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1.
Curr Opin Biotechnol ; 61: 198-208, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32035346

RESUMO

The implementation of omics technologies such as genomics, proteomics and transcriptomics has revolutionized our understanding of microbiomes, and shed light on the functional attributes and mechanisms of action underlying the ability of probiotics to impact host health and starter cultures to drive food fermentation. Recently, molecular machines from CRISPR-Cas systems have redefined the gene editing toolbox and democritized our ability to alter the genome of food microorganisms. An integrated approach in which CRISPR-based genome editing is informed by omics studies is poised to enable the engineering of microorganisms and the formulation of microbiomes impacting the food supply chain. Here, we highlight the current applications of omics technologies in food microorganisms and CRISPR-based genome editing technologies in bacteria, and discuss how this integrated approach enables effective engineering of food microbes to generate enhanced probiotic strains, develop novel biotherapeutics and alter microbial communities in food matrices.

2.
Lab Med ; 51(1): 41-46, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31185079

RESUMO

OBJECTIVE: To determine a method to reduce specimen hemolysis rates in pediatric blood specimens. METHODS: A total of 290 blood specimens from pediatric patients were classified into the capped group or uncapped group. The hemolysis index and levels of lactate dehydrogenase (LDH) were measured using an automated biochemical analyzer. Also, we performed a paired test to measure the concentration of free hemoglobin in specimens from 25 randomly selected healthy adult volunteers, using a direct spectrophotometric technique. RESULTS: The hemolytic rate of capped specimens was 2-fold higher than that of uncapped specimens. We found significant differences for LDH. Also, there was a significant difference in the concentration of free hemoglobin in the random-volunteers test. CONCLUSIONS: Eliminating the residual negative pressure of vacuum blood-collection tubes was effective at reducing the macrohemolysis and/or microhemolysis rate.

3.
Int J Legal Med ; 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31802193

RESUMO

Custodial deaths refer to the death of an individual who is in prison, a detention center, or a police station. The present study aims to retrospectively analyze cases of custodial deaths examined at Tongji Medico Legal Expertise Center in Hubei (TMECH). A total of 172 out of 5853 cases were screened at TMECH from January 1999 to December 2016. Male preponderance was observed in 172 cases (male-female ratio: 5:1). Natural deaths accounted for the majority (70.93%), followed by suicide (16.28%), accidents (3.49%), homicides (4.65%), and undetermined causes (4.65%). The most common natural cause was cardiovascular disease. Custodial deaths occurred more frequently in prisons and detention houses than in police cells (63%, 63%, and 46%, respectively). Among the 172 cases, 105 deaths occurred after resuscitation failure despite the individual being sent to the hospital. The average age across cases was 36.3 years, and 90% of the deceased were aged under 50 years. Since there is no officially reported data regarding the prevalence of causes and manners of custodial deaths in China, our analysis contributes to enhancing the understanding of such deaths in central China and serves as a reference for law enforcement to develop a prevention program to reduce incidents of mortality in custody.

4.
Proc Natl Acad Sci U S A ; 116(32): 15774-15783, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31341082

RESUMO

CRISPR-Cas systems are now widely used for genome editing and transcriptional regulation in diverse organisms. The compact and portable nature of class 2 single effector nucleases, such as Cas9 or Cas12, has facilitated directed genome modifications in plants, animals, and microbes. However, most CRISPR-Cas systems belong to the more prevalent class 1 category, which hinges on multiprotein effector complexes. In the present study, we detail how the native type I-E CRISPR-Cas system, with a 5'-AAA-3' protospacer adjacent motif (PAM) and a 61-nucleotide guide CRISPR RNA (crRNA) can be repurposed for efficient chromosomal targeting and genome editing in Lactobacillus crispatus, an important commensal and beneficial microbe in the vaginal and intestinal tracts. Specifically, we generated diverse mutations encompassing a 643-base pair (bp) deletion (100% efficiency), a stop codon insertion (36%), and a single nucleotide substitution (19%) in the exopolysaccharide priming-glycosyl transferase (p-gtf). Additional genetic targets included a 308-bp deletion (20%) in the prophage DNA packaging Nu1 and a 730-bp insertion of the green fluorescent protein gene downstream of enolase (23%). This approach enables flexible alteration of the formerly genetically recalcitrant species L. crispatus, with potential for probiotic enhancement, biotherapeutic engineering, and mucosal vaccine delivery. These results also provide a framework for repurposing endogenous CRISPR-Cas systems for flexible genome targeting and editing, while expanding the toolbox to include one of the most abundant and diverse systems found in nature.

