Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 271
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Proc Natl Acad Sci U S A ; 121(39): e2403510121, 2024 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-39288179

RESUMO

Multispecies microbial communities drive most ecosystems on Earth. Chemical and biological interactions within these communities can affect the survival of individual members and the entire community. However, the prohibitively high number of possible interactions within a microbial community has made the characterization of factors that influence community development challenging. Here, we report a Microbial Community Interaction (µCI) device to advance the systematic study of chemical and biological interactions within a microbial community. The µCI creates a combinatorial landscape made up of an array of triangular wells interconnected with circular wells, which each contains either a different chemical or microbial strain, generating chemical gradients and revealing biological interactions. Bacillus cereus UW85 containing green fluorescent protein provided the "target" readout in the triangular wells, and antibiotics or microorganisms in adjacent circular wells are designated the "variables." The µCI device revealed that gentamicin and vancomycin are antagonistic to each other in inhibiting the target B. cereus UW85, displaying weaker inhibitory activity when used in combination than alone. We identified three-member communities constructed with isolates from the plant rhizosphere that increased or decreased the growth of B. cereus. The µCI device enables both strain-level and community-level insight. The scalable geometric design of the µCI device enables experiments with high combinatorial efficiency, thereby providing a simple, scalable platform for systematic interrogation of three-factor interactions that influence microorganisms in solitary or community life.


Assuntos
Bacillus cereus , Interações Microbianas/fisiologia , Microbiota/fisiologia , Antibacterianos/farmacologia , Vancomicina/farmacologia , Rizosfera , Gentamicinas/farmacologia , Dispositivos Lab-On-A-Chip , Proteínas de Fluorescência Verde/metabolismo
2.
J Cell Sci ; 137(3)2024 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-38224139

RESUMO

Neutrophil-directed motility is necessary for host defense, but its dysregulation can also cause collateral tissue damage. Actinopathies are monogenic disorders that affect the actin cytoskeleton and lead to immune dysregulation. Deficiency in ARPC1B, a component of the Arp2/3 complex, results in vascular neutrophilic inflammation; however, the mechanism remains unclear. Here, we generated human induced pluripotent stem cell (iPSC)-derived neutrophils (denoted iNeutrophils) that are deficient in ARPC1B and show impaired migration and a switch from forming pseudopodia to forming elongated filopodia. We show, using a blood vessel on a chip model, that primary human neutrophils have impaired movement across an endothelium deficient in APRC1B. We also show that the combined deficiency of ARPC1B in iNeutrophils and endothelium results in further reduction in neutrophil migration. Taken together, these results suggest that ARPC1B in endothelium is sufficient to drive neutrophil behavior. Furthermore, the findings provide support for using the iPSC system to understand human neutrophil biology and model disease in a genetically tractable system.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina , Células-Tronco Pluripotentes Induzidas , Neutrófilos , Humanos , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Movimento Celular , Proteínas do Citoesqueleto , Células Endoteliais , Endotélio
3.
J Biol Chem ; 298(3): 101649, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35104504

RESUMO

RNA-binding proteins (RBPs) regulate the expression of large cohorts of RNA species to produce programmatic changes in cellular phenotypes. To describe the function of RBPs within a cell, it is key to identify their mRNA-binding partners. This is often done by crosslinking nucleic acids to RBPs, followed by chemical release of the nucleic acid fragments for analysis. However, this methodology is lengthy, which involves complex processing with attendant sample losses, thus large amounts of starting materials and prone to artifacts. To evaluate potential alternative technologies, we tested "exclusion-based" purification of immunoprecipitates (IFAST or SLIDE) and report here that these methods can efficiently, rapidly, and specifically isolate RBP-RNA complexes. The analysis requires less than 1% of the starting material required for techniques that include crosslinking. Depending on the antibody used, 50% to 100% starting protein can be retrieved, facilitating the assay of endogenous levels of RBPs; the isolated ribonucleoproteins are subsequently analyzed using standard techniques, to provide a comprehensive portrait of RBP complexes. Using exclusion-based techniques, we show that the mRNA-binding partners for RBP IGF2BP1 in cultured mammary epithelial cells are enriched in mRNAs important for detoxifying superoxides (specifically glutathione peroxidase [GPX]-1 and GPX-2) and mRNAs encoding mitochondrial proteins. We show that these interactions are functionally significant, as loss of function of IGF2BP1 leads to destabilization of GPX mRNAs and reduces mitochondrial membrane potential and oxygen consumption. We speculate that this underlies a consistent requirement for IGF2BP1 for the expression of clonogenic activity in vitro.


