Mapping the T cell epitopes of the M-type transmembrane phospholipase A2 receptor in primary membranous nephropathy.

The M-type phospholipase A2 receptor (PLA2R) is the major autoantigen of primary membranous nephropathy (MN). Despite many studies on B-cell epitopes recognized by antibodies, little is known about T-cell epitopes. Herein, we synthesized 123 linear peptides, each consisting of 15-22 amino acids with 8-12 amino acid overlaps, across ten domains of PLA2R. Their binding capacity to risk (DRB1∗1501, DRB1∗0301) and protective (DRB1∗0901, DRB1∗0701) HLA molecules was then assessed by flow cytometry. Proliferation of CD4+ T cells from patients with anti-PLA2R positive MN was analyzed after peptide stimulation. Cytokines produced by activated peripheral blood mononuclear cells were measured by cytometric bead arrays. We identified 17 PLA2R peptides that bound to both DRB1∗1501 and DRB1∗0301 molecules with high capacity. Some of these peptides showed decreased binding to heterozygous DRB1∗1501/0901 and DRB1∗0301/0701. Ten of the 17 peptides (CysR1, CysR10, CysR12, FnII-3, CTLD3-9, CTLD3-10, CTLD3-11, CTLD5-2-1, CTLD7-1 and CTLD7-2) induced significant proliferation of CD4+ T cells from patients with MN than cells from healthy individuals. Upon activation by these peptides, peripheral blood mononuclear cells from patients with MN produced higher levels of pro-inflammatory cytokines, predominantly IL-6, TNF-α, IL-10, IL-9 and IL-17. Thus, we mapped and identified ten peptides in the CysR, FnII, CTLD3, CTLD5, and CTLD7 domains of PLA2R as potential T-cell epitopes of MN. These findings are a first step towards developing peptide-specific immunotherapies.

Burden of visual impairment in mainland China: the Handan Eye Study and Beijing Eye Study.

PURPOSE: To determine the impact of visual impairment (VI) on health-related quality of life (HRQoL) and to compare the health burden of VI in different areas in mainland China. METHODS: A cohort of 6830 people from rural villages and a cohort of 3251 people from an urban city were included to receive comprehensive ophthalmologic examinations and complete the European Quality of Life-5 Dimensions 3 Levels (EQ-5D-3L) questionnaire. For urban and rural populations, a unified VI grouping standard was adopted: the eyes were classified into normal group, mild-moderate group, and severe group according to WHO standards, and then divided into 6 groups considering both eyes. We estimated the effects of VI on the EQ-5D index score using linear regression models and the association between VI and self-reported EQ-5D health problems using logistic regression models. Associations were assessed by the Spearman correlation coefficient. RESULTS: The prevalence of VI and the index scores of EQ-5D-3L for each subgroup of VI were higher for the rural cohort. In these two cohorts, the severity of VI in rural population (Spearman r = 0.205; p < 0.0001) and urban population (Spearman r = 0.164; p < 0.0001) is correlated with the EQ-5D index score. In the rural cohort, the difference in index scores with bilateral severe VI compared to those without VI, after adjusting for covariates, was - 0.053 for the rural cohort and - 0.084 for the urban cohort, respectively. In the rural cohort, the odds ratio for bilateral severe VI was 4.39 for mobility, 6.33 for self-care, and 5.88 for usual activities. The incidence of anxiety or depression and pain or discomfort in the urban cohort was greater; the OR for bilateral severe VI in the urban cohort was 4.75. CONCLUSIONS: VI has a negative impact on HRQoL in the rural and urban areas of China, especially in urban population. It is also more likely to cause anxiety or depression among the urban cohort, which deserves special attention.

Endophilin A2 regulates calcium-activated chloride channel activity via selective autophagy-mediated TMEM16A degradation.

TMEM16A Ca2+-activated chloride channel (CaCC) plays an essential role in vascular homeostasis. In this study we investigated the molecular mechanisms underlying downregulation of TMEM16A CaCC activity during hypertension. In cultured basilar artery smooth muscle cells (BASMCs) isolated from 2k2c renohypertesive rats, treatment with angiotensin II (0.125-1 µM) dose-dependently increased endophilin A2 levels and decreased TMEM16A expression. Similar phenomenon was observed in basilar artery isolated from 2k2c rats. We then used whole-cell recording to examine whether endophilin A2 could regulate TMEM16A CaCC activity in BASMCs and found that knockdown of endophilin A2 significantly enhanced CaCC activity, whereas overexpression of endophilin A2 produced the opposite effect. Overexpression of endophilin A2 did not affect the TMEM16A mRNA level, but markedly decreased TMEM16A protein level in BASMCs by inducing ubiquitination and autophagy of TMEM16A. Ubiquitin-binding receptor p62 (SQSTM1) could bind to ubiquitinated TMEM16A and resulted in a process of TMEM16A proteolysis in autophagosome/lysosome. These data provide new insights into the regulation of TMEM16A CaCC activity by endophilin A2 in BASMCs, which partly explains the mechanism of angiotensin-II-induced TMEM16A inhibition during hypertension-induced vascular remodeling.

