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A sensitive and rapid method for the simultaneous determination of nine anticoagulant rodenticides (ARs) in human blood is reported herein. The method involves phospholipid removal pretreatment for reduced matrix effect (ME) and detection with ultra-performance liquid chromatography coupled with tandem mass spectrometry. Satisfactory recoveries were achieved ranging from 80.6% to 113.1% for the nine analytes, with the intra-day relative standard deviations (RSDs) in the range of 3.4-7.9% and inter-day RSDs in the range of 4.1-8.3%, indicating good precision. Linear relationships with correlation coefficients above 0.998 (n = 6) were found in the range of 1-2,000 ng/mL. High sensitivity was achieved with limits of detection ranging from 0.02 to 0.3. The application of phospholipid removal step significantly optimized the ME, and the reduction of ME ranged from 6.1% to 15.5%. This method was successfully applied to the determination of ARs for blood samples from real forensic cases. These results prove that this method is reliable for rapid forensic and clinical diagnosis. The removal capabilities for five representative phospholipids that are abundant in blood were evaluated individually with Phree™ phospholipid removal plates. While significant capabilities for phospholipid removal were confirmed, the results showed that the removal capability for certain phospholipid could be improved.

Background: Leber congenital amaurosis (LCA) is a visual disease which is caused by RPE65 mutations and results in retinal degeneration and severe vision loss in early infancy. According to previous researches, mutations of the RPE65 gene account for 16% of all cases of LCA. This study aimed to identify RPE65 gene mutations in Chinese patients with LCA. Methods: We recruited 52 sporadic patients from Peking University Third Hospital in 2016 and applied Sanger sequencing to identify variants among exons responsible for the disease. The genomic DNAs from blood leukocytes of these patients were isolated, and the entire coding region of the RPE65 gene was amplified by polymerase chain reaction. We then determined the sequence of RPE65 using ABI 3100 Genetic Analyzer. Results: Our study identified that only 1 out of the 52 patients with LCA carried the previously unreported homozygosis missense mutation c1174A>C (T392P) of the RPE65 gene. However, the mutation was associated with the disease phenotype and not detected in 100 normal controls. Conclusions: Though we identified a novel missense mutation in the RPE65 gene that causes LCA, our result indicates that RPE65 mutations may not play a major role in the LCA patients in China since only 1 out of the 52 patients carried mutation in the RPE65 gene. Key words: Leber Congenital Amaurosis; Mutation; RPE65

A number of poisoning and suicide cases involving formamidine pesticides have been reported, thus developing a rapid and low cost determination method is crucial. In this work, a rapid, sensitive and low-cost method for the simultaneous determination of formamidine pesticides (amitraz, chlordimeform, formetanate) and their main metabolites, N-(2,4-dimethylphenyl)-N-methyl-formamidine, 2,4-dimethylformamidine, 2,4-dimethylaniline, 4-chloro-2-methylaniline and 3-hydroxyacetanilide in human blood by high-performance liquid chromatography with tandem mass spectrometry is developed. The application of columns with core-shell particles significantly reduced the analysis time. Very low LODs (0.01–0.04 μg L−1) were obtained for formamidine pesticides and their metabolites. The method was successfully applied to the analysis of human blood samples from a real forensic case. The significantly reduced analysis time, high sensitivity and low cost are the primary advantages of the developed method. This methodology provides important value for sensitive and rapid determination of residue pesticides and metabolites, study of residue pesticides behavior in human body, as well as application in real forensic cases.

Image segmentation is an important tool in image processing and can serve as an efficient front end to sophisticated algorithms and thereby simplify subsequent processing. In this paper, we present a pixel-based color image segmentation using Support Vector Machine (SVM) and Fuzzy C-Means (FCM). Firstly, the pixel-level color feature and texture feature of the image, which is used as input of the SVM model (classifier), are extracted via the local spatial similarity measure model and Steerable filter. Then, the SVM model (classifier) is trained by using FCM with the extracted pixel-level features. Finally, the color image is segmented with the trained SVM model (classifier). This image segmentation can not only take full advantage of the local information of the color image but also the ability of the SVM classifier. Experimental evidence shows that the proposed method has a very effective computational behavior and effectiveness, and decreases the time and increases the quality of color image segmentation in comparison with the state-of-the-art segmentation methods recently proposed in the literature.

