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1.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 2467-2470, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33018506

RESUMO

A mathematical model, in the form of a system of nonlinear ordinary differential equations, that describes the interaction between tumor cells and effective immune cells is proposed. An exact solution cannot be found to this system like many other nonlinear systems. Yet, approximate analytical solution is explored. This solution should have a large interval of convergence to be acceptable because the interaction can take many days to reach its steady state. Power series method is used to obtain a series solution. In this process, some auxiliary variables are used to transform the system of equations to polynomial form. However, this solution has a small radius of convergence, therefore, Padé approximant method is used to extend the domain of convergence. Hence, the obtained approximate analytical solution is valid over a large interval and has a remarkable accuracy when compared with numerical solution.


Assuntos
Neoplasias , Contagem de Células , Humanos , Modelos Teóricos
2.
Medicine (Baltimore) ; 99(40): e22242, 2020 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-33019399

RESUMO

BACKGROUND: To evaluate the clinical value of circulating tumor cell (CTC) detection in peripheral blood for the diagnosis and prognosis of hepatocellular carcinoma (HCC). METHODS: Public databases were searched, and a meta-analysis was performed to determine the specificity, sensitivity, negative- likelihood ratio (NLR) and positive-likelihood ratio (PLR), and diagnostic odds ratio (dOR) of CTC detection for the diagnosis of HCC. Hazard ratios (HRs) and 95% confidence intervals (CIs) were analyzed for the association of CTC detection with overall survival (OS) and HCC recurrence. The Meta-DiSc 1.4 and Review Manager 5.2 software programs were used for statistical analysis. RESULTS: Meta-analysis of 20 studies including 1191 patients showed that the specificity, sensitivity, NLR, PLR, and dOR of CTC testing for HCC diagnosis were 0.60 (95% CI = 0.57-0.63), 0.95 (95%CI = 0.93-0.96), 0.36 (95%CI = 0.28-0.48), 11.64 (95%CI = 5.85-23.14), and 38.94 (95%CI = 18.33-82.75), respectively. Meta-analysis of 18 studies including 1466 patients indicated that the OS of CTC-positive HCC patients was less than that of CTC-negative patients (HR = 2.31; 95% CI = 1.55-3.42; P < .01). Meta-analysis of 5 studies including 339 patients revealed that the presence of CTCs in peripheral blood significantly increased the risk of HCC recurrence (HR = 3.03, 95% CI = 1.89-4.86; P < .01). CONCLUSION: CTCs in peripheral blood may be a useful marker for HCC diagnosis. In addition, the prognosis of CTC-positive HCC patients was significantly worse than that of CTC-negative HCC patients. Therefore, further studies are warranted to confirm the clinical potential of CTC detection in peripheral blood in patients with primary HCC.


Assuntos
Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/patologia , Células Neoplásicas Circulantes/metabolismo , Biomarcadores Tumorais , Carcinoma Hepatocelular/sangue , Contagem de Células , Humanos , Neoplasias Hepáticas/sangue , Recidiva Local de Neoplasia , Células Neoplásicas Circulantes/patologia , Razão de Chances , Prognóstico , Modelos de Riscos Proporcionais , Curva ROC , Sensibilidade e Especificidade , Análise de Sobrevida
3.
Chem Biol Interact ; 330: 109251, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32888910

RESUMO

Cisplatin induces acute renal failure in humans and mice.Tubular apoptosis, necrosis and inflammation are the primary pathogenesis of cisplatin-induced acute kidney injury(AKI). We previously reported that the depletion of Numb from proximal tubules exacerbates tubular cells apoptosis in cisplatin-induced AKI, however, the role of Numb in tubular necrosis and renal inflammation in cisplatin-induced AKI remains unclear. A mouse model of AKI was produced by cisplatin intraperitoneally injection in mice from proximal tubule-specific depletion of Numb (PT-Nb-KO) and their wild-type littermates (PT-Nb-WT) respectively. Renal Numb expression was determined by Western blotting. Renal morphological damage was examined by hematoxylin and eosin staining (H&E staining). Tubular necrosis was evaluated by histological study and the protein level of renal Mixed lineage kinase domain-like protein (MLKL) which is a molecular marker of necrosis. Leukocyte infiltration and pro-inflammatory cytokines was determined by immunostaining and quantitative real-time PCR (qRT-PCR) respectively.The protein level of Numb was dramatically decreased in kidneys of PT-Nb-KO mice compared with PT-Nb-WT mice. After cisplatin injection, a significant increase of tubular injury score and the protein level of renal MLKL were detected in PT-Nb-KO mice compared with those in PT-Nb-WT. In addition, the number of F4/80-positve and CD3-positive cells, markers for macrophages and neutraphils respectively, showed significantly increased in kidneys from PT-Nb-KO mice compared with those in PT-Nb-WT mice. Consistently, the gene expression of pro-inflammatory cytokines including TNF-α and MCP-1 in the kidneys was higher in PT-Nb-KO mice than those in PT-Nb-WT mice. Numb play additional protective role in cisplatin-induced AKI through ameliorating tubular necrosis and renal inflammation besides attenuating cisplatin-induced tubular apoptosis.


