The prion protein regulates glutamate-mediated Ca2+ entry and mitochondrial Ca2+ accumulation in neurons

The cellular prion protein (PrPC) whose conformational misfolding leads to the production of deadly prions, has a still-unclarified cellular function despite decades of intensive research. Following our recent finding that PrPC limits Ca2+ entry via store-operated Ca2+ channels in neurons, we investigated whether the protein could also control the activity of ionotropic glutamate receptors (iGluRs). To this end, we compared local Ca2+ movements in primary cerebellar granule neurons and cortical neurons transduced with genetically encoded Ca2+ probes and expressing, or not expressing, PrPC. Our investigation demonstrated that PrPC downregulates Ca2+ entry through each specific agonist-stimulated iGluR and after stimulation by glutamate. We found that, although PrP-knockout (KO) mitochondria were displaced from the plasma membrane, glutamate addition resulted in a higher mitochondrial Ca2+ uptake in PrP-KO neurons than in their PrPC-expressing counterpart. This was because the increased Ca2+ entry through iGluRs in PrP-KO neurons led to a parallel increase in Ca2+-induced Ca2+ release via ryanodine receptor channels. These data thus suggest that PrPC takes part in the cell apparatus controlling Ca2+ homeostasis, and that PrPC is involved in protecting neurons from toxic Ca2+ overloads

Intracellular Ca2+ signalling: unexpected new roles for the usual suspect

Cytosolic Ca2+ signals are organized in complex spatial and temporal patterns that underlie their unique ability to regulate multiple cellular functions. Changes in intracellular Ca2+ concentration ([Ca2+](i)) are finely tuned by the concerted interaction of membrane receptors and ion channels that introduce Ca2+ into the cytosol, Ca2+-dependent sensors and effectors that translate the elevation in [Ca2+](i) into a biological output, and Ca2+-clearing mechanisms that return the [Ca2+](i) to pre-stimulation levels and prevent cytotoxic Ca2+ overload. The assortment of the Ca2+ handling machinery varies among different cell types to generate intracellular Ca2+ signals that are selectively tailored to subserve specific functions. The advent of novel high-speed, 2D and 3D time-lapse imaging techniques, single-wavelength and genetic Ca2+ indicators, as well as the development of novel genetic engineering tools to manipulate single cells and whole animals, has shed novel light on the regulation of cellular activity by the Ca2+ handling machinery. A symposium organized within the framework of the 72nd Annual Meeting of the Italian Society of Physiology, held in Bari on 14-16th September 2022, has recently addressed many of the unexpected mechanisms whereby intracellular Ca2+ signalling regulates cellular fate in healthy and disease states. Herein, we present a report of this symposium, in which the following emerging topics were discussed: 1) Regulation of water reabsorption in the kidney by lysosomal Ca2+ release through Transient Receptor Potential Mucolipin 1 (TRPML1); 2) Endoplasmic reticulum-to-mitochondria Ca2+ transfer in Alzheimer's disease-related astroglial dysfunction; 3) The non-canonical role of TRP Melastatin 8 (TRPM8) as a Rap1A inhibitor in the definition of some cancer hallmarks; and 4) Non-genetic optical stimulation of Ca2+ signals in the cardiovascular system

The Cytokine Nicotinamide Phosphoribosyltransferase (eNAMPT; PBEF; Visfatin) Acts as a Natural Antagonist of C-C Chemokine Receptor Type 5 (CCR5)

