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Background: Drug resistance mutations (DRMs) increasingly jeopardize paediatric HIV programmes in sub-Saharan Africa. As individual monitoring of DRMs and viral loads has limited availability, population data on DRMs are essential to determine first-line susceptibility. Paediatric data from sub-Saharan Africa are scarce and unavailable for Malawi. Objectives: To determine the prevalence of virological failure (VF) and DRMs among ART-naive HIV-infected Malawian children during the first year of first-line ART. Methods: In a prospective cohort of HIV-infected Malawian children, on first-line treatment, children were followed monthly; blood was collected for viral load testing (6 and 12 months) and genotypic resistance testing (12 months). VF was defined as at least one viral load >1000 copies/mL or death after 6 months of ART. DRMs were identified and susceptibility to NRTIs and NNRTIs was scored using the Stanford algorithm and by calculating genotypic susceptibility scores (GSSs). Results: VF occurred in 66% (23/35) of the children during 12 months of follow-up. DRMs were detected in 44% (15/34); all had NNRTI resistance and 12% (4/34) had dual-class NNRTI/NRTI resistance. Reduced susceptibility (DRMs and GSS <3) was seen in 41% (14/34) to their current first-line regimen. High-level resistance was most common for nevirapine [26% (9/34)]. Conclusions: In this first report on VF and DRMs in children on first-line ART in Malawi, the rates of VF and DRMs were alarmingly high. Paediatric HIV programmes in sub-Saharan Africa should emphasize programmatic evaluation of VF and include detection of DRMs to adjust and design adequate first- and second-line regimens and prevent widespread resistance in children.
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Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , HIV/efeitos dos fármacos , Mutação , Adolescente , Criança , Pré-Escolar , Feminino , Seguimentos , Genótipo , Técnicas de Genotipagem , HIV/genética , HIV/isolamento & purificação , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , Humanos , Lactente , Malaui/epidemiologia , Masculino , Prevalência , Estudos Prospectivos , Análise de Sequência de DNA , Falha de Tratamento , Carga ViralRESUMO
Reproductive technologies such as multiple ovulation and embryo transfer (MOET) and ovum pick-up (OPU) accelerate genetic improvement in dairy breeding schemes. To enhance the efficiency of embryo production, breeding values for traits such as number of oocytes (NoO) and number of MOET embryos (NoM) can help in selection of donors with high MOET or OPU efficiency. The aim of this study was therefore to estimate variance components and (genomic) breeding values for NoO and NoM based on Dutch Holstein data. Furthermore, a 10-fold cross-validation was carried out to assess the accuracy of pedigree and genomic breeding values for NoO and NoM. For NoO, 40,734 OPU sessions between 1993 and 2015 were analyzed. These OPU sessions originated from 2,543 donors, from which 1,144 were genotyped. For NoM, 35,695 sessions between 1994 and 2015 were analyzed. These MOET sessions originated from 13,868 donors, from which 3,716 were genotyped. Analyses were done using only pedigree information and using a single-step genomic BLUP (ssGBLUP) approach combining genomic information and pedigree information. Heritabilities were very similar based on pedigree information or based on ssGBLUP [i.e., 0.32 (standard error = 0.03) for NoO and 0.21 (standard error = 0.01) for NoM with pedigree, 0.31 (standard error = 0.03) for NoO, and 0.22 (standard error = 0.01) for NoM with ssGBLUP]. For animals without their own information as mimicked in the cross-validation, the accuracy of pedigree-based breeding values was 0.46 for NoO and NoM. The accuracies of genomic breeding values from ssGBLUP were 0.54 for NoO and 0.52 for NoM. These results show that including genomic information increases the accuracies. These moderate accuracies in combination with a large genetic variance show good opportunities for selection of potential bull dams.
