HIV-1 Nef binds the DOCK2-ELMO1 complex to activate rac and inhibit lymphocyte chemotaxis

The infectious cycle of primate lentiviruses is intimately linked to interactions between cells of the immune system. Nef, a potent virulence factor, alters cellular environments to increase lentiviral replication in the host, yet the mechanisms underlying these effects have remained elusive. Since Nef likely functions as an adaptor protein, we exploited a proteomic approach to directly identify molecules that Nef targets to subvert the signaling machinery in T cells. We purified to near homogeneity a major Nef-associated protein complex from T cells and identified by mass spectroscopy its subunits as DOCK2–ELMO1, a key activator of Rac in antigen- and chemokine-initiated signaling pathways, and Rac. We show that Nef activates Rac in T cell lines and in primary T cells following infection with HIV-1 in the absence of antigenic stimuli. Nef activates Rac by binding the DOCK2–ELMO1 complex, and this interaction is linked to the abilities of Nef to inhibit chemotaxis and promote T cell activation. Our data indicate that Nef targets a critical switch that regulates Rac GTPases downstream of chemokine- and antigen-initiated signaling pathways. This interaction enables Nef to influence multiple aspects of T cell function and thus provides an important mechanism by which Nef impacts pathogenesis by primate lentiviruses.     

Predictors of mortality of patients with acute respiratory failure secondary to chronic obstructive pulmonary disease admitted to an intensive care unit: A one year study

Background

Patients with acute exacerbation of chronic obstructive pulmonary disease (COPD) commonly require hospitalization and admission to intensive care unit (ICU). It is useful to identify patients at the time of admission who are likely to have poor outcome. This study was carried out to define the predictors of mortality in patients with acute exacerbation of COPD and to device a scoring system using the baseline physiological variables for prognosticating these patients.

Methods

Eighty-two patients with acute respiratory failure secondary to COPD admitted to medical ICU over a one-year period were included. Clinical and demographic profile at the time of admission to ICU including APACHE II score and Glasgow coma scale were recorded at the time of admission to ICU. In addition, acid base disorders, renal functions, liver functions and serum albumin, were recorded at the time of presentation. Primary outcome measure was hospital mortality.

Results

Invasive ventilation was required in 69 patients (84.1%). Fifty-two patients survived to hospital discharge (63.4%). APACHE II score at the time of admission to ICU {odds ratio (95 % CI): 1.32 (1.138–1.532); p < 0.001} and serum albumin (done within 24 hours of admission) {odds ratio (95 % CI): 0.114 (0.03-0.432); p = 0.001}. An equation, constructed using the adjusted odds ratio for the two parameters, had an area under the ROC curve of 91.3%. For the choice of cut-off, sensitivity, specificity, positive and negative predictive value for predicting outcome was 90%, 86.5%, 79.4% and 93.7%.

Conclusion

APACHE II score at admission and SA levels with in 24 hrs after admission are independent predictors of mortality for patients with COPD admitted to ICU. The equation derived from these two parameters is useful for predicting outcome of these patients.     

Markers of small cell lung cancer

Lung cancer is the number one cause of cancer death; however, no specific serum biomarker is available till date for detection of early lung cancer. Despite good initial response to chemotherapy, small-cell lung cancer (SCLC) has a poor prognosis. Therefore, it is important to identify molecular markers that might influence survival and may serve as potential therapeutic targets. The review aims to summarize the current knowledge of serum biomarkers in SCLC to improve diagnostic efficiency in the detection of tumor progression in lung cancer. The current knowledge on the known serum cytokines and tumor biomarkers of SCLC is emphasized. Recent findings in the search for novel diagnostic and therapeutic molecular markers using the emerging genomic technology for detecting lung cancer are also described. It is believed that implementing these new research techniques will facilitate and improve early detection, prognostication and better treatment of SCLC.     

A fractal analysis of analyte-estrogen receptor binding and dissociation kinetics using biosensors: environmental and biomedical effects

