ER and SOCE Ca2+ signals are not required for directed cell migration in human iPSC-derived microglia.

The central nervous system (CNS) is constantly surveilled by microglia, highly motile and dynamic cells deputed to act as the first line of immune defense in the brain and spinal cord. Alterations in the homeostasis of the CNS are detected by microglia that respond by extending their processes or - following major injuries - by migrating toward the affected area. Understanding the mechanisms controlling directed cell migration of microglia is crucial to dissect their responses to neuroinflammation and injury. We used a combination of pharmacological and genetic approaches to explore the involvement of calcium (Ca2+) signaling in the directed migration of human induced pluripotent stem cell (iPSC)-derived microglia challenged with a purinergic stimulus. This approach mimics cues originating from injury of the CNS. Unexpectedly, simultaneous imaging of microglia migration and intracellular Ca2+ changes revealed that this phenomenon does not require Ca2+ signals generated from the endoplasmic reticulum (ER) and store-operated Ca2+ entry (SOCE) pathways. Instead, we find evidence that human microglial chemotaxis to purinergic signals is mediated by cyclic AMP in a Ca2+-independent manner. These results challenge prevailing notions, with important implications in neurological conditions characterized by perturbation in Ca2+ homeostasis.

Widespread Production of Polyunsaturated Aldehydes by Benthic Diatoms of the North Pacific Ocean's Salish Sea.

Diatoms are key primary producers across marine, freshwater, and terrestrial ecosystems. They are responsible for photosynthesis and secondary production that, in part, support complex food webs. Diatoms can produce phytochemicals that have transtrophic ecological effects which increase their competitive fitness. Polyunsaturated aldehydes (PUAs) are one class of diatom-derived phytochemicals that are known to have allelopathic and anti-herbivory properties. The anti-herbivory capability of PUAs results from their negative effect on grazer fecundity. Since their discovery, research has focused on their production by pelagic marine diatoms, and their effects on copepod egg production, hatching success, and juvenile survival and development. Few investigations have explored PUA production by the prolific suite of benthic marine diatoms, despite their importance to coastal trophic systems. In this study, we tested eight species of benthic diatoms for the production of the bioactive PUAs 2,4-heptadienal, 2,4-octadienal, and 2,4-decadienal. Benthic diatom species were isolated from the Salish Sea, an inland sea within the North Pacific ecosystem. All species were found to be producers of at least two PUAs in detectable concentrations, with five species producing all three PUAs in quantifiable concentrations. Our results indicate that production of PUAs from Salish Sea benthic diatoms may be widespread, and thus these compounds may contribute to benthic coastal food web dynamics through heretofore unrecognized pathways. Future studies should expand the geographic scope of investigations into benthic diatom PUA production and explore the effects of benthic diatoms on benthic consumer fecundity.

ER and SOCE Ca2+ signals are not required for directed cell migration in human microglia.

The central nervous system (CNS) is constantly surveilled by microglia, highly motile and dynamic cells deputed to act as the first line of immune defense in the brain and spinal cord. Alterations in the homeostasis of the CNS are detected by microglia that respond by migrating toward the affected area. Understanding the mechanisms controlling directed cell migration of microglia is crucial to dissect their responses to neuroinflammation and injury. We used a combination of pharmacological and genetic approaches to explore the involvement of calcium (Ca2+) signaling in the directed migration of induced pluripotent stem cell (iPSC)-derived microglia challenged with a purinergic stimulus. This approach mimics cues originating from injury of the CNS. Unexpectedly, simultaneous imaging of microglia migration and intracellular Ca2+ changes revealed that this phenomenon does not require Ca2+ signals generated from the endoplasmic reticulum (ER) and store-operated Ca2+ entry (SOCE) pathways. Instead, we find evidence that human microglial chemotaxis to purinergic signals is mediated by cyclic AMP in a Ca2+-independent manner. These results challenge prevailing notions, with important implications in neurological conditions characterized by perturbation in Ca2+ homeostasis.

