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Bien que leur présence en STIM (sciences, technologies, ingénierie et mathématiques) ait augmenté de manière significative ces cinquante dernières années, les femmes demeurent largement sous-représentées dans ces secteurs à l’avenir florissant. Les nombreux facteurs contribuant à l’absence de parité dans les disciplines des STIM sont désormais bien documentés et, ces dernières années, de multiples acteurs ont mis en œuvre des campagnes de sensibilisation et de promotion afin d’encourager la participation des filles et des femmes aux STIM. Malgré ces efforts, le problème persiste et acquiert de plus en plus d’importance dans les débats actuels sur le genre et le travail. Dans une démarche comparative, cet article s’intéresse au déploiement de campagnes digitales visant à inspirer les jeunes filles et les femmes à emprunter la voie des sciences et technologies dans trois pays du Commonwealth : le Canada, l’Écosse et l’Inde. Il propose, tout d’abord, de revenir sur les raisons de l’importance grandissante de la question du genre et de la diversité en STIM. En veillant à replacer les campagnes dans leur contexte sociétal de production, il étudiera ensuite la manière dont chacune de ces campagnes permet aux femmes de reprendre le contrôle du récit et de contribuer à la déconstruction des stéréotypes de genre. Les campagnes choisies placent les questions de la représentation et de l’autoreprésentation des femmes en STIM au cœur du débat et donnent à voir comment les femmes en sciences et technologie perçoivent leur trajectoire ainsi que la culture de travail dans laquelle elles évoluent.

Moderate coolness is sensed by TRPM8 ion channels in peripheral sensory nerves, but the mechanism by which noxious cold is detected remains elusive. Here, we show that somatosensory and sympathetic neurons express two distinct mechanisms to detect noxious cold. In the first, inhibition by cold of a background outward current causes membrane depolarization that activates an inward current through voltage-dependent calcium (CaV ) channels. A second cold-activated mechanism is independent of membrane voltage, is inhibited by blockers of ORAI ion channels and by downregulation of STIM1, and is recapitulated in HEK293 cells by co-expression of ORAI1 and STIM1. Using total internal reflection fluorescence microscopy we found that cold causes STIM1 to aggregate with and activate ORAI1 ion channels, in a mechanism similar to that underlying store-operated calcium entry (SOCE), but directly activated by cold and not by emptying of calcium stores. This novel mechanism may explain the phenomenon of cold-induced vasodilation (CIVD), in which extreme cold increases blood flow in order to preserve the integrity of peripheral tissues.

Routinely, in membrane technology, the decay from radioactive particles or the bombardment of ions with MeV energy per nucleon have been employed for the production of narrow and long pores in membranes. Presently, the ion lithography is proposed to make the fabrication cost more affordable. It is prospective for the use of medium capacity accelerators making more feasible the fabrication of customized membranes. Thin polyethylene terephthalate foils have been patterned using 12 MeV O5+ ions and then processed to obtain good aspect ratio ion track pores in membranes. Pores of micrometric diameter with the following profiles were fabricated in the membranes: truncated cone, double conical, ideal cone, and cylindrical. Monitoring of the shape and size of pores has been attempted with a combination of Scanning Transmission Ion Microscope and a newly designed simulation program. This study is focused on the use of low-energy ions, accomplished in all laboratories, for the fabrication of membranes where the pores are not randomly traced and exhibit higher surface density and negligible overlapping than in membranes commonly manufactured. The good reproducibility and the ordered pore locations can be potentially utilized in applications such as microfluidics and organ-on-chip microsystems, where cells growing over porous substrates are used in simulation of biological barriers and transport processes.

The skin is a complex organ that acts as a protective layer against the external environment. It protects the internal tissues from harmful agents, dehydration, ultraviolet radiation and physical injury as well as conferring thermoregulatory control, sensation, immunological surveillance and various biochemical functions. The diverse cell types that make up the skin include 1) keratinocytes, which form the bulk of the protective outer layer; 2) melanocytes, which protect the body from ultraviolet radiation by secreting the pigment melanin; and 3) cells that form the secretory appendages: eccrine and apocrine sweat glands, and the sebaceous gland. Emerging evidence suggests that store-operated Ca2+ entry (SOCE), whereby depletion of intracellular Ca2+ stores triggers Ca2+ influx across the plasma membrane, is central to the normal physiology of these cells and thus skin function. Numerous skin pathologies including dermatitis, anhidrotic ectodermal dysplasia, hyperhidrosis, hair loss and cancer are now linked to dysfunction in SOCE proteins. Principal amongst these are the stromal interaction molecules (STIMs) that sense Ca2+ depletion and Orai channels that mediate Ca2+ influx. In this review, the roles of STIM, Orai and other store-operated channels are discussed in the context of keratinocyte differentiation, melanogenesis, and eccrine sweat secretion. We explore not only STIM1-Orai1 as drivers of SOCE, but also independent actions of STIM, and emerging signal cascades stemming from their activities. Roles are discussed for the elusive transient receptor potential canonical channel (TRPC) complex in keratinocytes, Orai channels in Ca2+-cyclic AMP signal crosstalk in melanocytes, and Orai isoforms in eccrine sweat gland secretion.

The purpose of this paper is to examine new possible ways of discrimination of neutron from gamma radiation in diamond detectors. A brand new patented diamond detector was designed and tested using proton beams of various energies accelerated using the Tandem Van de Graaf and Tandetron accelerators in experimental chambers of the Laboratory for ion beam interactions of the Ruđer Bošković Institute in Zagreb. In order to corroborate that the geometry design was optimal for fast neutron and gamma ray discrimination, simulations were made using Monte-Carlo simulation tools GRAS, GEANT4 and CERN’s scientific software framework ROOT as well as SRIM. Cilj ovog rada je ispitivanje novih mogućih načina diskriminacije neutronskog od gama zračenja u dijamantnim detektorima. Novi patentirani dijamantni detektor dizajniran je i ispitan pomoću protonskih snopova različitih energija ubrzanih korištenjem akceleratora Tandem Van de Graaf i Tandetron u eksperimentalnim komorama Laboratorija za interakcije ionskih snopova Instituta Ruđer Bošković u Zagrebu. Za potvrdu optimalnog dizajna geometrije napravljene su Monte-Carlo simulacije pomoću alata kao što su GRAS, Geant4 i ROOT za definiranje geometrije detektora, izvora i analizu, kao i SRIM programski paket.

