Direct modulation of TRPC ion channels by Gα proteins.

GPCR-Gi protein pathways are involved in the regulation of vagus muscarinic pathway under physiological conditions and are closely associated with the regulation of internal visceral organs. The muscarinic receptor-operated cationic channel is important in GPCR-Gi protein signal transduction as it decreases heart rate and increases GI rhythm frequency. In the SA node of the heart, acetylcholine binds to the M2 receptor and the released Gßγ activates GIRK (I(K,ACh)) channel, inducing a negative chronotropic action. In gastric smooth muscle, there are two muscarinic acetylcholine receptor (mAChR) subtypes, M2 and M3. M2 receptor activates the muscarinic receptor-operated nonselective cationic current (mIcat, NSCC(ACh)) and induces positive chronotropic effect. Meanwhile, M3 receptor induces hydrolysis of PIP2 and releases DAG and IP3. This IP3 increases intracellular Ca2+ and then leads to contraction of GI smooth muscles. The activation of mIcat is inhibited by anti-Gi/o protein antibodies in GI smooth muscle, indicating the involvement of Gαi/o protein in the activation of mIcat. TRPC4 channel is a molecular candidate for mIcat and can be directly activated by constitutively active Gαi QL proteins. TRPC4 and TRPC5 belong to the same subfamily and both are activated by Gi/o proteins. Initial studies suggested that the binding sites for G protein exist at the rib helix or the CIRB domain of TRPC4/5 channels. However, recent cryo-EM structure showed that IYY58-60 amino acids at ARD of TRPC5 binds with Gi3 protein. Considering the expression of TRPC4/5 in the brain, the direct G protein activation on TRPC4/5 is important in terms of neurophysiology. TRPC4/5 channels are also suggested as a coincidence detector for Gi and Gq pathway as Gq pathway increases intracellular Ca2+ and the increased Ca2+ facilitates the activation of TRPC4/5 channels. More complicated situation would occur when GIRK, KCNQ2/3 (IM) and TRPC4/5 channels are co-activated by stimulation of muscarinic receptors at the acetylcholine-releasing nerve terminals. This review highlights the effects of GPCR-Gi protein pathway, including dopamine, µ-opioid, serotonin, glutamate, GABA, on various oragns, and it emphasizes the importance of considering TRPC4/5 channels as crucial players in the field of neuroscience.

Change in Maxillary Sinus Mucosal Thickness in Patients with Preoperative Maxillary Sinus Mucosal Thickening as Assessed by Otolaryngologists: A Retrospective Study.

Background and Objectives: Maxillary sinus pathologic conditions may increase the risk of complications during posterior maxillary sinus augmentation surgery. The purpose of this study was to evaluate the changes in participants with preoperative maxillary sinus mucosal thickening and to assess this factor as a preoperative risk indicator for sinusitis after maxillary dental implantation. Materials and Methods: We compared the preoperative and postoperative maxillary sinus mucosal thickness (MSMT), the distance between the maxillary sinus ostium and sinus floor (MOD), and the MSMT/MOD ratio. The participants were divided into three groups (sinus augmentation, bone grafting, and no grafting). Results: The mean preoperative MSMT was 4.3 ± 2.0 mm, and the mean MSMT/MOD ratio was 0.13 ± 0.05. No postoperative sinusitis was observed in these patients, including cases caused by anatomical variations. The mean postoperative MSMT was 4.5 ± 2.3 mm, and the mean postoperative MSMT/MOD ratio was 0.15 ± 0.06. There was no statistically significant difference between the groups at each time point (p > 0.05). Conclusions: The study found no significant change in MSMT at post-treatment evaluation, even when considering different subgroups. It underscores the importance of preoperative maxillary sinus radiographic assessments and collaboration between dentists and otolaryngologists for better outcomes in patients with preoperative maxillary sinus mucosal thickening.

Molecular architecture of the Gαi-bound TRPC5 ion channel.

G-protein coupled receptors (GPCRs) and ion channels serve as key molecular switches through which extracellular stimuli are transformed into intracellular effects, and it has long been postulated that ion channels are direct effector molecules of the alpha subunit of G-proteins (Gα). However, no complete structural evidence supporting the direct interaction between Gα and ion channels is available. Here, we present the cryo-electron microscopy structures of the human transient receptor potential canonical 5 (TRPC5)-Gαi3 complexes with a 4:4 stoichiometry in lipid nanodiscs. Remarkably, Gαi3 binds to the ankyrin repeat edge of TRPC5 ~ 50 Å away from the cell membrane. Electrophysiological analysis shows that Gαi3 increases the sensitivity of TRPC5 to phosphatidylinositol 4,5-bisphosphate (PIP2), thereby rendering TRPC5 more easily opened in the cell membrane, where the concentration of PIP2 is physiologically regulated. Our results demonstrate that ion channels are one of the direct effector molecules of Gα proteins triggered by GPCR activation-providing a structural framework for unraveling the crosstalk between two major classes of transmembrane proteins: GPCRs and ion channels.

