Aurora A activates D-TACC–Msps complexes exclusively at centrosomes to stabilize centrosomal microtubules

Centrosomes are the dominant sites of microtubule (MT) assembly during mitosis in animal cells, but it is unclear how this is achieved. Transforming acidic coiled coil (TACC) proteins stabilize MTs during mitosis by recruiting Minispindles (Msps)/XMAP215 proteins to centrosomes. TACC proteins can be phosphorylated in vitro by Aurora A kinases, but the significance of this remains unclear. We show that Drosophila melanogaster TACC (D-TACC) is phosphorylated on Ser863 exclusively at centrosomes during mitosis in an Aurora A–dependent manner. In embryos expressing only a mutant form of D-TACC that cannot be phosphorylated on Ser863 (GFP-S863L), spindle MTs are partially destabilized, whereas astral MTs are dramatically destabilized. GFP-S863L is concentrated at centrosomes and recruits Msps there but cannot associate with the minus ends of MTs. We propose that the centrosomal phosphorylation of D-TACC on Ser863 allows D-TACC–Msps complexes to stabilize the minus ends of centrosome-associated MTs. This may explain why centrosomes are such dominant sites of MT assembly during mitosis

Conceptions of teaching quality in public higher education institutions in the Alentejo region

Este estudo teve como objectivos conhecer e comparar as concepções sobre qualidade de ensino de docentes e estudantes de estabelecimentos públicos de ensino superior da região do Alentejo. Tratou-se de um estudo qualitativo, que incluiu uma análise de conteúdo a uma questão aberta relativa ao modo como 427 estudantes e 103 docentes definiam qualidade de ensino. A análise de conteúdo baseou-se nos modelos teóricos disponíveis, nas actividades previstas para o ensino superior e, sobretudo, no conteúdo das respostas escritas dos participantes. Os resultados revelaram que as respostas de docentes e estudantes das três instituições, globalmente consideradas, não diferiam substancialmente umas das outras e que a qualidade de ensino era mais associada a aspectos de natureza pedagógica, preparação para o mercado de trabalho, articulação com a comunidade e qualificação dos docentes do que com aspectos associados à investigação

Pyomelanin synthesis in alternaria alternata inhibits DHN-Melanin synthesis and decreases cell wall chitin content and thickness

The genus Alternaria includes several of fungi that are darkly pigmented by DHNmelanin. These are pathogenic to plants but are also associated with human respiratory allergic diseases and with serious infections in immunocompromised individuals. The present work focuses on the alterations of the composition and structure of the hyphal cell wall of Alternaria alternata occuring under the catabolism of L-tyrosine and L-phenylalanine when cultured in minimal salt medium (MM). Under these growing conditions, we observed the released of a brown pigment into the culture medium. FTIR analysis demonstrates that the produced pigment is chemically identical to the pigment released when the fungus is grown in MM with homogentisate acid (HGA), the intermediate of pyomelanin, confirming that this pigment is pyomelanin. In contrast to other fungi that also synthesize pyomelanin under tyrosine metabolism, A. alternata inhibits DHN-melanin cell wall accumulation when pyomelanin is produced, and this is associated with reduced chitin cell wall content. When A. alternata is grown in MM containing L-phenylalanine, a L-tyrosine percursor, pyomelanin is synthesized but only at trace concentrations and A. alternata mycelia display an albino-like phenotype since DHN-melanin accumulation is inhibited. CmrA, the transcription regulator for the genes coding for the DHN-melanin pathway, is involved in the down-regulation of DHN-melanin synthesis when pyomelanin is being synthetized, since the CMRA gene and genes of the enzymes involved in DHN-melanin synthesis pathway showed a decreased expression. Other amino acids do not trigger pyomelanin synthesis and DHN-melanin accumulation in the cell wall is not affected. Transmission and scanning electron microscopy show that the cell wall structure and surface decorations are altered in L-tyrosine- and L-phenylalanine-grown fungi, depending on the pigment produced. In summary, growth in presence of L-tyrosine and L-phenylalanine leads to pigmentation and cell wall changes, which could be relevant to infection conditions where these amino acids are expected to be available.This study was partly supported by the FEDER funds through the Operational Programme Competitiveness Factors-COMPETE and national funds by FCT-Foundation for Science and Technology under the strategic projects UIDB/00285/2020 and UID/NEU/04539/2013 the European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme under project CENTRO-01-0145-FEDER-000012 and project CENTRO-01-0145-FEDER-022095:ViraVector, and through the COMPETE 2020—Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT—Fundação para a Ciência e a Tecnologia, under project UIDB/04539/2020, and the European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme: project CENTRO-01-0145-FEDER- 000012-HealthyAging 2020, the COMPETE 2020—Operational Programme for Competitiveness and Internationalisation, and the Portuguese national funds via FCT—Fundação para a Ciência e a Tecnologia, I.P.: project POCI-01-0145-FEDER- 007440. IF thank the Foundation for Science and Technology (FCT, Portugal) and FEDER under Program PT2020 for financial support to CIMO (UID/AGR/00690/2013), LB (SFRH/BPD/107855/2015) and MD (SFRH/BD/84485/2012) grants. To POCI-01-0145-FEDER-006984 (LA LSRE-LCM), funded by ERDF, through POCI-COMPETE2020 and FCT. AC was supported in part by 5R01HL059842, 5R01AI033774, 5R37AI033142, and 5R01AI052733.info:eu-repo/semantics/publishedVersio