Dijamant je poluvodički materijal širokog zabranjenog pojasa i izvrsnih mehaničkih svojstava. Posljednjih godina raste zanimanje za umjetno proizvedenim dijamantima visoke čistoće koji se primjenjuju kao čestični detektori u fizici visokih energija, što u radioterapiji kao dozimetri, a potencijalno i u kvantnim računalima u kojima se istražuje primjena NV (engl. nitrogen vacancy) centara pri izradi kvantnih bitova (engl. qubits). Upravo iz tih razloga veliki se napori ulažu u detaljnu karakterizaciju svojstava dijamanta poput: električne otpornosti, širina zabranjenog pojasa (engl. band gap), mobilnosti nosioca naboja, zaustavne moći, itd. U ovom radu je mjerena zaustavna moć za protonske snopove energija u rasponu od 1.6 do 6 MeV-a koji prolaze kroz tanki kristal sintetičkog dijamanta, realiziran kao samopodržavajuća membrana mikrometarske debljine. Najprije, kako bi se moglo precizno odrediti zaustavnu moć, bilo je potrebno odrediti debljinu same membrane te su za to korištena dvije različite metode: indirektno mjerenjem apsorpcije x-zraka u membrani tzv. PIXE (engl. particle-induced x-ray emission) metodom i direktno iz snimaka ruba membrane dobivenih pretražnom elektronskom mikroskopijom (engl. scanning electron microscopy, SEM). Nakon toga je izmjeren gubitak energije protonskog snopa pri prolasku kroz membranu tzv. STIM (engl. scanning transmission ion microscopy) metodom te je iz dobivenih mjerenja određena zaustavna moć protona MeV-skih energija u dijamantu. Dobiveni rezultati uspoređeni su s vrijednostima zaustavne mo´ci dobivene u Monte Carlo simulacijama unutar SRIM (engl. Stopping and Range of Ions in Matter) programskog paketa i eksperimentalnim rezultatima drugih autora. Naposlijetku, određen je energijski rasap u dijamantu te su rezultati također uspoređeni s vrijednostima dobivenim pomoću SRIM programskog paketa. Diamond is a semiconductor material with a wide band gap and excellent mechanical properties. In the last few years interest for high purity synthetic diamonds is growing due to its various applications ranging from high energy physics (particle detectors), radiotherapy (dosimeters) to quantum computing in which nitrogen vacancy (NV) centers in diamond are being researched as a possible way of creating qubits. For these reasons extensive efforts are being made to characterize the properties of synthetic diamond such as: electrical resistivity, band gap width, mobility of free charge carriers, stopping power, etc. In this work, stopping power for proton beams of energies ranging from 1.6 to 6 MeV pass through a thin crystal of synthetic diamond which is made as a self-supporting membrane of micrometer thickness. Firstly, in order to properly calculate the stopping power, the thickness of the membrane needed to be measured for which two different methods were used: indirectly by measuring the absorption of x-rays in the membrane via the so-called PIXE (particle induced x-ray emission) method and directly from pictures of the membrane’s edge obtained with scanning electron microscopy (SEM). After the membrane thickness was calculated, the energy loss inside the membrane was measured using the so-called STIM (scanning transmission ion microscopy) technique from which the stopping power for MeV protons in diamond was calculated. The stopping power for diamond was calculated and cross referenced with stopping power data obtained by Monte Carlo simulations from SRIM (Stopping and Range of Ions in Matter) and experimental data from other authors. Finally, energy straggling was calculated and cross referenced with values obtained via the SRIM program.

Advances in observing methods and the advent of instruments such as SINFONI, GPI etc allow to simultaneously produce high contrast images (HCI) and extract high resolution spectra (HRS) of exoplanet targets. This has produced multispectral images of the targets making it possible to simultaneously use the image and spectral dimensions of such data. Such data has been used in detection and characterization of multiple systems such as PDS70 (Haffert et al 2020., Christiaens 2020 etc.), HD142527 (Christiaens 2019) etc. Advanced data science techniques have been proposed to improve the current detection limit taking advantage of the large feature set provided by multispectral imaging. Machine learning (ML) has had a particularly high success rate in the imaging domain (e.g Dahlquist et al 2021, Gomez Gonzales et al 2018). However ML has proven ineffective when using spectra alone (e.g Fisher et al 2020) owing to the a large number of spectral channels that do not contribute discriminatory features from the star. Therefore, dimensionality reduction have been suggested in order to effectively harness HRS data. Consequently, this project investigates if after reducing the dimensionality of HRS, will the spatial diversity provided by the HCI improve the detection limit. We use SDI cubes from SINFONI. This consist of the HD142527 data cube and an empty data cube with injected companions. We implement dimensionality reduction by replacing the spectral dimension with a relative velocity dimension and the pixel values with cross correlation (CCF) values This produces a spatial CCF map consisting of correlated and uncorrelated pixels. Naturally, pixels which contain spectra closer to the template that it is correlated with have a higher value. However, this map is still contaminated by noise correlations and field rotation. It has been proven that the application of derotation and STIM algorithms with appropriate thresholding (Pairet et al 2019) to a standard ADI cube produces a reliable detection map. In our case we replace the ADI cube with a CCF cube and apply derotation+STIM. Choice of an appropriate threshold converts this STIM map into a detection map The thresholding in the STIM map has been shown to be somewhat noise dependent. In order to now harness the power of ML to our project we will replace the STIM+thresholding with an appropriate noise independent ML algorithm and summarize the improvement in detectability. &nbsp

Introduction. Functional gastrointestinal disorders (FGID) occupy one of the leading positions among intestinal pathologies. At present, scientific data indicate the lack of efficiency of existing methods of FGID treatment and the need for further research. This study is devoted to the assessment of the impact of STIM Lax and STIM treatment on clinical manifestations in patients with FC and FD.Aim of the study. Evaluation of the effectiveness of biologically active additives (BAA) STIM and STIM Laks in 39 patients with functional gastrointestinal disorders. Objectives of the study: to evaluate the effect of the drug on clinical symptoms according to an individual diary and using specialized questionnaires with a score of symptoms before and after treatment.Materials of the research. 39 patients divided into two groups were admitted to the research. Of these, 20 patients were with FC (4 men and 16 women; mean age – 40.3 ± 8.9 years). Monotherapy with STIM Laks was conducted (1 tablet 3 times a day for 30 days), and 19 FD patients (10 men and 9 women; mean age – 36.9 ± 14.1 years). Before and after the course of treatment a carbolene sample was conducted, stool form and frequency, intensity of flatulence, purring, transfusion, abdominal pain were clinically evaluated.Results. Therapy of FС and FD with STIM and STIM Laks effectively reduces the frequency and intensity of clinical symptoms, normalizes the transit time of the carbolene sample, the frequency of stool. Increase of flatulence was observed in 7 (17.9%) patients, its resolution within 1–3 days led to a decrease in the dose of the drug, without affecting the end result.Conclusions. This study indicates a high efficacy and good tolerability of treatment with STIM Laks and STIM in patients with FGID.

Studien syftade till att förena begreppsuppfattningar av black boxfenomenet, genom att härleda vad black box är enligt branschkunniga och undersöka vad de anser att problematiken grundar sig i. Problemområdet innefattar distribution av ekonomiskt kapital som inte fördelas korrekt. Bakgrunden till studien vilade i viljan att forma en hållbar musikindustri för mig och mina branschkollegor. Studien utformades av fyra forskningsfrågor som besvarades med metoden semistrukturerade intervjuer genom ett strategiskt urval av branschkunniga aktörer. De fem respondenterna intervjuades genom videosamtal för att skapa den deskriptiva empiriska data som studien eftersökte. Det övergripande resultatet påvisar att uppfattningarna består av orsakskedjor som ingrips av flera delkomponenter. Uppfattningarnas grund vilar i fenomenets uppkomst/historia, dess bakomliggande orsaker och vilket ekonomiska kapital som omfattas. Komponenterna bildar tillsammans begreppsuppfattningar som delvis handlar om att begreppet växt i och med det digitala skiftet som i sin tur påverkat hur data och information hanteras mellan kunder, rättighetsorganisationer, företag och aktörer i musikbranschen. Studiens resultat har bidragit till djupare förståelse av begreppet blackbox och kunskap om hur branschkunniga uppfattar problematiken, samt hur musikbranschen arbetar för en förbättring.

Quantitative nanoscale imaging using transmission He ion channelling contrast: Proof-of-concept and application to study isolated crystalline defects

Polymer membranes are conventionally prepared using high-energy particles from radioactive decay or by the bombardment of hundreds of MeVs energy ions. In both circumstances, tracks of damage are produced by particles/ions passing through the polymer, and successively, the damaged material is removed by chemical etching to create narrow pores. This process ensures nanosized pore diameter but with random placement, leading to non-uniform local pore density and low membrane porosity, which is necessary to reduce the risk of their overlapping. The present study is focused on the use of polyethylene terephthalate (PET) foils irradiated by 10.0 MeV carbon ions, easily achievable with ordinary ion accelerators. The ion irradiation conditions and the chemical etching conditions were monitored to obtain customized pore locations without pore overlapping in PET. The quality, shape, and size of the pores generated in the micromembranes can have a large impact on their applicability. In this view, the Scanning Transmission Ion Microscopy coupled with a computer code created in our laboratory was implemented to acquire new visual and quantitative insights on fabricated membranes.

