Exercise and Doxorubicin Modify Markers of Iron Overload and Cardiolipin Deficiency in Cardiac Mitochondria.

Doxorubicin (DOX) is a chemotherapeutic agent highly effective at limiting cancer progression. Despite the efficacy of this anticancer drug, the clinical use of DOX is limited due to cardiotoxicity. The cardiac mitochondria are implicated as the primary target of DOX, resulting in inactivation of electron transport system complexes, oxidative stress, and iron overload. However, it is established that the cardiac mitochondrial subpopulations reveal differential responses to DOX exposure, with subsarcolemmal (SS) mitochondria demonstrating redox imbalance and the intermyofibrillar (IMF) mitochondria showing reduced respiration. In this regard, exercise training is an effective intervention to prevent DOX-induced cardiac dysfunction. Although it is clear that exercise confers mitochondrial protection, it is currently unknown if exercise training mitigates DOX cardiac mitochondrial toxicity by promoting beneficial adaptations to both the SS and IMF mitochondria. To test this, SS and IMF mitochondria were isolated from sedentary and exercise-preconditioned female Sprague Dawley rats exposed to acute DOX treatment. Our findings reveal a greater effect of exercise preconditioning on redox balance and iron handling in the SS mitochondria of DOX-treated rats compared to IMF, with rescue of cardiolipin synthase 1 expression in both subpopulations. These results demonstrate that exercise preconditioning improves mitochondrial homeostasis when combined with DOX treatment, and that the SS mitochondria display greater protection compared to the IMF mitochondria. These data provide important insights into the molecular mechanisms that are in part responsible for exercise-induced protection against DOX toxicity.

Evaluation of Early Renal Involvement in Essential Hypertension by Measuring Urinary Biomarkers.

Hypertensive kidney damage results in glomerular as well as tubular dysfunction. Albuminuria is a well-known marker of glomerular damage. On the other hand, urinary uromodulin is increasingly considered as a potential biomarker of early tubular dysfunction. The aim of the study was to assess glomerular and tubular function of the kidney by measuring urinary albumin and uromodulin excretion in hypertensive subjects. This cross-sectional study was conducted from July 2018 to June 2019 in Hypertension Clinic of Dhaka Medical College Hospital, Dhaka and Kidney Care and Research Centre, Sonargaon, Narayanganj, Bangladesh. In this study 122 hypertensive subjects with age >30 years, duration of hypertension <5 years, without accelerated or malignant BP, absence of dipstick proteinuria and eGFR >60ml/min were included. There were also 33 normotensive individuals included as healthy controls. Albumin-creatinine ratio (uACR mg/g), urine uromodulin-creatinine ratio (uUMODµg/g), urinary sodium-creatinine ratio (mEq/g) and potassium-creatinine ratio (mEq/g) were measured from single morning spot urine sample. Urinary uromodulin levels were measured by ELISA method. The hypertensive and normotensive subjects were age matched 49.0±12.0 vs. 48.0±11.0, years (p=NS). The mean uACR was 29.0±65.0 versus 5.6±2.7mg/g, (p<0.001) respectively. The median uUMOD in hypertensive subjects was 3.38 (1.73-9.06) and in normotensives 3.85(2.28-5.69) µg/g (p=non significant). Multivariate analysis showed significant inverse association between diastolic blood pressure and urinary uromodulin excretion. A uUMOD cut-off of 2.9 (25th percentile) showed eGFR, urinary sodium and potassium excretions were significantly lower at low uromodulin group. The glomerular involvement was found in 21.0% of hypertensive subjects as evidenced by albuminuria. No difference was observed in urinary uromodulin level between hypertensive and normotensive subjects. Low urinary uromodulin level was associated with lower eGFR, Na+ and K+ excretion which indicate simultaneous tubular and glomerular involvement.

Anticancer L-Asparaginase and Phytoactive Compounds From Plant Solanum nigrum Against MDR (Methicillindrug resistant) Staphylococcus aureus and Fungal Isolates.