Silica nanomaterials induce organ injuries by Ca2+-ROS-initiated disruption of the endothelial barrier and triggering intravascular coagulation.

BACKGROUND: The growing use of silica nanoparticles (SiNPs) in many fields raises human toxicity concerns. We studied the toxicity of SiNP-20 (particle diameter 20 nm) and SiNP-100 (100 nm) and the underlying mechanisms with a focus on the endothelium both in vitro and in vivo. METHODS: The study was conducted in cultured human umbilical vein endothelial cells (HUVECs) and adult female Balb/c mice using several techniques. RESULTS: In vitro, both SiNP-20 and SiNP-100 decreased the viability and damaged the plasma membrane of cultured HUVECs. The nanoparticles also inhibited HUVECs migration and tube formation in a concentration-dependent manner. Both SiNPs induced significant calcium mobilization and generation of reactive oxygen species (ROS), increased the phosphorylation of vascular endothelial (VE)-cadherin at the site of tyrosine 731 residue (pY731-VEC), decreased the expression of VE-cadherin expression, disrupted the junctional VE-cadherin continuity and induced F-actin re-assembly in HUVECs. The injuries were reversed by blocking Ca2+ release activated Ca2+ (CRAC) channels with YM58483 or by eliminating ROS with N-acetyl cysteine (NAC). In vivo, both SiNP-20 and SiNP-100 (i.v.) induced multiple organ injuries of Balb/c mice in a dose (range 7-35 mg/kg), particle size, and exposure time (4-72 h)-dependent manner. Heart injuries included coronary endothelial damage, erythrocyte adhesion to coronary intima and coronary coagulation. Abdominal aorta injury exhibited intimal neoplasm formation. Lung injuries were smaller pulmonary vein coagulation, bronchiolar epithelial edema and lumen oozing and narrowing. Liver injuries included multifocal necrosis and smaller hepatic vein congestion and coagulation. Kidney injuries involved glomerular congestion and swelling. Macrophage infiltration occurred in all of the observed organ tissues after SiNPs exposure. SiNPs also decreased VE-cadherin expression and altered VE-cadherin spatial distribution in multiple organ tissues in vivo. The largest SiNP (SiNP-100) and longest exposure time exerted the greatest toxicity both in vitro and in vivo. CONCLUSIONS: SiNPs, administrated in vivo, induced multiple organ injuries, including endothelial damage, intravascular coagulation, and secondary inflammation. The injuries are likely caused by upstream Ca2+-ROS signaling and downstream VE-cadherin phosphorylation and destruction and F-actin remodeling. These changes led to endothelial barrier disruption and triggering of the contact coagulation pathway.

Ca2+/Calmodulin-Dependent Protein Kinase II γ-Dependent Serine727 Phosphorylation Is Required for TMEM16A Ca2+-Activated Cl- Channel Regulation in Cerebrovascular Cells.

BACKGROUND: TMEM16A is a critical component of Ca2+-activated chloride channels (CaCCs) and mediates basilar arterial smooth muscle cell (BASMC) proliferation in hypertensive cerebrovascular remodeling. CaMKII is a negative regulator of CaCC, and four CaMKII isoforms (α, ß, γ and δ) are expressed in vasculature; however, it is unknown which and how CaMKII isoforms affect TMEM16A-associated CaCC and BASMC proliferation.Methods and Results:Patch clamp and small interfering RNA (siRNA) knockdown of different CaMKII isoforms revealed that only CaMKIIγ inhibited native Ca2+-activated chloride currents (ICl.Ca) in BASMCs. The TMEM16A overexpression evoked TMEM16A Cl-current and inhibited angiotensin II (Ang II)-induced proliferation in BASMCs. The co-immunoprecipitation and pull-down assay indicated an interaction between CaMKIIγ and TMEM16A protein. TMEM16A Cl-current was modulated by CaMKIIγ phosphorylation at serine residues in TMEM16A. Serine525 and Serine727 in TMEM16A were mutated to alanine, and only mutation at Ser727 (S727A) reversed the CaMKIIγ inhibition of the TMEM16A Cl-current. Phosphomimetic mutation S727D markedly decreased TMEM16A Cl-current and reversed TMEM16A-mediated suppression of BASMC proliferation, mimicking the inhibitory effects of CaMKIIγ on TMEM16A. A significant increase in CaMKIIγ isoform content was observed in parallel to the decrease of TMEM16A and ICl.Cain basilar artery proliferative remodeling in Ang II-infused mice. CONCLUSIONS: Serine 727 phosphorylation in TMEM16A by CaMKIIγ provides a new mechanism for regulating TMEM16A CaCC activity and Ang II-induced BASMC proliferation.

