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1.
J Fluoresc ; 32(2): 521-531, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34989923

RESUMO

Tumor spheroid models have proven useful in the study of cancer cell responses to chemotherapeutic compounds by more closely mimicking the 3-dimensional nature of tumors in situ. Their advantages are often offset, however, by protocols that are long, complicated, and expensive. Efforts continue for the development of high-throughput assays that combine the advantages of 3D models with the convenience and simplicity of traditional 2D monolayer methods. Herein, we describe the development of a breast cancer spheroid image cytometry assay using T47D cells in Aggrewell™400 spheroid plates. Using the Celigo® automated imaging system, we developed a method to image and individually track thousands of spheroids within the Aggrewell™400 microwell plate over time. We demonstrate the use of calcein AM and propidium iodide staining to study the effects of known anti-cancer drugs Doxorubicin, Everolimus, Gemcitabine, Metformin, Paclitaxel and Tamoxifen. We use the image cytometry results to quantify the fluorescence of calcein AM and PI as well as spheroid size in a dose dependent manner for each of the drugs. We observe a dose-dependent reduction in spheroid size and find that it correlates well with the viability obtained from the CellTiter96® endpoint assay. The image cytometry method we demonstrate is a convenient and high-throughput drug-response assay for breast cancer spheroids under 400 µm in diameter, and may lay a foundation for investigating other three-dimensional spheroids, organoids, and tissue samples.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Ensaios de Triagem em Larga Escala/métodos , Citometria por Imagem/métodos , Esferoides Celulares/efeitos dos fármacos , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Fluoresceínas , Corantes Fluorescentes , Humanos , Propídio
2.
SLAS Discov ; 28(3): 65-72, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36758833

RESUMO

Solid tumors account for approximately 90% of all adult human cancers. As such, the development of novel cellular therapies has become of increasing importance to target solid tumor malignancies, such as prostate, lung, breast, bladder, colon, and liver cancers. One such cellular therapy relies on the use of chimeric antigen receptor T cells (CAR-T cells). CAR-T cells are engineered to target specific antigens on tumor cells. To date, there are six FDA-approved CAR-T cell therapies that have been utilized for hematologic B cell malignancies. Immune cell trafficking and immunosuppressive factors within the tumor microenvironment increase the relative difficulty in developing a robust CAR-T cell therapy against solid tumors. Therefore, it is critical to develop novel methodologies for high-throughput phenotypic and functional assays using 3D tumor spheroid models to assess CAR-T cell products against solid tumors. In this manuscript, we discuss the use of CAR-T cells targeted towards PSMA, an antigen that is found on prostate cancer tumor cells, the second most common cause of cancer deaths among men worldwide. We demonstrate the use of high-throughput, plate-based image cytometry to characterize CAR-T cell-mediated cytotoxic potency against 3D prostate tumor spheroids. We were able to kinetically evaluate the efficacy and therapeutic value of PSMA CAR-T cells by analyzing the cytotoxicity against prostate tumor spheroids. In addition, the CAR-T cells were fluorescently labeled to visually identify the location of the T cells as cytotoxicity occurs, which may provide more meaningful information for assessing the functionality of the CAR-T cells. The proposed image cytometry method can overcome limitations placed on traditional methodologies to effectively assess cell-mediated 3D tumor spheroid cytotoxicity and efficiently generate time- and dose-dependent results.


Assuntos
Neoplasias da Próstata , Receptores de Antígenos Quiméricos , Masculino , Humanos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Imunoterapia Adotiva/métodos , Linfócitos T/metabolismo , Citometria por Imagem/métodos , Microambiente Tumoral
3.
Foodborne Pathog Dis ; 9(9): 853-60, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22779701

