Methods to Minimize Confounding Effects of Hematocrit and Hemoglobin when using Dried Blood Spots

Dried blood spots (DBS) are an alternative method of collecting venous blood samples that can be used to measure blood biomarkers. Two confounding factors, hemoglobin and hematocrit, limit the validity of DBS in comparison to the gold standard serum sample. The saturation of biomarkers on DBS filter paper is affected by the sample’s hematocrit and hemoglobin. Also hemoglobin contamination is known to confounder for antibody binding in assay systems. The purpose of this study was two-fold: 1) to evaluate a DBS punching technique designed to limit the effects of hematocrit whilst minimizing sample volume and 2) to evaluate a novel device designed to remove hemoglobin from plasma during DBS collection (Seraform™). A bead-based multiplex assay of nine cardiovascular disease risk (CVD) biomarkers (C-reactive protein, fibrinogen, L-selectin, Haptoglobin, serum amyloid protein, von Willebrand factor, adipsin, α2-macroglobulin, and α1-acid glycoprotein) was measured and compared using the various DBS treatments. Outcomes were compared using linear regression analysis examining the R2 change with hematocrit and hemoglobin as covariates. Significance was set at

Whole-Retina Reduced Electrophysiological Activity in Mice Bearing Retina-Specific Deletion of Vesicular Acetylcholine Transporter

Background: Despite rigorous characterization of the role of acetylcholine in retinal development, long-term effects of its absence as a neurotransmitter are unknown. One of the unanswered questions is how acetylcholine contributes to the functional capacity of mature retinal circuits. The current study investigates the effects of disrupting cholinergic signalling in mice, through deletion of vesicular acetylcholine transporter (VAChT) in the developing retina, pigmented epithelium, optic nerve and optic stalk, on electrophysiology and structure of the mature retina. Methods & Results A combination of electroretinography, optical coherence tomography imaging and histological evaluation assessed retinal integrity in mice bearing retina-targeted (embryonic day 12.5) deletion of VAChT (VAChT(Six3-Cre-flox/flox)) and littermate controls at 5 and 12 months of age. VAChT(Six3-Cre-flox/flox) mice did not show any gross changes in nuclear layer cellularity or synaptic layer thickness. However, VAChT(Six3-Cre-flox/flox) mice showed reduced electrophysiological response of the retina to light stimulus under scotopic conditions at 5 and 12 months of age, including reduced a-wave, b-wave, and oscillatory potential (OP) amplitudes and decreased OP peak power and total energy. Reduced a-wave amplitude was proportional to the reduction in b-wave amplitude and not associated with altered a-wave 10%-90% rise time or inner and outer segment thicknesses. Significance This study used a novel genetic model in the first examination of function and structure of the mature mouse retina with disruption of cholinergic signalling. Reduced amplitude across the electroretinogram wave form does not suggest dysfunction in specific retinal cell types and could reflect underlying changes in the retinal and/or extraretinal microenvironment. Our findings suggest that release of acetylcholine by VAChT is essential for the normal electrophysiological response of the mature mouse retina

Consumption of a High-Fat Meal Alters Post-Prandial SIRT mRNA Expression in Blood Leukocytes