[Distribution Characteristics and Risk Assessment of Microplastics in Water of Different Functional Parks in Guilin].

The water environment capacity of urban parks is small, and their self-purification ability is poor. They are also more likely to be affected by microplastics (MPs), which cause an imbalance of the water micro-ecosystem. Based on the functional characteristics of parks (comprehensive park, community park, and ecological park), this study investigated the distribution characteristics of MPs in the water of Guilin parks through spot sampling, microscopic observation, and Fourier transform infrared spectroscopy. In addition, the pollution risk index and the pollution load index were used to evaluate the pollution risk of MPs.The results showed that the abundances of MPs in the park surface water and sediments ranged from 104.67-674.44 n·m-3 and 95.57-877.78 n·kg-1, respectively. There were four main shape types of MPs:fragments, fibers, films, and particles. MPs were dominated by fragments and fibers with small sizes (<1 mm). The polymers of MPs were polyethylene and polyethylene terephthalate. There were significant differences in the abundance of MPs in the water of different functional parks, and the abundance of MPs in comprehensive parks was the highest. The abundance of MPs in park water was closely related to the function of the park and the number of people entering the park. The pollution risk of MPs in the surface water of Guilin parks was low, whereas the pollution risk of MPs in sediments was relatively high. The results of this study indicated that tourism was an important source of MPs pollution in the water of Guilin City parks. The pollution risk of MPs in the water of Guilin City parks was mild. However, the pollution risk of MPs accumulated in small freshwater waters of urban parks requires continued attention.

Cost-Effectiveness Analysis of Camrelizumab Plus Chemotherapy vs. Chemotherapy Alone as the First-Line Treatment in Patients With IIIB-IV Non-Squamous Non-Small Cell Lung Cancer (NSCLC) Without EGFR and ALK Alteration from a Perspective of Health - Care System in China.

Objective: The CAMEL clinical trial (412 patients were randomly assigned to either camrelizumab plus chemotherapy (n = 205) or chemotherapy alone (n = 207)) demonstrated that camrelizumab plus chemotherapy (CC) improved the overall survival time (OS) and progression-free survival time (PFS) of patients with metastatic nonsquamous non-small cell lung cancer (non-sq NSCLC) without epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) mutations (EGFRm and ALKm) vs. chemotherapy (C) alone. Our objective was to conduct a cost-effectiveness analysis of CC vs. C from a perspective of health - care system in China with a lifetime horizon to identify whether it will be cost-effective. Materials and Methods: A partitioned survival model (PSM) was applied for patients with IIIB-IV non-sq NSCLC without EGFRm and ALKm. Transition parameters and proportions of three health states were derived from the CAMEL trial. The model was designed using a lifetime horizon, a 21-day cycle, and a 5% discount rate of costs and outcomes. It was deemed cost-effective in China if the incremental cost-effectiveness ratio (ICER) value is less than $32,457 per quality adjusted life-year (QALY). Deterministic and probabilistic sensitivity analyses were performed to verify the influence of parameter uncertainty on the results. Results: In the base-case analysis, we found that the ICER of CC compared with C is $-7,382.72/QALY which meant that CC had lower costs and better outcomes. The results of the sensitivity analyses demonstrated that the result was robust for the ICERs never transcending the willingness-to-pay (WTP) threshold. Conclusion: Camrelizumab plus chemotherapy is an obviously cost-effective therapeutic regime for patients of IIIB-IV non-sq NSCLC without EGFRm and ALKm in China at a $32,457 WTP threshold.

Distribution of IOP and its relationship with refractive error and other factors: the Anyang University Students Eye Study.

