Myeloperoxidase-derived oxidants inhibit sarco/endoplasmic reticulum Ca2+-ATPase activity, and perturb Ca2+ homeostasis in human coronary artery endothelial cells

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) plays a critical role in Ca2+ homeostasis via sequestration of this ion into the sarco/endoplasmic reticulum. The activity of this pump is inhibited by oxidants and impaired in ageing tissues and cardiovascular disease. We have shown previously that the myeloperoxidase- (MPO) derived oxidants HOCl and HOSCN target thiols and mediate cellular dysfunction. As SERCA contains Cys residues critical to ATPase activity, we hypothesized that HOCl and HOSCN might inhibit SERCA activity, via thiol oxidation, and increase cytosolic Ca2+ levels in human coronary artery endothelial cells (HCAEC). Exposure of sarcoplasmic reticulum vesicles to pre-formed or enzymatically-generated HOCl and HOSCN resulted in a concentration-dependent decrease in ATPase activity; this was also inhibited by the SERCA inhibitor thapsigargin. Decomposed HOSCN and incomplete MPO enzyme systems did not decrease activity. Loss of ATPase activity occurred concurrently with oxidation of SERCA Cys residues and protein modification. Exposure of HCAEC, with or without external Ca2+, to HOSCN or HOCl, resulted in a time- and concentration-dependent increase in intracellular Ca2+ under conditions that did not result in immediate loss of cell viability. Thapsigargin, but not inhibitors of plasma membrane or mitochondrial Ca2+ pumps/channels, completely attenuated the increase in intracellular Ca2+ consistent with a critical role for SERCA in maintaining endothelial cell Ca2+ homeostasis. Angiotensin II pre-treatment potentiated the effect of HOSCN at low concentrations. MPO-mediated modulation of intracellular Ca2+ levels may exacerbate endothelial dysfunction, a key early event in atherosclerosis, and be more marked in smokers due to their higher SCN− levels

“Nanostandardization” in action: implementing standardization processes in a multidisciplinary nanoparticle-based research and development project

Nanomaterials have attracted much interest in the medical field and related applications as their distinct properties in the nano-range enable new and improved diagnosis and therapies. Owing to these properties and their potential interactions with the human body and the environment, the impact of nanomaterials on humans and their potential toxicity have been regarded a very significant issue. Consequently, nanomaterials are the subject of a wide range of cutting-edge research efforts in the medical and related fields to thoroughly probe their potential beneficial utilizations and their more negative effects. We posit that the lack of standardization in the field is a serious shortcoming as it has led to the establishment of methods and results that do not ensure sufficient consistency and thus in our view can possibly result in research outputs that are not as robust as they should be. The main aim of this article is to present how NanoDiaRA, a large FP7 European multidisciplinary project that seeks to investigate and develop nanotechnology-based diagnostic systems, has developed and implemented robust, standardized methods to support research practices involving the engineering and manipulation of nanomaterials. First, to contextualize this research, an overview of the measures defined by different regulatory bodies concerning nano-safety is presented. Although these authorities have been very active in the past several years, many questions remain unanswered in our view. Second, a number of national and international projects that attempted to ensure more reliable exchanges of methods and results are discussed. However, the frequent lack of publication of procedures and protocols in research can often be a hindrance for sharing those good practices. Subsequently, the efforts made through NanoDiaRA to introduce standardized methods and techniques to support the development and utilization of nanomaterials are discussed in depth. A series of semi-structured interviews were conducted with the partners of this project, and the interviews were analyzed thematically to highlight the determined efforts of the researchers to standardize their methods. Finally, some recommendations are made towards the setting up of well-defined methods to support the high-quality work of collaborative nanoparticle-based research and development projects and to enhance standardization processes

The script concordance test in radiation oncology: validation study of a new tool to assess clinical reasoning

