Function and Regulation of the Calcium-Activated Chloride Channel Anoctamin 1 (TMEM16A).

Various human tissues express the calcium-activated chloride channel Anoctamin 1 (ANO1), also known as TMEM16A. ANO1 allows the passive chloride flux that controls different physiological functions ranging from muscle contraction, fluid and hormone secretion, gastrointestinal motility, and electrical excitability. Overexpression of ANO1 is associated with pathological conditions such as hypertension and cancer. The molecular cloning of ANO1 has led to a surge in structural, functional, and physiological studies of the channel in several tissues. ANO1 is a homodimer channel harboring two pores - one in each monomer - that work independently. Each pore is activated by voltage-dependent binding of two intracellular calcium ions to a high-affinity-binding site. In addition, the binding of phosphatidylinositol 4,5-bisphosphate to sites scattered throughout the cytosolic side of the protein aids the calcium activation process. Furthermore, many pharmacological studies have established ANO1 as a target of promising compounds that could treat several illnesses. This chapter describes our current understanding of the physiological roles of ANO1 and its regulation under physiological conditions as well as new pharmacological compounds with potential therapeutic applications.

Nutrition education on obesity and diabetes to medical students.

It is important for medical students to understand the relationship between nutrition, obesity, and diabetes to educate their patients in the future. However, medical training does not always include nutritional education. An experiential learning project was incorporated into the medical school curriculum as an effort to implement nutrition in the physiology course. First-year medical students (n = 140) received lectures on the regulation of blood glucose levels and their relationship to carbohydrates with different glycemic indexes (GI), obesity, and diabetes. Lectures were followed by a laboratory exercise where students calculated their body mass index (BMI), percentage body fat, and percentage muscle using a Bioelectrical Impedance Commercial Scale. While 63% of students had normal BMI, 31% were overweight or obese and 5% were underweight. A subgroup of 54 students tested different types of breakfasts with varying GI and provided blood samples at 0, 30, 60, 90, and 120 min. Their glucose responses were plotted based on the breakfast GI. Pre- and posttests were conducted to assess the teaching intervention where the Wilcoxon signed ranks test indicated that posttest ranks were significantly higher than pretest ranks (Z = -6.6, P < 0.001), suggesting the intervention was beneficial to students.

Phosphatidylinositol 4,5-bisphosphate, cholesterol, and fatty acids modulate the calcium-activated chloride channel TMEM16A (ANO1).

The TMEM16A-mediated Ca2+-activated Cl- current drives several important physiological functions. Membrane lipids regulate ion channels and transporters but their influence on members of the TMEM16 family is poorly understood. Here we have studied the regulation of TMEM16A by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), cholesterol, and fatty acids using patch clamp, biochemistry and fluorescence microscopy. We found that depletion of membrane PI(4,5)P2 causes a decline in TMEM16A current that is independent of cytoskeleton, but is partially prevented by removing intracellular Ca2+. On the other hand, supplying PI(4,5)P2 to inside-out patches attenuated channel rundown and/or partially rescued activity after channel rundown. Also, depletion (with methyl-ß-cyclodextrin M-ßCD) or restoration (with M-ßCD+cholesterol) of membrane cholesterol slows down the current decay observed after reduction of PI(4,5)P2. Neither depletion nor restoration of cholesterol change PI(4,5)P2 content. However, M-ßCD alone transiently increases TMEM16A activity and dampens rundown whereas M-ßCD+cholesterol increases channel rundown. Thus, PI(4,5)P2 is required for TMEM16A function while cholesterol directly and indirectly via a PI(4,5)P2-independent mechanism regulate channel function. Stearic, arachidonic, oleic, docosahexaenoic, and eicosapentaenoic fatty acids as well as methyl stearate inhibit TMEM16A in a dose- and voltage-dependent manner. Phosphatidylserine, a phospholipid whose hydrocarbon tails contain stearic and oleic acids also inhibits TMEM16A. Finally, we show that TMEM16A remains in the plasma membrane after treatment with M-ßCD, M-ßCD+cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction.

Analysis of transcription factors, microRNAs and cytokines involved in T lymphocyte differentiation in patients with tuberculosis after directly observed treatment short-course.

Tuberculosis (Tb) is an infectious disease in which the immune system plays an important role. MicroRNAs are involved in the development and maintenance of CD4 + T lymphocyte subpopulations. miR-326 regulates the differentiation to Th17 cells and miR-29 correlates with the Th1 response. The aim of this study was to determine the role of microRNAs, Transcription Factors, and cytokines in Th differentiation before and after the directly observed treatment short-course (DOTS). Peripheral blood mononuclear cells and serum from Tb patients were collected at times 0 (before therapy), 2 (after the intensive phase), and 6 months (after the holding phase). The cells were cultivated in presence or absence of ESAT-6 (10 µg/ml) and CFP-10 (10 µg/ml). Transcription Factor and microRNA expressions were analyzed by qPCR and cytokine production in both serum and culture supernatant using ELISA. A decrease in Th1 response with a diminishing in the relative expression of TBET and miR-29a at 2 and 6 months after the anti-Tb therapy (p < 0.01) were found. The miR-326 levels decreased after the intensive phase of the DOTS scheme. However, subdivision of the Tb patients according to gender, showed increased levels of miR-29a and miR-155 in females after the intensive phase of the therapeutic treatment when compared to time 0 and similar increased levels of miR-326 at time 6 versus time 0. In contrast, we observed a decrease in miR-326 levels in males at 6 months when compared to before therapy (time 0). In addition, high production of IL-17 in the culture supernatant was found at 2 and 6 months (p < 0.05) while in serum IL-17 was decreased. A positive correlation between IL-17 and RORC2 at time 6 was detected (p = 0.0202, r = 0.7880). In conclusion, these data suggest a reduction in Th1 and an induction of Th17 response after the anti-Tb therapy.

Abnormal expression and function of Dectin-1 receptor in type 2 diabetes mellitus patients with poor glycemic control (HbA1c>8%).

Dectin-1 is a key innate receptor involved in various cellular responses and may have a direct role in chronic inflammatory conditions such as type 2 diabetes mellitus. The aim of this work was to evaluate the expression and function of Dectin-1 in peripheral blood mononuclear cells from T2D patients. Dectin-1 expression was analyzed by flow cytometry and RT-PCR in monocytes and lymphocyte subpopulations from T2D patients (n=34) and healthy subjects (n=29). Functional assays were used to assess cytokine synthesis, ROS levels and oxidative stress ratio. We found increased expression (MFI) of Dectin-1 in monocytes from T2D patients. Significantly higher Dectin-1 expression was also detected in CD4(+) T, CD8(+) T, B cells and NK cells from T2D patients compared to controls. In contrast, monocytes from T2D patients with poor glycemic control (HbA1c>8%) showed a diminished percentage of Dectin-1(+)/TLR2(+) cells. Negative correlations between the percent of Dectin-1(+)/TLR2(+) cells and fasting plasma glucose levels (FPG) and HbA1c levels were found. A significant reduction in basal levels of IL-10 was observed in patients with poor glycemic control (HbA1c>8%) compared to patients with appropriate glycemic control (HbA1c≤6.5%) and healthy controls, an effect that was not observed in monocytes stimulated with zymosan. Higher ROS levels in zymosan-stimulated cells from patients with poor glycemic control positively correlated with FPG levels, and the oxidative stress ratio was higher in T2D cells compared with controls. Our data indicate that Dectin-1 may be involved in the abnormal immune responses that are observed in patients with T2D.