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1.
Transgenic Res ; 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32078126

RESUMO

Despite great values in many applications, heavy chain-only antibodies (HcAbs) are naturally only produced in camelids and sharks, which are not easy to access and handle. Production of the type of antibodies in small laboratory animals would remarkably facilitate their applications. We previously reported a mouse line in which the CH1 exon of mouse γ1 was deleted that could express heavy chain-only IgG1 antibodies. However, these mice showed an extremely weak IgG1 response to specific antigens when immunized, and we could only achieve single VH domains with low affinity to antigens using these mice. One possibility is that the mouse germline VH repertoire was not sufficient to support the expression of functional heavy chain-only antibodies. In this study, we report the generation of a rat line in which the CH1 exon of the γ2a gene was removed and the γ1 and γ2b genes were silenced. Although the genetically modified rats expressed heavy chain-only IgG2a, they also exhibited a very weak IgG2a response to antigen immunization. Panning of a phage library constructed using IgG2a VH segments amplified from immunized rats identified antigen-specific single VH antibodies, which also exhibited much lower affinity than that of commercial mAbs. Together with our previous report, this study suggests that the simple genetic removal of the CH1 exon does not guarantee the successful expression of functional heavy chain-only antibodies.

2.
Theranostics ; 10(3): 1454-1478, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31938074

RESUMO

Hair regeneration has long captured researchers' attention because alopecia is a common condition and current therapeutic approaches have significant limitations. Dermal papilla (DP) cells serve as a signaling center in hair follicles and regulate hair formation and cycling by paracrine secretion. Secreted EVs are important signaling mediators for intercellular communication, and DP-derived extracellular vesicles (DP-EVs) may play an important role in hair regeneration. However, the instability of EVs in vivo and their low long-term retention after transplantation hinder their use in clinical applications. Methods: Human DP-EVs were encapsulated in partially oxidized sodium alginate (OSA) hydrogels, yielding OSA-encapsulated EVs (OSA-EVs), which act as a sustained-release system to increase the potential therapeutic effect of DP-EVs. The ability of the OSA-EVs to protect protein was assessed. The hair regeneration capacity of OSA-EVs, as well as the underlying mechanism, was explored in hair organ culture and a mouse model of depilation. Results: The OSA-EVs were approximately 100 µm in diameter, and as the hydrogel degraded, DP-EVs were gradually released. In addition, the hydrogel markedly increased the stability of vesicular proteins and increased the retention of EVs in vitro and in vivo. The OSA-EVs significantly facilitated proliferation of hair matrix cells, prolonged anagen phase in cultured human hairs, and accelerated the regrowth of back hair in mice after depilation. These effects may be due to upregulation of hair growth-promoting signaling molecules such as Wnt3a and ß-catenin, and downregulation of inhibitory molecule BMP2. Conclusion: This study demonstrated that OSA hydrogels promote the therapeutic effects of DP-EVs, and indicate that our novel OSA-EVs could be used to treat alopecia.

3.
Blood ; 135(1): 41-55, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31697823

RESUMO

To study the mechanisms of relapse in acute lymphoblastic leukemia (ALL), we performed whole-genome sequencing of 103 diagnosis-relapse-germline trios and ultra-deep sequencing of 208 serial samples in 16 patients. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2) involved in drug response. Their prevalence was 17% in very early relapse (<9 months from diagnosis), 65% in early relapse (9-36 months), and 32% in late relapse (>36 months) groups. Convergent evolution, in which multiple subclones harbor mutations in the same drug resistance gene, was observed in 6 relapses and confirmed by single-cell sequencing in 1 case. Mathematical modeling and mutational signature analysis indicated that early relapse resistance acquisition was frequently a 2-step process in which a persistent clone survived initial therapy and later acquired bona fide resistance mutations during therapy. In contrast, very early relapses arose from preexisting resistant clone(s). Two novel relapse-specific mutational signatures, one of which was caused by thiopurine treatment based on in vitro drug exposure experiments, were identified in early and late relapses but were absent from 2540 pan-cancer diagnosis samples and 129 non-ALL relapses. The novel signatures were detected in 27% of relapsed ALLs and were responsible for 46% of acquired resistance mutations in NT5C2, PRPS1, NR3C1, and TP53. These results suggest that chemotherapy-induced drug resistance mutations facilitate a subset of pediatric ALL relapses.