5.
Drug Des Devel Ther ; 13: 1023-1032, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31114156

RESUMO

Aim: To investigate the anticancer effects of Jinlong capsule (JLC) against human glioblastoma cells and the possible underlying mechanism. Methods: Cell Counting Kit-8 and colony formation assay were adopted for the analysis of cell viability. Cell invasion and migration were evaluated by transwell and wound healing assays. Then, the expression level of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), S6 and phosphorylated S6 (p-S6) were determined by western blotting. Results: The results showed that JLC significantly inhibited human glioblastoma cell proliferation, invasion and migration in a dose-dependent manner. The expressions of p-mTOR and p-S6 were dramatically suppressed by JLC. Furtherly, inhibition of mTOR reduced the cell migration and invasion, while the mTOR agonist (MHY1485) could partially reverse the anti-migration and anti-invasion activity of JLC. Conclusion: The above results suggested that JLC would be a potential candidate for the treatment of glioblastoma.


Assuntos
Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Glioblastoma/tratamento farmacológico , Invasividade Neoplásica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Antineoplásicos/química , Cápsulas/química , Cápsulas/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Medicamentos de Ervas Chinesas/química , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Células Tumorais Cultivadas
6.
Cell Host Microbe ; 25(2): 273-284.e6, 2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30658906

RESUMO

The mammalian intestinal tract contains a complex microbial ecosystem with many lysogens, which are bacteria containing dormant phages (prophages) inserted within their genomes. Approximately half of intestinal viruses are derived from lysogens, suggesting that these bacteria encounter triggers that promote phage production. We show that prophages of the gut symbiont Lactobacillus reuteri are activated during gastrointestinal transit and that phage production is further increased in response to a fructose-enriched diet. Fructose and exposure to short-chain fatty acids activate the Ack pathway, involved in generating acetic acid, which in turn triggers the bacterial stress response that promotes phage production. L. reuteri mutants of the Ack pathway or RecA, a stress response component, exhibit decreased phage production. Thus, prophages in a gut symbiont can be induced by diet and metabolites affected by diet, which provides a potential mechanistic explanation for the effects of diet on the intestinal phage community.


Assuntos
Ácidos Graxos Voláteis/metabolismo , Frutose/metabolismo , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/virologia , Lactobacillus reuteri/metabolismo , Lactobacillus reuteri/virologia , Prófagos/crescimento & desenvolvimento , Animais , Lactobacillus reuteri/crescimento & desenvolvimento , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Estresse Fisiológico , Ativação Viral
7.
SLAS Technol ; 23(6): 631-640, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29787327

RESUMO

Arterial blood gas (ABG) analysis is important for acutely ill patients and should be performed by qualified laboratorians. The existing manual verifications are tedious, time-consuming, and prone to send wrong reports. Autoverification uses computer-based rules to verify clinical laboratory test results without manual review. To date, no data are available on the use of autoverification for ABG analysis. All autoverification rules were established according to AUTO10-A. Additionally, the rules were established using retrospective patient data, and then validated by actual clinical samples in a "live" environment before go-live. The average autoverification passing rate was 75.5%. The turnaround time (TAT) was reduced by 33.3% (27 min vs 18 min). Moreover, the error rate fell to 0.05% after implementation. Statistical analysis resulted in a kappa statistic of 0.92 ( p < 0.01), indicating close agreement between autoverification and senior technician verification, and the chi-square value was 22.4 ( p < 0.01), indicating that the autoverification error rate was lower than the manual verification error rate. Results showed that implementing autoverification rules with intelligent guidelines for ABG analysis of patients with critical illnesses could decrease the number of samples requiring manual verification, reduce TAT, and eliminate errors, allowing laboratorians to concentrate more time on abnormal samples, patient care, and collaboration with physicians.


