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Circular RNA (circRNA) is a non-coding RNA with a covalently closed loop structure and usually more stable than messenger RNA (mRNA). However, coding sequences (CDSs) following an internal ribosome entry site (IRES) in circRNAs can be translated, and this property has been recently utilized to produce proteins as novel therapeutic tools. However, it is difficult to produce large proteins from circRNAs because of the low circularization efficiency of lengthy RNAs. In this study, we report that we successfully synthesized circRNAs with the splint DNA ligation method using RNA ligase 1 and the splint DNAs, which contain complementary sequences to both ends of precursor linear RNAs. This method results in more efficient circularization than the conventional enzymatic method that does not use the splint DNAs, easily generating circRNAs that express relatively large proteins, including IgG heavy and light chains. Longer splint DNA (42 nucleotide) is more effective in circularization. Also, the use of splint DNAs with an adenine analog, 2,6-diaminopurine (DAP), increase the circularization efficiency presumably by strengthening the interaction between the splint DNAs and the precursor RNAs. The splint DNA ligation method requires 5 times more splint DNA than the precursor RNA to efficiently produce circRNAs, but our modified splint DNA ligation method can produce circRNAs using the amount of splint DNA which is equal to that of the precursor RNA. Our modified splint DNA ligation method will help develop novel therapeutic tools using circRNAs, to treat various diseases and to develop human and veterinary vaccines.
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Human adipose-derived mesenchymal stem cells (hAMSCs) are capable of immunomodulation and regeneration after neural injury. For these reasons, hAMSCs have been investigated as a promising stem cell candidate for stroke treatment. However, noninvasive experiments studying the effects of grafted stem cells in the host brain have not yet been reported. Cerebrospinal fluid (CSF), which can be collected without sacrificing the subject, is involved in physiological control of the brain and reflects the pathophysiology of various neurological disorders of the central nervous system (CNS). Following stem cell transplantation in a stroke model, quantitative analysis of CSF proteome changes can potentially reveal the therapeutic effect of stem cells on the host CNS. We examined hAMSC-secreted proteins obtained from serum-free culture medium by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which identified several extracellular matrix proteins, supporting the well-known active paracrine function of hAMSCs. Subsequently, we performed label-free quantitative proteomic analysis on CSF samples from rat stroke models intravenously injected with hAMSC (experimental) or phosphate buffered saline (control). In total, 524 proteins were identified; among them, 125 and 91 proteins were increased and decreased with hAMSC treatment, respectively. Furthermore, gene set enrichment analysis revealed three proteins, 14-3-3 theta, MAG, and neurocan, that showed significant increases in the hAMSC-treated model; these proteins are core members of neurotrophin signaling, nerve growth factor (NGF) signaling, and glycosaminoglycan metabolism, respectively. Subsequent histological and neurologic function experiments validated proliferative neurogenesis in the hAMSC-treated stroke model. We conclude that (i) intravenous injection of hAMSCs can induce neurologic recovery in a rat stroke model and (ii) CSF may reflect the therapeutic effect of hAMSCs. Additionally, proteins as 14-3-3 theta, MAG, and neurocan could be considered as potential CSF biomarkers of neuroregeneration. These CSF proteome profiling results would be utilized as valuable resource in further stroke studies.
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Transplante de Células-Tronco Mesenquimais/métodos , Proteoma/metabolismo , Acidente Vascular Cerebral/líquido cefalorraquidiano , Animais , Diferenciação Celular , Modelos Animais de Doenças , Humanos , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
The partner switching system (PSS) of the SigF regulatory pathway in Mycobacterium smegmatis has been previously demonstrated to include the anti-sigma factor RsbW (MSMEG_1803) and two anti-sigma factor antagonists RsfA and RsfB. In this study, we further characterized two additional RsbW homologs and revealed the distinct roles of three RsbW homologs [RsbW1 (MSMEG_1803), RsbW2 (MSMEG_6129), and RsbW3 (MSMEG_1787)] in the SigF PSS. RsbW1 and RsbW2 serve as the anti-sigma factor of SigF and the protein kinase phosphorylating RsfB, respectively, while RsbW3 functions as an anti-SigF antagonist through its protein interaction with RsbW1. Using relevant mutant strains, RsfB was demonstrated to be the major anti-SigF antagonist in M. smegmatis. The phosphorylation state of Ser-63 was shown to determine the functionality of RsfB as an anti-SigF antagonist. RsbW2 was demonstrated to be the only protein kinase that phosphorylates RsfB in M. smegmatis. Phosphorylation of Ser-63 inactivates RsfB to render it unable to interact with RsbW1. Our comparative RNA sequencing analysis of the wild-type strain of M. smegmatis and its isogenic Δaa 3 mutant strain lacking the aa 3 cytochrome c oxidase of the respiratory electron transport chain revealed that expression of the SigF regulon is strongly induced under respiration-inhibitory conditions in an RsfB-dependent way.
