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2.
J Clin Microbiol ; 2020 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-33148709

RESUMO

Background: Bacterial vaginosis (BV) is caused by the excessive and imbalanced growth of bacteria in vagina, affecting 30-50% of women in their lives. Gram stain followed by Nugent scoring based on bacterial morphotypes under the microscope (NS) has been considered the golden standard for BV diagnosis, which is often labor-intensive, time-consuming, and variable results from person to person.Methods: We developed and optimized a convolutional neural networks (CNN) model, and evaluated its ability to automatically identify and classify three categories of Nugent scores from microscope images. The CNN model was first established with a panel of microscopic images with Nugent scores determined by experts. The model was trained by minimizing the cross entropy loss function and optimized by using a momentum optimizer. The separate test sets of images collected from three hospitals were evaluated by the CNN models.Results: The CNN model consisted of 25 convolutional layers, 2 pooling layers, and a fully connected layer. The model obtained 82.4% sensitivity and 96.6% specificity on the 5,815 validation images when considered altered vaginal flora and BV as the positive samples, which was better than the top-level technologists and obstetricians in China. The ability of generalization for our model was strong that it obtained 75.1% accuracy of three categories of Nugent scores on the independent test set of 1082 images, which was 6.6% higher than the average of three technologists, who are with a bachelor degree in medicine and eligible making diagnostic decisions. When three technologists ran one specimen in triplicate, the precision of three categories of Nugent scores was 54.0%. 103 samples diagnosed by two technologists at different days showed repeatability of 90.3%.Conclusion: The CNN model over-performed human healthcare practitioners on accuracy and stability for three categories of Nugent scores diagnosis. The deep learning model may offer translational applications in automating diagnosis of bacterial vaginosis with proper supporting hardware.

3.
Front Microbiol ; 11: 594065, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33240248

RESUMO

The high incidence of bacterial vaginosis recurrence is common after treatment with an antibiotic agent and suggests the need for new treatments to prevent this. We conducted a randomized trial to evaluate the ability of maltose gel to treat bacterial vaginosis. Eighteen female rhesus macaques were randomly assigned, in a 2:1 ratio, to receive maltose gel or placebo gel by syringe to the fornix of the vagina for five consecutive days. We used 16S rRNA sequencing data from 70 swab samples of vaginal secretions in two groups in total on days 0, 3, and 5 after medication initiation and days 3 and 5 after medication withdrawal for the study of microbiome composition. We found that, in the placebo control group, there was no significant change in the composition and abundance of vaginal microbiota during the follow-up period. In the maltose gel test group, the abundance of Lactobacillus in the vagina microbiota increased gradually with the prolongation of the treatment time on Days 3 and 5 (ANOVA p = 6.99e-5 < 0.01) but began to decrease after the withdrawal of maltose gel, which was different from that of the control group. Correspondingly, the diversity and abundance of BV-related bacteria, Fusobacterium, Parvimonas, Mobiluncus, Campylobacter, Prevotella, and Sneathia, decreased on Day 0 to Day 5 of medication and increased after drug withdrawal in the maltose gel test group. The study confirms that maltose gel can facilitate the proliferation of Lactobacillus and promote the transition of the vaginal microbiota from BV-related bacteria dominant to Lactobacillus dominant in the rhesus macaque.

4.
Sheng Wu Gong Cheng Xue Bao ; 36(6): 1041-1050, 2020 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-32597055

RESUMO

In recent years, the demand of biologics has increased rapidly. Cell culture process with perfusion mode has become more and more popular due to its high productivity, good quality and high efficiency. In this paper, the unique operation and the details of process optimization for perfusion culture mode are discussed by comparing with traditional batch culture process. Meanwhile, the progress and strategies in the development and optimization of perfusion culture process in recent years are summarized to provide reference for the future development of mammalian cell perfusion culture technology.


