Biology subjects

Helassa, N.

Publications and source records attributed to Helassa, N..

9 recordsLinked to original sources

bioRxiv2026

Integrated Transcriptomic Profiling Reveals MAPK14-Related Inflammatory Signalling Associated with Postoperative Atrial Fibrillation

Postoperative atrial fibrillation (POAF) affects up to half of patients after cardiac surgery and raises long-term risk of stroke and death. Why only some patients develop it, despite similar surgical exposure, remains poorly understood. We studied a pilot cohort of 20 patients undergoing cardiac surgery in Northwest England, comparing those who developed POAF within four days (n=10) with those who did not (n=10). Circulating plasma RNA was profiled using the NanoString nCounter Cardiovascular Disease panel, and differentially expressed genes were mapped onto protein-protein interaction networks using STRING and Cytoscape. Candidate genes were then validated by RT-qPCR in the same cohort, alongside serial ELISA measurement of plasma MAPK14 protein. Peripheral blood monocyte, neutrophil and total white blood cell counts were also compared between the two groups on postoperative Days 1, 2 and 4. Screening identified MAPK14, COX6C and COX7B as elevated in POAF, with PTPN12 reduced. These genes clustered within a connected inflammatory and stress-response network, and pathway enrichment pointed to immune activation. RT-qPCR confirmed higher MAPK14, COX6C and COX7B expression in POAF patients. Plasma MAPK14 rose after surgery in both groups, with the largest increase in POAF patients on day two, though this did not reach significance. Peripheral blood monocyte counts were significantly higher in POAF patients than in those without POAF at all three postoperative timepoints, whereas neutrophil and total white blood cell counts did not differ significantly between groups. These early findings identify a circulating plasma RNA signature linked to MAPK14-related inflammatory signalling as a candidate marker of POAF susceptibility, warranting confirmation in a larger, adequately powered cohort.

physiology↗

bioRxiv2026

Lysosomal abundance in young and aged mouse hearts assessed by In Vivo Imaging Systems (IVIS) Lysotracker imaging and autophagy-related gene expression

Lysosomal function is essential for cardiac proteostasis and cellular health, yet its regulation during ageing remains poorly defined. We hypothesised that ageing alters both the abundance of acidic organelles and the machinery supporting their acidification. Using fluorescence-based In Vivo Imaging Systems (IVIS) with Lysotracker Red in young (2-4 months) and aged (18 months) mouse hearts, we quantified whole-heart acidic-vesicle signals and assessed expression of lysosomal and autophagy-related genes (Lamp2, Atp6v1a, Sqstm1, Cd63, Atg12, Nfe2l2, M6pr) by RT-qPCR. Whole-heart labelled Lysotracker fluorescence did not differ significantly between age groups, indicating preservation of the total acidic-vesicle pool. No changes in Atp6v1a and Lamp2 expression suggest acidification capacity and structural stability are maintained, whereas the minor, upregulation of Sqstm1 might indicate increased autophagic demand and altered vesicle trafficking, which warrants further investigation. No statistical significant changes in M6pr, Atg12, or Nfe2l2 were detected, suggesting transcriptional stability in enzyme trafficking, core autophagy, and oxidative stress pathways. Regionally, atria showed higher Lysotracker signal than ventricles, consistent with known enrichment of acidic vesicular stores in atrial physiology. These findings highlight the utility of IVIS imaging of Lysotracker-labelled hearts, providing rapid whole-organ assessment of acidic vesicle distribution, albeit with limited depth resolution. Complementary techniques such as RT-qPCR analysis is essential to interpret IVIS findings, enabling insight into underlying molecular changes in lysosomal and autophagy pathways during cardiac ageing.

physiology↗

bioRxiv2020

The vesicular release probability sets the strength of individual Schaffer collateral synapses

Information processing in the brain is controlled by quantal release of neurotransmitters, a tightly regulated process. From ultrastructural analysis, it is known that presynaptic boutons along single axons differ in the number of vesicles docked at the active zone. It is not clear whether the probability of these vesicles to get released (pves) is homogenous or also varies between individual boutons. Here, we optically measure evoked transmitter release at individual Schaffer collateral synapses at different calcium concentrations, using the genetically encoded glutamate sensor iGluSnFR. Fitting a binomial model to measured response amplitude distributions allowed us to extract the quantal parameters N, pves, and q. We find that Schaffer collateral boutons typically release single vesicles under low pves conditions and switch to multivesicular release in high calcium saline. Analyzing the variability of quantal parameters, we conclude that the vesicular release probability rather than the number of readily releasable vesicles or their transmitter content determines the potency of individual boutons.

neuroscience↗

bioRxiv2019

GenEPi: Piezo1-based fluorescent reporter for visualizing mechanical stimuli with high spatiotemporal resolution

