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Objective: To develop a quantitative evaluation software for three-dimensional morphology of pathological scars based on photo modeling technology, and to verify its accuracy and feasibility in clinical application. Methods: The method of prospective observational study was adopted. From April 2019 to January 2022, 59 patients with pathological scars (totally 107 scars) who met the inclusion criteria were admitted to the First Medical Center of Chinese PLA General Hospital, including 27 males and 32 females, aged 33 (26, 44) years. Based on photo modeling technology, a software for measuring three-dimensional morphological parameters of pathological scars was developed with functions of collecting patients' basic information, and scar photography, three-dimensional reconstruction, browsing the models, and generating reports. This software and the clinical routine methods (vernier calipers, color Doppler ultrasonic diagnostic equipment, and elastomeric impression water injection method measurement) were used to measure the longest length, maximum thickness, and volume of scars, respectively. For scars with successful modelling, the number, distribution of scars, number of patients, and the longest length, maximum thickness, and volume of scars measured by both the software and clinical routine methods were collected. For scars with failed modelling, the number, distribution, type of scars, and the number of patients were collected. The correlation and consistency of the software and clinical routine methods in measuring the longest length, maximum thickness, and volume of scars were analyzed by unital linear regression analysis and the Bland-Altman method, respectively, and the intraclass correlation coefficients (ICCs), mean absolute error (MAE), and mean absolute percentage error (MAPE) were calculated. Results: A total of 102 scars from 54 patients were successfully modeled, which located in the chest (43 scars), in the shoulder and back (27 scars), in the limb (12 scars), in the face and neck (9 scars), in the auricle (6 scars), and in the abdomen (5 scars). The longest length, maximum thickness, and volume measured by the software and clinical routine methods were 3.61 (2.13, 5.19) and 3.53 (2.02, 5.11) cm, 0.45 (0.28, 0.70) and 0.43 (0.24, 0.72) cm, 1.17 (0.43, 3.57) and 0.96 (0.36, 3.26) mL. The 5 hypertrophic scars and auricular keloids from 5 patients were unsuccessfully modeled. The longest length, maximum thickness, and volume measured by the software and clinical routine methods showed obvious linear correlation (with r values of 0.985, 0.917, and 0.998, P<0.05). The ICCs of the longest length, maximum thickness, and volume of scars measured by the software and clinical routine methods were 0.993, 0.958, and 0.999 (with 95% confidence intervals of 0.989-0.995, 0.938-0.971, and 0.998-0.999, respectively). The longest length, maximum thickness, and volume of scars measured by the software and clinical routine methods had good consistency. The Bland-Altman method showed that 3.92% (4/102), 7.84% (8/102), and 8.82% (9/102) of the scars with the longest length, maximum thickness, and volume respectively were outside the 95% consistency limit. Within the 95% consistency limit, 2.04% (2/98) scars had the longest length error of more than 0.5 cm, 1.06% (1/94) scars had the maximum thickness error of more than 0.2 cm, and 2.15% (2/93) scars had the volume error of more than 0.5 mL. The MAE and MAPE of the longest length, maximum thickness, and volume of scars measured by the software and clinical routine methods were 0.21 cm, 0.10 cm, 0.24 mL, and 5.75%, 21.21%, 24.80%, respectively. Conclusions: The quantitative evaluation software for three-dimensional morphology of pathological scars based on photo modeling technology can realize the three-dimensional modeling and measurement of morphological parameters of most pathological scars. Its measurement results were in good consistency with those of clinical routine methods, and the errors were acceptable in clinic. This software can be used as an auxiliary method for clinical diagnosis and treatment of pathological scars.