AIM: To investigate the distribution of intraocular pressure (IOP) and its relationship with refractive error and other factors in university students from Anyang, China. METHODS: A university-based study was conducted. Subjects were invited to complete ophthalmic examinations, including visual acuity, noncontact tonometry (NCT), cycloplegic autorefraction, and ocular biometry. Univariable and multivariable analyses were used to evaluate the associations between IOP and other factors. Only data from right eyes were used in analysis. RESULTS: A total of 7720 subjects aged 16 to 26 years old were included, and 2834 (36.4%) of the participants were male. The mean IOP of the right eye for all subjects was 15.52±3.20 mm Hg (95%CI: 15.45, 15.59). Using multivariate linear regression analysis, IOP was found to correlate significantly with younger age (P<0.001; standardized regression coefficient ß, -0.061; regression coefficient ß, -0.139; 95%CI: -0.18, -0.09), higher myopic refractive error (P=0.044; standardized ß, -0.060; regression coefficient ß, -0.770; 95%CI: -0.15, -0.002), higher central corneal thickness (P<0.001; standardized ß, 0.450; regression coefficient ß, 0.044; 95%CI: 0.04, 0.05), and shorter axial length (AL; P<0.001; standardized ß, -0.061; regression coefficient ß, -0.163; 95%CI: -0.25, -0.07). CONCLUSION: This study described the normal distribution of IOP. In Chinese university students aged 16-26y, higher IOP is associated with younger age, higher myopic refractive error, higher thickness of the central cornea, and shorter AL.

Central Retinal Vessel Trunk Caliber Changes After Short-term Intraocular Pressure Elevation.

PRECIS: After a short-term intraocular pressure (IOP) elevation, the central retinal vein caliber may be widened at lower IOP rise levels, while be compressed at higher IOP rise values. PURPOSE: The purpose of this study was to investigate changes in the calibers of the central retinal vein trunk (CRVT) and central retinal artery trunk (CRAT) trunk during a short-term elevation of IOP. METHODS: A prospective observational study. Acute primary angle-closure suspects underwent a dark room prone provocative test (DRPPT) for 2 hours. Before and at the end of the test, tonometry, swept-source optical coherence tomography, and nonmydriatic fundus photography were performed. The calibers of the CRVT and CRAT were measured on the fundus photos taken at baseline and at the end of the DRPPT. RESULTS: The study included 101 eyes (61 individuals; mean age: 54.8±9.3 y; range: 30 to 70 y) which showed an increase in IOP by 9.6±9.0 mm Hg (range: 2.3 to 46.7 mm Hg). From baseline to the end of the DRPPT, the mean CRVT caliber increased from 101.8±25.9 to 107.7±26.6 µm (P<0.001), while the CRAT caliber did not differ significantly (110.3±24.2 vs. 109.7±21.5 µm; P=0.54) during the test. The CRVT widening was larger in the subgroup with IOP rise of <6 mm Hg than in the subgroup with an IOP rise of 6 to 15 mm Hg, while in the subgroup with an IOP rise of >15 mm Hg the CRVT caliber did not change significantly (P=0.20) during the test. CONCLUSIONS: A physiological short-term IOP rise at lower levels of IOP elevation led to a widening of the CRVT, while at higher IOP values, the further IOP-rise may have compressed the retinal vein. Because of higher intraluminal pressure values, the retinal artery diameters were not affected by the IOP-rise.

TMEM16A ameliorates vascular remodeling by suppressing autophagy via inhibiting Bcl-2-p62 complex formation.

Rationale: Transmembrane member 16A (TMEM16A) is a component of calcium-activated chloride channels that regulate vascular smooth muscle cell (SMC) proliferation and remodeling. Autophagy, a highly conserved cellular catabolic process in eukaryotes, exerts important physiological functions in vascular SMCs. In the current study, we investigated the relationship between TMEM16A and autophagy during vascular remodeling. Methods: We generated a transgenic mouse that overexpresses TMEM16A specifically in vascular SMCs to verify the role of TMEM16A in vascular remodeling. Techniques employed included immunofluorescence, electron microscopy, co-immunoprecipitation, and Western blotting. Results: Autophagy was activated in aortas from angiotensin II (AngII)-induced hypertensive mice with decreased TMEM16A expression. The numbers of light chain 3B (LC3B)-positive puncta in aortas correlated with the medial cross-sectional aorta areas and TMEM16A expression during hypertension. SMC-specific TMEM16A overexpression markedly inhibited AngII-induced autophagy in mouse aortas. Moreover, in mouse aortic SMCs (MASMCs), AngII-induced autophagosome formation and autophagic flux were blocked by TMEM16A upregulation and were promoted by TMEM16A knockdown. The effect of TMEM16A on autophagy was independent of the mTOR pathway, but was associated with reduced kinase activity of the vacuolar protein sorting 34 (VPS34) enzyme. Overexpression of VPS34 attenuated the effect of TMEM16A overexpression on MASMC proliferation, while the effect of TMEM16A downregulation was abrogated by a VPS34 inhibitor. Further, co-immunoprecipitation assays revealed that TMEM16A interacts with p62. TMEM16A overexpression inhibited AngII-induced p62-Bcl-2 binding and enhanced Bcl-2-Beclin-1 interactions, leading to suppression of Beclin-1/VPS34 complex formation. However, TMEM16A downregulation showed the opposite effects. Conclusion: TMEM16A regulates the four-way interaction between p62, Bcl-2, Beclin-1, and VPS34, and coordinately prevents vascular autophagy and remodeling.

