Pro-inflammatory Cytokine Interleukin-6 is Upregulated in Early Stage Type 1 Diabetic Rats

Type 1 diabetes (T1DM) is an autoimmune disease characterized by systemic inflammation. T1DM patients are at a higher risk of cardiovascular disease and chronic inflammation, and this is reflected by increased levels of circulating pro-inflammatory cytokines IL-6, IL-1β, and TNF-α. However, the time point at which these pro-inflammatory cytokines become elevated is not known. PURPOSE: The purpose of this study was to determine the concentration of circulating pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in the early stage of T1DM. METHODS: We injected (i.p) 50mg/kg of Streptozotocin (STZ) or the vehicle (CTL) into male and female Sprague Dawley rats and waited one to three weeks before drawing blood. Blood was drawn from the carotid artery into a serum separator vacutainer, centrifuged, and then the serum was aliquoted into tubes and frozen at -80°C for subsequent batch analyses. A rat cytokine multiplex kit (RADPKMAG, EMD Millipore) was used to determine cytokine concentrations in the aliquoted samples. Samples were analyzed using a Luminex 200 instrument (Luminex Corp) according to manufacturer’s instructions. RESULTS: STZ rats had significantly higher blood glucose (CTL=200±15 mg/dl, n=14; STZ=523±14 mg/dl; n=14; p0.05) and TNF-α (STZ: 15.6±12 pg/ml, n=14; CTL: 14.3±9 pg/ml, n=13, p\u3e0.05) were not significantly different between STZ and CTL. CONCLUSION: We conclude that serum IL-6 concentrations are trending toward being greater in the early stage of T1DM. However, serum levels of IL-1β and TNF-α do not appear to be elevated at this stage of the disease. Further studies are needed to determine if concentrations of pro-inflammatory cytokines fluctuate during the progression of the disease

Autism in Glasgow: cumulative incidence and the effects of referral age, deprivation and geographical location

Background: Referrals to the Greater Glasgow Community Autism Team (CAT) made before the child's sixth birthday were analysed to obtain an estimation of the proportion of children in Greater Glasgow with childhood autism and investigate whether there were any variations in diagnosis rates, or in age at referral and diagnosis, depending on deprivation or geographical location. Methods: An analysis was made of the database recording referrals to Greater Glasgow CAT, between 2004 and 2007 inclusive, of children referred by age 6 years, comprising 584 cases. Cumulative incidence was calculated for childhood autism. Ages at referral and diagnosis were also analysed. Results: For this subset of children, there were 246 diagnosed cases of childhood autism, a cumulative incidence from 2004 until 2007 of 11.1 per year per 10 000 children aged 0–6 years. Of children with an eventual diagnosis of autism by age 6, 72% were referred by the age of 4 years. Deprivation was found to have an association with referral and diagnostic rates, with higher rates seen in the most deprived. There was geographical variation in the cumulative incidence of autism. Conclusion: Given that the populations were not known to differ in any manner that would lead to a true variation, the geographical variation in the cumulative incidence of autism in children up to 6 years in Greater Glasgow observed in this study is likely to represent differences in the care pathway between areas. Such differences may also explain the observed association with deprivation. Reasons for the variation are being explored

Detection of P2X3 in DRG Using an Automated Approach to Immunoblotting, Jess

Exaggerated cardiovascular (CV) responses to exercise can lead to adverse CV events. Previous studies have reported that P2X3 receptors, found on the peripheral endings of afferents, contribute to an exaggerated exercise pressor reflex in individuals with CV-related diseases. One way to investigate the role played by these receptors in CV pathophysiology is through immunoblotting. The Jess (Protein Simple) provides an automated option for the protein separation and immunoblotting of the traditional Western Blot and allows for total protein staining, an improvement over the use of loading controls to normalize for sample loading variability. PURPOSE: The purpose of this study was to develop a protocol, using the Jess, for quantifying P2X3 receptor protein expression in the L4 and L5 dorsal root ganglia (DRG) of healthy and type 1 diabetic rats. METHODS: Streptozotocin (STZ), 50 mg/kg, or a vehicle (CTL) was injected i.p into fasted Sprague Dawley rats (n=7 each group). After a minimum of 3 weeks, L4 and L5 DRG were excised, immediately placed in HBSS, and then stored at -80°C until subsequent analyses. For quantification, samples were lysed and protein was isolated (Macherey-Nagel) and then quantified (Qubit protein assay kit). For an initial Optimization Run a single test sample lysate was used; different protein concentrations (0.1 - 1.4 mg/ml) were tested against multiple Anti-P2X3 (Novus Biologicals) dilutions (1:25 – 1:250). Sample lysates (3 μl) and required reagents were loaded into a microplate as per manufacturer’s instructions and then the microplate and capillaries were loaded into the Jess. Over 3 hours; protein separation, antibody incubations, washes, and detection were all performed automatically within the Jess. The data from that run provided the optimal protein concentration and antibody dilution that were then used for the Sample Run, which involved running the CTL and STZ sample lysates. For the Sample Run, protein normalization reagent was added to the microplate in order to normalize for sample loading variability using total protein staining. RESULTS: Optimization Run - A protein concentration of 1.4 mg/ml and a dilution of 1:250 for the P2X3 antibody were found to be optimal. This determination was based on the combination of a low background signal from the antibody and a detectable target protein signal. Sample Run - We found that P2X3 receptor protein expression decreased in STZ rats compared to CTL rats (0.82 ± 0.09 vs 1.00 ± 0.19; n=7 both groups, p=0.03). CONCLUSION: Using the Jess, an automated protocol was developed that detected differences in P2X3 receptor protein expression in rats with and without diabetes. Advantages over the traditional Western Blot include: a run time of 3 hours, reduced user-associated variability thru automation, and the capability of using total protein staining to normalize for sample loading variability. Technological advances such as the Jess are a step towards addressing current rigor and reproducibility concerns