Assuntos
Cicatriz Hipertrófica , Queloide , Feminino , Humanos , Masculino , Povo Asiático , Cicatriz Hipertrófica/diagnóstico por imagem , Extremidades , Queloide/diagnóstico por imagem , Estudos Prospectivos , AdultoRESUMO
Objective: To evaluate the progress and influence factors of asymptomatic osteonecrosis of the femoral head(ONFH). Methods: MRI was performed on the contralateral hips of 174 patients with unilateral symptomatic ONFH who admitted at Department of Orthopaedics, the Second Affiliated Hospital of Xi'an Jiaotong University from January 2012 to December 2018. Eighty-three of 174 patients with unilateral ONFH were found suffering from contralateral ONFH(47.7%), of which 77 patients were followed up.There were 28 males and 49 females with age of 48.6 years (range: 21-73 years). The pathogenesis, ARCO classfication, areas and position of osteonecrosis were collected.Independent sample t test, χ(2) test, Fisher exact test, multivariate Logistic regression were used to analyze the potential influence factors. Results: Patients were followed up for 36.7 months. During the following up period, ARCO classification of 28 patients (36.4%) progressed.The progress of asymptomatic ONFH was not related to the gender, age and original ARCO classification, but related to the pathogenesis, position and area of osteonecrosis (all P<0.05). Conclusion: The progress of asymptomatic osteonecrosis is related to the pathogenesis, position and area of osteonecrosis,but most of asymptomatic ONFH will not progress.
Assuntos
Necrose da Cabeça do Fêmur/diagnóstico por imagem , Cabeça do Fêmur/diagnóstico por imagem , Adulto , Idoso , Progressão da Doença , Feminino , Necrose da Cabeça do Fêmur/classificação , Necrose da Cabeça do Fêmur/etiologia , Seguimentos , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries and is currently incurable due, in part, to difficulty in eliminating the leukemia cells protected by stromal microenvironment. Based on previous observations that CLL cells exhibit mitochondrial dysfunction and altered lipid metabolism and that carnitine palmitoyltransferases (CPT) have a major role in transporting fatty acid into mitochondria to support cancer cell metabolism, we tested several clinically relevant inhibitors of lipid metabolism for their ability to eliminate primary CLL cells. We discovered that perhexiline, an antiangina agent that inhibits CPT, was highly effective in killing CLL cells in stromal microenvironment at clinically achievable concentrations. These effective concentrations caused low toxicity to normal lymphocytes and normal stromal cells. Mechanistic study revealed that CLL cells expressed high levels of CPT1 and CPT2. Suppression of fatty acid transport into mitochondria by inhibiting CPT using perhexiline resulted in a depletion of cardiolipin, a key component of mitochondrial membranes, and compromised mitochondrial integrity, leading to rapid depolarization and massive CLL cell death. The therapeutic activity of perhexiline was further demonstrated in vivo using a CLL transgenic mouse model. Perhexiline significantly prolonged the overall animal survival by only four drug injections. Our study suggests that targeting CPT using an antiangina drug is able to effectively eliminate leukemia cells in vivo, and is a novel therapeutic strategy for potential clinical treatment of CLL.
Assuntos
Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Perexilina/farmacologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Cardiolipinas/metabolismo , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Expressão Gênica , Glucose/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/mortalidade , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Modelos Biológicos , Consumo de Oxigênio , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Quantitative real-time PCR is the most sensitive technique for gene expression analysis. Data normalization is essential to correct for potential errors incurred in all steps from RNA isolation to PCR amplification. The commonly accepted approach for normalization is the use of reference gene. Until now, no suitable reference genes have been available for data normalization of gene expression in milk somatic cells of lactating yaks across lactation. In the present study, we evaluated the transcriptional stability of 10 candidate reference genes in milk somatic cells of lactating yak, including ACTB, B2M, GAPDH, GTP, MRPL39, PPP1R11, RPS9, RPS15, UXT, and RN18S1. Four genes, RPS9, PPP1R11, UXT, and MRPL39, were identified as being the most stable genes in milk somatic cells of lactating yak. Using the combination of RPS9, PPP1R11, UXT, and MRPL39 as reference genes, we further assessed the relative expression of 4 genes of interest in milk somatic cells of yak across lactation, including ELF5, ABCG2, SREBF2, and DGAT1. Compared with expression in colostrum, the overall transcription levels of ELF5, ABCG2, and SREBF2 in milk were found to be significantly upregulated in early, peak, and late lactation, and significantly downregulated thereafter, before the dry period. A similar pattern was observed in the relative expression of DGAT1, but no significant difference was revealed in its expression in milk from late lactation compared with colostrum. Based on these results, we suggest that the geometric mean of RPS9, PPP1R11, UXT, and MRPL39 can be used for normalization of real-time PCR data in milk somatic cells of lactating yak, if similar experiments are performed.
