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1.
Biomedicines ; 12(4)2024 Apr 12.
Article in English | MEDLINE | ID: mdl-38672207

ABSTRACT

It is crucial to regulate N-methyl-D-aspartate (NMDA) function bivalently depending on the central nervous system (CNS) conditions. CNS disorders with NMDA hyperfunction are involved in the pathogenesis of neurotoxic and/or neurodegenerative disorders with elevated D-serine, one of the NMDA receptor co-agonists. On the contrary, NMDA-enhancing agents have been demonstrated to improve psychotic symptoms and cognition in CNS disorders with NMDA hypofunction. Serine racemase (SR), the enzyme regulating both D- and L-serine levels through both racemization (catalysis from L-serine to D-serine) and ß-elimination (degradation of both D- and L-serine), emerges as a promising target for bidirectional regulation of NMDA function. In this study, we explored using dimethyl malonate (DMM), a pro-drug of the SR inhibitor malonate, to modulate NMDA activity in C57BL/6J male mice via intravenous administration. Unexpectedly, 400 mg/kg DMM significantly elevated, rather than decreased (as a racemization inhibitor), D-serine levels in the cerebral cortex and plasma. This outcome prompted us to investigate the regulatory effects of dodecagalloyl-α-D-xylose (α12G), a synthesized tannic acid analog, on SR activity. Our findings showed that α12G enhanced the racemization activity of human SR by about 8-fold. The simulated and fluorescent assay of binding affinity suggested a noncooperative binding close to the catalytic residues, Lys56 and Ser84. Moreover, α12G treatment can improve behaviors associated with major CNS disorders with NMDA hypofunction including hyperactivity, prepulse inhibition deficit, and memory impairment in animal models of positive symptoms and cognitive impairment of psychosis. In sum, our findings suggested α12G is a potential therapeutic for treating CNS disorders with NMDA hypofunction.

2.
World J Gastrointest Surg ; 15(8): 1684-1692, 2023 Aug 27.
Article in English | MEDLINE | ID: mdl-37701706

ABSTRACT

BACKGROUND: The liver hemodynamic changes caused by portal hypertension (PH) are closely related to various complications such as gastroesophageal varices and portosys-temic shunts, which may lead to adverse clinical outcomes in these patients, so it is of great clinical significance to find treatment strategies with favorable clinical efficacy and low risk of complications. AIM: To study the clinical efficacy of total laparoscopic splenectomy (TLS) for PH and its influence on hepatic hemodynamics and liver function. METHODS: Among the 199 PH patients selected from October 2016 to October 2020, 100 patients [observation group (OG)] were treated with TLS, while the remaining 99 [reference group (RG)] were treated with open splenectomy (OS). We observed and compared the clinical efficacy, operation indexes [operative time (OT) and intraoperative bleeding volume], safety (intraperitoneal hemorrhage, ascitic fluid infection, eating disorders, liver insufficiency, and perioperative death), hepatic hemodynamics (diameter, velocity, and flow volume of the portal vein system), and liver function [serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), and serum total bilirubin (TBil)] of the two groups. RESULTS: The OT was significantly longer and intraoperative bleeding volume was significantly lesser in the OG than in the RG. Additionally, the overall response rate, postoperative complications rate, and liver function indexes (ALT, AST, and TBil) did not differ significantly between the OG and RG. The hepatic hemodynamics statistics showed that the pre- and postoperative blood vessel diameters in the two cohorts did not differ statistically. Although the postoperative blood velocity and flow volume reduced significantly when compared with the preoperative values, there were no significant inter-group differences. CONCLUSION: TLS contributes to comparable clinical efficacy, safety, hepatic hemodynamics, and liver function as those of OS in treating PH, with a longer OT but lesser intraoperative blood loss.