Assuntos
Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Animais , Técnicas de Cultura Celular por Lotes/tendências , Reatores Biológicos/normas , Células CHO , Cricetulus , Mamíferos , Perfusão
5.
Food Chem Toxicol ; 140: 111276, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32199966

RESUMO

Deoxynivalenol (DON), a toxic secondary metabolite produced by Fusarium species that mainly infests cereals such as wheat and corn, threatens human and livestock health. The present study describes the characterization of a novel bacterial strain, Pelagibacterium halotolerans ANSP101 which is capable of transforming DON to less-toxic product 3-keto-deoxynivalenol by the oxidation of the C3 hydroxyl group. Strain ANSP101 was isolated from a seawater sample from a depth of 55 m in Chinese Bohai sea. The strain was identified as Pelagibacterium halotolerans by morphology characterization and 16S rDNA gene sequencing. The DON degrading activity of strain ANSP101 was predominantly attributed to the bacterial cell lysate. Besides, the cell lysate was sensitive to sodium dodecyl sulfate, heat, and proteinase K treatment, indicating that the intracellular proteins or enzymes are responsible for the DON degradation. The optimal temperature and pH for the maximal degradation of DON were 40 °C and pH 8.0 by the cell lysate. These results provide the potential use of P. halotolerans ANSP101 as a detoxification agent for DON decontamination in cereals and feed.

7.
Infect Drug Resist ; 12: 3745-3754, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31824176

RESUMO

Background: Aerobic vaginitis (AV) is a newly defined type of bacterial vaginitis, but its pathogenesis is not yet clear. Streptococcus anginosus appears as an emerging pathogen in recent case reports, and colonizes in vagina of patients with AV. In this study, we investigate the pathogenesis of S. anginosus in AV. Materials and methods: (1) We collected 41 vaginal specimens from 21 healthy, fertile women with normal vaginal flora (NM), 10 with bacterial vaginosis (BV) and 10 with AV; their microbiome structure was analysed by 16S rRNA gene sequencing. (2) S. anginosus and vaginal epithelial cells were cocultured in vitro, and cytotoxicity was tested by an LDH kit. (3) The S. anginosus virulence gene sag was knocked out, and the cytotoxicity of the mutant in vaginal epithelial cells was tested. Results: (1) The microbiome structure of AV was dramatically different from that of BV and NM. The predominant genera of the three groups were Streptococcus spp., Gardnerella spp. and Lactobacillus spp. Streptococcus spp. were significantly more abundant in AV than in BV (95% CI [0.1391, 0.8676], P<0.01) and NM (95% CI [0.1391, 0.8676], P<0.01). (2) S. anginosus was the dominant species in AV (95% CI [0.04672, 0.1097], P<0.01). (3) The mean cytotoxicity of S. anginosus in vaginal epithelial cells was 58.34% for the wild type (WT) and 16.43% for the mutant; this difference was significant (95% CI [-60.55, -23.28], P<0.01). Conclusion: S. anginosus was the predominant microorganism in patients with AV in our study. S. anginosus caused vaginal epithelial cell lysis, indicating that S. anginosus is an AV pathogen. The S. anginosus virulence gene sag is vital for vaginal epithelial cell lysis.

8.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(7): 720-723, 2019 Jul 10.
Artigo em Chinês | MEDLINE | ID: mdl-31302920

RESUMO

OBJECTIVE: To explore the genetic cause of a neonate with congenital dysplasia, growth retardation through clinical evaluation, laboratory tests and next generation sequencing (NGS). METHODS: Peripheral blood samples were obtained from the child and his parents. Whole genomic DNA was extracted and subjected to NGS. Suspected mutation was predicted by bioinformatic tools and validated by Sanger sequencing. RESULTS: The child was found to carry a c.556G>A (p.E186K) mutation of the HDAC8 gene on the X chromosome, which was predicted to be pathogenic by Bioinformatic analysis. CONCLUSION: The patient was diagnosed as Cornelia de Lange syndrome 5 caused by the c.556G>A mutation of the HDAC8 gene.