Assuntos
Lesão Renal Aguda/patologia , Cisplatino/efeitos adversos , Inflamação/prevenção & controle , Proteínas de Membrana/fisiologia , Necrose/prevenção & controle , Proteínas do Tecido Nervoso/fisiologia , Animais , Contagem de Células , Citocinas/metabolismo , Modelos Animais de Doenças , Inflamação/etiologia , Túbulos Renais Proximais/patologia , Mastócitos , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Necrose/etiologia , Proteínas do Tecido Nervoso/deficiência , Neutrófilos , Proteínas Quinases/metabolismo
4.
Ecotoxicol Environ Saf ; 205: 111376, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32961488

RESUMO

Deoxynivalenol (DON) is extensively detected in many kinds of foods and feeds to harm human and animal health. This research aims to investigate the effect of chlorogenic acid (CGA) on alleviating inflammation and apoptosis of swine jejunal epithelial cells (IPEC-J2) triggered by DON. The results demonstrated that cell viability was decreased when DON concentrations increased or incubation time expanded. The pretreatment with CGA (40 µg/mL) for 1 h increased cell viability, decreased lactate dehydrogenase (LDH) release and apoptosis in cells triggered by DON at 0.5 µg/mL for 6 h, compared with the DON alone-treated cells. Moreover, the mRNA abundances of IL-8, IL-6, TNF-α, COX-2, caspase-3, Bax and ASCT2 genes, and protein expressions of COX-2, Bax and ASCT2 were significantly down-regulated; while the mRNA abundances of ZO-1, claudin-1, occludin, PePT1 and GLUT2 genes, and protein expressions of ZO-1, claudin-1 and PePT1 were significantly up-regulated in the CGA + DON group, compared with the DON alone group. This study indicated that CGA pretreatment alleviated cytotoxicity, inflammation and apoptosis in DON-triggered IPEC-J2 cells, and protected intestinal cell integrity from DON damages.


Assuntos
Ácido Clorogênico/farmacologia , Substâncias Protetoras/farmacologia , Tricotecenos/toxicidade , Animais , Apoptose/efeitos dos fármacos , Caspase 3 , Contagem de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ácido Clorogênico/metabolismo , Células Epiteliais/efeitos dos fármacos , Inflamação/metabolismo , Intestinos/efeitos dos fármacos , Ocludina/genética , Suínos
5.
Anticancer Res ; 40(10): 5679-5685, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988893

RESUMO

BACKGROUND/AIM: The presence of circulating tumor cells (CTC) has been reported to have an impact on prognosis in different tumor entities. Little is known about CTC morphology and heterogeneity. PATIENTS AND METHODS: In a multicenter setting, pre-therapeutic peripheral blood specimens were drawn from patients with non-metastatic esophageal adenocarcinoma (EAC). CTCs were captured by size-based filtration (ScreenCell®), subsequently Giemsa-stained and evaluated by two trained readers. The isolated cells were categorized in groups based on morphologic criteria. RESULTS: Small and large single CTCs, as well as CTC-clusters, were observed in 69.2% (n=81) of the 117 specimens; small CTCs were observed most frequently (59%; n=69), followed by large CTCs (40%; n=47) and circulating cancer-associated macrophage-like cells (CAMLs; 34.2%, n=40). Clusters were rather rare (12%; n=14). CTC/CAML were heterogeneous in the cohort, but also within one specimen. Neither the presence of the CTC subtypes/CAMLs nor the exact cell count were associated with the primary clinical TNM stage. CONCLUSION: Morphologically heterogenic CTCs and CAMLs are present in patients with non-metastatic, non-pretreated EAC.


