background

Respiratory rate (RR) is an important vital sign which is strongly correlated with in-hospital mortality. At the same time, RR is the most likely vital sign to be omitted when assessing a patient. We believe that one reason for this could be the difficulty in measure the RR, since it is not read off a monitor, but counted manually. Also there is the possibility of assessment bias and the inter-observer reliability becomes important. We therefore set out to investigate how the nursing staff counting the actual number of respirations per minute would agree with the nursing staff using a predefined ordinal scale.

Methods

For this prospective study, we recorded five videos of a young healthy man breathing approximately 5, 10, 15, 30 and 60 times per minute. The videos were shown in a random order to a suitable sample of the nursing staff. The participants were randomized into two groups; one to count the exact number of breaths per minute, and one to use a predefined ordinal scale.

Results

Comparing the exact number of breaths per minute, the Intra Class Coefficient (ICC) was 0.99 (95% CI: 0.97–1.00). Comparing the RR using the predefined scale, the overall Kappa Fleiss Coefficient was 0.75.

Conclusions

The inter-observer agreement was high when comparing the use of the actual number of breaths per minute and substantial when comparing the use of the predefined scale. This is the largest inter-observer study on RR to date. However, further studies on the use of scaled comparisons of RR are needed.     

Novel CAD-like enzymes from Escherichia coli K-12 as additional tools in chemical production

In analyzing the reductive power of Escherichia coli K-12 for metabolic engineering approaches, we identified YahK and YjgB, two medium-chain dehydrogenases/reductases subgrouped to the cinnamyl alcohol dehydrogenase family, as being important. Identification was achieved using a stepwise purification protocol starting with crude extract. For exact characterization, the genes were cloned into pET28a vector and expressed with N-terminal His tag. Substrate specificity studies revealed that a large variety of aldehydes but no ketones are converted by both enzymes. YahK and and YjgB strongly preferred NADPH as cofactor. The structure of YjgB was modeled using YahK as template for a comparison of the active center giving a first insight to the different substrate preferences. The enzyme activity for YahK, YjgB, and YqhD was determined on the basis of the temperature. YahK showed a constant increase in activity until 60 °C, whereas YjgB was most active between 37 and 50 °C. YqhD achieved the highest activity at 50 °C. Comparing YjgB and Yahk referring to the catalytic efficiency, YjgB achieved for almost all substrates higher rates (butyraldehyde 221 s^?1?mM^?1, benzaldehyde 1,305 s^?1?mM^?1). Exceptions are the two substrates glyceraldehydes (no activity for YjgB) and isobutyraldehyde (YjgB 0.26 s^?1?mM^?1) which are more efficiently converted by YahK (glyceraldehyde 2.8 s^?1?mM^?1, isobutyraldehyde 14.6 s^?1?mM^?1). YahK and even more so YjgB are good candidates for the reduction of aldehydes in metabolic engineering approaches and could replace the currently used YqhD.     

Remote sensing as a data source to analyse regional implications of genetically modified plants in agriculture—Oilseed rape (Brassica napus) in Northern Germany

Remote sensing permits the identification of locations and area extent where particular crops are cultivated across larger regions. This requires the availability of crop- and region-specific algorithms. We developed an approach to identify oilseed rape fields in Northern Germany. The remote sensing data sources and main processing steps are described. The derived cultivation density information provides an overview of the fine-scale spatial structure and crop neighbourhood relations in Northern Germany as one of the main oilseed rape cultivation regions in Europe. Geographical Information System (GIS) analyses involving buffer operations allowed to identify those parts of the region, where the highest interaction potential of GM crops and conventional crops would occur if GM varieties were admitted for cultivation. Cultivation density and field sizes in combination were used to indicate the interaction intensity as a marker for observation requirement (environmental monitoring) and for those potential risks that relate to density parameter. Conclusions can be drawn for the feasibility of coexistence measures when neighbouring farmers have to co-operate to keep separation distances between GM crop cultivation and conventional varieties. Together with other data sources, the results of satellite image analysis can be used as input data for up-scaling small-scale model results. Remote sensing data allow to specify, which field density parameter must be chosen to cover the regional variability of cultivation conditions, in particular the regional distribution of field sizes and field density.