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

The inflammasome in chronic complications of diabetes and related metabolic disorders

Diabetes mellitus (DM) ranks seventh as a cause of death worldwide. Chronic complications, including cardiovascular, renal, and eye disease, as well as DM-associated non-alcoholic fatty liver disease (NAFLD) account for most of the morbidity and premature mortality in DM. Despite continuous improvements in the management of late complications of DM, significant gaps remain. Therefore, searching for additional strategies to prevent these serious DM-related conditions is of the utmost importance. DM is characterized by a state of low-grade chronic inflammation, which is critical in the progression of complications. Recent clinical trials indicate that targeting the prototypic pro-inflammatory cytokine interleukin-1β (IL-1 β) improves the outcomes of cardiovascular disease, which is the first cause of death in DM patients. Together with IL-18, IL-1β is processed and secreted by the inflammasomes, a class of multiprotein complexes that coordinate inflammatory responses. Several DM-related metabolic factors, including reactive oxygen species, glyco/lipoxidation end products, and cholesterol crystals, have been involved in the pathogenesis of diabetic kidney disease, and diabetic retinopathy, and in the promoting effect of DM on the onset and progression of atherosclerosis and NAFLD. These metabolic factors are also well-established danger signals capable of regulating inflammasome activity. In addition to presenting the current state of knowledge, this review discusses how the mechanistic understanding of inflammasome regulation by metabolic danger signals may hopefully lead to novel therapeutic strategies targeting inflammation for a more effective treatment of diabetic complications

Diabetes and pancreatic cancer-a dangerous liaison relying on carbonyl stress

Both type 2 (T2DM) and type 1 (T1DM) diabetes mellitus confer an increased risk of pancreatic cancer in humans. The magnitude and temporal trajectory of the risk conferred by the two forms of diabetes are similar, suggesting a common mechanism. Carbonyl stress is a hallmark of hyperglycemia and dyslipidemia, which accompanies T2DM, prediabetes, and obesity. Accumulating evidence demonstrates that diabetes promotes pancreatic ductal adenocarcinoma (PDAC) in experimental models of T2DM, a finding recently confirmed in a T1DM model. The carbonyl stress markers advanced glycation end-products (AGEs), the levels of which are increased in diabetes, were shown to markedly accelerate tumor development in a mouse model of Kras-driven PDAC. Consistently, inhibition of AGE formation by trapping their carbonyl precursors (i.e., reactive carbonyl species, RCS) prevented the PDAC-promoting effect of diabetes. Considering the growing attention on carbonyl stress in the onset and progression of several cancers, including breast, lung and colorectal cancer, this review discusses the mechanisms by which glucose and lipid imbalances induce a status of carbonyl stress, the oncogenic pathways activated by AGEs and their precursors RCS, and the potential use of carbonyl-scavenging agents and AGE inhibitors in PDAC prevention and treatment, particularly in high-risk diabetic individuals

Diabetic complications and oxidative stress: A 20‐year voyage back in time and back to the future

Twenty years have passed since Brownlee and colleagues proposed a single unifying mechanism for diabetic complications, introducing a turning point in this field of research. For the first time, reactive oxygen species (ROS) were identified as the causal link between hyperglycemia and four seemingly independent pathways that are involved in the pathogenesis of diabetes-associated vascular disease. Before and after this milestone in diabetes research, hundreds of articles describe a role for ROS, but the failure of clinical trials to demonstrate antioxidant benefits and some recent experimental studies showing that ROS are dispensable for the pathogenesis of diabetic complications call for time to reflect. This twenty‐year journey focuses on the most relevant literature regarding the main sources of ROS generation in diabetes and their role in the pathogenesis of cell dysfunction and diabetic complications. To identify future research directions, this review discusses the evidence in favor and against oxidative stress as an initial event in the cellular biochemical abnormalities induced by hyperglycemia. It also explores possible alternative mechanisms, including carbonyl stress and the Warburg effect, linking glucose and lipid excess, mitochondrial dysfunction, and the activation of alternative pathways of glucose metabolism leading to vascular cell injury and inflammation

DaDoEval @ EVALITA 2020: Same-genre and cross-genre dating of historical documents

