Agarose-dextran gels as synthetic analogs of glomerular basement membrane: water permeability

Novel agarose-dextran hydrogels were synthesized and their suitability as experimental models of glomerular basement membrane was examined by measuring their Darcy (hydraulic) permeabilities (kappa). Immobilization of large dextran molecules in agarose was achieved by electron beam irradiation. Composite gels were made with agarose volume fractions (phi(a)) of 0.04 or 0.08 and dextran volume fractions (phi(d)) ranging from 0 to 0.02 (fiber volume/gel volume), using either of two dextran molecular weights (500 or 2000). At either agarose concentration and for either size of dextran, kappa decreased markedly as the amount of dextran was increased. Statistically significant deviations from the value of kappa for pure agarose were obtained for remarkably small volume fractions of dextran: phi(d) > or = 0.0003 for phi(a) = 0.04 and phi(d) > or = 0.001 for phi(a) = 0.08. The Darcy permeabilities were much more sensitive to phi(d) than to phi(a), and were as much as 26 times smaller than those of pure agarose. Although phi(d) was an important variable, dextran molecular weight was not. The effects of dextran addition on kappa were described fairly well using simple structural idealizations. At high agarose concentrations, the dextran chains behaved as fine fibers interspersed among coarse agarose fibrils, whereas, at low concentrations, the dextran molecules began to resemble spherical obstacles embedded in agarose gels. The ability to achieve physiologically relevant Darcy permeabilities with these materials (as low as 1.6 nm2) makes them an attractive experimental model for glomerular basement membrane and possibly other extracellular matrices.     

Engagement with HIV Prevention Treatment and Care among Female Sex Workers in Zimbabwe: a Respondent Driven Sampling Survey

Objective(S)

To determine the HIV prevalence and extent of engagement with HIV prevention and care among a representative sample of Zimbabwean sex workers working in Victoria Falls, Hwange and Mutare.

Design

Respondent driven sampling (RDS) surveys conducted at each site.

Methods

Sex workers were recruited using respondent driven sampling with each respondent limited to recruiting 2 peers. Participants completed an interviewer-administered questionnaire and provided a finger prick blood sample for HIV antibody testing. Statistical analysis took account of sampling method.

Results

870 women were recruited from the three sites. HIV prevalence was between 50 and 70%. Around half of those confirmed HIV positive were aware of their HIV status and of those 50-70% reported being enrolled in HIV care programmes. Overall only 25-35% of those with laboratory-confirmed HIV were accessing antiretroviral therapy. Among those reporting they were HIV negative, 21-28% reported having an HIV test in the last 6 months. Of those tested HIV negative, most (65-82%) were unaware of their status. Around two-thirds of sex workers reported consistent condom use with their clients. As in other settings, sex workers reported high rates of gender based violence and police harassment.

Conclusions

This survey suggests that prevalence of HIV is high among sex workers in Zimbabwe and that their engagement with prevention, treatment and care is sub-optimal. Intensifying prevention and care interventions for sex workers has the potential to markedly reduce HIV and social risks for sex workers, their clients and the general population in Zimbabwe and elsewhere in the region.     

Toward ecologically explicit null models of nestedness

A community is nested when species assemblages in less rich sites form nonrandom subsets of those at richer sites. Conventional null models used to test for statistically nonrandom nestedness are under- or over-restrictive because they do not sufficiently isolate ecological processes of interest, which hinders ecological inference. We propose a class of null models that are ecologically explicit and interpretable. Expected values of species richness and incidence, rather than observed values, are used to create random presence-absence matrices for hypothesis testing. In our examples, based on six datasets, expected values were derived either by using an individually based random placement model or by fitting empirical models to richness data as a function of environmental covariates. We describe an algorithm for constructing unbiased null matrices, which permitted valid testing of our null models. Our approach avoids the problem of building too much structure into the null model, and enabled us to explicitly test whether observed communities were more nested than would be expected for a system structured solely by species-abundance and species-area or similar relationships. We argue that this test or similar tests are better determinants of whether a system is truly nested; a nested system should contain unique pattern not already predicted by more fundamental ecological principles such as species-area relationships. Most species assemblages we studied were not nested under these null models. Our results suggest that nestedness, beyond that which is explained by passive sampling processes, may not be as widespread as currently believed. These findings may help to improve the utility of nestedness as an ecological concept and conservation tool.     

