a fascinating and neglected pathogen

Hepatitis delta virus (HDV) is the etiologic agent of the most severe form of virus hepatitis in humans. Sharing some structural and functional properties with plant viroids, the HDV RNA contains a single open reading frame coding for the only virus protein, the Delta antigen. A number of unique features, including ribozyme activity, RNA editing, rolling-circle RNA replication, and redirection for a RNA template of host DNA-dependent RNA polymerase II, make this small pathogen an excellent model to study virus-cell interactions and RNA biology. Treatment options for chronic hepatitis Delta are scarce and ineffective. The disease burden is perhaps largely underestimated making the search for new, specific drugs, targets, and treatment strategies an important public health challenge. In this review we address the main features of virus structure, replication, and interaction with the host. Virus pathogenicity and current treatment options are discussed in the light of recent developments.publishersversionpublishe

Structural and nucleic acid binding properties of hepatitis delta virus small antigen

AIM: To further characterize the structure and nucleic acid binding properties of the 195 amino acid small delta antigen, S-HDAg, a study was made of a truncated form of S-HDAg, comprising amino acids 61-195 (∆60HDAg), thus lacking the domain considered necessary for dimerization and higher order multimerization. METHODS: Circular dichroism, and nuclear magnetic resonance experiments were used to assess the structure of ∆60HDAg. Nucleic acid binding properties were investigated by gel retardation assays. RESULTS: Results showed that the truncated ∆60HDAg protein is intrinsically disordered but compact, whereas the RNA binding domain, comprising residues 94-146, adopts a dynamic helical conformation. We also found that ∆60HDAg fails to multimerize but still contains nucleic acid binding activity, indicating that multimerization is not essential for nucleic acid binding. Moreover, in agreement with what has been previously reported for full-length protein, no apparent specificity was found for the truncated protein regarding nucleic acid binding. CONCLUSION: Taken together these results allowed concluding that ∆60HDAg is intrinsically disordered but compact; ∆60HDAg is not a multimer but is still capable of nucleic acid binding albeit without apparent specificity.publishersversionpublishersversionpublishe

Desenvolvimento de um método para determinação de Vitamina B12 em alimentos, por cromatografia líquida acoplada a espectrometria de massas (LC-MS/MS)

O objetivo deste trabalho centrou-se na otimização do método de extração e do método cromatográfico utilizados com o objetivo de determinar os teores de vitamina B12 em amostras fortificadas (bebidas vegetais de soja) e não fortificadas (cavala crua). A utilização de colunas de imunoafinidade como método de clean-up contribuiu para a otimização do método e, consequentemente, para a determinação dos diferentes vitâmeros nas diferentes matrizes. Nas bebidas vegetais fortificadas com cianocobalamina, matrizes mais simples, foi possível a identificação da cianocobalamina presente, estimando-se teores entre os 0,25 e 0,66 μg/100 mL, resultados muito semelhantes aos obtidos por ultracentrifugação. Para as cavalas, matrizes mais complexas, foram testados dois métodos de extração: com e sem cianeto de sódio, com o objetivo de determinar as diferentes formas naturais de vitamina B12 bem como o seu teor total. Os resultados obtidos mostraram que a hidroxocobalamina é o vitâmero predominante na amostra, representando entre 35 e 58% do teor total de vitamina B12, ao contrário da cianocobalamina que se encontra em teores vestigiais. Já na determinação do teor total de vitamina B12, expresso em teor de cianocobalamina, o valor estimado é de aproximadamente 13,9 μg/ 100 g. Foram ainda avaliados alguns parâmetros para a validação do método. A linearidade foi verificada para a gama de concentração das curvas de calibração em estudo e, embora a homogeneidade de variâncias não se tenha verificado, a análise de materiais de referência permitiu avaliar a exatidão, tendo sido obtido um z-score < 2. O desenvolvimento deste trabalho representa assim uma contribuição para a otimização e desenvolvimento do método para quantificar a vitamina B12, podendo considerar-se os resultados obtidos, embora seja necessária a realização de mais testes, promissores.The aim of the present work was focused on the otimization of the extraction and chromatographic method used for the purpose of determining the vitamin B12 content in fortified (soy based vegetable drinks) and unfortified (raw mackerel) samples. The immunoaffinity columns used as clean-up method in the optimization process showed not only to be effective but also allowed the determination of different vitamers on matrices. In the fortified soy based vegetable drinks (simpler matrices) it was possible to identify the presence of cyanocobalamin, with levels estimated between 0,25 and 0,66 μg/100 mL, being similar to those obtained by ultracentrifugation. In the mackerel samples (more complex matrices) two extraction methods were tested: with and without sodium cyanide, in order to determine the different natural forms of vitamin B12 as well as their total content. The results show that hydroxocobalamin is the major form in this sample and it represents 35 to 58% of the total vitamin B12 content, cyanocobalamin on the contrary is found in residual levels. The total content of vitamin B12, expressed by content of cyanocobalamin is estimated to be approximately 13,9 μg/100 mL. Several parameters for the validation of the method were also evaluated. Linearity of calibration curve was verified in the range of concentrations of studied samples and, although the homogeneity of variances was not verified, the analysis of reference materials allowed to evaluate the accuracy, with a z-score < 2. In summary, the present work is a contribution to development and optimization method for the determination of vitamin B12