Assuntos
Glândulas Mamárias Animais , Glândulas Mamárias Humanas , Proteínas de Ligação a RNA , Animais , Células Epiteliais/metabolismo , Feminino , Humanos , Imunoprecipitação , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/metabolismo , RNA/metabolismo , RNA Mensageiro , Proteínas de Ligação a RNA/metabolismo
4.
J Cell Sci ; 134(21)2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34622930

RESUMO

Leukocyte extravasation into inflamed tissue is a complex process that is difficult to capture as a whole in vitro. We employed a blood-vessel-on-a-chip model in which human endothelial cells were cultured in a tube-like lumen in a collagen-1 matrix. The vessels are leak tight, creating a barrier for molecules and leukocytes. Addition of inflammatory cytokine TNF-α (also known as TNF) caused vasoconstriction, actin remodelling and upregulation of ICAM-1. Introducing leukocytes into the vessels allowed real-time visualization of all different steps of the leukocyte transmigration cascade, including migration into the extracellular matrix. Individual cell tracking over time distinguished striking differences in migratory behaviour between T-cells and neutrophils. Neutrophils cross the endothelial layer more efficiently than T-cells, but, upon entering the matrix, neutrophils display high speed but low persistence, whereas T-cells migrate with low speed and rather linear migration. In conclusion, 3D imaging in real time of leukocyte extravasation in a vessel-on-a-chip enables detailed qualitative and quantitative analysis of different stages of the full leukocyte extravasation process in a single assay. This article has an associated First Person interview with the first authors of the paper.


Assuntos
Células Endoteliais , Migração Transendotelial e Transepitelial , Endotélio Vascular , Humanos , Leucócitos , Neutrófilos
5.
FASEB J ; 36(10): e22540, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36083096

RESUMO

The tumor microenvironment (TME) is a complex network of non-malignant cells and stroma that perform a wide array of vital roles in tumor growth, immune evasion, metastasis, and therapeutic resistance. These highly diverse roles have been shown to be critically important to the progression of cancers and have already shown potential as therapeutic targets. Therefore, there has been a tremendous push to elucidate the pathways that underlie these roles and to develop new TME-directed therapies for cancer treatment. Unfortunately, TME-focused research has been limited by a lack of translational in vitro culture platforms that can model this highly complex niche and can support the integrated analysis of cell biology and function. In the current study, we investigate whether an independently developed reconfigurable microfluidic platform, known as Stacks, can address the critical need for translational multi-cellular tumor models and integrated analytics in TME research. We present data on multi-cellular culture of primary human cells in Stacks as well as the orthogonal analysis of cellular polarization, differentiation, migration, and cytotoxicity in this reconfigurable system. These expanded capabilities of Stacks are highly relevant to the cancer research community with the potential to enhance clinical translation of pre-clinical TME studies and to yield novel biological insight into TME crosstalk, metastasis, and responses to novel drug combinations or immune therapies.


Assuntos
Neoplasias , Microambiente Tumoral , Técnicas de Cultura de Células , Humanos , Microfluídica , Neoplasias/patologia
6.
Prostate ; 82(7): 836-849, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35226381