<p>Abstract</p> <p>Background</p> <p>The Script Concordance test (SCT) is a reliable and valid tool to evaluate clinical reasoning in complex situations where experts' opinions may be divided. Scores reflect the degree of concordance between the performance of examinees and that of a reference panel of experienced physicians. The purpose of this study is to demonstrate SCT's usefulness in radiation oncology.</p> <p>Methods</p> <p>A 90 items radiation oncology SCT was administered to 155 participants. Three levels of experience were tested: medical students (n = 70), radiation oncology residents (n = 38) and radiation oncologists (n = 47). Statistical tests were performed to assess reliability and to document validity.</p> <p>Results</p> <p>After item optimization, the test comprised 30 cases and 70 questions. Cronbach alpha was 0.90. Mean scores were 51.62 (± 8.19) for students, 71.20 (± 9.45) for residents and 76.67 (± 6.14) for radiation oncologists. The difference between the three groups was statistically significant when compared by the Kruskall-Wallis test (p < 0.001).</p> <p>Conclusion</p> <p>The SCT is reliable and useful to discriminate among participants according to their level of experience in radiation oncology. It appears as a useful tool to document the progression of reasoning during residency training.</p

Chemically-Induced RAT Mesenchymal Stem Cells Adopt Molecular Properties of Neuronal-Like Cells but Do Not Have Basic Neuronal Functional Properties

Induction of adult rat bone marrow mesenchymal stem cells (MSC) by means of chemical compounds (β-mercaptoethanol, dimethyl sulfoxide and butylated hydroxyanizole) has been proposed to lead to neuronal transdifferentiation, and this protocol has been broadly used by several laboratories worldwide. Only a few hours of MSC chemical induction using this protocol is sufficient for the acquisition of neuronal-like morphology and neuronal protein expression. However, given that cell death is abundant, we hypothesize that, rather than true neuronal differentiation, this particular protocol leads to cellular toxic effects. We confirm that the induced cells with neuronal-like morphology positively stained for NF-200, S100, β-tubulin III, NSE and MAP-2 proteins. However, the morphological and molecular changes after chemical induction are also associated with an increase in the apoptosis of over 50% of the plated cells after 24 h. Moreover, increased intracellular cysteine after treatment indicates an impairment of redox circuitry during chemical induction, and in vitro electrophysiological recordings (patch-clamp) of the chemically induced MSC did not indicate neuronal properties as these cells do not exhibit Na+ or K+ currents and do not fire action potentials. Our findings suggest that a disruption of redox circuitry plays an important role in this specific chemical induction protocol, which might result in cytoskeletal alterations and loss of functional ion-gated channels followed by cell death. Despite the neuronal-like morphology and neural protein expression, induced rat bone marrow MSC do not have basic functional neuronal properties, although it is still plausible that other methods of induction and/or sources of MSC can achieve a successful neuronal differentiation in vitro

Global wealth disparities drive adherence to COVID-safe pathways in head and neck cancer surgery

Peer reviewe

Evidence for redox sensing by a human cardiac calcium channel

Ion channels are critical to life and respond rapidly to stimuli to evoke physiological responses. Calcium influx into heart muscle occurs through the ion conducting α1C subunit (Cav1.2) of the L-type Ca2+ channel. Glutathionylation of Cav1.2 results in increased calcium influx and is evident in ischemic human heart. However controversy exists as to whether direct modification of Cav1.2 is responsible for altered function. We directly assessed the function of purified human Cav1.2 in proteoliposomes. Truncation of the C terminus and mutation of cysteines in the N terminal region and cytoplasmic loop III-IV linker did not alter the effects of thiol modifying agents on open probability of the channel. However mutation of cysteines in cytoplasmic loop I-II linker altered open probability and protein folding assessed by thermal shift assay. We find that C543 confers sensitivity of Cav1.2 to oxidative stress and is sufficient to modify channel function and posttranslational folding. Our data provide direct evidence for the calcium channel as a redox sensor that facilitates rapid physiological responses

