Effects of a calcium supplement on bone mineral density in male cyclists.

AIM: The purpose of this study was to maintain or improve bone density in male road cyclists through provision of calcium and vitamin D3 supplementation ingested before cycling. METHODS: Participants were male cyclists (N=17), with a mean (±SD) age of 42.7 (9.4) years. Measurements of lumbar spine and hip areal bone mineral density (aBMD) were performed at the start and end of a cycling season. Cyclists were randomized into the calcium supplement (CAL) or the control group (CON) group based on lumbar spine T-scores. The CAL group was instructed to consume 1600 mg calcium and 1000 IU vitamin D3 prior to cycling for the 5-month period. RESULTS: Femoral trochanter aBMD significantly decreased during the 5 month cycling season. There was no difference in aBMD between CAL and CON groups. CONCLUSION: Negative effects of competitive cycling on aBMD in hip structures can be observed within 5 months. Calcium and vitamin D3 ingested prior to cycling does not ameliorate this effect. This proof of concept paper provides evidence that more work is needed to find mechanisms to protect cyclists from destructive bone loss in hip structures.

Effect of flow rate, humidifier dome and water volume on maximising heated, humidified gas use for neonatal resuscitation.

AIM: Dry, cold gas is used for neonatal resuscitation, contributing to low admission temperatures and exacerbation of lung injury. Recently, a method of heating and humidifying neonatal resuscitation gases has become available. We aimed to determine the optimal flow rate, humidifier chamber and water volume needed to reach 36°C, and near 100% humidity at the patient T-piece in the shortest possible time. METHOD: A T-piece resuscitator was connected via a heated patient circuit to a humidifier chamber. Trials were performed using different gas flow rates (6, 8 and 10L/min), humidification chambers (MR290, MR225) and water volumes (30g, 108g). Temperature was recorded at the humidifier chamber (T1), distal temperature probe (T2) and the T-piece (T3) over a 20min period at 30s intervals. A test lung was added during one trial. RESULTS: No significant difference existed between flow rates 8L/min and 10L/min (p=0.091, p=0.631). T3 reached 36°C and remained stable at 360s (8L/min, MR225, 30mL); near 100% RH was reached at 107s (10L/min, MR225, 30mL). T3 and humidity reached and remained stable at 480s (10L/min, MR290, 30mL). Target temperature and humidity was not reached with the test lung. CONCLUSIONS: It is possible to deliver heated, humidified gases in neonatal resuscitation in a clinically acceptable timeframe. We suggest the set-up to achieve optimal temperature and humidity for resuscitation purposes is 10L/min of gas flow, a MR290 humidification chamber, and 30mL of water.

Key harm reduction interventions and their impact on the reduction of risky behavior and HIV incidence among people who inject drugs in low-income and middle-income countries.

PURPOSE OF REVIEW: HIV infection rates continue to rise among people who inject drugs (PWID) in many lower- and middle-income countries (LMICs). Although progress is being made in prevention and care for PWID in some settings, coverage of essential services remains low. This article reviews the evidence for the benefits of scaling up key interventions as a combination prevention and treatment package for PWID. RECENT FINDINGS: WHO defined a comprehensive package of nine interventions for PWID, of which the following four have evidence for effectiveness in reducing HIV incidence: needle and syringe programs (NSP), medication-assisted therapy (MAT), antiretroviral therapy (ART), and HIV counseling and testing (HCT). Coverage of these interventions among PWID in LMICs varies from low (≤20%) to medium (>20-60%). At least a 60% coverage is likely to be required to reduce HIV incidence. Evidence from LMIC contexts suggests that NSP and MAT can reduce high-risk injecting behavior, HCT can reduce risky sexual behavior and ART can plausibly have preventive benefit among PWID for onward parenteral transmission with clearer evidence that antiretroviral therapy (ARV) can prevent onward sexual transmission. Modeling analysis suggests that compared with current low coverage, a scale-up of these four interventions in combination would be a beneficial and cost-effective approach. SUMMARY: The continuation of significant HIV incidence among PWID in LMIC settings is avoidable with the implementation of immediate scale-up of key harm reduction and ARV treatment interventions. Policymakers should address the structural and resource allocation barriers to allow this scale-up to occur.