4.
J Colloid Interface Sci ; 559: 65-75, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31610306

RESUMO

Electroactive nanofibrous scaffold is a vital tool for the study of the various biological research fields from bioelectronics to regenerative medicine, which can provide cell preferable 3D nanofiber architecture and programmed electrical signal. However, intrinsic non-biodegradability is a major problem that hinders its widespread application in the clinic. Herein, we designed, synthesized, and characterized shell/core poly (3,4-ethylenedioxythiophene) (PEDOT)/chitosan (CS) nanofibers by combining the electrospinning and recrystallization processes. Upon incorporating a trace amount of PEDOT (1.0 wt%), the resultant PEDOT/CS nanofibers exhibited low interfacial charge transfer impedance, high electrochemical stability, high electrical conductivity (up to 0.1945 S/cm), and ultrasensitive piezoelectric property (output voltage of 22.5 mV by a human hair prodding). With such unique electrical and conductive properties, PEDOT/CS nanofibers were further applied to brain neuroglioma cells (BNCs) to stimulate their adhesion, proliferation, growth, and development under an optimal external electrical stimulation (ES) and a pulse voltage of 400 mV/cm. ES-responsive PEDOT/CS nanofibers indeed promoted BNCs growth and development as indicated by a large number and density of axons. The synergetic interplay between external ES and piezoelectric voltage demonstrates new PEDOT-based nanofibers as implantable electroactive scaffolds for numerous applications in nerve tissue engineering, human health monitoring, brain mantle information extraction, and degradable microelectronic devices.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Quitosana/química , Condutividade Elétrica , Nanofibras/química , Polímeros/química , Testes de Impedância Acústica/métodos , Axônios/metabolismo , Materiais Biocompatíveis/química , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cristalização , Estimulação Elétrica/métodos , Glioma/metabolismo , Humanos
5.
Immunobiology ; : 151889, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31812342

RESUMO

The genomic organization of goat immunoglobulin light chains (Igλ and Igκ) loci were annotated based on the goat genome database. The goat Igλ chain located on chromosome 17 contains at least 35 Vλ gene fragments (seven potential functional genes, one ORF and 27 pseudogenes), two Jλ-Cλ clusters arranged in a Vλ(35)-Jλ2-Cλ1-Jλ1-Cλ2 pattern, with another Cλ3 on scaffold. The Igκ locus included 11 Vκ (five potential functional genes, two ORFs and four pseudogene fragments), three Jκ genes and a single Cκ gene ordered in Vκ(35)-Jκ(3)-Cκ pattern on chromosome 11. By analyzing the clonies of Igλ and Igκ, we further found Vλ2 (26.23 %) &Vλ3 (73.11 %), Vκ2 (52.07 %) &Vκ4 (46.15 %) were predominately used in the expression of λ and κ chains respectively. λ chain showed more abundance in connective diversity than κ chain. Besides, somatic hypermutation with higher frequency in both immunoglobulin light chains was the major mechanism for the goat repertoire diversity. These results demonstrated goat immunoglobulin light chain variable region genome loci and repertoire diversity.

6.
BMC Complement Altern Med ; 19(1): 309, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31718632

RESUMO

BACKGROUND: Sheng Mai San (SMS) has been proven to exhibit cardio-protective effects. This study aimed to explore the molecular mechanisms of SMS on hyperglycaemia (HG)-induced apoptosis in H9C2 cells. METHODS: HG-induced H9C2 cells were established as the experimental model, and then treated with SMS at 25, 50, and 100 µg/mL. H9C2 cell viability and apoptosis were quantified using MTT and Annexin V-FITC assays, respectively. Furthermore, Bcl-2/Bax signalling pathway protein expression and Fas and FasL gene expression levels were quantified using western blotting and RT-PCR, respectively. RESULTS: SMS treatments at 25, 50, 100 µg/mL significantly improved H9C2 cell viability and inhibited H9C2 cell apoptosis (p < 0.05). Compared to the HG group, SMS treatment at 25, 50, and 100 µg/mL significantly downregulated p53 and Bax expression and upregulated Bcl-2 expression (p < 0.05). Moreover, SMS treatment at 100 µg/mL significantly downregulated Fas and FasL expression level (p < 0.05) when compared to the HG group. CONCLUSION: SMS protects H9C2 cells from HG-induced apoptosis probably by downregulating p53 expression and upregulating the Bcl-2/Bax ratio. It may also be associated with the inhibition of the Fas/FasL signalling pathway.