Assuntos
Artérias , Automação Laboratorial/métodos , Gasometria/métodos , Gasometria/normas , Sistemas de Informação em Laboratório Clínico/normas , Estado Terminal , Humanos , Estudos Retrospectivos
8.
Medicine (Baltimore) ; 97(7): e9941, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29443781

RESUMO

RATIONALE: Acute epiglottitis is a potentially life-threaten disease, which makes it more challenging to save the life for doctors. Unexpected deaths in custody are a primary cause of concern for the forensic community and doctor worldwide. PATIENT CONCERNS: We present a case of a 44-year-old male detainee who was clinically suspected of dying of acute epiglottitis. The man experienced failure of resuscitation and died after admitted to a hospital. DIAGNOSES: The autopsy, toxicological testing, the test of immunoglobulin E and bacterial culture suggested the patient died of acute epiglottitis. INTERVENTIONS: The bacterial culture was performed to imprecisely identify the cause of death. OUTCOMES: The bacterial culture of the patient's heart blood and nasal and throat swabs showed the presence of the pathogenic microorganism Haemophilus influenza type B. LESSONS: We aim to provide a reference to the medical and forensic community and remind the local law enforcement agencies on the problems present within the correctional healthcare system through this case report. Additionally, we also aim to increase the current knowledge and understanding on custodial deaths caused by natural diseases.


Assuntos
Morte Súbita/etiologia , Epiglotite/diagnóstico , Infecções por Haemophilus/diagnóstico , Prisioneiros , Doença Aguda , Adulto , Autopsia , Epiglotite/virologia , Evolução Fatal , Humanos , Masculino
9.
Int J Infect Dis ; 59: 110-117, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28416440

RESUMO

OBJECTIVES: To investigate PI3K-Akt-mTOR signaling pathway changes and the proliferation of FoxP3+Treg cells in patients with active tuberculosis. METHODS: We isolated PBMCs and CD4+CD25+FoxP3+Treg cells from peripheral blood collected from patients with active tuberculosis and healthy controls. We compared the proportion and MFI of PI3K-Akt-mTOR pathway components and PTEN by flow cytometry using specific cell-surface and intracellular markers. Moreover, we detected the specific secretory proteins ESAT-6 and Ag85B, cytokines IL-10, TGF-ß1 and IL-35 in serum by ELISA. RESULTS: Compared with healthy controls, the proportions of CD3+Akt+, CD3+p-Akt+, CD3+mTOR+, CD3+p-mTOR+ and CD3+PTEN+ cells, in the T lymphocyte population of patients with active tuberculosis, were decreased (p<0.05), while CD3+FoxP3+ cells were increased (p=0.013). Similarly, for CD4+CD25+FoxP3+Treg cells, the proportions of Akt+ cells, p-Akt+ cells, mTOR+ cells, p-mTOR+ cells and PTEN+ cells were decreased (p<0.05) in patients with active tuberculosis. Compared with healthy controls, the levels of ESAT-6 and Ag85B were higher in patients with active tuberculosis (p<0.001). Levels of IL-10 and TGF-ß1 were higher (p<0.001), whereas the level of IL-35 was lower (p<0.001). CONCLUSION: The PI3K-Akt-mTOR signaling pathway in T lymphocytes and CD4+CD25+FoxP3+Treg cells was inhibited, which could explain why M.tuberculosis can induce FoxP3+Treg cell to expand.


Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Tuberculose Pulmonar/metabolismo , Adulto , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Humanos , Interleucina-10/metabolismo , Masculino , Mycobacterium tuberculosis , PTEN Fosfo-Hidrolase/metabolismo , Tuberculose Pulmonar/imunologia
10.
Hemoglobin ; 40(2): 138-42, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26865073

RESUMO

Thalassemia is one of the most prevalent inherited disease in southern China. However, there have been only a few epidemiological studies of thalassemia in the Chaoshan region of Guangdong Province, People's Republic of China (PRC). A total of 6231 unrelated subjects in two main geographical cities of the Chaoshan region was analyzed for thalassemia. Seven hundred and thirty-six cases of suspected thalassemia carriers with microcytosis [mean corpuscular volume (MCV) <82.0 fL] were found by complete blood cell (CBC) count, and were tested by reverse dot-blot gene chip to reveal a total of 331 mutant chromosomes, including 278 α-thalassemia (α-thal) alleles and 53 ß-thalassemia (ß-thal) alleles. The most common α-thal mutations were the Southeast Asian (- -(SEA)), followed by the -α(3.7) (rightward) and -α(4.2) (leftward) deletions. The two most common ß-thal mutations were HBB: c.316-197C>T and HBB: c.126_129delCTTT, accounting for 69.81% of the ß-thal defects in the studied individuals. In addition, a rare mutation, Cap +1 (A>C) (HBB: c.-50A>C) was described for the first time in the Chaoshan region. Our results gave a heterozygote frequency of 5.31% for common α- and ß-thal in the Chaoshan region, and also indicated a higher prevalence of thalassemia with a heterozygote frequency of 6.29% in Chaozhou, followed by Shantou (3.37%). This study provided a detailed prevalence and molecular characterization of thalassemia in the Chaoshan region, and will be valuable for developing a strategy for prevention of thalassemia and reducing excessive health care costs in this area.


Assuntos
Talassemia/epidemiologia , Talassemia/genética , Alelos , China/epidemiologia , Frequência do Gene , Genótipo , Geografia , Hemoglobinas Anormais/genética , Humanos , Mutação , Vigilância da População , Prevalência
11.
J Lab Autom ; 21(5): 642-51, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26311059

RESUMO

Autoverification is a process of using computer-based rules to verify clinical laboratory test results without manual review. But to date, there are few published articles on the use of autoverification over the course of years in a clinical laboratory. In our study, we firstly described the development and implementation of autoverification rules for enzyme-linked immunosorbent assay (ELISA) results of hepatitis B virus (HBV) serological markers in a clinical immunology laboratory. We designed the autoverification rules for HBV by using Boolean logic on five clinically used serological markers in accordance with the framework of AUTO-10A, issued by the American Clinical Laboratory Standards Institute in 2006. The rules were written into the laboratory information system (LIS) and installed in the computer, so we could use the LIS to screen the test results. If the results passed the autoverification rules, they could be sent to doctors immediately. To evaluate the autoverification rules, we applied the real-time data of 11,585 patients with the autoverification rules. The autoverification rate of the five HBV serological markers was 79.5%. Furthermore, the turnaround time (TAT) was reduced by 38% (78 minutes vs. 126 minutes). The error rate was nearly eliminated. These results show that using LIS with autoverification rules can shorten TAT, enhance efficiency, and reduce manual review errors.


Assuntos
Automação Laboratorial/métodos , Biomarcadores/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Hepatite B/diagnóstico , Testes Sorológicos/métodos , Humanos
12.
PLoS One ; 9(8): e102243, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25089872

