Tuning the formation and rupture of single ligand-receptor bonds by hyaluronan-induced repulsion

We used a combination of laminar flow chamber and reflection interference microscopy to study the formation and rupture of single bonds formed between Fc-ICAM-1 attached to a substrate and anti-ICAM-1 carried by micrometric beads in the presence of a repulsive hyaluronan (HA) layer adsorbed onto the substrate. The absolute distance between the colloids and the surface was measured under flow with an accuracy of a few nanometers. We could verify the long-term prediction of classical lubrication theory for the movement of a sphere near a wall in a shear flow. The HA polymer layer exerted long-range repulsive steric force on the beads and the hydrodynamics at the boundary remained more or less unchanged. By incubating HA at various concentrations, the thickness of the layer, as estimated by beads most probable height, was tuned in the range 20-200 nm. Frequency of bond formation was decreased by more than one order of magnitude by increasing the thickness of the repulsive layer, while the lifetime of individual bonds was not affected. This study opens the way for further quantitative studies of the effect of molecular environment and separation distance on ligand-receptor association and dissociation.     

Performance evaluation of a reversed-phase,high-performance liquid chromatographic assay of valproic acid involving a “solvent demixing” extraction procedure and precolumn derivatisation

As previously shown by others, the antiepileptic drug valproic acid can be assayed in biological fluids by reversed-phase, high-performance liquid chromatography after derivatisation with a bromomethyl aryl ketone through crown-ether catalysis. It is possible to extract the drug directly into acetonitrile, the solvent used for its derivatisation: when an excess of some salt such as NaCl is added to a mixture of plasma and acetonitrile, the organic solvent separates and valproic acid is extracted into it with a high recovery yield. This “solvent demixing” extraction method has shown excellent reproducibility, as well as promising versatility. Derivatisation, still in acetonitrile, using bromomethyl naphthyl ketone and 15-crown-5 allowed us to get rid of the current heating step without markedly increasing the delay of reaction. Chromatography was performed on a C-18 bonded stationary phase with acetonitrile—water as mobile phase, cyclohexanecarboxylic acid as internal standard and ultraviolet spectrophotometric detection. Statistical analysis of results shows 80% recovery of extraction, good linearity and an inter-extract variation coefficient of 4%, the last mainly ascribable to chromatographic measurements. Recovery is readily improved by increasing the amount of acetonitrile, which was equal to that of plasma in our experiments, since the high sensitivity of detection can tolerate the resulting decrease of valproic acid concentration in the extract.     

Rapid inhibition of Ca2+ influx by neurosteroids in murine embryonic sensory neurones

The non-genomic role of neuroactive steroids on [Ca2+]i transients induced by GABA receptor activation was investigated in cultured dorsal root ganglia (DRG) neurones at embryonic stage E13. [Ca2+]i measurements were performed with Fura-2 fast fluorescence microfluorimetry. Application of the GABAA receptor agonist muscimol (Musci) evoked an increase in [Ca2+]i, confirming the excitatory effect of GABA at this embryonic stage. The muscimol-induced [Ca2+]i response was inhibited by progesterone (Proges) and its primary metabolite allopregnanolone (Allo) in a rapid, reversible and dose-dependent manner. These calcium transients were suppressed in the absence of external Ca2+ or in the presence of Ni2+ + Cd2+ suggesting an involvement of voltage-activated Ca2+ channels. In contrast, none of these steroids affected the resting [Ca2+]i nor exhibited any inhibitory effect on 50 mM KCl-induced [Ca2+]i increases. In view of the well-established potentiation of GABAA receptor by direct binding of neurosteroids, the inhibitory effects described in this study seem to involve distinct mechanisms. This new inhibitory effect of progesterone is observed at low and physiological concentrations, is rapid and independent of RU38486, an antagonist of the classic progesterone receptor, probably involving a membrane receptor. Using RT-PCR, we demonstrated the expression of progesterone receptor membrane component 1 (Pgrmc1), encoding 25-Dx, a membrane-associated progesterone binding protein in DRG neurones at different stages of development. In conclusion, we describe for the first time a rapid effect of progestins on embryonic DRG neurones involving an antagonistic effect of progesterone and allopregnanolone on GABAA receptors.     