Jacques Lacan, psicólogo barrado / Jacques Lacan, psychologue barré / Jacques Lacan, barred psychologist

Esse artigo revisa uma série de contradições entre a disciplina da psicologia e a obra de Jacques Lacan. Psicologia descrita aqui como o domínio acadêmico e profissional da teoria e prática desenvolvida na cultura ocidental, especificamente anglo-estadunidense, de descrever e explicar os processos mentais e do comportamento. Lacan é caracterizado como uma referência para a elaboração de seu trabalho teórico e clínico, com o foco principal em seus próprios escritos. O argumento principal é que há uma incompatibilidade fundamental entre a obra de Lacan e as visões psicológicas do sujeito como indivíduo e, portanto, as tentativas de equivaler as duas tradições são mal concebidas. Isso significa que os psicólogos que buscam por respostas em Lacan devem questionar os pressupostos subjacentes sobre a teoria e a metodologia em sua disciplina se eles estão dispostos a levar a sua obra a sério. A incompatibilidade entre Lacan e a psicologia também tem importantes consequências para os psicólogos clínicos que possam querer adotar ideias da tradição lacaniana, visto que destaca os perigos que a psicologia reserva para a psicanálise caso as teorias e metodologias psicológicas sejam aceitas de bom grado. O mote de Lacan como "psicólogo barrado" é designado para enfatizar esses argumentos bem como a concepção distintiva do sujeito que implica sua obra

Cet article examine une série de contradictions entre la discipline de la psychologie et l'œuvre de Jacques Lacan. La psychologie est décrite ici comme le domaine académique et professionnel de la théorie et de la pratique développées dans la culture occidentale, plus précisément anglo-américaine, pour décrire et expliquer les processus mentaux et comportementaux. Lacan est caractérisé comme une référence pour l'élaboration de son travail théorique et clinique, en se concentrant principalement sur ses propres écrits. L'argument principal est qu'il existe une incompatibilité fondamentale entre l'œuvre de Lacan et les conceptions psychologiques du sujet en tant qu'individu, et donc que les tentatives d'équivaloir les deux traditions sont mal conçues. Cela signifie que les psychologues qui cherchent des réponses chez Lacan doivent remettre en question les présupposés sous-jacents à la théorie et à la méthodologie de leur discipline s'ils sont disposés à prendre son travail au sérieux. L'incompatibilité entre Lacan et la psychologie a également d'importantes conséquences pour les psychologues cliniciens qui pourraient souhaiter adopter des idées de la tradition lacanienne, car elle met en évidence les dangers que la psychologie représente pour la psychanalyse si les théories et méthodologies psychologiques sont acceptées de bon gré. Le slogan de Lacan en tant que "psychologue exclu" est utilisé pour souligner ces arguments ainsi que la conception distinctive du sujet qui implique son œuvre

This paper reviews a series of contradictions between the discipline of psychology and the work of Jacques Lacan. Psychology here is the academic and professional domain of theory and practice developed in Western, specifically Anglo-US American, culture to describe and explain behavioural and mental processes. Lacan is characterized with reference to the elaboration of his theoretical and clinical work, with the focus primarily on his own writings. The main argument is that there is a fundamental incompatibility between Lacan's work and psychological views of the individual subject, and therefore attempts to assimilate the two traditions are misconceived. This means that psychologists looking to Lacan for answers must question underlying assumptions about theory and methodology in their discipline if they are to take his work seriously. The incompatibility between Lacan and psychology also has important consequences for clinical psychologists who may wish to adopt ideas from the Lacanian tradition, for it highlights the dangers that psychology holds for psychoanalysis if psychological theories and methodologies are taken on good coin. Themotif of Lacan as 'barred psychologist' is designed to emphasize these arguments as well as the distinctiveaccount of the human subject that his work entails

PIEZO1-HaloTag hiPSCs: Bridging Molecular, Cellular and Tissue Imaging.