Low concentrations of tricyclic antidepressants stimulate TRPC4 channel activity by acting as an opioid receptor ligand.

Traditionally prescribed for mood disorders, tricyclic antidepressants (TCAs) have shown promising therapeutic effects on chronic neuralgia and irritable bowel syndrome. However, the mechanism by which these atypical effects manifest is unclear. Among the proposed mechanisms is the well-known pain-related inhibitory G-protein coupled receptor, namely the opioid receptor (OR). Here, we confirmed that TCA indeed stimulates OR and regulates the gating of TRPC4, a downstream signaling of the Gi-pathway. In an ELISA to quantify the amount of intracellular cAMP, a downstream product of OR/Gi-pathway, treatment with amitriptyline (AMI) showed a decrease in [cAMP]i similar to that of the µOR agonist. Next, we explored the binding site of TCA by modeling the previously revealed ligand-bound structure of µOR. A conserved aspartate residue of ORs was predicted to participate in salt bridge interaction with the amine group of TCAs, and in aspartate-to-arginine mutation, AMI did not decrease the FRET-based binding efficiency between the ORs and Gαi2. As an alternative way to monitor the downstream signaling of Gi-pathway, we evaluated the functional activity of TRPC4 channel, as it is well known to be activated by Gαi. TCAs increased the TRPC4 current through ORs, and TCA-evoked TRPC4 activation was abolished by an inhibitor of Gαi2 or its dominant-negative mutant. As expected, TCA-evoked activation of TRPC4 was not observed in the aspartate mutants of OR. Taken together, OR could be proclaimed as a promising target among numerous binding partners of TCA, and TCA-evoked TRPC4 activation may help to explain the nonopioid analgesic effect of TCA.NEW & NOTEWORTHY Endogenous opioid systems modulate pain perception, but concerns about opioid-related substance misuse limit their use. This study has raised TRPC4 channel as a candidate target for alternative analgesics, tricyclic antidepressants (TCAs). TCAs have been shown to bind to and activate opioid receptors (ORs), leading to downstream signaling pathways involving TRPC4. The functional selectivity and biased agonism of TCA towards TRPC4 in dependence on OR may provide a better understanding of its efficacy or side effects.

Vitamin E Enhances Cell Viability and the Osteogenic Differentiation of Cell Spheroids Made of Gingiva-Derived Stem Cells.

Background and Objectives: Vitamin E is reported to expedite new bone formation in animal models, and this has led to a decrease in the time needed for treatment. In this study, human gingiva-derived stem cell-derived spheroids were examined to determine the effects of vitamin E on cell survival, osteogenic differentiation, and mineralization. Materials and Methods: Human gingiva-derived stem cells were used to create spheroids, which were then cultivated with vitamin E at doses of 0, 0.1, 1, 10, and 100 ng/mL. The morphological examination and the qualitative and quantitative vitality of the cells were assessed. Alizarin Red S staining and alkaline phosphatase activity assays were performed on days 7 and 14 to evaluate the osteogenic differentiation. The expression levels of RUNX2 and COL1A1 were assessed using a real-time polymerase chain reaction. Results: The addition of vitamin E did not appear to alter the spheroid's shape at the measured quantities without altering the diameter. During the culture time, the majority of the cells in the spheroids fluoresced green. Regardless of concentration, there were substantial increases in cell viability in the vitamin E-loaded groups on day 7 (p < 0.05). On day 14, the Alizarin Red S staining was statistically higher in the 1 ng/mL group compared to the unloaded control (p < 0.05). The addition of vitamin E to the culture enhanced the mRNA expression levels of RUNX2, OCN, and COL1A1 based on the real-time polymerase chain reaction data. Conclusions: We draw the conclusion that vitamin E may be used to promote the osteogenic differentiation of stem cell spheroids in light of these data.

Negative self-regulation of transient receptor potential canonical 4 by the specific interaction with phospholipase C-δ1.

Transient receptor potential canonical (TRPC) channels are non-selective calcium-permeable cation channels. It is suggested that TRPC4ß is regulated by phospholipase C (PLC) signaling and is especially maintained by phosphatidylinositol 4,5-bisphosphate (PIP2). In this study, we present the regulation mechanism of the TRPC4 channel with PIP2 hydrolysis which is mediated by a channel-bound PLCδ1 but not by the GqPCR signaling pathway. Our electrophysiological recordings demonstrate that the Ca2+ via an open TRPC4 channel activates PLCδ1 in the physiological range, and it causes the decrease of current amplitude. The existence of PLCδ1 accelerated PIP2 depletion when the channel was activated by an agonist. Interestingly, PLCδ1 mutants which have lost the ability to regulate PIP2 level failed to reduce the TRPC4 current amplitude. Our results demonstrate that TRPC4 self-regulates its activity by allowing Ca2+ ions into the cell and promoting the PIP2 hydrolyzing activity of PLCδ1.