Assuntos
Apoptose/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Hiperglicemia/fisiopatologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Hiperglicemia/tratamento farmacológico , Hiperglicemia/genética , Hiperglicemia/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
7.
Vet Immunol Immunopathol ; 215: 109913, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31420069

RESUMO

The development of a rapid and efficient system to generate porcine monoclonal antibodies (mAbs) is an important step toward the discovery of critical neutralizing targets for designing rational vaccines against porcine viruses. In this study, we established a platform for producing porcine mAbs based on single cell technologies. First, we singled out an optimal donor from 507 pigs based on serum antibody neutralizing activity against porcine reproductive and respiratory syndrome virus (PRRSV). After identifying the contribution of IgG to the neutralizing activity, single CD45R+IgG+Ag+ B cells were sorted from peripheral blood mononuclear cells (PBMCs). Single B cell RT-PCR was performed using primers designed to cover the germline repertoire of the porcine VH/VL gene segments. Paired VH/VLs were cloned into a eukaryotic expression vector and transfected into 293T cells. We demonstrate that full-length porcine mAbs were produced, and antigen-specific mAbs were obtained after further validation. The approach reported in this study can be applied to generate porcine mAbs against any given antigen and may help with the screening of neutralizing antibodies against porcine pathogens.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Suínos/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Especificidade de Anticorpos , Linfócitos B/imunologia , Células HEK293 , Humanos , Região Variável de Imunoglobulina/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Transfecção , Recombinação V(D)J
8.
Molecules ; 24(3)2019 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-30744185

RESUMO

Sampling for DUS test of flower colors should be fixed at the stages and sites that petals are fully colored, and besides, flower colorations are uniform among individuals and stable for a period of time to allow testers to get consistent results. It remains a problem since spatial and temporal flower colorations are reported a lot but their change traits are little discussed. In this study, expression state, uniformity and stability of color phenotypes, anthocyanin contents, and gene expression levels were taken into account based on measurements at 12 development stages and three layers (inner, middle, and outer petals) of two varieties of Ranunculus asiaticus L. to get their best sampling. Our results showed that, outer petals of L9⁻L10 (stage 9⁻stage 10 of variety 'Jiaoyan zhuanhong') and C5⁻C6 (stage 5⁻stage 6 of variety 'Jiaoyan yanghong') were the best sampling, respectively. For DUS test, it is suggested to track flower colorations continuously to get the best sampling as well as representative colors since different cultivars had different change traits, and moreover, full expression of color phenotypes came later and lasted for a shorter duration than those of anthocyanin contents and gene expressions. Our innovation exists in following two points. Firstly, a model of change dynamic was introduced to illustrate the change traits of flower colorations, anthocyanin contents, and gene expressions. Secondly, genes used for expression analysis were screened on account of tentative anthocyanins, which were identified based on comparison between liquid chromatography⁻mass spectrometry (LC⁻MS) results and molecular mass and mass fragment pattern (M²) of each putative anthocyanin and their fragments deduced in our previous study. Gene screening in this regard may also be interest for other non-model plant genera with little molecular background.


Assuntos
Flores/genética , Regulação da Expressão Gênica de Plantas , Pigmentação/genética , Ranunculus/genética , Antocianinas/metabolismo , Cromatografia Líquida de Alta Pressão , Metabolismo Energético , Flores/metabolismo , Perfilação da Expressão Gênica , Humanos , Espectrometria de Massas , Fenótipo , Característica Quantitativa Herdável , Ranunculus/metabolismo
9.
FASEB J ; 33(3): 4525-4537, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30702927