RESUMO

Broiler digestive tract fungal communities have gained far less scrutiny than that given corresponding bacterial communities. Attention given poultry-associated fungi have focused primarily on feed-associated toxin-producers, yeast, and yeast products. The current project focused on the use of pyrosequencing and denaturing gradient gel electrophoresis (DGGE) to identify and monitor broiler digestive fungal communities. Eight different treatments were included. Four controls were an Uninfected-Unmedicated Control, an Unmedicated-Infected Control, the antibiotic bacitracin methylene disalicylate plus the ionophore monensin as Positive Control, and the ionophore monensin alone as a Negative Control. Four treatments were two probiotics (BC-30 and Calsporin) and two specific essential oil blends (Crina Poultry Plus and Crina Poultry AF). All chickens except the Unmedicated-Uninfected Control were given, at 15 days of age, a standard oral Eimeria inoculum of sporulated oocysts. Ileal and cecal digesta were collected at pre-Eimeria infection at 14 days of age and at 7 days post-Eimeria infection at 22 days of age. Extracted cecal DNA was analyzed by pyrosequencing to examine the impact of diet supplements and Eimeria infection on individual constituents in the fungal community, while DGGE was used to compare more qualitative changes in ileal and cecal communities. Pyrosequencing identified three phyla, seven classes, eight orders, 13 families, 17 genera, and 23 fungal species. Ileal and cecal DGGE patterns showed fungal communities were clustered mainly into pre- and post-infection patterns. Post-infection Unmedicated-Uninfected patterns were clustered with pre-infection groups demonstrating a strong effect of Eimeria infection on digestive fungal populations. These combined techniques offered added versatility towards unraveling the effects of enteropathogen infection and performance enhancing feed additives on broiler digestive microflora.


Assuntos
Galinhas/microbiologia , Coccidiose/veterinária , Fungos/isolamento & purificação , Intestinos/microbiologia , Óleos Voláteis/uso terapêutico , Doenças das Aves Domésticas/dietoterapia , Probióticos/uso terapêutico , Animais , Animais Endogâmicos , Ceco/crescimento & desenvolvimento , Ceco/microbiologia , Galinhas/crescimento & desenvolvimento , Análise por Conglomerados , Coccidiose/dietoterapia , Coccidiose/microbiologia , Coccidiose/parasitologia , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Eletroforese em Gel de Gradiente Desnaturante/veterinária , Eimeria/patogenicidade , Fungos/classificação , Fungos/genética , Gastroenterite/dietoterapia , Gastroenterite/microbiologia , Gastroenterite/parasitologia , Gastroenterite/veterinária , Íleo/crescimento & desenvolvimento , Íleo/microbiologia , Intestinos/crescimento & desenvolvimento , Masculino , Tipagem Molecular/veterinária , Técnicas de Tipagem Micológica/veterinária , Filogenia , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/parasitologia , RNA Ribossômico/química , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Análise de Sequência de DNA/veterinária
4.
J Food Sci ; 84(6): 1501-1512, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31116418

RESUMO

Susceptibility profiles were determined for 111 Campylobacter coli strains obtained in 1998 to 1999 and 2015 from market age pigs and pork chops against 22 disinfectants and 9 antimicrobials. Resistance to tetracycline (TET) was observed in 44.4% of 1998 to 1999 strains, and the antibiotic resistance profile was TET. But strains obtained in 2015 from swine and retail pork chops had 75% TET resistance and the antibiotic resistance profile was TET, followed by azithromycin-erythromycin-TET-telithromycin-clindamycin. Antimicrobial resistance increased in 2015 strains. All strains were resistant to triclosan, and 84.1% and 95.8% of strains in 1998 to 1999 and 2015, respectively, were chlorhexidine resistant. All strains were susceptible to benzalkonium chloride. There was a shift toward higher susceptibility to chlorhexidine, triclosan, P-128, OdoBan, CPB, and CPC in 2015 swine and pork chop strains compared with 1998 to 1999 strains. The disinfectants Tek-Trol and providone-iodine, tris(hydroxylmethyl)nitromethane (THN) and formaldehyde demonstrated the highest susceptibilities. Didecyldimethylammonium chloride (C10AC) appeared to be about equally effective as benzyldimethyltetradecylammonium chloride (C14BAC) for inhibiting C. coli, and both were more effective than C8AC and C12BAC, but C16BAC was not efficient at inhibiting C. coli. The BACs, C12BAC and C14BAC, were the most effective ingredients in DC&R. Also, C12BAC and C14BAC, or these two in synergy with C10AC were responsible for inhibition of C. coli at high P-128 MICs. No cross-resistance was observed between antibiotics and disinfectants. The continued use of THN and formaldehyde in DC&R should be evaluated since these components are not effective, and their inclusion adds unwanted chemicals in the environment. PRACTICAL APPLICATION: Campylobacter species cause diarrheal disease throughout the world. Disinfectants are often used on the farm, in veterinary medicine, by the food processing industry, in restaurants, and in consumer's homes. Limited information is available in the literature showing how disinfectants or disinfectant components may affect the many different foodborne pathogens, and, specifically, Campylobacter coli studied here. The knowledge generated in this study concerning the interactions of a broad array of disinfectants against C. coli may well affect the types of disinfectants and disinfectant formulations allowable for use by medical personnel, producers, food processors, restaurants, and consumers.