Assuntos
Adenocarcinoma/sangue , Biomarcadores Tumorais/sangue , Neoplasias Esofágicas/sangue , Células Neoplásicas Circulantes/metabolismo , Adenocarcinoma/patologia , Contagem de Células , Separação Celular , Neoplasias Esofágicas/patologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/patologia , Prognóstico
6.
Ann Hematol ; 99(10): 2405-2416, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32813071

RESUMO

Predictive factors of response to hypomethylating agents (HMA) in elderly acute myeloid leukemia (AML) patients remain unclear in the real-life setting and no direct comparison between azacitidine (AZA) and decitabine (DEC) has been carried out. We retrospectively evaluated 110 AML patients treated with HMA (78 AZA, 32 DEC) as first-line therapy outside of clinical trials. Median age was 75 years (range 58-87). The median overall survival (OS) of the entire cohort was 8.0 months (95% CI 6.1-10), without significant differences among the subgroups: AZA 8.8 months vs DEC 6.3 months (p = 0.291). HMA treatment yielded an overall response rate (ORR) of 40% (AZA 37% vs DEC 47%, p = 0.237). A stable disease (SD) after 4 HMA cycles was not associated with a worse survival outcome compared with an early optimal response. Factors independently associated with a better OS were transfusion independence during treatment (p = 0.049), achievement of an optimal response to treatment (p < 0.001), and a baseline hemoglobin level ≥ 9.25 (p = 0.018). A bone marrow (BM) blast count ≥ 30% (p < 0.001) and a therapy-related AML (p = 0.008) remain poor survival predictors. Of the available biologic features, an adverse risk category according to the ELN classification was significantly associated with a shorter survival over the intermediate risk category (p = 0.034). Disease progression remains the primary cause of death. Infectious complications were more severe (p = 0.036) and occurred earlier (p = 0.006) in the DEC group compared with that of the AZA group. In conclusion, clinical prognostic factors associated to response and survival have been identified without significant associations concerning overall outcomes between the two HMAs.


Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/uso terapêutico , Metilação de DNA/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , Decitabina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/patologia , Causas de Morte , Contagem de Células , DNA de Neoplasias/química , Progressão da Doença , Intervalo Livre de Doença , Feminino , Hemoglobinas/análise , Humanos , Infecções/etiologia , Infecções/mortalidade , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Resultado do Tratamento
7.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(4): 1357-1362, 2020 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-32798426

RESUMO

OBJECTIVE: To explore the method for inducing the differentiation of bone marrow cells into megakaryocytes in vitro so as to use for evaluating the activity of traditional Chinese medicines. METHODS: The bone marrow cells were separated from femurs and tibias of mice. The experiments were divided into 4 groups: control (no adding cytokines), TPO (adding 50 ng/ml TPO), TPO+SCF (50 ng/ml+50 ng/ml) and TPO+SCF+IL-6+IL-9 (50 ng/ml+50 ng/ml+20 ng/ml+20 ng/ml). The bone marrow cells in 4 groups were cultured in vitro for 6 d. Then the cell growth status was observed by the inverted microscopy, and the cell count was detected by using the automatic cell counter. The ratio and absolute count of megakaryocytes were detected by flow cytometry. RESULTS: Compared with control, three induction methods could stimulate the differentiation of bone marrow cells into megakaryocytes in vitro. TPO could slightly enhance the differentiation of bone marrow cells into megakaryocytes. Both the combination of TPO and SCF, and the combination of TPO, SCF, IL-6 and IL-9 could intensively stimulate proliferation of bone morrow cells and promote the differentiation of bone marrow cells into megakaryocytes. The addition of IL-6 and IL-9 could decrease the proliferation of non-megakaryocytes, but promote the differentiation of bone marrow cells into megakaryocytes. CONCLUSION: The optimized differentiation of bone marrow cells into megakaryocytes has been completed by co-induction regimen of TPO, SCF, IL-6 and IL-9, which can be used to screen and evaluate traditional Chinese medicines promoting formation of platelets.