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Cruzamento/métodos , Transferência Embrionária/veterinária , Oócitos/citologia , Linhagem , Seleção Genética , Animais , Bovinos , Contagem de Células/veterinária , Transferência Embrionária/estatística & dados numéricos , Feminino , Genoma , Genômica , Genótipo , Masculino , Modelos GenéticosRESUMO
Introduction: Determining whether a neurovascular conflict (NVC) involving the anterior visual pathway (AVP) and a non-diseased intracranial artery is amenable for microvascular decompression is challenging. Moreover, it is unclear whether microvascular decompression of the optic nerve is an effective therapy. Research question: What are the outcomes of different treatment strategies for NVCs involving the AVP and a non-diseased intracranial artery? Material and methods: Data on patients with symptomatic NVCs involving the AVP and a non-diseased intracranial artery was collected and included treatment and outcome parameters. The case series was drafted in accordance with the CARE guidelines. Results: Three patients aged 53,53 and 55 visited our out-patient clinic with a suspected symptomatic NVC between the optic nerve and a non-diseased intracranial artery. A conservative treatment was opted for in the first patient aimed at treating her glaucoma, with temporary improvement of symptoms. Microvascular decompression of the optic nerve was performed in two patients. One operated patient developed post-operative complications resulting in posterior circulation perfusion decline, while the other experienced a worse tunnel vision with a decrease in visual acuity. Discussion and conclusion: The diagnosis of a symptomatic NVC between the AVP and a non-diseased intracranial artery should be considered with caution, i.e. after exclusion of all other causes. Microvascular decompression can be performed but does not necessarily improve symptoms. A better understanding of the pathophysiological mechanisms underlying these NVCs is warranted to determine the benefit of microvascular decompression of the optic nerve.
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Up to now, only a few brief procedures for purifying white spot syndrome virus (WSSV) have been described. They were mainly based on sucrose, NaBr and CsCl density gradient centrifugation. This work describes for the first time the purification of WSSV through iodixanol density gradients, using virus isolated from infected tissues and haemolymph of Penaeus vannamei (Boone). The purification from tissues included a concentration step by centrifugation (2.5 h at 60,000 g) onto a 50% iodixanol cushion and a purification step by centrifugation (3 h at 80,000 g) through a discontinuous iodixanol gradient (phosphate-buffered saline, 5%, 10%, 15% and 20%). The purification from infected haemolymph enclosed a dialysis step with a membrane of 1,000 kDa (18 h) and a purification step through the earlier iodixanol gradient. The gradients were collected in fractions and analysed. The number of particles, infectivity titre (in vivo), total protein and viral protein content were evaluated. The purification from infected tissues gave WSSV suspensions with a very high infectivity and an acceptable purity, while virus purified from haemolymph had a high infectivity and a very high purity. Additionally, it was observed that WSSV has an unusually low buoyant density and that it is very sensitive to high external pressures.
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Centrifugação com Gradiente de Concentração , Penaeidae/virologia , Ácidos Tri-Iodobenzoicos/química , Vírus da Síndrome da Mancha Branca 1/isolamento & purificação , Animais , Hemolinfa/virologia , Carga Viral , Proteínas Virais/análise , Vírus da Síndrome da Mancha Branca 1/fisiologiaRESUMO
As adhesion and translocation through fish gut enterocytes of the pathogen Vibrio (Listonella) anguillarum are not well investigated, the effective cause of disease and mortality outbreaks in larval sea bass, Dicentrarchus labrax, suffering from vibriosis is unknown. We detected V. anguillarum within the gut of experimentally infected gnotobiotic sea bass larvae using transmission electron microscopy and immunogold labelling. Intact bacteria were observed in close contact with the apical brush border in the gut lumen. Enterocytes contained lysosomes positive for protein A-gold particles suggesting intracellular elimination of bacterial fragments. Shed intestinal cells were regularly visualized in the gut lumen in late stages of exposure. Some of the luminal cells showed invagination and putative engulfment of bacterial structures by pseudopod-like formations. The engulfed structures were positive for protein A-colloidal gold indicating that these structures were V. anguillarum. Immunogold positive thread-like structures secreted by V. anguillarum suggested the presence of outer membrane vesicles (MVs) hypothesizing that MVs are potent transporters of active virulence factors to sea bass gut cells suggestive for a substantial role in biofilm formation and pathogenesis. We put forward the hypothesis that MVs are important in the pathogenesis of V. anguillarum in sea bass larvae.