A fractal analysis is used to model the binding and dissociation kinetics between analytes in solution and estrogen receptors (ERs) immobilized on a sensor chip of a surface plasmon resonance (SPR) biosensor. The influence of different ligands is also analyzed. A better understanding of the kinetics provides physical insights into the interactions, and suggests means by which appropriate interactions (to promote correct signaling) and inappropriate interactions such as with xenoestrogens (to minimize inappropriate and deleterious to health signaling) may be better controlled. The fractal approach is applied to analyte–ER interaction data available in the literature. The units for the different parameters (rate coefficients and affinities) in fractal-type kinetics are different from those obtained in classical kinetics. Numerical values obtained for the binding and the dissociation rate coefficients are linked to the degree of roughness or heterogeneity (fractal dimension, D_f) present on the biosensor chip surface. In general, the binding and the dissociation rate coefficients are very sensitive to the degree of heterogeneity on the surface. A single-fractal analysis is adequate in some cases. In others (that exhibit complexities in the binding or the dissociation curves) a dual-fractal analysis is required to obtain a better fit. This has biomedical and environmental implications in that the dissociation (and the binding) rate coefficient may be used to alleviate (deleterious effects) or enhance (beneficial effects) by selective modulation of the surface. The affinity values obtained in the analysis are consistent with the numbers required to (a) promote signaling between the correct analyte and the estrogen receptor, and (b) minimize the signaling between xenoestrogens and the estrogen receptor.     

Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.     

Novel branched poly(ethylenimine)-cholesterol water-soluble lipopolymers for gene delivery

A novel water-soluble lipopolymer was synthesized by linking cholesteryl chloroformate to the secondary amino groups of branched poly(ethylenimine) (PEI) of 1,800 and 10,000 Da. Conjugation through PEI secondary amines gives this newly synthesized lipopolymer (abbreviated as PEI-Chol) special advantage over our previously synthesized lipopolymers, which utilized the primary amino groups for conjugation, as the primary amino groups have a significant role in DNA condensation. Also, significantly, only one cholesterol molecule was grafted onto each PEI molecule (confirmed by (1)H NMR and MALDI-TOF mass spectrometry), leaving enough space for the steric interactions of the PEI's primary amines with the DNA. The PEI-Chol lipopolymer was characterized for the critical micellar concentration (cmc), buffer capacity, DNA condensation (by band retardation and circular dichroism), in vitro transfection efficiency, and cell viability. The cmcs of PEI-Chol 1,800 and PEI-Chol 10,000 were 496.6 and 1,330.5 microg/mL, respectively. The acid-base titration indicated high buffering capacity of the polymers around the pH range of 5-7, which indicated their potential for buffering in the acidic pH environment of the endosomes. The band retardation studies indicated that efficient condensation of the plasmid DNA could be achieved using these lipopolymers. The circular dichroism spectra indicated a change in DNA conformation and adoption of lower energy state upon condensation with these lipopolymers when an N/P ratio of 2.5/1 or above was formulated. The mean particle size of these complexes was in the range 110-205 nm, except for the complexes prepared using PEI of 1,800 Da, which had a mean particle size of 384 +/- 300 nm. The zeta potential of DNA complexes prepared using PEI-Chol 1,800, PEI-Chol 10,000 and PEI of 1,800, 10,000, and 25,000 Da at an N/P ratio of 15/1 was in the range 23-30 mV and was dependent on the N/P ratios. The in vitro transfection of PEI-Chol/pCMS-EGFP complexes in Jurkat cells showed high levels of expressed Green Fluorescent Protein (GFP) with little toxicity as determined by flow cytometry. These novel water-soluble lipopolymers provided good transfection efficiency with other desirable characteristics such as water solubility, free primary amino groups for efficient DNA condensation and high buffer capacity that indicated the possibility of efficient endosomal release.     

An analysis of turbulent shear stresses in leakage flow through a bileaflet mechanical prostheses

In this work, estimates of turbulence were made from pulsatile flow laser Doppler velocimetry measurements using traditional phase averaging and averaging after the removal of cyclic variation. These estimates were compared with estimates obtained from steady leakage flow LDV measurements and an analytical method. The results of these studies indicate that leakage jets which are free and planar in shape may be more unstable than other leakage jets, and that cyclic variation does not cause a gross overestimation of the Reynolds stresses at large distances from the leakage jet orifice.     

Mechanism of incomplete mitral leaflet coaptation--interaction of chordal restraint and changes in mitral leaflet coaptation geometry. Insight from in vitro validation of the premise of force equilibrium

Clinically observed incomplete mitral leaflet coaptation was reproduced in vitro by altering the balance of the chordal tethering and chordal coapting force components. Mitral leaflet coaptation geometry was distorted by changes of the spatial relations between the papillary muscles and the mitral valve as well as hemodynamics. Mitral leaflet malalignment was accentuated by a redistribution of the chordal tethering and coapting force components. For the overall assessment of systolic mitral leaflet configuration in functional mitral regurgitation it is important to consider the interaction between chordal restraint and an altered mitral leaflet coaptation geometry.