The acute toxic effects of platinum nanoparticles on ion channels, transmembrane potentials of cardiomyocytes in vitro and heart rhythm in vivo in mice.

BACKGROUND: Platinum nanoparticles (PtNPs) have been considered a nontoxic nanomaterial and been clinically used in cancer chemotherapy. PtNPs can also be vehicle exhausts and environmental pollutants. These situations increase the possibility of human exposure to PtNPs. However, the potential biotoxicities of PtNPs including that on cardiac electrophysiology have been poorly understood. METHODS: Ion channel currents of cardiomyocytes were recorded by patch clamp. Heart rhythm was monitored by electrocardiogram recording. Morphology and characteristics of PtNPs were examined by transmission electron microscopy, dynamic light scattering and electrophoretic light scattering analyses. RESULTS: In cultured neonatal mice ventricular cardiomyocytes, PtNPs with diameters 5 nm (PtNP-5) and 70 nm (PtNP-70) concentration-dependently (10-9 - 10-5 g/mL) depolarized the resting potentials, suppressed the depolarization of action potentials and delayed the repolarization of action potentials. At the ion channel level, PtNPs decreased the current densities of INa, IK1 and Ito channels, but did not affect the channel activity kinetics. In vivo, PtNP-5 and PtNP-70 dose-dependently (3-10 mg/kg, i.v.) decreased the heart rate and induced complete atrioventricular conduction block (AVB) at higher doses. Both PtNP-5 and PtNP-70 (10-9 - 10-5 g/mL) did not significantly increase the generation of ROS and leak of lactate dehydrogenase (LDH) from cardiomyocytes within 5 mins after exposure except that only very high PtNP-5 (10-5 g/mL) slightly increased LDH leak. The internalization of PtNP-5 and PtNP-70 did not occur within 5 mins but occurred 1 hr after exposure. CONCLUSION: PtNP-5 and PtNP-70 have similar acute toxic effects on cardiac electrophysiology and can induce threatening cardiac conduction block. These acute electrophysiological toxicities of PtNPs are most likely caused by a nanoscale interference of PtNPs on ion channels at the extracellular side, rather than by oxidative damage or other slower biological processes.

Design, methodology, and preliminary results of the follow-up of a population-based cohort study in rural area of northern China: Handan Eye Study.

BACKGROUND: Handan Eye Study (HES), a large population-based cohort study in rural area of northern China, was one of the few studies focusing on the major eye diseases of rural Chinese population. The aim of this study was to introduce the design, methodology and to assess the data quality of the follow-up phase of HES. METHODS: All participants were recruited in Yongnian county of Handan city between 2012 and 2013. Main outcomes were measured by visual quality scales and ocular examinations. We performed the Chi-square test to make comparison of categorical data among groups, One-way analysis of variance and Kruskal-Wallis test was applied to make comparison of continuous data among groups, a post-hoc test was done to make further pairwise comparison. Inter-class correlation coefficients (ICCs) and Kappa coefficients were used to evaluate the consistency between different operators. Logistic regression was used to explore the influence factors of death, odds ratio (OR) and 95% confidence interval (CI) were used to estimate the effect size of each influence factor. RESULTS: The follow-up rate was 85.3%. Subjects were classified into three groups: the follow-up group (n = 5394), the loss to follow-up group (n = 929), and the dead group (n = 507), comparison of their baseline information was done. Compared with the other two groups, age of the dead group (66.52 ±â€Š10.31 years) was the oldest (Z = 651.293, P < 0.001), male proportion was the highest (59.0%) (χ = 42.351, P < 0.001), only 65.9% of the dead finished middle school education (Z = 205.354, P < 0.001). The marriage percentage, body mass index (BMI), best-corrected visual acuity (BCVA), and intra-ocular pressure of the dead group was the lowest either. Spherical equivalent error (SER) of the dead group was the highest. Besides, history of smoking, hypertension, diabetes, and heart disease were more common in the dead group. Multivariate analysis showed that age (OR = 1.901, 95% CI: 1.074-1.108), gender (OR = 0.317, 95% CI: 0.224-0.448), and BCVA (OR = 0.282, 95% CI: 0.158-0.503) were associated with death. While between the follow-up group and the loss to follow-up group, there was only difference on age, gender, BMI, systolic blood pressure and SER. The Cronbach coefficients of all scales used in the follow-up were ≥0.63 and the cumulative variances were ≥0.61, indicating good reliability and validity. The ICCs and Kappa coefficients between different operators were ≥0.69. CONCLUSIONS: HES has a high follow-up rate and a low risk of loss to follow-up bias. Age, gender, and BCVA are influence factors of death. Specifically, male subjects are at a higher risk of death than female, age is a risk factor of death while BCVA is a protective factor for death.

