A cell-free bioassay for the detection of androgens.

Androgens remain abused performance-enhancing drugs in sports. Technologies based on mass spectrometry can detect all forms of androgens but fail if the androgen represents a novel structure. A bioassay detects androgens based on function rather than structure. To date, there has been limited adoption of cell-based in vitro bioassays as a screening tool for nontargeted androgen detection because they require expert personnel and specialized equipment to perform. We now describe the development of a cell-free version of an androgen in vitro bioassay. Stage 1 involved in vitro transcription/translation reactions (IVTT) using a DNA template encoding an enhancer/androgen response element (ARE) regulatory region upstream of a minimal promoter that drives expression of a reporter protein. The assay detected testosterone across the concentration range of 106.7 to 0.0144 ng/ml (3.7 × 10-7 to 5 × 10-11 M), with an EC50 of 6.63 ng/ml (23 nM). To reduce complexity, Stages 2-4 of development included just in vitro transcription (IVT) reactions, whereby the output was an RNA molecule. Stage 2 involved directly labelling the RNA molecule with fluorophore-labelled nucleotide triphosphates, Stage 3 involved reverse transcription-polymerase chain reaction (PCR) of the RNA molecule, and Stage 4 utilized an RNA aptamer, Mango II, as its RNA output. The Stage 4 product detected testosterone across the range of 106.7-0.0001 ng/ml (3.7 × 10-7 to 5 × 10-13 M), with an EC50 of 0.04 ng/ml (0.155 nM). Further to this, we show that the Stage 4 product can detect other androgenic molecules. Relative to cell-based bioassays, the Stage 4 product is easy to perform and could be developed into a routine, high-throughput, nontargeted androgen screen.

Unravelling androgens in sport: Altrenogest shows strong activation of the androgen receptor in a mammalian cell bioassay.

Altrenogest is a commonly used progestogen for the suppression of oestrus and associated distracting behaviours that interfere with training and performance of female racehorses. The steroid is derived from 19-nor testosterone and is structurally similar to the anabolic androgenic steroid, trenbolone. In this study, the relative androgen potency of altrenogest was determined by a kidney (HEK293) cell androgen bioassay. The HEK293 bioassay shows that in its pure form, altrenogest has a high relative potency compared with testosterone but is not as strong as ß-trenbolone. Our results also show that altrenogest is able to activate the androgen receptor at the concentrations relevant to the administration regime of racehorses and retains its activity ex vivo. Thus, we show unequivocally that altrenogest, a progestogen used widely in female racehorses, acts as a strong androgen in a mammalian cell bioassay.

Evaluation of the Effects of Synovial Multipotent Cells on Deep Digital Flexor Tendon Repair in a Large Animal Model of Intra-Synovial Tendinopathy.

Intra-synovial tendon injuries are a common orthopedic problem with limited treatment options. The synovium is a specialized connective tissue forming the inner encapsulating lining of diarthrodial joints and intra-synovial tendons. It contains multipotent mesenchymal stromal cells that render it a viable source of progenitors for tendon repair. This study evaluated the effects of autologous implantation of cells derived from normal synovium (synovial membrane cells [SMCs]) in augmenting repair in an ovine model of intra-synovial tendon injury. For this purpose, synovial biopsies were taken from the right digital flexor tendon sheath following creation of a defect to the lateral deep digital flexor tendon. Mononuclear cells were isolated by partial enzymatic digestion and assessed for MSC characteristics. Cell tracking and tendon repair were assessed by implanting 5 × 106 cells into the digital flexor tendon sheath under ultrasound guidance with the effects evaluated using magnetic resonance imaging and histopathology. Synovial biopsies yielded an average 4.0 × 105 ± 2.7 × 105 SMCs that exhibited a fibroblastic morphology, variable osteogenic, and adipogenic responses but were ubiquitously strongly chondrogenic. SMCs displayed high expression of CD29 with CD271NEGATIVE and MHC-IILOW cell-surface marker profiles, and variable expression of CD73, CD90, CD105, CD166, and MHC-I. Implanted SMCs demonstrated engraftment within the synovium, though a lack of repair of the tendon lesion over 24 weeks was observed. We conclude healthy synovium is a viable source of multipotent cells, but that the heterogeneity of synovium underlies the variability between different SMC populations, which while capable of engraftment and persistence within the synovium exhibit limited capacity of influencing tendon repair. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 38:128-138, 2020.