Introduction. Sirtuins (SIRT) are protein deacetylases, hypothesized to regulate the transcription of various genes involved in the prevention of atherogenesis and diet induced obesity. Previous research from our laboratory has demonstrated that consumption of a single, high-fat meal increases various CVD risk factors for up to 5-h post-prandial. Given the importance of SIRT to metabolic disorders, it is reasonable to speculate that a single, high-fat meal also disrupts SIRT. Methods. The purpose of this study was to determine the effect of a high-fat meal (75% of daily kcals & 80% of daily fat needs), on SIRT mRNA expression in blood leukocytes during a 5-h post-prandial period. Men and women (N=17) were recruited to report to the lab following an overnight fast. Venous blood samples were collected prior to the meal, 1, 3, and 5-h post-meal. White buffy coat aliquot was frozen in RNALater solution. At the end of the study samples were thawed and RNA was isolated using a phenol/chloroform method. RNA was reverse transcribed and mRNA expression for SIRT 1-7 was determined using a Taqman qPCR technique with 18S rRNA as a normalizer, under standard PCR cycling conditions. An additional aliquot of serum was used to measure triglyceride, total cholesterol, and glucose responses were measured using enzymatic assays on an automated chemistry analyzer (ChemWell T; P.C., FL). Data was analyzed using a RM ANOVA with P\u3c0.05. Results. Consistent with previous results, the meal caused an increase in triglycerides, total cholesterol and glucose that reached peaked values at 3-h post-prandial. We also observed significant expression changes in the mRNA of the SIRT 1 (P=0.02) and SIRT 6 (P=0.03) during the 5-h post-prandial period. Both SIRT 1 and SIRT 6 showed the greatest decreased expression at 3-h post-prandial compared to baseline, 51.8% and 46.2% respectfully. Conclusion. To our knowledge, this is the 1st study to report that consumption of a high-fat meal transiently alters SIRT mRNA expression consistent with changes in serum triglyceride and glucose concentration. More research is needed to understand how transient, post-prandial changes in SIRT mRNA expression contribute to increased disease risk

Sex Differences in Change in Skin Temperature When Exercising in a Hot, Humid Environment

The risk for heat-related illness is increased when exercising in a hot, humid environment. In an effort to protect the athlete, body temperature is measured continuously while exercising in extreme environments. Currently, researchers and laboratory personnel employ the use of mean skin temperature to monitor athlete safety; however, this measurement fails to consider localized changes in temperature that may arise as a function of sex and exercise time. Therefore, the purpose of this study was to examine potential sex differences in the change in skin temperature at 17 different upper body locations while exercising in a hot, humid environment. Young men and women were recruited and completed a 60-min walk/jog interval protocol in a hot (34.1 ± 1 °C), humid (64 ± 8%) environment while skin temperature was continuously measured. To account for differences that may have arisen due to differing workloads between men and women, energy expenditure and metabolic heat production were calculated after the completion of exercise. Data was analyzed either a repeated-measures ANOVA (change in skin temperature) or t-test­ (energy expenditure and metabolic heat production). Location of interaction effects was determined using a Fisher’s Least Significant Difference test. Significance was set a p\u3c0.05 for all statistical testing. There was no difference between men and women in total energy expenditure; however, men were found to have a higher metabolic heat production. Women had a higher change in skin temperature at three locations on the back (left upper, right upper, and right mid-back). Conversely, there were no differences at any time point between men and women in the change in core temperature from baseline measurements. This study highlights the need to further investigate sex differences in cooling mechanisms while exercising in a hot, humid environment

Consumption of a high-fat meal increased monocyte adhesion molecule expression and oxLDL phagocytosis: implications for cardiovascular disease risk?

Macrophage-derived foam cells are the predominant component of arterial plaques in the early stages of atherosclerosis. The deposition of arterial plaques is effected by several factors that are influenced by a person’s daily nutritional habits. One factor that poses a major risk for plaque development is high levels of plasma LDL resulting from the consumption of a high-fat meal. In order to understand how an individuals’ diet effects arterial plaque deposition via the process of foam cell formation, we measured the acute response in circulating monocyte activity after consuming a high-fat meal. Samples were acquired on a FlowSight (EMD Millipore) equipped with 405, 488, 642, and 785 nm lasers. Samples were analyzed in IDEAS software to identify pro-inflammatory (CD14+/16+) and classic (CD14+/16-) monocytes. We measured monocyte concentration, adhesion molecule expression, scavenger R expression, and oxLDL phagocytosis for 5 h postprandial. We found that consuming a high-fat meal caused an increase in pro-inflammatory monocyte concentration, adhesion molecule expression, monocyte phagocytosis of oxLDL, and CD36 expression in pro-inflammatory monocytes. These results suggest that consuming a high-fat meal increases the potential of monocytes to become foam cells for at least 5 h postprandial

