Interrogation of human microglial phagocytosis by CRISPR genome editing.

Background: Microglia are an integral part of central nervous system, but our understanding of microglial biology is limited due to the challenges in obtaining and culturing primary human microglia. HMC3 is an important cell line for studying human microglia because it is readily accessible and straightforward to maintain in standard laboratories. Although HMC3 is widely used for microglial research, a robust genetic method has not been described. Here, we report a CRISPR genome editing platform, by the electroporation of Cas9 ribonucleoproteins (Cas9 RNP) and synthetic DNA repair templates, to enable rapid and precise genetic modifications of HMC3. For proof-of-concept demonstrations, we targeted the genes implicated in the regulation of amyloid beta (Aß) and glioblastoma phagocytosis in microglia. We showed that CRISPR genome editing could enhance the phagocytic activities of HMC3. Methods: We performed CRISPR gene knockout (KO) in HMC3 by the electroporation of pre-assembled Cas9 RNP. Co-introduction of DNA repair templates allowed site-specific knock-in (KI) of an epitope tag, a synthetic promoter and a fluorescent reporter gene. The editing efficiencies were determined genotypically by DNA sequencing and phenotypically by immunofluorescent staining and flow cytometry. The gene-edited HMC3 cells were examined in vitro by fluorescent Aß and glioblastoma phagocytosis assays. Results: Our platform enabled robust single (>90%) and double (>70%) KO without detectable off-target editing by high throughput DNA sequencing. We also inserted a synthetic SFFV promoter to efficiently upregulate the expression of endogenous CD14 and TREM2 genes associated with microglial phagocytosis. The CRISPR-edited HMC3 showed stable phenotypes and enhanced phagocytosis of fluorescence-labeled Aß1-42 peptides. Confocal microscopy further confirmed the localization of Aß1-42 aggregates in the acidified lysosomes. HMC3 mutants also changed the phagocytic characteristic toward apoptotic glioblastoma cells. Conclusion: CRISPR genome editing by Cas9 RNP electroporation is a robust approach to genetically modify HMC3 for functional studies such as the interrogation of Aß and tumor phagocytosis, and is readily adoptable to investigate other aspects of microglial biology.

Association Between Air Pollution and the Risk of Uveitis: A Nationwide, Population-Based Cohort Study.

Previous studies have revealed an association between ocular surface disorders and air pollution, few studies have focused on the risk of uveitis. We aimed to investigate whether air pollution increases the risk of uveitis. We used the Taiwan Longitudinal Health Insurance Database (LHID) and the Taiwan Air Quality Monitoring Database (TAQMD) to conduct a retrospective cohort study. Air pollutant concentrations, including those of carbon dioxide (CO2), were grouped into four levels according to quartiles. The outcome was the incidence of uveitis, as defined in the International Classification of Diseases, Ninth Revision. We used univariable and multivariable Cox proportional hazard regression models to calculate the adjusted hazard ratios (aHRs) and determine the potential risk factors of uveitis. Overall, 175,489 subjects were linked to their nearby air quality monitoring stations. We found that for carbon monoxide, the aHRs of uveitis risk for the Q3 and Q4 levels were 1.41 (95% confidence interval (CI) = 1.23-1.61) and 2.19 (95% CI = 1.93-2.47), respectively, in comparison with those for the Q1 level. For nitric oxide, the aHRs for the Q3 and Q4 levels were 1.46 (95% CI = 1.27-1.67) and 2.05 (95% CI = 1.81-2.32), respectively. For nitrogen oxide (NOx), the aHRs for the Q2, Q3, and Q4 levels were 1.27 (95% CI = 1.11-1.44), 1.34 (95% CI = 1.16-1.53), and 1.85 (95% CI = 1.63-2.09), respectively. For total hydrocarbon (THC), the aHRs for the Q2, Q3, and Q4 levels were 1.42 (95% CI = 1.15-1.75), 3.80 (95% CI = 3.16-4.57), and 5.02 (95% CI = 4.19-6.02), respectively. For methane (CH4), the aHRs for the Q3 and Q4 levels were 1.94 (95% CI = 1.60-2.34) and 7.14 (95% CI = 6.01-8.48), respectively. In conclusion, air pollution was significantly associated with incidental uveitis, especially at high THC and CH4 levels. Furthermore, the uveitis risk appeared to increase with increasing NOx and THC levels.