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BACKGROUND: Mannitol increases blood-brain barrier permeability and can improve the efficiency of systemically administered stem cells by facilitating stem cell entry from the periphery into the injured brain. The aim of this study was to elucidate the neuroprotective effects of a combination of mannitol pretreatment and stem cell transplantation on stroke-induced neural injury. METHODS: The experimental rats were randomly assigned to three groups 24 h after middle cerebral artery occlusion and reperfusion. One group received intravenous (IV) injections of phosphate-buffered saline (vehicle), another group received IV injections of human adipose-derived stem cells (hADSCs), and the last group received IV injections of hADSCs 10 min after IV mannitol injections. Neurobehavioral functions and infarct volume were compared. Immunohistochemistry (IHC) analyses were performed using antibodies against ionized calcium binding adapter-1 (IBA-1), rat endothelial antigen-1 (RECA-1), and bromodeoxyuridine/doublecortin (BrdU/DCX). RESULTS: PKH-26 labeling revealed no difference in the number of stem cells that had migrated into the injured brain, and hADSC transplantation did not improve the infarct volume. However, neurobehavioral functions improved in the mannitol group. IHC showed higher numbers of RECA-1-positive cells in the peri-infarcted brain and BrdU-/DCX-colocalized cells in the subventricular zone in the mannitol group. IBA-1-positive cell number decreased in the hADSC-only and mannitol-pretreatment groups compared with the vehicle group even though there was no difference between the former two groups. CONCLUSION: Combinatorial treatment with mannitol and hADSC transplantation may have better therapeutic potential than hADSC monotherapy for ischemic stroke.
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Manitol , Acidente Vascular Cerebral , Animais , Movimento Celular , Modelos Animais de Doenças , Proteína Duplacortina , Infarto da Artéria Cerebral Média/tratamento farmacológico , Ratos , Transplante de Células-Tronco , Acidente Vascular Cerebral/tratamento farmacológicoRESUMO
Excellent mechanical properties of carbon-fiber-reinforced plastic material (CFRP) demonstrates many possibilities in industries using lightweight materials, but unlike isotropic materials, such as iron, aluminum, and magnesium, they show direction-sensitive properties, which makes it difficult to apply for them. The sensitivity of a modal damping coefficient of a CFRP material over the direction of carbon fiber was examined on spectral input patterns in recent research, but the effect of temperature was not considered up to now. To overcome this, uniaxial vibration tests were conducted using five simple specimens with different direction of carbon fiber in a CFRP specimen, the frequency response functions were experimentally determined and the modal damping coefficients were calculated. It was revealed that the resonance point and the modal damping of the specimen changed according to the change in temperature condition. Based on the experimental results, it was demonstrated that the theoretical frequency response function of the carbon composite material is a function of temperature, and it was confirmed that the nonlinear characteristic of the modal damping was the smallest under the 0 degree of direction of carbon fiber.