(1) Background: Extracellular nicotinamide phosphoribosyltrasferase (eNAMPT) is released by various cell types with pro-tumoral and pro-inflammatory properties. In cancer, eNAMPT regulates tumor growth through the activation of intracellular pathways, suggesting that it acts through a putative receptor, although its nature is still elusive. It has been shown, using surface plasma resonance, that eNAMPT binds to the C-C chemokine receptor type 5 (CCR5), although the physiological meaning of this finding is unknown. The aim of the present work was to characterize the pharmacodynamics of eNAMPT on CCR5. (2) Methods: HeLa CCR5-overexpressing stable cell line and B16 melanoma cells were used. We focused on some phenotypic effects of CCR5 activation, such as calcium release and migration, to evaluate eNAMPT actions on this receptor. (3) Results: eNAMPT did not induce ERK activation or cytosolic Ca2+-rises alone. Furthermore, eNAMPT prevents CCR5 internalization mediated by Rantes. eNAMPT pretreatment inhibits CCR5-mediated PKC activation and Rantes-dependent calcium signaling. The effect of eNAMPT on CCR5 was specific, as the responses to ATP and carbachol were unaffected. This was strengthened by the observation that eNAMPT inhibited Rantes-induced Ca2+-rises and Rantes-induced migration in a melanoma cell line. (4) Conclusions: Our work shows that eNAMPT binds to CCR5 and acts as a natural antagonist of this receptor

Point singularity array with metasurfaces

Phase singularities are loci of darkness surrounded by monochromatic light in a scalar field, with applications in optical trapping, super-resolution imaging, and structured light-matter interactions. Although 1D singular structures, such as optical vortices, are the most common due to their robust topological properties, uncommon 0D (point) and 2D (sheet) singular structures can be generated by wavefront-shaping devices such as metasurfaces. Here, using the design flexibility of metasurfaces, we deterministically position ten identical point singularities in a cylindrically symmetric field generated by a single illumination source. The phasefront is inverse-designed using phase gradient maximization with an automatically-differentiable propagator. This process produces tight longitudinal intensity confinement. The singularity array is experimentally realized with a 1 mm diameter TiO2 metasurface. One possible application is blue-detuned neutral atom trap arrays, for which this light field would enforce 3D confinement and a potential depth around 0.22 mK per watt of incident trapping laser power. Metasurface-enabled point singularity engineering may significantly simplify and miniaturize the optical architecture required to produce super-resolution microscopes and dark traps

Efimov physics beyond three particles

Efimov physics originally refers to a system of three particles. Here we review recent theoretical progress seeking for manifestations of Efimov physics in systems composed of more than three particles. Clusters of more than three bosons are tied to each Efimov trimer, but no independent Efimov physics exists there beyond three bosons. The case of a few heavy fermions interacting with a lighter atom is also considered, where the mass ratio of the constituent particles plays a significant role. Following Efimov's study of the (2+1) system, the (3+1) system was shown to have its own critical mass ratio to become Efimovian. We show that the (4+1) system becomes Efimovian at a mass ratio which is smaller than its sub-systems thresholds, giving a pure five-body Efimov effect. The (5+1) and (6+1) systems are also discussed, and we show the absence of 6- and 7-body Efimov physics there

Liver fibrosis and accelerated immune dysfunction (immunosenescence) among HIV-infected Russians with heavy alcohol consumption - an observational cross-sectional study

The multifactorial mechanisms driving negative health outcomes among risky drinkers with HIV may include immunosenescence. Immunosenescence, aging of the immune system, may be accentuated in HIV and leads to poor outcomes. The liver regulates innate immunity and adaptive immune tolerance. HIV-infected people have high prevalence of liver-related comorbidities. We hypothesize that advanced liver fibrosis/cirrhosis is associated with alterations in T-cell subsets consistent with immunosenescence. ART-naïve people with HIV with a recent history of heavy drinking were recruited into a clinical trial of zinc supplementation. Flow cytometry was used to characterize T-cell subsets. The two primary dependent variables were CD8+ and CD4+ T-cells expressing CD28-CD57+ (senescent cell phenotype). Secondary dependent variables were CD8+ and CD4+ T-cells expressing CD45RO + CD45RA- (memory phenotype), CD45RO-CD45RA+ (naïve phenotype), and the naïve phenotype to memory phenotype T-cell ratio (lower ratios associated with immunosenescence). Advanced liver fibrosis/cirrhosis was defined as FIB-4 > 3.25, APRI≥1.5, or Fibroscan measurement ≥10.5 kPa. Analyses were conducted using multiple linear regression adjusted for potential confounders. Mean age was 34 years; 25% female; 88% hepatitis C. Those with advanced liver fibrosis/cirrhosis (N = 25) had higher HIV-1 RNA and more hepatitis C. Advanced liver fibrosis/cirrhosis was not significantly associated with primary or secondary outcomes in adjusted analyses. Advanced liver fibrosis/cirrhosis was not significantly associated with these senescent T-cell phenotypes in this exploratory study of recent drinkers with HIV. Future studies should assess whether liver fibrosis among those with HIV viral suppression and more advanced, longstanding liver disease is associated with changes in these and other potentially senescent T-cell subsets

