RESUMO
In the paper, we present an integrated flow cytometer with a 2D array of magnetic sensors based on dual-frequency oscillators in a 65-nm CMOS process, with the chip packaged with microfluidic controls. The sensor architecture and the presented array signal processing allows uninhibited flow of the sample for high throughput without the need for hydrodynamic focusing to a single sensor. To overcome the challenge of sensitivity and specificity that comes as a trade off with high throughout, we perform two levels of signal processing. First, utilizing the fact that a magnetically tagged cell is expected to excite sequentially an array of sensors in a time-delayed fashion, we perform inter-site cross-correlation of the sensor spectrograms that allows us to suppress the probability of false detection drastically, allowing theoretical sensitivity reaching towards sub-ppM levels that is needed for rare cell or circulating tumor cell detection. In addition, we implement two distinct methods to suppress correlated low frequency drifts of singular sensors-one with an on-chip sensor reference and one that utilizes the frequency dependence of the susceptibility of super-paramagnetic magnetic beads that we deploy as tags. We demonstrate these techniques on a 7 ×7 sensor array in 65 nm CMOS technology packaged with microfluidics with magnetically tagged dielectric particles and cultu lymphoma cancer cells.
Assuntos
Citometria de Fluxo , Humanos , Citometria de Fluxo/instrumentação , Desenho de Equipamento , Semicondutores , Dispositivos Lab-On-A-Chip , Processamento de Sinais Assistido por Computador/instrumentaçãoRESUMO
Extensive research for the last two decades has significantly contributed to understanding the roles of lamins in the maintenance of nuclear architecture and genome organization which is drastically modified in neoplasia. It must be emphasized that alteration in lamin A/C expression and distribution is a consistent event during tumorigenesis of almost all tissues of human bodies. One of the important signatures of a cancer cell is its inability to repair DNA damage which befalls several genomic events that transform the cells to be sensitive to chemotherapeutic agents. This genomic and chromosomal instability is the most common feature found in cases of high-grade ovarian serous carcinoma. Here, we report elevated levels of lamins in OVCAR3 cells (high-grade ovarian serous carcinoma cell line) in comparison to IOSE (immortalised ovarian surface epithelial cells) and, consequently, altered damage repair machinery in OVCAR3. We have analysed the changes in global gene expression as a sequel to DNA damage induced by etoposide in ovarian carcinoma where lamin A is particularly elevated in expression and reported some differentially expressed genes associated with pathways conferring cellular proliferation and chemoresistance. We hereby establish the role of elevated lamin A in neoplastic transformation in the context of high-grade ovarian serous cancer through a combination of HR and NHEJ mechanisms.
Assuntos
Cistadenocarcinoma Seroso , Neoplasias Ovarianas , Feminino , Humanos , Apoptose , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Cistadenocarcinoma Seroso/genética , Lamina Tipo A/metabolismo , Neoplasias Ovarianas/genéticaRESUMO
Early diabetes research is hampered by limited availability, variable quality, and instability of human pancreatic islets in culture. Little is known about the human ß cell secretome, and recent studies question translatability of rodent ß cell secretory profiles. Here, we verify representativeness of EndoC-ßH1, one of the most widely used human ß cell lines, as a translational human ß cell model based on omics and characterize the EndoC-ßH1 secretome. We profiled EndoC-ßH1 cells using RNA-seq, data-independent acquisition, and tandem mass tag proteomics of cell lysate. Omics profiles of EndoC-ßH1 cells were compared to human ß cells and insulinomas. Secretome composition was assessed by data-independent acquisition proteomics. Agreement between EndoC-ßH1 cells and primary adult human ß cells was â¼90% for global omics profiles as well as for ß cell markers, transcription factors, and enzymes. Discrepancies in expression were due to elevated proliferation rate of EndoC-ßH1 cells compared to adult ß cells. Consistently, similarity was slightly higher with benign nonmetastatic insulinomas. EndoC-ßH1 secreted 783 proteins in untreated baseline state and 3135 proteins when stressed with nontargeting control siRNA, including known ß cell hormones INS, IAPP, and IGF2. Further, EndoC-ßH1 secreted proteins known to generate bioactive peptides such as granins and enzymes required for production of bioactive peptides. EndoC-ßH1 secretome contained an unexpectedly high proportion of predicted extracellular vesicle proteins. We believe that secretion of extracellular vesicles and bioactive peptides warrant further investigation with specialized proteomics workflows in future studies.
