12 research outputs found
Alteração da resposta regulatória do Rab7 e do Rab9 no subtipo MM1 e VV2 da doença Creutzfeldt-Jakob
Mestrado em Biologia Molecular e CelularThe present study was undertaken to identify proteins interacting with PrPC that could provide new insights into its physiological functions and pathological role. We performed a target search for lysosomal network protein, Rab7a and Rab9, in frontal cortex and cerebellum of human brain from patients with sCJD-MM1 and sCJD-VV2. The intracellular level of Rab7a was increased significantly, when compared with healthy age-matched control. Interactions of PrPC and Rab7a/Rab9 were further investigated by using confocal laser scanning microscopy. Immunofluorescence results suggested potential interactions of Rab7a and PrPC. siRNA against the Rab7a gene was used to knockdown the expression of Rab7a protein in primary cell culture of cortical neurons from wild type mice. This depleted Rab7a resulted an impairment of PrPC trafficking leading to an accumulation of PrPC in the endocytosis pathway. Furthermore, interactions of Tau and Rab7a were investigated by using western blot analysis and confocal laser scanning microscopy. Cell cultures of cortex of wildtype mice were treated with siRNA-Tau, siRNA-Rab7 and control siRNA followed by immunofluorescence. The results of immunofluorescence suggested potential interaction of Tau and Rab7a. Cells lines treated with siRNA-Tau, the intracellular levels of Rab7a and Rab9 significantly increases and their localization is also modified. When we transfected this cells lines with siRNA-rab7a the accumulation of Tau decreases in cytosolic region and their localization was also modified when compared with control cells. In conclusion, this study may help to understand and characterize the subtype specific disease progression in CJD cases. Furthermore, it could be a step ahead to development of new treatment strategies for diseases subtype specific manner.O presente estudo foi levado a cabo com o intuito de identificar possĂveis proteĂnas que interajam com PrPC que possam fornecer novos conhecimentos sobre as suas funçÔes fisiolĂłgicas assim como no papel patolĂłgico. O presente estudo incidiu na investigação dos nĂveis das proteĂnas endossomais Rab7a e Rab9 em amostras do cĂłrtex frontal e cerebelo de pacientes diagnosticados com sCJD-MM1 e sCJD-VV2. Observamos um aumento significativo dos nĂveis intracelulares do Rab7a em pacientes diagnosticados com sCJD-MM1 e sCJD-VV2 quando comparadas com amostras de pacientes na mesma faixa etĂĄria sem a doença (controlo). As interaçÔes entre PrPC e as proteĂnas Rab7a e Rab9 foram posteriormente estudadas com o auxĂlio Ă microscopia confocal. Os resultados da imunofluorescĂȘncia sugeriram potenciais interaçÔes entre as proteĂnas Rab7a com o PrPC. O siRNA-Rab7 foi usado para diminuir a expressĂŁo da proteĂna Rab7a (âKnockdownâ) na cultura primĂĄria de cĂ©lulas do cĂłrtex de ratos saudĂĄveis. Na cultura de cĂ©lulas do cĂłrtex tratadas com o siRNA-Rab7, foi observado um emparelhamento da via endocĂtica. Seguidamente investigamos possĂveis interaçÔes da proteĂna Tau com a proteĂna Rab7a, recorrendo ao western blot e a microscopia confocal. Culturas de cĂ©lulas do cĂłrtex de ratos saudĂĄveis foram tratadas com siRNA-Tau, siRNA-Rab7a e siRNA. Os resultados da imunofluorescĂȘncia das diferentes culturas celulares sugeriram uma potencial interação entre as proteĂnas Tau e Rab7a. Nas linhas celulares tratadas com siRNA-Tau os nĂveis intracelulares das proteĂnas Rab7a e Rab9 aumentaram significativamente, assim como, foi tambĂ©m observada a alteração da sua localização. Nas cĂ©lulas tratadas com siRNA-Rab7a observamos uma diminuição da acumulação da proteĂna Tau na regiĂŁo citosĂłlica. Em conclusĂŁo, este trabalho pode ajudar a perceber e a caracterizar a progressĂŁo da doença nos subtipos especĂficos, no caso de sCJD. Contudo, este trabalho poderĂĄ ser tambĂ©m um passo Ă frente para o desenvolvimento de novas estratĂ©gias terapĂȘuticas para os subtipos sCJD-MM1 e sCJD-VV2
Optimization of the Real-Time Quaking-Induced Conversion Assay for Prion Disease Diagnosis