RESUMO

It has been shown that 5-amino-4-imidazolecarboxamide riboside (AICAr) can inhibit cell proliferation and induce apoptosis in childhood acute lymphoblastic leukemia (ALL) cells. Although AICAr could regulate cellular energy metabolism by activating AMPK, the cytotoxic mechanisms of AICAr are still unclear. Here, we knocked out TP53 or PRKAA1 gene (encoding AMPKα1) in NALM-6 and Reh cells by using the clustered regularly interspaced short palindromic repeats/Cas9 system and found that AICAr-induced proliferation inhibition was independent of AMPK activation but dependent on p53. Liquid chromatography-mass spectrometry analysis of nucleotide metabolites indicated that AICAr caused an increase in adenosine triphosphate, deoxyadenosine triphosphate, and deoxyguanosine triphosphate levels by up-regulating purine biosynthesis, while AICAr led to a decrease in cytidine triphosphate, uridine triphosphate, deoxycytidine triphosphate, and deoxythymidine triphosphate levels because of reduced phosphoribosyl pyrophosphate production, which consequently impaired the pyrimidine biosynthesis. Ribonucleoside triphosphate (NTP) pool imbalances suppressed the rRNA transcription efficiency. Furthermore, deoxy-ribonucleoside triphosphate (dNTP) pool imbalances induced DNA replication stress and DNA double-strand breaks, followed by cell cycle arrest and apoptosis in ALL cells. Exogenous uridine could rebalance the NTP and dNTP pools by supplementing pyrimidine and then attenuate AICAr-induced cytotoxicity. Our data indicate that RNA transcription inhibition and DNA replication stress induced by NTP and dNTP pool imbalances might play a key role in AICAr-mediated cytotoxic effects on ALL cells, suggesting a potential clinical application of AICAr in future ALL therapy.-Du, L., Yang, F., Fang, H., Sun, H., Chen, Y., Xu, Y., Li, H., Zheng, L., Zhou, B.-B. S. AICAr suppresses cell proliferation by inducing NTP and dNTP pool imbalances in acute lymphoblastic leukemia cells.

10.
J Cell Biol ; 218(5): 1653-1669, 2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-30808704

RESUMO

How morphogenetic signals are prepared for intercellular dispersal and signaling is fundamental to the understanding of tissue morphogenesis. We discovered an intracellular mechanism that prepares Drosophila melanogaster FGF Branchless (Bnl) for cytoneme-mediated intercellular dispersal during the development of the larval Air-Sac-Primordium (ASP). Wing-disc cells express Bnl as a proprotein that is cleaved by Furin1 in the Golgi. Truncated Bnl sorts asymmetrically to the basal surface, where it is received by cytonemes that extend from the recipient ASP cells. Uncleavable mutant Bnl has signaling activity but is mistargeted to the apical side, reducing its bioavailability. Since Bnl signaling levels feedback control cytoneme production in the ASP, the reduced availability of mutant Bnl on the source basal surface decreases ASP cytoneme numbers, leading to a reduced range of signal/signaling gradient and impaired ASP growth. Thus, enzymatic cleavage ensures polarized intracellular sorting and availability of Bnl to its signaling site, thereby determining its tissue-specific intercellular dispersal and signaling range.

11.
Methods Mol Biol ; 1863: 29-45, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30324591

RESUMO

Conserved morphogenetic signaling proteins disperse across tissues to generate signal and signaling gradients, which in turn are considered to assign positional coordinates to the recipient cells. Recent imaging studies in Drosophila model have provided evidence for a "direct-delivery" mechanism of signal dispersion that is mediated by specialized actin-rich signaling filopodia, named cytonemes. Cytonemes establish contact between the signal-producing and target cells to directly exchange and transport the morphogenetic proteins. Although an increasing amount of evidence supports the critical role of these specialized signaling structures, imaging these highly dynamic 200 nm-thin structures in the complex three-dimensional contour of living tissues is challenging. Here, we describe the imaging methods that we optimized for studying cytonemes in Drosophila embryos.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/ultraestrutura , Embrião não Mamífero/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Pseudópodes/ultraestrutura , Animais , Comunicação Celular , Drosophila melanogaster/metabolismo , Embrião não Mamífero/metabolismo , Feminino , Masculino , Morfogênese , Pseudópodes/metabolismo , Transdução de Sinais
12.
J Vis Exp ; (139)2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30295654

RESUMO

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression. Precise spatiotemporal expression of the gene in targeted tissues is critical for unbiased interpretation of cell/gene activity. Therefore, developing a method for generating exclusive cell/tissue-specific driver lines is essential. Here we present a method to generate highly tissue-specific targeted expression system by employing a "Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated" (CRISPR/Cas)-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented here for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.