Dissociating the Neural Correlates of Intra-Item and Inter-Item Working-Memory Binding

Background

Integration of information streams into a unitary representation is an important task of our cognitive system. Within working memory, the medial temporal lobe (MTL) has been conceptually linked to the maintenance of bound representations. In a previous fMRI study, we have shown that the MTL is indeed more active during working-memory maintenance of spatial associations as compared to non-spatial associations or single items. There are two explanations for this result, the mere presence of the spatial component activates the MTL, or the MTL is recruited to bind associations between neurally non-overlapping representations.

Methodology/Principal Findings

The current fMRI study investigates this issue further by directly comparing intrinsic intra-item binding (object/colour), extrinsic intra-item binding (object/location), and inter-item binding (object/object). The three binding conditions resulted in differential activation of brain regions. Specifically, we show that the MTL is important for establishing extrinsic intra-item associations and inter-item associations, in line with the notion that binding of information processed in different brain regions depends on the MTL.

Conclusions/Significance

Our findings indicate that different forms of working-memory binding rely on specific neural structures. In addition, these results extend previous reports indicating that the MTL is implicated in working-memory maintenance, challenging the classic distinction between short-term and long-term memory systems.     

TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome

Velo-cardio-facial syndrome (VCFS)/DiGeorge syndrome (DGS) is a human disorder characterized by a number of phenotypic features including cardiovascular defects. Most VCFS/DGS patients are hemizygous for a 1.5-3.0 Mb region of 22q11. To investigate the etiology of this disorder, we used a cre-loxP strategy to generate mice that are hemizygous for a 1.5 Mb deletion corresponding to that on 22q11. These mice exhibit significant perinatal lethality and have conotruncal and parathyroid defects. The conotruncal defects can be partially rescued by a human BAC containing the TBX1 gene. Mice heterozygous for a null mutation in Tbx1 develop conotruncal defects. These results together with the expression patterns of Tbx1 suggest a major role for this gene in the molecular etiology of VCFS/DGS.     

Priming and activation of mouse macrophages by trehalose 6,6'-dicorynomycolate vesicles from Corynebacterium glutamicum

Vesicles consisting of pure trehalose dicorynomycolate (TDCM), the corynebacterial analog of the most studied mycobacterial glycolipid 'cord factor', were isolated from Corynebacterium glutamicum cells by mild detergent treatment; these induced in vivo a macrophage priming similar to that obtained with mycobacterial-derived trehalose dimycolate. In vitro, both TDCM and bacterial lipopolysaccharide (LPS) induced in macrophages the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha), endotoxin tolerance, and were primed for an enhanced secondary NO response to LPS. Interferon-gamma pretreatment did not influence the LPS-induced TNF-alpha response, but considerably increased the TDCM-induced response.     

AIDS-Related Stigma and Mental Disorders among People Living with HIV: A Cross-Sectional Study in Cambodia

Background

AIDS-related stigma and mental disorders are the most common conditions in people living with HIV (PLHIV). We therefore conducted this study to examine the association of AIDS-related stigma and discrimination with mental disorders among PLHIV in Cambodia.

Methods

A two-stage cluster sampling method was used to select 1,003 adult PLHIV from six provinces. The People Living with HIV Stigma Index was used to measure stigma and discrimination, and a short version of general health questionnaire (GHQ-12) was used to measure mental disorders. Multivariate logistic regression analysis was conducted.

Results

The reported experiences of discrimination in communities in the past 12 months ranged from 0.8% for reports of being denied health services to 42.3% for being aware of being gossiped about. Internal stigma was also common ranging from 2.8% for avoiding going to a local clinic and/or hospital to 59.6% for deciding not to have (more) children. The proportions of PLHIV who reported fear of stigma and discrimination ranged from 13.9% for fear of being physically assaulted to 34.5% for fear of being gossiped about. The mean score of GHQ-12 was 3.2 (SD = 2.4). After controlling for several potential confounders, higher levels of mental disorders (GHQ-12≥ 4) remained significantly associated with higher levels of experiences of stigma and discrimination in family and communities (AOR = 1.9, 95% CI = 1.4–2.6), higher levels of internal stigma (AOR = 1.7, 95% CI = 1.2–2.3), and higher levels of fear of stigma and discrimination in family and communities (AOR = 1.5, 95% CI = 1.1–2.2).