Assuntos
Síndrome de Cornélia de Lange/genética , Histona Desacetilases/genética , Proteínas Repressoras/genética , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recém-Nascido , Masculino , Mutação
10.
Biosci Trends ; 13(1): 58-69, 2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30773525

RESUMO

Immunotherapy might be an effective treatment in extrahepatic cholangiocarcinoma (eCCA), a tumor with extremely limited therapeutic options. Our study is to characterize the programmed death ligand-1 (PD-L1) protein expression and cancer microenvironment profiles in surgically resected eCCA samples. PD-L1 positivity was observed on tumor cells (32.3%) as well as on tumor-associated macrophages (74.2%). PD-L1 expression by eCCA correlated significantly with immune parameters such as intra-tumoral CD3+ tumor infiltrating lymphocytes (TILs) density (P = 0.002), intra-tumoral CD8+ TILs density (P < 0.001), and the expression pattern of human leukocyte antigen (HLA) class I (P < 0.001). Immunofluorescence showed that PD-L1 positive tumor cells were adjacent to PD-1 positive cells and the stroma covered with interferon-γ. Correlation with clinicopathological parameters and survival analyses revealed that PD-L1 positivity in eCCA was related to the absence of venous invasion (P = 0.030), improved overall survival (P = 0.020) and progressionfree survival (P = 0.011). HLA class I molecules defect, which is an important mechanism of immune evasion, was frequently observed in eCCA (50.0%) and was associated with a decreased number of intra-tumoral CD8+ TIL density (P = 0.028). Additionally, the presence of unusually high numbers of tumor-associated macrophages (TAMs) subsets M2 in most of eCCA (74.2%) was noted. Our study indicated that PD-L1 expression in association with intra-tumoral TILs infiltration and HLA class I expression in 32.3% of the eCCA reflects an active immune microenvironment potentially responsive to PD-1/PD-L1 inhibitors. In addition, the combination of macrophage-targeting agents may provide therapeutic synergy for future immunotherapy.


Assuntos
Antígeno B7-H1/metabolismo , Neoplasias dos Ductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Idoso de 80 Anos ou mais , Neoplasias dos Ductos Biliares/imunologia , Neoplasias dos Ductos Biliares/mortalidade , Neoplasias dos Ductos Biliares/patologia , China/epidemiologia , Colangiocarcinoma/imunologia , Colangiocarcinoma/mortalidade , Colangiocarcinoma/patologia , Estudos de Coortes , Feminino , Humanos , Linfócitos do Interstício Tumoral/fisiologia , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Microambiente Tumoral , Adulto Jovem
11.
Biotechnol J ; 14(3): e1800315, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30144348

RESUMO

Metabolomics is an essential discipline in industrial biotechnology. Sample preparation approaches dramatically influence data quality and, ultimately, interpretation and conclusions from metabolomic experiments. However, standardized protocols for highly reproducible metabolic datasets are limited, especially for the fungal cell factory Aspergillus niger. Here, an improved liquid chromatography-tandem mass spectrometry-based pipeline for A. niger metabolomics is developed. It is found that fast filtration with liquid nitrogen is more suitable for cell quenching, causing minimal disruption to cell integrity, and improved intracellular metabolite recovery when compared to cold methanol quenching approaches. Seven solutions are evaluated for intracellular metabolite extraction, and found acetonitrile/water (1:1, v/v) at -20 °C, combined with boiling ethanol extraction protocols, showed unbiased metabolite profiling. This improved methodology is applied to unveil the dynamic metabolite profile of one citrate over-producing A. niger isolate under citrate fermentation. Citrate precursors, especially pyruvate, oxaloacetate, and malate, are maintained at a relatively high intracellular level, which can be necessary for high citrate synthesis flux. Glutamine shows a similar trend compared to citrate production, suggesting glutamine may be involved in intracellular pH homeostasis. Taken together, this study delivers a highly standardized and improved metabolomics methodology and paves the way for systems metabolic engineering in biotechnologically important fungi.


Assuntos
Aspergillus niger/metabolismo , Metaboloma/fisiologia , Metabolômica/métodos , Manejo de Espécimes/métodos , Acetonitrilos/química , Biotecnologia/métodos , Cromatografia Líquida/métodos , Etanol/química , Fermentação/fisiologia , Filtração/métodos , Glutamina/química , Engenharia Metabólica/métodos , Metanol/química , Espectrometria de Massas em Tandem/métodos , Água/química
12.
Metab Eng ; 49: 220-231, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30048680

