Development of a novel FRET immunosensor technique

We report on a novel technique to develop an optical immunosensor based on fluorescence resonance energy transfer (FRET). IgG antibodies were labeled with acceptor fluorophores while one of three carrier molecules (protein A, protein G, or F(ab')2 fragment) was labeled with donor fluorophores. The carrier molecule was incubated with the antibody to allow specific binding to the Fc portion. The labeled antibody-protein complex was then exposed to specific and nonspecific antigens, and experiments were designed to determine the 'in solution' response. The paper reports the results of three different donor-acceptor FRET pairs, fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate, Texas Red/Cy5, and Alexa Fluor 546/Alexa Fluor 594. The effects of the fluorophore to protein conjugation ratio (F/P ratio) and acceptor to donor fluorophore ratios between the antibody and protein (A/D ratio) were examined. In the presence of specific antigens, the antibodies underwent a conformational change, resulting in an energy transfer from the donor to the acceptor fluorophore as measured by a change in fluorescence. The non-specific antigens elicited little or no changes. The Alexa Fluor FRET pair demonstrated the largest change in fluorescence, resulting in a 35% change. The F/P and A/D ratio will affect the efficiency of energy transfer, but there exists a suitable range of A/D and F/P ratios for the FRET pairs. The feasibility of the FRET immunosensor technique was established; however, it will be necessary to immobilize the complexes onto optical substrates so that consistent trends can be obtained that would allow calibration plots.     

Endosomal phosphatidylinositol 3‐phosphate controls synaptic vesicle cycling and neurotransmission

Neural circuit function requires mechanisms for controlling neurotransmitter release and the activity of neuronal networks, including modulation by synaptic contacts, synaptic plasticity, and homeostatic scaling. However, how neurons intrinsically monitor and feedback control presynaptic neurotransmitter release and synaptic vesicle (SV) recycling to restrict neuronal network activity remains poorly understood at the molecular level. Here, we investigated the reciprocal interplay between neuronal endosomes, organelles of central importance for the function of synapses, and synaptic activity. We show that elevated neuronal activity represses the synthesis of endosomal lipid phosphatidylinositol 3‐phosphate [PI(3)P] by the lipid kinase VPS34. Neuronal activity in turn is regulated by endosomal PI(3)P, the depletion of which reduces neurotransmission as a consequence of perturbed SV endocytosis. We find that this mechanism involves Calpain 2‐mediated hyperactivation of Cdk5 downstream of receptor‐ and activity‐dependent calcium influx. Our results unravel an unexpected function for PI(3)P‐containing neuronal endosomes in the control of presynaptic vesicle cycling and neurotransmission, which may explain the involvement of the PI(3)P‐producing VPS34 kinase in neurological disease and neurodegeneration.     

Plasma Selenium, Zinc, Copper and Lipid Levels in Postmenopausal Turkish Women and Their Relation with Osteoporosis

It has been shown that the trace elements and lipids play role in the growth, development and maintenance of bones. We aimed to investigate serum selenium (Se), zinc (Zn), copper (Cu) and lipid (total cholesterol, triglyceride (TG), high density lipoprotein-cholesterol, low-density lipoprotein-cholesterol) levels in postmenopausal women with osteoporosis, osteopenia and in healthy controls, and to determine the relationship between Se, Zn, Cu and lipid parameters and bone mineral density (BMD). The study included 107 postmenopausal women; 35 healthy (group 1), 37 osteopenic (group 2) and 35 osteoporotic (group 3). The women in all three groups were carefully matched for body mass index (BMI). Serum concentrations of Se, Zn and Cu were measured by atomic absorption spectrophotometry. Plasma Se, Cu, Zn and lipid levels were similar in all groups (p?>?0.05). When we combined the women in each of the three groups, and considered them as one group (n?=?107) we found a positive correlation between BMI and lumbar vertebra BMD, femur neck BMD, femur total BMD; a positive correlation between TG and femur neck BMD, femur total BMD; a positive correlation between Zn and lumbar vertebra BMD (total T score) (p??0.05). Although BMI has a positive effect on BMD, trace elements and lipids, except Zn and TG, did not directly and correlatively influence BMD. Further studies are needed to clarify the role and relationship of trace elements and lipid parameters in postmenopausal osteoporosis.     

Genome-wide association of implantable cardioverter-defibrillator activation with life-threatening arrhythmias

Objectives

To identify genetic factors that would be predictive of individuals who require an implantable cardioverter-defibrillator (ICD), we conducted a genome-wide association study among individuals with an ICD who experienced a life-threatening arrhythmia (LTA; cases) vs. those who did not over at least a 3-year period (controls).

Background

Most individuals that receive implantable cardioverter-defibrillators never experience a life-threatening arrhythmia. Genetic factors may help identify who is most at risk.