RESUMO

BACKGROUND: Prostatic cancers include a diverse microenvironment of tumor cells, cancer-associated fibroblasts, and immune components. This tumor microenvironment (TME) is a known driving force of tumor survival after treatment, but the standard-of-care tissue freezing or fixation in pathology practice limit the use of available approaches/tools to study the TME's functionality in tumor resistance. Thus, there is a need for approaches that satisfy both clinical and laboratory endpoints for TME study. Here we present methods for clinical case identification, tissue processing, and analytical workflow that are compatible with standard histopathology while enabling molecular and functional interrogation of prostate TME components. METHODS: We first performed a small retrospective review to identify cases where submission of alternate prostate tissue slices and a parallel live tissue processing protocol complement traditional histopathology and enable viable multicompartment analysis of the TME. Then, we tested its compatibility with commonly employed methods to study the microenvironment including quantification of components both in situ and after tissue dissociation. We also evaluated tissue digestion conditions and cell isolation techniques to aid various molecular and functional endpoints. RESULTS: We identified Gleason Grade Group 3+ clinical cases where tumor volume was sufficient to allow slicing of unfixed tissue and distribution of alternating tissue slices to standard-of-care histopathology and viable multi-modal TME analyses. No single method was found that preserved cellular sub-types for all downstream readouts; instead, tissues were further divided so techniques could be catered to each endpoint. For instance, we show that incorporating the protease dispase into tissue dissociation improves viability for culture and functional analyses but hinders immune cell analysis by flow cytometry. We also found that flow activated cell sorting provides highly pure cell populations for quantitative reverse-transcription polymerase chain reaction and RNA-seq while isolation using antibody-labeled paramagnetic particles facilitated functional coculture experiments. CONCLUSIONS: The identification of candidate cases and use of these techniques enable translational research and the development of molecular and functional assays to facilitate prostate TME study without compromising standard-of-care histopathological diagnosis. This allows bridging clinical histopathology and further interrogation of the prostate TME and promises to advance our understanding of tumor biology and unveil new predictive and prognostic markers of prostate cancer progression.


Assuntos
Fibroblastos Associados a Câncer , Neoplasias da Próstata , Obtenção de Tecidos e Órgãos , Fibroblastos Associados a Câncer/patologia , Humanos , Masculino , Próstata/patologia , Neoplasias da Próstata/patologia , Microambiente Tumoral/fisiologia
7.
Chem Soc Rev ; 49(17): 6402-6442, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32760967

RESUMO

Microfluidic lumen-based systems are microscale models that recapitulate the anatomy and physiology of tubular organs. These technologies can mimic human pathophysiology and predict drug response, having profound implications for drug discovery and development. Herein, we review progress in the development of microfluidic lumen-based models from the 2000s to the present. The core of the review discusses models for mimicking blood vessels, the respiratory tract, the gastrointestinal tract, renal tubules, and liver sinusoids, and their application to modeling organ-specific diseases. We also highlight emerging application areas, such as the lymphatic system, and close the review discussing potential future directions.


Assuntos
Biomimética , Dispositivos Lab-On-A-Chip , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Materiais Biocompatíveis , Materiais Biomiméticos , Humanos
8.
Blood ; 132(17): 1818-1828, 2018 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-30143504

RESUMO

Neutrophil infiltration into tissues is essential for host defense and pathogen clearance. Although many of the signaling pathways involved in the transendothelial migration of neutrophils are known, the role of the endothelium in regulating neutrophil behavior in response to infection within interstitial tissues remains unclear. Here we developed a microscale 3-dimensional (3D) model that incorporates an endothelial lumen, a 3D extracellular matrix, and an intact bacterial source to model the host microenvironment. Using this system, we show that an endothelial lumen significantly increased neutrophil migration toward a source of Pseudomonas aeruginosa Surprisingly, we found neutrophils, which were thought to be short-lived cells in vitro, migrate for up to 24 hours in 3D in the presence of an endothelial lumen and bacteria. In addition, we found that endothelial cells secrete inflammatory mediators induced by the presence of P aeruginosa, including granulocyte-macrophage colony-stimulating factor (GM-CSF), a known promoter of neutrophil survival, and interleukin (IL)-6, a proinflammatory cytokine. We found that pretreatment of neutrophils with a blocking antibody against the IL-6 receptor significantly reduced neutrophil migration to P aeruginosa but did not alter neutrophil lifetime, indicating that secreted IL-6 is an important signal between endothelial cells and neutrophils that mediates migration. Taken together, these findings demonstrate an important role for endothelial paracrine signaling in neutrophil migration and survival.


