Spectrum of SLC20A2, PDGFRB, PDGFB, and XPR1 mutations in a large cohort of patients with primary familial brain calcification.

Primary familial brain calcification (PFBC) is a rare neurodegenerative disorder with four causative genes (SLC20A2, PDGFRB, PDGFB, and XPR1) that have been identified. Here, we aim to describe the mutational spectrum of four causative genes in a series of 226 unrelated Chinese PFBC patients. Mutations in four causative genes were detected in 16.8% (38/226) of PFBC patients. SLC20A2 mutations accounted for 14.2% (32/226) of all patients. Mutations in the other three genes were relatively rare, accounting for 0.9% (2/226) of all patients, respectively. Clinically, 44.8% of genetically confirmed patients (probands and relatives) were considered symptomatic. The most frequent symptoms were chronic headache, followed by movement disorders and vertigo. Moreover, the total calcification score was significantly higher in the symptomatic group compared to the asymptomatic group. Functionally, we observed impaired phosphate transport induced by seven novel missense mutations in SLC20A2 and two novel mutations in XPR1. The mutation p.D164Y in XPR1 might result in low protein expression through an enhanced proteasome pathway. In conclusion, our study further confirms that mutations in SLC20A2 are the major cause of PFBC and provides additional evidence for the crucial roles of phosphate transport impairment in the pathogenies of PFBC.

Safety, pharmacokinetics, and prevention effect of intraocular crocetin in proliferative vitreoretinopathy.

The study was designed to determine the safety and pharmacokinetics of intraocular crocetin and examine whether crocetin inhibits the development of proliferative vitreoretinopathy (PVR) in a rabbit model. In the toxicity study, the right eyes of rabbits were injected with 0.2 µmol or 0.4 µmol crocetin. The left eyes were injected with 0.1 ml phosphate buffered saline (PBS) containing the same concentration of DMSO. Fundus photography, optical coherence tomography (OCT), and electroretinogram (ERG) were obtained at baseline and 14 days. Afterward, the eyes were enucleated for histopathological analysis and terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) assay. In the pharmacokinetic study, the eyes received an intravitreous injection of 0.4 µmol crocetin to detect vitreous drug levels with HPLC at specific time points. In the efficacy study, PVR was induced with an intravitreal injection of ARPE-19 cells in rabbits. Then ten eyes were injected with 0.4 µmol crocetin, and the other 10 eyes received 0.1 ml PBS. Fundus photography, OCT and ERG were performed at days 3 and 7 and weekly for a total of 4 weeks after injection. Afterward, the eyes were enucleated and subjected to histological analysis and TUNEL staining. The results demonstrated no signs of retinal toxicity. Intravitreal injection of 0.4 µmol crocetin had a half-life of 4.231 h. Treatment with crocetin significantly inhibited the progression of PVR in parallel with a reduced expression of α-SMA, collagen fibers and Ki67. These results indicate that crocetin is an effective and safe inhibitor of PVR in rabbit models.

Crocetin inhibits the proliferation, migration and TGF-ß2-induced epithelial-mesenchymal transition of retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells, the major cell type in the fibrotic membrane of proliferative vitreoretinopathy, display enhanced proliferative and migratory capacities and epithelial-mesenchymal transition (EMT). In this study, we investigated the potential impact of crocetin on the proliferation, migration and EMT of cultured ARPE-19 cells. The cells were treated with crocetin alone or in combination with transforming growth factor-ß2 (TGF-ß2). Cell proliferation was examined using the CCK-8 assay. Cell cycle distribution was analyzed by flow cytometry after propidium iodide staining. The expression levels of proliferating cell nuclear antigen (PCNA), p21 and p53 were examined by Western blot analysis. Cell migration was assessed by in vitro scratch and Transwell assays. Real-time PCR, Western blotting and immunofluorescence were used to assess EMT features. Treatment of ARPE-19 cells with crocetin (50-200µM) significantly inhibited their proliferation and migration in a concentration- and time-dependent manner. Crocetin induced G1 arrest, reduced PCNA protein expression and increased the p21 and p53 accumulation in ARPE-19 cells. Crocetin inhibited TGF-ß2-induced EMT in ARPE-19 cells by maintaining the expression of E-cadherin and ZO-1 and by reducing the expression of vimentin and α-SMA through the suppression of phosphorylation of p38. These results indicate that crocetin is an effective inhibitor of the proliferation, migration and TGF-ß2-mediated EMT of ARPE-19 cells.

Analysis of gene expression and functional characterization of XPR1: a pathogenic gene for primary familial brain calcification.