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Bovinos/fisiologia , Regulação da Expressão Gênica/fisiologia , Lactação/fisiologia , Animais , Colostro , Feminino , Lactação/genética , Leite/citologia , Reação em Cadeia da Polimerase , Gravidez , RNA/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
MicroRNA are approximately 18- to 22-nucleotide nonprotein coding molecules that play important roles in the regulation of gene expression at the posttranscriptional level. In the present study, we assessed the suitability of 8 noncoding small RNA as normalizers for microRNA (miR) quantitative analysis in milk somatic cells of lactating yaks, including 3 small nuclear RNA (snRNA; RNU1A, RNU5A, and RNU6B), 3 small nucleolar RNA (snoRNA; SNORA73A, Z30, and SNORA74A), 1 rRNA (5S), and 1 transfer RNA (Met-tRNA). The snRNA RNU1A, RNU5A, and SNORA73A were identified as the most stable references in milk somatic cells of lactating yaks. Also, a minimum of 3 reference RNA (RNU1A, RNU5A, and SNORA73A) were required for the normalization of microRNA expression data in milk somatic cells of the lactating yak. We further evaluated the suitability of the combination of RNU1A, RNU5A, and SNORA73A as reference RNA in milk somatic cells of lactating yaks via detecting the relative expression of miR 16b, miR 21-5p, miR 145, and miR 155 as microRNA of putative interest. In comparison to the colostrum period, on the whole, the expressions of the 4 microRNA were found to be upregulated at an early period and, thereafter, a declining pattern was exhibited from early to final periods in all microRNA investigated. Based on the results from this study, we recommend that the combination of RNU1A, RNU5A, and SNORA73A can be used as normalizers for microRNA quantitative analysis in future longitudinal studies on milk somatic cells of lactating yaks in relation to lactation.
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Bovinos , Células/química , MicroRNAs/análise , Leite/citologia , Animais , Contagem de Células/veterinária , Feminino , Expressão Gênica , Lactação/fisiologia , MicroRNAs/genética , RNA Nuclear Pequeno , RNA Nucleolar Pequeno , Reação em Cadeia da Polimerase em Tempo Real/veterináriaAssuntos
Perfilação da Expressão Gênica , Cabras/genética , Prolactina/genética , Pele/citologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Biologia Computacional , DNA Complementar/genética , DNA Complementar/metabolismo , Feminino , Regulação da Expressão Gênica , Cabras/metabolismo , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Prolactina/metabolismo , Estrutura Terciária de Proteína , Pele/metabolismoRESUMO
Osteopontin (OPN) is a secreted phosphorylated glycoprotein. It has an important role in mammary gland development and lactation, as well as, is thought to be a potential candidate gene for lactation traits. In the present work, we isolated and characterized a full-length open reading frame (ORF) of yak OPN cDNA from lactating mammary tissue, and examined its expression pattern in mammary gland during different stages of lactation, as well as, the recombinant OPN protein of yak was expressed successfully in E. coli. The sequencing results indicated that the isolated cDNA was 1132-bp in length containing a complete ORF of 837-bp. It encoded a precursor protein of yak OPN consisting of 278 amino acid with a signal peptide of 16 amino acids. Yak OPN has a predicted molecular mass of 29285.975 Da and an isoelectric point of 4.245. It had an identity of 65.50-99.16% in cDNA, identity of 52.06-98.56% and similarity of 65.40-98.56% in deduced amino acids with the corresponding sequences of cattle, buffalo, sheep, goat, pig, human, and rabbit. The phylogenetic analysis indicated that yak OPN had the closest evolutionary relationship with that of cattle, and next buffalo. In mammary gland, yak OPN was generally transcribed in a declining pattern from colostrum period to dry period with an apparent increase of OPN expression being present in the late period of lactation compared with peak period of lactation. Western blot analysis indicated that His-tagged yak OPN protein expressed in E. coli could be recognized not only by an anti-His-tag antibody but also by an anti-human OPN antibody. These results from the present work provided a foundation for further insight into the role of OPN gene in yak lactation.