Assuntos
Quimiotaxia de Leucócito/fisiologia , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Interleucina-6/biossíntese , Neutrófilos/metabolismo , Células Endoteliais/imunologia , Endotélio Vascular/imunologia , Humanos , Interleucina-6/imunologia , Neutrófilos/imunologia , Comunicação Parácrina/fisiologia , Pseudomonas aeruginosa , Migração Transendotelial e Transepitelial/fisiologia
9.
FASEB J ; 33(7): 8623-8633, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31002529

RESUMO

Aromatase inhibitors are the preferred treatment for certain women with estrogen receptor (ER)-positive breast cancer, but evidence suggests that women with obesity experience aromatase inhibitor resistance at higher rates. To compare how stromal cells derived from women who are lean or obese influence response to the aromatase inhibitor (anastrazole), we incorporated patient-derived stroma in a previously characterized MCF7-derived in vitro duct model. Coculture with adipose stromal cells enabled the metabolism of testosterone (T) to E2, which induced estrogen response element activity, epithelial proliferation, and hyperplasia in MCF7 cells. The effects of T were inhibited by the ER antagonist tamoxifen and aromatase inhibitor anastrazole and were increased by the aromatase inducer dexamethasone. Primary mammary adipose stromal cells derived from women with obesity displayed increased aromatase mRNA compared with lean controls. MCF7-derived ducts cocultured with obese stromal cells exhibited higher maximal aromatization-induced ER transactivation and reduced anastrazole sensitivity, a difference not seen in 2-dimensional coculture. Finally, tamoxifen was more effective than anastrazole at reducing aromatization-induced ER transactivation and proliferation. These findings suggest that patient-specific responses to hormone therapies can be modeled and studied organotypically in vitro and add to evidence advocating obesity as a parameter to consider when identifying treatments for patients with ER-positive breast cancer.-Morgan, M. M., Arendt, L. M., Alarid, E. T., Beebe, D. J., Johnson, B. P. Mammary adipose stromal cells derived from obese women reduce sensitivity to the aromatase inhibitor anastrazole in an organotypic breast model.


Assuntos
Adipócitos/efeitos dos fármacos , Anastrozol/farmacologia , Inibidores da Aromatase/farmacologia , Neoplasias da Mama/tratamento farmacológico , Mama/efeitos dos fármacos , Obesidade/metabolismo , Células Estromais/efeitos dos fármacos , Adipócitos/metabolismo , Mama/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura/métodos , Estrogênios/metabolismo , Feminino , Humanos , Células MCF-7 , Receptores de Estrogênio/metabolismo , Células Estromais/metabolismo , Tamoxifeno/farmacologia
10.
Nature ; 507(7491): 181-9, 2014 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-24622198

RESUMO

Microfluidics, a technology characterized by the engineered manipulation of fluids at the submillimetre scale, has shown considerable promise for improving diagnostics and biology research. Certain properties of microfluidic technologies, such as rapid sample processing and the precise control of fluids in an assay, have made them attractive candidates to replace traditional experimental approaches. Here we analyse the progress made by lab-on-a-chip microtechnologies in recent years, and discuss the clinical and research areas in which they have made the greatest impact. We also suggest directions that biologists, engineers and clinicians can take to help this technology live up to its potential.


Assuntos
Pesquisa Biomédica/métodos , Pesquisa Biomédica/tendências , Técnicas Analíticas Microfluídicas , Microfluídica , Animais , Líquidos Corporais/química , Ensaios de Migração Celular , Quimiotaxia , Testes Diagnósticos de Rotina , Descoberta de Drogas , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/tendências , Microfluídica/instrumentação , Microfluídica/métodos , Microfluídica/estatística & dados numéricos , Microfluídica/tendências
11.
Proc Natl Acad Sci U S A ; 114(43): E9076-E9085, 2017 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-29073104

RESUMO

High-risk human papillomaviruses (HPVs) infect epithelial cells and are causally associated with cervical cancer, but HPV infection is not sufficient for carcinogenesis. Previously, we reported that estrogen signaling in the stromal tumor microenvironment is associated with cervical cancer maintenance and progression. We have now determined how HPV oncogenes and estrogen treatment affect genome-wide host gene expression in laser-captured regions of the cervical epithelium and stroma of untreated or estrogen-treated nontransgenic and HPV-transgenic mice. HPV oncogene expression in the cervical epithelium elicited significant gene-expression changes in the proximal stromal compartment, and estrogen treatment uniquely affected gene expression in the cervical microenvironment of HPV-transgenic mice compared with nontransgenic mice. Several potential estrogen-induced paracrine-acting factors were identified in the expression profile of the cervical tumor microenvironment. The microenvironment of estrogen-treated HPV-transgenic mice was significantly enriched for chemokine/cytokine activity and inflammatory and immune functions associated with carcinogenesis. This inflammatory signature included several proangiogenic CXCR2 receptor ligands. A subset of the same CXCR2 ligands was likewise increased in cocultures of early-passage cells from human cervical samples, with levels highest in cocultures of cervical fibroblasts and cancer-derived epithelial cells. Our studies demonstrate that high-risk HPV oncogenes profoundly reprogram the tumor microenvironment independently of and synergistically with estrogen. These observations illuminate important means by which HPVs can cause cancer through alterations in the tumor microenvironment.