Aging is an unstoppable process and begins shortly after birth. Each cell of the organism is affected by the irreversible process, not only with equal density but also at varying ages and with different speed. Therefore, aging can also be understood as an adaptation to a continually changing cellular environment. One of these very prominent changes in age affects Ca2+ signaling. Especially immune cells highly rely on Ca2+-dependent processes and a strictly regulated Ca2+ homeostasis. The intricate patterns of impaired immune cell function may represent a deficit or compensatory mechanisms. Besides, altered immune function through Ca2+ signaling can profoundly affect the development of age-related disease. This review attempts to summarize changes in Ca2+ signaling due to channels and receptors in T cells and beyond in the context of aging.

Pulmonary arterial hypertension (PAH) is a severe and multifactorial disease. PAH pathogenesis mostly involves pulmonary arterial endothelial and pulmonary arterial smooth muscle cell (PASMC) dysfunction, leading to alterations in pulmonary arterial tone and distal pulmonary vessel obstruction and remodeling. Unfortunately, current PAH therapies are not curative, and therapeutic approaches mostly target endothelial dysfunction, while PASMC dysfunction is under investigation. In PAH, modifications in intracellular Ca2+ homoeostasis could partly explain PASMC dysfunction. One of the most crucial actors regulating Ca2+ homeostasis is store-operated Ca2+ channels, which mediate store-operated Ca2+ entry (SOCE). This review focuses on the main actors of SOCE in human and experimental PASMC, their contribution to PAH pathogenesis, and their therapeutic potential in PAH.

Orai channels belong to the calcium release-activated calcium (CRAC) channel family. Orai channels are responsible for the influx of extracellular Ca2+ that is triggered by Ca2+ depletion from the endoplasmic reticulum (ER); this function is essential for many types of non-excitable cells. Extensive structural and functional studies have advanced the knowledge of the molecular mechanism by which Orai channels are activated. However, the gating mechanism that allows Ca2+ permeation through Orai channels is less well explained. Here, we reviewed and summarized the existing structural studies of Orai channels. We detailed the structural features of Orai channels, described structural comparisons of their closed and open states, and finally proposed a “push–pull” model of Ca2+ permeation.

The solute carrier family 10 member SLC10A7 is a negative regulator of intracellular calcium signaling (RCAS). In cell culture, SLC10A7 expression is negatively correlated with store-operated calcium entry (SOCE) via the plasma membrane. SLC10A7-deficient cells have significantly increased calcium influx after treatment with thapsigargin for depletion of ER calcium stores, whereas SLC10A7/RCAS overexpression limits calcium influx. Genetic variants in the human SLC10A7 gene are associated with skeletal dysplasia and amelogenesis imperfecta and reveal loss of function on cellular calcium influx. More recently, an additional disease-related genetic variant (P303L) as well as some novel genetic variants (V235F, T221M, I136M, L210F, P285L, and G146S) have been identified. In the present study, these variants were expressed in HEK293 cells to study their subcellular localization and their effect on cellular calcium influx. All variants were properly sorted to the ER compartment and closely co-localized with the STIM protein, a functional component of SOCE. The variants P303L and L210F showed significantly reduced effects on cellular calcium influx compared to the wild type but still maintained some degree of residual activity. This might explain the milder phenotype of patients bearing the P303L variant and might indicate disease potential for the newly identified L210F variant. In contrast, all other variants behaved like the wild type. In conclusion, the occurrence of variants in the SLC10A7 gene should be considered in patients with skeletal dysplasia and amelogenesis imperfecta. In addition to the already established variants, the present study identifies another potential disease-related SLC10A7/RCAS variant, namely, L210F, which seems to be most frequent in South Asian populations.

Current knowledge on store-operated Ca2+ entry (SOCE) regarding its localization, kinetics, and regulation is mostly derived from studies performed in non-excitable cells. After a long time of relative disinterest in skeletal muscle SOCE, this mechanism is now recognized as an essential contributor to muscle physiology, as highlighted by the muscle pathologies that are associated with mutations in the SOCE molecules STIM1 and Orai1. This review mainly focuses on the peculiar aspects of skeletal muscle SOCE that differentiate it from its counterpart found in non-excitable cells. This includes questions about SOCE localization and the movement of respective proteins in the highly organized skeletal muscle fibers, as well as the diversity of expressed STIM isoforms and their differential expression between muscle fiber types. The emerging evidence of a phasic SOCE, which is activated during EC coupling, and its physiological implication is described as well. The specific issues related to the use of SOCE modulators in skeletal muscles are discussed. This review highlights the complexity of SOCE activation and its regulation in skeletal muscle, with an emphasis on the most recent findings and the aim to reach a current picture of this mesmerizing phenomenon.

Mechanosensitive ion channels are widely expressed in the cardiovascular system. They translate mechanical forces including shear stress and stretch into biological signals. The most prominent biological signal through which the cardiovascular physiological activity is initiated or maintained are intracellular calcium ions (Ca2+). Growing evidence show that the Ca2+ entry mediated by mechanosensitive ion channels is also precisely regulated by a variety of key proteins which are distributed in the cell membrane or endoplasmic reticulum. Recent studies have revealed that mechanosensitive ion channels can even physically interact with Ca2+ regulatory proteins and these interactions have wide implications for physiology and pathophysiology. Therefore, this paper reviews the cross-talk between mechanosensitive ion channels and some key Ca2+ regulatory proteins in the maintenance of calcium homeostasis and its relevance to cardiovascular health and disease.

Ca2+ ion channels are critical in a variety of physiological events, including cell growth, differentiation, gene transcription and apoptosis. One such essential entry pathway for calcium into the cell is the Ca2+ release-activated Ca2+ (CRAC) channel. It consists of the Ca2+ sensing protein, stromal interaction molecule 1 (STIM1) located in the endoplasmic reticulum (ER) and a Ca2+ ion channel Orai in the plasma membrane. The Orai channel family includes three homologues Orai1, Orai2 and Orai3. While Orai1 is the “classical” Ca2+ ion channel within the CRAC channel complex and plays a universal role in the human body, there is increasing evidence that Orai2 and Orai3 are important in specific physiological and pathophysiological processes. This makes them an attractive target in drug discovery, but requires a detailed understanding of the three Orai channels and, in particular, their differences. Orai channel activation is initiated via Ca2+ store depletion, which is sensed by STIM1 proteins, and induces their conformational change and oligomerization. Upon STIM1 coupling, Orai channels activate to allow Ca2+ permeation into the cell. While this activation mechanism is comparable among the isoforms, they differ by a number of functional and structural properties due to non-conserved regions in their sequences. In this review, we summarize the knowledge as well as open questions in our current understanding of the three isoforms in terms of their structure/function relationship, downstream signaling and physiology as well as pathophysiology.

Simple Summary Glioblastoma is a high-grade primary brain tumor that contains a subpopulation of cells called glioblastoma stem cells, which are responsible for tumor initiation, growth and recurrence after treatment. Recent transcriptomic studies have highlighted that calcium pathways predominate in glioblastoma stem cells. Calcium channels have the ability to transduce signals from the microenvironment and are therefore ideally placed to control cellular behavior. Using multiple approaches, we demonstrate in five different primary cultures, previously derived from surgical specimens, that glioblastoma stem cells express store-operated channels (SOC) that support calcium entry into these cells. Pharmacological inhibition of SOC dramatically reduces cell proliferation and stem cell self-renewal in these cultures. By identifying SOC as a critical mechanism involved in the maintenance of the stem cell population in glioblastoma, our study will contribute to the framework for the identification of new therapies against this deadly tumor. Abstract Glioblastoma is the most frequent and deadly form of primary brain tumors. Despite multimodal treatment, more than 90% of patients experience tumor recurrence. Glioblastoma contains a small population of cells, called glioblastoma stem cells (GSC) that are highly resistant to treatment and endowed with the ability to regenerate the tumor, which accounts for tumor recurrence. Transcriptomic studies disclosed an enrichment of calcium (Ca2+) signaling transcripts in GSC. In non-excitable cells, store-operated channels (SOC) represent a major route of Ca2+ influx. As SOC regulate the self-renewal of adult neural stem cells that are possible cells of origin of GSC, we analyzed the roles of SOC in cultures of GSC previously derived from five different glioblastoma surgical specimens. Immunoblotting and immunocytochemistry experiments showed that GSC express Orai1 and TRPC1, two core SOC proteins, along with their activator STIM1. Ca2+ imaging demonstrated that SOC support Ca2+ entries in GSC. Pharmacological inhibition of SOC-dependent Ca2+ entries decreased proliferation, impaired self-renewal, and reduced expression of the stem cell marker SOX2 in GSC. Our data showing the ability of SOC inhibitors to impede GSC self-renewal paves the way for a strategy to target the cells considered responsible for conveying resistance to treatment and tumor relapse.

