Anion and cation permeabilities of the mouse TMEM16F/ANO6 calcium-activated channel

TMEM16F/ANO6 is widely expressed in different tissues where it plays important physiological roles, such as the regulation of blood coagulation, bone mineralization and apoptosis. TMEM16F plays two different biological roles in cells: it acts both as scramblase and as ion channel. Both of these functions are activated by the increase of intracellular Ca2+ concentration. In physiological conditions the distribution of cellular membrane lipids is polarized, meaning that the composition of the outer leaflet is different than that of the inner one. When the intracellular Ca2+ increases, the scramblases are activated. Scramblases are integral membrane proteins responsible for the translocation of phospholipids between the two leaflets of a lipid bilayer upon their electrochemical gradient, abolishing the asymmetry of the bilayer. This is a key process in many physiological and pathological contests because the exposure of particular lipids at the cell surface induces several transduction signalling cascades. Many studies investigated in details the molecular mechanisms of TMEM16F lipid scrambling and identified a specific domain of the protein which is essential for the lipid transport. The mechanisms of TMEM16F ion channel activity are less clear and, in addition, several studies reported conflicting results about the ionic selectivity of the TMEM16F-mediated current. These data were partially obtained using different methodologies and analysis making difficult to reconcile the discrepancies. In particular, studies reporting that TMEM16F is more permeable for anions were performed only using the whole-cell recordings, a technique that does not allow a direct comparison of the results with or without intracellular calcium. In contrast, results showing a higher permeability for cations derive from recordings from excised patches in the inside-out configuration. Here, we decided to investigate the ionic selectivity of TMEM16F using both configurations of the patch clamp technique, whole-cell and inside-out excised patch, using the same solutions, in order to directly compare the results. Moreover, we investigated the properties of the Q559K mutant that has been reported to alter the ionic selectivity of TMEM16F in inside-out patches. First of all, our study revealed a characteristic which is shared between the configurations. Indeed, TMEM16F heterologous expression in HEK 293T cells generates a large outward rectifying Ca2+-activated current both in whole-cell and inside-out configuration which is due to the voltage-dependent gating of the channel. On the other hand, we found consistent differences in terms of channel time-dependent activation and selectivity. In fact, although in inside-out experiments the Ca2+-dependent activation is fast and the TMEM16F-mediated current is activated in few ms, in whole-cell recordings a full activation requires up to 4 minutes. Similar results are obtained also with Q559K mutant. In inside-out patches the Ca2+ concentration for half-maximal current activation (K1/2) is 42 \ub5M at +60 mV, while the Hill coefficient is >2. Q559K mutant shows a significant reduction of Ca2+ sensitivity with a K1/2 almost 5-folds higher and a reduction of Hill coefficient to 1.4, indicating a possible alteration of the gating mechanism. To determine the channel selectivity, we decided to calculate the relative permeability between Na+ and Cl\uaf (PNa/PCl) using the dilution method both in whole-cell and inside-out configuration. We found that TMEM16F-mediated current is highly non-selective but there are differences depending on the configuration of the recordings. Indeed, in whole-cell both TMEM16F wild type and Q559K mutant have a PNa/PCl around 0.5 indicating a slight preference for Cl\uaf permeation. In contrast, in inside-out experiments the Q559K mutant retains a higher permeability for Cl\uaf, while TMEM16F wild type channel shows a higher permeability for Na+ with a PNa/PCl reaching 3.6. These results have two major implications: First, the discrepancies found between the recordings in whole-cell and inside-out, could suggest the presence of a cellular mechanism factor which controls TMEM16F selectivity. When the patch is excised this cellular mechanism and its controlling activity are lost. Second, the residue in position 559 could be responsible to control the selectivity among cations and anions when this cellular mechanism is lost