Assuntos
Estrogênios/metabolismo , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/patologia , Proteínas Repressoras/genética , Neoplasias do Colo do Útero/virologia , Animais , Quimiocinas/genética , Quimiocinas/metabolismo , Técnicas de Cocultura , Citocinas/genética , Citocinas/metabolismo , Estrogênios/farmacologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Camundongos Transgênicos , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Proteínas Repressoras/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia
12.
Int J Mol Sci ; 21(23)2020 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-33260673

RESUMO

Tumor-specific metabolic adaptations offer an interesting therapeutic opportunity to selectively destroy cancer cells. However, solid tumors also present gradients of nutrients and waste products across the tumor mass, forcing tumor cells to adapt their metabolism depending on nutrient availability in the surrounding microenvironment. Thus, solid tumors display a heterogenous metabolic phenotype across the tumor mass, which complicates the design of effective therapies that target all the tumor populations present. In this work, we used a microfluidic device to study tumor metabolic vulnerability to several metabolic inhibitors. The microdevice included a central chamber to culture tumor cells in a three-dimensional (3D) matrix, and a lumen in one of the chamber flanks. This design created an asymmetric nutrient distribution across the central chamber, generating gradients of cell viability. The results revealed that tumor cells located in a nutrient-enriched environment showed low to no sensitivity to metabolic inhibitors targeting glycolysis, fatty acid oxidation, or oxidative phosphorylation. Conversely, when cell density inside of the model was increased, compromising nutrient supply, the addition of these metabolic inhibitors disrupted cellular redox balance and led to tumor cell death.


Assuntos
Dispositivos Lab-On-A-Chip , Microfluídica , Modelos Biológicos , Neoplasias/metabolismo , Contagem de Células , Humanos , Células MCF-7 , Necrose , Neoplasias/patologia , Hipóxia Tumoral
13.
Anal Chem ; 91(18): 11848-11855, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31411020

RESUMO

The extraction of bioanalytes is the first step in many diagnostic and analytical assays. However, most bioanalyte extraction methods require extensive dilution-based washing processes that are not only time-consuming and laborious but can also result in significant sample loss, limiting their applications in rare sample analyses. Here, we present a method that enables the efficient extraction of multiple different bioanalytes from rare samples (down to 10 cells) without washing-centrifugation-assisted immiscible fluid filtration (CIFF). CIFF utilizes centrifugal force to drive the movement of analyte-bound glass microbeads from an aqueous sample into an immiscible hydrophobic solution to perform an efficient, simple, and nondilutive extraction. The method can be performed using conventional polymerase chain reaction (PCR) tubes with no requirement of specialized devices, columns, or instruments, making it broadly accessible and cost-effective. The CIFF process can effectively remove approximately 99.5% of the aqueous sample in one extraction with only 0.5% residual carryover, whereas a traditional "spin-down and aspirate" operation results in a higher 3.6% carryover. Another unique aspect of CIFF is its ability to perform two different solid-phase bioanalytes extractions simultaneously within a single vessel without fractionating the sample or performing serial extractions. Here we demonstrate efficient mRNA and DNA extraction from low-input samples (down to 10 cells) with slightly higher to comparable recovery compared to a traditional column-based extraction technique and the simultaneous extraction of two different proteins in the same tube using CIFF.