L-asparaginase is used in chemotherapy for acute lymphoblastic leukemia and other cancers. L-asparaginase derived from bacterial source triggers immune responses. The current study investigates Solanum nigrum as a novel and latent source of L-asparaginase to minimize immunological reactions. The antitumor activity of SN methanol extract was determined using the potato disc assay. InterPro Chimera and InterPro were used to predict the amino acid sequence of L-asparaginase and its anticancer activity. Purification of the enzyme was carried out to homogeneity of 1.51-fold with a recovery of 61.99%. At optimal conditions of 36.5°C, pH 8.6, and 8.5 g/mL substrate, fruit (crude extract) revealed an L-asparaginase titer of 48.23 U/mL. The molecular weight of the enzyme was calculated to be 32 ± 5 kDa using SDS PAGE. The fruit's total flavonoids and phenolic contents are 0.42 ± .030 g/mL and 94 ± 1.9 mg CAE, respectively. Anti-tumorigenic efficacy was determined to be 66% against Agrobacterium tumefaciens. Additionally, the extract possesses potent antifungal and antibacterial properties. Molecular docking provided the structural motifs and underlying interactions between L-asparaginase, N-acetylglucosamine, murine, and chitin. SN contains high levels of the enzyme L-asparaginase and phytochemicals, making it a potential source of anticancer drugs.

Age Related Changes in the Purkinje Cells in Human Cerebellar Cortex in Bangladeshi People.

This study was done to see the changes in the number of Purkinje cells per square mm in different age groups of Bangladeshi people. This cross-sectional descriptive type of study was done on total 40 postmortem human cerebellums, in the Department of Anatomy, Mymensingh Medical College, Bangladesh from July 2016 to June 2017. The specimens were collected from morgue in the department of Forensic Medicine, Mymensingh Medical College, by purposive sampling technique. All the specimens were grouped into four categories: Group A (20 to 29 years), Group B (30 to 39 years), Group C (40 to 49 years) and Group D (50 to 59 years). Paraffin blocks of cerebellum were cut at 4-5µm thickness and stained with routine "Haematoxylin and Eosin" (H & E) stain. Estimation of number of Purkinje cell was done by using the counting circle and examined under the light microscope. In the present study, the mean±SD number of Purkinje cell was found 128.67±25.15 per sq mm in Group A, 136.53±34.45 in Group B per sq mm, 135.55±29.44 per sq mm in Group C, 127.69±35.31 per sq mm in Group D.

The impact of low-intensity blood flow restriction endurance training on aerobic capacity, hemodynamics, and arterial stiffness.

BACKGROUND: The aim of this study was to determine the effects of short-term low-intensity blood flow restriction (BFR) endurance training (ET) programs on measures of aerobic capacity, hemodynamics, and arterial stiffness in healthy young males. METHODS: Thirty-nine healthy young recreationally active males participated in this short-term training study. They were randomly assigned to a high-intensity (HI; N.=11; trained at 60-70% of VO2 reserve [VO2R]), low-intensity (LI; N.=8; trained at 30-40% of VO2R), low-intensity with BFR (LI-BFR; N.=10; trained at 30-40% of VO2R with BFR) or a non-exercising control group (N.=10). The exercising subjects completed a 6-wk training protocol on a treadmill. Assessment of aerobic capacity (VO2max), hemodynamics and arterial stiffness were done before and after training. RESULTS: Statistical analyses revealed a significant condition main effect (P<0.05) for VO2max, indicating significant increase (P<0.05) in VO2max in LI-BFR group compared to control. There were no significant changes for resting heart rate (RHR), systolic blood pressure (SBP), diastolic blood pressure (DBP), carotid-radial pulse wave velocity (PWV), and carotid-femoral PWV (P>0.05). However, femoral-tibial PWV decreased significantly (P<0.05) from baseline to post-training. CONCLUSIONS: The results indicate that the application of BFR during ET may cause faster and/or greater adaptations in one or more physiological systems resulting in improved cardiorespiratory fitness.

Modelling of the adsorption of Pb, Cu and Ni ions from single and multi-component aqueous solutions by date seed derived biochar: Comparison of six machine learning approaches.