Synthesis of Hydrophilic and Amphiphilic Acryl Sucrose Monomers and Their Copolymerisation with Styrene, Methylmethacrylate and α- and β-Pinenes

Herein, we report the synthesis of monomethacryloyl sucrose esters, and their successful free radical homo- and co-polymerisation with styrene, methylmethacrylate, α-and β-pinene. The chemical, physical, structural and surface chemical properties of these polymers, containing a hydrophobic olefin backbone and hydrophilic sugar moieties as side chains, have been investigated. Biodegradation tests of the copolymer samples by a microbial fungal culture (Aspergillus niger) method showed good biodegradability. The chemical structure and surface chemistry of the synthesized homo- and co-polymers demonstrate their potential technological relevance as amphiphilic and biodegradable polymers

Using Tetracysteine-Tagged TDP-43 with a Biarsenical Dye To Monitor Real-Time Trafficking in a Cell Model of Amyotrophic Lateral Sclerosis.

TAR DNA-binding protein 43 (TDP-43) has been identified as the major constituent of the proteinaceous inclusions that are characteristic of most forms of amyotrophic lateral sclerosis (ALS) and ubiquitin positive frontotemporal lobar degeneration (FTLD). Wild type TDP-43 inclusions are a pathological hallmark of >95% of patients with sporadic ALS and of the majority of familial ALS cases, and they are also found in a significant proportion of FTLD cases. ALS is the most common form of motor neuron disease, characterized by progressive weakness and muscular wasting, and typically leads to death within a few years of diagnosis. To determine how the translocation and misfolding of TDP-43 contribute to ALS pathogenicity, it is crucial to define the dynamic behavior of this protein within the cellular environment. It is therefore necessary to develop cell models that allow the location of the protein to be defined. We report the use of TDP-43 with a tetracysteine tag for visualization using fluorogenic biarsenical compounds and show that this model displays features of ALS observed in other cell models. We also demonstrate that this labeling procedure enables live-cell imaging of the translocation of the protein from the nucleus into the cytosol

Degradation of pollutants in water by Fenton-like oxidation over LaFe-catalysts: Optimization by experimental design