Metformin Use Was Associated With Reduced Risk of Incidental Sjögren's Syndrome in Patients With Type 2 Diabetes: A Population-Based Cohort Study.

Purpose: Previous studies have shown that metformin exhibits an anti-inflammatory effect and may decrease the risk of incidental diabetes. But the effect of metformin on incidental Sjögren's syndrome is unknown. The aim of the study was to examine the association between metformin exposure and Sjögren's syndrome in diabetic patients. Methods: The dataset in this retrospective cohort study was obtained from the National Health Insurance Research Database (2000-2013) in Taiwan. In total, 15,098 type 2 diabetic patients under metformin treatment and an equivalent number without metformin treatment matched for comparison were included. The primary endpoint was the incidence of Sjogren's syndrome. Univariate and multivariate Cox proportional hazards models were used for data analysis. A subgroup analysis and sensitivity test were also performed. Results: The incidence rate of Sjögren's syndrome in non-metformin controls was 40.83 per 100,000 person-years and 16.82 per 100,000 person-years in metformin users. The adjusted hazard ratio (aHR) in diabetic patients under metformin treatment was 0.46 (95% CI, 0.23 to 0.92). In subgroup analysis, men had a lower risk of developing Sjögren's syndrome than women [aHR = 0.15, 95% CI = (0.05, 0.41)]. After prescribing metformin to type 2 diabetic patients aged 60 years or more, those patients had a lower risk of developing Sjögren's syndrome [aHR = 0.34, 95% CI = (0.12, 0.96)]. Conclusion: In this large population-based cohort study, metformin exposure was associated with a reduced risk of developing Sjögren's syndrome in type 2 diabetic patients.

[Searching for WDMS Candidates In SDSS-DR10 With Automatic Method].

The Sloan Digital Sky Survey (SDSS) has released the latest data (DR10) which covers the first APOGEE spectra. The massive spectra can be used for large sample research inscluding the structure and evolution of the Galaxy and multi-wave-band identi cation. In addition, the spectra are also ideal for searching for rare and special objects like white dwarf main-sequence star (WDMS). WDMS consist of a white dwarf primary and a low-mass main-sequence (MS) companion which has positive significance to the study of evolution and parameter of close binaries. WDMS is generally discovered by repeated imaging of the same area of sky, measuring light curves for objects or through photometric selection with follow-up observations. These methods require significant manual processing time with low accuracy and the real-time processing requirements can not be satisfied. In this paper, an automatic and efficient method for searching for WDMS candidates is presented. The method Genetic Algorithm (GA) is applied in the newly released SDSS-DR10 spectra. A total number of 4 140 WDMS candidates are selected by the method and 24 of them are new discoveries which prove that our approach of finding special celestial bodies in massive spectra data is feasible. In addition, this method is also applicable to mining other special celestial objects in sky survey telescope data. We report the identfication of 24 new WDMS with spectra. A compendium of positions, mjd, plate and fiberid of these new discoveries is presented which enrich the spectral library and will be useful to the research of binary evolution models.

(-)-Epigallocatechin-3-gallate inhibits VEGF expression induced by IL-6 via Stat3 in gastric cancer.

AIM: To demonstrate that (-)-Epigallocatechin-3-gallate (EGCG) inhibits vascular endothelial growth factor (VEGF) expression and angiogenesis induced by interleukin-6 (IL-6) via suppressing signal transducer and activator of transcription 3 (Stat3) activity in gastric cancer. METHODS: Human gastric cancer (AGS) cells were treated with IL-6 (50 ng/mL) and EGCG at different concentrations. VEGF, total Stat3 and activated Stat3 protein levels in the cell lyses were examined by Western blotting, VEGF protein level in the conditioned medium was measured by enzyme-linked immunosorbent assay, and the level of VEGF mRNA was evaluated by reverse transcription polymerase chain reaction (RT-PCR). Stat3 nuclear translocation was determined by Western blotting with nuclear extract, and Stat3-DNA binding activity was examined with Chromatin immunoprecipitation (ChIP) assay. IL-6 induced endothelial cell proliferation was measured with 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazoliumbromide assay, in vitro angiogenesis was determined with endothelial cell tube formation assay in Matrigel, and IL-6-induced angiogenesis in vitro was measured with Matrigel plug assay. RESULTS: There was a basal expression and secretion of VEGF in AGS cells. After stimulation with IL-6, VEGF expression was apparently up-regulated and a 2.4-fold increase was observed. VEGF secretion in the conditioned medium was also increased by 2.8 folds. When treated with EGCG, VEGF expression and secretion were dose-dependently decreased. IL-6 also increased VEGF mRNA expression by 3.1 folds. EGCG treatment suppressed VEGF mRNA expression in a dose-dependent manner. EGCG dose-dependently inhibited Stat3 activation induced by IL-6, but did not change the total Stat3 expression. When treated with EGCG or AG490, VEGF expressions were reduced to the level or an even lower level in the tumor cells not stimulated with IL-6. However, PD98059 and LY294002 did not change VEGF expression induced by IL-6. EGCG inhibited Stat3 nucleus translocation, and Stat3-DNA binding activity was also markedly decreased by EGCG. Furthermore, EGCG inhibited IL-6 induced vascular endothelial cell proliferation and tube formation in vitro and angiogenesis in vitro. CONCLUSION: EGCG inhibits IL-6-induced VEGF expression and angiogenesis via suppressing Stat3 activity in gastric cancer, which has provided a novel mechanistic insight into the anti-angiogenic activity of EGCG.