Biochar is an effective material for the removal of heavy metals from wastewater. Operational conditions, such as metal initial concentration, temperature, contact time as well as the presence of competing ions can impact the effectiveness of the treatment process. While several models have been proposed for modelling the adsorption process, no model currently exists that accounts for the mutual interactions of key process parameters on the adsorption capacity in multi-solute systems. The aim of this study is to address this gap in knowledge by formulating a multi-input multi-output (MIMO) model, which takes into account the effect of mutual interactions of key factors while predicting heavy metals adsorption capacity of the biochar in single and multi-solute systems. In this study, we use machine learning models, specifically several ANN models, radial basis and gradient boosting algorithms to model the MIMO process. The results of our models provide highly accurate predictions (R2 > 0.99). The generalized regression network provided the best match to the experimental data. This approach can allow operators to predict how the adsorption system will respond to changes in the operations and hence provide them with a tool for process optimization.

Endoscopic tattooing for colorectal lesions: impact on quality of care and patient outcomes.

INTRODUCTION: The National Bowel Cancer Screening Programme guidelines advocate the use of endoscopic tattooing for suspected malignant lesions to assist identification and to facilitate laparoscopic resections. However, endoscopic tattooing practices are variable in endoscopic units, resulting in repeat endoscopy and delay in patient management. The aim of this study was to assess the adherence to tattoo protocol for significant colonic lesions at an endoscopy unit in a large district general hospital. MATERIALS AND METHODS: Prospectively collected data were analysed for 252 patients with significant colonic lesions between January 2017 and December 2018. Data were collected through reviewing patient's notes, histopathology findings and endoscopy reports. Data on lesions, complications, number and site of tattoo placed, and any repeat endoscopy for a tattoo were collected. RESULTS: Of the 252 patients, 88% (n = 222) had malignant and 12% (n = 30) had benign lesions. Only 58.7% (n = 148) of those patients who had colonoscopy had tattoo placement reported. Of these 148 cases, the report stated the distance of tattoo in relation to the lesion in only 46% (n = 68) of patients. Unfortunately, 14.3% (n = 36) of patients required repeat endoscopy to tattoo the lesions prior to surgery. CONCLUSIONS: Our study highlights the lack of uniformity of tattoo practice among endoscopists. Despite the National Bowel Cancer Screening Programme guidelines, a significant proportion of colorectal lesions are still not tattooed during their first endoscopy. Some patients had to have repeat endoscopy just for the purpose of tattooing. Active involvement and participation of all endoscopists in the colorectal and the complex polyp multidisciplinary teams may help to improve the tattoo service.

An atypical presentation of a Respiratory Epithelial Adenomatoid Hamartoma, a case report.

INTRODUCTION: Respiratory Epithelial Adenomatoid Hamartoma (REAH) is a benign disease that can resemble other malignant entities. Thus, it is essential to diagnose it accurately as the treatment approach differs, from radical surgeries in malignant cases, to a simple excision in hamartoma. We present an unusual case of bilateral REAH that was misdiagnosed, and hence it was treated aggressively. CASE REPORT: A 57-year-old male patient presented with anosmia, 2-years history of bilateral nasal obstruction, and was accompanied with a moderate headache. An impression of olfactory neuroblastoma was made after history taking physical examination, and imaging studies. The patient underwent Functional Endoscopic Sinus Surgery (FESS), excisional biopsy of the cribriform plate mass bilaterally, and superior septectomy. Histopathologic examination of the bilateral masses showed sinonasal polyposis with crypting of surface mucosa and pseudoglandular formation. A diagnosis of sinonasal polyps with REAH was established. The patient's nasal obstruction improved, with no recurrence of sinusitis ±â€¯polyposis. However, he still complains of anosmia after 2-years follow-up. CONCLUSION: Although REAH is a benign disease, it is critical to reach the correct diagnosis, in order to avoid aggressive treatment. Unfortunately, the preoperative investigations were not consistent with REAH, thus it was misdiagnosed and treated aggressively.

Human vitreous in proliferative diabetic retinopathy: Characterization and translational implications.