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OBJECTIVE: Milk fat globule-epidermal growth factor VIII (MFG-E8) may play a key role in inflammatory responses and has the potential to function as a neuroprotective agent for ameliorating brain injury in cerebral infarction. This study aimed to determine the role of MFG-E8 in brain injury in the subacute phase of cerebral ischemia in a rat model. METHODS: Focal cerebral ischemia was induced in rats by occluding the middle cerebral artery with the modified intraluminal filament technique. Twenty-four hours after ischemia induction, rats were randomly assigned to two groups and treated with either recombinant human MFG-E8 or saline. Functional outcomes were assessed using the modified Neurological Severity Score (mNSS), and infarct volumes were evaluated using histology. Anti-inflammation, angiogenesis, and neurogenesis were assessed using immunohistochemistry with antibodies against ionized calcium-binding adapter molecule 1 (Iba-1), rat endothelial cell antigen-1 (RECA-1), and bromodeoxyuridine (BrdU)/doublecortin (DCX), respectively. RESULTS: Our results showed that intravenous MFG-E8 treatment did not reduce the infarct volume; however, the mNSS test revealed that neurobehavioral deficits were significantly improved in the MFG-E8-treated group than in the vehicle group. Immunofluorescence staining revealed a significantly lower number of Iba-1-positive cells and higher number of RECA-1 in the periinfarcted brain region, and significantly higher numbers of BrdU- and DCX-positive cells in the subventricular zone in the MFG-E8-treated group than in the vehicle group. CONCLUSION: Our findings suggest that MFG-E8 improves neurological function by suppressing inflammation and enhancing angiogenesis and neuronal proliferation in the subacute phase of cerebral infarction.
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E2F3, a member of the E2F family, plays a critical role in cell cycle and proliferation by targeting downstream, retinoblastoma (RB) a tumor suppressor family protein. The purpose of this study, was to investigate the role and function of E2F3 in vivo. We examined phenotypic abnormalities, by deletion of the E2f3 gene in mice. Complete ablation of the E2F3 was fully penetrant, in the pure C57BL/6N background. The E2f3+/ - mouse embryo developed normally without fatal disorder. However, they exhibited reduced body weight, growth retardation, skeletal imperfection, and poor grip strength ability. Findings suggest that E2F3 has a pivotal role in muscle and bone development, and affect normal mouse growth.
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Desenvolvimento Ósseo/genética , Fator de Transcrição E2F3/genética , Desenvolvimento Embrionário/genética , Músculo Esquelético/crescimento & desenvolvimento , Animais , Apoptose/genética , Peso Corporal/genética , Ciclo Celular/genética , Proliferação de Células/genética , Embrião de Mamíferos , Humanos , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , FenótipoRESUMO
The mycobacterial SenX3-RegX3 two-component system consists of the SenX3 sensor histidine kinase and its cognate RegX3 response regulator. This system is a phosphorelay-based regulatory system involved in sensing environmental Pi levels and induction of genes required for Pi acquisition under Pi-limiting conditions. Here we demonstrate that overexpression of the kinase domain of Mycobacterium tuberculosis PknB (PknB-KDMtb) inhibits the transcriptional activity of RegX3 of both M. tuberculosis and Mycobacterium smegmatis (RegX3Mtb and RegX3Ms, respectively). Mass spectrometry results, along with those of in vitro phosphorylation and complementation analyses, revealed that PknB kinase activity inhibits the transcriptional activity of RegX3Mtb through phosphorylation events at Thr-100, Thr-191, and Thr-217. Electrophoretic mobility shift assays disclosed that phosphorylation of Thr-191 and Thr-217 abolishes the DNA-binding ability of RegX3Mtb and that Thr-100 phosphorylation likely prevents RegX3Mtb from being activated through conformational changes induced by SenX3-mediated phosphorylation. We propose that the convergence of the PknB and SenX3-RegX3 signaling pathways might enable mycobacteria to integrate environmental Pi signals with the cellular replication state to adjust gene expression in response to Pi availability.
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Proteínas de Bactérias/metabolismo , Fosfotransferases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Fosforilação , Fosfotransferases/genética , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/fisiologia , Rifabutina/metabolismo , Transdução de Sinais/genéticaRESUMO
Precise control of the oxidation state of transition-metal oxides, such as copper, is important for high selectivity of CO2 reduction in an aqueous condition to compete with the reduction of water. The phase of copper oxide nanofibers was controlled by predictive synthesis, which controls the nanoscale gas-solid reaction by considering thermodynamics and kinetics. The driving force of the phase transformation between the different oxidation states of copper oxide is calculated by comparing the Gibbs free energy of each of the oxidation states. From the calculation, the kinetically processable window for the fabrication of Cu2O in which monophase Cu2O can be fabricated in a reasonable reaction time scale is discovered. Herein, we report the monophase Cu2O nanofiber photocathode, which photoelectrochemically converted CO2 into methanol with over 90% selectivity in an aqueous electrolyte, and a hierarchical structure is developed to optimize the photoactivity and stability of the electrode. Our work suggests a rational design of the calcination strategy for precisely controlling the oxidation states of transition metals that can be applied to various applications in which the phase of the materials plays an important role.