Room temperature photo-response of titanium supersaturated silicon at energies over the bandgap

Silicon samples were implanted with high Ti doses and subsequently processed with the pulsed-laser melting technique. The electronic transport properties in the 15–300 K range and the room temperature spectral photoresponse at energies over the bandgap were measured. Samples with Ti concentration below the insulator-metal (I-M) transition limit showed a progressive reduction of the carrier lifetime in the implanted layer as Ti dose is increased. However, when the Ti concentration exceeded this limit, an extraordinary recovery of the photoresponse was measured. This result supports the theory of intermediate band materials and is of utmost relevance for photovoltaic cells and Si-based detectors

Using Dry Blood Spots to Evaluate Serum Cytokines and Chemokines in Humans via Multiplex Technology

Introduction: Dried Blood Spot (DBS) analysis has been used routinely since 1963 for the assessment of metabolic diseases in neonates; however, recent efforts have focused on the refinement and validation of DBS for other patient populations. The purpose of this study was to adapt existing DBS methods to analyze 38 serum cytokines/chemokines in human subjects. Validation will be completed by comparing DBS to serum for a given analyte. Methods: After providing informed consent, subjects (N=21) provided a finger-stick DBS or venous serum sample using standard technique. Subjects were apparently healthy, non-obese, and had no known disease. Finger-stick capillary blood samples were collected on Whatman 903 Protein Saver Card (Maidstone, U.K.) and frozen with desiccant in sealed bags prior to elution. DBS samples (2, 6 mm punches) were eluted and transferred through 96-well Multiscreen and Ultracel plates (EMD Millipore) using elution buffer (PBS with 0.5 M NaCL and 0.1% Tween-20) for 16-18 h in the refrigerator. The resultant DBS elute was resuspended in 90 mL of nuclease-free water. Serum samples were thawed overnight in the refrigerator to prevent protein loss. Prior to Milliplex analysis, protein and lipid content were analyzed using a IR-based spectrometer (EMD Millipore Direct Detect). DBS solutions were also analyzed for hemoglobin concentration. Samples were analyzed in pairs to determine the concentration 38 cytokines/chemokines using a Magnetic Multiplex Kit (Milliplex Map Kit High Sensitivity Human Cytokine; Billerica ,MA), A minimum of 50 beads of each targeted analyte were collected on a Luminex MagPix (Austin, TX) and analyzed using Milliplex Analyst Software. Bi-variate correlations were completed in SPSS to compare DBS with serum. Results: Of the 38 markers, 13 were measurable in both DBS and serum, and 3 of these were significantly correlated with each other: Eotaxin (.755), MDC (.446), IP-10 (.831). Based on a post-hoc sample size analysis we need to expand our sample size by approximately 40 subjects in order to establish significant correlations the other 10 DBS measurable cytokines/chemokines. Conclusion: Utilization of DBS may make it possible to obtain previously unobtainable blood samples in a field setting. Despite the potential for DBS all serum markers have not been validated in this sample source. An increase in validated biomarkers detectable in DBS could make the decision to utilize DBS over venipuncture easier. Now that we have identified changes in resting cytokines/chemokines, the next logical step is to evaluate the effect of exercise

Histamine H-3 Receptors Decrease Dopamine Release in the Ventral Striatum by Reducing the Activity of Striatal Cholinergic Interneurons