Anion and cation permeability of the mouse tmem16f calcium-activated channel

TMEM16F is involved in several physiological processes, such as blood coagulation, bone development and virus infections. This protein acts both as a Ca2+-dependent phospholipid scram-blase and a Ca2+-activated ion channel but several studies have reported conflicting results about the ion selectivity of the TMEM16F-mediated current. Here, we have performed a detailed side-by-side comparison of the ion selectivity of TMEM16F using the whole-cell and inside-out excised patch configurations to directly compare the results. In inside-out configuration, Ca2+-dependent activation was fast and the TMEM16F-mediated current was activated in a few milliseconds, while in whole-cell recordings full activation required several minutes. We determined the relative permeability between Na+ and Cl¯ (PNa /PCl ) using the dilution method in both configurations. The TMEM16F-mediated current was highly nonselective, but there were differences depending on the configuration of the recordings. In whole-cell recordings, PNa /PCl was approximately 0.5, indicating a slight preference for Cl¯ permeation. In contrast, in inside-out experiments the TMEM16F channel showed a higher permeability for Na+ with PNa /PCl reaching 3.7. Our results demonstrate that the time dependence of Ca2+ activation and the ion selectivity of TMEM16F depend on the recording configuration

Due visioni della responsabilità sociale dell'impresa, con una applicazione alla società benefit

Il lavoro analizza due diverse concezioni della CSR e tenta una applicazione all'istituto della societ\ue0 benefit.The paper analyzes two different conceptions of CSR and attempts an application to the benefit corporation

Functional evaluation in orthodontic surgical treatment: long-term stability and predictability

Background: The introduction of electromyographic and kinesiographic technology in orthodontics allows to obtain objective data regarding the functional aspects of the mandibular movements and the masticatory muscular activity. It is then important to be able to correlate the data obtained by instrumental activity with the clinical ones. The aim of this study consists to analyse the post ortodontic surgical stability through instrumental evaluation of the masticatory muscles and mandibular movements. Method: 30 patients undergo electromyographic and kinesiographic evaluations through all the surgical orthodontic iter and were than followed during other 4 years. JMP software was used to analyze and correlate the electromyographic and knesioographic data during treatment and during the follow up. Results: A linear correlations between some functional objective values collected from the examinations at the beginning and during therapy and the follow up one has been demonstrated. Conclusion: It is important to submit patients in surgical ortodontic treatment to instrumental analysis which can evidence how masticatory function and mandibular movements are performed. It is also important to highlight some functional values also from the beginning of the treatment because an alteration of such values can be related to a better or worse postsurgical rehabilitation

SECCA procedure for anal incontinence and antibiotic treatment: a case report of anal abscess

Background: Fecal Incontinence (FI) can seriously affect quality of life. The treatment of fecal incontinence starts conservatively but in case of failure, different surgical approaches may be proposed to the patient. Recently several not invasive approaches have been developed. One of these is the radiofrequency (RF) energy application to the internal anal sphincter. Case presentation: We report a rare case of an anal abscess related to a SECCA procedure in a 66-year-old woman affected by gas and FI for twenty years. Conclusions: The complications post-SECCA procedure reported in literature are generally not serious and often self-limited, such as bleeding or anal pain. This is a case of an anal abscess. We suggest that this finding could consolidate the importance of administering antibiotic therapy to patients and to run a full course of at least 6 days rather than a short-term (24 h) therapy, with the aim to minimize the incidence of this complication

Generation of donor-specific Tr1 cells to be used after kidney transplantation and definition of the timing of their in vivo infusion in the presence of immunosuppression

Background: Operational tolerance is an alternative to lifelong immunosuppression after transplantation. One strategy to achieve tolerance is by T regulatory cells. Safety and feasibility of a T regulatory type 1 (Tr1)-cell-based therapy to prevent graft versus host disease in patients with hematological malignancies has been already proven. We are now planning to perform a Tr1-cell-based therapy after kidney transplantation. Methods: Upon tailoring the lab-grade protocol to patients on dialysis, aims of the current work were to develop a clinical-grade compatible protocol to generate a donor-specific Tr1-cell-enriched medicinal product (named T10 cells) and to test the Tr1-cell sensitivity to standard immunosuppression in vivo to define the best timing of cell infusion. Results: We developed a medicinal product that was enriched in Tr1 cells, anergic to donor-cell stimulation, able to suppress proliferation upon donor- but not third-party stimulation in vitro, and stable upon cryopreservation. The protocol was reproducible upon up scaling to leukapheresis from patients on dialysis and was effective in yielding the expected number of T10 cells necessary for the planned infusions. The tolerogenic gene signature of circulating Tr1 cells was minimally compromised in kidney transplant recipients under standard immunosuppression and it eventually started to recover 36weeks post-transplantation, providing rationale for selecting the timings of the cell infusions. Conclusions: These data provide solid ground for proceeding with the trial and establish robust rationale for defining the correct timing of cell infusion during concomitant immunosuppressive treatment