Assuntos
Antibacterianos/farmacologia , Campylobacter coli/efeitos dos fármacos , Desinfetantes/farmacologia , Carne Vermelha/microbiologia , Animais , Compostos de Benzalcônio/farmacologia , Campylobacter coli/genética , Campylobacter coli/isolamento & purificação , Clindamicina/farmacologia , Farmacorresistência Bacteriana , Eritromicina/farmacologia , Contaminação de Alimentos/análise , Testes de Sensibilidade Microbiana , Suínos , Tetraciclina/farmacologia
5.
Cell Stem Cell ; 23(3): 396-411.e8, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30146411

RESUMO

As somatic cells are converted into induced pluripotent stem cells (iPSCs), their chromatin is remodeled to a pluripotent configuration with unique euchromatin-to-heterochromatin ratios, DNA methylation patterns, and enhancer and promoter status. The molecular machinery underlying this process is largely unknown. Here, we show that embryonic stem cell (ESC)-specific factors Dppa2 and Dppa4 play a key role in resetting the epigenome to a pluripotent state. They are induced in reprogramming intermediates, function as a heterodimer, and are required for efficient reprogramming of mouse and human cells. When co-expressed with Oct4, Klf4, Sox2, and Myc (OKSM) factors, Dppa2/4 yield reprogramming efficiencies that exceed 80% and accelerate reprogramming kinetics, generating iPSCs in 2 to 4 days. When bound to chromatin, Dppa2/4 initiate global chromatin decompaction via the DNA damage response pathway and contribute to downregulation of somatic genes and activation of ESC enhancers, all of which enables an efficient transition to pluripotency. Our work provides critical insights into how the epigenome is remodeled during acquisition of pluripotency.


Assuntos
Reprogramação Celular , Epigênese Genética , Proteínas Nucleares/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , Fatores de Transcrição
6.
Cell Stem Cell ; 22(2): 235-251.e9, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29337181

RESUMO

Polycomb group proteins regulate self-renewal and differentiation in many stem cell systems. When assembled into two canonical complexes, PRC1 and PRC2, they sequentially deposit H3K27me3 and H2AK119ub histone marks and establish repressive chromatin, referred to as Polycomb domains. Non-canonical PRC1 complexes retain RING1/RNF2 E3-ubiquitin ligases but have unique sets of accessory subunits. How these non-canonical complexes recognize and regulate their gene targets remains poorly understood. Here, we show that the BCL6 co-repressor (BCOR), a member of the PRC1.1 complex, is critical for maintaining primed pluripotency in human embryonic stem cells (ESCs). BCOR depletion leads to the erosion of Polycomb domains at key developmental loci and the initiation of differentiation along endoderm and mesoderm lineages. The C terminus of BCOR regulates the assembly and targeting of the PRC1.1 complex, while the N terminus contributes to BCOR-PRC1.1 repressor function. Our findings advance understanding of Polycomb targeting and repression in ESCs and could apply broadly across developmental systems.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Complexos Multiproteicos/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Cromatina/metabolismo , Proteínas F-Box/metabolismo , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/metabolismo , Metilação , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas , Domínios Proteicos , Proteínas Proto-Oncogênicas/química , Proteínas Repressoras/química
7.
PLoS One ; 13(8): e0202100, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30096155

RESUMO

Campylobacter coli is a bacterial species that is a major cause of diarrheal disease worldwide, and Campylobacter spp. are among the top 5 foodborne pathogens in the United States. During food production organic acids (OAs) are often used to remove bacteria from animal carcasses. The interactions of six OAs with 111 C. coli strains obtained from swine and retail pork chops were studied by determining the molar minimum inhibitory concentrations (MICMs) of the C. coli strains, and the pH at the MICMs. The Henderson-Hasselbalch equation was used to calculate the concentrations of the undissociated and dissociated OAs at the MICMs of the C. coli strains. The results for the 111 different C. coli strains obtained from different locations were treated as a single group for each OA since many of the C. coli strains behaved similarly to each different OA. Inhibition of C. coli was not dependent on pH or on the undissociated OA species, but C. coli inhibition correlated with the dissociated OA species. Therefore, if the concentration of the dissociated OAs decreases from optimum, one may then expect that C. coli bacteria would escape disinfection. The concentration of the dissociated OA should be carefully controlled in a carcass wash. We suggest maintaining a concentration of the dissociated acetic, butyric, citric, formic, lactic and propionic acids at 29, 23, 11, 35, 22 and 25 mM, respectively, when using a carcass wash with these OAs to remove C. coli bacteria. However, due to C. coli utilization of acetate, formate, lactate and propionate, these four OAs may not be the best choice to use for a carcass wash to remove C. coli contamination. Of the six OAs, citric acid was the most efficient at inhibiting C. coli.