PIEZO1 channels play a critical role in numerous physiological processes by transducing diverse mechanical stimuli into electrical and chemical signals. Recent studies underscore the importance of endogenous PIEZO1 activity and localization in regulating mechanotransduction. To enable physiologically and clinically relevant human-based studies, we genetically engineered human induced pluripotent stem cells (hiPSCs) to express a HaloTag fused to endogenous PIEZO1. Combined with super-resolution imaging, our chemogenetic approach allows precise visualization of PIEZO1 in various cell types. Further, the PIEZO1-HaloTag hiPSC technology allows non-invasive monitoring of channel activity via Ca2+-sensitive HaloTag ligands, with temporal resolution approaching that of patch clamp electrophysiology. Using lightsheet imaging of hiPSC-derived neural organoids, we also achieve molecular scale PIEZO1 imaging in three-dimensional tissue samples. Our advances offer a novel platform for studying PIEZO1 mechanotransduction in human cells and tissues, with potential for elucidating disease mechanisms and development of targeted therapeutics.

Intracellular Injection of Brain Extracts from Alzheimer's Disease Patients Triggers Unregulated Ca2+ Release from Intracellular Stores That Hinders Cellular Bioenergetics.

Strong evidence indicates that amyloid beta (Aß) inflicts its toxicity in Alzheimer's disease (AD) by promoting uncontrolled elevation of cytosolic Ca2+ in neurons. We have previously shown that synthetic Aß42 oligomers stimulate abnormal intracellular Ca2+ release from the endoplasmic reticulum stores, suggesting that a similar mechanism of Ca2+ toxicity may be common to the endogenous Aßs oligomers. Here, we use human postmortem brain extracts from AD-affected patients and test their ability to trigger Ca2+ fluxes when injected intracellularly into Xenopus oocytes. Immunological characterization of the samples revealed the elevated content of soluble Aß oligomers only in samples from AD patients. Intracellular injection of brain extracts from control patients failed to trigger detectable changes in intracellular Ca2+. Conversely, brain extracts from AD patients triggered Ca2+ events consisting of local and global Ca2+ fluorescent transients. Pre-incubation with either the conformation-specific OC antiserum or caffeine completely suppressed the brain extract's ability to trigger cytosolic Ca2+ events. Computational modeling suggests that these Ca2+ fluxes may impair cells bioenergetic by affecting ATP and ROS production. These results support the hypothesis that Aß oligomers contained in neurons of AD-affected brains may represent the toxic agents responsible for neuronal malfunctioning and death associated with the disruption of Ca2+ homeostasis.

KRAP is required for diffuse and punctate IP3-mediated Ca2+ liberation and determines the number of functional IP3R channels within clusters.

KRas-induced actin-interacting protein (KRAP) has been identified as crucial for the appropriate localization and functioning of the inositol trisphosphate receptors (IP3Rs) that mediate Ca2+ release from the endoplasmic reticulum. Here, we used siRNA knockdown of KRAP expression in HeLa and HEK293 cells to examine the roles of KRAP in the generation of IP3-mediated local Ca2+ puffs and global, cell-wide Ca2+ signals. High resolution Ca2+ imaging revealed that the mean amplitude of puffs was strongly reduced by KRAP knockdown, whereas the Ca2+ flux during openings of individual IP3R channels was little affected. In both control and KRAP knockdown cells the numbers of functional channels in the clusters underlying puff sites were stochastically distributed following a Poisson relationship, but the mean number of functional channels per site was reduced by about two thirds by KRAP knockdown. We conclude that KRAP is required for activity of IP3R channels at puff sites and stochastically 'licenses' the function of individual channels on a one-to-one basis, rather than determining the functioning of the puff site as a whole. In addition to puff activity ('punctate' Ca2+ release), global, cell-wide Ca2+ signals evoked by higher levels of IP3 are further composed from a discrete 'diffuse' mode of Ca2+ release. By applying fluctuation analysis to isolate the punctate component during global Ca2+ signals, we find that KRAP knockdown suppresses to similar extents punctate and diffuse Ca2+ release in wild-type cells and in HEK293 cells exclusively expressing type 1 and type 3 IP3Rs. Thus, KRAP appears essential for the functioning of the IP3Rs involved in diffuse Ca2+ release as well as the clustered IP3Rs that generate local Ca2+ puffs.

TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia.

The membrane protein TREM2 (Triggering Receptor Expressed on Myeloid cells 2) regulates key microglial functions including phagocytosis and chemotaxis. Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer's disease (AD). Because abnormalities in Ca2+ signaling have been observed in several AD models, we investigated TREM2 regulation of Ca2+ signaling in human induced pluripotent stem cell-derived microglia (iPSC-microglia) with genetic deletion of TREM2. We found that iPSC-microglia lacking TREM2 (TREM2 KO) show exaggerated Ca2+ signals in response to purinergic agonists, such as ADP, that shape microglial injury responses. This ADP hypersensitivity, driven by increased expression of P2Y12 and P2Y13 receptors, results in greater release of Ca2+ from the endoplasmic reticulum stores, which triggers sustained Ca2+ influx through Orai channels and alters cell motility in TREM2 KO microglia. Using iPSC-microglia expressing the genetically encoded Ca2+ probe, Salsa6f, we found that cytosolic Ca2+ tunes motility to a greater extent in TREM2 KO microglia. Despite showing greater overall displacement, TREM2 KO microglia exhibit reduced directional chemotaxis along ADP gradients. Accordingly, the chemotactic defect in TREM2 KO microglia was rescued by reducing cytosolic Ca2+ using a P2Y12 receptor antagonist. Our results show that loss of TREM2 confers a defect in microglial Ca2+ response to purinergic signals, suggesting a window of Ca2+ signaling for optimal microglial motility.

Termination of Ca2+ puffs during IP3-evoked global Ca2+ signals.

We previously described that cell-wide cytosolic Ca2+ transients evoked by inositol trisphosphate (IP3) are generated by two modes of Ca2+ liberation from the ER; 'punctate' release via an initial flurry of transient Ca2+ puffs from local clusters of IP3 receptors, succeeded by a spatially and temporally 'diffuse' Ca2+ liberation. Those findings were derived using statistical fluctuation analysis to monitor puff activity which is otherwise masked as global Ca2+ levels rise. Here, we devised imaging approaches to resolve individual puffs during global Ca2+ elevations to better investigate the mechanisms terminating the puff flurry. We find that puffs contribute about 40% (∼90 attomoles) of the total Ca2+ liberation, largely while the global Ca2+ signal rises halfway to its peak. The major factor terminating punctate Ca2+ release is an abrupt decline in puff frequency. Although the amplitudes of large puffs fall during the flurry, the amplitudes of more numerous small puffs remain steady, so overall puff amplitudes decline only modestly (∼30%). The Ca2+ flux through individual IP3 receptor/channels does not measurably decline during the flurry, or when puff activity is depressed by pharmacological lowering of Ca2+ levels in the ER lumen, indicating that the termination of punctate release is not a simple consequence of reduced driving force for Ca2+ liberation. We propose instead that the gating of IP3 receptors at puff sites is modulated such that their openings become suppressed as the bulk [Ca2+] in the ER lumen falls during global Ca2+ signals.

Piezo1 channels restrain regulatory T cells but are dispensable for effector CD4+ T cell responses.

T lymphocytes encounter complex mechanical cues during an immune response. The mechanosensitive ion channel, Piezo1, drives inflammatory responses to bacterial infections, wound healing, and cancer; however, its role in helper T cell function remains unclear. In an animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we found that mice with genetic deletion of Piezo1 in T cells showed diminished disease severity. Unexpectedly, Piezo1 was not essential for lymph node homing, interstitial motility, Ca2+ signaling, T cell proliferation, or differentiation into proinflammatory T helper 1 (TH1) and TH17 subsets. However, Piezo1 deletion in T cells resulted in enhanced transforming growth factor-ß (TGFß) signaling and an expanded pool of regulatory T (Treg) cells. Moreover, mice with deletion of Piezo1 specifically in Treg cells showed significant attenuation of EAE. Our results indicate that Piezo1 selectively restrains Treg cells, without influencing activation events or effector T cell functions.

MR method for measuring microscopic histologic soft tissue textures.