Assuntos
Interleucina-3 , Megacariócitos , Animais , Células da Medula Óssea , Contagem de Células , Diferenciação Celular , Divisão Celular , Células Cultivadas , Camundongos , Fator de Células-Tronco , Trombopoetina
8.
Water Res ; 183: 116102, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32745672

RESUMO

We report the existence and resuscitation of viable but nonculturable (VBNC) Escherichia coli O157:H7 cells in drinking water induced by the common point-of-use disinfection treatments of boiling or microwaving. Tap water and saline samples containing E. coli O157:H7 culturable cells from a bovine isolate or two clinical isolates were boiled (1, 10, or 15 min) on a hot plate or microwaved (1.5 min) to reach boiling. No culturable E. coli O157:H7 cells were observed in the treated samples using conventional plating methods. In samples boiled for 1 or 10 min, two viability assays separately detected that 2-5.5% of the cells retained an intact membrane, while 28 to 87 cells out of the initial 108 cells retained both measurable intracellular esterase activity and membrane integrity. In samples boiled for 15 min, no viable cells were detected. The microwaved samples contained 6-10% of cells with an intact membrane, while 21 to 108 cells out of the initial 108 cells retained both membrane integrity and esterase activity. The number of viable cells retaining both metabolic activity and membrane integrity were consistent in all samples, supporting the survival of a small number of E. coli O157:H7 cells in the VBNC state after boiling for 1 or 10 min or microwaving. Furthermore, the VBNC E. coli O157:H7 cells regained growth at 37 °C in culture media containing autoinducers produced by common non-pathogenic E. coli, commonly present in the human intestine, and norepinephrine. The resuscitated cells were culturable on conventional plates and expressed mRNA encoding the E. coli O157 lipopolysaccharide gene (rfbE) and the H7 flagellin gene (fliC). This study highlights potential concerns for public health risk management of VBNC E. coli O157:H7 in drinking water disinfected by heat treatment at point-of-use. The public health significance of these concerns warrants further investigation.


Assuntos
Escherichia coli O157 , Animais , Bovinos , Contagem de Células , Contagem de Colônia Microbiana , Meios de Cultura , Humanos , Micro-Ondas , Água
9.
PLoS One ; 15(8): e0237615, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32780761

RESUMO

The impact of cow mammary gland diseases on the quality of colostrum is not conclusively defined; research results are conflicting. However, it is widely believed that mastitis lowers the level of immunoglobulins and the quality of the colostrum produced. Therefore, the aim of this study was to determine the influence of somatic cell counts (SCC) on the colostrum immunostimulating and chemical components. The experiment was conducted on an experimental organic dairy farm in which a herd of approximately 250 cows was kept in a freestall housing system, with the average performance exceeding 6,000 kg of milk per lactation. Colostrum and milk samples were taken individually from each cow seven times during the experiment: from the first to second day after calving-twice per day, and from the third to fifth day after calving-once per day. Therefore, after preliminary analyses, the cows were divided into two groups based on the cytological quality of their colostrum at the first collection: 1. SCC ≤400,000 cells/ml (good quality colostrum; GCC- 18 cows), 2. SCC ≥ 400,000 cells/ml (low quality colostrum; LCC- 22 cows). The study found almost double the concentration of immunoglobulins and essential fatty acids in first milking colostrum in the GCC group than in colostrum from the LCC group. In addition, an increase in the concentration of lysozyme in first milking colostrum was associated with a decrease in the concentration of immunoglobulins. In addition, the increase in the level of lysozyme was associated with a decrease in the concentration of immunoglobulins. In conclusion, the SCC of first milking colostrum can be used as an indicator of colostrum quality.


Assuntos
Contagem de Células/veterinária , Colostro/metabolismo , Indústria de Laticínios/métodos , Lactação/metabolismo , Leite/química , Leite/citologia , Animais , Bovinos , Feminino , Gravidez
10.
J Med Life ; 13(2): 241-248, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32742521

RESUMO

Cell culture is one of the most commonly used techniques in the production of biological products. Many physical and chemical parameters may affect cell growth and proliferation. This study was conducted to investigate the effect of chemical components as supplements using the experimental design method, which aimed at reducing the number of experiments. For this purpose, supplements including chemical components using four levels, with three replications in suspension and batch culture conditions, were examined for 72 hours using the Taguchi experimental design method. From the experiments, it was concluded that the culture media composition had a significant impact on final cell count and pH. High concentrations of different media composition alone were insufficient to ensure higher cell count. According to the results, this insufficiency was associated with an increase of 20% in the number of final cells. In the majority of cultures, the number of final cells at 48 hours increased relative to the number of final cells at 24 hours after culturing the cells.