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Here we demonstrated that the inhibition of electron flux through the respiratory electron transport chain (ETC) by either the disruption of the gene for the major terminal oxidase (aa3 cytochrome c oxidase) or treatment with KCN resulted in the induction of ald encoding alanine dehydrogenase in Mycobacterium smegmatis A decrease in functionality of the ETC shifts the redox state of the NADH/NAD+ pool toward a more reduced state, which in turn leads to an increase in cellular levels of alanine by Ald catalyzing the conversion of pyruvate to alanine with the concomitant oxidation of NADH to NAD+ The induction of ald expression under respiration-inhibitory conditions in M. smegmatis is mediated by the alanine-responsive AldR transcriptional regulator. The growth defect of M. smegmatis by respiration inhibition was exacerbated by inactivation of the ald gene, suggesting that Ald is beneficial to M. smegmatis in its adaptation and survival under respiration-inhibitory conditions by maintaining NADH/NAD+ homeostasis. The low susceptibility of M. smegmatis to bcc1 complex inhibitors appears to be, at least in part, attributable to the high expression level of the bd quinol oxidase in M. smegmatis when the bcc1-aa3 branch of the ETC is inactivated.IMPORTANCE We demonstrated that the functionality of the respiratory electron transport chain is inversely related to the expression level of the ald gene encoding alanine dehydrogenase in Mycobacterium smegmatis Furthermore, the importance of Ald in NADH/NAD+ homeostasis during the adaptation of M. smegmatis to severe respiration-inhibitory conditions was demonstrated in this study. On the basis of these results, we propose that combinatory regimens including both an Ald-specific inhibitor and respiration-inhibitory antitubercular drugs such as Q203 and bedaquiline are likely to enable a more efficient therapy for tuberculosis.
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Alanina Desidrogenase/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Mycobacterium smegmatis/enzimologia , Consumo de Oxigênio/fisiologia , Alanina Desidrogenase/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Imidazóis/farmacologia , Testes de Sensibilidade Microbiana , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , NAD/metabolismo , Piperidinas/farmacologia , Piridinas/farmacologiaRESUMO
The DevSR (DosSR) two-component system, which is a major regulatory system involved in oxygen sensing in mycobacteria, plays an important role in hypoxic induction of many genes in mycobacteria. We demonstrated that overexpression of the kinase domain of Mycobacterium tuberculosis (Mtb) PknB inhibited transcriptional activity of the DevR response regulator in Mycobacterium smegmatis and that this inhibitory effect was exerted through phosphorylation of DevR on Thr180 within its DNA-binding domain. Moreover, the purified kinase domain of Mtb PknB significantly phosphorylated RegX3, NarL, KdpE, TrcR, DosR, and MtrA response regulators of Mtb that contain the Thr residues corresponding to Thr180 of DevR in their DNA-binding domains, implying that transcriptional activities of these response regulators might also be inhibited when the kinase domain of PknB is overexpressed.
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Hipóxia Celular/genética , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Oxigênio/química , Oxigênio/metabolismo , Fosforilação , Protamina Quinase/genética , Protamina Quinase/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Tuberculose/genética , Tuberculose/microbiologiaRESUMO
Vertical stacking and lateral growth of molybdenum disulfide (MoS2 ) are controlled with remarkable precision, and MoS2 nanotubes are directly converted from nanofibers. Predictive synthesis is enabled by identifying the specific thermodynamic region where the Boudouard reaction becomes favored. It reveals how the chemical potential of each species in the MoSCO system can predict phase behaviors.
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Organogel-based stretchable electronic conductors exhibit electrical conduction even under a large stretching deformation of 300% without electrochemical reactions at DC voltages. The resistance change with stretching is almost strain-insensitive up to 50% strain and it remains at each deformation up to 1000 fatigue cycle. The polymeric conductive paths of PEDOT: PSS are well preserved during the mechanical deformation.