Siglec-14 Enhances NLRP3-Inflammasome Activation in Macrophages.

Pathogenic microorganisms are sensed by the inflammasome, resulting in the release of the pro-immune and proinflammatory cytokine interleukin-1ß (IL-1ß). In humans, the paired sialic acid-binding Ig-like lectin receptors Siglec-5 (inhibitory) and Siglec-14 (activating) have been shown to have reciprocal roles in regulating macrophage immune responses, but their interaction with IL-1ß signaling and the inflammasome has not been characterized. Here we show that in response to known inflammasome activators (ATP, nigericin) or the sialic acid-expressing human bacterial pathogen group B Streptococcus (GBS), the presence of Siglec-14 enhances, whereas Siglec-5 reduces, inflammasome activation and macrophage IL-1ß release. Human THP-1 macrophages stably transfected with Siglec-14 exhibited increased caspase-1 activation, IL-1ß release and pyroptosis after GBS infection, in a manner blocked by a specific inhibitor of nucleotide-binding domain leucine-rich repeat protein 3 (NLRP3), a protein involved in inflammasome assembly. Another leading pathogen, Streptococcus pneumoniae, lacks sialic acid but rather prominently expresses a sialidase, which cleaves sialic acid from macrophages, eliminating cis- interactions with the lectin receptor, thus attenuating Siglec-14 induced IL-1ß secretion. Vimentin, a cytoskeletal protein released during macrophage inflammatory activation is known to induce the inflammasome. We found that vimentin has increased interaction with Siglec-14 compared to Siglec-5, and this interaction heightened IL-1ß production by Siglec-14-expressing cells. Siglec-14 is absent from some humans because of a SIGLEC5/14 fusion polymorphism, and we found increased IL-1ß expression in primary macrophages from SIGLEC14+/+ individuals compared to those with the SIGLEC14-/+ and SIGLEC14-/- genotypes. Collectively, our results identify a new immunoregulatory role of Siglec-14 as a positive regulator of NLRP3 inflammasome activation.

Canadian-Australasian Randomised trial of screening kidney transplant candidates for coronary artery disease-A trial protocol for the CARSK study.

Transplantation is the preferred treatment for patients with kidney failure, but the need exceeds the supply of transplantable kidneys, and patients routinely wait >5 years on dialysis for a transplant. Coronary artery disease (CAD) is common in kidney failure and can exclude patients from transplantation or result in death before or after transplantation. Screening asymptomatic patients for CAD using noninvasive tests prior to wait-listing and at regular intervals (ie, annually) after wait-listing until transplantation is the established standard of care and is justified by the need to avoid adverse patient outcomes and loss of organs. Patients with abnormal screening tests undergo coronary angiography, and those with critical stenoses are revascularized. Screening is potentially harmful because patients may be excluded or delayed from transplantation, and complications after revascularization are more frequent in this population. CARSK will test the hypothesis that eliminating screening tests for occult CAD after wait-listing is not inferior to regular screening for the prevention of major adverse cardiac events defined as the composite of cardiovascular death, nonfatal myocardial infarction, urgent revascularization, and hospitalization for unstable angina. Secondary outcomes include the transplant rate, safety measures, and the cost-effectiveness of screening. Enrolment of 3,306 patients over 3 years is required, with patients followed for up to 5 years during wait-listing and for 1 year after transplantation. By validating or refuting the use of screening tests during wait-listing, CARSK will ensure judicious use of health resources and optimal patient outcomes.

ß2 -Adrenoceptors indirectly support impaired ß1 -adrenoceptor responsiveness in the isolated type 2 diabetic rat heart.