Intracellular Ca2+ distribution is a tightly regulated process. Numerous Ca2+ chelating, storage, and transport mechanisms are required to maintain normal cellular physiology. Ca2+-binding proteins, mainly calmodulin and calbindins, sequester free intracellular Ca2+ ions and apportion or transport them to signaling hubs needing the cations. Ca2+ channels, ATP-driven pumps, and exchangers assist the binding proteins in transferring the ions to and from appropriate cellular compartments. Some, such as the endoplasmic reticulum, mitochondria, and lysosomes, act as Ca2+ repositories. Cellular Ca2+ homeostasis is inefficient without the active contribution of these organelles. Moreover, certain key cellular processes also rely on inter-organellar Ca2+ signaling. This review attempts to encapsulate the structure, function, and regulation of major intracellular Ca2+ buffers, sensors, channels, and signaling molecules before highlighting how cancer cells manipulate them to survive and thrive. The spotlight is then shifted to the slow pace of translating such research findings into anticancer therapeutics. We use the PubMed database to highlight current clinical studies that target intracellular Ca2+ signaling. Drug repurposing and improving the delivery of small molecule therapeutics are further discussed as promising strategies for speeding therapeutic development in this area.

Inositol 1,4,5-trisphosphate receptors (IP3R) are the most widely expressed intracellular Ca2+ release channels. Their activation by IP3 and Ca2+ allows Ca2+ to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca2+] may directly regulate cytosolic effectors or fuel Ca2+ uptake by other organelles, while the decrease in ER luminal [Ca2+] stimulates store-operated Ca2+ entry (SOCE). We are close to understanding the structural basis of both IP3R activation, and the interactions between the ER Ca2+-sensor, STIM, and the plasma membrane Ca2+ channel, Orai, that lead to SOCE. IP3Rs are the usual means through which extracellular stimuli, through ER Ca2+ release, stimulate SOCE. Here, we review evidence that the IP3Rs most likely to respond to IP3 are optimally placed to allow regulation of SOCE. We also consider evidence that IP3Rs may regulate SOCE downstream of their ability to deplete ER Ca2+ stores. Finally, we review evidence that IP3Rs in the plasma membrane can also directly mediate Ca2+ entry in some cells.

This collaborative autoethnography examines how we (four students and a professor of community-engaged research) used our experiences to make sense of life during COVID‑19. Engaging in a collective approach to autoethnographic writing, we highlight different perspectives of a cultural moment shaped by the science denialism and untruths that define US governmental practices and approaches to the pandemic. We share how we are dealing with COVID‑19 discourses that run counter to the scientific foregrounding of our STEM (Science, Technology, Engineering, and Math) and health and medical sciences training, reflect on the role of the pandemic in shifting post-baccalaureate plans, navigate the lockdown while participating in racial justice protests in Minneapolis, and examine the experiences of being essential healthcare workers while in school. By situating these shifting ways of learning, teaching, and engaging as portents of the difficulties we may continue to face, we show the possibilities of narrative methods in imagining and implementing post-pandemic healthcare practices grounded on a praxis of community justice and collective care., Cette auto-ethnographie collaborative examine comment nous (quatre étudiants et un professeur de recherche communautaire) avons tiré parti de nos expériences pour donner un sens à la vie pendant la pandémie de COVID-19. À travers une approche collective de l’écriture auto-ethnographique, nous donnons à voir différentes perspectives d’un moment culturel marqué par le négationnisme scientifique et les contre-vérités propres aux pratiques et approches du gouvernement américain face à la pandémie. Nous montrons comment nous traitons les discours sur la COVID-19 qui vont à l’encontre de la centralité scientifique de notre formation en STIM (science, technologie, ingénierie et mathématiques) ou en sciences médicales et de la santé ; nous réfléchissons à la façon dont la pandémie nous oblige à modifier nos plans post-baccalauréat ; nous décrivons comment nous vivons le confinement tout en participant aux manifestations pour la justice raciale à Minneapolis ; et nous analysons nos expériences en tant que travailleurs de la santé essentiels pendant nos études. En abordant la transformation des méthodes d’apprentissage, d’enseignement et d’engagement comme le signe des difficultés auxquelles nous pourrions continuer à être confrontés, nous mettons en évidence les possibilités qu’offrent les méthodes narratives pour imaginer et mettre en oeuvre des pratiques de soins de santé post-pandémiques fondées sur une praxis de justice communautaire et de soins collectifs.

Significance Store-operated Ca2+ entry (SOCE) links Ca2+ release from endoplasmic reticulum (ER) to Ca2+ entry across the plasma membrane (PM). SOCE is unusual in requiring interaction between proteins in different membranes. STIM1, when it senses loss of ER Ca2+, unfurls domains that interact with Orai1 PM Ca2+ channels. The stoichiometry of the SOCE complex is contentious, but it determines the regulation and functional consequences of SOCE. We show that native complexes are likely to comprise a single Orai1 channel and a few STIM1 dimers, too few to cluster Orai1 channels. We suggest that SOCE may be digitally regulated by local ER depletion, and that local SOCE-evoked Ca2+ fluxes are small enough to allow substantial intracellular redistribution of Ca2+ through ER tunnels., Increases in cytosolic Ca2+ concentration regulate diverse cellular activities and are usually evoked by opening of Ca2+ channels in intracellular Ca2+ stores and the plasma membrane (PM). For the many signals that evoke formation of inositol 1,4,5-trisphosphate (IP3), IP3 receptors coordinate the contributions of these two Ca2+ sources by mediating Ca2+ release from the endoplasmic reticulum (ER). Loss of Ca2+ from the ER then activates store-operated Ca2+ entry (SOCE) by causing dimers of STIM1 to cluster and unfurl cytosolic domains that interact with the PM Ca2+ channel, Orai1, causing its pore to open. The relative concentrations of STIM1 and Orai1 are important, but most analyses of their interactions use overexpressed proteins that perturb the stoichiometry. We tagged endogenous STIM1 with EGFP using CRISPR/Cas9. SOCE evoked by loss of ER Ca2+ was unaffected by the tag. Step-photobleaching analysis of cells with empty Ca2+ stores revealed an average of 14.5 STIM1 molecules within each sub-PM punctum. The fluorescence intensity distributions of immunostained Orai1 puncta were minimally affected by store depletion, and similar for Orai1 colocalized with STIM1 puncta or remote from them. We conclude that each native SOCE complex is likely to include only a few STIM1 dimers associated with a single Orai1 channel. Our results, demonstrating that STIM1 does not assemble clusters of interacting Orai channels, suggest mechanisms for digital regulation of SOCE by local depletion of the ER.