In this paper we introduce the DaDoEval shared task at EVALITA 2020, aimed at automatically assigning temporal information to documents written in Italian. The evaluation exercise comprises three levels of temporal granularity, from coarse-grained to year-based, and includes two types of test sets, either having the same genre of the training set, or a different one. More specifically, DaDoEval deals with the corpus of Alcide De Gasperi's documents, providing both public documents and letters as test sets. Two systems participated in the competition, achieving results always above the baseline in all subtasks. As expected, coarse-grained classification into five periods is rather easy to perform automatically, while the year-based one is still an unsolved problem also due to the lack of enough training data for some years. Results showed also that, although De Gasperi's letters in our test set were written in standard Italian and in a style which was not too colloquial, cross-genre classification yields remarkably lower results than the same-genre setting

Expression pattern of Stomatin-domain proteins in the peripheral olfactory system

Recent data show that Stomatin-like protein 3 (STOML3), a member of the stomatin-domain family, is expressed in the olfactory sensory neurons (OSNs) where it modulates both spontaneous and evoked action potential firing. The protein family is constituted by other 4 members (besides STOML3): STOM, STOML1, STOML2 and podocin. Interestingly, STOML3 with STOM and STOML1 are expressed in other peripheral sensory neurons: dorsal root ganglia. In here, they functionally interact and modulate the activity of the mechanosensitive Piezo channels and members of the ASIC family. Therefore, we investigated whether STOM and STOML1 are expressed together with STOML3 in the OSNs and whether they could interact. We found that all three are indeed expressed in ONSs, although STOML1 at very low level. STOM and STOML3 share a similar expression pattern and STOML3 is necessary for STOM to properly localize to OSN cilia. In addition, we extended our investigation to podocin and STOML2, and while the former is not expressed in the olfactory system, the latter showed a peculiar expression pattern in multiple cell types. In summary, we provided a first complete description of stomatin-domain protein family in the olfactory system, highlighting the precise compartmentalization, possible interactions and, finally, their functional implications

Interactions between permeation and gating in the TMEM16B/anoctamin2 calcium-activated chloride channel

At least two members of the TMEM16/anoctamin family, TMEM16A (also known as anoctamin1) and TMEM16B (also known as anoctamin2), encode Ca2+-activated Cl- channels (CaCCs), which are found in various cell types and mediate numerous physiological functions. Here, we used whole-cell and excised inside-out patch-clamp to investigate the relationship between anion permeation and gating, two processes typically viewed as independent, in TMEM16B expressed in HEK 293T cells. The permeability ratio sequence determined by substituting Cl- with other anions (PX/PCl) was SCN- > I- > NO3- > Br- > Cl- > F- > gluconate. When external Cl- was substituted with other anions, TMEM16B activation and deactivation kinetics at 0.5 μM Ca2+ were modified according to the sequence of permeability ratios, with anions more permeant than Cl- slowing both activation and deactivation and anions less permeant than Cl- accelerating them. Moreover, replacement of external Cl- with gluconate, or sucrose, shifted the voltage dependence of steady-state activation (G-V relation) to more positive potentials, whereas substitution of extracellular or intracellular Cl- with SCN- shifted G-V to more negative potentials. Dose-response relationships for Ca2+ in the presence of different extracellular anions indicated that the apparent affinity for Ca2+ at +100 mV increased with increasing permeability ratio. The apparent affinity for Ca2+ in the presence of intracellular SCN- also increased compared with that in Cl-. Our results provide the first evidence that TMEM16B gating is modulated by permeant anions and provide the basis for future studies aimed at identifying the molecular determinants of TMEM16B ion selectivity and gating. © 2014 Betto et al

