RESUMO
The accurate detection and genotyping of high-risk human papillomavirus (HR-HPV) are critical for cervical cancer screening and epidemiological investigations. GeneFirst Papilloplex® HR-HPV is a new CE-IVD-marked real-time PCR test based on patented multiplex probe amplification technology. Papilloplex® HR-HPV provides the simultaneous detection and differentiation of 14 HR-HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68a/b) in a single closed-tube reaction ensuring rapid, cost-effective, and contamination-free results. In this study, the analytical performance characteristics in terms of the assay's sensitivity, specificity, range, reproducibility, and cross-reactivity were evaluated. Papilloplex® HR-HPV provided sensitive detection and differentiation of 14 HR-HPV types with highly reproducible results. The differential HR-HPV specificity and sensitivity were further confirmed through the participation in the WHO HPV Laboratory Network Proficiency Study (2014). Overall, GeneFirst Papilloplex® HR-HPV assay demonstrated a robust analytical performance with reproducible and reliable results in the detection of HR-HPV genotypes.
Assuntos
Técnicas de Diagnóstico Molecular/métodos , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , DNA Viral/genética , Detecção Precoce de Câncer , Feminino , Genótipo , Humanos , Infecções por Papillomavirus/virologia , Kit de Reagentes para Diagnóstico/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Skeletal muscle is a major site of metabolic activity and is the most abundant tissue in the human body. Age-related muscle atrophy (sarcopenia) and weakness, characterized by progressive loss of lean muscle mass and function, is a major contributor to morbidity and has a profound effect on the quality of life of older people. With a continuously growing older population (estimated 2 billion of people aged >60 by 2050), demand for medical and social care due to functional deficits, associated with neuromuscular ageing, will inevitably increase. Despite the importance of this 'epidemic' problem, the primary biochemical and molecular mechanisms underlying age-related deficits in neuromuscular integrity and function have not been fully determined. Skeletal muscle generates reactive oxygen and nitrogen species (RONS) from a variety of subcellular sources, and age-associated oxidative damage has been suggested to be a major factor contributing to the initiation and progression of muscle atrophy inherent with ageing. RONS can modulate a variety of intracellular signal transduction processes, and disruption of these events over time due to altered redox control has been proposed as an underlying mechanism of ageing. The role of oxidants in ageing has been extensively examined in different model organisms that have undergone genetic manipulations with inconsistent findings. Transgenic and knockout rodent studies have provided insight into the function of RONS regulatory systems in neuromuscular ageing. This review summarizes almost 30 years of research in the field of redox homeostasis and muscle ageing, providing a detailed discussion of the experimental approaches that have been undertaken in murine models to examine the role of redox regulation in age-related muscle atrophy and weakness.
Assuntos
Envelhecimento/metabolismo , Homeostase , Músculo Esquelético/fisiologia , Oxirredução , Fatores Etários , Animais , Animais Geneticamente Modificados , Antioxidantes/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Mitocôndrias/metabolismo , Modelos Animais , Neurônios Motores/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Junção Neuromuscular/metabolismo , Tamanho do Órgão , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). We show that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function.
Assuntos
Axônios/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Retículo Endoplasmático/metabolismo , Proteínas de Membrana Transportadoras/genética , Paraplegia Espástica Hereditária/genética , Animais , Transporte Axonal , Axônios/ultraestrutura , Modelos Animais de Doenças , Proteínas de Drosophila/deficiência , Drosophila melanogaster/classificação , Drosophila melanogaster/citologia , Drosophila melanogaster/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Expressão Gênica , Humanos , Larva/citologia , Larva/genética , Larva/metabolismo , Larva/ultraestrutura , Proteínas de Membrana Transportadoras/deficiência , Mutação , Filogenia , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Paraplegia Espástica Hereditária/metabolismo , Paraplegia Espástica Hereditária/patologiaRESUMO
OBJECTIVES: Williams-Beuren Syndrome (WBS) is a multisystem disorder caused by the deletion of contiguous genes on chromosome 7q11.23. Ophthalmologic abnormalities and deficits in visual motor integration are important features of WBS. Here we describe our experience with Brazilian WBS patients and their ophthalmologic features. METHODS: Sixteen patients with confirmed WBS went through thorough ophthalmologic examination. RESULTS: The most frequent ocular findings in our group of patients were stellate iris pattern (81.2%), hyperopic astigmatism (50%), hyperopia (37.5%), tortuosity of retinal vessel (37.5%) and strabismus (18.7%). CONCLUSIONS: This is the second report of ophthalmologic abnormalities in a group of Brazilian individuals with WBS. It is extremely valuable that specific populations are studied so that clinical diagnosis can be refined and management of patients can be driven to the most common presentations of the disease.