Methods

Patients with an ICD and extended follow-up were recruited from 34 clinical sites with the goal of oversampling those who had experienced LTA, with a cumulative 607 cases and 297 controls included in the analysis. A total of 1,006 Caucasian patients were enrolled during a time period of 13 months. Arrhythmia status of 904 patients could be confirmed and their genomic data were included in the analysis. In this cohort, there were 704 males, 200 females, and the average age was 73.3 years. We genotyped DNA samples using the Illumina Human660 W Genotyping BeadChip and tested for association between genotype at common variants and the phenotype of having an LTA.

Results and Conclusions

We did not find any associations reaching genome-wide significance, with the strongest association at chromosome 13, rs11856574 at P = 5×10⁻⁶. Loci previously implicated in phenotypes such as QT interval (measure of the time between the start of the Q wave and the end of the T wave as measured by electrocardiogram) were not found to be significantly associated with having an LTA. Although powered to detect such associations, we did not find common genetic variants of large effect associated with having a LTA in those of European descent. This indicates that common gene variants cannot be used at this time to guide ICD risk-stratification.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00664807","term_id":"NCT00664807"}}NCT00664807     

Comparative feeding ecology of shortfin mako, blue and thresher sharks in the California Current

This study describes the feeding ecology of three pelagic shark species in the California Current: shortfin mako (Isurus oxyrinchus); blue (Prionace glauca); and thresher (Alopias vulpinus) sharks. Stomach contents of sharks collected from 2002 to 2008 were identified to the lowest taxonomic level and analyzed using univariate and multivariate methods. Of 330 mako sharks sampled (53 to 248?cm fork length [FL]), 238 stomachs contained 42 prey taxa, with jumbo squid (Dosidicus gigas) and Pacific saury (Cololabis saira) representing the most important prey based on the geometric index of importance (GII). In addition, 158 blue sharks were sampled (76 to 248?cm FL) and 114 stomachs contained 38 prey taxa, with jumbo and Gonatus spp. squids representing the most important prey. Lastly, 225 thresher sharks were sampled (108 to 228?cm FL) and 157 stomachs contained 18 prey taxa with northern anchovy (Engraulis mordax) and Pacific sardine (Sardinops sagax) identified as the most important prey. Overall, mako sharks had the most diverse diet based upon Simpson??s diversity index (1/D) (8.43?±?1.16), feeding on many species of teleosts and cephalopods, followed by blue sharks (6.20?±?2.11) which consumed a wide range of prey (primarily cephalopods), while thresher sharks were most specialized (2.62?±?0.34), feeding primarily on coastal pelagic teleosts. Dietary overlap was lowest between blue and thresher sharks (S?rensen similarity index?=?0.321 and Simplified Morisita Horn index?=?0.006), and seasonal variability in diet was greatest for blue sharks (Simplified Morisita Horn index?=?0.260, Analysis of Similarity (ANOSIM) p?<?0.001). In addition, size class, and subregion were significant factors that affected diet of each species differently (ANOSIM p?<?0.001). Despite similarities in life history characteristics and spatial and temporal overlap in habitat, diets of these three common shark species are distinct in the California Current.     

Status of pandemic influenza vaccination and factors affecting it in pregnant women in Kahramanmaras, an eastern Mediterranean city of Turkey

Background

Pregnant women are a target group for receipt of influenza vaccine because there appears to be an elevated mortality and morbidity rate associated with influenza virus infection in pregnant women. The goal of this study is to determine the factors affecting the decisions of pregnant women in Turkey to be vaccinated or not for 2009 H1N1 influenza.

Methodology

We enrolled 314 of 522 (60.2%) pregnant women who attended to the antenatal clinics of the Medical Faculty of Kahramanmaras Sutcuimam University''s Department of Gynecology and Obstetrics between December 23, 2009, and February 1, 2010. We developed a 48-question survey which was completed in a face-to-face interview at the clinic with each pregnant woman.

Principal Findings

Of the 314 pregnant women, 27.4% were in the first trimester, 33.8% were in the second trimester, and 38.8% were in the third trimester. Twenty-eight pregnant women (8.9%) got vaccinated. Of all the women interviewed, 68.5% stated that they were comfortable with their decisions about the vaccine, 7.3% stated they were not comfortable, and 24.2% stated that they were hesitant about their decisions. The probability of receiving the 2009 H1N1 vaccine was 3.46 times higher among working women than housewives, 1.85 times higher among women who have a child than those who do not, and 1.29 times higher among women with a high-school education or higher than those with only a secondary-school education and below. Correct knowledge about the minimal risks associated with receipt of influenza vaccine were associated with a significant increase in the probability of receiving the 2009 H1N1 vaccine.

Conclusions/Significance

The number of pregnant women in the study group who received the 2009 H1N1 vaccine was very low (8.9%) and two-thirds of them stated that they were comfortable with their decisions concerning the vaccine. Our results may have implications for public health measures to increase the currently low vaccination rate among pregnant women. Further studies are required to confirm whether our findings generalize to other influenza seasons and other settings.