Assuntos
Bovinos/genética , DNA Complementar/genética , Perfilação da Expressão Gênica , Lactação/genética , Glândulas Mamárias Animais/metabolismo , Osteopontina/genética , Animais , Western Blotting , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Feminino , Regulação da Expressão Gênica , Humanos , Fases de Leitura Aberta/genética , Osteopontina/química , Osteopontina/metabolismo , Filogenia , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
BACKGROUND AND OBJECTIVE: The survival outcomes of patients with metastatic nasopharyngeal carcinoma (NPC) differ significantly between individuals. The aim of this study is to build a prognostic score model (PSM) incorporating circulating tumour markers for metastatic NPC in an epidemic area. METHODS: Seven hundred and ninety-nine patients with disseminated NPC were analysed retrospectively. Univariate and multivariable analyses were conducted using the Cox proportion hazards model. Factors analysed included patients' characteristics (gender, age group, performance status), circulating tumour-marker characteristics (Epstein-Barr virus (EBV) DNA level, EBV VCA-IgA level, lactate dehydrogenase (LDH) level, alkaline phosphatase (ALP) level), basic laboratory characteristics (leucocyte count, haemoglobin level, albumin level), and disease characteristics (presence of metastasis at presentation, disease-free interval, number of metastatic sites, specific metastatic sites). The PSM was built according to numerical score derived from the regression coefficients of each independent prognostic variable. The prognostic score of each patient was calculated by totalling up the scores of each independent variable. RESULTS: Independent prognostic factors included performance status, age, haemoglobin level, LDH level, ALP level and EBV DNA level. Three prognostic groups based on PSM were obtained: low risk (total score=0-4); intermediate risk (5-8); high risk (9-12). Median survivals of the three groups were 25.5, 15.1 and 7 months, respectively, (P<0.001). CONCLUSION: Clinical and laboratory characteristics can help guide the prognostication of patients with metastatic NPC in epidemic areas.
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Biomarcadores Tumorais/sangue , Carcinoma/sangue , Carcinoma/secundário , Neoplasias Nasofaríngeas/sangue , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Carcinoma/mortalidade , Criança , China/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Nasofaríngeas/mortalidade , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos , Fatores Sexuais , Análise de Sobrevida , Adulto JovemRESUMO
The trembling shear behavior of electrorheological (ER) fluids has been investigated by using a computer simulation method, and a shear-slide boundary model is proposed to understand this phenomenon. A thiourea-doped Ba-Ti-O ER fluid which shows a trembling shear behavior was first prepared and then systematically studied by both theoretical and experimental methods. The shear curves of ER fluids in the dynamic state were simulated with shear rates from 0.1 to 1000 s(-1) under different electric fields. The simulation results of the flow curves match the experimental results very well. The trembling shear curves are divided into four regions and each region can be explained by the proposed model.
Assuntos
Eletroquímica/métodos , Bário/química , Simulação por Computador , Eletrodos , Modelos Estatísticos , Oxigênio/química , Física/métodos , Reologia/métodos , Resistência ao Cisalhamento , Tioureia/química , Titânio/química , ViscosidadeRESUMO
The aim of the present work was to investigate single nucleotide polymorphism (SNP) of growth hormone receptor (GHR) gene exon 10, characterize the genetic variation in three Chinese indigenous goat breeds, and search for its potential association with cashmere traits. In this study, a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) protocol has been developed for rapid genotyping of the GHR gene in goats. One hundred seventy-eight goats from Liaoning Cashmere (96), Inner Mongolia White Cashmere (40), and Chengdu Grey (42) breeds in China were genotyped at GHR locus using the protocol developed. In all goat breeds investigated, a SNP in exon 10 of GHR gene has been identified by analyzing genomic DNA. The polymorphism consists of a single nucleotide substitution A â G, resulting in two alleles named, respectively, A and G based on the nucleotide at the position. The allele A was found to be more common in the animals investigated, and seems to be more consistent with cattle and zebu at this polymorphic site found in goats. The Hardy-Weinberg equilibrium of genotype distributions of GHR locus was verified in Liaoning Cashmere, and Inner Mongolia White Cashmere breeds. According to the classification of polymorphism information content (PIC), Chengdu Grey was less polymorphic than Liaoning Cashmere and Inner Mongolia White Cashmere breeds at this locus. The phylogenetic tree of different species based on the nucleotide sequences of GHR gene exon 10 is generally in agreement with the known species relationship. No significant association was found between the polymorphism revealed and the cashmere traits analyzed in present work.