Hsp70 Chaperones and Type I PRMTs Are Sequestered at Intranuclear Inclusions Caused by Polyalanine Expansions in PABPN1

Genomic instability at loci with tandem arrays of simple repeats is the cause for many neurological, neurodegenerative and neuromuscular diseases. When located in coding regions, disease-associated expansions of trinucleotide repeats are translated into homopolymeric amino acid stretches of glutamine or alanine. Polyalanine expansions in the poly(A)-binding protein nuclear 1 (PABPN1) gene causes oculopharyngeal muscular dystrophy (OPMD). To gain novel insight into the molecular pathophysiology of OPMD, we studied the interaction of cellular proteins with normal and expanded PABPN1. Pull-down assays show that heat shock proteins including Hsp70, and type I arginine methyl transferases (PRMT1 and PRMT3) associate preferentially with expanded PABPN1. Immunofluorescence microscopy further reveals accumulation of these proteins at intranuclear inclusions in muscle from OPMD patients. Recombinant PABPN1 with expanded polyalanine stretches binds Hsp70 with higher affinity, and data from molecular simulations suggest that expansions of the PABPN1 polyalanine tract result in transition from a disordered, flexible conformation to a stable helical secondary structure. Taken together, our results suggest that the pathological mutation in the PABPN1 gene alters the protein conformation and induces a preferential interaction with type I PRMTs and Hsp70 chaperones. This in turn causes sequestration in intranuclear inclusions, possibly leading to a progressive cellular defect in arginine methylation and chaperone activity

PABPN1 gene therapy for oculopharyngeal muscular dystrophy

International audienceOculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant, late-onset muscle disorder characterized by ptosis, swallowing difficulties, proximal limb weakness and nuclear aggregates in skeletal muscles. OPMD is caused by a trinucleotide repeat expansion in the PABPN1 gene that results in an N-terminal expanded polyalanine tract in polyA-binding protein nuclear 1 (PABPN1). Here we show that the treatment of a mouse model of OPMD with an adeno-associated virus-based gene therapy combining complete knockdown of endogenous PABPN1 and its replacement by a wild-type PABPN1 substantially reduces the amount of insoluble aggregates, decreases muscle fibrosis, reverts muscle strength to the level of healthy muscles and normalizes the muscle transcriptome. The efficacy of the combined treatment is further confirmed in cells derived from OPMD patients. These results pave the way towards a gene replacement approach for OPMD treatment

Maturation of mammalian H/ACA box snoRNAs: PAPD5-dependent adenylation and PARN-dependent trimming

Small nucleolar and small Cajal body RNAs (snoRNAs and scaRNAs) of the H/ACA box and C/D box type are generated by exonucleolytic shortening of longer precursors. Removal of the last few nucleotides at the 3' end is known to be a distinct step. We report that, in human cells, knock-down of the poly(A) specific ribonuclease (PARN), previously implicated only in mRNA metabolism, causes the accumulation of oligoadenylated processing intermediates of H/ACA box but not C/D box RNAs. In agreement with a role of PARN in snoRNA and scaRNA processing, the enzyme is concentrated in nucleoli and Cajal bodies. Oligo(A) tails are attached to a short stub of intron sequence remaining beyond the mature 3' end of the snoRNAs. The noncanonical poly(A) polymerase PAPD5 is responsible for addition of the oligo(A) tails. We suggest that deadenylation is coupled to clean 3' end trimming, which might serve to enhance snoRNA stability