Cycloplegic refraction by 1% cyclopentolate in young adults: is it the gold standard? The Anyang University Students Eye Study (AUSES).

AIMS: To document the difference between non-cycloplegic and cycloplegic refraction and explore its associated factors in Chinese young adults. METHODS: A school-based study including 7971 undergraduates was conducted in Anyang, Henan Province, China. Cycloplegia was achieved with two drops of 1% cyclopentolate and 1 drop of Mydrin P (Tropicamide 0.5%, phenylephrine HCl 0.5%) with a 5 min interval. Non-cycloplegic and cycloplegic refractions were measured by an autorefractor. A paired-sample t-test and Spearman correlation analysis were used for analysis with data from only the right eyes included.  RESULTS: Of the 7971 students examined, 7793 (97.8%) with complete data were included, aging 20.2±1.5 years. Male students accounted for 36.8%. Overall, there was a significant difference between non-cycloplegic and cycloplegic SE (spherical equivalent) of 0.83±0.81D (p<0.01). The difference was 1.80±1.11D, 1.26±0.93D and 0.69±0.69D for those with cycloplegic hyperopia, emmetropia and myopia, respectively (p<0.01 for all). Those with a hyperopic shift less than 0.25D and 0.5D accounted for 11.1% and 34.1%, respectively. A significant relationship was found between difference in SE and cycloplegic refraction (r=0.33, b=0.11, p<0.01). Without cycloplegia, prevalence of hyperopia and emmetropia would be underestimated by 6.2% (1.0% vs 7.2%) and 5.7% (3.8% vs 9.5%), respectively, with prevalence of myopia and high myopia overestimated by 12.1% (95.3% vs 83.2%) and 6.1% (17.2% vs 11.1%).  CONCLUSION: Lack of cycloplegia will lead to significant misclassification of myopia, emmetropia and hyperopia in Chinese young adults. Cycloplegia is therefore essential for this age-group in epidemiological studies.

The acute toxic effects of silver nanoparticles on myocardial transmembrane potential, INa and IK1 channels and heart rhythm in mice.

This study focused on the potential toxicity of silver nanoparticles (AgNPs) on cardiac electrophysiology which is rarely investigated. We found that AgNPs (10-9-10-6 g/ml) concentration-dependently depolarized the resting potential, diminished the action potential, and finally led to loss of excitability in mice cardiac papillary muscle cells in vitro. In cultured neonatal mice cardiomyocytes, AgNPs (10-9-10-7 g/ml) concentration-dependently decreased the Na+ currents (INa), accelerated the activation, and delayed the inactivation and recovery of Na+ channels from inactivation within 5 min. AgNPs at 10-8 g/ml also rapidly decreased the inwardly rectifying K+ currents (IK1) and delayed the activation of IK1 channels. Intravenous injection of AgNPs at 3 mg/kg only decreased the heart rate, while at ≥4 mg/kg sequentially induced sinus bradycardia, complete atrio-ventricular conduction block, and cardiac asystole. AgNPs at 10-10-10-6 g/ml did not increase reactive oxygen species (ROS) generation and only at 10-6 g/ml mildly induced lactate dehydrogenase (LDH) release in the cardiomyocytes within 5 min. Endocytosis of AgNPs by cardiomyocytes was not observed within 5 min, but was observed 1 h after exposing to AgNPs. Comparative Ag+ (≤0.02% of the AgNPs) could not induce above toxicities. We conclude that AgNPs exert rapid toxic effects on myocardial electrophysiology and induce lethal bradyarrhythmias. These acute toxicities are likely due to direct effects of AgNPs on ion channels at the nano-scale level, but not caused by Ag+, ROS, and membrane injury. These findings provide warning to the nanomedical practice using AgNPs.

Safety of femtosecond laser-assisted cataract surgery: assessment of aqueous humour and lens capsule.