Sendai virus-mediated CFTR gene transfer to the airway epithelium.

The potential for gene therapy to be an effective treatment for cystic fibrosis has been hampered by the limited gene transfer efficiency of current vectors. We have shown that recombinant Sendai virus (SeV) is highly efficient in mediating gene transfer to differentiated airway epithelial cells, because of its capacity to overcome the intra- and extracellular barriers known to limit gene delivery. Here, we have identified a novel method to allow the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA sequence to be inserted within SeV (SeV-CFTR). Following in vitro transduction with SeV-CFTR, a chloride-selective current was observed using whole-cell and single-channel patch-clamp techniques. SeV-CFTR administration to the nasal epithelium of cystic fibrosis (CF) mice (Cftr(G551D) and Cftr(tm1Unc)TgN(FABPCFTR)#Jaw mice) led to partial correction of the CF chloride transport defect. In addition, when compared to a SeV control vector, a higher degree of inflammation and epithelial damage was found in the nasal epithelium of mice treated with SeV-CFTR. Second-generation transmission-incompetent F-deleted SeV-CFTR led to similar correction of the CF chloride transport defect in vivo as first-generation transmission-competent vectors. Further modifications to the vector or the host may make it easier to translate these studies into clinical trials of cystic fibrosis.

Tenofovir and abacavir combination therapy: lessons learned from an urban clinic population.

Regimens containing abacavir (ABC), tenofovir (TDF), and lamivudine (3TC) have recently been demonstrated to have high failure rates. This poses a clinical dilemma of how to manage patients currently being treated with other regimens containing tenofovir/abacavir. We evaluated the outcomes of tenofovir/abacavir regimens in our clinical practice through a retrospective review of 2655 charts. Two hundred patients (7%) were on a tenofovir/abacavir-containing regimen. Fifty-nine patients met the criteria for analysis and were grouped into three groups: (1) antiretroviral naïve, (2) virally suppressed patients switched to TDF/ABC, and (3) patients with failure of their first antiretroviral regimen. Rates of viral suppression in the naïve, switch, and first-failure groups were 95%, 86%, and 46%, respectively. In the first-failure group, viral suppression was 66% without and 18% with a preexisting M184V. A composite analysis of the groups revealed a success rate of 86% when the regimen contained zidovudine (ZDV) and 62% when it did not. No K65R mutations were noted. These findings support continued caution in the use of TDF/ABC in combination. However, these data suggest that this combination may be successfully used in selected situations such as in combination with ZDV. In patients already virally suppressed on a TDF/ABC-containing regimen, considerations include continuing the regimen or adding zidovudine, in the attempt to protect against the development of a K65R mutation and/or virologic failure, versus changing a stable regimen.

A simple and inexpensive particle agglutination test to distinguish recent from established HIV-1 infection.

OBJECTIVES: We sought to modify the Serodia HIV-1/HIV-2 particle agglutination assay (PA), a simple and cost-effective HIV assay that is used globally for the detection of HIV antibodies, as a sensitive/less sensitive test (S/LS) to identify recently infected individuals and to estimate HIV incidence. METHODS: The Serodia PA test was modified as an S/LS test (PA-LS) by using HIV antigen-coated gelatin particles at a dilution of 1:68 and a specific diluent, and calibrated using 37 HIV clade B seroconversion panels (309 samples) from Trinidad and from a commercial source that were tested at dilution intervals from 1:10 to 1:80,000. The greatest sensitivity for correctly classifying samples from recent and established infections was determined by receiver operator curve (ROC) analysis. RESULTS: At a 1:40,000 sample dilution and a days post-seroconversion cutoff of 190 days, the PA-LS test yielded a 97% sensitivity for classifying recent and established infection samples. Furthermore, at a 1:20,000 dilution, the positive predictive value for correctly identifying recently infected individuals was 99%. The PA-LS test offers a 30-44-fold cost saving over currently available S/LS tests. CONCLUSION: A modified, low cost and simple-to-perform PA test is appropriate for use in resource-limited countries, and has exhibited excellence in distinguishing recent from established HIV infection.

Use of ultrasound to enhance nonviral lung gene transfer in vivo.