RESUMO

ß-thalassemia is a common inherited disorder worldwide including southern China, and at least 45 distinct ß-thalassemia mutations have been identified in China. High-resolution melting (HRM) assay was recently introduced as a rapid, inexpensive and effective method for genotyping. However, there was no systemic study on the diagnostic capability of HRM to identify ß-thalassemia. Here, we used an improved HRM method to screen and type 12 common ß-thalassemia mutations in Chinese, and the rapidity and reliability of this method was investigated. The whole PCR and HRM procedure could be completed in 40 min. The heterozygous mutations and 4 kinds of homozygous mutations could be readily differentiated from the melting curve except c.-78A>G heterozygote and c.-79A>G heterozygote. The diagnostic reliability of this HRM assay was evaluated on 756 pre-typed genomic DNA samples and 50 cases of blood spots on filter paper, which were collected from seven high prevalent provinces in southern China. If c.-78A>G heterozygote and c.-79A>G heterozygote were classified into the same group (c.-78&79 A>G heterozygote), the HRM method was in complete concordance with the reference method (reverse dot blot/DNA-sequencing). In a conclusion, the HRM method appears to be an accurate and sensitive method for the rapid screening and identification of ß-thalassemia mutations. In the future, we suggest this technology to be used in neonatal blood spot screening program. It could enlarge the coverage of ß-thalassemia screening program in China. At the same time, its value should be confirmed in prospectively clinical and epidemiological studies.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Testes Genéticos , Técnicas de Genotipagem/métodos , Mutação/genética , Desnaturação de Ácido Nucleico/genética , Talassemia beta/genética , China , DNA/sangue , DNA/genética , Geografia , Humanos , Reprodutibilidade dos Testes , Talassemia beta/sangue
13.
Mol Biol Rep ; 40(4): 3073-82, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23275194

RESUMO

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited disorder worldwide including southern China. G6PD gene mutations cause deficiency of the enzyme and a large spectrum of diseases. High-resolution DNA melting (HRM) assay was recently introduced as a rapid, inexpensive and effective method for genotyping. But there was a shortcoming of this method that hemizygous and homozygous genotypes were not easily distinguished from wild-types. Here we used improved HRM method for a small-scale screening of G6PD-deficient variants among people of Meizhou region. Then all amplicons were ascertained by direct DNA sequencing. These results indicated that HRM method was a major technical advance for G6PD mutations screening.


Assuntos
Técnicas de Genotipagem/métodos , Deficiência de Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/genética , Desnaturação de Ácido Nucleico , Grupo com Ancestrais do Continente Asiático , China , Deficiência de Glucosefosfato Desidrogenase/diagnóstico , Humanos , Mutação , Análise de Sequência de DNA
14.
Blood Cells Mol Dis ; 48(2): 86-90, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22197394

RESUMO

Thalassemia is the commonest inherited autosomal recessive disorders of hemoglobin in southern China. We developed and evaluated a reverse dot blot (RDB) assay combined with flow-through hybridization technology platform for the rapid and simultaneous identification of 5 types of α-thalassemia and 16 types of ß-thalassemia common in Chinese. Reliable genotyping of wild-type and thalassemic genomic DNA samples was achieved by means of a gene chip on which allele-specific oligonucleotide probes were immobilized on a nylon membrane. This method involved two multiplex PCR amplification systems of α-thalassemia and ß-thalassemia and one time of hybridization. The whole procedure starting from blood sampling to the identification of thalassemia genotype required less than 4h. The diagnostic reliability of this reverse dot blot assay was evaluated on 427 samples (387 cases of thalassemia and 40 healthy persons) by using direct DNA sequence analysis and gap-PCR in a blind study. These samples included 377 cases of blood, 7 cases of amniotic fluid, 18 cases of chorionic villus, and 25 cases of cord blood. The RDB gene chip was in complete concordance with the reference method. The reverse dot blot assay was a simple, rapid, accurate, and cost-effective method to identify common thalassemia genotypes in the Chinese population.


Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , Talassemia alfa/diagnóstico , Talassemia beta/diagnóstico , Grupo com Ancestrais do Continente Asiático/genética , Sequência de Bases , China , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Mutação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , alfa-Globinas/genética , Talassemia alfa/genética , Globinas beta/genética , Talassemia beta/genética
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