PURPOSE: To investigate the effect of femtosecond laser-assisted cataract surgery (FLACS) on aqueous humour and lens capsule. METHODS: This prospective randomized comparative study enrolled 19 eyes that underwent FLACS as the trial group and 20 eyes that underwent conventional phacoemulsification as the control group. The femtosecond laser platform (LLS-fs 3D; LensAR, Orlando, FL, USA) was used to generate capsulotomy (laser energy 8 µJ) and lens fragmentation (laser energy 10 µJ). Morphology of the cutting edge and cells of anterior capsule was assessed by light microscopy. The proteins in the aqueous humour were identified by mass spectrometry (Ultraflex III TOF/TOF; Bruker Dalton, Bremen, Germany). Electrolyte in the aqueous humour was detected by a chemistry analyzer (Aeroset Clinical Chemistry Analyzer; Abbott Laboratories, Abbott Park, IL, USA). RESULTS: The cutting edge of anterior capsule was saw-tooth-shaped under magnification of 200× and 400× in the trial group, while it was smooth in the control group. Intact cells were found in the boundary area next to the cutting edge of anterior capsule in both groups. ß-Crystallin B1, γ-crystallin S and transferrin were detected in the aqueous humour in the trial group. The concentrations of K+ , Na+ and Cl- in the aqueous humour in the trial group differed significantly from those in the control group (p = 0.02, 0.03 and 0.04, respectively). CONCLUSION: Femtosecond laser-assisted cataract surgery (FLACS) causes release of transferrin and crystallin from lens to aqueous humour and results in significant changes in the concentrations of K+ , Na+ and Cl- in aqueous humour. However, these changes due to FLACS have no clinical significance or toxicity.

Threonine532 phosphorylation in ClC-3 channels is required for angiotensin II-induced Cl(-) current and migration in cultured vascular smooth muscle cells.

BACKGROUND AND PURPOSE: Angiotensin II (AngII) induces migration and growth of vascular smooth muscle cell (VSMC), which is responsible for vascular remodelling in some cardiovascular diseases. Ang II also activates a Cl(-) current, but the underlying mechanism is not clear. EXPERIMENTAL APPROACH: The A10 cell line and primary cultures of VSMC from control, ClC-3 channel null mice and WT mice made hypertensive with AngII infusions were used. Techniques employed included whole-cell patch clamp, co-immunoprecipitation, site-specific mutagenesis and Western blotting, KEY RESULTS: In VSMC, AngII induced Cl(-) currents was carried by the chloride ion channel ClC-3. This current was absent in VSMC from ClC-3 channel null mice. The AngII-induced Cl(-) current involved interactions between ClC-3 channels and Rho-kinase 2 (ROCK2), shown by N- or C-terminal truncation of ClC-3 protein, ROCK2 siRNA and co-immunoprecipitation assays. Phosphorylation of ClC-3 channels at Thr(532) by ROCK2 was critical for AngII-induced Cl(-) current and VSMC migration. The ClC-3 T532D mutant (mutation of Thr(532) to aspartate), mimicking phosphorylated ClC-3 protein, significantly potentiated AngII-induced Cl(-) current and VSMC migration, while ClC-3 T532A (mutation of Thr(532) to alanine) had the opposite effects. AngII-induced cell migration was markedly decreased in VSMC from ClC-3 channel null mice that was insensitive to Y27632, an inhibitor of ROCK2. In addition, AngII-induced cerebrovascular remodelling was decreased in ClC-3 null mice, possibly by the ROCK2 pathway. CONCLUSIONS AND IMPLICATIONS: ClC-3 protein phosphorylation at Thr(532) by ROCK2 is required for AngII-induced Cl(-) current and VSMC migration that are involved in AngII-induced vascular remodelling in hypertension.

A simple and highly selective 2,2-diferrocenylpropane-based multi-channel ion pair receptor for Pb(2+) and HSO4(-).

A structurally simple, 2,2-diferrocenylpropane-based ion pair receptor 1 was synthesized and characterized by (1)H NMR, (13)C NMR, HRMS, elemental analyses, and single-crystal X-ray diffraction. The ion pair receptor 1 showed excellent selectivity and sensitivity towards Pb(2+) with multi-channel responses: a fluorescence enhancement (more than 42-fold), a notable color change from yellow to red, redox anodic shift (ΔE1/2 = 151 mV), while HSO4(-) promoted fluorescence enhancement when Pb(2+) or Zn(2+) was bonded to the cation binding-site. (1)H NMR titration and density functional theory were performed to reveal the sensing mechanism based on photo-induced electron transfer (PET).

Leptin receptor of the hind brain nuclei is involved in the conditioned taste preference of rats.