3.
J Alzheimers Dis Rep ; 6(1): 557-575, 2022.
Article in English | MEDLINE | ID: mdl-36275418

ABSTRACT

Background: Alzheimer's disease (AD) is a multifactorial neurodegenerative disease affecting many cellular pathways, including protein aggregation, mitochondrial dysfunction, oxidative stress (OS), and neuroinflammation. Currently, no effective treatment for AD exists. Objective: We aim to determine the effect of lithium benzoate (LiBen) in protecting neurons from amyloid-ß (Aß) or other neurotoxin insults. Methods: Primary rat cortical neurons co-treated with neurotoxins and LiBen were used to examine its effect in cell viability, reactive oxygen species (ROS) clearance, and mitochondrial functions by MTT, CellRox fluorescence staining, and seahorse assay. Then, Barnes maze and prepulse inhibition test were performed in APP/PS1 mice that received chronic LiBen treatment to assess its effect on cognitive protection. Oral bioavailability of LiBen was also assessed by pharmacokinetic study in rat plasma. Results: In this study, we discovered that LiBen can attenuate cellular ROS level, improve mitochondrial function, increase cell viability against multiple different insults of mitochondrial dysfunction, Aß accumulation, and neuroinflammation, and promote neurogenesis. We demonstrated that LiBen has advantages over lithium or sodium benzoate alone as LiBen displays superior neuroprotective efficacy and oral bioavailability than the other two agents when being applied either alone or in combination. Furthermore, chronic administration of LiBen showed protection for cognition as well as spatial memory and reduced the senile plaque deposition in brains of AD animal models. Conclusion: LiBen stands as a promising therapeutic agent for improving cognition and delaying the progression of AD.

4.
ACS Pharmacol Transl Sci ; 5(6): 400-412, 2022 Jun 10.
Article in English | MEDLINE | ID: mdl-37582235

ABSTRACT

The rampageous transmission of SARS-CoV-2 has been devastatingly impacting human life and public health since late 2019. The waves of pandemic events caused by distinct coronaviruses at present and over the past decades have prompted the need to develop broad-spectrum antiviral drugs against them. In this study, our Pentarlandir ultrapure and potent tannic acids (UPPTA) showed activities against two coronaviral strains, SARS-CoV-2 and HCoV-OC43, the earliest-known coronaviruses. The mode of inhibition of Pentarlandir UPPTA is likely to act on 3-chymotrypsin-like protease (3CLpro) to prevent viral replication, as supported by results of biochemical analysis, a 3CLpro assay, and a "gain-of-function" 3CLpro overexpressed cell-based method. Even in the 3CLpro overexpressed environment, Pentarlandir UPPTA remained its antiviral characteristic. Utilizing cell-based virucidal and cytotoxicity assays, the 50% effective concentrations (EC50) and 50% cytotoxicity concentration (CC50) of Pentarlandir UPPTA were determined to be ∼0.5 and 52.5 µM against SARS-CoV-2, while they were 1.3 and 205.9 µM against HCoV-OC43, respectively. In the pharmacokinetic studies, Pentarlandir UPPTA was distributable at a high level to the lung tissue with no accumulation in the body, although the distribution was affected by the food effect. With further investigation in toxicology, Pentarlandir UPPTA demonstrated an overall safe toxicology profile. Taking these findings together, Pentarlandir UPPTA is considered to be a safe and efficacious pancoronal antiviral drug candidate that has been advanced to clinical development.