We have assessed if high-frequency ultrasound (US) can enhance nonviral gene transfer to the mouse lung. Cationic lipid GL67/pDNA, polyethylenimine (PEI)/pDNA and naked plasmid DNA (pDNA) were delivered via intranasal instillation, mixed with Optison microbubbles, and the animals were then exposed to 1 MHz US. Addition of Optison alone significantly reduced the transfection efficiency of all three gene transfer agents. US exposure did not increase GL67/pDNA or PEI/pDNA gene transfer compared to Optison-treated animals. However, it increased naked pDNA transfection efficiency by approximately 15-fold compared to Optison-treated animals, suggesting that despite ultrasound being attenuated by air in the lung, sufficient energy penetrates the tissue to increase gene transfer. US-induced lung haemorrhage, assessed histologically, increased with prolonged US exposure. The left lung was more affected than the right and this was mirrored by a lesser increase in naked pDNA gene transfer, in the left lung. The positive effect of US was dependent on Optison, as in its absence US did not increase naked pDNA transfection efficiency. We have thus established proof of principle that US can increase nonviral gene transfer, in the air-filled murine lung.

Mechanism of efficient transfection of the nasal airway epithelium by hypotonic shock.

The main barrier to gene transfer in the airway epithelium is the low rate of apical endocytosis limiting naked DNA uptake. Deionized water is known to stimulate the exocytosis of numerous intracellular vesicles during hypotonic cell swelling, in order to expand plasma membrane and prevent cell lysis. This is followed by the phase of regulatory volume decrease (RVD), during which the excess plasma membrane is retrieved by intensive endocytosis. Here we show that the more hypotonic the DNA solution, the higher the transfection of the nasal tissue. P2 receptors are known to be involved in RVD and we demonstrate that some P2 agonists and a P2 antagonist impair transfection in a time-dependent manner. Our study strongly suggests that the nasal airway epithelial cells take up plasmid DNA in deionized water during RVD, within approximately half an hour. Our simple gene delivery system may constitute a promising method for respiratory tract gene therapy.

Effect of ethanol on the interaction between poly(vinylpyrrolidone) and sodium dodecyl sulfate.

The effect of ethanol on the interaction between the anionic surfactant sodium dodecyl sulfate (SDS) and the nonionic polymer poly(vinylpyrrolidone) (PVP) has been investigated using a range of techniques including surface tension, fluorescence, electron paramagnetic resonance (EPR), small-angle neutron scattering (SANS), and viscosity. Surface tension and fluorescence studies show that the critical micelle concentration (cmc) of the surfactant decreases to a minimum value around 15 wt % ethanol; that is, it follows the cosurfactant effect. However, in the presence of PVP, the onset of the interaction, denoted cmc(1), between the surfactant and the polymer is considerably less dependent on ethanol concentration. The saturation point, cmc(2), however, reflects the behavior of the cmc in that it decreases upon addition of ethanol. This results in a decrease in the amount of surfactant bound to the polymer [C(bound) = cmc(2) - cmc] at saturation. The viscosity of simple PVP solutions depends on ethanol concentration, but since SANS studies show that ethanol has no effect on the polymer conformation, the changes observed in the viscosity reflect the viscosity of the background solvent. There are significant increases in bulk viscosity when the surfactant is added, and these have been correlated with the polymer conformation extracted from an analysis of the SANS data and with the amount of polymer adsorbed at the micelle surface. Competition between ethanol and PVP to occupy the surfactant headgroup region exists; at low ethanol concentration, the PVP displaces the ethanol and the PVP/SDS complex resembles that formed in the absence of the ethanol. At higher ethanol contents, the polymer does not bind to the ethanol-rich micelle surface.

Custom skiing and trekking adaptations for a trans-tibial and trans-radial quadrilateral amputee.

A keen skier who is a trans-tibial and trans-radial quadrilateral amputee sought an improved adaptation for skiing from the Rehabilitation Engineering Service in Edinburgh. The unpredictable nature of the bending moments and loads that can be imposed on the prostheses during skiing raised concern about the suitability of standard prosthetic components for this purpose. The authors report a ski boot modification that incorporates mechanical protection for the standard prosthetic components and a description of the custom-adapted alpine trekking sticks used also as ski poles. Reference is made to the role of risk assessment, the design and manufacture in providing this type of custom-made rehabilitation device.