Assuntos
Sistemas CRISPR-Cas , Drosophila/genética , Edição de Genes/métodos , Animais , Animais Geneticamente Modificados , Drosophila/metabolismo , Regulação da Expressão Gênica , RNA Guia/genética
13.
Elife ; 72018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30328809

RESUMO

Gradients of signaling proteins are essential for inducing tissue morphogenesis. However, mechanisms of gradient formation remain controversial. Here we characterized the distribution of fluorescently-tagged signaling proteins, FGF and FGFR, expressed at physiological levels from the genomic knock-in alleles in Drosophila. FGF produced in the larval wing imaginal-disc moves to the air-sac-primordium (ASP) through FGFR-containing cytonemes that extend from the ASP to contact the wing-disc source. The number of FGF-receiving cytonemes extended by ASP cells decreases gradually with increasing distance from the source, generating a recipient-specific FGF gradient. Acting as a morphogen in the ASP, FGF activates concentration-dependent gene expression, inducing pointed-P1 at higher and cut at lower levels. The transcription-factors Pointed-P1 and Cut antagonize each other and differentially regulate formation of FGFR-containing cytonemes, creating regions with higher-to-lower numbers of FGF-receiving cytonemes. These results reveal a robust mechanism where morphogens self-generate precise tissue-specific gradient contours through feedback regulation of cytoneme-mediated dispersion.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Retroalimentação Fisiológica , Fatores de Crescimento de Fibroblastos/metabolismo , Especificidade de Órgãos , Alelos , Animais , Extensões da Superfície Celular , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Genoma de Inseto , Proteínas de Fluorescência Verde/metabolismo , Discos Imaginais/metabolismo , Imagem Tridimensional , Transporte Proteico , Transdução de Sinais , Asas de Animais/metabolismo
14.
J Cell Mol Med ; 22(12): 6202-6212, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30255549

RESUMO

Relapse-specific mutations in phosphoribosyl pyrophosphate synthetase 1 (PRPS1), a rate-limiting purine biosynthesis enzyme, confer significant drug resistances to combination chemotherapy in acute lymphoblastic leukemia (ALL). It is of particular interest to identify drugs to overcome these resistances. In this study, we found that PRPS1 mutant ALL cells specifically showed more chemosensitivity to 5-Fluorouracil (5-FU) than control cells, attributed to increased apoptosis of PRPS1 mutant cells by 5-FU. Mechanistically, PRPS1 mutants increase the level of intracellular phosphoribosyl pyrophosphate (PRPP), which causes the apt conversion of 5-FU to FUMP and FUTP in Reh cells, to promote 5-FU-induced DNA damage and apoptosis. Our study not only provides mechanistic rationale for re-targeting drug resistant cells in ALL, but also implicates that ALL patients who harbor relapse-specific mutations of PRPS1 might benefit from 5-FU-based chemotherapy in clinical settings.


Assuntos
Fluoruracila/farmacologia , Fosforribosil Pirofosfato/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Ribose-Fosfato Pirofosfoquinase/genética , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Humanos , Células Jurkat , Lentivirus/genética , Camundongos , Fosforribosil Pirofosfato/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia
15.
Shanghai Kou Qiang Yi Xue ; 27(2): 164-169, 2018 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-30146643