5.
J Anim Physiol Anim Nutr (Berl) ; 105(6): 1002-1013, 2021 Nov.
Article in English | MEDLINE | ID: mdl-33899975

ABSTRACT

The potential reproduction power of domestic animals is limited by a complicated follicular atresia process. P53, caspase-9 (Casp9), Bax, Bcl-2 and Fas play a crucial role in the ovarian mitochondrion-dependent apoptosis and death receptor pathway. In accordance with this study, the expression levels of Casp9, Bax, Bcl-2 and Fas were analysed in ovaries and oviducts of yak by immunohistochemistry (IHC). P53 and the above in ovarian granulosa cells (GCs) from atretic (3-6 mm) to healthy follicles (6-8 mm) and in oviducts were examined from the luteal phase to the follicular phase during the oestrous circle by Western blot (WB) and real-time PCR (RT-PCR). Results demonstrated that typical classic apoptotic factors Casp9, Bax, Bcl-2 and Fas were expressed in the cytoplasm and zonal pellucida of oocytes, primordial follicles, primary follicles, ovarian surface epithelium, ovarian GCs, granular lutein cells, surface epithelia in oviduct uterotubal junction and oviduct ampulla during the luteal phase. RT-PCR and WB revealed that P53 and Fas significantly increased in GCs of atretic follicles. P53 and Casp9 increased in oviduct epithelium during the luteal phase, but Fas was unchanged. A contrary tendency was noted in Bcl-2 and Bax expression. Overall, P53 and Fas play an essential role in inducing GC apoptosis, and Bax, Bcl-2, Casp9 and P53 are involved in oviduct epithelial regeneration in yak.


Subject(s)
Apoptosis , Follicular Atresia , Animals , Cattle , Female , Gene Expression , Granulosa Cells , Ovarian Follicle
6.
Ecotoxicol Environ Saf ; 206: 111329, 2020 Dec 15.
Article in English | MEDLINE | ID: mdl-32979722

ABSTRACT

The aim of the study was to investigate the protective effects of selenium yeast (SeY) against necroptosis triggered by Cd via inhibition of oxidative stress and MAPK pathway in the liver of chicken. Two hundred 120-day-old layers were randomly divided into four groups and raised for 120 days. The histopathological examination showed that necrosis characteristics were observed in Cd-exposed chicken livers. The exposure of Cd significantly reduced the activities of SOD, GSH-Px and CAT while improving MDA level in both serum and liver of chickens (P < 0.05), and induced oxidative stress. The MLKL, Rip1, RIP3, ERK, JNK and P38 mRNA expression of Cd group were significantly higher than other three groups (P < 0.01), and those in the Se + Cd group were significantly higher than control group and Se group (P < 0.01). However, the mRNA expression level of caspase8 of Cd was significantly lower than other three groups (P < 0.01), and that in the Se + Cd group was significantly higher than control group and Se group (P < 0.01), so the supplement of SeY could improve these situations. Similar results were also detected at the protein level. The results of the present study indicated that Cd could induce oxidative stress, activate MAPK pathway and evoke necroptosis damage in chicken livers, whereas SeY had protective effects in preventing this kind of Cd-induced injury by inhibition of oxidative stress and down-regulation MAPK pathway.


Subject(s)
Cadmium/toxicity , Hazardous Substances/toxicity , Protective Agents/pharmacology , Selenium/pharmacology , Animals , Cadmium/metabolism , Chickens/metabolism , Dietary Supplements , Liver/drug effects , Necroptosis , Oxidative Stress/drug effects , Saccharomyces cerevisiae/metabolism
7.
Mol Med Rep ; 17(5): 6472-6482, 2018 05.
Article in English | MEDLINE | ID: mdl-29512731

ABSTRACT

Research advances and analysis in the non­protein coding part of the human genome have suggested that microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are associated with tumor initiation, growth and metastasis. Accumulating studies have demonstrated that a class of miRNAs and lncRNAs are dysregulated in hepatocellular carcinoma (HCC) and closely associated with tumorigenesis, diagnosis and prognosis. In the present study, integrative analysis of published data on multi­level Gene Expression Omnibus (GEO) and a bioinformatics computational approach were used to predict regulatory mechanism networks among differentially expressed mRNAs, miRNAs, and lncRNAs. Firstly, nine microarray expression data sets of mRNAs, miRNAs, and lncRNAs associated with HCC were collected from GEO datasets. Secondly, a total of 628 mRNAs, 15 miRNAs, and 49 lncRNAs were differentially expressed in this integrative analysis. Following this, mRNA, miRNA and lncRNA regulatory or co­expression networks were constructed. From the construction of the regulatory networks, five miRNAs and ten lncRNAs were identified as key differentially expressed noncoding RNAs associated with HCC progression. Finally, the regulatory effects of ten lncRNAs and miRNAs were validated. The study provides a novel insight into the understanding of the transcriptional regulation of HCC, and differentially expressed lncRNAs targeted and regulated by miRNAs were identified and validated in HCC specimens and cell lines.