Transvaginal ultrasonography with vs without bowel preparation in the diagnosis of rectosigmoid endometriosis: prospective study

Objectives: The primary aim of this study was to compare the diagnostic accuracy of transvaginal sonography (TVS) with vs without bowel preparation (BP) in detecting the presence of rectosigmoid endometriosis. Secondary objectives were to compare the diagnostic accuracy of the two techniques in estimating infiltration of the submucosa, length of the largest rectosigmoid nodules, distance of the nodules from the anal verge and presence of multifocal disease. Methods: This was a prospective study of patients with symptoms of pelvic pain for more than 6 months and/or suspicion of endometriosis referred to our institution between October 2016 and April 2018. Participants underwent a first TVS without BP followed by TVS with BP within a time interval of 1 week to 3 months. The examinations were performed independently and blindly by two sonographers. Only patients who underwent laparoscopy within the 6 months following the second ultrasound examination were included. Ultrasound results using the two techniques were compared with surgical and histological findings. Results: Of the 262 patients included in the study, 118 had rectosigmoid endometriosis confirmed at surgery. There was no significant difference in accuracy between TVS with and that without BP in diagnosing the presence of rectosigmoid endometriosis (93.5% vs 92.3%; P = 0.453). No significant difference was observed in accuracy between TVS with and that without BP in diagnosing submucosal infiltration (88.8% vs 84.6%; P = 0.238) and multifocal disease (97.2% vs 95.2%; P = 0.727) in patients diagnosed sonographically with rectosigmoid endometriosis. The accuracy of TVS with BP was similar to that of TVS without BP in estimating the maximum diameter of the largest nodule (P = 0.644) and the distance between the more caudal rectosigmoid nodule and the anal verge (P = 0.162). Conclusion: BP does not improve the diagnostic performance of TVS in detecting rectosigmoid endometriosis and in assessing characteristics of endometriotic nodules

Rapamycin and Interleukin-10 Treatment Induces T Regulatory Type 1 Cells That Mediate Antigen-Specific Transplantation Tolerance

Islet transplantation is a cure for type 1 diabetes, but its potential is limited by the need for constant immunosuppression. One solution to this problem is the induction of transplantation tolerance mediated by T regulatory cells. T regulatory type 1 (Tr1) cells are characterized by their production of high levels of interleukin (IL)-10, which is crucial for their differentiation and suppressive function. We investigated the effects of IL-10 administered in combination with rapamycin on the induction of Tr1 cells that could mediate a state of tolerance in diabetic mice after pancreatic islet transplantation. The efficacy of this treatment was compared with IL-10 alone and standard immunosuppression. Stable long-term tolerance that was not reversible by alloantigen rechallenge was achieved only in mice treated with rapamycin plus IL-10. Tr1 cells that produced high levels of IL-10 and suppressed T-cell proliferation were isolated from splenocytes of rapamycin plus IL-10–treated mice after treatment withdrawal. In rapamycin plus IL-10–treated mice, endogenous IL-10 mediated an active state of tolerance, as was observed when the blockade of IL-10 activity rapidly induced graft rejection >100 days after transplantation. CD4+ T-cells from rapamycin plus IL-10–treated mice transferred antigen-specific tolerance in mice that received new transplants. Thus rapamycin plus IL-10 not only prevented allograft rejection but also induced Tr1 cells that mediated stable antigen-specific, long-term tolerance in vivo

A matrix factorization framework for jointly analyzing multiple nonnegative data

Nonnegative matrix factorization based methods provide one of the simplest and most effective approaches to text mining. However, their applicability is mainly limited to analyzing a single data source. In this paper, we propose a novel joint matrix factorization framework which can jointly analyze multiple data sources by exploiting their shared and individual structures. The proposed framework is flexible to handle any arbitrary sharing configurations encountered in real world data. We derive an efficient algorithm for learning the factorization and show that its convergence is theoretically guaranteed. We demonstrate the utility and effectiveness of the proposed framework in two real-world applications–improving social media retrieval using auxiliary sources and cross-social media retrieval. Representing each social media source using their textual tags, for both applications, we show that retrieval performance exceeds the existing state-of-the-art techniques. The proposed solution provides a generic framework and can be applicable to a wider context in data mining wherever one needs to exploit mutual and individual knowledge present across multiple data sources