Primary familial brain calcification (PFBC) is a neuropsychiatric disorder characterized by bilateral cerebral calcification with diverse neurologic or psychiatric symptoms. Recently, XPR1 variation has accounted for PFBC as another new causative gene. However, little is known about the distribution and basic function of XPR1 and its interaction with the other three pathogenic genes for PFBC (SLC20A2, PDGFRB and PDGFB). The aim of this study was to further clarify the role of XPR1 in PFBC brain pathology. As a result, gene expression profiles showed that XPR1 mRNA was widely expressed throughout the mouse brain. Cerebellum and striatum, most commonly affected in PFBC, contained a higher level of XPR1 protein than other brain regions. Additionally, XPR1 deficiency seriously affected Pi efflux and XPR1 mutations seemed to have an effect through haploinsufficiency mechanism. The immunoprecipitation and immunohistochemical studies demonstrated that XPR1 could interact with PDGFRB and might form a complex on the cell membrane. These results suggested that XPR1 played a fundamental role in the maintenance of cellular phosphate balance in the brain. This provided us with a novel perspective on understanding the pathophysiology of PFBC. The expression networks and interaction with the known pathogenic genes could shed new light on additional candidate genes for PFBC.

Novel mutations of PDGFRB cause primary familial brain calcification in Chinese families.

Four causative genes, including solute carrier family 20 member 2 (SLC20A2), platelet-derived growth factor receptor b (PDGFRB), platelet-derived growth factor b (PDGFB)and xenotropic and polytropic retrovirus receptor 1 (XPR1), have been identified to cause primary familial brain calcification (PFBC). However, PDGFRB mutations seem to be quite rare and no PDGFRB mutations have been reported in Chinese PFBC patients. A total of 146 PFBC patients including 12 families and 134 sporadic patients were recruited in this study. All of them were previously tested negative for the SLC20A2. Mutational analyses of the entire exons and exon-intron boundaries of PDGFRB were carried out by direct gene sequencing. In silico analyses of the identified variants were conducted using Mutation Taster, PolyPhen-2 and Sorts Intolerant From Tolerant. Two heterozygous variants, c.3G>A and c.2209G>A, of the PDGFRB gene were revealed in two PFBC families, respectively. These two variants were not observed in 200 healthy controls. The variant c.3G>A was located in exon 2 and affected the initiation codon of the PDGFRB gene. The variant c.2209G>A resulted in amino-acid substitutions of aspartic acid to asparagine at position 737. Both of these two variants co-segregated with the disease phenotype (variant carriers in Family 1: I1, II2 and II3; variant carriers in Family 2: I2 and II8), suggesting a pathogenic impact of these variants. The prevalence of PDGFRB mutations in Chinese PFBC patients seems to be quite low, indicating that PDGFRB is not a major causative gene of PFBC in Chinese population.

Mutation screening of PDGFB gene in Chinese population with primary familial brain calcification.

BACKGROUND: Until recently, primary familial brain calcification (PFBC) has been determined by four genes, SLC20A2, PDGFRB, PDGFB and XPR1. No studies have been carried out to analyze the gene mutation of PDGFB in Chinese population. OBJECTIVE: To screen mutations of PDGFB gene in a large cohort of Chinese PFBC patients with no SLC20A2 mutations. METHODS: We recruited 192 PFBC patients, including 21 index cases and 171 sporadic cases, in our study. Peripheral venous blood samples of all included participants were collected for genomic DNA extraction. The coding sequence of PDGFB was amplified by polymerase chain reaction (PCR) followed by direct sequencing. The potential effects of the identified variants on protein function were assessed by bioinformatics analysis. RESULTS: Three missense variants (c.35G>T, c.232C>T, and c.610C>A) and one nonsense variant (c.220G>T) of PDGFB were identified in five sporadic PFBC patients. The variant c.35G>T was found in 2 healthy controls from the same ethnic background, whereas c.220G>T, c.232C>T and c.610C>A were absent from 500 controls. c.220G>T (p.E74*) produced a stop codon in the place of the glutamicacid residue number 74. c.232C>T (p.R78C) occurred at highly conserved regions and were predicted as damaging by at least two computational predictive programs, suggesting that this variant were likely to have a causal role in PFBC. Although variant c.610C>A (p.P204T) also occurred at a highly conserved region, it was predicted to be most likely benign by two computational predictive programs, suggesting an uncertain role of this variant on PFBC. CONCLUSIONS: The present study identified one likely pathogenic variant (p.E74*) and two variants of uncertain significance (p.R78C and p.P204T) in PDGFB. Further studies of PDGF-B functional expression for these variants are still required to confirm the pathogenic effect.

Multilocus sequencing typing of Pseudomonas aeruginosa isolates and analysis of potential pathogenicity of typical genotype strains from occupational oxyhelium saturation divers.