Subject(s)
Carcinoma, Hepatocellular/metabolism , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Liver Neoplasms/metabolism , MicroRNAs/biosynthesis , RNA, Long Noncoding/biosynthesis , RNA, Messenger/biosynthesis , RNA, Neoplasm/biosynthesis , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , MicroRNAs/genetics , RNA, Long Noncoding/genetics , RNA, Messenger/genetics , RNA, Neoplasm/genetics
8.
Biochem Biophys Res Commun ; 490(3): 920-926, 2017 08 26.
Article in English | MEDLINE | ID: mdl-28651931

ABSTRACT

A growing amount of literature has indicated that long non-coding RNAs (lncRNAs) are important factors in hepatocellular carcinoma (HCC) progression. However, the significance of lncRNAs in the progression and prognosis of liver cancer is largely unknown. In the present study, upregulated lncRNA LOC90784 was identified through integrative analysis of GSE58043 and GSE55191. Furthermore, associations between LOC90784 expression and the clinicopathological characteristics of patients were analyzed with a validated cohort 1 and the Cancer Genome Atlas (TCGA) cohort 2. We investigated the mechanisms by which this highly expressed lncRNA promotes HCC proliferation, invasion and migration via qRT-PCR, fluorescence in situ hybridization (FISH) staining, siRNA transfection, cell proliferation assays, Transwell and colony formation assays, flow cytometry analysis and Western blot. The results showed that LOC90784 expression levels were significantly higher in HCC cell lines and tissues and mainly localized in the cytoplasm. Knockdown of lncRNA LOC90784 expression inhibited proliferation and induced apoptosis and cell cycle arrest by promoting Bax and repressing CDK4 and Cyclin D1 protein expression; it also inhibited invasion and migration by repressing MMP2 and MMP9 expression in HCC cells. LOC90784 overexpression was associated with poor clinical features in the two cohorts and poor overall survival rates in HCC patients with clear resection margins (R0) in cohort 2. These results indicated that LOC90784 upregulation may be a critical oncogene and potential new biomarker in HCC.


Subject(s)
Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , Liver/pathology , Neoplasm Invasiveness/pathology , RNA, Long Noncoding/genetics , Apoptosis , Carcinoma, Hepatocellular/genetics , Cell Line , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , Humans , Liver/metabolism , Liver Neoplasms/genetics , Neoplasm Invasiveness/genetics , Up-Regulation
9.
Genome Res ; 27(5): 813-823, 2017 05.
Article in English | MEDLINE | ID: mdl-28360230

ABSTRACT

The most polymorphic part of the human genome, the MHC, encodes over 160 proteins of diverse function. Half of them, including the HLA class I and II genes, are directly involved in immune responses. Consequently, the MHC region strongly associates with numerous diseases and clinical therapies. Notoriously, the MHC region has been intractable to high-throughput analysis at complete sequence resolution, and current reference haplotypes are inadequate for large-scale studies. To address these challenges, we developed a method that specifically captures and sequences the 4.8-Mbp MHC region from genomic DNA. For 95 MHC homozygous cell lines we assembled, de novo, a set of high-fidelity contigs and a sequence scaffold, representing a mean 98% of the target region. Included are six alternative MHC reference sequences of the human genome that we completed and refined. Characterization of the sequence and structural diversity of the MHC region shows the approach accurately determines the sequences of the highly polymorphic HLA class I and HLA class II genes and the complex structural diversity of complement factor C4A/C4B It has also uncovered extensive and unexpected diversity in other MHC genes; an example is MUC22, which encodes a lung mucin and exhibits more coding sequence alleles than any HLA class I or II gene studied here. More than 60% of the coding sequence alleles analyzed were previously uncharacterized. We have created a substantial database of robust reference MHC haplotype sequences that will enable future population scale studies of this complicated and clinically important region of the human genome.