Site-directed mutagenesis of amino acids in the cytoplasmic loop 6/7 of Na,K-ATPase.

The loop between transmembrane helices 6 and 7 (L6/7) of P-type ATPases has been suggested to be important for the functional linkage of ion binding and enzyme phosphorylation or to be a site of initial cation binding. To investigate the role of L6/7 in Na,K-ATPase, alanine substitutions were made for charged and conserved residues in L6/7 of the human alpha1 subunit and the proteins were expressed in yeast for analysis. All mutants except the triple mutant E825A/E828A/D830A bound ouabain. Although the equilibrium dissociation constant for ouabain binding by most mutants was similar to the wild-type value, the K(d) of R837A for ouabain binding was approximately 15-fold higher than the wild-type K(d). (18)O exchange measurements indicated that the apparent affinity of this mutant for Pi was reduced about 3-fold. The concentration dependence of KCl inhibition of ouabain binding or of NaCl inhibition of ouabain binding revealed 2-4-fold changes in the apparent affinity for cations in the E825A, E828A, and R837A mutants. The E825A and E828A mutants lost the ability to bind ouabain after extraction with 0.1% SDS or after brief heating, indicating that these mutations affected the stability of the enzyme. The ATPase activity of the other mutants was measured after extraction of crude yeast membranes with 0.1% SDS. For all mutants except R834A, R837A, and R848A, the activity was at least 50% of wild-type activity.

Improved classification of recent HIV-1 infection by employing a two-stage sensitive/less-sensitive test strategy.

Current serologic techniques for the classification of recent HIV-1 infection produce some misclassifications, and, together with the loss to follow-up of individuals, results in decreased enrollment of HIV-infected persons into appropriate intervention programs. We report on the development of a sensitive/less sensitive (S/LS) test strategy that includes a rapid assay to quickly identify persons most likely to have recent infection, followed by an enzyme immunoassay (EIA) with exquisite specificity. The Uni-Gold Recombigen HIV rapid assay (UG; Trinity Biotech, Dublin, Ireland) was procedurally-modified and calibrated as an LS test to differentiate recent (<133 days) from established HIV infections using 178 samples from persons with known dates of infection. This method correctly classified 83.0% of recent infections, but with a high misclassification rate of persons with established infection. By performing the rapid test followed by a modified S/LS EIA, the positive predictive value of the combined results for recent infections was increased to 100%. This two-stage testing algorithm can result in an increased efficiency for the enrollment of recent infection cases over a standard EIA S/LS method alone due to provisional enrollment during an initial testing visit, and because of an increased accuracy for identifying truly recent infections. We conclude that the rapid S/LS assay provides a tool for capturing recent infection cases quickly and is particularly valuable in resource-limited settings, and that the two-stage strategy provides a more accurate identification of persons with recent HIV infection.

Development of segments and appendages in embryos of the desert scorpion Paruroctonus mesaensis (Scorpiones: Vaejovidae).

The scanning electron microscope was used to study the changing features of scorpion embryos from the blastula through early stages in the development of appendages. The earliest scorpion fossils (Silurian period) have structures more advanced than the embryos herein, so the possibility is considered that these embryos still retain and display some features indicative of evolutionary patterns in adult pre-Silurian ancestors. The blastodisc stage is followed by a knob-like germinal center that gives rise to most of the embryo body. The germinal center elongates on the ventral surface of the spherical yolk mass. The broad cephalic lobe is first delineated from the following pedipalpal segment. The limbbuds for the pedipalps and anterior walking legs appear, as additional segments are added at a growth zone at the rear of the embryo body. Initially, in the cephalic lobe there are no limbbuds; then the cheliceral buds emerge from the posterior part of the lobe. The stomodeum appears first in the anterior half of the cephalic lobe, but an oral groove forms and the mouth is displaced posteriorly within the groove. This repositioning allows space anteriorly for invagination (semilunar grooves) of epithelium for the brain and medial eyes. The mouth is directed ventrally in all stages of this study. The widespread chelicerae are initially posterior to the mouth, but later move anterior and dorsal to it. Small limbbud bulges on mesosomal segments disappear later and never become protruding appendages. Metasomal segments are produced free from the yolk surface in a ventral flexure beneath the embryo body. The telson starts as two spherical lobes, but later elongates and tapers distally, not yet developing the sharp sting (aculeus) seen in Silurian and all subsequent scorpions. The walking legs are digitigrade, as in most fossil aquatic scorpions. Segments are delineated in the appendages; the chelicerae and pedipalps are divided distally for chela (claw) formation. Bilateral swellings (limbbuds) on the third abdominal segment become larger than the others, indicating the site of pectine formation. The early fin-like pectines are somewhat posterior in the mesosoma, suggesting ancestral swimming, maneuvering, and balancing for the elongate abdomen. The pectinal surface is initially smooth but later transverse striations increase the surface area as a possible respiratory adaptation. Pectinal teeth (present in Silurian and all subsequent scorpions) and forward movement and merging of anterior abdominal segments are not yet evident in embryos of this study.