Assuntos
Oftalmopatias/diagnóstico , Síndrome de Williams/diagnóstico , Adolescente , Adulto , Astigmatismo/diagnóstico , Brasil/epidemiologia , Criança , Pré-Escolar , Elastina/genética , Oftalmopatias/epidemiologia , Oftalmopatias/genética , Feminino , Humanos , Hiperopia/diagnóstico , Hibridização in Situ Fluorescente , Doenças da Íris/diagnóstico , Quinases Lim/genética , Perda de Heterozigosidade , Masculino , Repetições de Microssatélites , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Vasos Retinianos/patologia , Estrabismo/diagnóstico , Síndrome de Williams/epidemiologia , Síndrome de Williams/genéticaRESUMO
Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5-0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH), which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR) assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36: PRKCZ and SKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescent in situ hybridization (FISH), and/or multiplex ligation-dependent probe amplification (MLPA) all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR of PRKCZ and SKI is a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 1/genética , Reação em Cadeia da Polimerase em Tempo Real/economia , Análise Custo-Benefício , Feminino , Marcadores Genéticos , Humanos , Masculino , Técnicas de Diagnóstico Molecular/economia , Monossomia/diagnósticoRESUMO
Detection of human microdeletion and microduplication syndromes poses significant burden on public healthcare systems in developing countries. With genome-wide diagnostic assays frequently inaccessible, targeted low-cost PCR-based approaches are preferred. However, their reproducibility depends on equally efficient amplification using a number of target and control primers. To address this, the recently described technique called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR) was shown to reliably detect four human syndromes by quantifying DNA amplification in an internally controlled PCR reaction. Here, we confirm its utility in the detection of eight human microdeletion syndromes, including the more common WAGR, Smith-Magenis, and Potocki-Lupski syndromes with 100% sensitivity and 100% specificity. We present selection, design, and performance evaluation of detection primers using variety of approaches. We conclude that MQF-PCR is an easily adaptable method for detection of human pathological chromosomal aberrations.
Assuntos
Transtornos Cromossômicos/diagnóstico , Variações do Número de Cópias de DNA , Reação em Cadeia da Polimerase/economia , Sequência de Bases , Estudos de Casos e Controles , Aberrações Cromossômicas , Transtornos Cromossômicos/genética , Primers do DNA/genética , Feminino , Testes Genéticos/economia , Genoma Humano , Humanos , Masculino , Técnicas de Diagnóstico Molecular/economia , Curva ROCRESUMO
Because of economic limitations, the cost-effective diagnosis of patients affected with rare microdeletion or microduplication syndromes is a challenge in developing countries. Here we report a sensitive, rapid, and affordable detection method that we have called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR). Our procedure is based on the finding of genomic regions with high homology to segments of the critical microdeletion/microduplication region. PCR amplification of both using the same primer pair, establishes competitive kinetics and relative quantification of amplicons, as happens in microsatellite-based Quantitative Fluorescence PCR. We used patients with two common microdeletion syndromes, the Williams-Beuren syndrome (7q11.23 microdeletion) and the 22q11.2 microdeletion syndromes and discovered that MQF-PCR could detect both with 100% sensitivity and 100% specificity. Additionally, we demonstrated that the same principle could be reliably used for detection of microduplication syndromes, by using patients with the Lubs (MECP2 duplication) syndrome and the 17q11.2 microduplication involving the NF1 gene. We propose that MQF-PCR is a useful procedure for laboratory confirmation of the clinical diagnosis of microdeletion/microduplication syndromes, ideally suited for use in developing countries, but having general applicability as well.