Subject(s)
Complement C4/genetics , Genes, MHC Class II , Genes, MHC Class I , Haplotypes , Mucins/genetics , Polymorphism, Genetic , Animals , Cell Line , Contig Mapping/methods , Contig Mapping/standards , Genome, Human , Genomics/methods , Genomics/standards , Humans , Open Reading Frames , Pan troglodytes/genetics , Reference Standards
10.
Tumour Biol ; 37(11): 14813-14824, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27638830

ABSTRACT

MicroRNAs (miRNAs) play important roles in the regulation of various tumor biological processes including proliferation and apoptosis. MiR-377 has been implicated in many types of cancer, whereas its expressional feature and potential biological function in pancreatic ductal adenocarcinoma (PDAC) remains unclear. In this study, we scanned the global miRNA expression profiles in PDAC from The Cancer Genome Atlas (TCGA) and found miR-377 was down-regulated significantly in PDAC. Then, its expression was measured in both pancreatic cancer tissues and cells; the data showed that miR-377 was de-regulated and inversely correlated with pathologic parameters of tumor growth or metastasis. We generated PDAC cell lines with stable overexpression or inhibition of miR-377, and our results indicated that miR-377 up-regulation significantly promoted cell viability, proliferation, and migration in PDAC cells, and also induced cell apoptosis and cell cycle arrest simultaneously. Binding-site predictions by bioinformatics showed that Pim-3 might be a potential target of miR-377. Luciferase reporter assay ulteriorly identified that miR-377 suppressed Pim-3 expression by binding the 3'-UTR. In tumor tissues, we also showed that the Pim-3 expression was inversely correlated with that of miR-377. Furthermore, stable ectopic miR-377 expression in pancreatic cancer cell lines suppressed Pim-3 expression, leading to the attenuation of Bad phosphorylation level at its Ser112 and promoting cell apoptosis. Overall, these results reveal that miR-377 may have tumor growth suppression function by down-regulating Pim-3 kinase expression to inhibit both pancreatic tumor growth and migration, and induce cell apoptosis. Hence, miR-377 may be a potential diagnostic marker and therapeutic target.


Subject(s)
Apoptosis/genetics , Carcinoma, Pancreatic Ductal/genetics , MicroRNAs/genetics , Pancreatic Neoplasms/genetics , Protein Serine-Threonine Kinases/biosynthesis , Proto-Oncogene Proteins/biosynthesis , 3' Untranslated Regions/genetics , Biomarkers, Tumor/genetics , Carcinoma, Pancreatic Ductal/pathology , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Cell Survival/genetics , Female , Gene Expression Regulation, Neoplastic/genetics , HEK293 Cells , Humans , Male , MicroRNAs/biosynthesis , Middle Aged , Pancreatic Neoplasms/pathology , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics , bcl-Associated Death Protein/metabolism , Pancreatic Neoplasms
11.
J Vet Med Sci ; 77(12): 1617-24, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26268666

ABSTRACT

MicroRNAs (miRNAs) are a class of short endogenous, single-stranded, non-coding small RNA molecules, about 19-25 nucleotides in length that regulate gene expression at the translation level and influence many physiological process, such apoptosis, metabolism, signal transduction, and occurrence and development of diseases. In this study, we constructed a library from the ovine luteal phase ovary by using next-generation sequencing technology (Solexa high-throughput sequencing technique) and identified 267 novel miRNAs by bioinformatics. One of the novel miRNAs (ovis_aries_ovary-m0033_3p), which expressed in the sheep ovary and testis, was confirmed by real time PCR and northern blot. Ovis_aries_ovary-m0033_3p was 21 nucleotides in length and located on chromosome 12, and it had 100% similarity to hsa-miR-214-3p, mmu-miR-214-3p, dre-miR-214and ssc-miR-214. Meanwhile, the pre-miRNA was 82 nucleotides in length and had a standard hairpin stem-loop structure. From the consistency of the sequence and structure, we speculated that ovis_aries_ovary-m0033_3p had a function similar to hsa-miR-214-3p, which is involved in the fine regulation of cell survival, embryonic development, breeding activities and resistance to ovarian cancer, so we defined it as oar-miR-214-3p. These experimental results will enrich the miRNA database for ovis aries and provide the basis for researching the regulation mechanism of miRNA in relation to breeding activities of seasonal breeding animals.