Increases in cytosolic Ca concentration regulate diverse cellular activities and are usually evoked by opening of Ca channels in intracellular Ca stores and the plasma membrane (PM). For the many signals that evoke formation of inositol 1,4,5-trisphosphate (IP), IP receptors coordinate the contributions of these two Ca sources by mediating Ca release from the endoplasmic reticulum (ER). Loss of Ca from the ER then activates store-operated Ca entry (SOCE) by causing dimers of STIM1 to cluster and unfurl cytosolic domains that interact with the PM Ca channel, Orai1, causing its pore to open. The relative concentrations of STIM1 and Orai1 are important, but most analyses of their interactions use overexpressed proteins that perturb the stoichiometry. We tagged endogenous STIM1 with EGFP using CRISPR/Cas9. SOCE evoked by loss of ER Ca was unaffected by the tag. Step-photobleaching analysis of cells with empty Ca stores revealed an average of 14.5 STIM1 molecules within each sub-PM punctum. The fluorescence intensity distributions of immunostained Orai1 puncta were minimally affected by store depletion, and similar for Orai1 colocalized with STIM1 puncta or remote from them. We conclude that each native SOCE complex is likely to include only a few STIM1 dimers associated with a single Orai1 channel. Our results, demonstrating that STIM1 does not assemble clusters of interacting Orai channels, suggest mechanisms for digital regulation of SOCE by local depletion of the ER. We acknowledge support from a Wellcome Senior Investigator Award 101844 (to C.W.T.) and the Biotechnology and Biological Sciences Research Council UK (grant code, BB/P005330/1). We thank Sumita Chakraborty for contributions to gene editing of STIM1.

Background Simultaneous infection with multiple malaria parasite strains is common in high transmission areas. Quantifying the number of strains per host, or the multiplicity of infection (MOI), provides additional parasite indices for assessing transmission levels but it is challenging to measure accurately with current tools. This paper presents new laboratory and analytical methods for estimating the MOI of Plasmodium falciparum. Methods Based on 24 single nucleotide polymorphisms (SNPs) previously identified as stable, unlinked targets across 12 of the 14 chromosomes within P. falciparum genome, three multiplex PCRs of short target regions and subsequent next generation sequencing (NGS) of the amplicons were developed. A bioinformatics pipeline including B4Screening pathway removed spurious amplicons to ensure consistent frequency calls at each SNP location, compiled amplicons by SNP site diversity, and performed algorithmic haplotype and strain reconstruction. The pipeline was validated by 108 samples generated from cultured-laboratory strain mixtures in different proportions and concentrations, with and without pre-amplification, and using whole blood and dried blood spots (DBS). The pipeline was applied to 273 smear-positive samples from surveys conducted in western Kenya, then providing results into StrainRecon Thresholding for Infection Multiplicity (STIM), a novel MOI estimator. Results The 24 barcode SNPs were successfully identified uniformly across the 12 chromosomes of P. falciparum in a sample using the pipeline. Pre-amplification and parasite concentration, while non-linearly associated with SNP read depth, did not influence the SNP frequency calls. Based on consistent SNP frequency calls at targeted locations, the algorithmic strain reconstruction for each laboratory-mixed sample had 98.5% accuracy in dominant strains. STIM detected up to 5 strains in field samples from western Kenya and showed declining MOI over time (q Conclusion The combined approach of new multiplex PCRs and NGS, the unique bioinformatics pipeline and STIM could identify 24 barcode SNPs of P. falciparum correctly and consistently. The methodology could be applied to field samples to reliably measure temporal changes in MOI.

There are growing indications for the involvement of calcium stores in the plastic properties of neurons and particularly in dendritic spines of central neurons. The store-operated calcium entry (SOCE) channels are assumed to be activated by the calcium sensor stromal interaction molecule (STIM)which leads to activation of its associated Orai channel. There are two STIM species, and the differential role of the two in SOCE is not entirely clear. In the present study, we were able to distinguish between transfected STIM1, which is more mobile primarily in young neurons, and STIM2 which is less mobile and more prominent in older neurons in culture. STIM1 mobility is associated with spontaneous calcium sparks, local transient rise in cytosolic [Ca2+]i, and in the formation and elongation of dendritic filopodia/spines. In contrast, STIM2 is associated with older neurons, where it is mobile and moves into dendritic spines primarily when cytosolic [Ca2+]i levels are reduced, apparently to activate resident Orai channels. These results highlight a role for STIM1 in the regulation of [Ca2+]i fluctuations associated with the formation of dendritic spines or filopodia in the developing neuron, whereas STIM2 is associated with the maintenance of calcium entry into stores in the adult neuron.

Current knowledge on store-operated Ca2+ entry (SOCE) regarding its localization, kinetics, and regulation is mostly derived from studies performed in non-excitable cells. After a long time of relative disinterest in skeletal muscle SOCE, this mechanism is now recognized as an essential contributor to muscle physiology, as highlighted by the muscle pathologies that are associated with mutations in the SOCE molecules STIM1 and Orai1. This review mainly focuses on the peculiar aspects of skeletal muscle SOCE that differentiate it from its counterpart found in non-excitable cells. This includes questions about SOCE localization and the movement of respective proteins in the highly organized skeletal muscle fibers, as well as the diversity of expressed STIM isoforms and their differential expression between muscle fiber types. The emerging evidence of a phasic SOCE, which is activated during EC coupling, and its physiological implication is described as well. The specific issues related to the use of SOCE modulators in skeletal muscles are discussed. This review highlights the complexity of SOCE activation and its regulation in skeletal muscle, with an emphasis on the most recent findings and the aim to reach a current picture of this mesmerizing phenomenon.

Stromal interaction molecules (STIMs), including STIM1 and STIM2, are single-pass transmembrane proteins that are located predominantly in the endoplasmic reticulum (ER). They serve as calcium ion (Ca2+) sensors within the ER. In the central nervous system (CNS), they are involved mainly in Orai-mediated store-operated Ca2+ entry (SOCE). The key molecular components of the SOCE pathway are well-characterized, but the molecular mechanisms that underlie the regulation of this pathway need further investigation. Numerous intracellular target proteins that are located in the plasma membrane, ER, cytoskeleton, and cytoplasm have been reported to play essential roles in concert with STIMs, such as conformational changes in STIMs, their translocation, the stabilization of their interactions with Orai, and the activation of other channels. The present review focuses on numerous regulators, such as Homer, SOCE-associated regulatory factor (SARAF), septin, synaptopodin, golli proteins, partner of STIM1 (POST), and transcription factors and proteasome inhibitors that regulate STIM-Orai interactions in the CNS. Further we describe novel roles of STIMs in mediating Ca2+ influx via other than Orai pathways, including TRPC channels, VGCCs, AMPA and NMDA receptors, and group I metabotropic glutamate receptors. This review also summarizes recent findings on additional molecular targets of STIM proteins including SERCA, IP3Rs, end-binding proteins (EB), presenilin, and CaMKII. Dysregulation of the SOCE-associated toolkit, including STIMs, contributes to the development of neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease, and Huntington's disease), traumatic brain injury, epilepsy, and stroke. Emerging evidence points to the role of STIM proteins and several of their molecular effectors and regulators in neuronal and glial physiology and pathology, suggesting their potential application for future therapeutic strategies.

Aging is an unstoppable process and begins shortly after birth. Each cell of the organism is affected by the irreversible process, not only with equal density but also at varying ages and with different speed. Therefore, aging can also be understood as an adaptation to a continually changing cellular environment. One of these very prominent changes in age affects Ca2+ signaling. Especially immune cells highly rely on Ca2+-dependent processes and a strictly regulated Ca2+ homeostasis. The intricate patterns of impaired immune cell function may represent a deficit or compensatory mechanisms. Besides, altered immune function through Ca2+ signaling can profoundly affect the development of age-related disease. This review attempts to summarize changes in Ca2+ signaling due to channels and receptors in T cells and beyond in the context of aging.

