Flow Cytometry Analysis of Tissue Factor Expression in Human Platelets.

A subset of circulating human platelets stores Tissue Factor (TF) intracellularly, the key activator of the blood coagulation cascade and thrombus formation. Upon platelet activation, TF is exposed on the cell membrane, where it binds to FVII, ultimately leading to thrombin generation. Considering that (1) levels of TF-positive platelets increase in various clinical settings, contributing to the patient's prothrombotic phenotype, and (2) different drugs can modulate platelet-associated TF expression, a standardized method for assessing TF-positive platelets is valuable, as its evaluation has been controversial in the past.

Sequential Moderate Hypofractionated Boost in Breast Cancer Patients: A Monoinstitutional Analysis of Late Side Effects.

Background/aim: In breast cancer (BC) patients who have received breast-conserving surgery, moderate hypofractionation is standard of care for whole-breast irradiation (HF-WBI). On the other hand, the fractionation schedule for the boost is less well defined. A previous prospective study of our group aimed at evaluating acute and late cutaneous and subcutaneous side effects related to a sequential hypofractionated boost (HB) in patients who had received HF-WBI.

Inactivation of hypocretin receptor-2 signaling in dopaminergic neurons induces hyperarousal and enhanced cognition but impaired inhibitory control.

Hypocretin/Orexin (HCRT/OX) and dopamine (DA) are both key effectors of salience processing, reward and stress-related behaviors and motivational states, yet their respective roles and interactions are poorly delineated. We inactivated HCRT-to-DA connectivity by genetic disruption of Hypocretin receptor-1 (Hcrtr1), Hypocretin receptor-2 (Hcrtr2), or both receptors (Hcrtr1&2) in DA neurons and analyzed the consequences on vigilance states, brain oscillations and cognitive performance in freely behaving mice. Unexpectedly, loss of Hcrtr2, but not Hcrtr1 or Hcrtr1&2, induced a dramatic increase in theta (7-11 Hz) electroencephalographic (EEG) activity in both wakefulness and rapid-eye-movement sleep (REMS).

Interplay of Ca and K signals in cell physiology and cancer.

The cytoplasmic Ca concentration and the activity of K channels on the plasma membrane regulate cellular processes ranging from mitosis to oriented migration. The interplay between Ca and K signals is intricate, and different cell types rely on peculiar cellular mechanisms. Derangement of these mechanisms accompanies the neoplastic progression.

Integrins regulate hERG1 dynamics by girdin-dependent Gαi3: signaling and modeling in cancer cells.

The hERG1 potassium channel is aberrantly over expressed in tumors and regulates the cancer cell response to integrin-dependent adhesion. We unravel a novel signaling pathway by which integrin engagement by the ECM protein fibronectin promotes hERG1 translocation to the plasma membrane and its association with β1 integrins, by activating girdin-dependent Gαi3 proteins and protein kinase B (Akt). By sequestering hERG1, β1 integrins make it avoid Rab5-mediated endocytosis, where unbound channels are degraded.

High throughput clone screening on overexpressed hERG1 and Kv1.3 potassium channels using ion channel reader (ICR) label free technology.

Pharmacological studies aimed at the development of newly synthesized drugs directed against ion channels (as well as genetic studies of ion channel mutations) involve the development and use of transfected cells. However, the identification of the best clone, in terms of transfection efficiency, is often a time consuming procedure when performed through traditional methods such as manual patch-clamp. On the other hand, the use of other faster techniques, such as for example the IF, are not informative on the effective biological functionality of the transfected ion channel(s).

Head-to-Head Comparison of Tissue Factor-Dependent Procoagulant Potential of Small and Large Extracellular Vesicles in Healthy Subjects and in Patients with SARS-CoV-2 Infection.

The relative contribution of small (sEVs) and large extracellular vesicles (lEVs) to the total plasma procoagulant potential is not yet well defined. Thus, we compared total and TF-sEVs and -lEVs isolated from healthy subjects and COVID-19 patients during the acute phase of the infection and after symptom remission in terms of (1) vesicle enumeration using nanoparticle tracking assay, imaging flow cytometry, and TF immunofluorescence localization in a single-vesicle analysis using microarrays; (2) cellular origin; and (3) TF-dependent Xa generation capacity, as well as assessing the contribution of the TF inhibitor, TFPI. In healthy subjects, the plasma concentration of CD9/CD63/CD81 sEVs was 30 times greater than that of calcein lEVs, and both were mainly released by platelets.

Cell Surface Platelet Tissue Factor Expression: Regulation by P2Y and Link to Residual Platelet Reactivity.

Background: ADP-induced platelet activation leads to cell surface expression of several proteins, including TF (tissue factor). The role of ADP receptors in platelet TF modulation is still unknown. We aimed to assess the (1) involvement of P2Y and P2Y receptors in ADP-induced TF exposure; (2) modulation of TF-platelets in anti-P2Y-treated patients with coronary artery disease.

Immunosuppressive effects of new thiophene-based K1.3 inhibitors.

Voltage-gated potassium channel K1.3 inhibitors have been shown to be effective in preventing T-cell proliferation and activation by affecting intracellular Ca homeostasis. Here, we present the structure-activity relationship, K1.

Nicotinic acetylcholine receptors and epilepsy.