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BACKGROUND: Chromosomal abnormalities are common in embryos produced in vitro and cause implantation failure, miscarriage, and serious medical problems in infants. Because preimplantation genetic screening (PGS) is increasingly being used to detect aneuploidy in embryos with the purpose of improving implantation rates after IVF (in vitro fertilization), we aimed to validate the usefulness of array CGH for the preimplantation genetic screening (PGS) of embryos at the blastocyst stage of development. RESULTS: A total of 150 blastocysts were biopsied from couples undergoing IVF and analyzed using array CGH. We found that 54.5% (73/134) of the blastocysts were euploid embryos, whereas 45.5% of the embryos (61/134) had chromosomal abnormalities. Multiple chromosome abnormality was most frequently observed (34.4%), and dual aneuploidy was observed in 26.2% of the embryos. Monosomy (21.3%) appeared more frequently than trisomy (18%). CONCLUSION: Chromosomal microarray analysis provided clinically significant cytogenetic information regarding the frequency and variety of chromosomal abnormalities observed in embryos at the blastocyst stage, suggesting that this is a useful tool for comprehensive aneuploidy screening in IVF.
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BACKGROUND: Array comparative genomic hybridization (CGH) is a powerful tool for detecting unbalanced chromosomal alterations. To validate the usefulness of array CGH in newborn screening, we examined 20,126 unselected infants. In addition, the number of newborns analyzed with array CGH is the largest one ever reported. FINDINGS: A total of 20,126 unselected newborns were investigated with array CGH and cytogenetic analyses. The analyses revealed 87 cases with chromosome abnormalities. Of these, 53 cases had significant chromosome aneuploidies, including trisomy 13, trisomy 21, 47,XXY or 45,X, and the other 34 cases presented partial chromosomal deletions or duplications. CONCLUSIONS: In this study, we show that array CGH is an appropriate tool for the screening of chromosomal abnormalities in newborns, especially for the infants without distinct clinical features.
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The enhancement of the electrical conductivity by doping is important in hematite (α-Fe(2)O(3)) photoanodes for efficient solar water oxidation. However, in spite of many successful demonstrations using extrinsic dopants, such as Sn, Ti, and Si, the achieved photocurrent is still lower than the practical requirement. There is still lack of our understanding of how intrinsic oxygen defects can change the photocurrent and interact with the extrinsic dopants. In this study, we systematically investigate the interplay of oxygen vacancies and extrinsic Sn dopants in the context of photoanodic properties. As a result, we demonstrate that the controlled generation of oxygen vacancies can activate the photoactivity of pure hematite remarkably and further enhance the Sn doping effects synergistically. Furthermore, the correlated behavior of oxygen vacancies and Sn dopants is closely linked to the variation of electrical conductance and results in the optimum concentration region to show the high photocurrent and low onset voltage.
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Leucine-responsive regulatory protein (Lrp) is known to be an indirect activator of type 1 fimbriae synthesis in Salmonella enterica serovar Typhimurium via direct regulation of FimZ, a direct positive regulator for type 1 fimbriae production. Using RT-PCR, we have shown previously that fimA transcription is dramatically impaired in both lrp-deletion (Δlrp) and constitutive-lrp expression (lrp(C)) mutant strains. In this work, we used chromosomal P(fimA)-lacZ fusions and yeast agglutination assays to confirm and extend our previous results. Direct binding of Lrp to P(fimA) was shown by an electrophoretic mobility shift assay (EMSA) and DNA footprinting assay. Site-directed mutagenesis revealed that the Lrp-binding motifs in P(fimA) play a role in both activation and repression of type 1 fimbriae production. Overproduction of Lrp also abrogates fimZ expression. EMSA data showed that Lrp and FimZ proteins independently bind to P(fimA) without competitive exclusion. In addition, both Lrp and FimZ binding to P(fimA) caused a hyper retardation (supershift) of the DNA-protein complex compared to the shift when each protein was present alone. Nutrition-dependent cellular Lrp levels closely correlated with the amount of type 1 fimbriae production. These observations suggest that Lrp plays important roles in type 1 fimbriation by acting as both a positive and negative regulator and its effect depends, at least in part, on the cellular concentration of Lrp in response to the nutritional environment.