OBJECTIVE: Conditioned taste preference (CTP) is a taste learning reflex by which an animal learns to prefer a substance which tastes not well and has been studied with much interest in recent years. However, the neural substrates of CTP are less known. This study aimed to determine the possible neural path- ways of CTP and whether serum leptin level and the leptin receptor (OB-Rb) in the hind brain are involved following CTP formation. METHODS: We established CTP of quinine in rats with a 2-bottle preference test. The serum leptin concentrations were detected, the expression of c-fos in the rat brain was tested to determine the nuclei in relation with establishment of CTR Finally, the OB-Rb mRNA expression was examined by RT-qPCR assay in parabrachial nucleus (PBN) and the nucleus of the solitary tract (NST) of the hind brain. RESULTS: Compared with control group, the level of serum leptin was higher in the CTP group (4.58 ± 0.52 vs 1.67 ± 0.25 µg/L, P < 0.01); increased c-fos positive cells were found in the anterior hypothalamus (AH, 221.75 ± 4.96 vs. 178.50 ± 6.63 cells/mm², P < 0.05), the basal lateral amygdala (BLA, 70.75 ± 6.17 vs 56.50 ± 3.62 cells/ mm², P < 0.05) and the nucleus of the solitary tract (NST, 41.25 ± 1.32 vs 32.50 ± 1.02 cells/mm², P < 0.05). But in ventromedial nucleus of the hypothalamus (VMH, 20.75 ± 2.73 vs 38.5 ± 1.54 per 1 mm², P < 005), PBN (21.50 ± 2.24 vs 36.25 ± 1.49 cells/mm², P < 0.05) and the central nucleus of the amygdala (CeA, 22.25 ± 1.53 vs 35.50 ± 2.11 cells/mm², P < 0.05), the number of c-fos positive cells was decreased in the CTP group. In addition, we found OB-Rb mRNA expression in PBN of CTP group rats was higher than that of control group (0.95 ± 0.055 vs 0.57 ± 0.034, P < 0.05), while there was no significant difference of OB-Rb mRNA expression in NST between the two groups. CONCLUSION: Nuclei AH, BLA, NST, VMH, PBN and CeA participate in the formation of CTP. Leptin and its receptor in PBN may be involved in the formation and maintenance of CTP.

[Clinical application of acupuncture and moxibustion to the treatment of gouty arthritis and analysis on their therapeutic effects].

In the present paper, the authors make a summary on the clinical application of acu-moxibustion therapy in the treatment of gouty arthritis in recent 10 years. Acupuncture needles often used are filiform needle, red-hot needle, moxibustion-warmed needle and three-edged needle. Clinical studies have showed that acupuncture therapy has a definite efficacy in relieving gouty arthritis and has its clinical characteristics, such as faster efficacy, lower relapse rate, etc. in comparison with medication.

[Effect of electroacupuncture on caspase-9 expression in hippocampus and neuroethology in hyperlipemia rats with concurrent cerebral ischemia].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on hippocampal apoptosis protein caspase-9 expression and neuroethology in hyperlipemia plus cerebral ischemia (HL-CI) rats. METHODS: Seventy male SD rats were randomized into control, hyperlipemia (HL), CI, HL-CI, CI + EA, HL-CI + EA I , and HL-CI + EA II groups, with 10 cases in each. HL model was established by feeding the animals with high fat forage for 6 weeks and CI model was established by occlusion of the unilateral middle cerebral artery. EA (1-3 mA, 15 Hz) was applied to bilateral "Sanyinjiao" (SP 6) and "Fenglong" (ST 40) for 20 min every time; and "Baihui" (GV 20) and "Shuigou" (GV 26) were punctured and stimulated by twirling the acupuncture needle with hand continuously for 1 min. Acupuncture was given once daily for 17 days (beginning from the 10th day on before CI) in HL-CI + EA I group, and for 7 days (beginning after CI) in HL-CI + EA II group. The expression of hippocampal Caspase-9 was detected with immunohistochemistry. The score of neuroethology was also measured according to modified Bederson's method. RESULTS: In comparison with normal control and hyperlipemia model groups, Caspase-9 immune reaction (IR) positive cells in the hippocampus in HL-CI group increased significantly (P<0.01). After acupuncture Caspase-9 IR-positive cells decreased remarkably. In comparison with group HL-CI + EA II, Caspase-9 IR-positive cells decreased significantly in HL-CI + EA I (P<0.01). The score of neuroethology also degraded obviously. CONCLUSION: Acupuncture can improve neuroethology symptom, lessen over expression of hippocampal Caspase-9, and prevent CI injury in hyperlipemia rats with concurrent CI.