PURPOSE: Provide a direct, non-invasive diagnostic measure of microscopic tissue texture in the size scale between tens of microns and the much larger scale measurable by clinical imaging. This paper presents a method and data demonstrating the ability to measure these microscopic pathologic tissue textures (histology) in the presence of subject motion in an MR scanner. This size range is vital to diagnosing a wide range of diseases. THEORY/METHODS: MR micro-Texture (MRµT) resolves these textures by a combination of measuring a targeted set of k-values to characterize texture-as in diffraction analysis of materials, performing a selective internal excitation to isolate a volume of interest (VOI), applying a high k-value phase encode to the excited spins in the VOI, and acquiring each individual k-value data point in a single excitation-providing motion immunity and extended acquisition time for maximizing signal-to-noise ratio. Additional k-value measurements from the same tissue can be made to characterize the tissue texture in the VOI-there is no need for these additional measurements to be spatially coherent as there is no image to be reconstructed. This method was applied to phantoms and tissue specimens including human prostate tissue. RESULTS: Data demonstrating resolution <50 µm, motion immunity, and clearly differentiating between normal and cancerous tissue textures are presented. CONCLUSION: The data reveal textural differences not resolvable by standard MR imaging. As MRµT is a pulse sequence, it is directly translatable to MRI scanners currently in clinical practice to meet the need for further improvement in cancer imaging.

Evaluation of indicators supporting reproducibility and transparency within cardiology literature.

OBJECTIVES: It has been suggested that biomedical research is facing a reproducibility issue, yet the extent of reproducible research within the cardiology literature remains unclear. Thus, our main objective was to assess the quality of research published in cardiology journals by assessing for the presence of eight indicators of reproducibility and transparency. METHODS: Using a cross-sectional study design, we conducted an advanced search of the National Library of Medicine catalogue for publications in cardiology journals. We included publications published between 1 January 2014 and 31 December 2019. After the initial list of eligible cardiology publications was generated, we searched for full-text PDF versions using Open Access, Google Scholar and PubMed. Using a pilot-tested Google Form, a random sample of 532 publications were assessed for the presence of eight indicators of reproducibility and transparency. RESULTS: A total of 232 eligible publications were included in our final analysis. The majority of publications (224/232, 96.6%) did not provide access to complete and unmodified data sets, all 229/232 (98.7%) failed to provide step-by-step analysis scripts and 228/232 (98.3%) did not provide access to complete study protocols. CONCLUSIONS: The presentation of studies published in cardiology journals would make reproducing study outcomes challenging, at best. Solutions to increase the reproducibility and transparency of publications in cardiology journals is needed. Moving forward, addressing inadequate sharing of materials, raw data and key methodological details might help to better the landscape of reproducible research within the field.

Regulatory T cells suppress Th17 cell Ca2+ signaling in the spinal cord during murine autoimmune neuroinflammation.

T lymphocyte motility and interaction dynamics with other immune cells are vital determinants of immune responses. Regulatory T (Treg) cells prevent autoimmune disorders by suppressing excessive lymphocyte activity, but how interstitial motility patterns of Treg cells limit neuroinflammation is not well understood. We used two-photon microscopy to elucidate the spatial organization, motility characteristics, and interactions of endogenous Treg and Th17 cells together with antigen-presenting cells (APCs) within the spinal cord leptomeninges in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Th17 cells arrive before the onset of clinical symptoms, distribute uniformly during the peak, and decline in numbers during later stages of EAE. In contrast, Treg cells arrive after Th17 cells and persist during the chronic phase. Th17 cells meander widely, interact with APCs, and exhibit cytosolic Ca2+ transients and elevated basal Ca2+ levels before the arrival of Treg cells. In contrast, Treg cells adopt a confined, repetitive-scanning motility while contacting APCs. These locally confined but highly motile Treg cells limit Th17 cells from accessing APCs and suppress Th17 cell Ca2+ signaling by a mechanism that is upstream of store-operated Ca2+ entry. Finally, Treg cell depletion increases APC numbers in the spinal cord and exaggerates ongoing neuroinflammation. Our results point to fundamental differences in motility characteristics between Th17 and Treg cells in the inflamed spinal cord and reveal three potential cellular mechanisms by which Treg cells regulate Th17 cell effector functions: reduction of APC density, limiting access of Th17 cells to APCs, and suppression of Th17 Ca2+ signaling.