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Proteínas de Fímbrias/genética , Fímbrias Bacterianas , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteína Reguladora de Resposta a Leucina/fisiologia , Salmonella typhimurium/metabolismo , Antígenos de Bactérias/metabolismo , Proteínas de Fímbrias/biossíntese , Proteínas de Fímbrias/metabolismo , Proteína Reguladora de Resposta a Leucina/metabolismo , Ligação ProteicaRESUMO
BACKGROUND: Array comparative genomic hybridization (CGH) is currently the most powerful method for detecting chromosomal alterations in pre and postnatal clinical cases. In this study, we developed a BAC based array CGH analysis platform for detecting whole genome DNA copy number changes including specific micro deletion and duplication chromosomal disorders. Additionally, we report our experience with the clinical implementation of our array CGH analysis platform. Array CGH was performed on 5080 pre and postnatal clinical samples from patients referred with a variety of clinical phenotypes. RESULTS: A total of 4073 prenatal cases (4033 amniotic fluid and 40 chorionic villi specimens) and 1007 postnatal cases (407 peripheral blood and 600 cord blood) were studied with complete concordance between array CGH, karyotype and fluorescence in situ hybridization results. Among 75 positive prenatal cases with DNA copy number variations, 60 had an aneuploidy, seven had a deletion, and eight had a duplication. Among 39 positive postnatal cases samples, five had an aneuploidy, 23 had a deletion, and 11 had a duplication. CONCLUSIONS: This study demonstrates the utility of using our newly developed whole-genome array CGH as first-tier test in 5080 pre and postnatal cases. Array CGH has increased the ability to detect segmental deletion and duplication in patients with variable clinical features and is becoming a more powerful tool in pre and postnatal diagnostics.
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SNP markers provide the primary data for population structure analysis. In this study, we employed whole-genome autosomal SNPs as a marker set (54,836 SNP markers) and tested their possible effects on genetic ancestry using 320 subjects covering 24 regional groups including Northern (=16) and Southern (=3) Asians, Amerindians (=1), and four HapMap populations (YRI, CEU, JPT, and CHB). Additionally, we evaluated the effectiveness and robustness of 50K autosomal SNPs with various clustering methods, along with their dependencies on recombination hotspots (RH), linkage disequilibrium (LD), missing calls and regional specific markers. The RH- and LD-free multi-dimensional scaling (MDS) method showed a broad picture of human migration from Africa to North-East Asia on our genome map, supporting results from previous haploid DNA studies. Of the Asian groups, the East Asian group showed greater differentiation than the Northern and Southern Asian groups with respect to Fst statistics. By extension, the analysis of monomorphic markers implied that nine out of ten historical regions in South Korea, and Tokyo in Japan, showed signs of genetic drift caused by the later settlement of East Asia (South Korea, Japan and China), while Gyeongju in South East Korea showed signs of the earliest settlement in East Asia. In the genome map, the gene flow to the Korean Peninsula from its neighboring countries indicated that some genetic signals from Northern populations such as the Siberians and Mongolians still remain in the South East and West regions, while few signals remain from the early Southern lineages.
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Fluxo Gênico/genética , Povo Asiático , China , Genótipo , Humanos , Japão , Polimorfismo de Nucleotídeo Único/genética , República da CoreiaRESUMO
Levan fructotransferase (LFTase) preferentially catalyzes the transfructosylation reaction in addition to levan hydrolysis, whereas other levan-degrading enzymes hydrolyze levan into a levan-oligosaccharide and fructose. Based on sequence comparisons and enzymatic properties, the fructosyl transfer activity of LFTase is proposed to have evolved from levanase. In order to probe the residues that are critical to the intramolecular fructosyl transfer reaction of the Microbacterium sp. AL-210 LFTase, an error-prone PCR mutagenesis process was carried out, and the mutants that led to a shift in activity from transfructosylation towards hydrolysis of levan were screened by the DNS method. After two rounds of mutagenesis, TLC and HPLC analyses of the reaction products by the selected mutants revealed two major products; one is a di-D-fructose- 2,6':6,2'-dianhydride (DFAIV) and the other is a levanbiose. The newly detected levanbiose corresponds to the reaction product from LFTase lacking transferring activity. Two mutants (2-F8 and 2-G9) showed a high yield of levanbiose (38-40%) compared with the wild-type enzyme, and thus behaved as levanases. Sequence analysis of the individual mutants responsible for the enhanced hydrolytic activity indicated that Asn-85 was highly involved in the transfructosylation activity of LFTase.