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Bass/imunologia , Enterócitos/fisiologia , Doenças dos Peixes/imunologia , Intestinos/citologia , Intestinos/microbiologia , Listonella/classificação , Animais , Vida Livre de Germes , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Larva/imunologia , Listonella/fisiologiaRESUMO
Crop improvement is a key innovation area in the pursuit of sustainable food systems. However, realising its potential requires integration of the needs and priorities of all agri-food chain stakeholders. In this study, we provide a multi-stakeholder perspective on the role of crop improvement in future-proofing the European food system. We engaged agri-business, farm- and consumer-level stakeholders, and plant scientists through an online survey and focus groups. Four of each group's top five priorities were shared and related to environmental sustainability goals (water, nitrogen and phosphorus efficiency, and heat stress). Consensus was identified on issues including considering existing alternatives to plant breeding (e.g. management strategies), minimising trade-offs, and addressing geographical variation in needs. We conducted a rapid evidence synthesis on the impacts of priority crop improvement options, highlighting the urgent need for further research examining downstream sustainability impacts to identify concrete targets for plant breeding innovation as a food systems solution.
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Melhoramento Vegetal , Grupos Focais , FazendasRESUMO
BACKGROUND: Persistent subretinal fluid after rhegmatogenous retinal detachment (RRD) surgery is responsible for delayed recovery, and may affect the final visual outcome. Cause, consequences, and treatment remain elusive. DESIGN: Literature review and case series. METHODS: We reviewed the pathophysiological principles and therapeutic options from the literature, and we report the results from a subretinal fluid cytology study. Nine eyes from nine patients with macula-involving RRD underwent surgical repair. The cellular content of subretinal fluid (SRF) was studied by electron microscopy and anti-rhodopsin immunostaining. All eyes were assessed postoperatively with optical coherence tomography for the detection of persistent submacular fluid (PSF) (Ethics Committee Ghent University Hospital, registration number B6702006169). RESULTS: Certain patient characteristics as well as surgical methods were implicated. PSF appears to occur more frequently in patients with longstanding detachments treated with buckling surgery. Several therapeutic options have been suggested but safety and efficacy remain unclear. We found PSF in three eyes on postoperative OCT scans, which corresponded to the three cell-rich subretinal samples. CONCLUSIONS: PSF after successful RRD repair seems to be related to fluid composition. We hypothesize, in the absence of an effective treatment, that a modified surgical drainage, including a washout of the subretinal space, could evacuate the subretinal fluid more completely, and may prevent this complication.
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Complicações Pós-Operatórias , Descolamento Retiniano/cirurgia , Segmento Externo da Célula Bastonete/ultraestrutura , Líquido Sub-Retiniano/citologia , Idoso , Idoso de 80 Anos ou mais , Drenagem , Feminino , Humanos , Imuno-Histoquímica , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Descolamento Retiniano/fisiopatologia , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Recurvamento da Esclera , Tomografia de Coerência Óptica , Acuidade Visual/fisiologia , VitrectomiaRESUMO
The location and cell damage caused by Vibrio anguillarum, the causative agent of classical vibriosis, within the developing gut of the newly hatched sea bass, Dicentrarchus labrax (L.), is unknown. A gnotobiotic sea bass model was used to investigate the early interactions of V. anguillarum with sea bass larvae. In the present study, germ-free sea bass larvae were orally exposed to a V. anguillarum HI-610 pathogen labelled with the green fluorescent protein (GFP-HI-610) and sampled at regular intervals. Pathogenic colonization of gut enterocytes was observed 2 h post-exposure (p.e.) and onwards, whereas bacteria within the swim bladder were visualized 48 h p.e and onwards. Ultrastructural findings demonstrated direct bacterial contact with the host cell in the oesophageal mucosa and putative attachment to microvilli of mid- and hindgut enterocytes. The present findings form a starting point for studies assessing the impact of potential candidates (probiotics, prebiotics, antimicrobial peptides) to mitigate bacterial virulence.