Diabetic retinopathy (DR) is one of the leading causes of visual impairment in the working-age population. DR is a progressive eye disease caused by long-term accumulation of hyperglycaemia-mediated pathological alterations in the retina of diabetic patients. DR begins with asymptomatic retinal abnormalities and may progress to advanced-stage proliferative diabetic retinopathy (PDR), characterized by neovascularization or preretinal/vitreous haemorrhages. The vitreous, a transparent gel that fills the posterior cavity of the eye, plays a vital role in maintaining ocular function. Structural and molecular alterations of the vitreous, observed during DR progression, are consequences of metabolic and functional modifications of the retinal tissue. Thus, vitreal alterations reflect the pathological events occurring at the vitreoretinal interface. These events are caused by hypoxic, oxidative, inflammatory, neurodegenerative, and leukostatic conditions that occur during diabetes. Conversely, PDR vitreous can exert pathological effects on the diabetic retina, resulting in activation of a vicious cycle that contributes to disease progression. In this review, we recapitulate the major pathological features of DR/PDR, and focus on the structural and molecular changes that characterize the vitreal structure and composition during DR and progression to PDR. In PDR, vitreous represents a reservoir of pathological signalling molecules. Therefore, in this review we discuss how studying the biological activity of the vitreous in different in vitro, ex vivo, and in vivo experimental models can provide insights into the pathogenesis of PDR. In addition, the vitreous from PDR patients can represent a novel tool to obtain preclinical experimental evidences for the development and characterization of new therapeutic drug candidates for PDR therapy.

N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe (BOC2) inhibits the angiogenic activity of heparin-binding growth factors.

The peptides N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe (BOC2) and BOC-Met-Leu-Phe (BOC1) are widely used antagonists of formyl peptide receptors (FPRs), BOC2 acting as an FPR1/FPR2 antagonist whereas BOC1 inhibits FPR1 only. Extensive investigations have been performed by using these FPR antagonists as a tool to assess the role of FPRs in physiological and pathological conditions. Based on previous observations from our laboratory, we assessed the possibility that BOC2 may exert also a direct inhibitory effect on the angiogenic activity of vascular endothelial growth factor-A (VEGF-A). Our data demonstrate that BOC2, but not BOC1, inhibits the angiogenic activity of heparin-binding VEGF-A165 with no effect on the activity of the non-heparin-binding VEGF-A121 isoform. Endothelial cell-based bioassays, surface plasmon resonance analysis, and computer modeling indicate that BOC2 may interact with the heparin-binding domain of VEGF-A165, thus competing for heparin interaction and preventing the binding of VEGF-A165 to tyrosine kinase receptor VEGFR2, its phosphorylation and downstream signaling. In addition, BOC2 inhibits the interaction of a variety of heparin-binding angiogenic growth factors with heparin, including fibroblast growth factor 2 (FGF2) whose angiogenic activity is blocked by the compound. Accordingly, BOC2 suppresses the angiogenic potential of human tumor cell lines that co-express VEGF-A and FGF2. Thus, BOC2 appears to act as a novel multi-heparin-binding growth factor antagonist. These findings caution about the interpretation of FPR-focusing experimental data obtained with this compound and set the basis for the design of novel BOC2-derived, FPR independent multi-target angiogenesis inhibitors.

3D endothelial cell spheroid/human vitreous humor assay for the characterization of anti-angiogenic inhibitors for the treatment of proliferative diabetic retinopathy.