Assuntos
Técnicas de Cultura de Células/métodos , Vírus da Febre Aftosa/imunologia , Rim/citologia , Vacinas Virais/imunologia , Aminoácidos/farmacologia , Animais , Contagem de Células , Células Cultivadas , Cricetinae , Vírus da Febre Aftosa/efeitos dos fármacos , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Polietilenoglicóis/química , Proteínas/farmacologia , Vitaminas/farmacologia
12.
PLoS One ; 15(8): e0237947, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833961

RESUMO

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) accounting for 85% of all lung cancer cases. Inflammation has been proven to be one of the characteristics of malignant tumors. Chronic inflammatory response mediated by cytokines in the tumor microenvironment is an important factor in tumorigenesis. The purpose of this study was to observe and evaluate the value of red blood cell distribution width (RDW), neutrophil-to-lymphocyte ratio (NLR), and hemoglobin-to-red blood cell distribution width ratio (HRR) in the progression of NSCLC. METHODS: A total of 245 patients with NSCLC, 97 patients with benign pulmonary nodules, and 94 healthy volunteers were included in this study. Factors, such as age, gender, smoking history, histological type, lymph node metastasis, distant metastasis, TNM stage, and differentiation degree were statistically analyzed. The correlation of RDW, NLR, and HRR of patients with NSCLC with other clinical experimental parameters were also analyzed. Then, the diagnostic value of RDW, NLR, and HRR in the progression of NSCLC was evaluated. RESULTS: RDW, NLR, and HRR could be used to distinguish patients with NSCLC from healthy controls (p < 0.05). In addition, only the RDW in the NSCLC group with III-IV stage was significantly different from that in the benign pulmonary nodules group (p = 0.033), while NLR and HRR could significantly distinguish patients with NSCLC and benign pulmonary nodules (p < 0.001). RDW and NLR were positively correlated with NSCLC stage, whereas HRR was negatively correlated with NSCLC stage. RDW, NLR, and HRR were also significantly associated with the differentiation degree of NSCLC (p < 0.05). The ROC curve analysis showed that the combination of RDW with NLR, HRR, and CEA could show significantly higher diagnostic value than any one marker alone (AUC = 0.925, 95% CI: 0.897-0.954, and sensitivity and specificity of 79.60% and 93.60%, respectively). CONCLUSION: RDW, NLR, and HRR can be utilized as simple and effective biomarkers for the diagnosis and evaluation of NSCLC progression.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Progressão da Doença , Eritrócitos/citologia , Hemoglobinas/metabolismo , Neoplasias Pulmonares/imunologia , Linfócitos/citologia , Neutrófilos/citologia , Idoso , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/patologia , Estudos de Casos e Controles , Contagem de Células , Feminino , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
13.
Anticancer Res ; 40(8): 4701-4706, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727795

RESUMO

BACKGROUND/AIM: Bovine mastitis is caused by the invasion and propagation of pathogenic microorganisms into the udder and mammary gland tissues of cattle. In this study, the therapeutic effect of a low-molecular-weight whey protein (LMW-WP) on bovine mastitis was evaluated. MATERIALS AND METHODS: LMW-WP was orally, intraperitoneally, and vaginally administered to bovine with mastitis. The number of somatic cells in milk was measured 24 h before the administration of LMW-WP. The effect of LMW-WP on cytokine production was measured with a microarray that evaluates the expression of cytokines. RESULTS: In the group that received 1,000 mg intraperitoneally, the somatic cell count was reduced to less than 400,000 at the shipment standard value in three of the four udders, indicating 75% efficacy. The group that received 1,000 mg by vaginal administration showed 67% efficacy. It was confirmed that LMW-WP increased the production of cytokines such as IL-5, IL-6, IL-9, IL-12, MCP-1, and VEGF in mouse macrophage cells, but it did not show any antibacterial activity. CONCLUSION: LMW-WP may be an effective therapeutic agent for bovine mastitis.