BACKGROUND: Pseudomonas aeruginosa (P. aeruginosa) is a common microbe isolated from divers with ear and skin infections. To obtain the epidemic characters of the occurrence of the P. aeruginosa infection, multilocus sequence typing (MLST) was used to assess the genetic background of different strains isolated from divers involved in saturation diving. METHODS: A total of 64 P. aeruginosa strains from naval divers were sequenced by multilocus sequence typing using seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA and trpE). The results were analyzed based on the P. aeruginosa international MLST database to obtain the allelic profiles and sequence types (STs). MLST data were analyzed by Bionumerics 4.0 (http: // pubmlst.org/mlstanalyse) using LIAN and eBURST. Twenty-eight strains with the typical genotype were selected for further analysis of pathogenic characteristics by Caenorhabditis elegans (C. elegans) fast killing model. RESULTS: Data from MLST revealed a high STs diversity among the strains. Of the 64 strains, 53 strains were assigned to 19 STs, and the remaining 11 clones could not be assigned. ST274 accounted for 18.5% (12/64), and ST260 accounted for 15.62% (10/64). C. elegans killing assay showed that all the test strains had distinct virulent properties as compared with the negative control group. Clone 503-1 had the highest virulence and clone 54 had the lowest virulence as compared with the positive clinical group. CONCLUSION: The P. aeruginosa strains carried by the occupational diver groups in Chinese regions have characteristically dominant STs, and have a relatively strong virulence as compared with the standard strain and the clinically isolated positive control strain.

[Regulation of antigen specific Th17 cells differentiation in experimental autoimmune uveitis].

OBJECTIVE: To assess the regulation of antigen specific Th17 cells differentiation in experimental autoimmune uveitis (EAU). METHODS: A randomized controlled trials research. EAU model was made through subcutaneous injection of interphotoreceptor retinoid-binding protein (IRBP) at backs and bellies. Mouse splenic CD4 T cells were collected on the fourteenth day and cultured in vitro in the cell culture plates containing IRBP antigen for 72 hours under different conditions: control group, TGF-beta group, IL-6 group, IL-23 group, TGF-beta + IL-6 group, TGF-beta + IL-6 + IL-23 group, IL-27 group, all transretinoic acid (ATRA) group. Cells and the clear liquid were collected. Then Th17 cells, IL-17 and other cells, cytokines were assessed by flow cytometry and ELISA. All data were analyzed by Student's t test. RESULTS: Flow cytometric detection and ELISA experimental results showed:IRBP polypeptide alone mainly induced Th17 and Th1 response. Addition of TGF-beta and IL-6 induced the differentiation of antigen specific Th17 cells. Percentage of Th17 cells increased from 7.55% to 13.08% (t = -2.842, P = 0.048), and the concentration of IL-17 in cell culture fluid increased from 50.66 microg/L to 164.12 microg/L (t = -9.493, P = 0.009). Percentage of Th1 cells reduced from 6.33% to 3.43% (t = 6.059, P = 0.004). Percentage of Treg cells reduced from 4.96% to 1.52% (t = 5.683, P = 0.005); Furthermore, addition of IL-23 could enhance Th17 cells differentiation induced by TGF-beta and IL-6. Compared with IRBP polypeptide alone group, percentage of Th17 cells increased from 7.55% to 18.37% (t = -3.329, P = 0.029), and percentage of Th1 and Th2 cells reduced obviously (t = 7.410, P = 0.002; t = -3.863, P = 0.018). While IL-27 suppressed the differentiation of Th17 cells. Percentage of Th17 cells reduced from 7.55% to 1.92% (t = 4.425, P = 0.041), while percentage of Treg cells increased from 4.96% to 9.98% (t = -5.073, P = 0.015); In ATRA group, percentage of Th17 cells reduced from 7.55% to 4.06% (t = 2.163, P = 0.099). CONCLUSIONS: Antigen specific Th17 differentiation is distinct from Th1 and Th2 cells. TGF-beta, IL-6 and IL-23 are the factors responsible for promoting the differentiation and development of Th17 subset, whereas IL-27 has inhibitory effects.

Different activation of ERK1/2 and p38 with hyperbaric oxygen in dorsal root ganglion.

Prolonged hyperbaric oxygen exposure causes pulmonary and nervous system toxicity, although hyperbaric oxygen treatment has been used to treat a broad spectrum of ailments. In the current study, animals have been exposed to 100% oxygen at a pressure of 2.3 atmospheres absolute (ATA) for two, six and 10 hours or 0.23 MPa normoxic hyperbaric nitrogen (N2-O2 mixture, oxygen partial pressure = 21 kPa) for 10 hours. Then we investigated whether ERK1/2 and p38 had been activated in the dorsal root ganglion (DRG) by hyperbaric conditions. Using Western blot analysis, we found that the phosphorylation levels of ERK1/2 (phospho-ERK1/2) increased significantly (p < 0.05, n = 3 for each group) in the six-hour treatment of 100% oxygen at a pressure of 2.3 ATA. The phosphorylation levels of p38 (phospho-p38) increased significantly (p < 0.05, n = 3 for each group) in the 10-hour treatment of 100% oxygen at a pressure of 2.3 ATA--which was consistent with time course changes of an apoptosis marker, cleavage caspase-3--while the phospho-p38 decreased in the 10 hours of N2-O2 mixture. These results demonstrate that the ERK1/2 and p38 have been differently activated in the DRG by prolonged hyperbaric oxygen exposure.