RESUMO

PURPOSE: To explore the characteristics of distribution of WNT10A gene rs10177996 polymorphism between Han and Uygur populations in Xinjiang area. METHODS: A cross-sectional survey on 154 Han individuals in Urumqi area and 134 Uygur individuals in Kashgar area was performed. Buccal epithelial cells were harvested using Cotton swab scraping, and DNA was extracted by special kit. After screening, the corresponding SNP segments of qualified samples were propagated by PCR. Dideoxy-mediated chain termination method was used for gene sequencing, and then, genotyping was conducted with corresponding software. Statistical analysis of genetic data was performed by SPSS 23.0 software package. RESULTS: Among Uygur nationality in Kashgar area, the frequencies of CC, CT, TT genetypes in rs10177996 were 8.21%, 30.60% and 61.19%, respectively. The allele frequency of C was 23.51% and T was 76.49%. Among Han nationality in Urumqi area, the frequencies on CC, CT, TT genetypes of rs10177996 were 9.74%, 43.51% and 46.75%, respectively. The allele frequency of C was 31.49% and T was 68.51%. When compared with Han nationality, the frequency of TT was significantly higher in Uygur nationality(P=0.046). When compared with European, the frequency of TT was significantly lower in Uygur nationality (P=0.05). When compared with European, the frequency of TT was significantly lower in Han nationality(P<0.01). Compared with European, the distribution on C allele frequency was significantly higher, the distribution on T allele frequency was significantly lower in Han nationality (P=0.033). However, there was no significant difference between Han nationality in Urumqi area and Uygur nationality in Kashgar area (P>0.05), and, between Uygur nationality in Kashgar area and European (P>0.05). Meanwhile, there was no significant difference in gender between Uygur nationality in Kashgar area and Han nationality in Urumqi area (P>0.05). CONCLUSIONS: The distributions of WNT10A gene rs10177996 SNP among Han nationality in Urumqi area, Uygur nationality in Kashgar area and the reported European population are obviously different.


Assuntos
Genótipo , Polimorfismo Genético , Proteínas Wnt , Grupo com Ancestrais do Continente Asiático , China , Estudos Transversais , Grupos Étnicos , Frequência do Gene , Humanos , Proteínas Wnt/genética
16.
Immunobiology ; 223(11): 599-607, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30025710

RESUMO

Based on the goat genome database, we have annotated the genomic organization of the goat immunoglobulin heavy chain variable region. The goat IgH locus is present on seven genome scaffolds, and contains ten VH, three DH and six JH segments. After the exclusion of three shorter segments, the VH genes were divided into two gene families based on sequence similarity. By analyzing the IgH cDNA sequences, we further identified that VH2 (54.2%), DH1 (61.7%) and JH1 (60.5%) segments were most frequently utilized in the expression of the immunoglobulin variable region, and that point mutations introduced by somatic hypermutation were the major mutation present in these expressed variable region. Compared with human and horses, DH-DH fusion occurred at a higher frequency in goat V(D)J recombination. These results provided variable insights into goat immunoglobulin heavy chain variable region genome loci and repertoire diversity.


Assuntos
DNA Complementar/genética , Genoma/genética , Cabras/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Mutação/genética , Animais , Evolução Molecular , Cavalos/imunologia , Humanos
17.
Biomed Res Int ; 2018: 1761865, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29862255

RESUMO

Platelet rich plasma (PRP) is a concentrate of autologous platelets which contain enrichment growth factors (GFs). However, the addition of exogenous anticoagulant and procoagulant may result in clinical side effects and raise the price of PRP. Herein, we report a novel method named temperature controlled PRP (t-PRP), in which exogenous additives are dispensable in the preparation and activation process. Human blood samples were processed by a two-step centrifugation process under hypothermic conditions (4°C) to obtain t-PRP and rewarming up to 37°C to activate t-PRP. Contemporary PRP (c-PRP) was processed as the control. t-PRP showed a physiological pH value between 7.46 and 7.48 and up to 6.58 ± 0.45-fold significantly higher platelet concentration than that of whole blood compared with c-PRP (4.06-fold) in the preparation process. Meanwhile, t-PRP also maintained a stable GF level between plasma and PRP. After activation, t-PRP demonstrated natural fiber scaffolding, which trapped more platelet and GFs, and exhibited a slow release and degradation rate of GFs. In addition, t-PRP exhibited the function of promoting wound healing. t-PRP is a novel and convenient method for the preparation and activation of PRP without any additives. Compared to c-PRP, t-PRP reflects more physiologic characteristics while maintaining high quality.