Ocular coloboma is a developmental defect of the eye and is due to abnormal or incomplete closure of the optic fissure. This disorder displays genetic and clinical heterogeneity. Using a positional cloning approach, we identified a mutation in the ATP-binding cassette (ABC) transporter ABCB6 in a Chinese family affected by autosomal-dominant coloboma. The Leu811Val mutation was identified in seven affected members of the family and was absent in six unaffected members from three generations. A LOD score of 3.2 at θ = 0 was calculated for the mutation identified in this family. Sequence analysis was performed on the ABCB6 exons from 116 sporadic cases of microphthalmia with coloboma (MAC), isolated coloboma, and aniridia, and an additional mutation (A57T) was identified in three patients with MAC. These two mutations were not present in the ethnically matched control populations. Immunostaining of transiently transfected, Myc-tagged ABCB6 in retinal pigment epithelial (RPE) cells showed that it localized to the endoplasmic reticulum and Golgi apparatus of RPE cells. RT-PCR of ABCB6 mRNA in human cell lines and tissue indicated that ABCB6 is expressed in the retinae and RPE cells. Using zebrafish, we show that abcb6 is expressed in the eye and CNS. Morpholino knockdown of abcb6 in zebrafish produces a phenotype characteristic of coloboma and replicates the clinical phenotype observed in our index cases. The knockdown phenotype can be corrected with coinjection of the wild-type, but not mutant, ABCB6 mRNA, suggesting that the phenotypes observed in zebrafish are due to insufficient abcb6 function. Our results demonstrate that ABCB6 mutations cause ocular coloboma.

Background The PARK2 gene was recently identified as a causative gene for autosomal recessive early-onset Parkinson's disease (EOPD). Studies on how specific PARK2 mutations are manifested on different genetic backgrounds may benefit prognosis and clinical management. Until now, there have been no reports of PARK2 mutations in a Uyghur family with EOPD. Methods We identified a large Uyghur EOPD family with PARK2 mutations, and analyzed genealogical, clinical, and genetic data from the family. Results Three of 15 members were diagnosed with EOPD, and two point mutations, c.951G>C (p.G284R) and c.924C>T (p.R275W), were found in six family members. Among the mutation-positive members, the three affected members were compound heterozygote, while the three unaffected members were single heterozygote. Conclusion This is the first report describing a Uyghur family with PARK2 mutations. The compound heterozygous mutation c.951G>C (p.G284R) and c.924C>T (p.R275W) is the pathogenic factor in this EOPD Uyghur family.

Current therapy of malignant glioma in clinic is unsatisfactory with poor patient compliance due to low therapeutic efficiency and strong systemic side effects. Herein, we combined chemo-photothermal targeted therapy of glioma within one novel multifunctional drug delivery system. A targeting peptide (IP)-modified mesoporous silica-coated graphene nanosheet (GSPI) was successfully synthesized and characterized, and first introduced to the drug delivery field. A doxorubicin (DOX)-loaded GSPI-based system (GSPID) showed heat-stimulative, pH-responsive, and sustained release properties. Cytotoxicity experiments demonstrated that combined therapy mediated the highest rate of death of glioma cells compared to that of single chemotherapy or photothermal therapy. Furthermore, the IP modification could significantly enhance the accumulation of GSPID within glioma cells. These findings provided an excellent drug delivery system for combined therapy of glioma due to the advanced chemo-photothermal synergistic targeted therapy and good drug release properties of GSPID, which could effectively avoid frequent and invasive dosing and improve patient compliance.

Background: Congenital nystagmus (CN) is characterized by conjugated, spontaneous, and involuntary ocular oscillations. It is an inherited disease and the most common inheritance pattern is X-linked CN. In this study, our aim is to identify the disease-causing mutation in a large sixth-generation Chinese family with X-linked CN. Methods: It has been reported that mutations in four-point-one, ezrin, radixin, moesin domain-containing 7 gene (FRMD7) and G protein-coupled receptor 143 gene (GPR143) account for the majority patients of X-linked nystagmus. We collected 8 ml blood samples from members of a large sixth-generation pedigree with X-linked CN and 100 normal controls. FRMD7 and GPR143 were scanned by polymerase chain reaction (PCR)-based DNA sequencing assays, and multiplex PCR assays were applied to detect deletions. Results: We identified a previously unreported deletion covering 7 exons in GPR143 in a Chinese family. The heterozygous deletion from exon 3 to exon 9 of GPR143 was detected in all affected males in the family, while it was not detected in other unaffected relatives or 100 normal controls. Conclusions: This is the first report of molecular characterization in GPR143 gene in the CN family. Our results expand the spectrum of GPR143 mutations causing CN and further confirm the role of GPR143 in the pathogenesis of CN.

To investigate potential mutation of PHOX2A (or ARIX) gene in a Chinese family affected with congenital fibrosis of extraocular muscles tyep 2 (CFEOM2).Genomic DNA was obtained from affected and unaffected members of the family. With an ABI PRSIM Linkage Mapping Set-MD10 kit, selected markers flanking the PHOX2A locus were used for linkage analysis. Exons of PHOX2 gene were amplified and sequenced. A total of 100 normal subjects were recruited as controls.Genetic linkage was found at 11q13 between D11S4151 and D11S1320 and the PHOX2A gene. DNA sequencing has identified a heterozygous mutation in the exon 2 of the gene (227T to G, N76K). The same mutation was not found in the unaffected and 100 normal controls.A mutation of the PHOX2A gene 227T to G is responsible for the onset of congenital fibrosis of extraocular muscles type 2 in this Chinese family.

