RESUMEN
A cost-effective, viral nucleic acid (NA) isolation kit based on NAxtra magnetic nanoparticles was developed at the Norwegian University of Science and Technology in response to the shortage of commercial kits for isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA during the coronavirus disease 2019 (COVID-19) pandemic. This method showed comparable sensitivity to available kits at significantly reduced cost, making its application for other biological sources an intriguing prospect. Thus, based on this low-cost nucleic acid extraction technology, we developed a simple, low- and high-throughput, efficient method for isolation of high-integrity total NA, DNA and RNA from mammalian cell lines (monolayer) and organoids (3D-cultures). The extracted NA are compatible with downstream applications including (RT-)qPCR and next-generation sequencing. When automated, NA isolation can be performed in 14 min for up to 96 samples, yielding similar quantities to available kits.
Asunto(s)
COVID-19 , Nanopartículas de Magnetita , Animales , Humanos , ARN Viral/análisis , SARS-CoV-2/genética , ADN , Sensibilidad y Especificidad , Mamíferos/genéticaRESUMEN
The year of 2020 was profoundly marked by a global pandemic caused by a strain of coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19). To control disease spread, a key strategy adopted by many countries was the regular testing of individuals for infection. This led to the rapid development of diagnostic testing technologies. In Norway, within a week, our group developed a test kit to quickly isolate viral RNA and safely detect SARS-CoV-2 infection with sensitivity comparable to available kits. Herein, the procedure employed for the detection of SARS-CoV-2 in swab samples from patients using the NTNU-COVID-19 test kit is described in detail. This procedure, based on NAxtra magnetic nanoparticles and an optimized nucleic acid extraction procedure, is robust, reliable, and straightforward, providing high-quality nucleic acids within 14 min. The NAxtra protocol is adaptable and was further validated for extraction of DNA and RNA from other types of viruses. A comparison of the protocol on different liquid handling systems is also presented. Due to the simplicity and low cost of this method, implementation of this technology to diagnose virus infections on a clinical setting would benefit health care systems, promoting sustainability.
Asunto(s)
COVID-19 , Nanopartículas de Magnetita , Ácidos Nucleicos , Humanos , COVID-19/diagnóstico , SARS-CoV-2/genética , Prueba de COVID-19 , ARN Viral/genética , Sensibilidad y EspecificidadRESUMEN
Alkylating drugs are among the most often used chemotherapeutics. While cancer cells frequently develop resistance to alkylation treatments, detailed understanding of mechanisms that lead to the resistance is limited. Here, by using genome-wide CRISPR-Cas9 based screen, we identify transcriptional Mediator complex subunit 13 (MED13) as a novel modulator of alkylation response. The alkylation exposure causes significant MED13 downregulation, while complete loss of MED13 results in reduced apoptosis and resistance to alkylating agents. Transcriptome analysis identified cyclin D1 (CCND1) as one of the highly overexpressed genes in MED13 knock-out (KO) cells, characterized by shorter G1 phase. MED13 is able to bind to CCND1 regulatory elements thus influencing the expression. The resistance of MED13 KO cells is directly dependent on the cyclin D1 overexpression, and its down-regulation is sufficient to re-sensitize the cells to alkylating agents. We further demonstrate the therapeutic potential of MED13-mediated response, by applying combinatory treatment with CDK8/19 inhibitor Senexin A. Importantly, the treatment with Senexin A stabilizes MED13, and in combination with alkylating agents significantly reduces viability of cancer cells. In summary, our findings identify novel alkylation stress response mechanism dependent on MED13 and cyclin D1 that can serve as basis for development of innovative therapeutic strategies.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Ciclina D1/genética , Complejo Mediador/fisiología , Sistemas CRISPR-Cas , Línea Celular , Línea Celular Tumoral , Ciclina D1/metabolismo , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Daño del ADN , Resistencia a Antineoplásicos , Regulación de la Expresión Génica , Humanos , Complejo Mediador/metabolismo , Regulación hacia ArribaRESUMEN
As of June 2020, the number of people infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) continues to skyrocket, with more than 6.7 million cases worldwide. Both the World Health Organization (WHO) and United Nations (UN) has highlighted the need for better control of SARS-CoV-2 infections. However, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against SARS-CoV-2 can be time-consuming and costly. Convalescent sera and safe-in-man broad-spectrum antivirals (BSAAs) are readily available treatment options. Here, we developed a neutralization assay using SARS-CoV-2 strain and Vero-E6 cells. We identified the most potent sera from recovered patients for the treatment of SARS-CoV-2-infected patients. We also screened 136 safe-in-man broad-spectrum antivirals against the SARS-CoV-2 infection in Vero-E6 cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. We found that a combination of orally available virus-directed nelfinavir and host-directed amodiaquine exhibited the highest synergy. Finally, we developed a website to disseminate the knowledge on available and emerging treatments of COVID-19.
Asunto(s)
Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Pruebas de Neutralización/métodos , Neumonía Viral/tratamiento farmacológico , Amodiaquina/farmacología , Animales , COVID-19 , Células CACO-2 , Línea Celular Tumoral , Chlorocebus aethiops , Infecciones por Coronavirus/terapia , Quimioterapia Combinada , Emetina/farmacología , Células HEK293 , Células HT29 , Homoharringtonina/farmacología , Humanos , Sueros Inmunes/inmunología , Inmunización Pasiva/métodos , Indoles , Nelfinavir/farmacología , Pandemias , Piranos/farmacología , Pirroles/farmacología , SARS-CoV-2 , Células Vero , Sueroterapia para COVID-19RESUMEN
While genotyping studies are scavenging for suitable samples to analyze, large serum collections are currently left unused as they are assumed to provide insufficient amounts of DNA for array-based genotyping. Long-term stored serum is considered to be difficult to genotype since preanalytical treatments and storage effects on DNA yields are not well understood. Successful genotyping of such samples has the potential to activate large biobanks for future genome-wide association studies (GWAS). We aimed to evaluate genotyping of ultralow amounts of DNA from samples stored up to 45 years in the Janus Serum Bank with two commercially available platforms. 64 samples, with various preanalytical treatments, were genotyped on the Axiom Array from Thermo Fisher Scientific and a subset of 24 samples with slightly higher yield were genotyped on the HumanCoreExome array from Illumina. Our results showed that about 80% of the serum samples produced call rates with the Axiom arrays that would be satisfactory in GWAS. The mean DNA yield was 5.8 ng as measured with PicoGreen, 3-6% of recommended yield. The failed samples had on average lower input amounts of DNA. All serum samples genotyped on the HumanCoreExome with a standard and FFPE protocol produced GWAS satisfactory call rates, with mean 97.57% and 98.35% call rates, respectively. The mean yield was 10.65 ng, 6% of the recommendations. Successful array-based genotyping of ultralow DNA yields from serum samples stored up to 45 years is possible. These results demonstrate the potential to activate large serum biobank collections for future studies.
Asunto(s)
Conservación de la Sangre/efectos adversos , ADN/química , Secuenciación del Exoma/métodos , Técnicas de Genotipaje/métodos , Bancos de Sangre , ADN/sangre , ADN/genética , ADN/normas , Pruebas Genéticas/métodos , Pruebas Genéticas/normas , Estudio de Asociación del Genoma Completo/métodos , Estudio de Asociación del Genoma Completo/normas , Técnicas de Genotipaje/normas , Humanos , Secuenciación del Exoma/normasRESUMEN
BACKGROUND: Gene expression profiling from blood is sensitive to technology choices. For example, the main blood RNA collection systems-the PAXgene and Tempus tubes-differently influence RNA expression signatures. The aim of this study was to establish a common RNA isolation protocol for these two systems and investigate if it could reduce the differences in gene expression between them. RESULTS: We collected identical blood samples on the PAXgene and Tempus systems and retrieved blood samples from two independent biobanks-NOWAC and HUNT3-which are based on PAXgene and Tempus, respectively. High-quality RNA was extracted from both sampling systems by using their original protocols and our common modified protocol, and were profiled on Illumina microarrays. Regardless of the protocol used, we found most of the measured transcripts to be differently affected by the two sampling systems. However, our modified protocol reduced the number of transcripts that were significantly differentially expressed between PAXgene and Tempus by approximately 50%. Expression differences between PAXgene and Tempus were highly reproducible both between protocols and between different independent sample sets (Pearson correlation 0.563-0.854 across 47323 probes). Moreover, the modified protocol increased the microRNA output of the system with lowest microRNA yield, the PAXgene system. CONCLUSIONS: Most transcripts are affected by the choice of sampling system, but these effects are highly reproducible between independent samples. We propose that by running a control experiment with samples on both systems in parallel with biologically relevant samples, researchers may adjust for technical differences between the sampling systems.
Asunto(s)
Bancos de Sangre , Recolección de Muestras de Sangre/métodos , Perfilación de la Expresión Génica/métodos , ARN/genética , Recolección de Muestras de Sangre/instrumentación , Perfilación de la Expresión Génica/instrumentación , Humanos , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN/sangre , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The oxyntic proliferative isthmus zone contains the main stem/progenitor cells that provide for physiological renewal of the distinct mature cell lineages in the oxyntic epithelium of the stomach. These cells are also proposed to be the potential cells-of-origin of gastric cancer, although little is known about their molecular characteristics and specific biological markers are lacking. In this study, we developed a method for serial section-navigated laser microdissection to isolate cells from the proliferative isthmus zone of rat gastric oxyntic mucosa for genome-wide microarray gene expression analysis. Enrichment analysis showed a distinct gene expression profile for the isthmus zone, with genes regulating intracellular processes such as the cell cycle and ribosomal activity. The profile was also related to stem cell transcriptional networks and stomach neoplasia. Genes expressed uniquely in the isthmus zone were associated with E2F transcription factor 1 (E2F1), which participates in the self-renewal of stem cells and in gastric carcinogenesis. One of the unique genes was Aspm [Asp (abnormal spindle) homologue, microcephaly-associated (Drosophila)]. Here we show ASPM in single scattered epithelial cells located in the proliferative isthmus zone of rat, mouse and human oxyntic mucosa, which do not seem to be actively dividing. The ASPM-expressing cells are mainly mature cell marker-deficient, except for a limited overlap with cells with neuroendocrine and tuft cell features. Further, both ASPM and E2F1 were expressed in human gastric cancer cell lines and increased and correlated in human gastric adenocarcinomas compared to non-tumour mucosa, as shown by expression profile analyses and immunohistochemistry. The association between ASPM and the transcription factor E2F1 in gastric tissue is relevant, due to their common involvement in crucial cell fate-regulatory mechanisms. Our results thus introduce ASPM as a novel possible oxyntic stem/progenitor cell marker that may be involved in both normal gastric physiology and gastric carcinogenesis.
Asunto(s)
Adenocarcinoma/patología , Mucosa Gástrica/citología , Células Madre Neoplásicas/patología , Proteínas del Tejido Nervioso/biosíntesis , Neoplasias Gástricas/patología , Animales , Biomarcadores de Tumor/análisis , Western Blotting , Proteínas de Unión a Calmodulina/biosíntesis , Técnica del Anticuerpo Fluorescente , Estudio de Asociación del Genoma Completo , Humanos , Hibridación in Situ , Captura por Microdisección con Láser , Ratones , Células Parietales Gástricas/patología , Células Madre/citología , TranscriptomaRESUMEN
BACKGROUND: Pemetrexed, a multi-folate inhibitor combined with a platinum compound is the first-line treatment of malignant mesothelioma, but median survival is still one year. Intrinsic and acquired resistance to pemetrexed is common, but its biological basis is obscure. Here we report for the first time a genome-wide profile of acquired resistance in the tumour from an exceptional case with advanced pleural mesothelioma and almost six years survival after 39 cycles of second-line pemetrexed/carboplatin treatment. METHODOLOGY AND PRINCIPAL FINDINGS: Genome-wide analysis with Illumina BeadChip Kit of 25,000 genes was performed on mRNA from pre-treatment and post-resistance biopsies from this individual as well on case and control samples from our previously published study (in total 17 samples). Cell specific expression of proteins encoded by selected genes were analysed by immunohistochemistry. Serial serum levels of CA125, CYFRA21-1 and SMRP levels were examined. TS protein, the main target of pemetrexed was overexpressed. Proteins and genes related to DNA damage response, elongation and telomere extension and repair related directly and indirectly to platinum resistance were overexpressed, as the CHK1 protein and the genes CHEK2, LIG3, POLD1, POLA2, FANCD2, PRPF19, RECQ5 respectively, the last two not previously described in mesothelioma. We observed a down-regulation of leukocyte transendothelial migration and cell adhesion molecules pathways. Silencing of NT5C in two mesothelioma cell lines did not sensitize the cells to Pemetrexed. Proposed resistance markers are TS, KRT7/ CK7, TYMP/ thymidine phosphorylase and down-regulated SPARCL1 and CDKN1B. Moreover, comparison of the primary expression of the sensitive versus a primary resistant case showed multi-fold overexpressed DNA repair, cell cycle, cytokinesis, and spindle formation in the latter. Serum CA125 and SMRP reflected the clinical and radiological course and tumour burden. CONCLUSIONS: Genome-wide microarray of mesothelioma pre- and post-resistance biopsies indicated a novel resistance signature to pemetrexed/carboplatin that deserve validation in a larger cohort.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Glutamatos/farmacología , Guanina/análogos & derivados , Mesotelioma/tratamiento farmacológico , Platino (Metal)/farmacología , 5'-Nucleotidasa/metabolismo , Adulto , Biopsia/métodos , Adhesión Celular , Estudios de Cohortes , Reparación del ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Estudio de Asociación del Genoma Completo , Guanina/farmacología , Humanos , Inmunohistoquímica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , Pemetrexed , Tomografía Computarizada por Rayos X/métodos , Resultado del TratamientoRESUMEN
The regulated endocrine-specific protein 18 (RESP18) has previously been localized to different endocrine cells and neurons, in particular the pituitary gland and hypothalamus. It is found in the lumen of the endoplasmic reticulum and is degraded at the post-ER pre-Golgi compartment, and a role in processing of secreted peptides has been hypothesized. The present study examines localization of RESP18 in the gastrointestinal mucosa of rats by immunohistochemistry, and expression and regulation in response to hypergastrinemia induced by acid inhibition (pantoprazole), gastrin antagonism (YF476), fasting-refeeding and octreotide by mRNA measurements. RESP18 was mainly found in the gastric mucosa, but could also be detected in a few, scattered cells in the lower small intestine and in colon. In the antral mucosa, all RESP18 immunoreactivity was localized to ghrelin-producing A-like cells and gastrin-producing G-cells. In the corpus mucosa, a significant fraction, but not all of the RESP18 immunoreactive cells, were A-like cells. In both antrum and corpus, Resp18 mRNA seemed to vary similarly with the activation of the A-like cells, and in the antrum also with stimulation of the G-cells. This study demonstrates, for the first time, the localization of RESP18 to specific neuroendocrine cells of the gastrointestinal mucosa and that it seems to be regulated synchronously with the peptides secreted from these cells. This suggests that Resp18 may indeed have a functional role in the synthesis or storage of these gastrointestinal peptides.
Asunto(s)
Mucosa Gástrica/metabolismo , Células Secretoras de Gastrina/metabolismo , Regulación de la Expresión Génica , Proteínas del Tejido Nervioso/metabolismo , 2-Piridinilmetilsulfinilbencimidazoles/farmacología , Animales , Benzodiazepinonas/farmacología , Ayuno/metabolismo , Conducta Alimentaria , Gastrinas/antagonistas & inhibidores , Gastrinas/farmacología , Ghrelina/metabolismo , Inmunohistoquímica , Intestino Delgado/metabolismo , Proteínas del Tejido Nervioso/genética , Octreótido/administración & dosificación , Octreótido/farmacología , Pantoprazol , Compuestos de Fenilurea/farmacología , Antro Pilórico/fisiología , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Estómago/citologíaRESUMEN
We show that the gastric hormone gastrin induces the expression of the prosurvival secretory clusterin (sCLU) in rat adenocarcinoma cells. Clusterin mRNA was still upregulated in the presence of the protein synthesis inhibitor cycloheximide, although at a lower level. This indicates that gastrin induces clusterin transcription independently of de novo protein synthesis but requires de novo protein synthesis of signal transduction pathway components to achieve maximal expression level. Luciferase reporter assay indicates that the AP-1 transcription factor complex is involved in gastrin-mediated activation of the clusterin promoter. Gastrin-induced clusterin expression and subsequent secretion is dependent on sustained treatment, because removal of gastrin after 1-2 h abolished the response. Neutralization of secreted clusterin by a specific antibody abolished the antiapoptotic effect of gastrin on serum starvation-induced apoptosis, suggesting that extracellular clusterin is involved in gastrin-mediated inhibition of apoptosis. The clusterin response to gastrin was validated in vivo in hypergastrinemic rats, showing increased clusterin expression in the oxyntic mucosa, as well as higher levels of clusterin in plasma. In normal rat oxyntic mucosa, clusterin protein was strongly expressed in chromogranin A-immunoreactive neuroendocrine cells, of which the main cell type was the histidine decarboxylase-immunoreactive enterochromaffin-like (ECL) cell. The association of clusterin with neuroendocrine differentiation was further confirmed in human gastric ECL carcinoids. Interestingly, in hypergastrinemic rats, clusterin-immunoreactive cells formed distinct groups of diverse cells at the base of many glands. Our results suggest that clusterin may contribute to gastrin's growth-promoting effect on the oxyntic mucosa.
Asunto(s)
Adenocarcinoma/metabolismo , Clusterina/biosíntesis , Gastrinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Células Parietales Gástricas/metabolismo , Regulación hacia Arriba , Adenocarcinoma/patología , Animales , Apoptosis/efectos de los fármacos , Tumor Carcinoide/química , Tumor Carcinoide/metabolismo , Línea Celular Tumoral , Cromogranina A/análisis , Clusterina/antagonistas & inhibidores , Clusterina/sangre , Clusterina/genética , Clusterina/metabolismo , Células Enterocromafines/efectos de los fármacos , Femenino , Histidina Descarboxilasa/metabolismo , Humanos , Células Neuroendocrinas/química , Células Neuroendocrinas/efectos de los fármacos , Neoplasias Pancreáticas/patología , Células Parietales Gástricas/patología , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Neoplasias Gástricas/inducido químicamente , Neoplasias Gástricas/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
OBJECTIVE: Ischaemic preconditioning may protect the intestine from subsequent prolonged ischaemia. This study evaluates whether a much longer initial ischaemia, encountered clinically, may modify intestinal resistance to further ischaemia in a pig model. MATERIAL AND METHODS: After cross-clamping of the superior mesenteric artery for 1 h, the intestine was either reperfused for 8 h or a second cross-clamping for 1 h was performed at 4 h of reperfusion. Based on microarray analysis of intestinal samples at 1, 4 and 8 h of reperfusion, mRNA of selected genes was measured with QRT-PCR. RESULTS: The first ischaemic period caused exfoliation of surface epithelial cells from the basement membrane comprising about 90 % of the villi tips, a marked increase in permeability and depletion of ATP. The second ischaemic challenge caused about 30 % less denudation of the basement membrane (p = 0.008), no increase in permeability (p = 0.008) and less depletion of ATP (p = 0.039). mRNAs for superoxide dismutase 2, heat shock proteins and signal transducer and activator of transcription 3, which may protect against ischaemia/reperfusion injury, were up-regulated throughout the reperfusion period. mRNAs for matrix metalloproteinase 1, connexin 43 and peripheral myelin 22, which may be associated with cell migration or tight junctions, showed a particular up-regulation at 4 h of reperfusion. CONCLUSION: One hour of initial ischaemia followed by 4 h of reperfusion is associated with increased intestinal resistance to further ischaemia. The differential regulation of genes identified in this study provides working hypotheses for mechanisms behind this observation.
Asunto(s)
Mucosa Intestinal/patología , Isquemia/patología , Animales , Absorción Intestinal , Mucosa Intestinal/irrigación sanguínea , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Flujo Sanguíneo RegionalRESUMEN
Previous studies show that octreotide LAR causes regression of gastric ECL-cell carcinoids, reducing both number and size of tumours. This study examines the molecular mechanisms behind the antiproliferative effect of octreotide on the oxyntic mucosa. Female rats received octreotide LAR for 21 days. Serum gastrin was measured and tissue samples for RNA extraction and histology collected from the oxyntic mucosa. Affymetrix analysis showed regulated genes related to apoptosis and proliferation, and a large group of regulated growth-related transcription factors. Verification by real time qRT-PCR showed a high degree of consistency to the microarray results. Supporting the molecular results, histomorphometry showed significant decreases in the number of gastric glands, cells per gland and length of glands, and a tendency towards increased apoptosis and decreased proliferation. Thus, octreotide exerts a negative effect on oxyntic mucosal growth, and induces extensive gene expression changes relevant to growth regulation.
Asunto(s)
Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Mucosa Gástrica/metabolismo , Fármacos Gastrointestinales/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Octreótido/farmacología , Animales , Tumor Carcinoide/tratamiento farmacológico , Tumor Carcinoide/metabolismo , Femenino , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Sprague-Dawley , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/metabolismoRESUMEN
The aim of the present work is to identify molecular markers that allow classification of gastric carcinoma with respect to important clinicopathological parameters. Gastric adenocarcinomas were subjected to cDNA microarray analysis with a 2.504 gene probe set. Using the Rosetta rough-set based learning system, good classifiers were generated for gene-expression based prediction of intestinal or diffuse growth pattern according to Laurén's classification and presence of lymph node metastases. To our knowledge, this is the first study on gastric carcinoma in which molecular classification has been achieved for more than one clinicopathological parameter based on microarray gene expression profiles.