Assuntos
Centrifugação/métodos , Fracionamento Químico/métodos , DNA/isolamento & purificação , Filtração/métodos , RNA Mensageiro/isolamento & purificação , Fracionamento Químico/instrumentação , Humanos , Reação em Cadeia da Polimerase/instrumentação , Proteínas/isolamento & purificação , Propriedades de Superfície , Células THP-1
14.
Molecules ; 24(23)2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31801265

RESUMO

Luminal geometries are common structures in biology, which are challenging to mimic using conventional in vitro techniques based on the use of Petri dishes. In this context, microfluidic systems can mimic the lumen geometry, enabling a large variety of studies. However, most microfluidic models still rely on polydimethylsiloxane (PDMS), a material that is not amenable for high-throughput fabrication and presents some limitations compared with other materials such as polystyrene. Thus, we have developed a microfluidic device array to generate multiple bio-relevant luminal structures utilizing polystyrene and micro-milling. This platform offers a scalable alternative to conventional microfluidic devices designed in PDMS. Additionally, the use of polystyrene has well described advantages, such as lower permeability to hydrophobic molecules compared with PDMS, while maintaining excellent viability and optical properties. Breast cancer cells cultured in the devices exhibited high cell viability similar to PDMS-based microdevices. Further, co-culture experiments with different breast cell types showed the potential of the model to study breast cancer invasion. Finally, we demonstrated the potential of the microfluidic array for drug screening, testing chemotherapy drugs and photodynamic therapy agents for breast cancer.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Microfluídica , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Desenho de Equipamento , Humanos , Microfluídica/métodos
15.
Mol Carcinog ; 57(4): 559-566, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29350431

RESUMO

MicroRNAs (miRNAs), small 22-25 nucleotide non-coding RNAs, play important roles in cellular and tumor biology. However, characterizing miRNA function remains challenging due to an abundance of predicted targets and an experimental bottleneck in identifying biologically relevant direct targets. Here, we developed a novel technique (miFAST) to identify direct miRNA target genes. Using miFAST, we confirmed several previously reported miR-340 target genes and identified five additional novel direct miR-340 targets in melanoma cells. This methodology can also be efficiently applied for the global characterization of miRNA targets. Utilizing miFAST to characterize direct miRNA targetomes will further our understanding of miRNA biology and function.


Assuntos
Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Biologia Molecular/métodos , Linhagem Celular Tumoral , Perfilação da Expressão Gênica/instrumentação , Humanos , Biologia Molecular/instrumentação , Reprodutibilidade dos Testes
16.
PLoS Pathog ; 12(9): e1005884, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27622514

RESUMO

Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix.


Assuntos
Biofilmes/crescimento & desenvolvimento , Candida albicans/fisiologia , Armadilhas Extracelulares/imunologia , Hifas/imunologia , Neutrófilos/imunologia , Animais , Candida albicans/ultraestrutura , Feminino , Humanos , Hifas/ultraestrutura , Masculino , Neutrófilos/ultraestrutura , Ratos
17.
Electrochim Acta ; 286: 205-211, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31130739

RESUMO

We have successfully integrated techniques for controlling cell adhesion and performing electrochemical differential pulse voltammetry (DPV) through the use of digitally controlled microfluidics and patterned transparent indium tin oxide electrode arrays to enable rapid and sensitive enumeration of cancer cells in a scalable microscale format. This integrated approach leverages a dual-working electrode (WE) surface to improve the specificity of the detection system. Here, one of the WE surfaces is functionalized with anti-Melanocortin 1 Receptor antibodies specific to melanoma cancer cells, while the other WE acts as a control (i.e., without antibody), for detecting non-specific interactions between cells and the electrode. The method is described and shown to provide effective detection of melanoma cells at concentrations ranging between 25 to 300 cells per 20 µL sample volume after a 5 min incubation and 15 s of DPV measurements. The estimated limit of detection was ~17 cells. The sensitivity and specificity of the assay were quantified using addition of large fractions of non-target cells and resulted in a detection reproducibility of ~97%. The proposed approach demonstrates a unique integration of electrochemical sensing and microfluidic cell adhesion technologies with multiple advantages such as label-free detection, short detection times, and low sample volumes. Next steps for this platform include testing with patient samples and use of other cell-surface biomarkers for detection and enumeration of circulating tumor cells in prostate, breast, and colon cancer.