Assuntos
Plasma Rico em Plaquetas/química , Adulto , Feminino , Humanos , Masculino
18.
J Ultrasound Med ; 37(8): 2043-2052, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29399851

RESUMO

OBJECTIVES: The aim of the study was to assess the feasibility of ultrasound strain imaging in characterizing the biceps brachii muscle in chronic poststroke spasticity. METHODS: We prospectively analyzed strain imaging data from bilateral biceps brachii muscles in 8 healthy volunteers and 7 patients with poststroke chronic spasticity. Axial deformations of the biceps brachii muscle and overlying subcutaneous tissue were produced by external compression using a sandbag (1.0 kg) attached to a transducer. The lengthening and shortening of the biceps brachii muscle and subcutaneous tissue were produced by manual passive elbow extension (from 90° to 0°) and flexion (from 0° to 90°), respectively. We used offline 2-dimensional speckle tracking to estimate axial and longitudinal strain ratios (biceps brachii strain/subcutaneous tissue strain), and the longitudinal tissue velocity of the biceps brachii muscle. Statistical analyses included analysis of variance for testing differences in strain imaging parameters among healthy, nonspastic, and spastic biceps brachii muscles, the Bonferroni correction for further testing differences in US strain imaging among paired groups (healthy versus spastic, nonspastic versus spastic, and healthy versus nonspastic), and the Pearson correlation coefficient for assessing the intraobserver reliability of performing strain imaging in stroke survivors. RESULTS: The differences in strain imaging parameters between healthy and spastic and between nonspastic and spastic biceps brachii muscles were significant at both 90° elbow flexion and maximal elbow extension (P < .01). There was no significant difference in axial strain ratios at 90° of elbow flexion or longitudinal tissue velocities between healthy and nonspastic muscles (P > .05). The intraobserver reliability of performing strain imaging in stroke survivors was good (r = 0.85; P < .01). CONCLUSIONS: Ultrasound strain imaging seems to be feasible for characterizing the biceps brachii muscle in chronic poststroke spasticity.


Assuntos
Espasticidade Muscular/diagnóstico por imagem , Espasticidade Muscular/fisiopatologia , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/fisiopatologia , Acidente Vascular Cerebral/complicações , Ultrassonografia/métodos , Adulto , Idoso , Braço/diagnóstico por imagem , Braço/fisiopatologia , Doença Crônica , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Espasticidade Muscular/etiologia , Estudos Prospectivos , Amplitude de Movimento Articular , Reprodutibilidade dos Testes
19.
Ultrasound Med Biol ; 44(9): 1931-1940, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29398131

RESUMO

We prospectively investigated the feasibility of using quantitative ultrasound imaging (QUI) to assess the biceps brachii muscle (BBM) in individuals with chronic post-stroke spasticity. To quantify muscle echogenicity and stiffness, we measured QUI parameters (gray-scale pixel value and shear wave velocity [SWV, m/s]) of the BBM in three groups: 16 healthy BBMs; 12 post-stroke, non-spastic BBMs; and 12 post-stroke, spastic BBMs. The QUI results were compared with the Modified Ashworth Scale and Tardieu Scale. A total of 20 SWVs were measured in each BBM, once at elbow in 90° flexion and again at maximally achievable extension using acoustic radiation force impulse imaging. BBM pixel value was measured in gray-scale images captured at 90° elbow flexion using ImageJ software. Statistical analyses included analysis of variance for examining the difference in SWV and pixel values among the three groups; Bonferroni correction for testing the difference in SWV and pixel values in a paired group; t-test for examining the difference in SWV values measured at two elbow angles; and Pearson correlation coefficient for analyzing the correlation of QUI to Modified Ashworth Scale and Tardieu Scale. SWV significantly differed between spastic BBMs and non-spastic or healthy BBMs. For pixel values, each of the three groups significantly differed from the others at elbow 90° flexion. The difference in SWV measured between the two elbow angles was also significant (p <0.01). A strong negative correlation was found between SWV and passive range of motion (R2 = -0.88, p <0.0001) in spastic upper limbs. These results suggest that the use of QUI is feasible in quantitative assessment of spastic BBM.


Assuntos
Técnicas de Imagem por Elasticidade/métodos , Espasticidade Muscular/diagnóstico por imagem , Espasticidade Muscular/fisiopatologia , Reabilitação do Acidente Vascular Cerebral/métodos , Acidente Vascular Cerebral/fisiopatologia , Extremidade Superior/diagnóstico por imagem , Adulto , Idoso , Doença Crônica , Estudos de Avaliação como Assunto , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/diagnóstico por imagem , Estudos Prospectivos , Amplitude de Movimento Articular , Reprodutibilidade dos Testes , Ultrassonografia , Extremidade Superior/fisiopatologia
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