Subject(s)
Computational Biology/methods , MicroRNAs/isolation & purification , Ovary/metabolism , Sheep , Animals , Female , Gene Expression Profiling , Gene Expression Regulation , High-Throughput Nucleotide Sequencing/methods , Real-Time Polymerase Chain Reaction
12.
Genome Biol Evol ; 5(7): 1376-92, 2013.
Article in English | MEDLINE | ID: mdl-23814129

ABSTRACT

Domestic chickens are excellent models for investigating the genetic basis of phenotypic diversity, as numerous phenotypic changes in physiology, morphology, and behavior in chickens have been artificially selected. Genomic study is required to study genome-wide patterns of DNA variation for dissecting the genetic basis of phenotypic traits. We sequenced the genomes of the Silkie and the Taiwanese native chicken L2 at ∼23- and 25-fold average coverage depth, respectively, using Illumina sequencing. The reads were mapped onto the chicken reference genome (including 5.1% Ns) to 92.32% genome coverage for the two breeds. Using a stringent filter, we identified ∼7.6 million single-nucleotide polymorphisms (SNPs) and 8,839 copy number variations (CNVs) in the mapped regions; 42% of the SNPs have not found in other chickens before. Among the 68,906 SNPs annotated in the chicken sequence assembly, 27,852 were nonsynonymous SNPs located in 13,537 genes. We also identified hundreds of shared and divergent structural and copy number variants in intronic and intergenic regions and in coding regions in the two breeds. Functional enrichments of identified genetic variants were discussed. Radical nsSNP-containing immunity genes were enriched in the QTL regions associated with some economic traits for both breeds. Moreover, genetic changes involved in selective sweeps were detected. From the selective sweeps identified in our two breeds, several genes associated with growth, appetite, and metabolic regulation were identified. Our study provides a framework for genetic and genomic research of domestic chickens and facilitates the domestic chicken as an avian model for genomic, biomedical, and evolutionary studies.


Subject(s)
Chickens/genetics , Genetic Variation , Genome , Animals , Breeding , Chickens/physiology , DNA Copy Number Variations , INDEL Mutation , Phenotype , Polymorphism, Single Nucleotide , Quantitative Trait Loci , Sequence Alignment , Sequence Analysis, DNA
13.
Genome Res ; 16(9): 1136-48, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16899659

ABSTRACT

Array-CGH is a powerful tool for the detection of chromosomal aberrations. The introduction of high-density SNP genotyping technology to genomic profiling, termed SNP-CGH, represents a further advance, since simultaneous measurement of both signal intensity variations and changes in allelic composition makes it possible to detect both copy number changes and copy-neutral loss-of-heterozygosity (LOH) events. We demonstrate the utility of SNP-CGH with two Infinium whole-genome genotyping BeadChips, assaying 109,000 and 317,000 SNP loci, to detect chromosomal aberrations in samples bearing constitutional aberrations as well tumor samples at sub-100 kb effective resolution. Detected aberrations include homozygous deletions, hemizygous deletions, copy-neutral LOH, duplications, and amplifications. The statistical ability to detect common aberrations was modeled by analysis of an X chromosome titration model system, and sensitivity was modeled by titration of gDNA from a tumor cell with that of its paired normal cell line. Analysis was facilitated by using a genome browser that plots log ratios of normalized intensities and allelic ratios along the chromosomes. We developed two modes of SNP-CGH analysis, a single sample and a paired sample mode. The single sample mode computes log intensity ratios and allelic ratios by referencing to canonical genotype clusters generated from approximately 120 reference samples, whereas the paired sample mode uses a paired normal reference sample from the same individual. Finally, the two analysis modes are compared and contrasted for their utility in analyzing different types of input gDNA: low input amounts, fragmented gDNA, and Phi29 whole-genome pre-amplified DNA.


