Identification of Domains and Amino Acids Essential to the Collagen Galactosyltransferase Activity of GLT25D1

Collagen is modified by hydroxylation and glycosylation of hydroxylysine residues. This glycosylation is initiated by the β1,O galactosyltransferases GLT25D1 and GLT25D2. The structurally similar protein cerebral endothelial cell adhesion molecule CEECAM1 was previously reported to be inactive when assayed for collagen glycosyltransferase activity. To address the cause of the absent galactosyltransferase activity, we have generated several chimeric constructs between the active human GLT25D1 and inactive human CEECAM1 proteins. The assay of these chimeric constructs pointed to a short central region and a large C-terminal region of CEECAM1 leading to the loss of collagen galactosyltransferase activity. Examination of the three DXD motifs of the active GLT25D1 by site-directed mutagenesis confirmed the importance of the first (amino acids 166–168) and second motif (amino acids 461–463) for enzymatic activity, whereas the third one was dispensable. Since the second DXD motif is incomplete in CEECAM1, we have restored the motif by introducing the substitution S461D. This change did not restore the activity of the C-terminal region, thereby showing that additional amino acids were required in this C-terminal region to confer enzymatic activity. Finally, we have introduced the substitution Q471R-V472M-N473Q-P474V in the CEECAM1-C-terminal construct, which is found in most animal GLT25D1 and GLT25D2 isoforms but not in CEECAM1. This substitution was shown to partially restore collagen galactosyltransferase activity, underlining its importance for catalytic activity in the C-terminal domain. Because multiple mutations in different regions of CEECAM1 contribute to the lack of galactosyltransferase activity, we deduced that CEECAM1 is functionally different from the related GLT25D1 protein

Abacavir, efavirenz, didanosine, with or without hydroxyurea, in HIV-infected adults failing initial nucleoside/protease inhibitor-containing regimens

BACKGROUND: Hydroxyurea (HU) is an immunomodulatory agent that has been documented to enhance the antiretroviral activity of nucleoside reverse transcriptase inhibitors, such as abacavir (ABC) and didanosine (ddI), and would be expected to improve virologic efficacy. METHODS: A 48-week, phase IV, multicenter, open-label, proof-of-concept clinical trial was conducted to evaluate second-line, protease inhibitor (PI)-sparing therapy with ABC/efavirenz (EFV)/ddI plus HU or without HU in HIV-infected subjects failing to achieve HIV-1 RNA ≤ 400 copies/mL after ≥ 16 weeks of treatment with lamivudine/zidovudine or lamivudine/stavudine, plus 1 or 2 PIs. Subjects were assigned to ABC (300 mg twice daily)/ EFV (600 mg once daily)/ ddI (400 mg once daily) plus HU (500 mg twice daily) (n = 30) or this regimen without HU (n = 24). RESULTS: Baseline mean HIV-1 RNA was 3.86 log(10 )copies/mL and CD4+ cell count was 345 cells/mm(3). A similar percentage of subjects in the non-HU arm (58%) and HU arm (53%) completed the study. Intent-to-treat: missing = failure analysis showed no differences in proportions of subjects in the non-HU and HU arms achieving undetectable plasma HIV-1 RNA levels at week 24 (<400 copies/mL: 58% [14/24] vs 57% [17/30], P = 0.899; <50 copies/mL (50% [12/24] vs 47% [14/30], P = 0.780). Median change from baseline in CD4+ cell count in the non-HU and HU arms at week 48 was +114 cells/mm(3 )and -63 cells/mm(3 )(P = 0.007), respectively. Both regimens were generally well tolerated, although more subjects in the HU arm withdrew prematurely from the study due to adverse events (23% vs 4%). Four cases of possible ABC-related hypersensitivity were observed. CONCLUSION: ABC/EFV/ddI was an effective and well-tolerated second-line regimen for nucleoside/PI-experienced HIV-infected subjects. The addition of HU blunted the CD4+ cell response, did not appear to enhance antiviral activity, and resulted in more treatment-limiting adverse events

Molecular phylogeny and timing of diversification in Alpine Rhithrogena (Ephemeroptera: Heptageniidae).