Histamine H-3 receptors are widely distributed Gi-coupled receptors whose activation reduces neuronal activity and inhibits release of numerous neurotransmitters. Although these receptors are abundantly expressed in the striatum, their modulatory role on activity-dependent dopamine release is not well understood. Here, we observed that histamine H-3 receptor activation indirectly diminishes dopamine overflow in the ventral striatum by reducing cholinergic interneuron activity. Acute brain slices from C57BL/6 or channelrhodopsin-2-transfected DAT-cre mice were obtained, and dopamine transients evoked either electrically or optogenetically were measured by fast-scan cyclic voltammetry. The H-3 agonist alpha-methylhistamine significantly reduced electrically-evoked dopamine overflow, an effect blocked by the nicotinic acetylcholine receptor antagonist dihydro-beta-erythroidine, suggesting involvement of cholinergic interneurons. None of the drug treatments targeting H-3 receptors affected optogenetically evoked dopamine overflow, indicating that direct H-3-modulation of dopaminergic axons is unlikely. Next, we used qPCR and confirmed the expression of histamine H-3 receptor mRNA in cholinergic interneurons, both in ventral and dorsal striatum. Activation of H-3 receptors by alpha-methylhistamine reduced spontaneous firing of cholinergic interneurons in the ventral, but not in the dorsal striatum. Resting membrane potential and number of spontaneous action potentials in ventral-striatal cholinergic interneurons were significantly reduced by alpha-methylhistamine. Acetylcholine release from isolated striatal synaptosomes, however, was not altered by alpha-methylhistamine. Together, these results indicate that histamine H-3 receptors are important modulators of dopamine release, specifically in the ventral striatum, and that they do so by decreasing the firing rate of cholinergic neurons and, consequently, reducing cholinergic tone on dopaminergic axons. (C) 2018 IBRO. Published by Elsevier Ltd. All rights reserved.Peer reviewe

Measuring Adiposity in Patients: The Utility of Body Mass Index (BMI), Percent Body Fat, and Leptin

Background: Obesity is a serious disease that is associated with an increased risk of diabetes, hypertension, heart disease, stroke, and cancer, among other diseases. The United States Centers for Disease Control and Prevention (CDC) estimates a 20 % obesity rate in the 50 states, with 12 states having rates of over 30%. Currently, the body mass index (BMI) is most commonly used to determine adiposity. However, BMI presents as an inaccurate obesity classification method that underestimates the epidemic and contributes to failed treatment. In this study, we examine the effectiveness of precise biomarkers and duel-energy x-ray absorptiometry (DXA) to help diagnose and treat obesity. Methodology/Principal Findings: A cross-sectional study of adults with BMI, DXA, fasting leptin and insulin results wer

Body Composition, Symptoms, and Survival in Advanced Cancer Patients Referred to a Phase I Service

Background: Body weight and body composition are relevant to the outcomes of cancer and antineoplastic therapy. However, their role in Phase I clinical trial patients is unknown. Methods: We reviewed symptom burden, body composition, and survival in 104 patients with advanced cancer referred to a Phase I oncology service. Symptom burden was analyzed using the MD Anderson Symptom Assessment Inventory(MDASI); body composition was evaluated utilizing computerized tomography(CT) images. A body mass index (BMI)$25 kg/m 2 was considered overweight. Sarcopenia, severe muscle depletion, was assessed using CT-based criteria. Results: Most patients were overweight (n = 65, 63$25 kg/m 2. Sarcopenic patients were older and less frequently African-American. Symptom burden did not differ among patients classified according to BMI and presence of sarcopenia. Median (95% confidence interval) survival (days) varied according to body composition: 215 (71–358) (BMI,25 kg/m 2; sarcopenic), 271 (99–443) (BMI,25 kg/m 2; non-sarcopenic), 484 (286–681) (BMI$25 kg/m 2; sarcopenic); 501 d (309–693) (BMI$25 kg/m 2; non-sarcopenic). Higher muscle index and gastrointestinal cancer diagnosis predicted longer survival in multivariate analysis after controlling for age, gender, performance status, and fat index. Conclusions: Patients referred to a Phase I clinic had a high frequency of sarcopenia and a BMI$25 kg/m 2, independent o