Store-operated calcium channels (SOCs) are widely expressed in excitatory and non-excitatory cells where they mediate significant store-operated calcium entry (SOCE), an important pathway for calcium signaling throughout the body. While the activity of SOCs has been well studied in non-excitable cells, attention has turned to their role in neurons and glia in recent years. In particular, the role of SOCs in the nervous system has been extensively investigated, with links to their dysregulation found in a wide variety of neurological diseases from Alzheimer’s disease (AD) to pain. In this review, we provide an overview of their molecular components, expression, and physiological role in the nervous system and describe how the dysregulation of those roles could potentially lead to various neurological disorders. Although further studies are still needed to understand how SOCs are activated under physiological conditions and how they are linked to pathological states, growing evidence indicates that SOCs are important players in neurological disorders and could be potential new targets for therapies. While the role of SOCE in the nervous system continues to be multifaceted and controversial, the study of SOCs provides a potentially fruitful avenue into better understanding the nervous system and its pathologies.

The Ca2+ sensor STIM1 and the Ca2+ channel Orai1 that form the store-operated Ca2+ (SOC) channel complex are key targets for drug development. Selective SOC inhibitors are currently undergoing clinical evaluation for the treatment of auto-immune and inflammatory responses and are also deemed promising anti-neoplastic agents since SOC channels are linked with enhanced cancer cell progression. Here, we describe an investigation of the site of binding of the selective inhibitor Synta66 to the SOC channel Orai1 using docking and molecular dynamics simulations, and live cell recordings. Synta66 binding was localized to the extracellular site close to the transmembrane (TM)1 and TM3 helices and the extracellular loop segments, which, importantly, are adjacent to the Orai1-selectivity filter. Synta66-sensitivity of the Orai1 pore was, in fact, diminished by both Orai1 mutations affecting Ca2+ selectivity and permeation of Na+ in the absence of Ca2+. Synta66 also efficiently blocked SOC in three glioblastoma cell lines but failed to interfere with cell viability, division and migration. These experiments provide new structural and functional insights into selective drug inhibition of the Orai1 Ca2+ channel by a high-affinity pore blocker.

International audience; The intracellular Ca2+ concentration is mainly controlled by Ca2+ channels. These channels form complexes with K+ channels, which function to amplify Ca2+ flux. In cancer cells, voltage-gated/voltage-dependent Ca2+ channels and non-voltage-gated/voltage-independent Ca2+ channels have been reported to interact with K+ channels such as Ca2+-activated K+ channels and voltage-gated K+ channels. These channels are activated by an increase in cytosolic Ca2+ concentration or by membrane depolarization, which induces membrane hyperpolarization, increasing the driving force for Ca2+ flux. These complexes, composed of K+ and Ca2+ channels, are regulated by several molecules including lipids (ether lipids and cholesterol), proteins (e.g. STIM), receptors (e.g. S1R/SIGMAR1), and peptides (e.g. LL-37) and can be targeted by monoclonal antibodies, making them novel targets for cancer research.

International audience; The intracellular Ca2+ concentration is mainly controlled by Ca2+ channels. These channels form complexes with K+ channels, which function to amplify Ca2+ flux. In cancer cells, voltage-gated/voltage-dependent Ca2+ channels and non-voltage-gated/voltage-independent Ca2+ channels have been reported to interact with K+ channels such as Ca2+-activated K+ channels and voltage-gated K+ channels. These channels are activated by an increase in cytosolic Ca2+ concentration or by membrane depolarization, which induces membrane hyperpolarization, increasing the driving force for Ca2+ flux. These complexes, composed of K+ and Ca2+ channels, are regulated by several molecules including lipids (ether lipids and cholesterol), proteins (e.g. STIM), receptors (e.g. S1R/SIGMAR1), and peptides (e.g. LL-37) and can be targeted by monoclonal antibodies, making them novel targets for cancer research.

Simple Summary Store-operated calcium channels constituted from the proteins Orai and STIM are important targets for development of new drugs, especially for the treatment of auto-immune diseases. Also, interference with channel function is linked to reduced cancer cell progression, making these channels potential targets for anti-cancer drug development. Therefore, inhibitors need to be evaluated for both their binding selectivity and their potential to interfere with cancer progression. Here, we investigated the inhibitor Synta66 and determined its site of binding via both patch clamp recordings and computational approaches and evaluated its potency as anti-cancer agent in glioblastoma multiforme cells. Our findings show that Synta66 is a highly selective ligand to the Orai1 pore and efficiently blocks store operated calcium entry in glioblastoma cells. Still, in the tested cell lines, Synta66 did not reduce cell viability. We therefore suggest Synta66 as a precise tool to observe interference of store-operated Orai1 channel function in vitro and of resulting downstream effects. Abstract The Ca2+ sensor STIM1 and the Ca2+ channel Orai1 that form the store-operated Ca2+ (SOC) channel complex are key targets for drug development. Selective SOC inhibitors are currently undergoing clinical evaluation for the treatment of auto-immune and inflammatory responses and are also deemed promising anti-neoplastic agents since SOC channels are linked with enhanced cancer cell progression. Here, we describe an investigation of the site of binding of the selective inhibitor Synta66 to the SOC channel Orai1 using docking and molecular dynamics simulations, and live cell recordings. Synta66 binding was localized to the extracellular site close to the transmembrane (TM)1 and TM3 helices and the extracellular loop segments, which, importantly, are adjacent to the Orai1-selectivity filter. Synta66-sensitivity of the Orai1 pore was, in fact, diminished by both Orai1 mutations affecting Ca2+ selectivity and permeation of Na+ in the absence of Ca2+. Synta66 also efficiently blocked SOC in three glioblastoma cell lines but failed to interfere with cell viability, division and migration. These experiments provide new structural and functional insights into selective drug inhibition of the Orai1 Ca2+ channel by a high-affinity pore blocker.

Cancer is caused by excessive cell proliferation and a propensity to avoid cell death, while the spread of cancer is facilitated by enhanced cellular migration, invasion, and vascularization. Cytosolic Ca2+ is central to each of these important processes, yet to date, there are no cancer drugs currently being used clinically, and very few undergoing clinical trials, that target the Ca2+ signalling machinery. The aim of this review is to highlight some of the emerging evidence that targeting key components of the Ca2+ signalling machinery represents a novel and relatively untapped therapeutic strategy for the treatment of cancer.

Background Openers of mitochondrial adenosine triphosphate-dependent potassium (mKATP) channels like diazoxide increase reactive oxygen species (ROS) production in cardiac cells and reduce Ca2+ elevations produced by ischemia–reperfusion, protecting the heart from damage. In this study we tested the hypothesis that opening mKATP channels regulates expression of the major components of store-operated Ca2+ entry (SOCE) STIM1 and Orai1. Results Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot experiments showed that diazoxide increased expression of STIM1 and Orai1 at the mRNA and protein levels, respectively, in adult rat cardiomyocytes. Immunofluorescence analyses revealed that diazoxide also disrupted the striated distribution pattern of STIM1. These effects were prevented by the ROS scavenger N-acetyl cysteine (NAC), the mKATP channel antagonist 5-hydroxydecanoate (5-HD), or the protein synthesis inhibitor cycloheximide (CHX). Confocal microscopy revealed that diazoxide also led to nuclear translocation of the transcription factors c-Fos and NFκB, which was also blocked by NAC or 5-HD. Finally, the MAPK pathway inhibitor UO126 attenuated diazoxide-induced upregulation of STIM1 and Orai1 expression. Conclusions Our results suggest that opening mitochondrial potassium ATP channels with diazoxide upregulates the expression of STIM1 and Orai1 by de novo synthesis by a mechanism that involves NFkB, c-Fos, and ROS via MAPK/ERK signaling.

Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca2+ sensor protein that induces permeation of Orai Ca2+ channels upon endoplasmic reticulum Ca2+-store depletion. A drop in luminal Ca2+ causes partial unfolding of the N-terminal STIM1 domains and thus initial STIM1 activation. We compared the STIM1 structure upon Ca2+ depletion from our molecular dynamics (MD) simulations with a recent 2D NMR structure. Simulation- and structure-based results showed unfolding of two &alpha, helices in the canonical and in the non-canonical EF-hand. Further, we structurally and functionally evaluated mutations in the non-canonical EF-hand that have been shown to cause tubular aggregate myopathy. We found these mutations to cause full constitutive activation of Ca2+-release-activated Ca2+ currents (ICRAC) and to promote autophagic processes. Specifically, heterologously expressed STIM1 mutations in the non-canonical EF-hand promoted translocation of the autophagy transcription factors microphthalmia-associated transcription factor (MITF) and transcription factor EB (TFEB) into the nucleus. These STIM1 mutations additionally stimulated an enhanced production of autophagosomes. In summary, mutations in STIM1 that cause structural unfolding promoted Ca2+ down-stream activation of autophagic processes.

The common currency of epithelial differentiation and homeostasis is calcium, stored primarily in the endoplasmic reticulum, rationed according to need, and replenished from the extracellular milieu via store-operated calcium entry (SOCE). This currency is disbursed by the IP3 receptor in response to diverse extracellular signals. The rate of release is governed by regulators of proliferation, autophagy, survival, and programmed cell death, the strength of the signal leading to different outcomes. Intracellular calcium acts chiefly through intermediates such as calmodulin that regulates growth factor receptors such as epidermal growth factor receptor (EGFR), actin polymerization, and adherens junction assembly and maintenance. Here we review this machinery and its role in differentiation, then consider how cancer cells subvert it to license proliferation, resist anoikis, and enable metastasis, either by modulating the level of intracellular calcium or its downstream targets or effectors such as EGFR, E-cadherin, IQGAP1, TMEM16A, CLCA2, and TRPA1. Implications are considered for the roles of E-cadherin and growth factor receptors in circulating tumor cells and metastasis. The discovery of novel, cell type-specific modulators and effectors of calcium signaling offers new possibilities for cancer chemotherapy.

Store-operated Ca2+ entry (SOCE) contributes to Ca2+ refilling of endoplasmic reticulum (ER), but also provides Ca2+ influx involved in physiological and pathological signalling functions. Upon depletion of Ca2+ store, the sensor protein stromal interaction molecule (STIM) activates Orai1, forming an ion-conducting pore highly selective for Ca2+. SOCE-associated regulatory factor (SARAF) associates with STIM1 to facilitate a slow form of Ca2+-dependent inactivation of SOCE or interacts with Orai1 to stimulate SOCE in STIM1-independent manner. We have investigated whether cerebral ischemic damage and neuroprotection conferred by ischemic preconditioning (PC) in mouse are associated with changes in the expression of the molecular components of SOCE. Ischemic PC induced by 15-min occlusion of the middle cerebral artery (MCAo) resulted in significant amelioration of histological and functional outcomes produced, 72 h later, by a more severe ischemia (1 h MCAo). Neither ischemia, nor PC affected the expression of Orai1 in the frontoparietal cortex. However, the number of Orai1-immunopositive cells, mostly corresponding to Ly-6G+ neutrophils, was significantly elevated in the blood after the ischemic insult, regardless of previous PC. The expression of Stim1 and SARAF, mainly localised in NeuN-immunopositive neurons, was reduced in the ischemic cortex. Interestingly, neuroprotection by ischemic PC prevented the reduction of SARAF expression in the lesioned cortex and this could be interpreted as a compensatory mechanism to restore ER Ca2+ refilling in neurons in the absence of STIM1. Thus, preventing SARAF downregulation may represent a pivotal mechanism implicated in neuroprotection provided by ischemic PC and should be exploited as an original target for novel stroke therapies.

Voltage-dependent Ca2+ channels and store-operated Ca2+ channels (SOCs) are the major routes of Ca2+ entry into mammalian cells. Previously, we reported that pharmacological preconditioning (PPC) leads to a decrease in the amplitude of L-type calcium channel current in the heart. In this study, we examined PPC-associated changes in SOC function. We measured adult cardiomyocyte membrane currents using the whole-cell patch-clamp technique, and we evaluated reactive oxygen species (ROS) production and intracellular Ca2+ levels in cardiomyocytes using fluorescent probes. Diazoxide (Dzx) and thapsigargin (Tg) were used to induce PPC and to deplete internal stores of Ca2+, respectively. Ca2+ store depletion generated inward currents with strong rectification, which were suppressed by the SOC blocker GSK-7975-A. These currents were completely abolished by PPC, an effect that could be countered with 5-hydroxydecanoate (5-HD; a selective mitochondrial ATP-sensitive K+ channel blocker), an intracellular mitochondrial energizing solution, or Ni2+ [a blocker of sodium–calcium exchanger (NCX)]. Buffering of ROS and intracellular Ca2+ also prevented PPC effects on SOC currents. Refilling of intracellular stores was largely suppressed by PPC, as determined by measuring intracellular Ca2+ with a fluorescent Ca2+ indicator. These results indicate that influx of Ca2+ through SOCs is inhibited by their ROS and Ca2+-dependent inactivation during PPC and that NCX is a likely source of PPC-inactivating Ca2+. We further showed that NCX associates with Orai1. Down-regulation of SOCs by PPC may play a role in cardioprotection following ischemia–reperfusion.

Transmission imaging in the helium ion microscope allows to measure mass-thickness contrast and reveal crystallographic information. We recently customized a microchannel plate followed by a delay line readout structure especially for this application. This system can correlate the scanning transmission ion image to the angular distribution of the transmitted ions. An in-vacuum linear support is used to place the detector at different distances from the sample, adjusting the maximum collection angle. Post-processing allows the reconstruction of images for selected scattering angles. The first results show images with nanometer resolution, material contrast, and identification of sub-surface features in biological tissues. This work has been supported by the H2020 Project npSCOPE under grant number 720964.

Stimulation Test (STIM) as a preliminary step before the main lie detecting test (called as polygraph test) has being performed on a traditional contacting lie detector (polygraph). The purpose of this STIM test is to ensure the reliability of the test by showing the accuracy of the testing result to the subject, and to check the basic principle of sensor attachment or the subject's physiological response. According to the Korean Supreme Court's precedent (No. 83 Do 712) regarding polygraph tests, as he made a precondition for capacity of evidence that there must be a certain change in psychological state if subject lies, and whether subject is lying or not will be judged accurately by the physiological response and so on. Stimulation test (STIM) that can guarantee reliability of lie detection using Video-polygraph based on Vibraimage technology should be preceded as well. With 50 STIM test video data used in traditional contacting polygraph tests, we developed a program for Stimulation (STIM) test using Video-polygraph application based on Vibraimage technology.

The role of Ca2+ as a signalling molecule is essential to fundamental cellular functions such as proliferation, cell growth, gene expression or the secretion of bioactive molecules. Intracellular Ca2+ homeostasis is maintained via regulatory systems such as store operated Ca2+ channels (SOCC), which are the key players of store-operated Ca2+ entry (SOCE). Orai1 proteins build hexameric Ca2+ selective channels in the plasma membrane. Upon activation by STIM1, which acts as a Ca2+ sensor protein in the ER, the Ca2+release activated Ca2+ (CRAC) channels allow the influx of Ca2+ ions into the cell, replenishing the intracellular Ca2+ store within the ER. Throughout years of research several disease-related STIM1 and Orai1 mutations have been identified. This thesis focuses on three cancer associated Orai1 mutants, taken from the cBioPortal database. The mutants A122T (malignant mixed Mullerian tumours of the uterus), N147S (germinal center B-cell type cancer) and P164H (lung adenocarcinoma) were investigated for their impact on pore structure at pore lining residues R91, F99 and E106 by utilizing a cysteine cross-linking approach. Furthermore, the electrophysiological channel characteristics were measured via whole cell patch clamp recordings, protein localization was verified by fluorescence microscopy and a NFAT screen was carried out to measure possible modulations of gene transcription downstream the Ca2+ signalling cascade. submitted by Sascha Berlansky BSc Universität Linz, Masterarbeit, 2020 (VLID)5215287