NEW FINDINGS: What is the central question of this study? Are there specific contributions of ß1 - and ß2 -adrenoceptor subtypes to the impaired ß-adrenoceptor responsiveness of the type 2 diabetic heart? What is the main finding and its importance? In hearts isolated from the Zucker diabetic fatty rat model of type 2 diabetes, we showed that the ß1 -adrenoceptors are the main subtype to regulate heart rate, contraction and relaxation. Notably, the ß2 -adrenoceptor subtype actions seem to support function in the diabetic heart indirectly. ABSTRACT: Impaired ß-adrenoceptor (ß-AR) responsiveness causes cardiac vulnerability in patients with type 2 diabetes, but the independent contributions of ß1 - and ß2 -AR subtypes to ß-AR-associated cardiac dysfunction in diabetes are unknown. Our aim was to determine the specific ß1 - and ß2 -AR responsiveness of heart rate (HR), contraction and relaxation in the diabetic heart. Isolated Langendorff-perfused hearts of Zucker type 2 diabetic fatty (ZDF) rats were stimulated with the ß-AR agonist isoprenaline (1 × 10-11 to 3 × 10-8  mol l-1 ) with or without the selective ß1 -AR antagonist CGP20712A (3 × 10-8  mol l-1 ) or the ß2 -AR antagonist ICI-118,551 (5 × 10-8  mol l-1 ), and HR, contraction and relaxation were measured. Diabetic hearts showed lower basal HR (non-diabetic 216 ± 17 beats min-1 versus diabetic 151 ± 23 beats min-1 , P < 0.05). However, the ß-AR-induced increase in HR was similar and was completely blocked by the ß1 -AR antagonist, but not by the ß2 -AR antagonist. The ß-AR-induced increase in contraction and acceleration of relaxation was impaired in diabetic hearts, completely blocked by the ß1 -AR antagonist and partly impaired by the ß2 -AR antagonist. Western blots revealed 41% higher phosphorylation levels of AMP kinase (AMPK), a key regulator of cardiac energy metabolism, in diabetic hearts (non-diabetic 1.62 ± 0.19 a.u. versus diabetic 2.30 ± 0.25 a.u., P < 0.05). In conclusion, the ß1 -AR is the main subtype regulating chronotropic, inotropic and lusitropic ß-AR responses in the healthy heart and the type 2 diabetic heart. The ß2 -AR subtype indirectly supports the ß1 -AR functional response in the diabetic heart. This suggests that ß2 -ARs could be an indirect target to improve the function of the heart in type 2 diabetes.

Bone marrow mesenchymal stem cells do not enhance intra-synovial tendon healing despite engraftment and homing to niches within the synovium.

BACKGROUND: Intra-synovial tendon injuries display poor healing, which often results in reduced functionality and pain. A lack of effective therapeutic options has led to experimental approaches to augment natural tendon repair with autologous mesenchymal stem cells (MSCs) although the effects of the intra-synovial environment on the distribution, engraftment and functionality of implanted MSCs is not known. This study utilised a novel sheep model which, although in an anatomically different location, more accurately mimics the mechanical and synovial environment of the human rotator cuff, to determine the effects of intra-synovial implantation of MSCs. METHODS: A lesion was made in the lateral border of the lateral branch of the ovine deep digital flexor tendon within the digital sheath and 2 weeks later 5 million autologous bone marrow MSCs were injected under ultrasound guidance into the digital sheath. Tendons were recovered post mortem at 1 day, and 1-2, 4, 12 and 24 weeks after MSC injection. For the 1-day and 1-2-week groups, MSCs labelled with fluorescent-conjugated magnetic iron-oxide nanoparticles (MIONs) were tracked with MRI, histology and flow cytometry. The 4, 12 and 24-week groups were implanted with non-labelled cells and compared with saline-injected controls for healing. RESULTS: The MSCs displayed no reduced viability in vitro to an uptake of 20.0 ± 4.6 pg MIONs per cell, which was detectable by MRI at minimal density of ~ 3 × 104 cells. Treated limbs indicated cellular distribution throughout the tendon synovial sheath but restricted to the synovial tissues, with no MSCs detected in the tendon or surgical lesion. The lesion was associated with negligible morbidity with minimal inflammation post surgery. Evaluation of both treated and control lesions showed no evidence of healing of the lesion at 4, 12 and 24 weeks on gross and histological examination. CONCLUSIONS: Unlike other laboratory animal models of tendon injury, this novel model mimics the failed tendon healing seen clinically intra-synovially. Importantly, however, implanted stem cells exhibited homing to synovium niches where they survived for at least 14 days. This phenomenon could be utilised in the development of novel physical or biological approaches to enhance localisation of cells in augmenting intra-synovial tendon repair.

Cardiac ß-adrenergic responsiveness of obese Zucker rats: The role of AMPK.