Subject(s)
Chromosome Aberrations , Genomics/methods , Oligonucleotide Array Sequence Analysis/methods , Cell Line, Tumor , Chromosomes, Human/metabolism , DNA/metabolism , Female , Genome, Human , Genotype , Humans , In Situ Hybridization, Fluorescence , Loss of Heterozygosity , Male , Polymorphism, Single Nucleotide
14.
Methods Enzymol ; 410: 359-76, 2006.
Article in English | MEDLINE | ID: mdl-16938560

ABSTRACT

We have developed an array-based whole-genome genotyping (WGG) assay (Infinium) using our BeadChip platform that effectively enables unlimited multiplexing and unconstrained single nucleotide polymorphism (SNP) selection. A single tube whole-genome amplification reaction is used to amplify the genome, and loci of interest are captured by specific hybridization of amplified gDNA to 50-mer probe arrays. After target capture, SNPs are genotyped on the array by a primer extension reaction in the presence of hapten-labeled nucleotides. The resultant signal is amplified during staining and the array is read out on a high-resolution confocal scanner. We have employed our high-density BeadChips supporting up to 288,000 bead types to create an array that can query over 100,000 SNPs using the Infinium assay. In addition, we have developed an automated BeadChip processing platform using Tecan's GenePaint slide processing system. Hybridization, washing, array-based primer extension, and staining are performed directly in Tecan's capillary gap Te-Flow chambers. This automation process increases assay robustness and throughput greatly while enabling laboratory information management system control of sample tracking.


Subject(s)
Genome , Oligonucleotide Array Sequence Analysis/methods , Animals , Genotype , Humans
15.
Nat Methods ; 3(1): 31-3, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16369550

ABSTRACT

We describe an efficient, accurate and robust whole-genome genotyping (WGG) assay based on a two-color, single-base extension (SBE), single-nucleotide polymorphism (SNP)-scoring step. We report genotyping results for biallelic International HapMap quality control (QC) SNPs using a single probe per locus. We show scalability, throughput and accuracy of the system by resequencing homozygous loci from our 100k Human-1 Genotyping BeadChip.


Subject(s)
DNA Primers/chemistry , Genome, Human/genetics , Genomics/methods , Polymorphism, Single Nucleotide , Genomics/economics , Genotype , Humans , Nucleotides/chemistry , Reproducibility of Results , Sensitivity and Specificity
16.
Mutat Res ; 573(1-2): 70-82, 2005 Jun 03.
Article in English | MEDLINE | ID: mdl-15829238

ABSTRACT

We have developed a flexible, accurate and highly multiplexed SNP genotyping assay for high-throughput genetic analysis of large populations on a bead array platform. The novel genotyping system combines high assay conversion rate and data quality with >1500 multiplexing, and Array of Arrays formats. Genotyping assay oligos corresponding to specific SNP sequences are each linked to a unique sequence (address) that can hybridize to its complementary strand on universal arrays. The arrays are made of beads located in microwells of optical fiber bundles (Sentrix Array Matrix) or silicon slides (Sentrix BeadChip). The optical fiber bundles are further organized into a matrix that matches a 96-well microtiter plate. The arrays on the silicon slides are multi-channel pipette compatible for loading multiple samples onto a single silicon slide. These formats allow many samples to be processed in parallel. This genotyping system enables investigators to generate approximately 300,000 genotypes per day with minimal equipment requirements and greater than 1.6 million genotypes per day in a robotics-assisted process. With a streamlined and comprehensive assay, this system brings a new level of flexibility, throughput, and affordability to genetic research.