Mucus altering agents as adjuncts for nonviral gene transfer to airway epithelium.

Nonviral vectors have been shown to be a safe and valid alternative to recombinant viruses for gene therapy of cystic fibrosis (CF). Nevertheless, gene transfer efficiency needs to be increased before clinical efficacy is likely in man. One barrier to increased efficacy is normal airway mucus. Using an ex vivo model of sheep tracheal epithelium, we show that this barrier can, in part, be overcome by treatment with the mucolytic agents, Nacystelyn or N-acetylcysteine using either a cationic lipid or a cationic polymer as the gene transfer agent. Further, in vivo application of either Nacystelyn or the anticholinergic glycopyrrolate, both clinically used agents, resulted in increased reporter gene expression in the mouse lung, but no significant correction of the bioelectric defect in CF null mice. These results, whilst unlikely to be sufficient in themselves to achieve clinically relevant gene therapy, may be a further useful step in the attainment of this goal.

All human Na(+)-K(+)-ATPase alpha-subunit isoforms have a similar affinity for cardiac glycosides.

Three alpha-subunit isoforms of the sodium pump, which is the receptor for cardiac glycosides, are expressed in human heart. The aim of this study was to determine whether these isoforms have distinct affinities for the cardiac glycoside ouabain. Equilibrium ouabain binding to membranes from a panel of different human tissues and cell lines derived from human tissues was compared by an F statistic to determine whether a single population of binding sites or two populations of sites with different affinities would better fit the data. For all tissues, the single-site model fit the data as well as the two-site model. The mean equilibrium dissociation constant (K(d)) for all samples calculated using the single-site model was 18 +/- 6 nM (mean +/- SD). No difference in K(d) was found between nonfailing and failing human heart samples, although the maximum number of binding sites in failing heart was only approximately 50% of the number of sites in nonfailing heart. Measurement of association rate constants and dissociation rate constants confirmed that the binding affinities of the different human alpha-isoforms are similar to each other, although calculated K(d) values were lower than those determined by equilibrium binding. These results indicate both that the affinity of all human alpha-subunit isoforms for ouabain is similar and that the increased sensitivity of failing human heart to cardiac glycosides is probably due to a reduction in the number of pumps in the heart rather than to a selective inhibition of a subset of pumps with different affinities for the drugs.

Ouabain and substrate affinities of human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells.

Human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) heterodimers were expressed individually in yeast, and ouabain binding and ATP hydrolysis were measured in membrane fractions. The ouabain equilibrium dissociation constant was 13-17 nM for alpha(1)beta(1) and alpha(3)beta(1) at 37 degrees C and 32 nM for alpha(2)beta(1), indicating that the human alpha-subunit isoforms have a similar high affinity for cardiac glycosides. K(0.5) values for antagonism of ouabain binding by K(+) were ranked in order as follows: alpha(2) (6.3 +/- 2.4 mM) > alpha(3) (1.6 +/- 0.5 mM) approximately alpha(1) (0.9 +/- 0.6 mM), and K(0.5) values for Na(+) antagonism of ouabain binding to all heterodimers were 9.5-13.8 mM. The molecular turnover for ATP hydrolysis by alpha(1)beta(1) (6,652 min(-1)) was about twice as high as that by alpha(3)beta(1) (3,145 min(-1)). These properties of the human heterodimers expressed in yeast are in good agreement with properties of the human Na(+)-K(+)-ATPase expressed in Xenopus oocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-D Horisberger, L Lelievie, and K Geering. J Biol Chem 275: 1976-1986, 2000). In contrast to Na(+) pumps expressed in Xenopus oocytes, the alpha(2)beta(1) complex in yeast membranes was significantly less stable than alpha(1)beta(1) or alpha(3)beta(1), resulting in a lower functional expression level. The alpha(2)beta(1) complex was also more easily denatured by SDS than was the alpha(1)beta(1) or the alpha(3)beta(1) complex.