The real-time quaking-induced conversion (RT-QuIC) assay is a highly reproducible and robust methodology exhibiting an excellent pre-mortem diagnostic accuracy for prion diseases. However, the protocols might be time-consuming and improvement of the detection technology is needed. In the present study, we investigated the influence of a pre-analytical cerebrospinal fluid (CSF) treatment with proteinase K (PK) on the kinetic of the RT-QuIC signal response. For this purpose, we added PK at different concentrations in RT-QuIC reactions seeded with Creutzfeldt-Jakob disease (sCJD) CSF. We observed that a mild pre-analytical PK treatment of CSF samples resulted in an increased seeding efficiency of the RT-QuIC reaction. Quantitative seeding parameters, such as a higher area under the curve (AUC) value or a shorter lag phase indicated a higher conversion efficiency after treatment. The diagnostic accuracy resulting from 2 mu g/ml PK treatment was analyzed in a retrospective study, where we obtained a sensitivity of 89%. Additionally, we analyzed the agreement with the previously established standard RT-QuIC protocol without PK treatment in a prospective study. Here, we found an overall agreement of 94% to 96%. A Cohen's kappa of 0.9036 (95% CI: 0.8114-0.9958) indicates an almost perfect agreement between both protocols. In conclusion, the outcome of our study can be used for a further optimization of the RT-QuIC assay in particular for a reduction of the testing time
Influenza seroprotection correlates with predominant circulating viruses during 2014/15 and 2015/16 seasons in Portugal
Rede Portuguesa de LaboratĂłrios para o DiagnĂłstico da GripeBACKGROUND: Population immune profile for influenza is highly affected by circulating influenza viruses, thus changing the risk of infection for influenza. This study aims to assess influenza immunity in the Portuguese population by age groups, during 2014 and 2015 and establish a relationship between seroprotection and circulating influenza viruses in 2014/15 and 2015/16 seasons.
METHODS: Two cross-sectional studies were developed based on a convenience serum sample collected in June 2014 (n=626) and July 2015 (n=675) in hospitals from mainland and Azores and Madeira.Serums equally represent all age groups. Antibody titers were evaluated by HI assay for strains recommended for seasonal influenza vaccine northern hemisphere,2014/15 and 2015/2016. Seroprevalences were estimated for each strain by age group and the association with seasonal cumulative influenza-like illness (ILI) rates for influenza virus during both seasons was analised.
RESULTS: In June 2014 the highest seroprotection was observed for influenza A(H3) (39.0%; 95% CI: 36.2-43.8%) and A(H1)pdm09 (29.7; 95% CI: 26.3-33.4%), with higher levels in children 5-14 years old. In 2014/2015 a dominant circulation of influenza B/Yamagata was observed with high incidence rates in individuals under 65 years old, the ones that had lower seroprotection. Although before the start of the season high protection for A(H3) was observed, the circulation of the new drift A(H3) strains had gained an immunological advantage,in accordance with A(H3) elevated incidence rates observed during 2014/15. In July 2015 the highest seroprotection was observed for influenza B/ Yamagata (55.1%; 95% CI: 51.4-58.9%), 2.4 times the estimated 2014.This increase was even more pronounced in younger (†4 years old), 6.3 times increase in 2015.This fact is in agreement with the predominant influenza B virus detected and the high ILI incidence rate observed in children during 2014/2015 epidemic. Seroprotection levels for influenza A in July 2015 were not significantly different from 2014.During 2015/16 season, influenza A(H1N1)pdm09 was predominant, with high incidence rate in < 65 year old. Influenza B/Victoria lineage,although detected at low levels increased in frequency, in agreement with the lowest level of seroprotection detected in the general population before the start of 2015/2016 season (21.8%; 95% CI: 18.7-24.0%).
CONCLUSIONS There was a correlation between virus circulation, incidence rates for each age group and the previous seroprotection for seasonal influenza viruses.Our study highlights the value of measuring the serological profile for influenza to establishe risk groups for infection for which an increase preventive measures, including vaccination, should be fostered.info:eu-repo/semantics/publishedVersio
SARS-CoV-2 introductions and early dynamics of the epidemic in Portugal
Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by
the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration
with more than 50 laboratories distributed nationwide.
Methods By applying recent phylodynamic models that allow integration of individual-based
travel history, we reconstructed and characterized the spatio-temporal dynamics of SARSCoV-2 introductions and early dissemination in Portugal.
Results We detected at least 277 independent SARS-CoV-2 introductions, mostly from
European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland),
which were consistent with the countries with the highest connectivity with Portugal.
Although most introductions were estimated to have occurred during early March 2020, it is
likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the
first cases were confirmed.
