Regulation of type I-interferon responses in the human epidermal melanocyte cell line SKMEL infected by the Ross River alphavirus.

Melanocytes are melanin-producing cells and with emerging innate immune functions including the expression of antiviral interferon-type I cytokines. We herein ascertained the susceptibility of the human melanocytes to Ross River alphavirus (RRV) infection and analyzed the subsequent immune responses. We demonstrated for the first time that (1) SKMEL-28 melanocyte cell line was susceptible to RRV infection and displaying major cytopathic activities and (2) RRV interfered with the interferon-type I response by altering nuclear translocation of pSTAT1 and pSTAT2 in infected SKMEL-28. These results suggest that the human melanoma cell line SKMEL-28 is a valuable model to analyze the mechanisms involved in severe skin manifestations and melanocyte's immunity at the portal of entry of major infection by arboviruses.

Biochemical evidence for Ca2+-independent functional activation of hPLSCR1 at low pH.

Human phospholipid scramblase 1 (hPLSCR1) is a Ca2+-dependent protein known to scramble phospholipids in the plasma membrane resulting in loss of membrane asymmetry. It has been reported that hPLSCR1 exhibits Ca2+- independent activity at low pH. However, the conformational changes induced at low pH leading to functional activation are not known. Our results showed that recombinant hPLSCR1 was functionally activated at low pH, which is similar to the behavior of natively extracted hPLSCR1. Tryptophan fluorescence measurements showed a decrease in Ca2+-binding affinity at low pH, although not at pH 5.5. Far and near UV-CD revealed that low pH induced structural changes, with a significant increase in the ß-sheet content of the protein. At the physiological level, decreased hPLSCR1 expression was observed after a period of exposure to low pH. The effect occurred at the promoter level. The expression levels of hPLSCR1 directly correlated with the sensitivity of HEK293 to apoptosis. Based on these results, we conclude that the mechanisms of Ca2+- and pH-induced functional activation of hPLSCR1 are different and that hPLSCR1 expression regulated by low pH could provide insights into the role of hPLSCR1 in cancer progression.

Snail interacts with hPLSCR1 promoter and down regulates its expression in IMR-32.

Human phospholipid scramblase 1 (hPLSCR1) is a proapoptotic protein whose expression is deregulated in a variety of cancers cells. However till date the transcription regulation of hPLSCR1 is unknown. Transcriptional regulation of hPLSCR1 was studied by cloning the 5'-flanking region of hPLSCR1. Luciferase assays revealed that -1525 to -1244 region of hPLSCR1 was found to regulate its promoter activity. A putative Snail transcription factor (TF) binding site was found within the regulatory region of the promoter. Snail binding was found to down regulate the expression of hPLSCR1 both at the transcriptional and translational levels. Snail knock down using Snail-shRNA confirmed that down regulation of hPLSCR1 by Snail was specific. Point mutation studies confirm that the predicted Snail TF binds to -1123 to -1117 site. ChIP assay further confirms the physical interaction of Snail with hPLSCR1 promoter. This is the first report showing the transcriptional regulation of hPLSCR1 expression by Snail TF and its possible implications in cancer progression.

Factors Associated with the Development of High Nutrition Risk: Data from the Canadian Longitudinal Study on Aging.

This study aimed to determine which social network, demographic, and health-indicator variables were able to predict the development of high nutrition risk in Canadian adults at midlife and beyond, using data from the Canadian Longitudinal Study on Aging. Multivariable binomial logistic regression was used to examine the predictors of the development of high nutrition risk at follow-up, 3 years after baseline. At baseline, 35.0 per cent of participants were at high nutrition risk and 42.2 per cent were at high risk at follow-up. Lower levels of social support, lower social participation, depression, and poor self-rated healthy aging were associated with the development of high nutrition risk at follow-up. Individuals showing these factors should be screened proactively for nutrition risk.

Biochemical evidence for energy-independent flippase activity in bovine epididymal sperm membranes: an insight into membrane biogenesis.

During the maturation process spermatozoa undergo a series of changes in their lateral and horizontal lipid profiles. However, lipid metabolism in spermatozoa is not clearly understood for two reasons: i) the mature spermatozoa are devoid of endoplasmic reticulum, which is the major site of phospholipid (PL) synthesis in somatic cells, and ii) studies have been superficial due to the difficulty in culturing spermatozoa. We hypothesize that spermatozoa contain biogenic membrane flippases since immense changes in lipids occur during spermatogenic differentiation. To test this, we isolated spermatozoa from bovine epididymides and reconstituted the detergent extract of sperm membranes into proteoliposomes. In vitro assays showed that proteoliposomes reconstituted with sperm membrane proteins exhibit ATP-independent flip-flop movement of phosphatidylcholine (PC), phosphatidylserine, and phosphatidylglycerol. Half-life time of PC flipping was found to be ∼3.2±1 min for whole sperm membrane, which otherwise would have taken ∼11-12 h in the absence of protein. Further biochemical studies confirm the flip-flop movement to be protein-mediated, based on its sensitivity to protease and protein-modifying reagents. To further determine the cellular localization of flippases, we isolated mitochondria of spermatozoa and checked for ATP-independent flippase activity. Interestingly, mitochondrial membranes showed flip-flop movement but were specific for PC with half-life time of ∼5±2 min. Our results also suggest that spermatozoa have different populations of flippases and that their localization within the cellular compartments depends on the type of PL synthesis.