Fourth-generation enzyme-linked immunosorbent assay for the simultaneous detection of human immunodeficiency virus antigen and antibody

The VIDAS HIV DUO Ultra, a fourth-generation immunoassay under development for the simultaneous detection of human immunodeficiency virus type 1 (HIV-1) p24 antigen and antibodies to HIV-1 and HIV-2, was evaluated. The enzyme-linked fluorescence immunoassay, performed on the automated VIDAS instrument, is claimed to detect early and established HIV infection. The assay was challenged with a total of 2,847 samples that included 74 members of 10 seroconversion panels, 9 p24 antigen-only-reactive members of a panel of group M clades, 503 consecutively collected samples from individuals seeking care in the University of Maryland Medical System, 1,010 samples from U.S. blood donors, 1,141 samples from patients in a high-incidence population in Trinidad, 83 samples from a clinic for sexually transmitted diseases in the Bahamas, 10 confirmed HIV-1 group O samples, and 16 confirmed HIV-2 samples from the Cote d'Ivoire. Reference tests were U.S. Food and Drug Administration-licensed HIV antibody screening, p24 antigen tests, HIV confirmatory assays, and the Roche Diagnostics Amplicor HIV-1 Monitor. The VIDAS HIV DUO Ultra demonstrated 100% sensitivity and 99.5% specificity overall, with a 99.7% specificity in low-risk individuals. The analytical sensitivity, as assessed by seroconversion panels and p24 antigen in samples, was equivalent to the sensitivity of the reference assays used to characterize these panels. The VIDAS HIV DUO Ultra is accurate, offers potential advantages over conventional HIV testing for time and cost savings, has walk-away capability, and correctly identifies both early and established HIV infections.

18O-exchange evidence that mutations of arginine in a signature sequence for P-type pumps affect inorganic phosphate binding.

We have proposed a model for part of the catalytic site of P-type pumps in which arginine in a signature sequence functions like lysine in P-loop-containing enzymes that catalyze adenosine 5'-triphosphate hydrolysis [Smirnova, I. N., Kasho, V. N., and Faller, L. D. (1998) FEBS Lett. 431, 309-314]. The model originated with evidence from site-directed mutagenesis that aspartic acid in the DPPR sequence of Na,K-ATPase binds Mg(2+) [Farley, R. A., et al. (1997) Biochemistry 36, 941-951]. It was developed by assuming that the catalytic domain of P-type pumps evolved from enzymes that catalyze phosphoryl group transfer. The functions of the positively charged amino group in P-loops are to bind substrate and to facilitate nucleophilic attack upon phosphorus by polarizing the gamma-phosphorus-oxygen bond. To test the prediction that the positively charged guanidinium group of R596 in human alpha(1) Na,K-ATPase participates in phosphoryl group transfer, the charge was progressively decreased by site-directed mutagenesis. Mutants R596K, -Q, -T, -M, -A, -G, and -E were expressed in yeast membranes, and their ability to catalyze phosphorylation with inorganic phosphate was evaluated by following (18)O exchange. R596K, in which the positive charge is retained, resembled the wild type. Substitution of a negative charge (R596E) resulted in complete loss of activity. The remaining mutants with uncharged side chains had both lowered affinity for inorganic phosphate and altered phosphate isotopomer distributions, consistent with increased phosphate-off rate constants compared to that of the wild type. Therefore, mutations of R596 strengthen our hypothesis that the oppositely charged side chains of the DPPR peptide in Na,K-ATPase form a quaternary complex with magnesium phosphate.