NEW FINDINGS: What is the central question of the study? Is the reduced signalling of AMP-activated protein kinase (AMPK), a key regulator of energy homeostasis in the heart, responsible for the reduced ß-adrenergic responsiveness of the heart in obesity? What is the main finding and its importance? Inhibition of AMPK in isolated hearts prevented the reduced cardiac ß-adrenergic responsiveness of obese rats, which was accompanied by reduced phosphorylation of AMPK, a proxy of AMPK activity. This suggests a direct functional link between ß-adrenergic responsiveness and AMPK signalling in the heart, and it suggests that AMPK might be an important target to restore the ß-adrenergic responsiveness in the heart in obesity. ABSTRACT: The obesity epidemic impacts heavily on cardiovascular health, in part owing to changes in cardiac metabolism. AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis in the heart and is regulated by ß-adrenoceptors (ß-ARs) in normal conditions. In obesity, chronic sympathetic overactivation leads to impaired cardiac ß-AR responsiveness, although it is unclear whether AMPK signalling, downstream of ß-ARs, contributes to this dysfunction. Therefore, we aimed to determine whether reduced AMPK signalling is responsible for the reduced ß-AR responsiveness in obesity. In isolated hearts of lean and obese Zucker rats, we tested ß-AR responsiveness to the ß1 -AR agonist isoprenaline (ISO, 1 × 10-10 to 5 × 10-8  m) in the absence and presence of the AMPK inhibitor, compound C (CC, 10 µm). The ß1 -AR expression and AMPK phosphorylation were assessed by Western blot. ß-Adrenergic responsiveness was reduced in the hearts of obese rats (logEC50 of ISO-developed pressure dose-response curves: lean -8.53 ± 0.13 × 10x  m versus obese -8.35 ± 0.10 × 10x  m ; P < 0.05 lean versus obese, n = 6 per group). This difference was not apparent after AMPK inhibition (logEC50 of ISO-developed pressure curves: lean CC -8.19 ± 0.12 × 10x  m versus obese CC 8.17 ± 0.13 × 10x  m, P < 0.05, n = 6 per group). ß1 -Adrenergic receptor expression and AMPK phosphorylation were reduced in hearts of obese rats (AMPK at Thr172 : lean 1.73 ± 0.17 a.u. versus lean CC 0.81 ± 0.13 a.u., and obese 1.18 ± 0.09 a.u. versus obese CC 0.81 ± 0.16 a.u., P < 0.05, n = 6 per group). Thus, a direct functional link between ß-adrenergic responsiveness and AMPK signalling in the heart exists, and AMPK might be an important target to restore the reduced cardiac ß-adrenergic responsiveness in obesity.

Challenges and impossibilities of 'standing alongside' in an intolerable context: Learning from refugees and volunteers in the Calais camp.

This article describes the experience of setting up a psychosocial and therapeutic support project in the French Calais refugee camp, by a group of family therapists and clinical psychologists from the United Kingdom. This came about in response to reports of a humanitarian crisis unfolding on our doorstep, with the British government's lack of support for the growing numbers of refugees gathering along the UK border with France. The project involved working alongside other agencies in the camp to provide psychosocial and resilience-based therapeutic support to unaccompanied young people, women, children and their families and also to many volunteers in the camp. The process of setting up the work is described, as well as the challenges and dilemmas of offering an intervention in extremely unsafe and insanitary conditions, where for most the experience of trauma was ongoing. The project was informed by systemic-narrative practice and community/liberation psychology, which incorporate the political and social context. A narrative framework offered a way of drawing on people's strengths and resources, rooted in their cultural and social histories and helping them connect with preferred identities, which we found to be essential in the context of ongoing crisis.

Estrogen Receptor Control of Atherosclerotic Calcification and Smooth Muscle Cell Osteogenic Differentiation.