Nutrition risk varies according to social network type: data from the Canadian Longitudinal Study on Aging.

OBJECTIVE: There were two primary objectives, namely: (1) to determine the social network types that Canadian adults aged 45 and older belong to and (2) to discover if social network type is associated with nutrition risk scores and the prevalence of high nutrition risk. DESIGN: A retrospective cross-sectional study. SETTING: Data from the Canadian Longitudinal Study on Aging (CLSA). PARTICIPANTS: 17 051 Canadians aged 45 years and older with data from baseline and first follow-up of the CLSA. RESULTS: CLSA participants could be classified into one of seven different social network types that varied from restricted to diverse. We found a statistically significant association between social network type and nutrition risk scores and percentage of individuals at high nutrition risk at both time points. Individuals with restricted social networks had lower nutrition risk scores and are more likely to be at nutrition risk, whereas individuals with diverse social networks had higher nutrition risk scores and are less likely to be at nutrition risk. CONCLUSIONS: Social network type was associated with nutrition risk in this representative sample of Canadian middle-aged and older adults. Providing adults with opportunities to deepen and diversify their social networks may decrease the prevalence of nutrition risk. Individuals with more restricted networks should be proactively screened for nutrition risk.

Biochemical and functional characterization of human phospholipid scramblase 4 (hPLSCR4).

Human phospholipid scramblase 4 (hPLSCR4), an isoform of the scramblase family, is a type II single-pass transmembrane protein whose function remains unknown. To understand its role, recombinant hPLSCR4 was obtained by cloning the ORF into a pET28 a(+) vector and overexpressed in Escherichia coli. Functional assay showed that Ca2+, Mg2+, and Zn2+ activate hPLSCR4 and mediate scrambling activity independent of the phospholipid head group. Far-UV-CD and fluorescence spectroscopy revealed that Ca2+ and Mg2+ binding induces conformation change in hPLSCR4, exposing hydrophobic patches of the protein, and Ca2+ has more affinity than Mg2+ and Zn2+. Stains-all studies further confirm that hPLSCR4 is a Ca2+-binding protein. Point mutation (Asp290→Ala) in hPLSCR4 decreased the Ca2+-binding affinity as well as Tb3+ luminescence, suggesting residues of the predicted Ca2+-binding motif are involved in Ca2+ binding. Functional reconstitution with (Asp290→Ala) mutant led to ~50% and ~40% decrease in scramblase activity in the presence of Ca2+ and Mg2+, respectively.

Recovery of functionally active recombinant human phospholipid scramblase 1 from inclusion bodies using N-lauroyl sarcosine.

Human phospholipid scramblase (hPLSCR1) is a transmembrane protein involved in rapid bidirectional scrambling of phospholipids across the plasma membrane in response to elevated intracellular calcium (Ca(2+)) levels. Overexpression of recombinant hPLSCR1 in Escherichia coli BL21 (DE3) leads to its deposition in inclusion bodies (IBs). N-lauroyl sarcosine was used to solubilize IBs and to recover functionally active hPLSCR1 from them. Protein was purified to homogeneity by nickel-nitrilotriacetic acid (Ni(2+)-NTA) affinity chromatography and was >98% pure. Functional activity of the purified protein was validated by in vitro reconstitution studies, ~18% of 7-nitrobenz-2-oxa-1, 3-diazol-4-yl-phosphatidylcholine (NBD-PC) phospholipids was translocated across the lipid bilayer in the presence of Ca(2+) ions. Far ultraviolet circular dichroism (UV-CD) studies reveal that the secondary structure of protein is predominantly an α-helix, and under nondenaturing conditions, the protein exists as a monomer. Here we describe a method to purify recombinant membrane protein with higher yield than previously described methods involving renaturation techniques.

Hospital federations and human resources for health: trends and proposals for improvement.

Health system performance depends on a performing health workforce. In many health care systems, hospital federations are key players as they represent actors, which are important in terms number of qualified health workers employed, and of the volume of services provided. In addressing the "crisis of human resources for health" (HRH) they are part of the problem as well as the solution.