Assuntos
Cruzamento , Variação Genética , Cabras/genética , Receptores da Somatotropina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , China , Feminino , Frequência do Gene/genética , Loci Gênicos/genética , Genótipo , Masculino , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Receptores da Somatotropina/química , Alinhamento de Sequência , Carneiro Doméstico/genéticaRESUMO
BACKGROUND: extranodal natural killer (NK)/T-cell lymphoma (ENKL) is a heterogeneous entity with poor survival, requiring risk stratification in affected patients. We proposed absolute lymphocyte count (ALC) as a new prognostic factor in ENKL. PATIENTS AND METHODS: we retrospectively analyzed 128 patients newly diagnosed with ENKL. Independent prognostic factors of survival were determined by Cox regression analysis. RESULTS: patients with low ALC (<1.0 × 10(9)/l) at diagnosis tended to have more adverse clinical features. Patients with high ALC (≥1.0 × 10(9)/l) at diagnosis had better overall survival (OS; P < 0.0001) and progression-free survival (PFS; P<0.0001), and achieved higher complete remission rates (P=0.001). Multivariate analysis with known prognostic factors showed that ALC, B symptoms and advanced stage were independent predictors for OS and PFS. Using the International Prognostic Index, Prognostic Index for Peripheral T-cell lymphoma unspecified, or Korean Prognostic Index for nasal NK/T-cell lymphoma, the majority of patients were in the low-risk category (with no or one adverse factor). ALC was helpful to differentiate the low-risk patients with different survival outcomes (P < 0.0001). CONCLUSIONS: our data suggest that ALC at diagnosis is a novel, powerful predictor of prognosis in ENKL. Immune status at diagnosis might have an important influence on survival in patients with ENKL.
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Linfoma Extranodal de Células T-NK/sangue , Linfoma de Células T/sangue , Neoplasias Nasais/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Adulto JovemRESUMO
κ-Casein is one of the major proteins in the milk of mammals. It plays an important role in determining the size and specific function of milk micelles. We have previously identified and characterized a genetic variant of yak κ-casein by evaluating genomic DNA. Here, we isolate and characterize a yak κ-casein cDNA harboring the full-length open reading frame (ORF) from lactating mammary gland. Total RNA was extracted from mammary tissue of lactating female yak, and the κ-casein cDNA were synthesized by RT-PCR technique, then cloned and sequenced. The obtained cDNA of 660-bp contained an ORF sufficient to encode the entire amino acid sequence of κ-casein precursor protein consisting of 190 amino acids with a signal peptide of 21 amino acids. Yak κ-casein has a predicted molecular mass of 19,006.588 Da with a calculated isoelectric point of 7.245. Compared with the corresponding sequences in GenBank of cattle, buffalo, sheep, goat, Arabian camel, horse, and rabbit, yak κ-casein sequence had identity of 64.76-98.78% in cDNA, and identity of 44.79-98.42% and similarity of 53.65-98.42% in deduced amino acids, revealing a high homology with the other livestock species. Based on κ-casein cDNA sequences, the phylogenetic analysis indicated that yak κ-casein had a close relationship with that of cattle. This work might be useful in the genetic engineering researches for yak κ-casein.