OBJECTIVE: Vascular calcification is associated with increased risk of myocardial infarction and stroke. The objective of this work was to examine the ability of 17ß-estradiol (E2) to stimulate calcification of vascular smooth muscle cells (VSMC) in vivo, using aged apolipoprotein E-null mice with advanced atherosclerotic lesions, and subsequently to explore underlying mechanisms in vitro. APPROACH AND RESULTS: Silastic E2 capsules were implanted into male and female apolipoprotein E-null mice aged 34 weeks. Plaque and calcified area were measured in the aortic sinus and innominate artery after 8 weeks. Immunohistochemical analysis examined expression of the estrogen receptors (estrogen receptor alpha and estrogen receptor beta [ERß]). VSMC expression of osteogenic markers was examined using digital polymerase chain reaction. Advanced atherosclerotic lesions were present in all mice at the end of 8 weeks. In both male and female mice, E2 increased calcified area in a site-specific manner in the aortic sinus independently of plaque growth or lipid levels and occurred in association with a site-specific decrease in the proportion of ERß-positive intimal cells. Calcified lesions expressed collagen I and bone sialoprotein, with decreased matrix Gla protein. In vitro, E2 suppressed ERß expression and increased VSMC mineralization, demonstrating increased collagen I and II, osteocalcin and bone sialoprotein, and reduced matrix Gla protein and osteopontin. Antagonism or RNA silencing of estrogen receptor alpha, ERß, or both further increased VSMC mineralization. CONCLUSIONS: We have demonstrated that E2 can drive calcification in advanced atherosclerotic lesions by promoting the differentiation of VSMC to osteoblast-like cells, a process which is augmented by inhibition of estrogen receptor alpha or ERß activity.

Chronic bilateral renal denervation reduces cardiac hypertrophic remodelling but not ß-adrenergic responsiveness in hypertensive type 1 diabetic rats.

NEW FINDINGS: What is the central question of this study? Can bilateral renal denervation, an effective antihypertensive treatment in clinical and experimental studies, improve cardiac ß-adrenoceptor responsiveness in a diabetic model with underlying hypertension? What is the main finding and its importance? Bilateral renal denervation did not affect ß-adrenergic responsiveness in the diabetic hypertensive rat heart, but denervation reduced the hypertension-induced concentric hypertrophic remodelling. This suggests that the positive haemodynamic changes induced by renal denervation are most likely to reflect an attenuation of sympathetic effects on the systemic vasculature and/or the renal function rather than direct sympathetic modulation of the heart. Bilateral renal denervation (BRD) has been shown to normalise blood pressure in clinical and experimental studies of hypertension by reducing systemic sympathetic output. This study determined the effect of BRD on cardiac ß-adrenoceptor (AR) responsiveness in a diabetic model with underlying hypertension using the transgenic (mRen-2)27 rats. Bilateral renal denervation or sham surgeries were conducted repeatedly at 3, 6 and 9 weeks in Ren-2 rats with or without streptozotocin (STZ)-induced diabetes (4 × n = 7); Sprague-Dawley rats (n = 6) served as control animals. Cardiac function was determined in isolated hearts at 18 weeks of age. Normalised left ventricular developed pressure and relaxation was recorded in response to incremental concentrations of the ß-AR agonist isoprenaline (from 10-10 to 10-7 m) or the ß3 -AR agonist BRL37344 (from 10(-13) to 10(-6 ) m). Expression levels of ß1 -AR were determined by Western blot. Both inotropic and lusitropic ß-AR responsiveness was reduced in the hypertensive diabetic hearts, but these responses were unaltered after BRD. Expression levels of ß1 -AR were increased after BRD (Sham, 0.85 ± 0.11 versus 1.01 ± 0.05 a.u.; BRD, 1.45 ± 0.11 versus 1.46 ± 0.07 a.u.; Ren-2 versus Ren-2 STZ, P < 0.05 versus Sham). No effect of ß3 -AR agonist stimulation with BRL37344 was observed. Interestingly, BRD increased left ventricular diastolic volume in both the Ren-2 and the Ren-2 STZ groups. Bilateral renal denervation did not restore the attenuated cardiac ß-AR responsiveness in the diabetic hypertensive rats, but it reduced the extent of hypertension-induced concentric hypertrophic remodelling. Thus, the haemodynamic protection offered by renal denervation appears to reflect an attenuated sympathetic innervation of the systemic vasculature and/or kidney rather than a direct cardiac effect.

Increased Efferent Cardiac Sympathetic Nerve Activity and Defective Intrinsic Heart Rate Regulation in Type 2 Diabetes.