Quantification Method of P2X3 Receptors in Rat DRG Neurons: Western Blotting

Skeletal muscle contractions are known to evoke pressor and cardioaccelerator responses in part by stimulating P2X3 receptors found on the peripheral endings of afferents. In diabetic patients, this pressor response is exaggerated. What is currently not known is whether P2X3 receptors play a role in evoking this exaggerated response. PURPOSE: The purpose of this project was to quantify P2X3 receptors in the L4 and L5 dorsal root ganglia (DRG) neurons in both healthy and type 1 diabetic rats using western blot analysis. METHODS: We injected 50 mg/kg streptozotocin (STZ) or the vehicle (CTL) i.p in fasted female and male Sprague Dawley rats and then waited at least 7 days for the rats to become diabetic. We then performed a laminectomy in the anesthetized rats to expose the spinal cord and roots. Using a dissecting microscope, we removed the L4 and L5 DRG from the spinal column. The DRG are the cell bodies of the peripheral afferents found in the hindlimb musculature. The DRG were placed in HBSS (is this buffer?) and stored at -80°C until analysis. For quantification, samples were lysed and proteins were isolated using the NucleoSpin RNA/Protein Kit (Macherey-Nagel, Bethlehem, PA, USA). A Qubit 3.0 Fluorometer was used to quantify the protein concentration of each sample so that equal protein concentrations could then be loaded onto a Bolt Bis-Tris (4-12%) gel. Following electrophoresis, the proteins were transferred to a membrane before being probed with a rabbit polyclonal P2X3 antibody (Alomone Labs), followed by an anti-rabbit secondary antibody conjugated to alkaline phosphatase (Life Technologies). The membrane was then exposed using a ChemiDoc XRS and the results analyzed using BioRad’s Quantity One imaging software. RESULTS: We were able to detect P2X3 receptor proteins. When compared with a molecular weight ladder, P2X3 receptor proteins were 54kDa, which is similar to the molecular weight of P2X3 receptors quantified in other studies. CONCLUSION: This method of quantifying P2X3 receptors in DRG neurons allows for a comparison between non-diabetic and diabetic rats. Further analyses are required to determine whether the quantity of P2X3 receptors in L4 and L5 DRG neurons is different in diabetic rats compared to non-diabetic rats

Quantum Criticality in an Organic Magnet

Exchange interactions between $S=\frac{1}{2}$ sites in piperazinium hexachlorodicuprate produce a frustrated bilayer magnet with a singlet ground state. We have determined the field-temperature phase diagram by high field magnetization and neutron scattering experiments. There are two quantum critical points: $H_{c1}=7.5$ T separates a quantum paramagnet phase from a three dimensional, antiferromagnetically-ordered state while $H_{c2}=37$ T marks the onset of a fully polarized state. The ordered phase, which we describe as a magnon Bose-Einstein condensate (BEC), is embedded in a quantum critical regime with short range correlations. A low temperature anomaly in the BEC phase boundary indicates that additional low energy features of the material become important near $H_{c1}$.Comment: 4 pages, 4 figures, submitted to Phys. Rev. Lett. Replaced original text with additional conten

Magnetic tomography for lead acid batteries

© 2017 The AuthorsThis paper explores the inverse problem approach for finding the current distribution within an electrochemical cell from magnetic field measurements. Current distribution is shown to be a useful measurement for diagnosis of cells and development of cell design. Existing current distribution measurement methods are discussed to provide context and motivation for the work. Magnetic field measurements can be obtained non-invasively and contain information about the current distribution, which is extracted using an appropriate solver. Experimental results are presented which test the effectiveness of a particular inverse problem solver, using both simulated and real magnetic field measurements. The solver presented is based upon one found in literature, but with novel problem-specific modifications. Errors in conductance values in the forward model definition are simulated in order to quantify their effect on solution quality. A modification to the solver is proposed to improve robustness against these model errors. This results in improved solution quality when using real measured data from a resistor-wire model of a cell, and simulated data from a model which more accurately represents the conductance of the cell plate grid and active mass