Proliferative diabetic retinopathy (PDR) represents a main cause of acquired blindness. Despite the recognition of the key role exerted by vascular endothelial growth factor (VEGF) in the pathogenesis of PDR, limitations to anti-VEGF therapies do exist. Thus, rapid and cost-effective angiogenesis assays are crucial for the screening of anti-angiogenic drug candidates for PDR therapy. In this context, evaluation of the angiogenic potential of PDR vitreous fluid may represent a valuable tool for preclinical assessment of angiostatic molecules. Here, vitreous fluid obtained from PDR patients after pars plana vitrectomy was used as a pro-angiogenic stimulus in a 3D endothelial cell spheroid/human vitreous assay. The results show that PDR vitreous is able to stimulate the sprouting of fibrin-embedded HUVEC spheroids in a time- and dose-dependent manner. A remarkable variability was observed among 40 individual vitreous fluid samples in terms of sprouting-inducing activity that was related, at least in part, to defined clinical features of the PDR patient. This activity was hampered by various extracellular and intracellular signaling pathway inhibitors, including the VEGF antagonist ranibizumab. When tested on 20 individual vitreous fluid samples, the inhibitory activity of ranibizumab ranged between 0 and 100% of the activity measured in the absence of the drug, reflecting a variable contribution of angiogenic mediators distinct from VEGF. In conclusion, the 3D endothelial cell spheroid/human vitreous assay represents a rapid and cost-effective experimental procedure suitable for the evaluation of the anti-angiogenic activity of novel extracellular and intracellular drug candidates, with possible implications for the therapy of PDR.

Genetic Differentiation of the Wheat Leaf Rust Fungus Puccinia triticina in Pakistan and Genetic Relationship to Other Worldwide Populations.

Collections of Puccinia triticina, the wheat leaf rust pathogen, were obtained from Pakistan in 2008, 2010, 2011, 2013, and 2014. Collections were also obtained from Bhutan in 2013. Single uredinial isolates were derived and tested for virulence phenotype to 20 lines of Thatcher wheat that differ for single leaf rust resistance genes, and for molecular genotype with 23 simple-sequence repeat (SSR) primers. Twenty-four virulence phenotypes were described among the 89 isolates tested for virulence. None of the isolates had virulence to Thatcher lines with Lr9, Lr24, or Lr18. Virulence to most of the other Thatcher lines was over 50%. The two most common virulence phenotypes, FHPSQ and KHPQQ, had virulence to Lr16, Lr17, and Lr26. Twenty-seven SSR genotypes were found among the 38 isolates tested for molecular variation. The SSR genotypes had high levels of observed heterozygosity and significant correlation with virulence phenotype, which indicated clonal reproduction. Cluster analysis and principal component plots indicated three groups of SSR genotypes that also varied significantly for virulence. Isolates with MBDSS and MCDSS virulence phenotypes from Pakistan and Bhutan were highly related for SSR genotype and virulence to isolates from Turkey, Europe, Central Asia, the Middle East, North America and South America, indicating the possible migration of the rust fungus between continental regions.

Inflammation and N-formyl peptide receptors mediate the angiogenic activity of human vitreous humour in proliferative diabetic retinopathy.

AIMS/HYPOTHESIS: Angiogenesis and inflammation characterise proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus. However, the impact of inflammation on the pathogenesis of PDR neovascularisation has not been elucidated. Here, we assessed the capacity of PDR vitreous fluid to induce pro-angiogenic/proinflammatory responses in endothelium and the contribution of the inflammation-related pattern recognition N-formyl peptide receptors (FPRs) in mediating these responses. METHODS: Pooled and individual pars plana vitrectomy-derived PDR vitreous fluid ('PDR vitreous') samples were assessed in endothelial cell proliferation, motility, sprouting and morphogenesis assays, and for the capacity to induce proinflammatory transcription factor activation, reactive oxygen species production, intercellular junction disruption and leucocyte-adhesion molecule upregulation in these cells. In vivo, the pro-angiogenic/proinflammatory activity of PDR vitreous was tested in murine Matrigel plug and chick embryo chorioallantoic membrane (CAM) assays. Finally, the FPR inhibitors Boc-Phe-Leu-Phe-Leu-Phe (Boc-FLFLF) and Ac-L-Arg-Aib-L-Arg-L-Cα(Me)Phe-NH2 tetrapeptide (UPARANT) were evaluated for their capacity to affect the biological responses elicited by PDR vitreous. RESULTS: PDR vitreous activates a pro-angiogenic/proinflammatory phenotype in endothelial cells. Accordingly, PDR vitreous triggers a potent angiogenic/inflammatory response in vivo. Notably, the different capacity of individual PDR vitreous samples to induce neovessel formation in the CAM correlates with their ability to recruit infiltrating CD45+ cells. Finally, the FPR inhibitor Boc-FLFLF and the novel FPR antagonist UPARANT inhibit neovessel formation and inflammatory responses triggered by PDR vitreous in the CAM assay. CONCLUSIONS/INTERPRETATION: This study provides evidence that inflammation mediates the angiogenic activity of PDR vitreous and paves the way for the development of FPR-targeting anti-inflammatory/anti-angiogenic approaches for PDR therapy.