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Bass/microbiologia , Doenças dos Peixes/microbiologia , Vida Livre de Germes , Interações Hospedeiro-Patógeno , Vibrioses/veterinária , Vibrio/fisiologia , Sacos Aéreos/microbiologia , Animais , Enterócitos/microbiologia , Esôfago/microbiologia , Proteínas de Fluorescência Verde , Larva/microbiologia , Vibrio/patogenicidade , Vibrioses/microbiologiaRESUMO
Background: Blood flow restriction (BFR) training uses a cuff to partially occlude venous blood flow and improve musculoskeletal training outcomes. Over the past 25 years, numerous studies have demonstrated its relative safety and efficacy. Objectives: Blood flow restriction training is under review by the Health Professions Council of South Africa due to safety and ethical concerns. The objective of this roundtable discussion is to gain better insight into the current use and perception of blood flow restriction training in South Africa. Formation of panel: The expert panel had experience with the use of BFR training and included one representative from each of the following professions, namely, sports medicine, physiotherapy and biokinetics. Discussion: The panellists provided their unique perspectives on BFR training, whilst reaching a relative consensus on its safety, screening, efficacy, and appropriate use. Agreement on appropriate loading and occlusion pressure protocols during different phases of rehabilitation was less clear. Conclusion: Although BFR is a safe and effective modality, the development of evidence-based protocols among different health professionals in South Africa is required to ensure good clinical practice.
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Background: The online intervention Runfitcheck was developed to stimulate injury-preventive behaviour among adult novice runners. Objectives: This study evaluated the effectiveness of Runfitcheck on injury-preventive behaviour among adult novice runners. Methods: A randomised controlled trial was conducted among adult novice runners. The intervention group had access to the Runfitcheck intervention, the control group performed their running activities as usual. One, three, and five months after enrolment, participants reported retrospectively what they had done regarding injury-preventive behaviour (operationalised as (i) using a (personalised) training schedule; (ii) performing strength and technique exercises; and (iii) performing a warm-up routine prior to running). Relative Risks (RR) and 95% Confidence Interval (95%CI) were used to analyse behavioural change. Results: The intervention group (n=715) searched more often for information about a warm-up routine (RR 1.211; 95%CI 1.080-1.357), and added more often strength exercises to their warm-up routine (RR 1.228; 95%CI 1.092-1.380). The intervention group performed more often running technique exercises compared to the control group (n=696) (RR 1.134; 95%CI 1.015-1.267), but less often strength exercises (RR 0.865 (95%CI 0.752-0.995). Within the group of runners that did not perform any warm-up routine at enrolment (n=272), the intervention group started to perform a regular warm-up routine more often than the control group (RR 1.461; 95%CI 1.084-1.968). No significant results were found for using a training schedule. Conclusion: The online intervention Runfitcheck was effective in stimulating aspects of injury-preventive behaviour in adult novice runners, mostly related to a warm-up routine. Trial registration: NL6225, Registered April 24th 2007 - Retrospectively registered, https://www.trialregister.nl/trial/6225.
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Methods currently used to restore bone defects in human and veterinary orthopaedics are often not satisfactory. This is especially the case in the healing of large, irregular defects which result in the formation of tissues with inferior qualities compared to the original structures. For these reasons, several new approaches are currently being explored to improve bone healing capacities in different situations. This review will examine the different techniques used to enhance bone regeneration, highlighting both experimental and clinically applicable methods with regard to veterinary orthopaedics.