Study on the Relevance of Metabolic Syndrome and Incidence of Gastric Cancer in Korea.

(1) Background: This study aimed to determine the relevance between stages of metabolic syndrome (MS) progression and the incidence of gastric cancer utilizing a big data cohort for the national health checkup. (2) Methods: There were 7,785,098 study subjects, and three stages of metabolic syndrome were categorized using the health checkup results from 2009. Incidence of gastric cancer was traced and observed from the date of the health insurance benefit claim in 2009 until 31 December, 2016, and Cox hazard-proportional regression was performed to determine the risk of gastric cancer incidence based on the stage of progression for metabolic syndrome. (3) Results: Hazard ratio (HR) incidence rate for the MS group was 2.31 times higher than the normal group (95% CI 2.22⁻2.40) after adjustment (Model 4). The HR incidence rate of gastric cancer for the pre-MS group was 1.08 times higher (95% CI 1.04⁻1.12) than the normal group, while the HR incidence rate of gastric cancer for the MS group was 1.26 times higher (95% CI 1.2⁻1.32). (4) Conclusions: Causal relevance observed in this study between metabolic syndrome and incidence of gastric cancer was high. Promotion and education for active responses in the general population and establishment of appropriate metabolic syndrome management systems to prevent gastric cancer are needed.

Association of Education and Smoking Status on Risk of Diabetes Mellitus: A Population-Based Nationwide Cross-Sectional Study.

Background: Exposure to smoke, including environmental tobacco smoke (ETS), is a well-known risk factor for diabetes. Low socioeconomic status, especially lack of education, is also a risk factor for diabetes. Therefore, we assessed the association of demographic, socioeconomic, clinical, and behavior risk factor-related variables and smoking status, including ETS exposure, with the prevalence of diabetes. Methods: Data were from the 2007-2013 Korea National Health and Nutritional Evaluation Survey (KNHANES). Multivariable logistic regression examined associations between various lifestyle and health factors and the prevalence of diabetes while controlling for potential confounding variables. Subgroup analysis was performed according to smoking status to determine factors associated with diabetes. Results: Of 19,303 individuals analyzed, 1325 (11.4%) had diabetes. Greater average age, male sex, lower educational level, unemployment, and coexisting health problems were significantly associated with diabetes. Individuals with only elementary, middle, or high school level education had significantly greater odds ratios (p < 0.05) compared to college graduates; smokers and nonsmokers exposed to ETS had significantly greater OR (p < 0.05) than nonsmokers unexposed to ETS. Subgroup analysis of diabetics according to smoking status revealed significant associations (p < 0.05) for diabetic nonsmokers exposed to ETS with female sex, single status, elementary level education, urban residence, National Health Insurance (NHI), hypertension, a lack of alcohol intake, and a lack of moderate physical activity. For diabetic smokers, there were significant associations (p < 0.05) with elementary education, urban residence, a lack of moderate physical activity, a lack of alcohol intake, and NHI. Conclusions: The results suggested that smoking status, as well as ETS exposure, was associated with a higher prevalence of diabetes, especially in populations with less education. Thus, we should direct efforts for controlling diabetes toward individuals with lower levels of education and those who are smokers and nonsmokers exposed to ETS.

Relationship between BMI, body image, and smoking in Korean women as determined by urine cotinine: results of a nationwide survey.

BACKGROUND: This study examined the influence of body mass index (BMI), subjective body perception (SBP), and the differences between BMI and SBP influence on smoking among women. METHODS: This study used the Korea National Health and Nutrition Examination Survey IV-2, 3 2008-2009. A urinary cotinine test was administered to 5485 women at least 19 years of age. Individuals whose cotinine level was at least 50 ng/mL were categorized as smokers. A multiple logistic regression analysis was performed to estimate the extent to which body-related variables affect female smoking. RESULTS: Women with a lower BMI who perceived themselves to be normal or very fat were 2.09 times (1.14-3.83) more likely to smoke than women with a normal BMI and SBP. Women who were never married with a low BMI and thin SBP were 3.11 times (1.47-6.55) more likely to smoke than women with a normal BMI and SBP. Married women with a high BMI who considered themselves very fat were 0.63 times (0.43-0.94) less likely to smoke than women with a normal BMI and SBP. In contrast, divorced and widowed women with a low or normal BMI who considered themselves very fat were 26.1 times (1.35-507.3) more likely to smoke. CONCLUSIONS: Discrepancies between the objective physical condition (BMI) and the subjective body image (SBP) influence the female smoking rate. To reduce the number of female smokers, public education on the association between smoking behavior and weight issues is needed, especially among women with low BMI and distorted weight perception.