Functional expression of TMEM16A in taste bud cells

Key points: Taste transduction occurs in taste buds in the tongue epithelium. The Ca2+-activated Cl– channels TMEM16A and TMEM16B play relevant physiological roles in several sensory systems. Here, we report that TMEM16A, but not TMEM16B, is expressed in the apical part of taste buds. Large Ca2+-activated Cl− currents blocked by Ani-9, a selective inhibitor of TMEM16A, are measured in type I taste cells but not in type II or III taste cells. ATP indirectly activates Ca2+-activated Cl– currents in type I cells through TMEM16A channels. These results indicate that TMEM16A is functional in type I taste cells and contribute to understanding the largely unknown physiological roles of these cells. Abstract: The Ca2+-activated Cl– channels TMEM16A and TMEM16B have relevant roles in many physiological processes including neuronal excitability and regulation of Cl– homeostasis. Here, we examined the presence of Ca2+-activated Cl– channels in taste cells of mouse vallate papillae by using immunohistochemistry and electrophysiological recordings. By using immunohistochemistry we showed that only TMEM16A, and not TMEM16B, was expressed in taste bud cells where it largely co-localized with the inwardly rectifying K+ channel KNCJ1 in the apical part of type I cells. By using whole-cell patch-clamp recordings in isolated cells from taste buds, we measured an average current of −1083 pA at −100 mV in 1.5 μm Ca2+ and symmetrical Cl– in type I cells. Ion substitution experiments and blockage by Ani-9, a specific TMEM16A channel blocker, indicated that Ca2+ activated anionic currents through TMEM16A channels. We did not detect any Ca2+-activated Cl– currents in type II or III taste cells. ATP is released by type II cells in response to various tastants and reaches type I cells where it is hydrolysed by ecto-ATPases. Type I cells also express P2Y purinergic receptors and stimulation of type I cells with extracellular ATP produced large Ca2+-activated Cl− currents blocked by Ani-9, indicating a possible role of TMEM16A in ATP-mediated signalling. These results provide a definitive demonstration that TMEM16A-mediated currents are functional in type I taste cells and provide a foundation for future studies investigating physiological roles for these often-neglected taste cells

TrkB signaling directs the incorporation of newly generated periglomerular cells in the adult olfactory bulb.

In the adult rodent brain, the olfactory bulb (OB) is continuously supplied with new neurons which survival critically depends on their successful integration into pre-existing networks. Yet, the extracellular signals that determine the selection which neurons will be ultimately incorporated into these circuits are largely unknown. Here, we show that immature neurons express the catalytic form of the brain-derived neurotrophic factor receptor TrkB [full-length TrkB (TrkB-FL)] only after their arrival in the OB, at the time when inte-gration commences. To unravel the role of TrkB signaling in newborn neurons, we conditionally ablated TrkB-FL in mice via Cre expression in adult neural stem and progenitor cells. TrkB-deficient neurons displayed a marked impairment in dendritic arborization and spine growth. By selectively manipulating the signaling pathways initiated by TrkB in vivo, we identified the transducers Shc/PI3K to be required for dendritic growth, whereas the activation of phospholipase C-was found to be responsible for spine formation. Further-more, long-term genetic fate mapping revealed that TrkB deletion severely compromised the survival of new dopaminergic neurons, leading to a substantial reduction in the overall number of adult-generated periglomerular cells (PGCs), but not of granule cells (GCs). Surprisingly, this loss of dopaminergic PGCs was mirrored by a corresponding increase in the number of calretinin PGCs, suggesting that distinct subsets of adult-born PGCs may respond differentially to common extracellular signals. Thus, our results identify TrkB signaling to be essential for balancing the incorporation of defined classes of adult-born PGCs and not GCs, reflecting their different mode of integration in the OB. \ua9 2013 the authors

Never Retreat, Never Retract: Argumentation Analysis for Political Speeches

In this work, we apply argumentation mining techniques, in particular relation prediction, to study political speeches in monological form, where there is no direct interaction between opponents. We argue that this kind of technique can effectively support researchers in history, social and political sciences, which must deal with an increasing amount of data in digital form and need ways to automatically extract and analyse argumentation patterns. We test and discuss our approach based on the analysis of documents issued by R. Nixon and J. F. Kennedy during 1960 presidential campaign. We rely on a supervised classifier to predict argument relations (i.e., support and attack), obtaining an accuracy of 0.72 on a dataset of 1,462 argument pairs. The application of argument mining to such data allows not only to highlight the main points of agreement and disagreement between the candidates' arguments over the campaign issues such as Cuba, disarmament and health-care, but also an in-depth argumentative analysis of the respective viewpoints on these topics