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Fraturas Ósseas/metabolismo , Fraturas Ósseas/terapia , Artropatias/veterinária , Ortopedia/veterinária , Regeneração/fisiologia , Animais , Substitutos Ósseos , Artropatias/terapia , Ortopedia/tendênciasRESUMO
The alpha-catenin molecule links E-cadherin/ beta-catenin or E-cadherin/plakoglobin complexes to the actin cytoskeleton. We studied several invasive human colon carcinoma cell lines lacking alpha-catenin. They showed a solitary and rounded morphotype that correlated with increased invasiveness. These round cell variants acquired a more normal epithelial phenotype upon transfection with an alpha-catenin expression plasmid, but also upon treatment with the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Video registrations showed that the cells started to establish elaborated intercellular junctions within 30 min after addition of TPA. Interestingly, this normalizing TPA effect was not associated with alpha-catenin induction. Classical and confocal immunofluorescence showed only minor TPA-induced changes in E-cadherin staining. In contrast, desmosomal and tight junctional proteins were dramatically rearranged, with a conversion from cytoplasmic clusters to obvious concentration at cell-cell contacts and exposition at the exterior cell surface. Electron microscopical observations revealed the TPA-induced appearance of typical desmosomal plaques. TPA-restored cell-cell adhesion was E-cadherin dependent as demonstrated by a blocking antibody in a cell aggregation assay. Addition of an antibody against the extracellular part of desmoglein-2 blocked the TPA effect, too. Remarkably, the combination of anti-E-cadherin and anti-desmoglein antibodies synergistically inhibited the TPA effect. Our studies show that it is possible to bypass the need for normal alpha-catenin expression to establish tight intercellular adhesion by epithelial cells. Apparently, the underlying mechanism comprises upregulation of desmosomes and tight junctions by activation of the PKC signaling pathway, whereas E-cadherin remains essential for basic cell-cell adhesion, even in the absence of alpha-catenin.
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Proteínas do Citoesqueleto/deficiência , Desmossomos/química , Desmossomos/enzimologia , Proteína Quinase C/metabolismo , Transativadores , Antígenos de Superfície/metabolismo , Caderinas/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Tamanho Celular/fisiologia , Neoplasias do Colo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , DNA Complementar , Detergentes , Humanos , Microscopia Eletrônica , Solubilidade , Acetato de Tetradecanoilforbol/farmacologia , Junções Íntimas/química , Junções Íntimas/enzimologia , Transfecção , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/enzimologia , Células Tumorais Cultivadas/ultraestrutura , alfa Catenina , beta CateninaRESUMO
Piglet mortality has a negative impact on animal welfare and public acceptance. Moreover, the number of weaned piglets per sow mainly determines the profitability of piglet production. Increased litter sizes are associated with lower birth weights and piglet survival. Decreased survival rates and performance of piglets make the control of diseases and infections within pig production even more crucial. Consequently, selection for immunocompetence becomes an important key aspect within modern breeding programmes. However, the phenotypic recording of immune traits is difficult and expensive to realize within farm routines. Even though immune traits show genetic variability, only few examples exist on their respective suitability within a breeding programme and their relationships to economically important production traits. The analysis of immune traits for an evaluation of immunocompetence to gain a generally improved immune response is promising. Generally, in-depth knowledge of the genetic background of the immune system is needed to gain helpful insights about its possible incorporation into breeding programmes. Possible physiological drawbacks for enhanced immunocompetence must be considered with regards to the allocation theory and possible trade-offs between the immune system and performance. This review aims to discuss the relationships between the immunocompetence of the pig, piglet survival as well as the potential of these traits to be included into a breeding strategy for improved robustness.