Assuntos
Macrófagos/efeitos dos fármacos , Mastite Bovina/tratamento farmacológico , Proteínas do Soro do Leite/farmacologia , Animais , Antibacterianos/farmacologia , Bovinos , Contagem de Células/métodos , Linhagem Celular , Citocinas/metabolismo , Feminino , Glândulas Mamárias Animais/metabolismo , Mastite Bovina/microbiologia , Camundongos , Leite/metabolismo , Células RAW 264.7
14.
PLoS One ; 15(7): e0232559, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32658922

RESUMO

PRESENILIN 2 (PSEN2) is one of the genes mutated in early onset familial Alzheimer's disease (EOfAD). PSEN2 shares significant amino acid sequence identity with another EOfAD-related gene PRESENILIN 1 (PSEN1), and partial functional redundancy is seen between these two genes. However, the complete range of functions of PSEN1 and PSEN2 is not yet understood. In this study, we performed targeted mutagenesis of the zebrafish psen2 gene to generate a premature termination codon close downstream of the translation start with the intention of creating a null mutation. Homozygotes for this mutation, psen2S4Ter, are viable and fertile, and adults do not show any gross psen2-dependent pigmentation defects, arguing against significant loss of γ-secretase activity. Also, assessment of the numbers of Dorsal Longitudinal Ascending (DoLA) interneurons that are responsive to psen2 but not psen1 activity during embryogenesis did not reveal decreased psen2 function. Transcripts containing the S4Ter mutation show no evidence of destabilization by nonsense-mediated decay. Forced expression in zebrafish embryos of fusions of psen2S4Ter 5' mRNA sequences with sequence encoding enhanced green fluorescent protein (EGFP) indicated that the psen2S4Ter mutation permits utilization of cryptic, novel downstream translation start codons. These likely initiate translation of N-terminally truncated Psen2 proteins lacking late endosomal/lysosomal localization sequences and that obey the "reading frame preservation rule" of PRESENILIN EOfAD mutations. Transcriptome analysis of entire brains from a 6-month-old family of wild type, heterozygous and homozygous psen2S4Ter female siblings revealed profoundly dominant effects on gene expression likely indicating changes in ribosomal, mitochondrial, and anion transport functions.


Assuntos
Códon de Terminação/genética , Perfilação da Expressão Gênica , Mitocôndrias/genética , Mutação , Presenilina-2/genética , Ribossomos/genética , Proteínas de Peixe-Zebra/genética , Alelos , Animais , Contagem de Células , Homozigoto , Hipóxia/genética , Neurônios/citologia , Estabilidade de RNA/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética
15.
Int J Nanomedicine ; 15: 4441-4452, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606688

RESUMO

Purpose: The present study focuses on threshold levels for cytotoxicity after long-term and repetitive exposure for HUVEC as a model for the specific microvascular endothelial system. Furthermore, possible genotoxic effects and functional impairment caused by ZnO NPs in HUVEC are elucidated. Methods: Thresholds for cytotoxic effects are determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Annexin V assay. To demonstrate DNA damage, single-cell microgel electrophoresis (comet) assay is performed after exposure to sub-cytotoxic concentrations of ZnO NPs. The proliferation assay, dot blot assay and capillary tube formation assay are also carried out to analyze functional impairment. Results: NPs showed to be spherical in shape with an average size of 45-55 nm. Long-term exposure as well as repetitive exposure with ZnO NPs exceeding 25 µg/mL lead to decreased viability in HUVEC. In addition, DNA damage was indicated by the comet assay after long-term and repetitive exposure. Twenty-four hours after long-term exposure, the proliferation assay does not show any difference between negative control and exposed cells. Forty-eight hours after exposure, HUVEC show an inverse concentration-related ability to proliferate. The dot blot assay provides evidence that ZnO NPs lead to a decreased release of VEGF, while capillary tube formation assay shows restriction in the ability of HUVEC to build tubes and meshes as a first step in angiogenesis. Conclusion: Sub-cytotoxic concentrations of ZnO NPs lead to DNA damage and functional impairment in HUVEC. Based on these data, ZnO NPs may affect neo-angiogenesis. Further investigation based on tissue cultures is required to elucidate the impact of ZnO NPs on human cell systems.