Conclusions Here we conclude that the earlier implementation of measures could have
minimized the number of introductions and subsequent virus expansion in Portugal. This
study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance.We gratefully acknowledge to Sara Hill and Nuno Faria (University of Oxford) and
Joshua Quick and Nick Loman (University of Birmingham) for kindly providing us with
the initial sets of Artic Network primers for NGS; Rafael Mamede (MRamirez team,
IMM, Lisbon) for developing and sharing a bioinformatics script for sequence curation
(https://github.com/rfm-targa/BioinfUtils); Philippe Lemey (KU Leuven) for providing
guidance on the implementation of the phylodynamic models; Joshua L. Cherry
(National Center for Biotechnology Information, National Library of Medicine, National
Institutes of Health) for providing guidance with the subsampling strategies; and all
authors, originating and submitting laboratories who have contributed genome data on
GISAID (https://www.gisaid.org/) on which part of this research is based. The opinions
expressed in this article are those of the authors and do not reflect the view of the
National Institutes of Health, the Department of Health and Human Services, or the
United States government. This study is co-funded by Fundação para a CiĂȘncia e Tecnologia
and AgĂȘncia de Investigação ClĂnica e Inovação BiomĂ©dica (234_596874175) on
behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study
come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by
COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation
(POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal
Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL
2020 Partnership Agreement, through the European Regional Development Fund
(ERDF), and by Fundação para a CiĂȘncia e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio
Strategies to Stabilize High Energetic States of Pharmaceutical Drug
International audienc
Stabilizing Unstable Amorphous Menthol through Inclusion in Mesoporous Silica Hosts
International audienc
Unraveling the genetic background of individuals with a clinical familial hypercholesterolemia phenotype
Familial hypercholesterolemia (FH) is a common genetic disorder of lipid metabolism caused by pathogenic/likely pathogenic variants in LDLR, APOB, and PCSK9 genes. Variants in FH-phenocopy genes (LDLRAP1, APOE, LIPA, ABCG5, and ABCG8), polygenic hypercholesterolemia, and hyperlipoprotein (a) [Lp(a)] can also mimic a clinical FH phenotype. We aim to present a new diagnostic tool to unravel the genetic background of clinical FH phenotype. Biochemical and genetic study was performed in 1,005 individuals with clinical diagnosis of FH, referred to the Portuguese FH Study. A next-generation sequencing panel, covering eight genes and eight SNPs to determine LDL-C polygenic risk score and LPA genetic score, was validated, and used in this study. FH was genetically confirmed in 417 index cases: 408 heterozygotes and 9 homozygotes. Cascade screening increased the identification to 1,000 FH individuals, including 11 homozygotes. FH-negative individuals (phenotype positive and genotype negative) have Lp(a) >50Â mg/dl (30%), high polygenic risk score (16%), other monogenic lipid metabolism disorders (1%), and heterozygous pathogenic variants in FH-phenocopy genes (2%). Heterozygous variants of uncertain significance were identified in primary genes (12%) and phenocopy genes (7%). Overall, 42% of our cohort was genetically confirmed with FH. In the remaining individuals, other causes for high LDL-C were identified in 68%. Hyper-Lp(a) or polygenic hypercholesterolemia may be the cause of the clinical FH phenotype in almost half of FH-negative individuals. A small part has pathogenic variants in ABCG5/ABCG8 in heterozygosity that can cause hypercholesterolemia and should be further investigated. This extended next-generation sequencing panel identifies individuals with FH and FH-phenocopies, allowing to personalize each personâs treatment according to the affected pathway
Characterisation of microbial attack on archaeological bone
As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved
Clinical manifestations of intermediate allele carriers in Huntington disease
Objective: There is controversy about the clinical consequences of intermediate alleles (IAs) in Huntington disease (HD). The main objective of this study was to establish the clinical manifestations of IA carriers for a prospective, international, European HD registry. Methods: We assessed a cohort of participants at risk with <36 CAG repeats of the huntingtin (HTT) gene. Outcome measures were the Unified Huntington's Disease Rating Scale (UHDRS) motor, cognitive, and behavior domains, Total Functional Capacity (TFC), and quality of life (Short Form-36 [SF-36]). This cohort was subdivided into IA carriers (27-35 CAG) and controls (<27 CAG) and younger vs older participants. IA carriers and controls were compared for sociodemographic, environmental, and outcome measures. We used regression analysis to estimate the association of age and CAG repeats on the UHDRS scores. Results: Of 12,190 participants, 657 (5.38%) with <36 CAG repeats were identified: 76 IA carriers (11.56%) and 581 controls (88.44%). After correcting for multiple comparisons, at baseline, we found no significant differences between IA carriers and controls for total UHDRS motor, SF-36, behavioral, cognitive, or TFC scores. However, older participants with IAs had higher chorea scores compared to controls (p 0.001). Linear regression analysis showed that aging was the most contributing factor to increased UHDRS motor scores (p 0.002). On the other hand, 1-year follow-up data analysis showed IA carriers had greater cognitive decline compared to controls (p 0.002). Conclusions: Although aging worsened the UHDRS scores independently of the genetic status, IAs might confer a late-onset abnormal motor and cognitive phenotype. These results might have important implications for genetic counseling. ClinicalTrials.gov identifier: NCT01590589
Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries
Background
Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks.
Methods
The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned.
Results
A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P < 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31).
Conclusion
Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)