Mechanosensing is a ubiquitous process to translate external mechanical stimuli into biological responses during development, homeostasis, and disease. However, non-invasive investigation of cellular mechanosensing in complex and intact live tissue remains challenging. Here, we developed GenEPi, a genetically-encoded fluorescent intensiometric reporter for mechanical stimuli based on Piezo1, an essential mechanosensitive ion channel found in vertebrates. We show that GenEPi has high specificity and spatiotemporal resolution for Piezo1-dependent mechanical stimuli, exemplified by resolving repetitive mechanical stimuli of spontaneously contracting cardiomyocytes within microtissues, in a non-invasive manner.

cell biology↗

bioRxiv2019

Kinetic mechanisms of fast glutamate sensing by fluorescent protein probes

Protein-based fluorescent glutamate sensors have the potential for real-time monitoring of synaptic and cellular glutamate concentration changes, however even the fastest currently available sensors response times of 2-3 ms are too slow for accurate reporting of the post-synaptic AMPA receptor function in physiological conditions. We have developed probes based on the bacterial periplasmic glutamate/aspartate binding protein with either an endogenously fluorescent protein or a synthetic fluorophore as the indicator of glutamate binding: affinity variants of iGluSnFR termed iGluh, iGlum and iGlul covering a range of Kd-s (5.8 M, 2.1 mM and 50 mM, respectively) and a novel fluorescently labelled indicator, Fl-GluBP with a Kd of 9.7 M are presented. The fluorescence response kinetics of all the probes are consistent with two-step mechanisms involving ligand binding and rate limiting isomerisation, however the contribution in each step to the total fluorescence enhancement and kinetic paths to the final state are diverse. In contrast to the previously characterised ultrafast indicators iGluu and iGluf, for which fluorescence enhancement occurred only in the rate limiting isomerisation step, the sensors described here all have biphasic binding kinetics with a significant fraction of the fluorescence increase evoked by glutamate binding which, in the case of iGluh and Fl-GluBP, occurs with a diffusion limited rate constant. The above genetically encoded and chemically labelled fluorescent glutamate sensor variants demonstrate how single amino acid changes around the binding site introduce structural heterogeneity affecting the kinetic mechanism of interactions with glutamate. Through their broad affinity range and mechanistic variety, the probes contribute to a novel toolkit for monitoring processes of glutamate neurotransmission and cellular homeostasis.\n\nSTATEMENT OF SIGNIFICANCEGlutamate is a major excitatory neurotransmitter, important in synaptic plasticity e.g. memory formation. Although predicted to clear rapidly from the synaptic cleft following presynaptic release, optical monitoring of glutamate neurotransmission has only become possible with the advent of fluorescent, protein-based indicators. Understanding their biophysical properties is important for quantification of the observed processes. Here we report the biophysical characterisation of a number of glutamate indicator variants based on the bacterial periplasmic glutamate/aspartate binding protein, revealing the subtle differences in their kinetic pathway caused by structural alteration of the glutamate binding protein by point mutations. Diffusion limited glutamate binding indicated by a novel chemically labelled probe hints at the mechanism that underlies the rapid response of the AMPA receptor.

biophysics↗

bioRxiv2018

Glutamate uptake and release at single adult corticostriatal synapses of normal and Huntington mice

Changes in the balance between glutamate (Glu) release and uptake may stimulate synaptic reorganization and even synapse loss. In the case of neurodegeneration, a mismatch between astroglial Glu uptake and presynaptic Glu release could be detected if both parameters were assessed independently and at a single synapse level. This has now become possible due to a new imaging assay with the genetically encoded ultrafast Glu sensor iGluu. We report findings from individual corticostriatal synapses in acute slices prepared from mice aged >1 year. Contrasting patterns of short-term plasticity and a size criterion identified 2 classes of terminals, presumably corresponding to the previously defined IT and PT synapses. The latter exhibited a higher degree of frequency potentiation/residual Glu accumulation and were selected for our first iGluu single synapse study in Q175 mice, a model of Huntingtons disease (HD). It was found that in HD the time constant of perisynaptic [Glu] decay (TauD, as indicator of uptake) and the peak iGluu amplitude (as indicator of release) were prolonged and reduced, respectively. Treatment of WT preparations with the astrocytic Glu uptake blocker TFB-TBOA (100 nM) mimicked the TauD changes in homozygotes (HOM). Considering the largest TauD values encountered in WT, about 40% of PT terminals tested in Q175 heterozygotes (HET) can be classified as dysfunctional. Moreover, HD but not WT synapses exhibited a positive correlation between TauD and the peak amplitude of iGluu. Finally, EAAT2 immunoreactivity was reduced next to corticostriatal terminals. Thus, astrocytic Glu transport remains a promising target for therapeutic intervention. SIGNIFICANCE STATEMENTAlterations in astrocytic Glu uptake can play a role in synaptic plasticity and neurodegeneration. Until now, sensitivity of synaptic responses to pharmacological transport block and the resulting activation of NMDA receptors were regarded as reliable evidence for a mismatch between synaptic uptake and release. But the latter parameters are interdependent. Using a new genetically encoded sensor to monitor [Glu] at individual corticostriatal synapses we can now quantify the time constant of perisynaptic [Glu] decay (as indicator of uptake) and the maximal [Glu] elevation next to the active zone (as indicator of Glu release). The results provide a positive answer to the hitherto unresolved question whether neurodegeneration (e.g. Huntingtons disease) associates with a glutamate uptake deficit at tripartite excitatory synapses.