Assuntos
Ácidos/farmacologia , Campylobacter coli/efeitos dos fármacos , Compostos Orgânicos/farmacologia , Campylobacter coli/isolamento & purificação , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana
8.
J Food Prot ; 76(1): 6-17, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23317851

RESUMO

The disinfectant and antibiotic susceptibility profiles of 344 Escherichia coli O157:H7 strains from cattle carcasses, feces, and hides and ground beef from the United States were determined. A low prevalence of antibiotic resistance was observed (14%). The highest prevalences of resistance were to sulfisoxazole (10.5%), tetracycline (9.9%), streptomycin (7%), and chloramphenicol (4.9%). Four strains were resistant to eight antibiotics (two strains from ground beef and one strain each from hide and preevisceration carcass swabs of cull cattle at harvest). Pulsed-field gel electrophoresis analysis of the E. coli O157:H7 strains revealed two major groups (designated 1 and 2) composed of 17 and 20 clusters, respectively. Clusters 1A, 1B, 1C, and 1G.1 were associated with multidrug-resistant strains. There was no observed correlation between disinfectant resistance and antibiotic resistance. Sixty-nine (20%) of the 344 strains were resistant to chlorhexidine or benzalkonium chloride or the MICs of benzyldimethyldodecylammonium chloride were elevated. Inducible resistance was observed at elevated concentrations of antibiotics (1.4%) and disinfectants (6.1%). The highest rate of disinfectant inducible resistance was to OdoBan, quaternary ammonium chlorides, and the surface disinfectants F25, FS512, and MG, which are used in dairies, restaurants, and food processing plants. High MICs (1,024 to 4,096 m g/ml) of acetic, lactic, and citric acids were found. The decreasing order of acid potency based on molar MICs (MICs(molar)) was acetic, citric, and lactic acid. The correlation of the concentration of dissociated organic acids and MICs(molar) strongly suggests that the observed inhibition of E. coli O157:H7 was primarily due to dissociated forms of the acids.


Assuntos
Antibacterianos/farmacologia , Bovinos/microbiologia , Farmacorresistência Bacteriana , Escherichia coli O157/efeitos dos fármacos , Manipulação de Alimentos/métodos , Produtos da Carne/microbiologia , Animais , Contagem de Colônia Microbiana , Desinfetantes/farmacologia , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana Múltipla , Fezes/microbiologia , Contaminação de Alimentos/análise , Contaminação de Alimentos/prevenção & controle , Cabelo/microbiologia , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Pele/microbiologia , Estados Unidos
9.
Ann Thorac Surg ; 78(5): e81-2, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15511418

RESUMO

Percutaneous coronary interventions have progressed from angioplasty and intracoronary stents to the more aggressive rotational atherectomy devices. These technically complex procedures are typically performed on the more diseased anatomy, which causes the likelihood of complications to be more common. In this report, we present our experience with three unusual complications and their surgical management.


Assuntos
Aterectomia Coronária/efeitos adversos , Aterectomia/efeitos adversos , Implante de Prótese Vascular , Vasos Coronários/lesões , Complicações Intraoperatórias/cirurgia , Idoso , Angioplastia Coronária com Balão , Tamponamento Cardíaco/etiologia , Tamponamento Cardíaco/cirurgia , Reestenose Coronária/cirurgia , Vasos Coronários/cirurgia , Emergências , Feminino , Corpos Estranhos/cirurgia , Coração Auxiliar , Humanos , Balão Intra-Aórtico , Masculino , Pessoa de Meia-Idade , Marca-Passo Artificial , Pericardiocentese , Choque Cardiogênico/etiologia , Choque Cardiogênico/cirurgia , Stents
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