ER-luminal [Ca2+] regulation of InsP3 receptor gating mediated by an ER-luminal peripheral Ca2+-binding protein.

Modulating cytoplasmic Ca2+ concentration ([Ca2+]i) by endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+-release channels is a universal signaling pathway that regulates numerous cell-physiological processes. Whereas much is known regarding regulation of InsP3R activity by cytoplasmic ligands and processes, its regulation by ER-luminal Ca2+ concentration ([Ca2+]ER) is poorly understood and controversial. We discovered that the InsP3R is regulated by a peripheral membrane-associated ER-luminal protein that strongly inhibits the channel in the presence of high, physiological [Ca2+]ER. The widely-expressed Ca2+-binding protein annexin A1 (ANXA1) is present in the nuclear envelope lumen and, through interaction with a luminal region of the channel, can modify high-[Ca2+]ER inhibition of InsP3R activity. Genetic knockdown of ANXA1 expression enhanced global and local elementary InsP3-mediated Ca2+ signaling events. Thus, [Ca2+]ER is a major regulator of InsP3R channel activity and InsP3R-mediated [Ca2+]i signaling in cells by controlling an interaction of the channel with a peripheral membrane-associated Ca2+-binding protein, likely ANXA1.

IP3 mediated global Ca2+ signals arise through two temporally and spatially distinct modes of Ca2+ release.

The 'building-block' model of inositol trisphosphate (IP3)-mediated Ca2+ liberation posits that cell-wide cytosolic Ca2+ signals arise through coordinated activation of localized Ca2+ puffs generated by stationary clusters of IP3 receptors (IP3Rs). Here, we revise this hypothesis, applying fluctuation analysis to resolve Ca2+ signals otherwise obscured during large Ca2+ elevations. We find the rising phase of global Ca2+ signals is punctuated by a flurry of puffs, which terminate before the peak by a mechanism involving partial ER Ca2+ depletion. The continuing rise in Ca2+, and persistence of global signals even when puffs are absent, reveal a second mode of spatiotemporally diffuse Ca2+ signaling. Puffs make only small, transient contributions to global Ca2+ signals, which are sustained by diffuse release of Ca2+ through a functionally distinct process. These two modes of IP3-mediated Ca2+ liberation have important implications for downstream signaling, imparting spatial and kinetic specificity to Ca2+-dependent effector functions and Ca2+ transport.

Noise analysis of cytosolic calcium image data.

Cellular Ca2+ signals are often constrained to cytosolic micro- or nano-domains where stochastic openings of Ca2+ channels cause large fluctuations in local Ca2+ concentration (Ca2+ 'noise'). With the advent of TIRF microscopy to image the fluorescence of Ca2+-sensitive probes from attoliter volumes it has become possible to directly monitor these signals, which closely track the gating of plasmalemmal and ER Ca2+-permeable channels. Nevertheless, it is likely that many physiologically important Ca2+ signals are too small to resolve as discrete events in fluorescence recordings. By analogy with noise analysis of electrophysiological data, we explore here the use of statistical approaches to detect and analyze such Ca2+ noise in images obtained using Ca2+-sensitive indicator dyes. We describe two techniques - power spectrum analysis and spatio-temporal correlation - and demonstrate that both effectively identify discrete, spatially localized calcium release events (Ca2+ puffs). Moreover, we show they are able to detect localized noise fluctuations in a case where discrete events cannot directly be resolved.

Myosin-II mediated traction forces evoke localized Piezo1-dependent Ca2+ flickers.