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Bem-Estar do Animal , Imunocompetência , Suínos/fisiologia , Animais , Animais Recém-Nascidos , Cruzamento , Feminino , Tamanho da Ninhada de Vivíparos , Mortalidade , Fenótipo , Suínos/genética , Suínos/imunologia , DesmameRESUMO
The mammalian innate immune system senses viral infection by recognizing viral signatures and activates potent antiviral responses. Besides the interferon (IFN) response, there is accumulating evidence that RNA silencing or RNA interference (RNAi) serves as an antiviral mechanism in mammalian cells. Mammalian viruses encode IFN antagonists to counteract the IFN response in infected cells. A number of IFN antagonists are also capable of blocking RNAi in infected cells and therefore serve as RNA-silencing suppressors. Virus replication in infected cells is restricted by these innate antiviral mechanisms, which may kick in earlier than the viral antagonistic or suppressor protein can accumulate. The yield of virus vaccines and viral gene delivery vectors produced in mammalian producer cells may therefore be suboptimal. To investigate whether blocking of the innate antiviral responses in mammalian cells leads to increased viral vector production, we expressed a number of immunity suppressors derived from plant and mammalian viruses in human cells. We measured that the yield of infectious human immunodeficiency virus-1 particles produced in these cells was increased 5- to 10-fold. In addition, the production of lentiviral and adenoviral vector particles was increased 5- to 10-fold, whereas Sindbis virus particle production was increased approximately 100-fold. These results can be employed for improving the production of viral gene transfer vectors and viral vaccine strains.
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Terapia Genética/métodos , Vetores Genéticos/imunologia , Interferons/antagonistas & inibidores , Vacinas Virais/imunologia , Viroses/imunologia , Replicação Viral , Adenoviridae/fisiologia , Animais , Bovinos , Linhagem Celular , Expressão Gênica , HIV-1/fisiologia , Humanos , Imunidade Inata , Interferência de RNA , Complexo de Inativação Induzido por RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sindbis virus/fisiologia , Transfecção/métodosRESUMO
Embryonic stem (ES) cells have the potential to differentiate into various cell types of the three germ layers. They are therefore a useful cell source for transplantation and tissue engineering. In the present paper, we studied the influences of ascorbic acid (AA), dexamethasone (Dex), and 17beta-estradiol (E(2)) on the osteogenic differentiation of ES cells. Differentiation into the osteoblastic phenotype was demonstrated by the appearance of osteoblastic markers such as alkaline phosphatase (ALP), the transcription factor core binding factor alpha 1 (Cbfa1), and osteocalcin, which were detected by immunohistochemistry. Bone nodule formation, including the deposition of collagen fibrils and matrix mineralization, was studied by transmission electron microscopy. In all our cultures, a progressive upregulation of ALP activity was observed, followed by a decline after 21 d of culture. Cbfa1 was first detected after 14 d in culture and increased during the culture time. The addition of E(2) resulted in a decrease in the formation of bone-like nodules in the embryoid bodies (EBs) compared with the EBs cultured in the presence of AA and AA supplemented with Dex. An increased osteocalcin concentration was observed in the EBs cultured with Dex and E(2) compared with the EBs cultured in a control medium. EBs cultured in the presence of E(2) resulted in a culture with a high amount of osteoblast-like cells not entrapped in bone-like nodules, creating the possibility to obtain a purified osteoblast population for bone tissue engineering.