Assuntos
Caseínas/genética , Bovinos/genética , Glândulas Mamárias Animais/química , Filogenia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Análise por Conglomerados , Primers do DNA/genética , DNA Complementar/genética , Feminino , Lactação , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
The effect of the boundary friction coefficient on the rheological properties of the electrorheological (ER) fluids in quasistatic and dynamic states is investigated by computer simulation. The relation between the shear stress and the boundary friction coefficient in quasistatic and dynamic states is discussed qualitatively and quantitatively, and the trend matches the previously reported experimental results well. The flow curves of ER fluids, under different friction coefficients, are calculated, and it is found that the friction coefficient affects the flow curves. In two dimensions, the transitions in structure corresponding to the shear stress variations are presented to understand the mechanism of ER fluids.
RESUMO
Osmotherapy with 10% hypertonic saline (HS) alleviates cerebral edema through osmotic force. Aquaporin-4 (AQP4) has been reported to be implicated in the pathogenesis of cerebral edema resulting from a variety of brain injury. This study aimed to determine if 10% hypertonic saline ameliorates cerebral edema through downregulation of AQP4 expression in the perivascular astrocytes in the ischemic cerebral edema. Adult male Sprague-Dawley (SD) rats were subjected to permanent right-sided middle cerebral artery occlusion (MCAO) and treated with a continuous i.v. infusion of 10% HS. Brain water content (BWC) analyzed by wet-to-dry ratios in the ischemic hemisphere of SD rats was attenuated after 10% HS treatment. This was coupled with the reduction of neuronal apoptosis in the peri-ischemic brain tissue. Concomitantly, downregulated expression of AQP4 in the perivascular astrocytes after 10% HS treatment was observed. Our results suggest that in addition to its osmotic force, 10% HS exerts anti-edema effects possibly through downregulation of AQP4 expression in the perivascular astrocytes. The reduction of brain edema after 10% HS administration can prevent ischemic brain damage.
Assuntos
Aquaporina 4/biossíntese , Astrócitos/metabolismo , Edema Encefálico/terapia , Solução Salina Hipertônica/farmacologia , Animais , Apoptose , Aquaporina 4/genética , Encéfalo/metabolismo , Encéfalo/patologia , Edema Encefálico/etiologia , Edema Encefálico/metabolismo , Regulação para Baixo , Infarto da Artéria Cerebral Média/complicações , Masculino , Neurônios/patologia , Osmose , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Solução Salina Hipertônica/uso terapêuticoRESUMO
Yak milk contains a greater percentage of protein and has better quality than bovine milk. There has been an increasing focus on yak milk and milk products during the last few years. In the present study, a PCR-based assay was developed for the specific identification of bovine milk in yak milk by designing 3 primers targeting the mitochondrial ND1 gene. The use of 3 primers in a single PCR reaction set yielded 2 amplification fragments of 293 and 190 bp from bovine milk DNA, whereas only 1 amplification fragment of 293 bp was obtained in yak milk DNA. The technique was applied to raw and heat-treated binary mixtures of yak and bovine milks and enabled the specific detection of bovine milk with a detection limit of 0.1%. The assay developed is sensitive, fast, and straightforward, and it might be useful in the quality control of yak milk and milk products.
