Intracellular Ca2+ regulating proteins in vascular smooth muscle cells are altered with type 1 diabetes due to the direct effects of hyperglycemia

<p>Abstract</p> <p>Background</p> <p>Diminished calcium (Ca²⁺) transients in response to physiological agonists have been reported in vascular smooth muscle cells (VSMCs) from diabetic animals. However, the mechanism responsible was unclear.</p> <p>Methodology/Principal Findings</p> <p>VSMCs from autoimmune type 1 Diabetes Resistant Bio-Breeding (DR-BB) rats and streptozotocin-induced rats were examined for levels and distribution of inositol trisphosphate receptors (IP₃R) and the SR Ca²⁺pumps (SERCA 2 and 3). Generally, a decrease in IP₃R levels and dramatic increase in ryanodine receptor (RyR) levels were noted in the aortic samples from diabetic animals. Redistribution of the specific IP₃R subtypes was dependent on the rat model. SERCA 2 was redistributed to a peri-nuclear pattern that was more prominent in the DR-BB diabetic rat aorta than the STZ diabetic rat. The free intracellular Ca²⁺in freshly dispersed VSMCs from control and diabetic animals was monitored using ratiometric Ca²⁺sensitive fluorophores viewed by confocal microscopy. In control VSMCs, basal fluorescence levels were significantly higher in the nucleus relative to the cytoplasm, while in diabetic VSMCs they were essentially the same. Vasopressin induced a predictable increase in free intracellular Ca²⁺in the VSMCs from control rats with a prolonged and significantly blunted response in the diabetic VSMCs. A slow rise in free intracellular Ca²⁺in response to thapsigargin, a specific blocker of SERCA was seen in the control VSMCs but was significantly delayed and prolonged in cells from diabetic rats. To determine whether the changes were due to the direct effects of hyperglycemica, experiments were repeated using cultured rat aortic smooth muscle cells (A7r5) grown in hyperglycemic and control conditions. In general, they demonstrated the same changes in protein levels and distribution as well as the blunted Ca²⁺responses to vasopressin and thapsigargin as noted in the cells from diabetic animals.</p> <p>Conclusions/Significance</p> <p>This work demonstrates that the previously-reported reduced Ca²⁺signaling in VSMCs from diabetic animals is related to decreases and/or redistribution in the IP₃R Ca²⁺channels and SERCA proteins. These changes can be duplicated in culture with high glucose levels.</p

Disorder-Induced Resistive Anomaly Near Ferromagnetic Phase Transitions

We show that the resistivity rho(T) of disordered ferromagnets near, and above, the Curie temperature T_c generically exhibits a stronger anomaly than the scaling-based Fisher-Langer prediction. Treating transport beyond the Boltzmann description, we find that within mean-field theory, d\rho/dT exhibits a |T-T_c|^{-1/2} singularity near T_c. Our results, being solely due to impurities, are relevant to ferromagnets with low T_c, such as SrRuO3 or diluted magnetic semiconductors, whose mobility near T_c is limited by disorder.Comment: 5 pages, 3 figures; V2: with a few clarifications, as publishe

Varied Length Stokes Shift BODIPY-Based Fluorophores for Multicolor Microscopy

Multicolor microscopy tools necessary to localize and visualize the complexity of subcellular systems are limited by current fluorophore technology. While commercial fluorophores cover spectral space from the ultraviolet to the near infrared region and are optimized for conventional bandpass based fluorescence microscopy, they are not ideal for highly multiplexed fluorescence microscopy as they tend to have short Stokes shifts, restricting the number of fluorophores that can be detected in a single sample to four to five. Herein, we synthesized a library of 95 novel boron-dipyrromethene (BODIPY)- based fluorophores and screened their photophysical, optical and spectral properties for their utility in multicolor microscopy. A subset of our BODIPY-based fluorophores yielded varied length Stokes shifts probes, which were used to create a five-color image using a single excitation with confocal laser scanning microscopy for the first time. Combining these novel fluorophores with conventional fluorophores could facilitate imaging in up to nine to ten colors using linear unmixing based microscopy approaches

Optimizing mycobacteria molecular diagnostics: No decontamination! Human DNA depletion? Greener storage at 4 °C!