Assuntos
Células Secretoras de Insulina , Insulinoma , Neoplasias Pancreáticas , Linhagem Celular , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Insulinoma/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteoma/metabolismo , Secretoma , TranscriptomaRESUMO
Nuclear morphological features are potent determining factors for clinical diagnostic approaches adopted by pathologists to analyze the malignant potential of cancer cells. Considering the structural alteration of the nucleus in cancer cells, various groups have developed machine learning techniques based on variation in nuclear morphometric information like nuclear shape, size, nucleus-cytoplasm ratio and various non-parametric methods like deep learning have also been tested for analyzing immunohistochemistry images of tissue samples for diagnosing various cancers. We aim to correlate the morphometric features of the nucleus along with the distribution of nuclear lamin proteins with classical machine learning to differentiate between normal and ovarian cancer tissues. It has already been elucidated that in ovarian cancer, the extent of alteration in nuclear shape and morphology can modulate genetic changes and thus can be utilized to predict the outcome of low to a high form of serous carcinoma. In this work, we have performed exhaustive imaging of ovarian cancer versus normal tissue and developed a dual pipeline architecture that combines the matrices of morphometric parameters with deep learning techniques of auto feature extraction from pre-processed images. This novel Deep Hybrid Learning model, though derived from classical machine learning algorithms and standard CNN, showed a training and validation AUC score of 0.99 whereas the test AUC score turned out to be 1.00. The improved feature engineering enabled us to differentiate between cancerous and non-cancerous samples successfully from this pilot study.
Assuntos
Núcleo Celular/patologia , Neoplasias Ovarianas/diagnóstico , Algoritmos , Área Sob a Curva , Forma do Núcleo Celular/fisiologia , Tamanho do Núcleo Celular/fisiologia , Aprendizado Profundo , Diagnóstico por Imagem , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Aprendizado de Máquina , Redes Neurais de Computação , Lâmina Nuclear/fisiologia , Projetos PilotoRESUMO
Lamins are type V intermediate filament proteins which are ubiquitously present in all metazoan cells providing a platform for binding of chromatin and related proteins, thereby serving a wide range of nuclear functions including DNA damage repair. Altered expression of lamins in different subtypes of cancer is evident from researches worldwide. But whether cancer is a consequence of this change or this change is a consequence of cancer is a matter of future investigation. However changes in the expression levels of lamins is reported to have direct or indirect association with cancer progression or have regulatory roles in common neoplastic symptoms like higher nuclear deformability, increased genomic instability and reduced susceptibility to DNA damaging agents. It has already been proved that loss of A type lamin positively regulates cathepsin L, eventually leading to degradation of several DNA damage repair proteins, hence impairing DNA damage repair pathways and increasing genomic instability. It is established in ovarian cancer, that the extent of alteration in nuclear morphology can determine the degree of genetic changes and thus can be utilized to detect low to high form of serous carcinoma. In this review, we have focused on ovarian cancer which is largely caused by genomic alterations in the DNA damage response pathways utilizing proteins like RAD51, BRCA1, 53BP1 which are regulated by lamins. We have elucidated the current understanding of lamin expression in ovarian cancer and its implications in the regulation of DNA damage response pathways that ultimately result in telomere deformation and genomic instability.
Assuntos
Dano ao DNA , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Laminina/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Animais , Proteína BRCA1/química , Catepsina L/metabolismo , Núcleo Celular/metabolismo , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Instabilidade Genômica , Genômica , Humanos , Laminas/metabolismo , Camundongos , Domínios Proteicos , Rad51 Recombinase/química , Telômero/metabolismo , Resultado do Tratamento , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/químicaRESUMO
Background: Etoricoxib is a second-generation cyclooxygenase-2-inhibitor approved in 2012 for short-term treatment of pain associated with dental surgery. Objectives: To evaluate etoricoxib utilization in dental patients in the Nordic countries, including its off-label use. Methods: The entire populations of Denmark, Finland, Sweden and Norway with etoricoxib prescriptions written by dentists and dispensed in 2012-2014 were evaluated using national register data. Nationwide estimates of etoricoxib utilization were generated according to year, gender, age, dose and package size. Off-label use in paediatric patients, prescribed doses >90 mg/day or for dental contacts not associated with surgical procedures, and concomitant administration with anticoagulants were evaluated. Results: Utilization of etoricoxib for dental pain was low (1615 prescriptions: Finland, 907; Sweden, 359; Norway, 337; Denmark, 12). Overall, 70% of the prescriptions were without an associated dental procedure. Moreover, 58%, 55%, 10% and 58% of the prescriptions in Denmark, Finland, Sweden and Norway, respectively, were for >90 mg/day doses. Few paediatric prescriptions were dispensed (n < 10), and only a small overlap (n = 21) was observed between etoricoxib and anticoagulant prescriptions. Conclusions: Given the low overall number of prescriptions, it is unlikely that off-label use of etoricoxib within dentistry in the Nordic countries is an important public health concern.
Assuntos
Etoricoxib , Dor Pós-Operatória/tratamento farmacológico , Extração Dentária/efeitos adversos , Etoricoxib/uso terapêutico , Humanos , Países Escandinavos e NórdicosRESUMO
Bleb formation has been correlated with nonmuscle myosin II (NM-II) activity. Whether three isoforms of NM-II (NM-IIA, -IIB and -IIC) have the same or differential roles in bleb formation is not well understood. Here we report that ectopically expressed, GFP-tagged NM-II isoforms exhibit different types of membrane protrusions, such as multiple blebs, lamellipodia, combinations of both, or absence of any such protrusions in MCF-7 cells. Quantification suggests that 50% of NM-IIA-GFP-, 29% of NM-IIB-GFP-, and 19% of NM-IIC1-GFP-expressing MCF-7 cells show multiple bleb formation, compared with 36% of cells expressing GFP alone. Of interest, NM-IIB has an almost 50% lower rate of dissociation from actin filament than NM-IIA and -IIC1 as determined by FRET analysis both at cell and bleb cortices. We induced bleb formation by disruption of the cortex and found that all three NM-II-GFP isoforms can reappear and form filaments but to different degrees in the growing bleb. NM-IIB-GFP can form filaments in blebs in 41% of NM-IIB-GFP-expressing cells, whereas filaments form in only 12 and 3% of cells expressing NM-IIA-GFP and NM-IIC1-GFP, respectively. These studies suggest that NM-II isoforms have differential roles in the bleb life cycle.
Assuntos
Extensões da Superfície Celular/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Citoesqueleto de Actina/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Citoesqueleto/metabolismo , Humanos , Células MCF-7 , Isoformas de Proteínas , Pseudópodes/metabolismoRESUMO
Hsp70 aids in protein folding and directs misfolded proteins to the cellular degradation machinery. We describe discrete roles of Hsp70,SSA1 as an important quality-control machinery that switches functions to ameliorate the cellular environment. SSA1 facilitates folding/maturation of newly synthesized protein kinases by aiding their phosphorylation process and also stimulates ubiquitylation and degradation of kinases in regular protein turnover or during stress when kinases are denatured or improperly folded. Significantly, while kinases accumulate as insoluble inclusions upon SSA1 inhibition, they form soluble inclusions upon Hsp90 inhibition or stress foci during heat stress. This suggests formation of inclusion-specific quality-control compartments under various stress conditions. Up-regulation of SSA1 results in complete removal of these inclusions by the proteasome. Elevation of the cellular SSA1 level accelerates kinase turnover and protects cells from proteotoxic stress. Upon overexpression, SSA1 targets heat-denatured kinases toward degradation, which could enable them to recover their functional state under physiological conditions. Thus active participation of SSA1 in the degradation of misfolded proteins establishes an essential role of Hsp70 in deciding client fate during stress.