Despite recent advances in understanding the causes of epilepsy, especially the genetic, comprehending the biological mechanisms that lead to the epileptic phenotype remains difficult. A paradigmatic case is constituted by the epilepsies caused by altered neuronal nicotinic acetylcholine receptors (nAChRs), which exert complex physiological functions in mature as well as developing brain. The ascending cholinergic projections exert potent control of forebrain excitability, and wide evidence implicates nAChR dysregulation as both cause and effect of epileptiform activity.

Head-to-head comparison of four COVID-19 vaccines on platelet activation, coagulation and inflammation. The TREASURE study.

Introduction: Studies exploring alterations in blood coagulation and platelet activation induced by COVID-19 vaccines are not concordant. We aimed to assess the impact of four COVID-19 vaccines on platelet activation, coagulation, and inflammation considering also the immunization dose and the history of SARS-CoV-2 infection.

Methods: TREASURE study enrolled 368 consecutive subjects (161 receiving viral vector vaccines -ChAdOx1-S/Vaxzevria or Janssen- and 207 receiving mRNA vaccines -Comirnaty/Pfizer-BioNTech or Spikevax/Moderna).

The Interaction between hERG1 and β1 Integrins Modulates hERG1 Current in Different Pathological Cell Models.

Ion channels are implicated in various diseases, including cancer, in which they modulate different aspects of cancer progression. In particular, potassium channels are often aberrantly expressed in cancers, a major example being provided by hERG1. The latter is generally complexed with β1 integrin in tumour cells, and such a molecular complex represents a new druggable hub.

Migraine in Patients Undergoing PFO Closure: Characterization of a Platelet-Associated Pathophysiological Mechanism: The LEARNER Study.

The association between migraine and patent foramen ovale (PFO) has been documented. We aimed to investigate platelet activation, prothrombotic phenotype, and oxidative stress status of migraineurs with PFO on 100 mg/day aspirin, before and 6 months after PFO closure. Data show that, before PFO closure, expression of the classical platelet activation markers is comparable in patients and aspirin-treated healthy subjects.

The β2 nicotinic subunit linked to sleep-related epilepsy differently affects fast-spiking and regular spiking somatostatin-expressing neurons in murine prefrontal cortex.

Mutant subunits of the neuronal nicotinic ACh receptor (nAChR) can cause Autosomal Dominant Sleep-related Hypermotor Epilepsy (ADSHE), characterized by frontal seizures during non-rapid eye movement (NREM) sleep. We studied the cellular bases of the pathogenesis in brain slices from mice conditionally expressing the ADSHE-linked β2 nAChR subunit. β2 mice displayed minor structural alterations, except for a ~10% decrease of prefrontal cortex thickness.

Dynamics and physiological meaning of complexes between ion channels and integrin receptors: the case of Kv11.1.

The cellular functions are regulated by a complex interplay of diffuse and local signals. Studying the latter is challenging, but experimental work in cell physiology has led to recognize that understanding a cell's dynamics requires a deep comprehension of local fluctuations of cytosolic regulators. Macromolecular complexes are major determinants of local signaling.

Alterations in platelets during SARS-CoV-2 infection.

Coronavirus disease 2019 (COVID-19) is a pandemic syndrome caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. SARS-CoV-2 infection induces a process of inflammation and thrombosis supported by an altered platelet activation state. This platelet activation is peculiar being characterized by the formation of platelet-leukocytes rather than platelet-platelet aggregates and by an increased procoagulant potential supported by elevated levels of TF positive platelets and microvesicles.

An early Sox2-dependent gene expression programme required for hippocampal dentate gyrus development.

The hippocampus is a brain area central for cognition. Mutations in the human SOX2 transcription factor cause neurodevelopmental defects, leading to intellectual disability and seizures, together with hippocampal dysplasia. We generated an allelic series of Sox2 conditional mutations in mouse, deleting Sox2 at different developmental stages.

Data describing the effects of potassium channels modulators on outward currents measured in human lymphoma cell lines.

In the present work, applying the whole-cell patch-clamp technique in voltage clamp mode, we have investigated the effects of different drugs, such as riluzole, Psora-4 and Tram-34, on the potassium currents in four human lymphoma cell lines. We focused on outward currents mediated by two potassium channels (Kv1.3 and KCa3.

Nicotinic Receptors in Sleep-Related Hypermotor Epilepsy: Pathophysiology and Pharmacology.

Sleep-related hypermotor epilepsy (SHE) is characterized by hyperkinetic focal seizures, mainly arising in the neocortex during non-rapid eye movements (NREM) sleep. The familial form is autosomal dominant SHE (ADSHE), which can be caused by mutations in genes encoding subunits of the neuronal nicotinic acetylcholine receptor (nAChR), Na-gated K channels, as well as non-channel signaling proteins, such as components of the gap activity toward rags 1 (GATOR1) macromolecular complex. The causative genes may have different roles in developing and mature brains.

Impaired flickering of the permeability transition pore causes SPG7 spastic paraplegia.

Background: Mutations of the mitochondrial protein paraplegin cause hereditary spastic paraplegia type 7 (SPG7), a so-far untreatable degenerative disease of the upper motoneuron with still undefined pathomechanism. The intermittent mitochondrial permeability transition pore (mPTP) opening, called flickering, is an essential process that operates to maintain mitochondrial homeostasis by reducing intra-matrix Ca and reactive oxygen species (ROS) concentration, and is critical for efficient synaptic function.

Methods: We use a fluorescence-based approach to measure mPTP flickering in living cells and biochemical and molecular biology techniques to dissect the pathogenic mechanism of SPG7.