Cavity filling mutations at the thyroxine-binding site dramatically increase transthyretin stability and prevent its aggregation.

More than a hundred different Transthyretin (TTR) mutations are associated with fatal systemic amyloidoses. They destabilize the protein tetrameric structure and promote the extracellular deposition of TTR as pathological amyloid fibrils. So far, only mutations R104H and T119M have been shown to stabilize significantly TTR, acting as disease suppressors. We describe a novel A108V non-pathogenic mutation found in a Portuguese subject. This variant is more stable than wild type TTR both in vitro and in human plasma, a feature that prevents its aggregation. The crystal structure of A108V reveals that this stabilization comes from novel intra and inter subunit contacts involving the thyroxine (T4) binding site. Exploiting this observation, we engineered a A108I mutation that fills the T4 binding cavity, as evidenced in the crystal structure. This synthetic protein becomes one of the most stable TTR variants described so far, with potential application in gene and protein replacement therapies.

Repositioning tolcapone as a potent inhibitor of transthyretin amyloidogenesis and associated cellular toxicity.

Transthyretin (TTR) is a plasma homotetrameric protein implicated in fatal systemic amyloidoses. TTR tetramer dissociation precedes pathological TTR aggregation. Native state stabilizers are promising drugs to treat TTR amyloidoses. Here we repurpose tolcapone, an FDA-approved molecule for Parkinson's disease, as a potent TTR aggregation inhibitor. Tolcapone binds specifically to TTR in human plasma, stabilizes the native tetramer in vivo in mice and humans and inhibits TTR cytotoxicity. Crystal structures of tolcapone bound to wild-type TTR and to the V122I cardiomyopathy-associated variant show that it docks better into the TTR T4 pocket than tafamidis, so far the only drug on the market to treat TTR amyloidoses. These data indicate that tolcapone, already in clinical trials for familial amyloid polyneuropathy, is a strong candidate for therapeutic intervention in these diseases, including those affecting the central nervous system, for which no small-molecule therapy exists.

Transthyretin chemical chaperoning by flavonoids: Structure-activity insights towards the design of potent amyloidosis inhibitors.

BACKGROUND: Many polyphenols have been proposed as broad-spectrum inhibitors of amyloid formation. To investigate structure-activity relationships relevant for the interaction of flavonoids with transthyretin (TTR), the protein associated with familial amyloid polyneuropathy (FAP), we compared the effects of major tea catechins and their larger polymers theaflavins, side-by-side, on TTR amyloid formation process. METHODS: Interaction of flavonoids with TTR and effect on TTR stability were assessed through binding assays and isoelectric focusing in polyacrylamide gel. TTR aggregation was studied, in vitro, by dynamic light scattering (DLS), transmission electron microscopy (TEM) and in cell culture, through cytotoxicity assays. RESULTS: Tested flavonoids bound to TTR and stabilized the TTR tetramer, with different potencies. The flavonoids also inhibited in vitro formation of TTR small oligomeric species and in cell culture inhibited pathways involving caspase-3 activation and ER stress that are induced by TTR oligomers. In all assays performed the galloyl esters presented higher potency to inhibit aggregation than the non-gallated flavonoids tested. CONCLUSIONS: Our results highlight the presence of gallate ester moiety as key structural feature of flavonoids in chemical chaperoning of TTR aggregation. Upon binding to the native tetramer, gallated flavonoids redirect the TTR amyloidogenic pathway into unstructured nontoxic aggregation assemblies more efficiently than their non-gallated forms. GENERAL SIGNIFICANCE: Our findings suggest that galloyl moieties greatly enhance flavonoid anti-amyloid chaperone activity and this should be taken into consideration in therapeutic candidate drug discovery.

Molecular tweezers targeting transthyretin amyloidosis.

Transthyretin (TTR) amyloidoses comprise a wide spectrum of acquired and hereditary diseases triggered by extracellular deposition of toxic TTR aggregates in various organs. Despite recent advances regarding the elucidation of the molecular mechanisms underlying TTR misfolding and pathogenic self-assembly, there is still no effective therapy for treatment of these fatal disorders. Recently, the "molecular tweezers", CLR01, has been reported to inhibit self-assembly and toxicity of different amyloidogenic proteins in vitro, including TTR, by interfering with hydrophobic and electrostatic interactions known to play an important role in the aggregation process. In addition, CLR01 showed therapeutic effects in animal models of Alzheimer's disease and Parkinson's disease. Here, we assessed the ability of CLR01 to modulate TTR misfolding and aggregation in cell culture and in an animal model. In cell culture assays we found that CLR01 inhibited TTR oligomerization in the conditioned medium and alleviated TTR-induced neurotoxicity by redirecting TTR aggregation into the formation of innocuous assemblies. To determine whether CLR01 was effective in vivo, we tested the compound in mice expressing TTR V30M, a model of familial amyloidotic polyneuropathy, which recapitulates the main pathological features of the human disease. Immunohistochemical and Western blot analyses showed a significant decrease in TTR burden in the gastrointestinal tract and the peripheral nervous system in mice treated with CLR01, with a concomitant reduction in aggregate-induced endoplasmic reticulum stress response, protein oxidation, and apoptosis. Taken together, our preclinical data suggest that CLR01 is a promising lead compound for development of innovative, disease-modifying therapy for TTR amyloidosis.