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Bovinos/fisiologia , Tecnologia de Alimentos/métodos , Leite/química , Reação em Cadeia da Polimerase/métodos , AnimaisRESUMO
BACKGROUND AND OBJECTIVE: 5-fluorouracil (5-FU) is still a widely used anticancer drug. More than 85% of the 5-FU administered is catabolized by dihydropyrimidine dehydrogenase (DPD) in the liver. However, mutations in the DPD gene have been found to be associated with low DPD activity causing severe complications. The purpose of this study was to determine the mutation frequency of four exons in Chinese cancer patients and the relationship between genotype and DPD activity. METHODS: Samples from 142 cancer patients were investigated in this study. The DPD activity was determined by reversed-phase HPLC. Exons 2, 13, 14 and 18 were amplified by polymerase chain reaction (PCR), sequenced and analysed from both sense and antisense directions. Nonparametric one-sample Kolmogorov-Smirnov test was used for distribution analysis; two independent samples t-test and one-way anova was performed for two groups and three groups analyses, respectively. RESULTS AND DISCUSSION: Plasma-DPD activities in the 142 cancer patients followed a Gaussian distribution. The mean plasma-DPD activity in women was lower than that in men (P = 0.006). Four mutations, 85T>C(DPYD*9A), 1627A>G(DPYD*5), 1896T>C and 2194G>A(DPYD*6), were found in the 142 cancer patients. The following mutations reported by others were not detected: 61C>T, 62G>A, 74A>G, 1601G>A(DPYD*4), 1679T>G(DPYD*13), 1714C>G, 1897delC(DPYD*3) and IVS 14 + 1G>A. No significant correlation was found between three mutations [85T>C(DPYD*9A), 1627A>G (DPYD*5) and 1896T>C], and DPD activity was found. CONCLUSION: No clear correlation between the mutations studied and DPD activity could be established in this study. However, larger-scale prospective studies are needed to better assess the reported genotype-phenotype correlations.
Assuntos
Antimetabólitos Antineoplásicos/metabolismo , Povo Asiático , Di-Hidrouracila Desidrogenase (NADP)/genética , Fluoruracila/metabolismo , Idoso , Análise de Variância , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , China , Cromatografia Líquida de Alta Pressão , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Éxons , Feminino , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Frequência do Gene , Genótipo , Humanos , Masculino , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Reação em Cadeia da Polimerase , Fatores SexuaisRESUMO
Angiogenesis plays a pivotal role in tumor growth, tissue invasion and metastasis. Endostatin is an angiogenesis inhibitor and has been shown to reduce tumor growth in animal models. However, therapy with recombinant endostatin protein was hampered by its short half-life and very-low yield of bioactive protein. We performed a phase I dose-escalation clinical trial using intratumoral injection of an adenovirus containing human endostatin gene (Ad-rhE; E10A; 10(10)-10(12) virus particles (vp)) in 15 patients with advanced solid tumors. We observed intratumoral injections of E10A without dose-limiting toxicity. Most frequently reported E10A-related adverse events were transient fever and local response. Distribution studies revealed that the vector was detected in the blood, throat and injection site, but rarely in the urine and stool. An increased endostatin expression was detected using enzyme immunoassay in serum in 13 of 14 treated patients throughout the period of treatment despite the presence of neutralizing antiadenovirus antibody. Median serum basic fibroblast growth factor levels decreased from 32.4 pg ml(-1) at baseline to 24.9 pg ml(-1) after 28 days of first treatment. Thus, direct intratumoral injection of up to 10(12) vp of E10A to patients is well tolerated and further studies are necessary to define and increase clinical efficacy.
Assuntos
Adenoviridae/genética , Endostatinas/farmacologia , Endostatinas/farmacocinética , Vetores Genéticos , Neoplasias/metabolismo , Adulto , Endostatinas/administração & dosagem , Endostatinas/genética , Feminino , Meia-Vida , Humanos , Injeções Intralesionais , Masculino , Pessoa de Meia-Idade , Neoplasias/irrigação sanguínea , Neoplasias/imunologia , Neovascularização Patológica , Distribuição TecidualRESUMO
Telomerase-negative cancer cells can maintain their telomeres by a recombination-mediated alternative lengthening of telomeres (ALT) process. We reported previously that sequestration of MRE11/RAD50/NBS1 complexes represses ALT-mediated telomere length maintenance, and suppresses formation of ALT-associated promyelocytic leukemia (PML) bodies (APBs). APBs are PML bodies containing telomeric DNA and telomere-binding proteins, and are observed only in a small fraction of cells within asynchronously dividing ALT-positive cell populations. Here, we report that methionine restriction caused a reversible arrest in G0/G1 phase of the cell cycle and reversible induction of APB formation in most cells within an ALT-positive population. We combined methionine restriction with RNA interference to test whether the following proteins are required for APB formation: PML body-associated proteins, PML and Sp100; telomere-associated proteins, TRF1, TRF2, TIN2 and RAP1; and DNA repair proteins, MRE11, RAD50, NBS1 and 53BP1. APB formation was not decreased by depletion of Sp100 (as reported previously) or of 53BP1, although 53BP1 partially colocalizes with APBs. Depletion of the other proteins suppressed APB formation. Because of the close linkage between ALT-mediated telomere maintenance and ability to form APBs, the eight proteins identified by this screen as being required for APB formation are also likely to be required for the ALT mechanism.