neuroscience↗

bioRxiv2018

A centrosome calcium signal is essential for mammalian cell mitosis

To generate a complex multicellular organism like a human requires enormous expansion in cell numbers and this is achieved predominantly through mitosis. Defects in mitosis can lead to premature ageing and cancer so understanding how it is regulated has important implications for human disease. Early data from plant and invertebrate model systems indicated that calcium (Ca2+) could influence mitosis. Here we explore this key question in the cell biology of mammalian cells by targeting high affinity genetically encoded Ca2+ sensors to mitosis specific subcellular locations. We reveal a prolonged yet spatially restricted Ca2+ signal at the centrosomes of mitotic cells using an actin-targeted Ca2+ sensor. Local depletion of Ca2+ at centrosomes using flash-photolysis of the caged Ca2+ chelator diazo-2 arrests mitosis and we provide evidence that this signal emanates from the endoplasmic reticulum. In summary, we characterize a centrosomal Ca2+ signal as a functionally essential input into mitosis. This extends our understanding of the complex regulatory network controlling cell division and pinpoints Ca2+ as an important controller of this fundamental process.

cell biology↗

bioRxiv2018

Lysosome exocytosis is required for mitosis

Mitosis, the accurate segregation of duplicated genetic material into what will become two new daughter cells, is accompanied by extensive membrane remodelling and membrane trafficking activities. Early in mitosis, adherent cells partially detach from the substratum, round up and their surface area decreases. This likely results from an endocytic uptake of plasma membrane material. As cells enter cytokinesis they re-adhere, flatten and exhibit an associated increase in surface area. The identity of the membrane donor for this phase of mitosis remains unclear. Here we show by biochemical and imaging approaches that lysosomes undergo exocytosis at telophase and that this requires the activity of phosphatidylinositol 4-kinase-III{beta}. Inhibition of lysosome exocytosis resulted in mitotic failure in a significant proportion of cells suggesting that this facet of lysosome physiology is essential and represents a new regulatory mechanism in mitosis.

cell biology↗

bioRxiv2017

Ultrafast glutamate sensors resolve synaptic short-term plasticity

Glutamatergic synapses display a rich repertoire of plasticity mechanisms on many different time scales, involving dynamic changes in the efficacy of transmitter release as well as changes in the number and function of postsynaptic glutamate receptors. The genetically encoded glutamate sensor iGluSnFR enables visualization of glutamate release from presynaptic terminals at frequencies up to [~]10 Hz. However, to resolve glutamate dynamics during high frequency bursts, faster indicators are required. Here we report the development of fast (iGluf) and ultrafast (iGluu) variants with comparable brightness, but increased Kd for glutamate (137 M and 600 M, respectively). Compared to iGluSnFR, iGluu has a 6-fold faster dissociation rate in vitro and 5-fold faster kinetics in synapses. Fitting a three-state model to kinetic data, we identify the large conformational change after glutamate binding as the rate-limiting step. In rat hippocampal slice culture stimulated at 100 Hz, we find that iGluu is sufficiently fast to resolve individual glutamate release events, revealing that glutamate is rapidly cleared from the synaptic cleft. Depression of iGluu responses during 100 Hz trains correlates with depression of postsynaptic EPSPs, indicating that depression during high frequency stimulation is purely presynaptic in origin. At individual boutons, the recovery from depression could be predicted from the amount of glutamate released on the second pulse (paired pulse facilitation/depression), demonstrating differential frequency-dependent filtering of spike trains at Schaffer collateral boutons.\n\nSignificance StatementExcitatory synapses convert presynaptic action potentials into chemical signals that are sensed by postsynaptic glutamate receptors. To eavesdrop on synaptic transmission, genetically encoded fluorescent sensors for glutamate have been developed. However, even the best available sensors lag behind the very fast glutamate dynamics in the synaptic cleft. Here we report the development of an ultrafast genetically encoded glutamate sensor, iGluu, which allowed us to image glutamate clearance and synaptic depression during 100 Hz spike trains. We found that only boutons showing paired-pulse facilitation were able to rapidly recover from depression. Thus, presynaptic boutons act as frequency-specific filters to transmit select features of the spike train to specific postsynaptic cells.

neuroscience↗