[Relationship of hypoxia-induced factor with tumor angiogenesis in early liver metastasis in colonic cancer].

OBJECTIVE: To study the relationships of hypoxia-induced factor (HIF) with tumor angiogenesis and early liver metastasis in colonic cancer. METHODS: Thirty three cases of colon cancer undergoing radical surgery were divided into two groups according to liver metastasis or not within half a year after operation. Expressions of HIF and vascular endothelial growth factor (VEGF) were examined using immunohistochemical method and tumor microvessel density (MVD) was measured in colonic cancer specimens. RESULTS: Fifteen cases developed early liver metastasis, while 18 did not. The positive rates of HIF and VEGF, and MVD were 86.7%, 66.7%, (57.9+/- 12.7)% respectively in the group with early liver metastasis, significantly higher than 44.4% (P< 0.05), 27.8% (P< 0.05) and (22.3+/- 10.2)% (P< 0.01) respectively in the group without early liver metastasis respectively. CONCLUSION: HIF can promote tumor angiogenesis in colonic cancer, and is closely related with early liver metastasis.

[A minimally invasive approach in the treatment of complicated anal fistula through spatium intermuscular of anal sphincter].

OBJECTIVE: To investigate the optimal operative approach for the complicated anal fistula. METHODS: One hundred and ninety-two cases with complicated anal fistula were randomly divided into minimally invasive operation group (through spatium intermuscular of anal sphincter) and fistula resection group. The operation time, bleeding time during and after operation, pain lasting time, healing time of incision, area of anal scar, anal malformation and function and post operative recurrence were observed and compared between the two groups. RESULTS: Compared to those of fistula resection group, the operative time was (36.5+/- 15.3)min, bleeding time during and after operation (2.0+/- 0.5)d, postoperative pain lasting time (1.5+/- 0.5)d, healing time of incision (18.5+/- 5.5)d in minimally invasive operation group. All were shortened (P< 0.05), and the incidence of anal malformation (5.2%, P< 0.01) and partial anal incontinence (2.1%, P< 0.01) was lower. There was no significant difference in postoperative recurrence between the two groups. CONCLUSIONS: The minimally invasive operation through spatium in termuscular of anal sphincter is superior to fistula resection on the management of complicated fistula.

Mild hypothermia protects liver against ischemia and reperfusion injury.

AIM: To determine whether mild hypothermia could protect liver against ischemia and reperfusion injury in pigs. METHODS: Twenty-four healthy pigs were randomly divided into normothermia, mild hypothermia and normal control groups. The experimental procedure consisted of temporary interruption of blood flow to total hepatic lobe for different lengths of time and subsequent reperfusion. Hepatic tissue oxygen pressure (PtiO2) and aspartate aminotransferase (AST) values were evaluated, and ultrastructural analysis was carried out for all samples. RESULTS: Serum AST was significantly lower, and hepatic PtiO2 values were significantly higher in the mild hypothermia group than in the normothermia group during liver ischemia-reperfusion periods (P=0.032, P=0.028). Meanwhile, the histopathologic injury of liver induced by ischemia-reperfusion was significantly improved in the mild hypothermia group, compared with that in the normothermia group. CONCLUSION: Mild hypothermia can protect the liver from ischemia-reperfusion injury in pigs.