INTRODUCTION Tuberculosis (TB) is an infectious disease caused by the group of bacterial pathogens Mycobacterium tuberculosis complex (MTBC) and is one of the leading causes of death worldwide. Timely diagnosis and treatment of drug-resistant TB is a key pillar of WHO's strategy to combat global TB. The time required to carry out drug susceptibility testing (DST) for MTBC via the classic culture method is in the range of weeks and such delays have a detrimental effect on treatment outcomes. Given that molecular testing is in the range of hours to 1 or 2 days its value in treating drug resistant TB cannot be overstated. When developing such tests, one wants to optimize each step so that tests are successful even when confronted with samples that have a low MTBC load or contain large amounts of host DNA. This could improve the performance of the popular rapid molecular tests, especially for samples with mycobacterial loads close to the limits of detection. Where optimizations could have a more significant impact is for tests based on targeted next generation sequencing (tNGS) which typically require higher quantities of DNA. This would be significant as tNGS can provide more comprehensive drug resistance profiles than the relatively limited resistance information provided by rapid tests. In this work we endeavor to optimize pre-treatment and extraction steps for molecular testing. METHODS We begin by choosing the best DNA extraction device by comparing the amount of DNA extracted by five commonly used devices from identical samples. Following this, the effect that decontamination and human DNA depletion have on extraction efficiency is explored. RESULTS The best results were achieved (i.e., the lowest Ct values) when neither decontamination nor human DNA depletion were used. As expected, in all tested scenarios the addition of decontamination to our workflow substantially reduced the yield of DNA extracted. This illustrates that the standard TB laboratory practice of applying decontamination, although being vital for culture-based testing, can negatively impact the performance of molecular testing. As a complement to the above experiments, we also considered the best Mycobacterium tuberculosis DNA storage method to optimize molecular testing carried out in the near- to medium-term. Comparing Ct values following three-month storage at 4 °C and at -20 °C and showed little difference between the two. DISCUSSION In summary, for molecular diagnostics aimed at mycobacteria this work highlights the importance of choosing the right DNA extraction device, indicates that decontamination causes significant loss of mycobacterial DNA, and shows that samples preserved for further molecular testing can be stored at 4 °C, just as well at -20 °C. Under our experimental settings, human DNA depletion gave no significant improvement in Ct values for the detection of MTBC

Hyperactivity in the Gunn rat model of neonatal jaundice: age-related attenuation and emergence of gait deficits

Background Neonatal jaundice resulting from elevated unconjugated bilirubin (UCB) occurs in 60–80% of newborn infants. Although mild jaundice is generally considered harmless, little is known about its long-term consequences. Recent studies have linked mild bilirubin-induced neurological dysfunction (BIND) with a range of neurological syndromes, including attention deficit-hyperactivity disorder. The goal of this study was to measure BIND across the lifespan in the Gunn rat model of BIND. Methods Using a sensitive force plate actometer, we measured locomotor activity and gait in jaundiced (jj) Gunn rats versus their non-jaundiced (Nj) littermates. Data were analyzed for young adult (3–4 months), early middle-aged (9–10 months), and late middle-aged (17–20 months) male rats. Results jj rats exhibited lower body weights at all ages and a hyperactivity that resolved at 17–20 months of age. Increased propulsive force and gait velocity accompanied hyperactivity during locomotor bouts at 9–10 months in jj rats. Stride length did not differ between the two groups at this age. Hyperactivity normalized and gait deficits, including decreased stride length, propulsive force, and gait velocity, emerged in the 17–20-month-old jj rats. Conclusions These results demonstrate that, in aging, hyperactivity decreases with the onset of gait deficits in the Gunn rat model of BIND

Hepatitis B screening practices and viral control among persons living with HIV in urban Senegal.

Chronic hepatitis B virus (HBV) infection affects >10% of the general population and is the leading cause of liver cirrhosis and cancer in West Africa. Despite current recommendations, HBV is often not tested for in clinical routine in the region. We included all people living with HIV (PLWH) in care between March and July 2019 at Fann University Hospital in Dakar (Senegal) and proposed hepatitis B surface antigen (HBsAg) test to those never tested. All HBsAg-positive underwent HIV and HBV viral load (VL) and liver stiffness measurement. We evaluated, using logistic regression, potential associations between patient characteristics and (a) HBV testing uptake; (b) HIV/HBV co-infection among individual HBsAg tested. We determined the proportion of co-infected who had HBV DNA >20 IU/ml on ART and sequenced HBV polymerase in those with HBV replication.of 1076 PLWH in care, 689 (64.0%) had never had an HBsAg test prior to our HBV testing intervention. Women and individuals >40 years old were less likely to have been previously tested. After HBV testing intervention,107/884 (12.1%) PLWH were HBsAg-positive. Seven of 58 (12.1%) individuals newly diagnosed with HIV/HBV co-infection had a detectable HBV VL, of whom five were HIV-suppressed. Two patients on ART including 3TC and AZT as backbone showed the presence of the triple resistance mutation 180M/204I/80V. In this Senegalese urban HIV clinic, the majority of patients on ART had never been tested for HBV infection. One in ten co-infected individuals had a detectable HBV VL despite HIV suppression, and 8% were not receiving a TDF-containing regimen

Associations between behavioral regulations and sedentary behavior among older adults