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Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Estradiol/farmacologia , Estrogênios/farmacologia , Osteoblastos/citologia , Fosfatase Alcalina/metabolismo , Animais , Linhagem da Célula/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Células-Tronco Embrionárias/metabolismo , Imuno-Histoquímica , Camundongos , Osteocalcina/metabolismoRESUMO
OBJECTIVES/BACKGROUND: In biological terms, progression means that malignancy increases as genetic mutations accumulate leading to increased proliferation and invasion capacity. By verifying the proliferation capacity, human telomerase reverse transcriptase (hTERT) expression and in vitro invasion, in a group of highly malignant glioblastomas, benign meningiomas and astrocytomas, at the initial stage of progression, we have analysed putative progression in vitro for proliferation and telomerase expression. MATERIALS AND METHODS: The relative proliferation status (visualized with Ki-67 antibodies) and presence of hTERT protein was analysed in 27 intracranial tumours (6 astrocytomas, 8 glioblastomas and 13 meningiomas) by immunohistochemistry on paraffin-embedded biopsy tissue, as well as on primary tumour-derived cell cultures. A confrontation model was used to analyse invasiveness in vitro. RESULTS: The mean proliferation indices were 22.3 (SD = 18.1) for glioblastomas and 2.1 (SD = 1.9) for low-grade (LG) astrocytomas. The group of benign meningiomas had a labelling index of 2.2 (SD = 2.7). Mean percentages of staining for hTERT varied between 36.5 (SD = 28.4) for glioblastomas and 10.2 (SD = 8.6) for LG astrocytomas. The group of benign meningiomas had a labelling index of 12.4 (SD = 19.2) for hTERT. A significant difference was seen for Ki-67 (P < 0.05) and hTERT (P < 0.001) in vivo versus in vitro. No difference was seen between the group of invasive and non-invasive tumour-derived cell cultures for the histopathological markers Ki-67 and hTERT (P > 0.05) in vitro. CONCLUSIONS: The elevated expression of hTERT and Ki-67 in vitro provides a potential prognostic tool for early detection of the progression of brain tumours. As tumour cells require telomerase for continued proliferation, the expression of hTERT may mark immortality, leading to indefinite life span. On the other hand, hTERT expression and cell proliferation are not linked directly to invasion in vitro.
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Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Proliferação de Células , Antígeno Ki-67/biossíntese , Neoplasias Meníngeas/patologia , Meningioma/patologia , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/análise , Criança , Progressão da Doença , Feminino , Humanos , Antígeno Ki-67/análise , Masculino , Meningioma/mortalidade , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Telomerase/análise , Células Tumorais CultivadasRESUMO
The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5' cap structure and a 3' poly(A) tail. We show that in a cell-free translation system derived from wheat germ, STNV RNA lacking the 600-nucleotide trailer is translated an order of magnitude less efficiently than full-size RNA. Deletion analyses positioned the translational enhancer domain (TED) within a conserved hairpin structure immediately downstream from the coat protein cistron. TED enhances translation when fused to a heterologous mRNA, but the level of enhancement depends on the nature of the 5' untranslated sequence and is maximal in combination with the STNV leader. The STNV leader and TED have two regions of complementarity. One of the complementary regions in TED resembles picornavirus box A, which is involved in cap-independent translation but which is located upstream of the coding region.
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Vírus de Plantas/genética , Biossíntese de Proteínas , RNA Viral/genética , Sequência de Bases , Capsídeo/biossíntese , Capsídeo/genética , DNA Viral/genética , Genes Virais , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos , Plantas Tóxicas , RNA Viral/química , RNA Viral/metabolismo , Mapeamento por Restrição , Deleção de Sequência , Nicotiana/microbiologia , Transcrição Gênica , Proteínas Estruturais Virais/genéticaRESUMO
Human linear chromosomes are capped by specialized DNA-protein structures called telomeres. The present study analysed the telomerase activity, hTERT protein and telomere length in meningiomas and gliomas in relation to their WHO grading. Fifty-three freshly dissected tumour biopsies were analysed for telomerase activity, hTERT protein expression and telomere length. Telomerase activity was examined in 41 of the 53 biopsies. Telomerase activity was detected in 3 of 35 (8.6%) screened meningiomas (1 benign, 1 atypical and 1 malignant meningioma). For hTERT expression, 56.4% of meningiomas were positive with a mean labelling index (hTERT LI) of 31.3% (SD=26.5) for the hTERT positive meningiomas. The mean telomere length for meningiomas was 6.983 kb (SD=1.969). For gliomas, no active telomerase was detected in 2 low-grade astrocytomas, whereas three of the four screened glioblastomas were positive for telomerase activity. The only hTERT protein positive astrocytoma had a mean labelling index of 9.0%. On the other hand, the hTERT LI for glioblastomas was 53.6% (SD=28.0). The two low-grade astrocytomas had a telomere length of 14.310 and 9.236 kb. The anaplastic astrocytoma had a telomere length of 4.903 kb and the glioblastomas 5.767 kb (SD=2.042). The normal meningeal and neuronal tissue is negative for telomerase activity and hTERT. The length was +/-10.000 kb. These results indicate that telomere shortening may be a critical step in pathogenesis of atypical and malignant meningiomas and gliomas. Critical telomere shortening in vitro was shown to activate telomerase.