Effects of mandibular advancement on upper airway dynamics in awake normal subjects: a pilot study with phrenic nerve stimulation.

BACKGROUND: Mandibular advancement (MA) can be an alternative to continuous positive airway pressure (CPAP) in the treatment of obstructive sleep apnea syndrome. This study was designed to describe its effects on upper airway mechanics. METHODS: Six awake healthy subjects (four men, 31+/-8 years, body mass index (BMI) 25+/-2kg/m2) were studied supine. Phrenic stimulation was used to measure pressure-flow relationships in response to diaphragm contractions without pre-activation of upper airway dilators during progressive MA. RESULTS: Phrenic stimulation consistently induced flow limitation (initial peak V'-t1, corresponding to the limiting pressure; reduction to a nadir V'-t2; reincrease to a second peak V'-t3). The upper airway resistances were negatively correlated with MA. Their values at 4mm were significantly lower than at baseline. Further MA reduced resistances. CONCLUSIONS: As with CPAP, MA in normal subjects decreases the propensity of the upper airway to collapse in response to a negative pressure pulse.

Mandibular advancement titration for obstructive sleep apnea: optimization of the procedure by combining clinical and oximetric parameters.

BACKGROUND: Oral appliances (OAs) have been used for the treatment of obstructive sleep apnea syndrome (OSAS), with different degrees of effectiveness having been shown in previous studies. But, in the absence of a consensual recommendation, the method of the determination of effective mandibular advancement varies from one study to another. STUDY OBJECTIVE: We prospectively evaluated an OA titration protocol based on a combined analysis of symptomatic benefit and oximetric recording to guide the progressive mandibular advancement. SETTING: University hospital sleep disorders center. PATIENTS: Forty patients with OSAS (mean [+/-SD] apnea-hypopnea index [AHI], 46 +/- 21 events per hour) found on baseline polysomnography, who were intolerant of nasal continuous positive airway pressure, completed all aspects of the study. METHODS: Two acrylic appliances connected by Herbst attachments were constructed. The mandible was advanced 1 mm every week until there was a resolution of the symptoms and a reduction in the oxygen desaturation index (ie, the number of desaturations yielding a > 3% fall in pulse oximetric saturation per hour of recording) [ODI] of <10 events per hour of recording or a maximum comfortable limit of advancement was obtained. The final response to OA was evaluated by full polysomnography recording. RESULTS: A complete response (ie, mean AHI, 5 +/- 3 events per hour; mean snoring reduction [SR], 91 +/- 13%; mean Epworth sleepiness scale [ESS] score, 5 +/- 3) was obtained in 63.6% of patients, and a limited response (ie, mean AHI, 21 +/- 11 events per hour; mean SR, 88 +/- 15%; mean ESS, 6 +/- 3) was obtained in 18.2% of patients. Twenty-five percent of mandibular advancements were motivated by an abnormal ODI (ie, 21 +/- 10 events per hour) despite resolution of the symptoms, while 20% were motivated by persistent symptoms with a normal ODI (ie, 6 +/- 2 events per hour). After a mean duration of 17 +/- 4 months, 34 patients declared that they had used the OA 5 +/- 2 days a week for 89 +/- 19% of their sleep time. CONCLUSIONS: A combination of the patient's subjective evaluation and oximetric score improves the effectiveness of the OA titration procedure.

One-night mandibular advancement titration for obstructive sleep apnea syndrome: a pilot study.

The effect of a dental appliance (DA) is usually evaluated in a single mandibular position reached after several weeks and corresponding to either improvement of symptoms or intolerance to any further advancement. The purpose of this study was to test the feasibility of one-night evaluation of the efficacy of a DA. The study population consisted of seven patients (six men) with obstructive sleep apnea syndrome (66.9 +/- 32.4 apneas and/or hypopneas per hour). Patients underwent two consecutive polysomnographies; first with a temporary DA (two arches connected by a hydraulic system) progressively adjusted during the night to correct sleep disordered breathing and second with a permanent DA (two arches connected by Herbst attachments) set to the effective degree of advancement during the titration night. All patients completed the protocol. The mean mandibular advancement reached during the titration night was 12.6 +/- 2.7 mm. Arousal was never observed during or for 60 seconds following advancement. The apnea-hypopnea index (AHI) was significantly reduced from 66.9 +/- 32.4 to 26.1 +/- 20.7 per hour during the titration night from the diagnostic night (p < 0.01). During the second night, the AHI was 19.6 +/- 20.2 per hour and was less than 20 per hour in 71.4% of patients and less than 10 per hour in 42.9% of patients. The efficacy of a DA can be evaluated during a single night of polysomnography.