Assuntos
Síndrome de Resistência a Andrógenos/diagnóstico , Deleção Cromossômica , Duplicação Cromossômica/genética , Síndrome de DiGeorge/diagnóstico , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/métodos , Síndrome de Williams/diagnóstico , Síndrome de Resistência a Andrógenos/genética , Síndrome de DiGeorge/genética , Fluorescência , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Síndrome de Williams/genéticaRESUMO
BACKGROUND: Williams-Beuren syndrome is a multisystemic genetic disorder caused by a contiguous gene deletion at 7q11.23. Keratoconus is a complex disease and it is suspected to have a genetic origin, although the specific gene responsible for keratoconus has not been identified. Although there are several ocular features in Williams-Beuren syndrome, keratoconus is not regularly described as part of this syndrome. PURPOSE: To report a new patient with keratoconus and Williams-Beuren syndrome. DISCUSSION: This is the third case of an association between Williams-Beuren syndrome and keratoconus. The authors believe that the Williams-Beuren syndrome chromosome region can be a possible target for further investigation as the genetic basis of keratoconus.
Assuntos
Cromossomos Humanos Par 7/genética , Elastina/genética , Deleção de Genes , Ceratocone/genética , Quinases Lim/genética , Síndrome de Williams/genética , Adulto , Topografia da Córnea , Éxons/genética , Humanos , Ceratocone/diagnóstico , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Síndrome de Williams/diagnósticoRESUMO
Leukocyte-like cells called hemocytes have key functions in Drosophila innate immunity. Three hemocyte types occur: plasmatocytes, crystal cells, and lamellocytes. In the absence of qimmune challenge, plasmatocytes are the predominant hemocyte type detected, while crystal cells and lamellocytes are rare. However, upon infestation by parasitic wasps, or in melanotic mutant strains, large numbers of lamellocytes differentiate and encapsulate material recognized as "non-self". Current models speculate that lamellocytes, plasmatocytes and crystal cells are distinct lineages that arise from a common prohemocyte progenitor. We show here that over-expression of the CoREST-interacting transcription factor Chn in plasmatocytes induces lamellocyte differentiation, both in circulation and in lymph glands. Lamellocyte increases are accompanied by the extinction of plasmatocyte markers suggesting that plasmatocytes are transformed into lamellocytes. Consistent with this, timed induction of Chn over-expression induces rapid lamellocyte differentiation within 18 hours. We detect double-positive intermediates between plasmatocytes and lamellocytes, and show that isolated plasmatocytes can be triggered to differentiate into lamellocytes in vitro, either in response to Chn over-expression, or following activation of the JAK/STAT pathway. Finally, we have marked plasmatocytes and show by lineage tracing that these differentiate into lamellocytes in response to the Drosophila parasite model Leptopilina boulardi. Taken together, our data suggest that lamellocytes arise from plasmatocytes and that plasmatocytes may be inherently plastic, possessing the ability to differentiate further into lamellocytes upon appropriate challenge.
Assuntos
Linhagem da Célula , Drosophila melanogaster/genética , Hemócitos/metabolismo , Macrófagos/metabolismo , Animais , Diferenciação Celular/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/parasitologia , Feminino , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hemócitos/citologia , Interações Hospedeiro-Parasita , Janus Quinases/genética , Janus Quinases/metabolismo , Larva/genética , Larva/metabolismo , Larva/parasitologia , Macrófagos/citologia , Masculino , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Vespas/fisiologiaRESUMO
In Drosophila, defense against foreign pathogens is mediated by an effective innate immune system, the cellular arm of which is composed of circulating hemocytes that engulf bacteria and encapsulate larger foreign particles. Three hemocyte types occur: plasmatocytes, crystal cells, and lamellocytes. The most abundant larval hemocyte type is the plasmatocyte, which is responsible for phagocytosis and is present either in circulation or in adherent sessile domains under the larval cuticle. The mechanisms controlling differentiation of plasmatocytes and their migration toward these sessile compartments are unclear. To address these questions we have conducted a misexpression screen using the plasmatocyte-expressed GAL4 driver Peroxidasin-GAL4 (Pxn-GAL4) and existing enhancer-promoter (EP) and EP yellow (EY) transposon libraries to systematically misexpress approximately 20% of Drosophila genes in larval hemocytes. The Pxn-GAL4 strain also contains a UAS-GFP reporter enabling hemocyte phenotypes to be visualized in the semitransparent larvae. Among 3412 insertions screened we uncovered 101 candidate hemocyte regulators. Some of these are known to control hemocyte development, but the majority either have no characterized function or are proteins of known function not previously implicated in hemocyte development. We have further analyzed three candidate genes for changes in hemocyte morphology, cell-cell adhesion properties, phagocytosis activity, and melanotic tumor formation.