Assuntos
Técnicas Genéticas , Telômero/genética , Telômero/metabolismo , Hidrolases Anidrido Ácido , Antígenos Nucleares/genética , Antígenos Nucleares/fisiologia , Autoantígenos/genética , Autoantígenos/fisiologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/fisiologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Fase G1 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteína Homóloga a MRE11 , Metionina/deficiência , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proteína da Leucemia Promielocítica , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Fase de Repouso do Ciclo Celular , Proteínas de Ligação a Telômeros/antagonistas & inibidores , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/fisiologia , Proteína 1 de Ligação a Repetições Teloméricas/antagonistas & inibidores , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/antagonistas & inibidores , Proteína 2 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/fisiologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
We previously isolated a mammalian gene STC1 that encodes a glycoprotein related to stanniocalcin (STC), a fish hormone that plays a major role in calcium homeostasis. However, the mammalian STC1 gene is expressed in a variety of adult tissues in contrast to fish where STC is expressed only in one unique gland, the corpuscles of Stannius. This suggested that STC1 may have wider autocrine/paracrine functions in mammals. In the present study, using immunocytochemistry, we showed that STC1 protein is localized in the developing bone and muscle of the mouse fetus. During endochondral bone formation, STC1 is found principally in prechondrocytes and prehypertrophic chondrocytes. During intramembranous bone formation STC1 is present in the mesenchyme that is about to undergo ossification. STC1 is also found in the myocardiocytes of the developing heart and at all stages of differentiation from myoblasts to myotube formation in developing skeletal muscle. The specific localization of STC1 to chondrocytes and muscle cells suggests a role for this protein in chondrogenic and myogenic differentiation.
Assuntos
Osso e Ossos/embriologia , Desenvolvimento Embrionário e Fetal , Glicoproteínas/fisiologia , Hormônios/fisiologia , Músculo Esquelético/embriologia , Animais , Osso e Ossos/química , Condrócitos/química , Coração Fetal/química , Glicoproteínas/análise , Hormônios/análise , Imuno-Histoquímica , Mesoderma/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Músculo Esquelético/químicaRESUMO
The disruption of the normal function and nuclear localization of the promyelocytic leukemia-associated protein (PML) may play a major role in the pathogenesis of acute promyelocytic leukemia. PML, which is concentrated in nuclear bodies (PML bodies), has been shown to have growth- and transformation-suppressive properties. In this study, we have examined the intranuclear distribution of PML in a conditionally immortalized human cell line (IDH4) in which both proliferation and immortalization are dependent on the presence of SV40-encoded large T-antigen (SV40T). Expression of SV40T is controlled by a dexamethasone (Dex)-inducible promotor. Suppression of SV40DT (Dex removal) in IDH4 cells causes G1 arrest and expression of the senescent phenotype. This is accompanied by a redistribution of PML in most cells from the usual pattern containing only spherical bodies to a pattern, containing large doughnut-like or fiber-like structures in addition to the spherical bodies. This change in pattern is reversed when phenotypically senescent IDH4 cells are stimulated to proliferate again by SV40T-induction. Moreover, we find that there is a similar change in the PML pattern between young and senescent or serum-starved young IMR90 human fibroblasts, from which IDH4 cells are derived. However, fewer serum-starved cells contain large PML bodies than senescent cells. Our observations suggest senescence, although it may be partly related to growth arrest. Using three-dimensional fluorescence digital imaging microscopy, we have found that the apparently doughnut-like PML structures have a cylindrical or egg-shaped form and that PML is concentrated to the outer shell of the structure.