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Neoplasias Encefálicas/patologia , Neoplasias Meníngeas/patologia , Meningioma/patologia , Telomerase/metabolismo , Telômero/patologia , Astrocitoma/enzimologia , Astrocitoma/genética , Astrocitoma/patologia , Biópsia , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Glioblastoma/enzimologia , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Neoplasias Meníngeas/enzimologia , Neoplasias Meníngeas/genética , Meningioma/enzimologia , Meningioma/genética , Telômero/ultraestruturaRESUMO
Histological and ultrastructural observations of dentin of two patients affected with rare types of type I collagen disorders are presented. In the first case, a homozygous nonsense mutation in ADAMTS2 (substitution of a codon for tryptophan by a stopcodon) causes type VIIC Ehlers-Danlos syndrome (EDS) with multiple tooth agenesis and focal dysplastic dentin defects. In the second case, a missense mutation in COL1A1 (substitution of arginine by cysteine) results in a type I EDS phenotype with clinically normal-appearing dentition. Tooth samples are investigated by using light microscopy (LM), transmission electron microscopy (TEM) and immunostaining for types I and III collagen, and tenascin. These are compared with samples from patients with types III and IV osteogenesis imperfecta (OI) in association with dentinogenesis imperfecta (DI), showing a consistently abnormal appearance of the dentin in all specimens, with variations being primarily those of degree of change. Similarities in histological changes include the alternating presence of normal and severe pathological areas in primary and secondary dentin, the latter being characterized by large canal-like structures in atubular areas. Ultrastructural evidence of pathological dentinogenesis include abnormal distribution, size and organization of collagen fibers, which may also be found in clinically unaffected teeth. The histological and ultrastructural changes seen can be explained on the basis of odontoblast dysfunction which may be secondary to the collagen defect, interfering with different levels of odontoblast cell function and intercellular communication. These observations on (ultra)structural dentin defects associated with the two novel gene mutations are the first ever reported.
Assuntos
Colágeno Tipo I/ultraestrutura , Dentina/ultraestrutura , Dentinogênese Imperfeita/genética , Osteogênese Imperfeita/genética , Colágeno Tipo I/genética , Dentina/patologia , Dentinogênese Imperfeita/patologia , Humanos , Microscopia Eletrônica , Mutação/genética , Osteogênese Imperfeita/patologia , Descoloração de Dente/genética , Descoloração de Dente/patologiaRESUMO
The satellite tobacco necrosis virus RNA is uncapped and requires a 3' translational enhancer domain (TED) for translation. Both in the wheat germ extract and in tobacco, TED stimulates in cis translation of heterologous, uncapped RNAs. In this study we investigated to what extent translation stimulation by TED depends on binding to wheat germ factors. We show that in vitro TED binds at least seven wheat germ proteins. Translation and crosslinking assays, to which TED or TED derivatives with reduced functionality were included as competitor, showed that TED function correlates with binding to a 28 kDa protein (p28). One particular condition of competition revealed that p28 binding is not obligatory for TED function. Under this condition, a 30 kDa protein (p30) binds to TED. Importantly, affinity of p30 correlates with functionality of TED. These results strongly suggest that TED has the capacity to stimulate translation by recruiting the translational machinery either via binding to p28 or via binding to p30.