RESUMO

Methanol is a promising feedstock for bioproduction of fuels and chemicals, thus massive efforts have been devoted to engineering non-native methylotrophic platform microorganisms to utilize methanol. Herein, we rationally designed and experimentally engineered the industrial workhorse Corynebacterium glutamicum to serve as a methanol-dependent synthetic methylotroph. The cell growth of the methanol-dependent strain relies on co-utilization of methanol and xylose, and most notably methanol is an indispensable carbon source. Due to the methanol-dependent characteristic, adaptive laboratory evolution was successfully applied to improving methanol utilization. The evolved mutant showed a 20-fold increase in cell growth on methanol-xylose minimal medium and utilized methanol and xylose with a high mole ratio of 3.83:1. 13C-labeling experiments demonstrated that the carbon derived from methanol was assimilated into intracellular building blocks, high-energy carriers, cofactors, and biomass (up to 63% 13C-labeling). By inhibiting cell wall biosynthesis, methanol-dependent glutamate production was also achieved, demonstrating the potential application in bioconversion of methanol into useful chemicals. Genetic mutations detected in the evolved strains indicate the importance of intracellular NAD+/NADH ratio, substrate uptake, and methanol tolerance on methanol utilization. This study reports significant improvement in the area of developing fully synthetic methylotrophs.


Assuntos
Corynebacterium glutamicum , Ácido Glutâmico/biossíntese , Engenharia Metabólica , Metanol/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Ácido Glutâmico/genética
13.
Appl Microbiol Biotechnol ; 102(16): 7113-7121, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29876603

RESUMO

Metabolomics has been a potential tool for strain improvement through analyzing metabolite changes in the context of different conditions. However, the availability of a universal metabolite profiling analysis is still a big challenge. In this study, we presented an optimized liquid chromatography-tandem mass spectrometry-based metabolomics methodology for Corynebacterium glutamicum, an important industrial workhorse. It was found that quenching the cellular metabolism with 5-fold volume of - 20 °C 40% methanol was highly recommended due to its lower cell damage rate and higher intracellular metabolite recovery rate. For extracting intracellular metabolites, ethanol/water (3:1, v/v) at 100 °C combined with acidic acetonitrile/water (1:1, v/v, with 0.1% formic acid) at - 20 °C achieved the unbiased metabolite profiling of C. glutamicum. The established methodology was then applied to investigate the intracellular metabolite differences between C. glutamicum ATCC 13032 and an mscCG-deleted mutant under biotin limitation condition. It was observed that in the presence of the functional L-glutamate exporter MscCG, biotin limitation led to accumulation of intracellular 2-oxoglutarate but not L-glutamate. Deletion of mscCG severely inhibited L-glutamate excretion and resulted in a dramatical increase of intracellular L-glutamate, which in turn affected the metabolite profile. The optimized metabolomics methodology holds promise for promoting studies on metabolic mechanism of C. glutamicum.


Assuntos
Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/metabolismo , Metabolômica/métodos , Transporte Biológico , Biotina/metabolismo , Corynebacterium glutamicum/genética , Ácido Glutâmico/metabolismo , Mutação , Espectrometria de Massas em Tandem/métodos
14.
J Am Chem Soc ; 138(16): 5308-15, 2016 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-27016183

RESUMO

Metal-organic frameworks (MOFs) with light-harvesting building blocks designed to mimic photosynthetic chromophore arrays in green plants provide an excellent platform to study exciton transport in networks with well-defined structures. A step-by-step exciton random hopping model made of the elementary steps of energy transfer between only the nearest neighbors is usually used to describe the transport dynamics. Although such a nearest neighbor approximation is valid in describing the energy transfer of triplet states via the Dexter mechanism, we found it inadequate in evaluating singlet exciton migration that occurs through the Förster mechanism, which involves one-step jumping over longer distance. We measured migration rates of singlet excitons on two MOFs constructed from truxene-derived ligands and zinc nodes, by monitoring energy transfer from the MOF skeleton to a coumarin probe in the MOF cavity. The diffusivities of the excitons on the frameworks were determined to be 1.8 × 10(-2) cm(2)/s and 2.3 × 10(-2) cm(2)/s, corresponding to migration distances of 43 and 48 nm within their lifetimes, respectively. "Through space" energy-jumping beyond nearest neighbor accounts for up to 67% of the energy transfer rates. This finding presents a new perspective in the design and understanding of highly efficient energy transport networks for singlet excited states.

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