The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitive

fAter being released from transcription sites, messenger ribonucleoprotein particles (mRNPs) must reach the nuclear pore complexes in order to be translocated to the cytoplasm. Whether the intranuclear movement of mRNPs results largely from Brownian motion or involves molecular motors remains unknown. Here we have used quantitative photobleaching techniques to monitor the intranuclear mobility of protein components of mRNPs tagged with GFP. The results show that the diffusion coefficients of the poly(A)-binding protein II (PABP2) and the export factor TAP are significantly reduced when these proteins are bound to mRNP complexes, as compared with nonbound proteins. The data further show that the mobility of wild-type PABP2 and TAP, but not of a point mutant variant of PABP2 that fails to bind to RNA, is significantly reduced when cells are ATP depleted or incubated at 22°C. Energy depletion has only minor effects on the intranuclear mobility of a 2,000-kD dextran (which corresponds approximately in size to 40S mRNP particles), suggesting that the reduced mobility of PABP2 and TAP is not caused by a general alteration of the nuclear environment. Taken together, the data suggest that the mobility of mRNPs in the living cell nucleus involves a combination of passive diffusion and ATP-dependent processes

Synopsys of the 3rd National Congress of Tropical Medicine and 1st Lusophone Congress of Vector-Borne Diseases

A medicina tropical tem vindo a assumir, cada vez mais, uma dimensão global. As patologias referidas como tropicais e restritas, durante muitos anos, a territórios tropicais têm vindo gradualmente a conquistar, cada vez mais, espaço geográfico em áreas anteriormente consideradas isentas destes flagelos. Atualmente, a Europa e os EUA debatem-se com surtos epidémicos de infeções por microrganismos patogénicos considerados tropicais e o impacto das doenças transmitidas por vetores na Saúde humana e veterinária encontra-se cada vez mais disseminado. O Instituto de Higiene e Medicina Tropical da Universidade NOVA de Lisboa organizou o 3º Congresso Nacional de Medicina Tropical e o 1º Congresso Lusófono de Doenças Transmitidas por Vetores, nos dias 20 e 21 de abril de 2015, tendo sido a maior parte dedicado às doenças transmitidas por vetores. A iniciativa contou com cerca de 300 participantes (oriundos de países da Lusofonia e ainda de outros centros científicos internacionais), com 57 comunicações orais e 41 posters. Este encontro teve como objetivo criar um espaço para a discussão e apresentação de trabalhos, desenvolvidos a nível nacional e internacional, nomeadamente sobre a prevenção, o controlo e a eliminação, assim como os vários desafios associados ao desenvolvimento de novas metodologias aplicadas ao diagnóstico e tratamento das doenças transmitidas por vetores. Currently, Tropical Medicine emerged as both an important medical specialty and scientific discipline assuming a preponderant global dimension. Until a few years ago, several tropical pathologies, which were exclusively associated with tropical regions, have been gradually expanding to other geographic areas previously considered free of these infections. Vector-borne diseases are included among this group of infections. Actually, Europe and the US struggle with disease outbreaks caused by pathogens primarily associated with tropical regions. In fact, the impact of vector-borne diseases in human and veterinary health is increasingly being disseminated worldwide. The Institute of Hygiene and Tropical Medicine, University NOVA of Lisbon organized the 3rd National Congress of Tropical Medicine and the 1st Congress of Portuguese Speaking Countries on Vector-borne Diseases, on 20th - 21st of April, 2015, dedicated to vector-borne diseases topics. The meeting was attended by about 300 participants from Portugal, Portuguese speaking countries, and other international scientific centers. Overall, 57 oral communications and 41 posters were presented on sessions that promoted great interest and scientific discussion, about the control, elimination, as well as the challenges associated with the development of novel methodologies applied to the diagnosis and treatment of several vector-borne diseases.publishersversionpublishe

Clastosome: a subtype of nuclear body enriched in 19S and 20S proteasomes, ubiquitin, and protein substrates of proteasome.

Nuclear bodies represent a heterogeneous class of nuclear structures. Herein, we describe that a subset of nuclear bodies is highly enriched in components of the ubiquitin-proteasome pathway of proteolysis. We coined the term clastosome (from the Greek klastos, broken and soma, body) to refer to this type of nuclear body. Clastosomes contain a high concentration of 1) ubiquitin conjugates, 2) the proteolytically active 20S core and the 19S regulatory complexes of the 26S proteasome, and 3) protein substrates of the proteasome. Although detected in a variety of cell types, clastosomes are scarce under normal conditions; however, they become more abundant when proteasomal activity is stimulated. In contrast, clastosomes disappear when cells are treated with proteasome inhibitors. Protein substrates of the proteasome that are found concentrated in clastosomes include the short-lived transcription factors c-Fos and c-Jun, adenovirus E1A proteins, and the PML protein. We propose that clastosomes are sites where proteolysis of a variety of protein substrates is taking place