Ryanodine receptor modification and regulation by intracellular Ca2+ and Mg2+ in healthy and failing human hearts.

RATIONALE: Heart failure is a multimodal disorder, of which disrupted Ca2+ homeostasis is a hallmark. Central to Ca2+ homeostasis is the major cardiac Ca2+ release channel - the ryanodine receptor (RyR2) - whose activity is influenced by associated proteins, covalent modification and by Ca2+ and Mg2+. That RyR2 is remodelled and its function disturbed in heart failure is well recognized, but poorly understood. OBJECTIVE: To assess Ca2+ and Mg2+ regulation of RyR2 from left ventricles of healthy, cystic fibrosis and failing hearts, and to correlate these functional changes with RyR2 modifications and remodelling. METHODS AND RESULTS: The function of RyR2 from left ventricular samples was assessed using lipid bilayer single-channel measurements, whilst RyR2 modification and protein:protein interactions were determined using Western Blots and co-immunoprecipitation. In all failing hearts there was an increase in RyR2 activity at end-diastolic cytoplasmic Ca2+ (100nM), a decreased cytoplasmic [Ca2+] required for half maximal activation (Ka) and a decrease in inhibition by cytoplasmic Mg2+. This was accompanied by significant hyperphosphorylation of RyR2 S2808 and S2814, reduced free thiol content and a reduced interaction with FKBP12.0 and FKBP12.6. Either dephosphorylation of RyR2 using PP1 or thiol reduction using DTT eliminated any significant difference in the activity of RyR2 from healthy and failing hearts. We also report a subgroup of RyR2 in failing hearts that were not responsive to regulation by intracellular Ca2+ or Mg2+. CONCLUSION: Despite different aetiologies, disrupted RyR2 Ca2+ sensitivity and biochemical modification of the channel are common constituents of failing heart RyR2 and may underlie the pathological disturbances in intracellular Ca2+ signalling.

Anti-Bacterial effect of Aqueous Garlic Extract (AGE) determined by Disc Diffusion Method against Escherichia coli.

The study was performed to determine the antibacterial effect of aqueous extract of garlic (Allium sativum) against standard strain of Escherichia coli ATCC 25922. An interventional study was conducted in Department of Pharmacology and Therapeutics in collaboration with Department of Microbiology, Mymensingh Medical College, Mymensingh. Antibacterial effect of AGE was determined by disc diffusion method. Sensitivity of AGE determined in disc diffusion and the zone of inhibition (ZOI) was 4 mm, 10 mm and 20 mm at 25 µg/10 µl, 50 µg/10 µl and 100 µg/10 µl concentrations respectively. From the findings it is clearly determined the extract has definite antibacterial effect upon Escherichia coli. Further studies are required to detect and isolate the active ingredients present in the Garlic extract as well as detail steps of mechanism responsible for antibacterial effect. Then their effects against the studied organism should be studied in vivo separately and its toxicity profile should also be taken into account.

Long-lived plasma cells are early and constantly generated in New Zealand Black/New Zealand White F1 mice and their therapeutic depletion requires a combined targeting of autoreactive plasma cells and their precursors.