Conclusions

AIDS-related stigma and discrimination among PLHIV in Cambodia are common and may have potential impacts on their mental health conditions. These findings indicate a need for community-based interventions to reduce stigma and discrimination in the general public and to help PLHIV to cope with this situation.     

Factors Associated with Inconsistent Condom Use among Men Who Have Sex with Men in Cambodia

Background

Compared to the general population, men who have sex with men (MSM) are at greater risk for HIV and less understood due to their more hidden and stigmatized nature. Moreover, the discrepancy in findings in the literature merits further investigations in MSM populations from different cultures and settings. We therefore conducted this study to explore factors associated with inconsistent condom use among high-risk MSM in Cambodia.

Methods

This cross-sectional study was conducted in 2014 among 367 MSM randomly selected from Battembang and Siem Reap using a two-stage cluster sampling method. A structured questionnaire was used for face-to-face interviews to collect information on characteristics of respondents, HIV testing history, self-perception of HIV risk, substance use, sexual behaviors, mental disorders, and HIV knowledge. Multivariable logistic regression analysis was performed to identify factors independently associated with inconsistent condom use.

Results

On average, 62.3% of respondents reported that they always used condoms over the past three months. The rates varied with types of sexual partners; the proportion of respondents who reported always using condoms was 55.1%, 64.2%, 75.9%, 73.0%, 78.1%, and 70.3%, for sexual partners who were girlfriends, boyfriends, female sex workers, male sex workers, female clients, or male clients, respectively. After adjustment, inconsistent condom use was significantly associated with age of ≥25 (AOR = 1.77, 95% CI = 1.09–2.86), self-rated quality of life as good or very good (AOR = 4.37, 95% CI = 1.79–5.67), self-perception of higher HIV risk compared to the general population (AOR = 2.37, 95% CI = 1.35–4.17), illicit drug use in the past three months (AOR = 5.76, 95% CI = 1.65–10.09), and reported consistent lubricant use when selling anal sex to men in the past three months (AOR = 2.85, 95% CI = 1.07–8.12).

Conclusions

We found risky sexual behaviors to be considerably high among MSM in this study, especially among those who used illicit drugs or were older than 25. HIV education and social marketing should be expanded and specifically designed for MSM to better educate on the increased risk of HIV with unprotected anal sex and illicit drug use as risk factors, and the importance of the use of both condoms and lubricant during anal intercourse.     

KIT is required for hepatic function during mouse post-natal development

Background

Targeted disruption of the murine 3β-hydroxysterol-Δ7-reductase gene (Dhcr7), an animal model of Smith-Lemli-Opitz syndrome, leads to loss of cholesterol synthesis and neonatal death that can be partially rescued by transgenic replacement of DHCR7 expression in brain during embryogenesis. To gain further insight into the role of non-brain tissue cholesterol deficiency in the pathophysiology, we tested whether the lethal phenotype could be abrogated by selective transgenic complementation with DHCR7 expression in the liver.

Results

We generated mice that carried a liver-specific human DHCR7 transgene whose expression was driven by the human apolipoprotein E (ApoE) promoter and its associated liver-specific enhancer. These mice were then crossed with Dhcr7+/- mutants to generate Dhcr7-/- mice bearing a human DHCR7 transgene. Robust hepatic transgene expression resulted in significant improvement of cholesterol homeostasis with cholesterol concentrations increasing to 80~90 % of normal levels in liver and lung. Significantly, cholesterol deficiency in brain was not altered. Although late gestational lung sacculation defect reported previously was significantly improved, there was no parallel increase in postnatal survival in the transgenic mutant mice.

Conclusion

The reconstitution of DHCR7 function selectively in liver induced a significant improvement of cholesterol homeostasis in non-brain tissues, but failed to rescue the neonatal lethality of Dhcr7 null mice. These results provided further evidence that CNS defects caused by Dhcr7 null likely play a major role in the lethal pathogenesis of Dhcr7 ^-/- mice, with the peripheral organs contributing the morbidity.