Salivary DNA methylation panel to diagnose HPV-positive and HPV-negative head and neck cancers

Background Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumours with a typical 5 year survival rate of INK4a, TIMP3, PCQAP/MED15) will allow us to diagnose HNSCC patients from a normal healthy control group as well as to discriminate between Human Papillomavirus (HPV)-positive and HPV-negative patients. Methods Methylation-specific PCR (MSP) was used to determine the methylation levels of RASSF1α, p16INK4a, TIMP3 and PCQAP/MED15 in DNA isolated from saliva. Statistical analysis was carried out using non-parametric Mann-Whitney’s U-test for individually methylated genes. A logistic regression analysis was carried out to determine the assay sensitivity when combing the five genes. Further, a five-fold cross-validation with a bootstrap procedure was carried out to determine how well the panel will perform in a real clinical scenario. Results Salivary DNA methylation levels were not affected by age. Salivary DNA methylation levels for RASSF1α, p16INK4a, TIMP3 and PCQAP/MED15 were higher in HPV-negative HNSCC patients (n = 88) compared with a normal healthy control group (n = 122) (sensitivity of 71 % and specificity of 80 %). Conversely, DNA methylation levels for these genes were lower in HPV-positive HNSCC patients (n = 45) compared with a normal healthy control group (sensitivity of 80 % and specificity of 74 %), consistent with the proposed aetiology of HPV-positive HNSCCs. Conclusions Salivary DNA tumour-suppressor methylation gene panel has the potential to detect early-stage tumours in HPV-negative HNSCC patients. HPV infection was found to deregulate the methylation levels in HPV-positive HNSCC patients. Large-scale double-blinded clinical trials are crucial before this panel can potentially be integrated into a clinical setting

Phenotypic and functional characterization of endothelial progenitor cells isolated from peripheral blood of renal cell carcinoma patients

Endothelial progenitor cells (EPCs) are mobilized from either bone marrow or arterial walls to restore blood perfusion to ischemic organs and establish the vascular network within growing tumors [1]. The Ca2+ machinery plays a key role in EPC activation and might serve a molecular target for novel therapies of highly angiogenic tumors, such as renal cell carcinoma (RCC) [1]. The Ca2+ toolkit is remodelled in EPCs isolated from RCC patients (RCC-EPCs) as respect to healthy donors [2]. The present study was undertaken to evaluate for the first time the functional properties of EPCs isolated from tumor patients by focusing on RCC-EPCs. We extended our analysis at microscopic level by monitoring the sub-cellular structure of RCC-EPCs relative to their Ca2+ signalling fingerprint. Our results showed a striking functional and ultrastructural difference between RCC-EPCs and their normal counterparts, which might be the basis for designing novel, more specific anti-angiogenic treatments