INTRODUCTION: Autoantibodies contribute significantly to the pathogenesis of systemic lupus erythematosus (SLE). Unfortunately, the long-lived plasma cells (LLPCs) secreting such autoantibodies are refractory to conventional immunosuppressive treatments. Although generated long before the disease becomes clinically apparent, it remains rather unclear whether LLPC generation continues in the established disease. Here, we analyzed the generation of LLPCs, including autoreactive LLPCs, in SLE-prone New Zealand Black/New Zealand White F1 (NZB/W F1) mice over their lifetime, and their regeneration after depletion. METHODS: Bromodeoxyuridine pulse-chase experiments in mice of different ages were performed in order to analyze the generation of LLPCs during the development of SLE. LLPCs were enumerated by flow cytometry and autoreactive anti-double-stranded DNA (anti-dsDNA) plasma cells by enzyme-linked immunospot (ELISPOT). For analyzing the regeneration of LLPCs after depletion, mice were treated with bortezomib alone or in combination with cyclophosphamide and plasma cells were enumerated 12 hours, 3, 7, 11 and 15 days after the end of the bortezomib cycle. RESULTS: Autoreactive LLPCs are established in the spleen and bone marrow of SLE-prone mice very early in ontogeny, before week 4 and before the onset of symptoms. The generation of LLPCs then continues throughout life. LLPC counts in the spleen plateau by week 10, but continue to increase in the bone marrow and inflamed kidney. When LLPCs are depleted by the proteasome inhibitor bortezomib, their numbers regenerate within two weeks. Persistent depletion of LLPCs was achieved only by combining a cycle of bortezomib with maintenance therapy, for example cyclophosphamide, depleting the precursors of LLPCs or preventing their differentiation into LLPCs. CONCLUSIONS: In SLE-prone NZB/W F1 mice, autoreactive LLPCs are generated throughout life. Their sustained therapeutic elimination requires both the depletion of LLPCs and the inhibition of their regeneration.

Multiple modes of ryanodine receptor 2 inhibition by flecainide.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) causes sudden cardiac death due to mutations in cardiac ryanodine receptors (RyR2), calsequestrin, or calmodulin. Flecainide, a class I antiarrhythmic drug, inhibits Na(+) and RyR2 channels and prevents CPVT. The purpose of this study is to identify inhibitory mechanisms of flecainide on RyR2. RyR2 were isolated from sheep heart, incorporated into lipid bilayers, and investigated by single-channel recording under various activating conditions, including the presence of cytoplasmic ATP (2 mM) and a range of cytoplasmic [Ca(2+)], [Mg(2+)], pH, and [caffeine]. Flecainide applied to either the cytoplasmic or luminal sides of the membrane inhibited RyR2 by two distinct modes: 1) a fast block consisting of brief substate and closed events with a mean duration of ∼1 ms, and 2) a slow block consisting of closed events with a mean duration of ∼1 second. Both inhibition modes were alleviated by increasing cytoplasmic pH from 7.4 to 9.5 but were unaffected by luminal pH. The slow block was potentiated in RyR2 channels that had relatively low open probability, whereas the fast block was unaffected by RyR2 activation. These results show that these two modes are independent mechanisms for RyR2 inhibition, both having a cytoplasmic site of action. The slow mode is a closed-channel block, whereas the fast mode blocks RyR2 in the open state. At diastolic cytoplasmic [Ca(2+)] (100 nM), flecainide possesses an additional inhibitory mechanism that reduces RyR2 burst duration. Hence, multiple modes of action underlie RyR2 inhibition by flecainide.

Autoantibodies from long-lived 'memory' plasma cells of NZB/W mice drive immune complex nephritis.

OBJECTIVES: We have previously shown that both short- and long-lived plasma cells (PCs) significantly contribute to autoantibody production in NZB/W mice as a model of lupus nephritis. The aim of this study was to determine the role of autoreactive long-lived (memory) PCs refractory to immunosuppression and B cell depletion in the pathogenesis of systemic lupus erythematosus. METHODS: Splenic CD138+ antibody-secreting cells (ASCs) from >6-month-old NZB/W mice with high titres of anti-dsDNA autoantibodies or from Balb/c mice 5 days after secondary immunisation with ovalbumin (OVA) were adoptively transferred to immunodeficient Rag1(-/-) mice, in which the development of nephritis was investigated by measuring proteinuria. Total IgG and IgM as well as anti-dsDNA and anti-OVA antibody levels were followed up by ELISA. After 21 weeks the recipient mice were sacrificed so that PCs in spleen and bone marrow could be analysed using ELISPOT and flow cytometry and renal immunohistology performed. RESULTS: The adoptive transfer of NZB/W and anti-OVA ASCs resulted in the continuous generation of anti-dsDNA antibodies and anti-OVA antibodies, respectively, exclusively by long-lived PCs that had homed to the spleen and bone marrow of recipient Rag1(-/-) mice. Rag1(-/-) mice generating autoantibodies including anti-dsDNA had reduced survival, proteinuria and immune complex nephritis with C1q, C3, IgG and IgM deposits 21 weeks after transfer. CONCLUSIONS: These findings demonstrate that autoantibodies exclusively secreted by long-lived (memory) PCs contribute to autoimmune pathology and should be considered as candidate targets for future therapeutic strategies.