Piezo channels transduce mechanical stimuli into electrical and chemical signals to powerfully influence development, tissue homeostasis, and regeneration. Studies on Piezo1 have largely focused on transduction of "outside-in" mechanical forces, and its response to internal, cell-generated forces remains poorly understood. Here, using measurements of endogenous Piezo1 activity and traction forces in native cellular conditions, we show that cellular traction forces generate spatially-restricted Piezo1-mediated Ca2+ flickers in the absence of externally-applied mechanical forces. Although Piezo1 channels diffuse readily in the plasma membrane and are widely distributed across the cell, their flicker activity is enriched near force-producing adhesions. The mechanical force that activates Piezo1 arises from Myosin II phosphorylation by Myosin Light Chain Kinase. We propose that Piezo1 Ca2+ flickers allow spatial segregation of mechanotransduction events, and that mobility allows Piezo1 channels to explore a large number of mechanical microdomains and thus respond to a greater diversity of mechanical cues.

Spatial-temporal patterning of Ca2+ signals by the subcellular distribution of IP3 and IP3 receptors.

The patterning of cytosolic Ca2+ signals in space and time underlies their ubiquitous ability to specifically regulate numerous cellular processes. Signals mediated by liberation of Ca2+ sequestered in the endoplasmic reticulum (ER) through inositol trisphosphate receptor (IP3R) channels constitute a hierarchy of events; ranging from openings of individual IP3 channels, through the concerted openings of several clustered IP3Rs to generate local Ca2+ puffs, to global Ca2+ waves and oscillations that engulf the entire cell. Here, we review recent progress in elucidating how this hierarchy is shaped by an interplay between the functional gating properties of IP3Rs and their spatial distribution within the cell. We focus in particular on the subset of IP3Rs that are organized in stationary clusters and are endowed with the ability to preferentially liberate Ca2+.

Applications of FLIKA, a Python-based image processing and analysis platform, for studying local events of cellular calcium signaling.

The patterning of cytosolic Ca2+ signals underlies their ubiquitous ability to specifically regulate numerous cellular processes. Advances in fluorescence microscopy have made it possible to image these signals with unprecedented temporal and spatial resolution. However, this is a double-edged sword, as the resulting enormous data sets necessitate development of software to automate image processing and analysis. Here, we describe Flika, an open source, graphical user interface program written in the Python environment that contains a suite of built-in image processing tools to enable intuitive visualization of image data and analysis. We illustrate the utility and power of Flika by three applications for studying cellular Ca2+ signaling: a script for measuring single-cell global Ca2+ signals; a plugin for the detection, localization and analysis of subcellular Ca2+ puffs; and a script that implements a novel approach for fluctuation analysis of transient, local Ca2+ fluorescence signals. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

A Systematic Review of the Reported Proportions of Diagnoses for Dizziness and Vertigo.

OBJECTIVES: To determine the typical proportions of diagnoses for patients presenting with dizziness or vertigo based on clinical speciality and to assess the change in proportions of diagnoses over time. DATA SOURCES: Following PRISMA guidelines, systematic searches of PubMed and CINAHL databases and follow-up reference searches were performed for articles published in English up to October 2016. STUDY SELECTION: Analysis of searches yielded 42 studies meeting the criteria of case series of adult patients with dizziness and/or vertigo presenting to general practice, emergency departments or specialist outpatient clinics. DATA EXTRACTION: Data comprising demographics, diagnostic cases, and the total number of cases were recorded and independently tested, followed by a risk of bias analysis. DATA SYNTHESIS: Sample size weighted proportions expressed as percentages with confidence intervals were calculated and compared using χ analysis and a reference proportion formed by the combination of Ear Nose and Throat and Neurotology case series published between 2010 and 2016. Analysis of diagnostic trends over time used Poisson regression with consideration for overdispersion. CONCLUSIONS: This systematic review of case series demonstrated significant differences in the proportions of diagnoses for patients presenting with dizziness or vertigo, depending on the specialty making the diagnosis. ENT proportions were dominated by BPPV, Psychogenic and Menière's disease diagnostic categories, whereas emergency proportions were dominated by Other, Cardiac, and Neurological categories. Analysis of case series proportions over time revealed increases in diagnoses such as Benign Paroxysmal Positional Vertigo and Vestibular Migraine, and a corresponding decrease in the diagnoses of Menière's disease.