Quantum spin correlations in an organometallic alternating sign chain

High resolution inelastic neutron scattering is used to study excitations in the organometallic magnet DMACuCl$_3$. The correct magnetic Hamiltonian describing this material has been debated for many years. Combined with high field bulk magnetization and susceptibility studies, the new results imply that DMACuCl$_3$ is a realization of the $S=1/2$ alternating antiferromagnetic-ferromagnetic (AFM-FM) chain. Coupled-cluster calculations are used to derive exchange parameters, showing that the AFM and FM interactions have nearly the same strength. Analysis of the scattering intensities shows clear evidence for inter-dimer spin correlations, in contrast to existing results for conventional alternating chains. The results are discussed in the context of recent ideas concerning quantum entanglement.Comment: 5 pages, 4 figures included in text. Submitted to APS Journal

Patient experiences of telephone outreach to enhance uptake of NHS Health Checks in more deprived communities and minority ethnic groups: A qualitative interview study

Background: The NHS Health Checks preventative programme aims to reduce cardiovascular morbidity across England. To improve equity in uptake, telephone outreach was developed in Bristol, involving community workers telephoning patients amongst communities potentially at higher risk of cardiovascular disease and/or less likely to take up a written invitation, to engage them with NHS Health Checks. Where possible, caller cultural background/main language is matched with that of the patient called. The call includes an invitation to book an NHS Health Check appointment, lifestyle questions from the Health Check, and signposting to lifestyle services. Objective: To explore the experiences of patients who received an outreach call. Design/Setting/Participants: Thematic analysis of semi-structured interviews with 24 patients (15 female), from seven primary care practices, who had received an outreach call. Results: The call increased participants’ understanding of NHS Health Checks and overcame anticipated difficulties with making an appointment. Half reported that they would not have booked if only invited by letter. The cultural identity/language skills of the caller were important in facilitating the interaction for some who might otherwise encounter language or cultural barriers. The inclusion of lifestyle questions and signposting prompted a minority to make lifestyle changes. Conclusions: Participants valued easily generalizable aspects of the intervention—a telephone invitation with ability to book during the call—and reported that it prompted acceptance of an NHS Health Check. A caller who shared their main language/cultural background was important for a minority of participants, and improved targeting of this would be beneficial

Acute Effect of Hyperglycemia on the Mechanoreflex and Metaboreflex

Recent studies in both humans and rodents have shown that the mechanoreflex and metaboreflex are exaggerated in type 2 diabetes mellitus (T2DM). Hyperglycemia is a main characteristic of T2DM and is known to cause damage to both cardiovascular and nervous system structures. However, the acute effect of the presence of hyperglycemia on the mechanoreflex and metaboreflex are not known. PURPOSE: To determine the acute effect of hyperglycemia on the mechanoreflex and metaboreflex. METHODS: Experiments were conducted after an overnight fast in unanesthetized, decerebrated healthy male and female Sprague-Dawley rats. The mechanoreflex was evoked by stretching the Achilles tendon for 30 s whereas the metaboreflex was evoked by locally injecting lactic acid (0.2ml, 24mM) into the hindlimb. Time and dosage for glucose infusion were selected based on a preliminary study that showed infusing 250 mg/ml of glucose solution for 15 min into the hindlimb circulation, with blood flow to and from the hindlimb restricted, would elevate local blood glucose concentration to the same degree as that seen in T2DM rats with an exaggerated exercise pressor reflex. To elicit an acute local hyperglycemic environment, while preventing an endogenous insulin response, somatostatin (3.9 ug/100 ul) was infused systemically and simultaneously with the local glucose infusion. Changes in mean arterial pressure (ΔMAP) and heart rate (ΔHR) in response to tendon stretch and lactic acid injection were measured and compared before and after infusion. RESULTS: We found that the peak pressor and cardioaccelerator responses to tendon stretch were not significantly affected by hyperglycemia (ΔMAP before: 12 ± 2 mmHg, after: 12 ± 3 mmHg, n=6, p\u3e0.05; ΔHR before: 10 ± 3 bpm; after: 10 ± 3 bpm, n=6, p\u3e0.05). Likewise, the pressor and cardioaccelerator responses to lactic acid were not significantly affected by hyperglycemia (ΔMAP before: 13 ± 2 mmHg, after: 16 ± 3 mmHg, n=10, p\u3e0.05; ΔHR before: 10 ± 2 bpm, after: 12 ± 5 bpm, n=10, p\u3e0.05). CONCLUSION: The acute presence of hyperglycemia in the local circulation of the hindlimb likely does not contribute to the exaggerated mechanoreflex or metaboreflex