Subject(s)
Genotype , Polymorphism, Single Nucleotide , DNA Methylation , Gene Expression Profiling , Humans , Oligonucleotide Array Sequence Analysis
17.
Clin Infect Dis ; 38(4): 483-9, 2004 Feb 15.
Article in English | MEDLINE | ID: mdl-14765339

ABSTRACT

This study analyzes single factors that affect the prognosis of severe acute respiratory syndrome (SARS) and establishes a prognosis model by multivariate analysis. We retrospectively analyzed the clinical features of SARS in 165 clinically confirmed severe cases. Both age and existence of other diseases before SARS were significantly correlated with prognosis (r=0.506 and r=0.457, respectively; P<.001). During the acute phase of SARS, lactate dehydrogenase level, degree of hypoxemia, respiratory rate, alpha -hydroxybutyric dehydrogenase level, creatine kinase isoenzyme-MB, platelet count, and number of involved lobes noted on chest radiographs, and so on, correlated markedly with the prognosis (r=0.257-0.788; P<.05). The multivariate prognosis regression model was associated with degree of hypoxemia and platelet count. The model was defined by the formula Py=1=es/(1+es), where S is [2.490 x degree of hypoxemia]-[0.050 x number of platelets], and it had a high sensitivity (91.67%), specificity (98.33%), and accuracy (96.42%). The model could be used to effectively judge the state of illness and the prognosis.


Subject(s)
Age Factors , Platelet Count , Severe Acute Respiratory Syndrome/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Creatine Kinase/metabolism , Female , Humans , Hydroxybutyrate Dehydrogenase/metabolism , Kidney Function Tests , L-Lactate Dehydrogenase/metabolism , Male , Middle Aged , Multivariate Analysis , Prognosis , Respiration , Retrospective Studies , Risk Factors , Sensitivity and Specificity , Severe Acute Respiratory Syndrome/enzymology , Severe Acute Respiratory Syndrome/metabolism
18.
Mol Cell Biochem ; 239(1-2): 173-80, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12479583

ABSTRACT

In vertebrate and invertebrate muscles, the expression of fatty acid binding proteins (FABP) is induced by long chain fatty acids. To identify the fatty acid response elements that mediate this up-regulation, the gene of the FABP expressed in locust flight muscle was cloned, and its upstream sequences analyzed for potential regulatory elements. Comparison with other muscle FABP promoters revealed the presence of a 19-bp imperfect inverted repeat sequence that contains two hexanucleotide half sites (AGTGGT and ATGGGA), interspersed by 3 nucleotides. The promoter activity was studied with reporter gene constructs in L6 myoblasts, in which H-FABP expression is stimulated by long-chain fatty acids in a similar manner as in adult cardiomyocytes. The 19 bp element, located 180 bp upstream of the transcription start site, was found to be essential for the fatty acid induction of gene expression, and gel shift analysis confirmed that this fatty acid response element is capable of binding nuclear proteins both from rat myoblasts and locust muscle in the presence of fatty acids. A similar, but reverse sequence that is present upstream of all mammalian H-FABP promoters may modulate the expression of the rat H-FABP gene.


Subject(s)
Carrier Proteins/genetics , Fatty Acids/metabolism , Gene Expression Regulation , Grasshoppers/genetics , Muscle, Skeletal/physiology , Neoplasm Proteins , Nerve Tissue Proteins , Response Elements , Tumor Suppressor Proteins , Animals , Base Sequence , Carrier Proteins/metabolism , Cells, Cultured , Fatty Acid-Binding Protein 7 , Fatty Acid-Binding Proteins , Humans , Male , Muscle, Skeletal/cytology , Myoblasts/cytology , Myoblasts/physiology , Protein Binding , Rats , Rats, Sprague-Dawley , Recombinant Fusion Proteins/metabolism , Sequence Alignment
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