REV1 Inhibition Enhances Radioresistance and Autophagy

SIMPLE SUMMARY: Cancer resistance to therapy continues to be the biggest challenge in treating patients. Targeting the mutagenic translesion synthesis (TLS) polymerase REV1 was previously shown to sensitize cancer cells to chemotherapy. In this study, we tested the ability of REV1 inhibitors to radiation therapy and observed a lack of radiosensitization. In addition, we observed REV1 inhibition to trigger an autophagy stress response. Because reduction of REV1 triggered autophagy and failed to radiosensitize cells, we hypothesize REV1 expression dynamics might link cancer cell response to radiation treatment through the potential induction of autophagy. ABSTRACT: Cancer therapy resistance is a persistent clinical challenge. Recently, inhibition of the mutagenic translesion synthesis (TLS) protein REV1 was shown to enhance tumor cell response to chemotherapy by triggering senescence hallmarks. These observations suggest REV1’s important role in determining cancer cell response to chemotherapy. Whether REV1 inhibition would similarly sensitize cancer cells to radiation treatment is unknown. This study reports a lack of radiosensitization in response to REV1 inhibition by small molecule inhibitors in ionizing radiation-exposed cancer cells. Instead, REV1 inhibition unexpectedly triggers autophagy, which is a known biomarker of radioresistance. We report a possible role of the REV1 TLS protein in determining cancer treatment outcomes depending upon the type of DNA damage inflicted. Furthermore, we discover that REV1 inhibition directly triggers autophagy, an uncharacterized REV1 phenotype, with a significant bearing on cancer treatment regimens

Treatment of patients with posttraumatic deformities of the thoracolumbar spine using Schwab 5 osteotomy through combined and posterior approaches

Objective. To analyze the results of surgical treatment of patients with rigid posttraumatic deformities of the thoracolumbar spine operated on using Schwab 5 osteotomy through combined and posterior approaches. Material and Methods. A retrospective cohort study was conducted. Study group included 60 patients (m/f = 25/35). Median age was 48 (26–58) years, median time since injury was 11 (9–14) months, and minimum follow-up period was 2 years. A two-stage intervention with resection of the vertebral body, correction of the deformity, and placement of an interbody implant through the anterior approach followed by final fixation through the posterior approach (VCR a+p group) was performed in 29 cases. Vertebral body resection through the posterior approach with correction of the deformity, installation of an interbody implant, and rigid transpedicular fixation (VCRp group) was performed in 31 cases. In all patients, the magnitude of correction was assessed, as well as the following parameters: frontal balance, sagittal balance, thoracic kyphosis, lumbar lordosis, pelvic incidence, sacral slope and pelvic tilt. The results of treatment were evaluated in dynamics by the level of pain syndrome (VAS) and quality of life (ODI), as well as based on the analysis of postoperative complications. Results. The groups were comparable in terms of gender, age, magnitude of the kyphotic component of the deformity, level of pain syndrome and degree of initial neurological deficit (p > 0.05). Correction of the deformity kyphotic component was significantly better in patients in the VCR p group compared to those in the VCRa+p group (p = 0.036). Both groups showed a significant decrease in the level of pain syndrome 3 months after surgery. However, further follow-up showed a tendency for back pain to increase on average one year after surgery in the VCRa+p group. A total of 67 complications were revealed in 40 (66.7 %) patients. Herewith, in the early postoperative period there were 55 complications in 31 patients, and in the late period there were 12 complications in 9 patients. Analysis of early complications showed a higher incidence of anemia (p = 0.002) and liquorrhea (p = 0.017) in the VCRp group compared to those in the VCRa+p group. The incidence of long-term complications did not differ significantly between groups (p = 0.866). An increase in back pain in the long-term period was observed in 12 (41.4 %) patients of the VCR a+p group and in 4 (12.9 %) patients of the VCR p group. Analysis of risk factors for this condition showed a tendency for back pain to increase in the long-term period in patients with residual local deformity against the background of low pelvic index values. Conclusion. Correction of the kyphotic component of deformity was significantly better in patients of the VCR p group, which was accompanied by greater surgical trauma and incidence of early postoperative complications. The tendency of patients’ quality of life to deteriorate in the long-term follow-up period seems to be related to the occurrence of pain syndrome in the lumbar spine in patients with residual kyphotic deformity against the background of initially low compensatory capabilities