Local motions in a benchmark of allosteric proteins

Allosteric proteins have been studied extensively in the last 40 years, but so far, no systematic analysis of conformational changes between allosteric structures has been carried out. Here, we compile a set of 51 pairs of known inactive and active allosteric protein structures from the Protein Data Bank. We calculate local conformational differences between the two structures of each protein using simple metrics, such as backbone and side-chain Cartesian displacement, and torsion angle change and rearrangement in residue-residue contacts. Thresholds for each metric arise from distributions of motions in two control sets of pairs of protein structures in the same biochemical state. Statistical analysis of motions in allosteric proteins quantifies the magnitude of allosteric effects and reveals simple structural principles about allostery. For example, allosteric proteins exhibit substantial conformational changes comprising about 20% of the residues. In addition, motions in allosteric proteins show strong bias toward weakly constrained regions such as loops and the protein surface. Correlation functions show that motions communicate through protein structures over distances averaging 10-20 residues in sequence space and 10-20 A in Cartesian space. Comparison of motions in the allosteric set and a set of 21 nonallosteric ligand-binding proteins shows that nonallosteric proteins also exhibit bias of motion toward weakly constrained regions and local correlation of motion. However, allosteric proteins exhibit twice as much percent motion on average as nonallosteric proteins with ligand-induced motion. These observations may guide efforts to design flexibility and allostery into proteins.     

Btn2, a Hook1 ortholog and potential Batten disease-related protein, mediates late endosome-Golgi protein sorting in yeast

BTN2 gene expression in the yeast Saccharomyces cerevisiae is up-regulated in response to the deletion of BTN1, which encodes the ortholog of a human Batten disease protein. We isolated Btn2 as a Snc1 v-SNARE binding protein using the two-hybrid assay and examined its role in intracellular protein trafficking. We show that Btn2 is an ortholog of the Drosophila and mammalian Hook1 proteins that interact with SNAREs, cargo proteins, and coat components involved in endosome-Golgi protein sorting. By immunoprecipitation, it was found that Btn2 bound the yeast endocytic SNARE complex (e.g., Snc1 and Snc2 [Snc1/2], Tlg1, Tlg2, and Vti1), the Snx4 sorting nexin, and retromer (e.g., Vps26 and Vps35). In in vitro binding assays, recombinant His(6)-tagged Btn2 bound glutathione S-transferase (GST)-Snc1 and GST-Vps26. Btn2-green fluorescent protein and Btn2-red fluorescent protein colocalize with Tlg2, Snx4, and Vps27 to a compartment adjacent to the vacuole that corresponds to a late endosome. The deletion of BTN2 blocks Yif1 retrieval back to the Golgi apparatus, while the localization of Ste2, Fur4, Snc1, Vps10, carboxypeptidases Y (CPY) and S (CPS), Sed5, and Sec7 is unaltered in btn2Delta cells. Yif1 delivery to the vacuole was observed in other late endosome-Golgi trafficking mutants, including ypt6Delta, snx4Delta, and vps26Delta cells. Thus, Btn2 facilitates specific protein retrieval from a late endosome to the Golgi apparatus, a process which may be adversely affected in patients with Batten disease.     

The development of tone in the smooth muscle of guinea-pig isolated tracheal preparations may be influenced by prostanoids released from the adjacent airway cartilage

Guinea-pig tracheal strip preparations containing cartilage, placed under an applied load , develop tone spontaneously. The finding that spontaneous tone is reduced by indomethacin suggests that one or more prostanoids are involved in the development of spontaneous tone in this species. In this study we examined the effects of removing the cartilage component of the preparations on changes in tone induced by indomethacin and isoproterenol. In contrast to preparations containing cartilage, tissues devoid of cartilage, did not develop tone after the application of an initial 1 g resting load. Indomethacin (1 μM) reduced resting tone by 0.62 ± 0.14 g in cartilage-containing tissues but, in contrast, reduced tone by only 0.03 ± 0.01 g in tissues devoid of cartilage. Furthermore, relaxation responses (0.38 ± 0.05 g) to isoproterenol (1 μM) could be produced in cartilage-containing preparations but not in cartilage-free preparations. Radioimmunoassays indicated that the release of PGE₂, PGF_2α and 6-keto PGF_1α, the end-product of PGI₂ breakdown, was diminished in preparations lacking cartilage. Thus, in guinea-pig airway preparations cartilage is apparently a source of sufficient prostanoids to induce spontaneous tone     

Cytokine stimulation promotes glucose uptake via phosphatidylinositol-3 kinase/Akt regulation of Glut1 activity and trafficking