BACKGROUND: Larvae of the Holarctic mayfly genus Rhithrogena Eaton, 1881 (Ephemeroptera, Heptageniidae) are a diverse and abundant member of stream and river communities and are routinely used as bio-indicators of water quality. Rhithrogena is well diversified in the European Alps, with a number of locally endemic species, and several cryptic species have been recently detected. While several informal species groups are morphologically well defined, a lack of reliable characters for species identification considerably hampers their study. Their relationships, origin, timing of speciation and mechanisms promoting their diversification in the Alps are unknown. RESULTS: Here we present a species-level phylogeny of Rhithrogena in Europe using two mitochondrial and three nuclear gene regions. To improve sampling in a genus with many cryptic species, individuals were selected for analysis according to a recent DNA-based taxonomy rather than traditional nomenclature. A coalescent-based species tree and a reconstruction based on a supermatrix approach supported five of the species groups as monophyletic. A molecular clock, mapped on the most resolved phylogeny and calibrated using published mitochondrial evolution rates for insects, suggested an origin of Alpine Rhithrogena in the Oligocene/Miocene boundary. A diversification analysis that included simulation of missing species indicated a constant speciation rate over time, rather than any pronounced periods of rapid speciation. Ancestral state reconstructions provided evidence for downstream diversification in at least two species groups. CONCLUSIONS: Our species-level analyses of five gene regions provide clearer definitions of species groups within European Rhithrogena. A constant speciation rate over time suggests that the paleoclimatic fluctuations, including the Pleistocene glaciations, did not significantly influence the tempo of diversification of Alpine species. A downstream diversification trend in the hybrida and alpestris species groups supports a previously proposed headwater origin hypothesis for aquatic insects

Optimal perceived timing: integrating sensory information with dynamically updated expectations

The environment has a temporal structure, and knowing when a stimulus will appear translates into increased perceptual performance. Here we investigated how the human brain exploits temporal regularity in stimulus sequences for perception. We find that the timing of stimuli that occasionally deviate from a regularly paced sequence is perceptually distorted. Stimuli presented earlier than expected are perceptually delayed, whereas stimuli presented on time and later than expected are perceptually accelerated. This result suggests that the brain regularizes slightly deviant stimuli with an asymmetry that leads to the perceptual acceleration of expected stimuli. We present a Bayesian model for the combination of dynamically-updated expectations, in the form of a priori probability of encountering future stimuli, with incoming sensory information. The asymmetries in the results are accounted for by the asymmetries in the distributions involved in the computational process

Systematic review of methods used in meta-analyses where a primary outcome is an adverse or unintended event

addresses: Peninsula College of Medicine and Dentistry, St Luke's Campus, University of Exeter, Exeter, UK. [email protected]: PMCID: PMC3528446types: Journal Article; Research Support, Non-U.S. Gov't© 2012 Warren et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Adverse consequences of medical interventions are a source of concern, but clinical trials may lack power to detect elevated rates of such events, while observational studies have inherent limitations. Meta-analysis allows the combination of individual studies, which can increase power and provide stronger evidence relating to adverse events. However, meta-analysis of adverse events has associated methodological challenges. The aim of this study was to systematically identify and review the methodology used in meta-analyses where a primary outcome is an adverse or unintended event, following a therapeutic intervention

In vitro reactivation of latent HIV-1 by cytostatic bis (thiosemicarbazonate) gold(III) complexes

BACKGROUND : A number of cytostatic agents have been investigated for the ability to reactivate latent viral reservoirs, which is a major prerequisite for the eradication of HIV-1 infection. Two cytostatic bis(thiosemicarbazonate) gold(III) complexes (designated 1 and 2) were tested for this potential in the U1 latency model of HIV-1 infection. METHODS : Cell viability in the presence or absence of 1 and 2 was determined using a tetrazolium dye and evidence of reactivation was assessed by p24 antigen capture following exposure to a latency stimulant, phorbol myristate acetate (PMA) and or test compounds. The latency reactivation mechanism was explored by determining the effect of the complexes on protein kinase C (PKC), histone deacetylases (HDAC) and proinflammatory cytokine production. RESULTS : The CC50 of the complexes in U1 cells were 0.53 ± 0.12 μM for 1 and 1.0 ± 0.4 μM for 2. In the absence of PMA and at non toxic concentrations of 0.2 and 0.5 μM, 1 and 2 significantly (p ≤ 0.02) reactivated virus in U1 cells by 2.7 and 2.3 fold respectively. In comparison, a 2.6 fold increase (p = 0.03) in viral reactivation was observed for hydroxyurea (HU), which was used as a cytostatic and latent HIV reactivation control. Viral reactivation was absent for the complexes during co-stimulation with PMA indicating the lack of an additive effect between the chemicals as well as an absence of inhibition of PMA induced HIV reactivation but for HU inhibition of the stimulant’s activity was observed (p = 0.01). Complex 1 and 2 activated PKC activity by up to 32% (p < 0.05) but no significant inhibition of HDAC was observed. Increases in TNF-α levels suggested that the reactivation of virus by the complexes may have been due to contributions from the latter and the activation of PKC. CONCLUSION : The ethyl group structural difference between 1 and 2 seems to influence bioactivity with lower active concentrations of 1, suggesting that further structural modifications should improve specificity. The cytostatic effect of 1 and 2 and now HIV reactivation from a U1 latency model is consistent with that of the cytostatic agent, HU. These findings suggest that the complexes have a potential dual (cytostatic and reactivation) role in viral “activation/elimination”.AuTEK Biomed (Mintek and Harmony Gold),Technology Innovation Agency (TIA) and the University of Pretoria.http://www.biomedcentral.com/bmcinfectdis/hb201