O momento da avaliação na intervenção precoce: o envolvimento da família estudo das qualidades psicométricas do ASQ-2 dos 30 aos 60 meses / The moment of assessment in early intervention: study of parental involvement of psychometric properties of ASQ-2 from 30 to 60 months

A relação entre pais e profissionais de saúde e de educação é fundamental para o desenvolvimento da criança. Esta se torna ainda mais essencial em crianças que apresentam risco de desenvolvimento. Essa mesma relação é valorizada numa abordagem centrada na família tendo implicações significativas nas práticas nos diferentes contextos e na sua aplicabilidade, exigindo que os profissionais assumam novos papéis e que aprendam novas competências no trabalho com as famílias. Estas competências concretizam-se nos primeiros contactos com a família desde o momento da avaliação até à efetiva prestação de serviços. A realização deste estudo surgiu da necessidade constatada de envolver os pais desde o primeiro momento do processo em intervenção precoce através de um instrumento formal e de simples aplicação. O ASQ-2 é constituído por 19 questionários divididos por diferentes idades entre os quatro e os 60 meses. Cada questionário é composto por 30 itens divididos pelas áreas de desenvolvimento comunicação, motricidade global, motricidade fina, resolução de problemas e pessoal-social. Este trabalho constitui a análise dos questionários dos 30 aos 60 meses para observar as qualidades psicométricas do ASQ, tendo sido aplicados a uma amostra de conveniência de 127 famílias do distrito de Braga. Foi possível verificar que o ASQ-2 traduzido apresenta resultados atraentes o que significa que poderá vir a ser utilizado pela população portuguesa de pais e profissionais (de saúde e de educação) que pretendam concretizar as suas dúvidas através de um instrumento formal de rastreio e de monitorização do desenvolvimento da criança.

The relationship between parents and health and education professionals is fundamental to the development of the child. This becomes even more important in children who are at risk. This relationship is valued in a family-centered approach and there are significant implications for practice in different contexts, requiring professionals to take on new roles and learn new skills in working with families. These skills come to the foreground during the first contacts with the family beginning at the time of assessment through to the effective provision of services. This study arose due to the perception that parents needed to be involved from the very beginning of the process in Early Intervention, by means of a formal instrument and simple application. The ASQ-2 is composed of 19 different questionnaires organized according to age intervals from 4 to 60 months. Each questionnaire comprises 30 items grouped according to the developmental areas of communication, gross motor, fine motor, problem solving and personal-social. This study presents the analysis of questionnaires from 30 to 60 months to observe the psychometric properties of the ASQ applied to a convenience sample of 127 households in the district of Braga. We concluded that the ASQ-2 presents attractive results suggesting that it could be used by the Portuguese population of parents and professionals (health and education) who wish to answer some questions using a formal screening and monitoring of child development instrument.

Thyroglobulin detection in fine-needle aspirates of cervical lymph nodes: a technique for the diagnosis of metastatic differentiated thyroid cancer.

BACKGROUND: Fine-needle aspiration cytology is frequently used for differential diagnosis of neck masses of unknown origin. Inconclusive and even false-negative results are not uncommon. AIM: To evaluate the utility of thyroglobulin (Tg) measurement in fine-needle aspirates (FNA-Tg) for detecting cervical lymph node (CLNs) metastases from differentiated thyroid carcinomas. METHODS: An ultrasound-guided fine-needle aspiration was done in 67 patients with 83 suspicious enlarged CLNs to obtain material for cytology and Tg measurement in the needle washout, using an immunometric chemiluminescent assay. Measurement of anti-Tg antibodies (FNA-TgAb) was also carried out in half of all the aspirates. Subjects were divided into two groups: one of 16 patients awaiting thyroidectomy and the other of 51 patients in follow-up after surgery. RESULTS: The first group of patients had positive FNA biopsy (FNAB-Tg) in 14 out of the 18 studied CLNs with a range of 3.2-43 352 ng/ml, while FNAB-cytology indicated metastasis in only 8 out of the 14 CLNs with positive histology. A total of 65 CLNs were studied in the follow-up group. Lymphadenectomy was performed in 23 patients and 28 aspirated CLNs were removed. Histology confirmed the diagnosis of metastasis suggested by FNAB-Tg in 20 CLNs and of reactive lymphadenitis in the remaining 8 CLNs. FNAB-cytology was positive in only 11 CLNs. Sensitivity of FNAB-Tg was not affected by the studied FNAB-TgAb. CONCLUSIONS: The FNAB-Tg achieved a sensitivity of 100% in both groups. FNAB-Tg is an easy and inexpensive technique which proved to increase the diagnostic of cytology in the early diagnosis of papillary carcinoma recurrence to CLN even in the presence of serum TgAb.