Elevated sympathetic nerve activity (SNA) coupled with dysregulated ß-adrenoceptor (ß-AR) signaling is postulated as a major driving force for cardiac dysfunction in patients with type 2 diabetes; however, cardiac SNA has never been assessed directly in diabetes. Our aim was to measure the sympathetic input to and the ß-AR responsiveness of the heart in the type 2 diabetic heart. In vivo recording of SNA of the left efferent cardiac sympathetic branch of the stellate ganglion in Zucker diabetic fatty rats revealed an elevated resting cardiac SNA and doubled firing rate compared with nondiabetic rats. Ex vivo, in isolated denervated hearts, the intrinsic heart rate was markedly reduced. Contractile and relaxation responses to ß-AR stimulation with dobutamine were compromised in externally paced diabetic hearts, but not in diabetic hearts allowed to regulate their own heart rate. Protein levels of left ventricular ß1-AR and Gs (guanine nucleotide binding protein stimulatory) were reduced, whereas left ventricular and right atrial ß2-AR and Gi (guanine nucleotide binding protein inhibitory regulatory) levels were increased. The elevated resting cardiac SNA in type 2 diabetes, combined with the reduced cardiac ß-AR responsiveness, suggests that the maintenance of normal cardiovascular function requires elevated cardiac sympathetic input to compensate for changes in the intrinsic properties of the diabetic heart.

Low dose of propranolol does not affect rat osteotomy healing and callus strength.

Experimental studies suggest that the ß-blocker propranolol stimulates bone formation but little work has investigated its effect on fracture healing. In this study, we examined if a low dose of propranolol, previously shown to be preventive against bone loss in rats, improves bone repair. Female Wistar rats were injected with saline or propranolol (0.1 mg/kg/day) (n = 20/group), 5 days a week for 8 weeks. Three weeks after the beginning of treatment, all rats underwent a mid-diaphyseal transverse osteotomy in the left femur. Radiographic analysis of ostetomy healing was performed 2 and 5 weeks after osteotomy. Rats were sacrificed at 5 weeks and femora collected for measurements of fracture strength by torsional testing, callus volume, and mineral content by micro-CT analysis and histology of fracture callus. Eighty nine percent of osteotomies achieved apparent radiological union by 5 weeks in both groups. Propranolol treatment did not significantly alter the torsional strength of the fractured femur compared with controls. The volume and mineralization of fracture callus at 5 weeks were not significantly different in both groups. Histology showed that endochondral ossification was not affected by propranolol. Altogether, our results demonstrate that propranolol using the regimen described does not significantly improve or inhibit rat osteotomy healing and mechanical strength.

Impaired relaxation despite upregulated calcium-handling protein atrial myocardium from type 2 diabetic patients with preserved ejection fraction.

BACKGROUND: Diastolic dysfunction is a key factor in the development and pathology of cardiac dysfunction in diabetes, however the exact underlying mechanism remains unknown, especially in humans. We aimed to measure contraction, relaxation, expression of calcium-handling proteins and fibrosis in myocardium of diabetic patients with preserved systolic function. METHODS: Right atrial appendages from patients with type 2 diabetes mellitus (DM, n = 20) and non-diabetic patients (non-DM, n = 36), all with preserved ejection fraction and undergoing coronary artery bypass grafting (CABG), were collected. From appendages, small cardiac muscles, trabeculae, were isolated to measure basal and ß-adrenergic stimulated myocardial function. Expression levels of calcium-handling proteins, sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) and phospholamban (PLB), and of ß1-adrenoreceptors were determined in tissue samples by Western blot. Collagen deposition was determined by picro-sirius red staining. RESULTS: In trabeculae from diabetic samples, contractile function was preserved, but relaxation was prolonged (Tau: 74 ± 13 ms vs. 93 ± 16 ms, non-DM vs. DM, p = 0.03). The expression of SERCA2a was increased in diabetic myocardial tissue (0.75 ± 0.09 vs. 1.23 ± 0.15, non-DM vs. DM, p = 0.007), whereas its endogenous inhibitor PLB was reduced (2.21 ± 0.45 vs. 0.42 ± 0.11, non-DM vs. DM, p = 0.01). Collagen deposition was increased in diabetic samples. Moreover, trabeculae from diabetic patients were unresponsive to ß-adrenergic stimulation, despite no change in ß1-adrenoreceptor expression levels. CONCLUSIONS: Human type 2 diabetic atrial myocardium showed increased fibrosis without systolic dysfunction but with impaired relaxation, especially during ß-adrenergic challenge. Interestingly, changes in calcium-handling protein expression suggests accelerated active calcium re-uptake, thus improved relaxation, indicating a compensatory calcium-handling mechanism in diabetes in an attempt to maintain diastolic function at rest despite impaired relaxation in the diabetic fibrotic atrial myocardium. Our study addresses important aspects of the underlying mechanisms of diabetes-associated diastolic dysfunction, which is crucial to developing new therapeutic treatments.