Purpose Screening of mutations in the fibrillin-1 (FBN1) gene in a Chinese family with autosomal dominant Marfan syndrome (MFS). Methods It has been reported that FBN1 mutations account for approximately 90% of Autosomal Dominant MFS. FBN1 mutations were analyzed in a Chinese family of 36 members including 13 MFS patients. The genomic DNAs from blood leukocytes of the patients and their relatives were isolated and the entire coding region of FBN1 was amplified by PCR. The sequence of FBN1 was dertermined with an ABI 3100 Genetic Analyzer. Results A previously unreported the missense mutation G214S (caused by a 640 A→G heterozygous change) in FBN1 was identified in the Chinese family. The mutation was associated with the disease phenotype in patients, but not detected in their relatives or in the 100 normal controls. Conclusions This is the first report of molecular characterization of FBN1 in the MFS family of Chinese origin. Our results expand the spectrum of FBN1 mutations causing MFS and further confirm the role of FBN1 in the pathogenesis of MFS. Direct sequencing of the mutation in FBN1 may be used for diagnosis of MFS.

To map the candidate gene by linkage analysis in a Chinese family with autosomal dominant congenital retinaochoroidal coloboma.A detailed clinical examination was performed for all patients in the family. The genomic DNA of all family members was extracted from peripheral blood leukocytes. Linkage analysis and genome-wide linkage screening was conducted using fluorescent detection of 398 microsatellite markers representing all autosomes at an average resolution of approximately 10 cM. Polymerase chain reaction was carried out to amplify all 398 microsatellite markers. The allele sizes were determined on ABI 3130-Avant genetic analyzer according to an internal size standard, and the results were analyzed using Genescan 3.1 and Genotyper 2.0 software.Linkage analysis showed the markers D2S2382-D2S301-D2S2244-D2S163 co-segregated with the disease locus in all affected members. The maximum Lod score was 3.01(D2S2382).The candidate region of the disease gene in the family was located in 2q34-2q35.

Mapping the mutation gene for a Chinese family with autosomal dominant cataract.It was a retrospective study. Thirty-two individuals in this family, including fifteen patients, eight normal siblings and nine spouses, were investigated and 8 ml blood was collected from each member under informed consent. Genomic DNA of all 29 members was isolated by standard protocol. A genome wide scan was performed after PCR amplification for microsatellite makers on autosomal chromosomes. LOD score was calculated by Linkage 5. 1 and GeneHunter software.Positive Lod score were obtained in 10 microsatellite makers (D20S186, D20S163, D20S915, D20S152, D20S98, D20S904, D20S875, D20S112, D20S1140, D20S432) on chromosome 20q, and the maximum LOD score with D20S904 was 6.02.Haplotype construction and multipoint analysis mapped the mutation gene in this inherited cataract family to the chromosome 20p12. 1-20p11.23 region between D20S186 and D20S912, which is an approximately 5.47 centimorgan length. This is the second congenital cataract locus linked to chromosome 20q.

To map and to identify the causal gene for autosomal dominant congenital cataract (ADCC) in a Chinese family.A four-generation family with a history of progressive congenital cataracts was investigated. Twenty-three members of the family were examined ophthalmologically. Blood samples were collected from twenty-nine family members for genetic linkage analysis. Two-point LOD scores were calculated. Multi-point linkage analysis and haplotype construction were performed to define the optimal cosegregating interval. Direct sequence analysis of the candidate gene, beaded filament structural protein 1, filensin (BFSP1) in the critical region was carried out.Fifteen family members were affected with autosomal dominant progressive congenital zonular nuclear cataract (ADPCZNC). The maximum two-point LOD Score of 6.02 was obtained for marker D20S904 (theta=0). The cataract locus in this family was mapped to chromosome 20p12.2-p11.23, a 9.34 Mb (16.37 cM) interval between markers D20S186 and D20S912. Although BFSP1 was in this critical region, we found no evidence that the condition in the family was caused by a BFSP1 mutation.We have mapped the genetic locus of ADPCZNC to chromosome 20p12.2-p11.23 in an ADCC family. This is the first time ADPCZNC was linked to this region.

Accessory auricular anomaly is a small excrescence of skin that contains elastic cartilage on different regions of the helix and the face. Previous work has shown that the genetic trait of some patients with the isolated symptom of accessory auricular anomaly is autosomal dominant. To map the gene for autosomal dominant accessory auricular anomaly (ADAAA), we investigated a Chinese family with 11 affected individuals. We performed linkage analysis with microsatellite markers spanning the whole human-genome in the family. The inheritance pattern of the ADAAA family was autosomal dominant with complete penetrance. Two-point linkage analysis revealed significant maximum LOD scores of 4.20(D14S990 and D14S264, sita = 0) in the family. Haplotype construction and multipoint linkage analysis also confirmed the locus and defined the isolated ADAAA locus to a 9.84 cM interval between the markers D14S283 and D14S297. Our study assigned an isolated ADAAA locus to 14q11.2-q12. This is the first ADAAA locus reported to date.