De har gjorts få studier på akvariesfiskars beteende. För att bemöta intresset för akvariefiskar så behöver vi veta mer om deras beteende i en artificiell miljö. Syftet med den här studien är att jämföra det utforskande beteendet hos vildfångade och odlade diamanttetror som är en vanlig akvariefisk. Hypotesen är att det finns en skillnad i deras beteende där vildfångade är mer djärva än de odlade. Grupper om fem individer av diamanttetra vildfångade och odlade släpptes ner i en ny artificiell miljö. De filmades i 20 minuter från de att de släpptes i. Testet upprepades 10 gånger med nya individer och för varje försök togs tiden det tog för varje individ att lämpa botten av akvariet och sedan mättes storleken på stimmen för varje grupp. Jag hittade en skillnad i hur lång tid fiskarna tog på sig för att lämna botten men det var ingen skillnad i höjd eller längd på stimmen. Detta visar att de vildfångade visade ett djärvare beteende än de odlade genom att lämna botten snabbare. Den här studien gav information om skillnaden i beteende för vildfångade och odlade diamanttetror och informationen kan användas för fortsatta studier kring akvariefisk. The behaviour of ornamental fish is not well studied. To meet the needs of ornamental fish hobbyists, we need to know more about the behavior of fish in artificial environments. The aim of this study is to compare the explorative behaviour of wild-captured and farmed fish in a common ornamental fish, the diamond tetra. The hypothesis is that there is a difference between the two groups behavior where the wild-captured tetras are bolder. Groups of five individuals of farmed respectively wild-captured diamond tetra were released in a new artificial environment where they were monitored for 20 minutes. The test was repeated 10 times with new individuals, and the time it took for them to leave the bottom of the aquarium was measured as well as the size of the shoal. I found a difference in the time it took for the individuals to leave the bottom, with the wild-captured tetras leaving the bottom earlier than the farmed fish. There were no differences in height or length of the shoal. Thus, the wild diamond tetra were bolder than the farmed tetras. The study gave information about the differences in behavior between wild-captured and farmed diamond tetra and can give information to further studies about ornamental fish.

Store-operated Ca2+ entry (SOCE) provides a major Ca2+ entry route in cancer cells. SOCE is mediated by the assembly of Stim and Orai proteins at endoplasmic reticulum (ER)-plasma membrane junctions upon depletion of the ER Ca2+ store. Additionally, Stim and Orai proteins underpin constitutive Ca2+ entry in a growing number of cancer cell types due to the partial depletion of their ER Ca2+ reservoir. Herein, we investigated for the first time the structure and function of SOCE in primary cultures of colorectal carcinoma (CRC) established from primary tumor (pCRC) and metastatic lesions (mCRC) of human subjects. Stim1-2 and Orai1-3 transcripts were equally expressed in pCRC and mCRC cells, although Stim1 and Orai3 proteins were up-regulated in mCRC cells. The Mn2+-quenching technique revealed that constitutive Ca2+ entry was significantly enhanced in pCRC cells and was inhibited by the pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3. The larger resting Ca2+ influx in pCRC was associated to their lower ER Ca2+ content as compared to mCRC cells. Pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3 prevented ER-dependent Ca2+ release, thereby suggesting that constitutive SOCE maintains ER Ca2+ levels. Nevertheless, pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3 did not affect CRC cell proliferation and migration. These data provide the first evidence that Stim and Orai proteins mediate constitutive Ca2+ entry and replenish ER with Ca2+ in primary cultures of CRC cells. However, SOCE is not a promising target to design alternative therapies for CRC.

The KRAS GTPase plays a fundamental role in transducing signals from plasma membrane growth factor receptors to downstream signalling pathways controlling cell proliferation, survival and migration. Activating KRAS mutations are found in 20% of all cancers and in up to 40% of colorectal cancers, where they contribute to dysregulation of cell processes underlying oncogenic transformation. Multiple KRAS-regulated cell functions are also influenced by changes in intracellular Ca2+ levels that are concurrently modified by receptor signalling pathways. Suppression of intracellular Ca2+ release mechanisms can confer a survival advantage in cancer cells, and changes in Ca2+ entry across the plasma membrane modulate cell migration and proliferation. However, inconsistent remodelling of Ca2+ influx and its signalling role has been reported in studies of transformed cells. To isolate the interaction between altered Ca2+ handling and mutated KRAS in colorectal cancer, we have previously employed isogenic cell line pairs, differing by the presence of an oncogenic KRAS allele (encoding KRASG13D), and have shown that reduced Ca2+ release from the ER and mitochondrial Ca2+ uptake contributes to the survival advantage conferred by oncogenic KRAS. Here we show in the same cell lines, that Store-Operated Ca2+ Entry (SOCE) and its underlying current, ICRAC are under the influence of KRASG13D. Specifically, deletion of the oncogenic KRAS allele resulted in enhanced STIM1 expression and greater Ca2+ influx. Consistent with the role of KRAS in the activation of the ERK pathway, MEK inhibition in cells with KRASG13D resulted in increased STIM1 expression. Further, ectopic expression of STIM1 in HCT 116 cells (which express KRASG13D) rescued SOCE, demonstrating a fundamental role of STIM1 in suppression of Ca2+ entry downstream of KRASG13D. These results add to the understanding of how ERK controls cancer cell physiology and highlight STIM1 as an important biomarker in cancerogenesis. ispartof: CELL CALCIUM vol:72 pages:70-80 ispartof: location:Netherlands status: published

Pulmonary arterial hypertension (PAH) is a severe and multifactorial disease. PAH pathogenesis mostly involves pulmonary arterial endothelial and pulmonary arterial smooth muscle cell (PASMC) dysfunction, leading to alterations in pulmonary arterial tone and distal pulmonary vessel obstruction and remodeling. Unfortunately, current PAH therapies are not curative, and therapeutic approaches mostly target endothelial dysfunction, while PASMC dysfunction is under investigation. In PAH, modifications in intracellular Ca2+ homoeostasis could partly explain PASMC dysfunction. One of the most crucial actors regulating Ca2+ homeostasis is store-operated Ca2+ channels, which mediate store-operated Ca2+ entry (SOCE). This review focuses on the main actors of SOCE in human and experimental PASMC, their contribution to PAH pathogenesis, and their therapeutic potential in PAH.

Orai channels belong to the calcium release-activated calcium (CRAC) channel family. Orai channels are responsible for the influx of extracellular Ca2+ that is triggered by Ca2+ depletion from the endoplasmic reticulum (ER); this function is essential for many types of non-excitable cells. Extensive structural and functional studies have advanced the knowledge of the molecular mechanism by which Orai channels are activated. However, the gating mechanism that allows Ca2+ permeation through Orai channels is less well explained. Here, we reviewed and summarized the existing structural studies of Orai channels. We detailed the structural features of Orai channels, described structural comparisons of their closed and open states, and finally proposed a “push–pull” model of Ca2+ permeation.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons (MNs), probably by a combination of cell- and non-cell-autonomous processes. The past decades have brought many important insights into the role of astrocytes in nervous system function and disease, including the implication in ALS pathogenesis possibly through the impairment of Ca2+-dependent astrocyte-MN cross-talk. In this respect, it has been recently proposed that altered astrocytic store-operated Ca2+ entry (SOCE) may underlie aberrant gliotransmitter release and astrocyte-mediated neurotoxicity in ALS. These observations prompted us to a thorough investigation of SOCE in primary astrocytes from the spinal cord of the SOD1(G93A) ALS mouse model in comparison with the SOD1(WT)-expressing controls. To this purpose, we employed, for the first time in the field, genetically-encoded Ca2+ indicators, allowing the direct assessment of Ca2+ fluctuations in different cell domains. We found increased SOCE, associated with decreased expression of the sarco-endoplasmic reticulum Ca2+-ATPase and lower ER resting Ca2+ concentration in SOD1(G93A) astrocytes compared to control cells. Such findings add novel insights into the involvement of astrocytes in ALS MN damage.