Control of sarcoplasmic reticulum Ca2+ release by stochastic RyR gating within a 3D model of the cardiac dyad and importance of induction decay for CICR termination.

The factors responsible for the regulation of regenerative calcium-induced calcium release (CICR) during Ca(2+) spark evolution remain unclear. Cardiac ryanodine receptor (RyR) gating in rats and sheep was recorded at physiological Ca(2+), Mg(2+), and ATP levels and incorporated into a 3D model of the cardiac dyad, which reproduced the time course of Ca(2+) sparks, Ca(2+) blinks, and Ca(2+) spark restitution. The termination of CICR by induction decay in the model principally arose from the steep Ca(2+) dependence of RyR closed time, with the measured sarcoplasmic reticulum (SR) lumen Ca(2+) dependence of RyR gating making almost no contribution. The start of CICR termination was strongly dependent on the extent of local depletion of junctional SR Ca(2+), as well as the time course of local Ca(2+) gradients within the junctional space. Reducing the dimensions of the dyad junction reduced Ca(2+) spark amplitude by reducing the strength of regenerative feedback within CICR. A refractory period for Ca(2+) spark initiation and subsequent Ca(2+) spark amplitude restitution arose from 1), the extent to which the regenerative phase of CICR can be supported by the partially depleted junctional SR, and 2), the availability of releasable Ca(2+) in the junctional SR. The physical organization of RyRs within the junctional space had minimal effects on Ca(2+) spark amplitude when more than nine RyRs were present. Spark amplitude had a nonlinear dependence on RyR single-channel Ca(2+) flux, and was approximately halved by reducing the flux from 0.6 to 0.2 pA. Although rat and sheep RyRs had quite different Ca(2+) sensitivities, Ca(2+) spark amplitude was hardly affected. This suggests that moderate changes in RyR gating by second-messenger systems will principally alter the spatiotemporal properties of SR release, with smaller effects on the amount released.

Multi-environment QTL analyses for drought-related traits in a recombinant inbred population of chickpea (Cicer arietinum L.).

A recombinant inbred line (RIL) population, comprising 181 lines derived from ILC588 × ILC3279, was evaluated in 10 environments across three locations with different moisture gradients. A drought resistance score (DRS) and three phenology traits-plant height (PLHT), days to flowering (DFLR), and days to maturity (MAT)-were recorded along with seven yield-related traits-grain yield (GY), biological yield (BY), harvest index (HI), the number of pods/3 plants (Pod), percentage of empty pods (%Epod), 100 seed weight (100 sw), and seed number/3 plants (SN). Two RILs (152, 162) showed the best GYs and DRSs under stressed and non-stressed environments. The quantitative trait loci (QTLs) analyses detected 93 significant QTLs (LOD ≥ 2.0) across the genome × environment interactions. The highest phenotypic variation (>24 %) was explained by the QTLDFLR in Terbol-11. Four common possible pleiotropic QTLs on LG3 and LG4 were identified as associated with DFLR, DRS, GY, MAT, HI, SN, and Pod. No significant epistatic interactions were found between these QTLs and the other markers. However, the QTL for DRS was detected as a conserved QTL in three late planting environments. The markers H6C-07 (on LG3) and H5G01 (on LG4) were associated with QTLs for many traits in all environments studied except two. The allele 'A' of marker H6C07 (from the tolerant parent ILC588) explained 80 % of the yield increase under late planting and 29.8 % of that under dry environments. Concentrating on LG3 and LG4 in molecular breeding programs for drought could speed up improvement for these traits.