18.
Biomacromolecules ; 18(3): 709-718, 2017 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-28157290

RESUMO

As a result of improved relevance to in vivo physiology, in vitro studies are increasingly performed in diverse, three-dimensional (3D) biomaterials. However, material-cell type pairing effects on cytokine availability remain unclear. We cultured five cell types in agarose, alginate, collagen, Matrigel, or RGD-functionalized polyethylene glycol (PEG) hydrogels. We measured 21 cytokines in the conditioned media, and we identified differences in measured cytokine levels that were cell-type- or material-dependent. We further evaluated our data using principal component analysis. Interestingly, component one identified two classes of biomaterials with characteristic cytokine expression levels. Component two identified cell-type-dependent differences in cytokines related to the wound response. Although elements of soluble cytokine availability are shared despite parameter differences, material and cellular properties variably influenced cytokine levels, underlining the influence of biomaterial-cell type pairings on in vitro assay outcomes. Relationships between material properties, cellular responses, and cytokine availability in 3D in vitro models warrant further investigation.


Assuntos
Materiais Biocompatíveis/química , Citocinas/biossíntese , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Meios de Cultura/química , Células Epiteliais/metabolismo , Humanos , Hidrogéis/química , Polietilenoglicóis/química , Análise de Componente Principal
19.
Proc Natl Acad Sci U S A ; 111(16): 5813-8, 2014 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-24711384

RESUMO

Asthma is a chronic inflammatory disorder that affects more than 300 million people worldwide. Asthma management would benefit from additional tools that establish biomarkers to identify phenotypes of asthma. We present a microfluidic solution that discriminates asthma from allergic rhinitis based on a patient's neutrophil chemotactic function. The handheld diagnostic device sorts neutrophils from whole blood within 5 min, and generates a gradient of chemoattractant in the microchannels by placing a lid with chemoattractant onto the base of the device. This technology was used in a clinical setting to assay 34 asthmatic (n = 23) and nonasthmatic, allergic rhinitis (n = 11) patients to establish domains for asthma diagnosis based on neutrophil chemotaxis. We determined that neutrophils from asthmatic patients migrate significantly more slowly toward the chemoattractant compared with nonasthmatic patients (P = 0.002). Analysis of the receiver operator characteristics of the patient data revealed that using a chemotaxis velocity of 1.55 µm/min for asthma yields a diagnostic sensitivity and specificity of 96% and 73%, respectively. This study identifies neutrophil chemotaxis velocity as a potential biomarker for asthma, and we demonstrate a microfluidic technology that was used in a clinical setting to perform these measurements.


Assuntos
Asma/sangue , Asma/patologia , Quimiotaxia de Leucócito , Neutrófilos/patologia , Adulto , Separação Celular , Expiração , Feminino , Humanos , Masculino , Microfluídica , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Adulto Jovem
20.
Anal Chem ; 88(20): 9902-9907, 2016 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-27598856

RESUMO

The simple, rapid magnetic manipulation of paramagnetic particles (PMPs) paired with the wide range of available surface chemistries has strongly positioned PMPs in the field of analyte isolation. One recent technology, sliding lid for immobilized droplet extractions (SLIDE), presents a simple, rapid alternative to traditional PMP isolation protocols. Rather than remove fluid from PMP-bound analyte, SLIDE directly removes the PMPs from the fluid. SLIDE collects the PMPs on a hydrophobic, removable surface, which allows PMPs to be captured from one well and then transferred and released into a second well. Despite several key advantages, SLIDE remains limited by its passive magnetic manipulation that only allows for a one-time capture-and-release of PMPs, preventing wash steps and limiting purity. Furthermore, the strategy employed by SLIDE constrains the position of the wells, thereby limiting throughput and integration into automated systems. Here, we introduce a new, mechanically and operationally simplistic magnetic manipulation system for integration with the SLIDE technology to overcome the previously stated limitations. This magnetic system is compatible with nearly any plate design, can be integrated into automated workflows, enables high-throughput formats, simplifies mechanical requirements, and is amenable to a range of analytes. Using this magnetic system, PMPs can be collected, released, and resuspended throughout multiple wells regardless of proximity. We demonstrate this system's capabilities to isolate whole cells, mRNA, and DNA, demonstrating up to a 28-fold improvement of purity via the multiwash protocols enabled by this magnetic technology.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA