RESUMO
Alzheimer's disease (AD) is a neurodegenerative disease that is known to accumulate amyloid-ß (Aß) and tau protein. Clinical studies have not identified pathogenesis mechanisms or produced an effective cure for AD. The Aß monoclonal antibody lecanemab reduces Aß plaque formation for the treatment of AD, but more studies are required to increase the effectiveness of drugs to reduce cognitive decline. The lack of AD therapy targets and evidence of an association with an acute neuroinflammatory response caused by several bacteria and viruses in some individuals has led to the establishment of the infection hypothesis during the last 10 years. How pathogens cross the blood-brain barrier is highly topical and is seen to be pivotal in proving the hypothesis. This review summarizes the possible role of the gut microbiome in the pathogenesis of AD and feasible therapeutic approaches and current research limitations.
Assuntos
Doença de Alzheimer , Microbioma Gastrointestinal , Doença de Alzheimer/microbiologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Humanos , Animais , Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/microbiologia , Proteínas tau/metabolismoRESUMO
Rapid changes in the number and flow cytometric behaviour of cells of E. coli taken from a stationary phase and inoculated into rich medium.Cells of E. coli were grown in LB medium, taken from a stationary phase of 2-4 h, and re-inoculated into fresh media at a concentration (105 ml-1 or lower) characteristic of bacteriuria. Flow cytometry was used to assess how quickly we could detect changes in cell size, number, membrane energization (using a carbocyanine dye) and DNA distribution. It transpired that while the lag phase observable macroscopically via bulk OD measurements could be as long as 4 h, the true lag phase could be less than 15-20 min, and was accompanied by many observable biochemical changes. Antibiotics to which the cells were sensitive affected these changes within 20 min of re-inoculation, providing the possibility of a very rapid antibiotic susceptibility test on a timescale compatible with a visit to a GP clinic. The strategy was applied successfully to genuine potential urinary tract infection (UTI) samples taken from a doctor's surgery. The methods developed could prove of considerable value in ensuring the correct prescription and thereby lowering the spread of antimicrobial resistance.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Citometria de Fluxo , Testes de Sensibilidade Microbiana/métodos , Infecções Urinárias/microbiologia , Antibacterianos/uso terapêutico , Bactérias/isolamento & purificação , Bacteriúria/tratamento farmacológico , Bacteriúria/microbiologia , Meios de Cultura , Humanos , Infecções Urinárias/tratamento farmacológicoRESUMO
A simple microfluidic fluorescence in situ hybridization (FISH) device allowing accurate analysis of interphase nuclei in 1 hr in narrow channels is presented. Photolithography and fluorosilicic acid etching were used to fabricate microfluidic channels (referred to as FISHing lines) that allowed analysis of 10 samples on a glass microscope slide 0.2 µl of sample volume was used to fill a micro-channel, which resembled a 250-fold reduction compared to conventional FISH. FISH signals were comparable to conventional FISH, with 50-fold less probe consumption and 10-fold less time. Cells were immobilized in single file in channels just exceeding the diameter of the cells, and were used for minimal residual disease (MRD) analysis. To test the micro-channels for application in FISH, MRD was simulated by mixing K562 cells (an established chronic myeloid leukemia cell line) carrying the BCR/ABL fusion gene across 1:1 to 1:1,000 Jurkat cells (an established acute lymphoblastic leukemia cell line). The limit of detection was seen to be 1:100 cells and 1:1,000 cells for FISHing lines and conventional FISH, respectively; however, the conventional method seemed to over-score the presence of K562 cells. This may in part be attributed to FISHing lines practically eliminating the chance of duplicate screening of cells and hastened the time of screening, enhancing scoring of all cells within the channels. This was compared to 1 in 500 cells on the slide being analyzed with the conventional FISH.
Assuntos
Neoplasias Hematológicas/diagnóstico , Hibridização in Situ Fluorescente/métodos , Tamanho Celular , Neoplasias Hematológicas/patologia , Humanos , Células Jurkat , Células K562 , Microfluídica , Neoplasia ResidualRESUMO
OBJECTIVE: Patients with systemic lupus erythematosus (SLE) have a higher rate of premature death compared to the general population, suggesting a phenotype of premature senescence in SLE. Telomere length can be used to assess overall biologic aging. This study was undertaken to address the hypothesis that patients with SLE have reduced telomere length. METHODS: Telomere length was measured cross-sectionally in whole blood from SLE patients and age-matched healthy female controls, using real-time quantitative polymerase chain reaction. SLE-related and cardiovascular risk factors were assessed. RESULTS: We compared telomere length in 63 SLE patients and 63 matched controls with a median age of 50.8 years (interquartile range [IQR] 37-59 years) and 49.9 years (IQR 32-60 years), respectively. The median relative telomere length in SLE patients was 0.97 (IQR 0.47-1.57), compared to 1.53 (IQR 0.82-2.29) in controls (P = 0.0008). We then extended our cohort to measure telomere length in 164 SLE patients. Shorter telomere length was associated with Ro antibodies (ß ± SE -0.36 ± 0.16; P = 0.023), and longer telomere length was associated with steroid therapy (0.29 ± 0.14; P = 0.046). We also noted an association of longer telomere length with increasing body mass index (ß ± SE 0.07 ± 0.01; P < 0.0001) and tobacco smoking (0.64 ± 0.26; P = 0.016), as well as with the presence of carotid plaque (0.203 ± 0.177; P = 0.032). CONCLUSION: Telomere length is shortened in SLE patients compared to controls and does not appear to be a reflection of disease activity or immune cell turnover. Subsets of patients such as those positive for Ro antibodies may be particularly susceptible to premature biologic aging. The predictive value of telomere length as a biomarker of future risk of damage/mortality in SLE requires longitudinal evaluation.
Assuntos
Aterosclerose/genética , Lúpus Eritematoso Sistêmico/genética , Obesidade/genética , Fumar/genética , Encurtamento do Telômero , Telômero/genética , Adulto , Anticorpos Antinucleares/análise , Aterosclerose/diagnóstico , Aterosclerose/epidemiologia , Comorbidade , Feminino , Glucocorticoides/uso terapêutico , Humanos , Lúpus Eritematoso Sistêmico/epidemiologia , Pessoa de Meia-Idade , Obesidade/epidemiologia , Fatores de Risco , Fumar/epidemiologia , Reino Unido/epidemiologiaRESUMO
Temozolomide (TMZ) is the standard chemotherapeutic agent for human malignant glioma, but intrinsic or acquired chemoresistance represents a major obstacle to successful treatment of this highly lethal group of tumours. Obtaining better understanding of the molecular mechanisms underlying TMZ resistance in malignant glioma is important for the development of better treatment strategies. We have successfully established a passage control line (D54-C10) and resistant variants (D54-P5 and D54-P10) from the parental TMZ-sensitive malignant glioma cell line D54-C0. The resistant sub-cell lines showed alterations in cell morphology, enhanced cell adhesion, increased migration capacities, and cell cycle arrests. Proteomic analysis identified a set of proteins that showed gradual changes in expression according to their 50% inhibitory concentration (IC(50)). Successful validation was provided by transcript profiling in another malignant glioma cell line U87-MG and its resistant counterparts. Moreover, three of the identified proteins (vimentin, cathepsin D and prolyl 4-hydroxylase, beta polypeptide) were confirmed to be upregulated in high-grade glioma. Our data suggest that acquired TMZ resistance in human malignant glioma is associated with promotion of malignant phenotypes, and our reported molecular candidates may serve not only as markers of chemoresistance but also as potential therapeutic targets in the treatment of TMZ-resistant human malignant glioma, providing a platform for future investigations.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Biomarcadores Tumorais/metabolismo , Dacarbazina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Apoptose , Western Blotting , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Adesão Celular , Ciclo Celular , Movimento Celular , Proliferação de Células , Dacarbazina/farmacologia , Eletroforese em Gel Bidimensional , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Temozolomida , CicatrizaçãoRESUMO
The pathogenesis of keloid formation is poorly understood. The fibroblasts in keloid patients continue to multiply even after initial wound repair and are characterized by a persistent dermal fibroproliferative reaction and excessive extracellular matrix production. Most studies concentrate on the type of collagen produced within keloids and the cytokines that dominate the disease. There have been considerably fewer studies in the expression of messenger RNA level in key cell cycle genes of the keloid fibroblast. The aim of this study was to measure the messenger RNA expression of the key regulators of cell cycle, cell cycle cyclins, and cyclin-dependent kinases, and their inhibitors.
Assuntos
Ciclo Celular , Fibroblastos/metabolismo , Queloide/patologia , RNA Mensageiro/metabolismo , Adolescente , Adulto , Estudos de Casos e Controles , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Despite the numerous scientific and technological advances made within the last decade the attrition rates for new drug discovery remain as high as 95% for anticancer drugs. Recent drug development has been in part guided by Lipinski's Rule of 5 (Ro5) even though many approved drugs do not comply to these rules. With Covid-19 vaccine development strategy dramatically accelerating drug development perhaps it is timely to question the generic drug development process itself to find a more efficient, cost effective, and successful approach. It is widely believed that drugs permeate cells via two methods: phospholipid bilayer diffusion and carrier mediated transporters. However, emerging evidence suggests that carrier mediated transport may be the primary mechanism of drug uptake and not diffusion as long believed. Computational biology increasingly assists drug design to achieve desirable absorption, distribution, metabolism, elimination and toxicity (ADMET) properties. Perfecting drug entry into target cells as a prerequisite to intracellular drug action is a logical and compelling route and is expected to reduce drug attrition rates, particularly gaining favour amongst chronic lifelong therapeutics. Novel drug development is rapidly expanding from the utilisation of beyond the rule of five (bRo5) to pulsatile drug delivery systems and fragment based drug design. Utilising transporters as drug targets and advocating bRo5 molecules may be the solution to increasing drug specificity, reducing dosage and toxicity and thus revolutionising drug development. This review explores the development of cell surface transporter exploitation in drug development and the relationship with improved therapeutic index.
RESUMO
The polymerase chain reaction has facilitated the ready analysis of nucleic acids. A next challenge requires the development of means to unravel the complexity of heterogeneous tissues. This has presented the task of producing massively parallelized quantitative nucleic acid data from the cellular constituents of tissues. The production of aqueous droplets in a two phase flow is shown to be readily and routinely facilitated by miniaturized fluidic devices. Droplets serve as ideal means to package a future generation of PCR, offering an enhanced handling potential by virtue of reactant containment, to concurrently eliminate both contamination and sample loss. This containment also enables the measurement of nucleic acids from populations of cells, or molecules by means of high throughput, single cell analysis. Details are provided for the production of a prototype micro-fluidic device which shows the production and stable flow of droplets which we suggest will be suitable for droplet-based continuous flow micro-fluidic PCR. Suggestions are also made as to the optimal fabrication techniques and the importance of device calibration.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Reação em Cadeia da Polimerase/métodos , Calibragem , Técnicas Analíticas Microfluídicas/métodos , Reação em Cadeia da Polimerase/instrumentação , TemperaturaRESUMO
Mapping the landscape of possible macromolecular polymer sequences to their fitness in performing biological functions is a challenge across the biosciences. A paradigm is the case of aptamers, nucleic acids that can be selected to bind particular target molecules. We have characterized the sequence-fitness landscape for aptamers binding allophycocyanin (APC) protein via a novel Closed Loop Aptameric Directed Evolution (CLADE) approach. In contrast to the conventional SELEX methodology, selection and mutation of aptamer sequences was carried out in silico, with explicit fitness assays for 44,131 aptamers of known sequence using DNA microarrays in vitro. We capture the landscape using a predictive machine learning model linking sequence features and function and validate this model using 5500 entirely separate test sequences, which give a very high observed versus predicted correlation of 0.87. This approach reveals a complex sequence-fitness mapping, and hypotheses for the physical basis of aptameric binding; it also enables rapid design of novel aptamers with desired binding properties. We demonstrate an extension to the approach by incorporating prior knowledge into CLADE, resulting in some of the tightest binding sequences.
Assuntos
Aptâmeros de Nucleotídeos/química , Evolução Molecular Direcionada/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Inteligência Artificial , Modelos Estatísticos , Ficocianina/metabolismo , Análise de Regressão , Análise de Sequência de DNARESUMO
Up until recently, it was believed that pharmaceutical drugs and their metabolites enter into the cell to gain access to their targets via simple diffusion across the hydrophobic lipid cellular membrane, at a rate which is based on their lipophilicity. An increasing amount of evidence indicates that the phospholipid bilayer-mediated drug diffusion is in fact negligible, and that drugs pass through cell membranes via proteinaceous membrane transporters or carriers which are normally used for the transportation of nutrients and intermediate metabolites. Drugs can be targeted to specific cells and tissues which express the relevant transporters, leading to the design of safe and efficacious treatments. Furthermore, transporter expression levels can be manipulated, systematically and in a high-throughput manner, allowing for considerable progress in determining which transporters are used by specific drugs. The ever-expanding field of miRNA therapeutics is not without its challenges, with the most notable one being the safe and effective delivery of the miRNA mimic/antagonist safely to the target cell cytoplasm for attaining the desired clinical outcome, particularly in miRNA-based cancer therapeutics, due to the poor efficiency of neo-vascular systems revolting around the tumour site, brought about by tumour-induced angiogenesis. This acquisition of resistance to several types of anticancer drugs can be as a result of an upregulation of efflux transporters expression, which eject drugs from cells, hence lowering drug efficacy, resulting in multidrug resistance. In this article, the latest available data on human microRNAs has been reviewed, together with the most recently described mechanisms for miRNA uptake in cells, for future therapeutic enhancements against cancer chemoresistance.
RESUMO
Alzheimer's disease (AD) is characterized by the presence of ß-amyloid plaques (Aß) and neurofibrillary tangles (NFTs) in the brain. The prevalence of the disease is increasing and is expected to reach 141 million cases by 2050. Despite the risk factors associated with the disease, there is no known causative agent for AD. Clinical trials with many drugs have failed over the years, and no therapeutic has been approved for AD. There is increasing evidence that pathogens are found in the brains of AD patients and controls, such as human herpes simplex virus-1 (HSV-1). Given the lack of a human model, the route for pathogen entry into the brain remains open for scrutiny and may include entry via a disturbed blood-brain barrier or the olfactory nasal route. Many factors can contribute to the pathogenicity of HSV-1, such as the ability of HSV-1 to remain latent, tau protein phosphorylation, increased accumulation of Aß invivo and in vitro, and repeated cycle of reactivation if immunocompromised. Intriguingly, valacyclovir, a widely used drug for the treatment of HSV-1 and HSV-2 infection, has shown patient improvement in cognition compared to controls in AD clinical studies. We discuss the potential role of HSV-1 in AD pathogenesis and argue for further studies to investigate this relationship.
Assuntos
Doença de Alzheimer , Herpes Simples , Herpesvirus Humano 1 , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides , Humanos , Emaranhados Neurofibrilares , Placa AmiloideRESUMO
Small hepatocellular carcinomas (HCCs) can be effectively cured by surgery with good clinical outcomes. However, the conventional AFP marker is ineffective in detecting small tumors. Here we employed a proteomic profiling approach to identify a candidate marker for HCC (Assuntos
Biomarcadores Tumorais/sangue
, Carcinoma Hepatocelular/sangue
, Neoplasias Hepáticas/sangue
, Proteômica/métodos
, Vimentina/sangue
, Adulto
, Idoso
, Área Sob a Curva
, Carcinoma Hepatocelular/diagnóstico
, Carcinoma Hepatocelular/patologia
, Linhagem Celular Tumoral
, Estudos de Coortes
, Diagnóstico Precoce
, Ensaio de Imunoadsorção Enzimática
, Feminino
, Humanos
, Neoplasias Hepáticas/diagnóstico
, Neoplasias Hepáticas/patologia
, Masculino
, Pessoa de Meia-Idade
, Sensibilidade e Especificidade
RESUMO
The absorbance at 260 nm (A(260)) is ubiquitously used for nucleic acid quantification. We show that following oxygenation, DNA solutions experience alterations in both spectral properties (hyperchromism in the UV region, lambda(max) 260 nm) and DNA conformation. The spectral changes caused by oxygen-DNA complexation are stable for at least several weeks at room temperature or several hours at 37 degrees C, but are also reversible by purging with nitrogen. Our data indicate that DNA in working solutions might already exist in the oxygen-complexed state, potentially confounding spectrophotometric analyses. Further, the presence of these complexes does not appear to impart cell toxicity in vitro or affect the biophysical functional behaviour (e.g. hybridisation) of DNA. Interestingly, our work also suggests that hybridisation could determine a release of bound oxygen, a phenomenon that could open the way to the use of such systems as oxygen carriers.
Assuntos
DNA/química , Oxigênio/química , Espectrofotometria/métodos , Animais , Bovinos , Linhagem Celular , Camundongos , Conformação de Ácido Nucleico , SalmãoRESUMO
BACKGROUND: Cidofovir is currently being used off-licence to treat different viral infections, such as benign low-risk human papillomavirus (HPV)-related recurrent respiratory papillomatosis (RRP). There are concerns over the safety of this practice as rat studies demonstrated a high malignant transformation rate. As yet, there are no clinical reports of cidofovir-induced malignant changes in humans. METHODS: Telomerase immortalised human keratinocytes (hTert) stably expressing E6 proteins from either low-risk HPV6b or high-risk HPV16 and vector control cells were treated with either low-dose (5 microg/ml) or higher dose (30 microg/ml) cidofovir for 2 days and the effects evaluated by clonogenic survival assays. Based on these results, gene expression microarray analysis was performed on cidofovir-treated low-risk E6 and vector cells before, during and after drug treatment, and the results verified by real-time PCR. RESULTS: Both low-risk and high-risk E6-expressing cells show significantly improved long-term survival compared with vector control cells when exposed to 5 microg/ml cidofovir for 2 days, (hTert T6E6 P=0.0007, hTert T16E6 P=0.00023 and hTert vector control P=0.62). Microarray and real-time PCR analyses of low-dose cidofovir-treated low-risk E6-expressing cells revealed changes in gene expression that are known to be associated with malignant progression, which were not observed in drug-treated vector control cells. CONCLUSIONS: This is the first report that cidofovir can both increase cell survival and induce alterations in gene expression that are known to be associated with malignant transformation in cells transduced only with the E6 gene from low-risk HPV. It is our belief that these data provide cause for concern over the off-license use of this drug to treat RRP.
Assuntos
Antivirais/efeitos adversos , Citosina/análogos & derivados , Organofosfonatos/efeitos adversos , Papiloma/tratamento farmacológico , Infecções por Papillomavirus/tratamento farmacológico , Neoplasias do Sistema Respiratório/tratamento farmacológico , Antivirais/administração & dosagem , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cidofovir , Citosina/administração & dosagem , Citosina/efeitos adversos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Uso Off-Label , Organofosfonatos/administração & dosagem , Papiloma/etiologia , Papiloma/metabolismo , Papillomaviridae/efeitos dos fármacos , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , RNA/biossíntese , Neoplasias do Sistema Respiratório/etiologia , Neoplasias do Sistema Respiratório/metabolismo , Fatores de RiscoRESUMO
PCR retains a pivotal role in making accessible marker nucleic acid sequences for ready analysis in cancer diagnosis. For certain cancers such as acute lymphoblastic leukaemia, the application of quantitative procedures to assess and subsequently direct therapy has given rise to the slowly maturing field of MRD (minimal residual disease) management. Although excellent protocols exist for performing these analyses, akin to all PCR procedures the limit of detection can vary markedly between laboratories. The present paper is an overview that describes how the analytical field relating to miniaturization is likely to identify the missing link that integrates sample processing with downstream PCR, analysis and eventual therapy. Miniaturized devices are suited to the multi-parallelized handling of defined numbers of cells, and PCR-based microfluidic procedures have become reasonably established. The integration of sample processing and PCR in microfluidic devices is beginning to offer reproducible quantitative data that relate the number of biomarker nucleic acids to the defined analysed cell or cells for meaningful clinical assessment. The application of MRD may, through integrated miniaturized PCR, become more reliable and routine with additional applications in defining disease threshold levels for other cancer types. These enabling integrated platforms may facilitate biomarker measurements to predict the response and outcome, which are also of current interest for personalized medical care.
Assuntos
Neoplasias/diagnóstico , Reação em Cadeia da Polimerase/métodos , Biomarcadores Tumorais/análise , Humanos , Técnicas Analíticas Microfluídicas , Neoplasias/genética , Ácidos Nucleicos/análiseRESUMO
Nucleic acids are routinely and readily analysed using the A(260)/A(280) ratio, although this method is known to be prone to erroneous results owing to contaminants in solution that absorb at similar wavelengths. The aim of the present review, while highlighting the problems and alternatives of using UV spectrophotometry for nucleic acid measurements, is to bring forth an observational result from our recent studies, namely that DO (dissolved oxygen) and nitrogen can alter the A(260) of aqueous DNA solutions. Our finding is of importance because DO is highly variable between protocols and storage conditions of DNA preparations. The physicochemical nature of the oxygen-DNA interactions is briefly discussed.
Assuntos
Ácidos Nucleicos/análise , Oxigênio/análise , Nitrogênio/análise , Soluções , Espectrofotometria Ultravioleta/métodosRESUMO
BACKGROUND: During the last decade, investigations have focused on revealing genes or proteins that are involved in HCC carcinogenesis using either genetic or proteomic techniques. However, these studies are overshadowed by a lack of good internal reference standards. The need to identify "housekeeping" markers, whose expression is stable in various experimental and clinical conditions, is therefore of the utmost clinical relevance in quantitative studies. This is the first study employed 2-DE analysis to screen for potential reference markers and aims to correlate the abundance of these proteins with their level of transcript expression. METHODS: A Chinese cohort of 224 liver tissues samples (105 cancerous, 103 non-tumourous cirrhotic, and 16 normal) was profiled using 2-DE analysis. Expression of the potential reference markers was confirmed by western blot, immunohistochemistry and real-time quantitative PCR. geNorm algorithm was employed for gene stability measure of the identified reference markers. RESULTS: The expression levels of three protein markers beta-actin (ACTB), heat shock protein 60 (HSP60), and protein disulphide isomerase (PDI) were found to be stable using p-values (p > 0.99) as a ranking tool in all 224 human liver tissues examined by 2-DE analysis. Of high importance, ACTB and HSP 60 were successfully validated at both protein and mRNA levels in human hepatic tissues by western blot, immunohistochemistry and real-time quantitative PCR. In addition, no significant correlation of these markers with any clinicopathological features of HCC and cirrhosis was found. Gene stability measure of these two markers with other conventionally applied housekeeping genes was assessed by the geNorm algorithm, which ranked ACTB and HSP60 as the most stable genes among this cohort of clinical samples. CONCLUSION: Our findings identified 2 reference markers that exhibited stable expression across human liver tissues with different conditions thus should be regarded as reliable reference moieties for normalisation of gene and protein expression in clinical research employing human hepatic tissues.
Assuntos
Actinas/metabolismo , Carcinoma Hepatocelular/metabolismo , Chaperonina 60/metabolismo , Eletroforese em Gel Bidimensional/normas , Neoplasias Hepáticas/metabolismo , Proteômica/normas , Actinas/genética , Carcinoma Hepatocelular/genética , Chaperonina 60/genética , Estudos de Coortes , Eletroforese em Gel Bidimensional/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Proteômica/métodos , Padrões de ReferênciaRESUMO
Closed loop aptameric directed evolution, (CLADE) is a technique enabling simultaneous discovery, evolution, and optimization of aptamers. It was previously demonstrated using a fluorescent protein, and here we extend its applicability with the generation of surface-bound aptamers for targets containing no natural fluorescence. Starting from a random population, in four generations CLADE produced a new aptamer to thrombin with high specificity and affinity. The best aptameric sequence was void of the set of four guanine repeats typifying thrombin aptamers and, thus, highlights the benefits of evolution performed in an environment closely mimicking the final diagnostic application.
Assuntos
Aptâmeros de Nucleotídeos/síntese química , Análise Serial de Proteínas/métodos , Técnica de Seleção de Aptâmeros/métodos , Ressonância de Plasmônio de Superfície/métodos , Trombina/análise , Aptâmeros de Nucleotídeos/metabolismo , Humanos , Ligação Proteica , Trombina/metabolismoRESUMO
The relationship between radiographic measures of severity of osteoarthritis (OA) and changes in gene expression in joints are not well characterised. In this study, the expression of 11 candidate genes was characterised by quantitative reverse transcriptase polymerase chain reaction in normal and OA cartilage and bone from the elbows of dogs with fragmented coronoid disease. The levels of expression of type I collagen alpha2 chain (COL1A2), type III collagen alpha1 chain (COL3A1), lumican (LUM), matrix metalloproteinase-2 (MMP2), -9 (MMP9) and -13 (MMP13) genes were increased and the expression of tissue inhibitor of metalloproteinase-2 (TIMP2) and cathepsin D (CTSD) genes were decreased in OA cartilage relative to normal cartilage. All differences correlated with radiographic measures of severity of OA. Levels of expression of COL1A2, MMP2, MMP9, MMP13 and TIMP1 were increased, whereas expression of TIMP2 was decreased in OA bone relative to normal bone. Cartilage gene expression may be correlated with the radiographic severity of OA.
Assuntos
Cartilagem Articular/metabolismo , Colágeno/genética , Doenças do Cão/diagnóstico por imagem , Doenças do Cão/genética , Osteoartrite/veterinária , RNA Mensageiro/análise , Animais , Sequência de Bases , Colágeno/metabolismo , Doenças do Cão/metabolismo , Doenças do Cão/patologia , Cães , Expressão Gênica , Perfilação da Expressão Gênica/veterinária , Metaloproteases/biossíntese , Metaloproteases/genética , Osteoartrite/diagnóstico por imagem , Osteoartrite/genética , Osteoartrite/metabolismo , Proteoglicanas/biossíntese , Proteoglicanas/genética , Radiografia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Índice de Gravidade de DoençaRESUMO
The prevailing approach to cellular molecular analyte investigations employs lysis. Using analogies with automobiles, we explain how current practise ridicules cellular individuality and meaningful variation. Single cell analysis and micro total analysis system (microTAS) prospects are discussed.