Cells require growth factors to support glucose metabolism for survival and growth. It is unclear, however, how noninsulin growth factors may regulate glucose uptake and glucose transporters. We show that the hematopoietic growth factor interleukin (IL)3, maintained the glucose transporter Glut1 on the cell surface and promoted Rab11a-dependent recycling of intracellular Glut1. IL3 required phosphatidylinositol-3 kinase activity to regulate Glut1 trafficking, and activated Akt was sufficient to maintain glucose uptake and surface Glut1 in the absence of IL3. To determine how Akt may regulate Glut1, we analyzed the role of Akt activation of mammalian target of rapamycin (mTOR)/regulatory associated protein of mTOR (RAPTOR) and inhibition of glycogen synthase kinase (GSK)3. Although Akt did not require mTOR/RAPTOR to maintain surface Glut1 levels, inhibition of mTOR/RAPTOR by rapamycin greatly diminished glucose uptake, suggesting Akt-stimulated mTOR/RAPTOR may promote Glut1 transporter activity. In contrast, inhibition of GSK3 did not affect Glut1 internalization but nevertheless maintained surface Glut1 levels in IL3-deprived cells, possibly via enhanced recycling of internalized Glut1. In addition, Akt attenuated Glut1 internalization through a GSK3-independent mechanism. These data demonstrate that intracellular trafficking of Glut1 is a regulated component of growth factor-stimulated glucose uptake and that Akt can promote Glut1 activity and recycling as well as prevent Glut1 internalization.     

Effects of creatine supplementation and three days of resistance training on muscle strength, power output, and neuromuscular function

Previous studies have demonstrated increases in peak torque (PT) and decreases in acceleration time (ACC) after only 2 days of resistance training, and other studies have reported improvements in isokinetic performance after 5 days of creatine supplementation. Consequently, there may be a combined benefit of creatine supplementation and short-term resistance training for eliciting rapid increases in muscle strength, which may be important for short-term rehabilitation and return-to-play for previously injured athletes. The purpose of this study, therefore, was to examine the effects of 3 days of isokinetic resistance training combined with 8 days of creatine monohydrate supplementation on PT, mean power output (MP), ACC, surface electromyography (EMG), and mechanomyography (MMG) of the vastus lateralis muscle during maximal concentric isokinetic leg extension muscle actions. Twenty-five men (mean age +/- SD = 21 +/- 3 years, stature = 177 +/- 6 cm, and body mass = 80 +/- 12 kg) volunteered to participate in this 9-day, double-blind, placebo-controlled study and were randomly assigned to either the creatine (CRE; n = 13) or placebo (PLA; n = 12) group. The CRE group ingested the treatment drink (280 kcal; 68 g carbohydrate; 10.5 g creatine), whereas the PLA group received an isocaloric placebo (70 g carbohydrate). Two servings per day (morning and afternoon) were administered in the laboratory on days 1-6, with only 1 serving on days 7-8. Before (pre; day 1) and after (post; day 9) the resistance training, maximal voluntary concentric isokinetic leg extensions at 30, 150, and 270 degrees x s(-1) were performed on a calibrated Biodex System 3 dynamometer. Three sets of 10 repetitions at 150 degrees x s(-1) were performed on days 3, 5, and 7. Peak torque increased (p = 0.005; eta(2) = 0.296), whereas ACC decreased (p < 0.001; eta(2) = 0.620), from pretraining to posttraining for both the CRE and PLA groups at each velocity (30, 150, and 270 degrees x s(-1)). Peak torque increased by 13% and 6%, whereas ACC decreased by 42% and 34% for the CRE and PLA groups, respectively, but these differences were not statistically significant (p > 0.05). There were no changes in MP, EMG, or MMG amplitude; however, EMG median frequency (MDF) increased, and MMG MDF increased at 30 degrees x s(-1), from pretraining to posttraining for both the CRE and PLA groups. These results indicated that 3 days of isokinetic resistance training was sufficient to elicit small, but significant, improvements in peak strength (PT) and ACC for both the CRE and PLA groups. Although the greater relative improvements in PT and ACC for the CRE group were not statistically significant, these findings may be useful for rehabilitation or strength and conditioning professionals who may need to rapidly increase the strength of a patient or athlete within 9 days.     

Validation of power measurement techniques in dynamic lower body resistance exercises

The objective of this study was to investigate the validity of power measurement techniques utilizing various kinematic and kinetic devices during the jump squat (JS), squat (S) and power clean (PC). Ten Division I male athletes were assessed for power output across various intensities: 0, 12, 27, 42, 56, 71, and 85% of one repetition maximum strength (1RM) in the JS and S and 30, 40, 50, 60, 70, 80, and 90% of 1RM in the PC. During the execution of each lift, six different data collection systems were utilized; (1) one linear position transducer (1-LPT); (2) one linear position transducer with the system mass representing the force (1-LPT+MASS); (3) two linear position transducers (2-LPT); (4) the force plate (FP); (5) one linear position transducer and a force plate (1-LPT+FP); (6) two linear position transducers and a force place (2-LPT+FP). Kinetic and kinematic variables calculated using the six methodologies were compared. Vertical power, force, and velocity differed significantly between 2-LPT+FP and 1-LPT, 1-LPT+MASS, 2-LPT, and FP methodologies across various intensities throughout the JS, S, and PC. These differences affected the load-power relationship and resulted in the transfer of the optimal load to a number of different intensities. This examination clearly indicates that data collection and analysis procedures influence the power output calculated as well as the load-power relationship of dynamic lower body movements.