Assuntos
Dano ao DNA , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Óxido de Zinco/toxicidade , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Humanos , Neovascularização Fisiológica/efeitos dos fármacos
16.
PLoS One ; 15(7): e0223395, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645024

RESUMO

Development of the cerebral cortex may be influenced by the composition of the maternal gut microbiota. To test this possibility, we administered probiotic Lactococcus lactis in drinking water to mouse dams from day 10.5 of gestation until pups reached postnatal day 1 (P1). Pups were assessed in a battery of behavioral tests starting at 10 weeks old. We found that females, but not males, exposed to probiotic during prenatal development spent more time in the center of the open field and displayed decreased freezing time in cue associated learning, compared to controls. Furthermore, we found that probiotic exposure changed the density of cortical neurons and increased the density of blood vessels in the cortical plate of P1 pups. Sex-specific differences were observed in the number of mitotic neural progenitor cells, which were increased in probiotic exposed female pups. In addition, we found that probiotic treatment in the latter half of pregnancy significantly increased plasma oxytocin levels in mouse dams, but not in the offspring. These results suggest that exposure of naïve, unstressed dams to probiotic may exert sex-specific long-term effects on cortical development and anxiety related behavior in the offspring.


Assuntos
Ansiedade/prevenção & controle , Córtex Cerebral/efeitos dos fármacos , Lactococcus lactis , Efeitos Tardios da Exposição Pré-Natal/psicologia , Probióticos/farmacologia , Animais , Animais Recém-Nascidos , Contagem de Células , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Medo , Feminino , Aprendizagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Ocitocina/metabolismo , Gravidez , Caracteres Sexuais
17.
PLoS One ; 15(7): e0235827, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32667933

RESUMO

Homogenization of the initial cell distribution is essential for effective cell development. However, there are few previous reports on efficient cell seeding methods, even though the initial cell distribution has a large effect on cell proliferation. Dense cell regions have an inverse impact on cell development, known as contact inhibition. In this study, we developed a method to homogenize the cell seeding density using secondary flow, or Ekman transportation, induced by orbital movement of the culture dish. We developed an orbital shaker device that can stir the medium in a 35-mm culture dish by shaking the dish along a circular orbit with 2 mm of eccentricity. The distribution of cells in the culture dish can be controlled by the rotational speed of the orbital shaker, enabling dispersion of the initial cell distribution. The experimental results indicated that the cell density became most homogeneous at 61 rpm. We further evaluated the cell proliferation after homogenization of the initial cell density at 61 rpm. The results revealed 36% higher proliferation for the stirred samples compared with the non-stirred control samples. The present findings indicate that homogenization of the initial cell density by Ekman transportation contributes to the achievement of higher cell proliferation.


Assuntos
Técnicas de Cultura de Células/instrumentação , Mioblastos/citologia , Animais , Contagem de Células , Técnicas de Cultura de Células/economia , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Desenho de Equipamento , Camundongos
18.
J Vis Exp ; (159)2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32478745

RESUMO

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.


Assuntos
Acústica/instrumentação , Técnicas de Cultura de Células/métodos , Animais , Células CHO , Contagem de Células , Cricetinae , Cricetulus , Nefelometria e Turbidimetria , Software , Temperatura
19.
J Vis Exp ; (159)2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32510518

RESUMO

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.


Assuntos
Neoplasias Pulmonares/patologia , Patologia/métodos , Cauda , Animais , Contagem de Células , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Humanos , Processamento de Imagem Assistida por Computador , Injeções Intravenosas , Camundongos , Metástase Neoplásica
20.
J Vis Exp ; (159)2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32510517

RESUMO

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, transports immune complexes, and participates in humoral and cellular immune responses. CR1 is present on the surface of many cell types, including erythrocytes, and exhibits polymorphisms in length, structure (Knops, or KN, blood group), and density. The average density of CR1 per erythrocyte (CR1/E) is 500 molecules per erythrocyte. This density varies from one individual to another (100-1,200 CR1/E) and from one erythrocyte to another in the same individual. We present here a robust flow cytometry method to measure the density of CR1/E, including in subjects expressing a low density, with the help of an amplifying immunostaining system. This method has enabled us to show the lowering of CR1 erythrocyte expression in diseases such as Alzheimer's disease (AD), systemic lupus erythematosus (SLE), AIDS, or malaria.


Assuntos
Eritrócitos/metabolismo , Citometria de Fluxo/métodos , Receptores de Complemento/sangue , Calibragem , Contagem de Células , Humanos , Análise de Regressão
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