Levels of sedentary behavior increase across the lifespan, making older adults the most sedentary segment of the population. Sedentary behavior is associated with many chronic health conditions including diabetes, cardiovascular disease, and all-cause mortality. Though attempts have been made to reduce or limit sedentary behavior through intervention, little is known about the motivational processes that may be contributing to sedentary time in older adults. It is important to recognize that although physical activity motivation has been extensively researched, physical activity and sedentary behavior are considered independent health behaviors and the same motivational processes that contribute to the upregulation of physical activity may not adequately explain the downregulation (i.e., limiting or reducing) of sedentary behavior. Therefore, the purpose of this thesis is to capture behavioral regulations (specified within Self-Determination Theory) to engage in physical activity and limiting sedentary behavior and examine associations between these behavioral regulations and average daily sedentary time in older adults. Older adults, age 60+ years, completed a baseline questionnaire indicating their behavioral regulations to limit sedentary behavior and to engage in physical activity, then wore an activity monitor for the following two weeks to collect their average daily sedentary time. Results regarding behavioral regulations to limit sedentary behavior indicated that only integrated (ß = -.203, p = .006) and intrinsic regulations (ß = -.185, p = .012) significantly and negatively predicted average daily sedentary time. When all behavioral regulations to limit sedentary behavior were included in the same model, no behavioral regulation significantly predicted average daily sedentary time. Results regarding behavioral regulations to engage in physical activity revealed that only integrated regulation significantly and negatively predicted average daily sedentary time (ß = -.205, p = .007). This negative association remained significant when all behavioral regulations to engage in physical activity were included in the same model (ß = .240, p = .032). This is one of the first studies to assess associations between behavioral regulations to limit sedentary behavior and to engage in physical activity and test their associations with average daily sedentary time among older adults. Results indicate distinct differences between behavioral regulations for limiting sedentary behavior and engaging in physical activity in predicting subsequent average daily sedentary time. Though across both sets of behavioral regulations more autonomous, self-determined behavioral regulations appeared to be associated with average daily behavior compared to more controlling behavioral regulations. Ultimately, this study fills an important knowledge gap by exploring associations between behavioral regulations to limit sedentary behavior and engage in physical activity and subsequent average daily device-based sedentary time. This work is an essential first step in developing effective interventions designed to limit or reduce sedentary behavior among older adults

Hysteresis, Avalanches, and Disorder Induced Critical Scaling: A Renormalization Group Approach

We study the zero temperature random field Ising model as a model for noise and avalanches in hysteretic systems. Tuning the amount of disorder in the system, we find an ordinary critical point with avalanches on all length scales. Using a mapping to the pure Ising model, we Borel sum the $6-\epsilon$ expansion to $O(\epsilon^5)$ for the correlation length exponent. We sketch a new method for directly calculating avalanche exponents, which we perform to $O(\epsilon)$. Numerical exponents in 3, 4, and 5 dimensions are in good agreement with the analytical predictions.Comment: 134 pages in REVTEX, plus 21 figures. The first two figures can be obtained from the references quoted in their respective figure captions, the remaining 19 figures are supplied separately in uuencoded forma

Dynamics of a ferromagnetic domain wall: avalanches, depinning transition and the Barkhausen effect

We study the dynamics of a ferromagnetic domain wall driven by an external magnetic field through a disordered medium. The avalanche-like motion of the domain walls between pinned configurations produces a noise known as the Barkhausen effect. We discuss experimental results on soft ferromagnetic materials, with reference to the domain structure and the sample geometry, and report Barkhausen noise measurements on Fe$_{21}$Co$_{64}$B$_{15}$ amorphous alloy. We construct an equation of motion for a flexible domain wall, which displays a depinning transition as the field is increased. The long-range dipolar interactions are shown to set the upper critical dimension to $d_c=3$, which implies that mean-field exponents (with possible logarithmic correction) are expected to describe the Barkhausen effect. We introduce a mean-field infinite-range model and show that it is equivalent to a previously introduced single-degree-of-freedom model, known to reproduce several experimental results. We numerically simulate the equation in $d=3$, confirming the theoretical predictions. We compute the avalanche distributions as a function of the field driving rate and the intensity of the demagnetizing field. The scaling exponents change linearly with the driving rate, while the cutoff of the distribution is determined by the demagnetizing field, in remarkable agreement with experiments.Comment: 17 RevTeX pages, 19 embedded ps figures + 1 extra figure, submitted to Phys. Rev.

Benefit of early commencement of growth hormone therapy in children with Prader-Willi syndrome

Prader-Willi syndrome (PWS) is a chromosomal disorder and growth failure is a common presentation. Growth hormone (GH) treatment is beneficial in PWS although the optimal age for starting GH is unknown. We investigated whether GH response in PWS was associated with the age of GH commencement by comparing 16 children who commenced GH before 3 years of age (early group) with 40 children who commenced GH after 3 years of age (late group) from the Ozgrow database. Height SDS, body mass index (BMI) SDS, bone age (BA)-chronological age (CA) ratio, change in height (Delta Ht) SDS and change in BMI during 4 years of GH treatment were compared between the groups. The early group had better height SDS and Delta Ht SIDS. BA delay was more pronounced in the early group but BA did not mature beyond CA with GH therapy in either group. Although the initial GH dose for the early group was lower than that of the late group, the former had better height outcome. The starting GH dose seen in the database is lower than the dose used by international centres