Effect of mixing on spontaneous struvite precipitation from semiconductor wastewater.

The objective of this study was to investigate on the effect of mixing intensity (G) and mixing duration (t(d)) on struvite precipitation in the chemical mechanical polishing (CMP) wastewater generated from the semiconductor manufacturing process. Batch-scale experiments revealed that struvite crystallization was affected by both G and t(d). The mixing effect was to enhance the mass transfer of solute to the crystals in the process, resulting in the improvement of struvite crystallization and growth. By forming struvite, removal efficiencies of N and P increased logarithmic with the multiple values of G and t(d), i.e., Gt(d). Insufficient mixing energy with the Gt(d) value less than 10(5) caused an increase in the formation potential of unexpected precipitate unlike to pure struvite, causing a decrease in removal efficiencies of N and P in the process. At the Gt(d) value over 10(6), struvite precipitation was not restricted by fluoride, of which high level inherently contained in the CMP wastewater. The study results can be taken into consideration in the design and operation of the struvite precipitation process for both nutrient (N and P) removal and recovery.

DNA damage-induced ubiquitylation of RFC2 subunit of replication factor C complex.

Many proteins involved in DNA replication and repair undergo post-translational modifications such as phosphorylation and ubiquitylation. Proliferating cell nuclear antigen (PCNA; a homotrimeric protein that encircles double-stranded DNA to function as a sliding clamp for DNA polymerases) is monoubiquitylated by the RAD6-RAD18 complex and further polyubiquitylated by the RAD5-MMS2-UBC13 complex in response to various DNA-damaging agents. PCNA mono- and polyubiquitylation activate an error-prone translesion synthesis pathway and an error-free pathway of damage avoidance, respectively. Here we show that replication factor C (RFC; a heteropentameric protein complex that loads PCNA onto DNA) was also ubiquitylated in a RAD18-dependent manner in cells treated with alkylating agents or H(2)O(2). A mutant form of RFC2 with a D228A substitution (corresponding to a yeast Rfc4 mutation that reduces an interaction with replication protein A (RPA), a single-stranded DNA-binding protein) was heavily ubiquitylated in cells even in the absence of DNA damage. Furthermore RFC2 was ubiquitylated by the RAD6-RAD18 complex in vitro, and its modification was inhibited in the presence of RPA. The inhibitory effect of RPA on RFC2 ubiquitylation was relatively specific because RAD6-RAD18-mediated ubiquitylation of PCNA was RPA-insensitive. Our findings suggest that RPA plays a regulatory role in DNA damage responses via repression of RFC2 ubiquitylation in human cells.

Development of a convenient in vitro fertilization method using interspecific hybrids between Oryzias latipes and Oryzias curvinotus.

Medaka is a small Asian freshwater teleost and has been an excellent model for fertilization studies for more than 50 years. Therefore, experimental procedures for in vitro fertilization (IVF) and cryopreservation of sperm are well established. In contrast, since the eggs or early embryos can not be cryopreserved, many females are killed to obtain unfertilized eggs for IVF. Recent progress in genomics is establishing medaka as a new model animal in functional genomics, and numerous mutant and transgenic strains have been established and stored as frozen sperm. Accumulated preserved resources require a simple and reliable recovery method for IVF. In this paper, we describe a method for obtaining a large number of unfertilized eggs without killing females, using sterile interspecific hybrids between Oryzias latipes and O. curvinotus. However, there is no report about the normality of offspring that were obtained by IVF using unfertilized eggs spawned in mating with the sterile hybrid male. In this paper, we have confirmed the reliability of the method regarding the influences on the next generation and also assessed conditions for efficient collection of unfertilized eggs. The method would be useful not only for fertilization studies but also for keeping transgenics and mutants, including a mutant library for a reverse genetic approach.

Enhancing struvite precipitation potential for ammonia nitrogen removal in municipal landfill leachate.

This study was conducted to improve struvite precipitation for NH4-N removal purpose in landfill leachate. For this purpose, we evaluated the effect of the feeding sequence of precipitating reagents (magnesium, orthophosphate, and buffering reagent) on NH4-N removal by forming struvite deposits. Struvite precipitation effectively proceeded by an addition of excess magnesium and phosphate sources followed by an addition of the buffering reagent, in which condition the local formation of inappropriate deposits or the contamination of the desired struvite was minimized. We also tested the effect of struvite addition as the seeding materials on NH4-N removal. Seed addition would increase the potential for the struvite crystal growth, which enhanced NH4-N removal performance in landfill leachate treatment.