Identifying the Site/S of Modification on Human L-type Calcium Channel Protein Isoforms During Oxidative Stress

Reactive oxygen species and calcium contribute to the development of cardiac hypertrophy. The L-type calcium channel (LTCC) is the major route for calcium influx into cardiac myocytes. Alterations in cellular redox state influence the activity of the LTCC. Exposing the native channel to the thiol-specific oxidising compound 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) increases peak inward current while the thiol reducing agent dithiothreitol (DTT) decreases basal channel activity. The purpose of this study was to identify the cysteines responsible for modulating channel function during oxidative stress. Human long and short N terminal (NT) isoforms of Cav1.2 (alpha subunit) were expressed in HEK cells. Cysteines present on the alpha interacting domain (AID) of the long NT isoform were mutated to a serine or an alanine. The channel protein was purified by histidine tag purification and incorporated in liposomes for functional analysis. Exposure of the long NT isoform to 200 μM DTNB increased open probability (Po) of the channel from 0.032 ± 0.003 to 0.075 ± 0.011 and subsequent exposure to 1 mM DTT decreased Po to 0.032 ± 0.014 (n = 12; p < 0.05). Similar results were obtained with the short NT isoform that lacks the first 46 amino acids of the N terminus (n = 5). This suggests that the NT domain is not the reactive region. In contrast, DTNB and DTT had little effect on Po of the LNT containing mutated cysteines in AID region (n = 9). The results of this study suggest that the cysteines on the alpha interacting domain of Cav1.2 are required for alterations in the function of the channel during oxidative stress

Identifying the Site/S of Modification on Human L-type Calcium Channel Protein Isoforms During Oxidative Stress

Reactive oxygen species and calcium contribute to the development of cardiac hypertrophy. The L-type calcium channel (LTCC) is the major route for calcium influx into cardiac myocytes. Alterations in cellular redox state influence the activity of the LTCC. Exposing the native channel to the thiol-specific oxidising compound 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) increases peak inward current while the thiol reducing agent dithiothreitol (DTT) decreases basal channel activity. The purpose of this study was to identify the cysteines responsible for modulating channel function during oxidative stress. Human long and short N terminal (NT) isoforms of Cav1.2 (alpha subunit) were expressed in HEK cells. Cysteines present on the alpha interacting domain (AID) of the long NT isoform were mutated to a serine or an alanine. The channel protein was purified by histidine tag purification and incorporated in liposomes for functional analysis. Exposure of the long NT isoform to 200 μM DTNB increased open probability (Po) of the channel from 0.032 ± 0.003 to 0.075 ± 0.011 and subsequent exposure to 1 mM DTT decreased Po to 0.032 ± 0.014 (n = 12; p < 0.05). Similar results were obtained with the short NT isoform that lacks the first 46 amino acids of the N terminus (n = 5). This suggests that the NT domain is not the reactive region. In contrast, DTNB and DTT had little effect on Po of the LNT containing mutated cysteines in AID region (n = 9). The results of this study suggest that the cysteines on the alpha interacting domain of Cav1.2 are required for alterations in the function of the channel during oxidative stress

Mitochondria at the Crossroads of Survival and Demise

Mitochondria are multifunctional organelles, and their structural and functional integrity is fundamental to cell life. In addition to their critical role in the production of ATP via oxidative phosphorylation and biosynthetic intermediates, mitochondria are also a major hub for cellular Ca2+ signaling. Moreover, mitochondria can actively or passively drive cellular demise. They can become the major source of reactive oxygen species (ROS) in pathological and physiological processes, and they are highly vulnerable to damage. Mitochondria represent a point of convergence for a variety of upstream cell death stimuli and undergo structural and functional remodeling with subsequent transmission of signals to downstream executioner proteins. The pathways include death stimuli such as dioxygen, metabolic perturbation, deprivation of survival factors, oxidative stress, Ca2+ overload, DNA damage, proteotoxic stress, and oncogene activation