Increased sympathetic drive during the onset of hypertension in conscious Cyp1a1-Ren2 rats.

Despite advances in our understanding concerning the pathology of hypertension, the mechanisms that underpin the origin of hypertension remain to be fully elucidated. This enigma is, at least in part, due to inherent limitations of various animal models of hypertension. Here, we show the genetically modified Cyp1a1-Ren2 rat model, in which the onset and severity of angiotensin II-dependent hypertension can be tightly controlled, as an effective model for investigating increased sympathetic drive for the onset of hypertension. Cyp1a1-Ren2 rats were surgically prepared with radiotelemetric transmitters for the continuous measurement of arterial blood pressure (ABP). ABP was recorded in freely moving rats that were fed with either normal rat chow or a diet containing indole-3-carbinol (0.225% w/w) for 7 days to induce hypertension. Structural morphology of and endothelial NO synthase (eNOS) protein expression in heart and/or vascular tissue were analyzed. Sympathetic tone was estimated using spectral analysis of heart rate variability. The progressive induction of hypertension over 7 days was matched with a parallel increase in sympathetic tone. By day 7 of hypertension, eNOS expression in the mesenteric artery was elevated. However, the elevated ABP, sympathetic tone, and eNOS had not elicited gross morphological remodeling of the heart or vasculature. Importantly, both the increase in sympathetic tone and overexpression of eNOS within the vasculature were reversed when ABP was returned to normal. We conclude that the Cyp1a1-Ren2 rat provides an effective model for investigating specific adverse and transient changes in central sympathetic modulation of arterial blood pressure during the early onset of angiotensin-dependent hypertension.

Finding a voice through 'The Tree of Life': a strength-based approach to mental health for refugee children and families in schools.

The Child and Family Refugee Service at the Tavistock Centre in London has run a series of 'Tree of Life' groups for both parents and children in schools. The groups were developed in response to a concern about the majority of psychological treatments, which focus predominantly on vulnerability factors in refugee populations, and the effect that this can have on those they are attempting to help. In addition, these are modelled on western assumptions, which do not adequately take account of culture. The Tree of Life groups have provided an alternative to traditional mental health services, which many refugee families find hard to access because of perceived stigma and lack of knowledge about what is on offer. The groups employed a strength-based narrative methodology, using the tree as a creative metaphor, which enabled parents and children to develop empowering stories about their lives, which were rooted in their cultural and social histories. From this secure base, participants were able to develop shared, culturally congruent solutions to their problems. The groups have been found to benefit parents and children alike, as well as the school communities in which they have taken place.

Conformational change and human cytochrome c function: mutation of residue 41 modulates caspase activation and destabilizes Met-80 coordination.

Cytochrome c is a highly conserved protein, with 20 residues identical in all eukaryotic cytochromes c. Gly-41 is one of these invariant residues, and is the position of the only reported naturally occurring mutation in cytochrome c (human G41S). The basis, if any, for the conservation of Gly-41 is unknown. The mutation of Gly-41 to Ser enhances the apoptotic activity of cytochrome c without altering its role in mitochondrial electron transport. Here we have studied additional residue 41 variants and determined their effects on cytochrome c functions and conformation. A G41T mutation decreased the ability of cytochrome c to induce caspase activation and decreased the redox potential, whereas a G41A mutation had no impact on caspase induction but the redox potential increased. All residue 41 variants decreased the pK (a) of a structural transition of oxidized cytochrome c to the alkaline conformation, and this correlated with a destabilization of the interaction of Met-80 with the heme iron(III) at physiological pH. In reduced cytochrome c the G41T and G41S mutations had distinct effects on a network of hydrogen bonds involving Met-80, and in G41T the conformational mobility of two Ω-loops was altered. These results suggest the impact of residue 41 on the conformation of cytochrome c influences its ability to act in both of its physiological roles, electron transport and caspase activation.