The effect of different parameters such as temperature, type of catalyst and hydrogen peroxide (H2O2) concentration on the degradation of pollutants in water by Fenton-like oxidation was studied by using the Box-Behnken design (BBD), an effective statistical model to design the experiments. Concerning the heterogeneous catalysts, three bimetallic catalysts with lanthanum (La) and iron (Fe) ion-exchanged into zeolites (NaY and ZSM5) and a natural clay from Morocco were prepared and used for Fenton-like oxidation of organic pollutants in water. Tartrazine (Tar, a food coloring compound known as E102) and caffeine (Caf, a stimulant drug present in popular beverages such as coffee and tea) were selected as pollutants due to their presence in several commercial products for daily consumption. The BBD model indicated that the optimum catalytic conditions for Fenton-like reaction with an initial pollutant concentration of 30ppm at pH 3.0 were T=40°C and 90mM of H2O2. The maximum conversion values achieved with the best catalyst, LaFeZSM5, were 96.6% for Tar after 180min and 51.0% for Caf after 300min of reaction. To increase the conversion of Caf, a modified zeolite electrode was used for electro Fenton-like oxidation without H2O2, at room temperature.O.A. thanks to ERASMUS + Program for the mobility Ph.D. grant and O.B. thanks to Fundação para Ciência e Tecnologia, Portugal (FCT) for his Ph.D. grant (SFRH/BD/140362/2018).This research work has been funded by national funds funded through FCT/MCTES (PIDDAC) over the projects: LA/P/0045/2020 (ALiCE), UIDB/50020/2020 and UIDP/50020/2020 (LSRE-LCM), UIDB/04469/2020 (CEB) and by LA/P/0029/2020 (LABBELS), Centre of Chemistry (UID/QUI/0686/2020) and project BioTecNorte (operation NORTE-01-0145-FEDER-000004), supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).info:eu-repo/semantics/publishedVersio

Clusterin protects neurons against intracellular proteotoxicity.

It is now widely accepted in the field that the normally secreted chaperone clusterin is redirected to the cytosol during endoplasmic reticulum (ER) stress, although the physiological function(s) of this physical relocation remain unknown. We have examined in this study whether or not increased expression of clusterin is able to protect neuronal cells against intracellular protein aggregation and cytotoxicity, characteristics that are strongly implicated in a range of neurodegenerative diseases. We used the amyotrophic lateral sclerosis-associated protein TDP-43 as a primary model to investigate the effects of clusterin on protein aggregation and neurotoxicity in complementary in vitro, neuronal cell and Drosophila systems. We have shown that clusterin directly interacts with TDP-43 in vitro and potently inhibits its aggregation, and observed that in ER stressed neuronal cells, clusterin co-localized with TDP-43 and specifically reduced the numbers of cytoplasmic inclusions. We further showed that the expression of TDP-43 in transgenic Drosophila neurons induced ER stress and that co-expression of clusterin resulted in a dramatic clearance of mislocalized TDP-43 from motor neuron axons, partially rescued locomotor activity and significantly extended lifespan. We also showed that in Drosophila photoreceptor cells, clusterin co-expression gave ER stress-dependent protection against proteotoxicity arising from both Huntingtin-Q128 and mutant (R406W) human tau. We therefore conclude that increased expression of clusterin can provide an important defense against intracellular proteotoxicity under conditions that mimic specific features of neurodegenerative disease

Diagnosis of metastases in patients with papillary thyroid cancer by the measurement of thyroglobulin in fine needle aspirate

The widespread use of neck ultrasonography (US) during the follow-up of patients with papillary thyroid carcinoma (PTC) has led to the discovery of small cervical lymph nodes (LN). Although US has a high sensitivity for diagnosing LN, fine needle aspiration biopsy (FNA) and measurement of thyroglobulin in fine needle aspirates (FNA-Tg) have proven to be invaluable tools. The aim of this study is to determine the sensitivity of the combined use of neck US, FNA biopsy and FNA-Tg for diagnosis of cervical lymph nodes. We have studied 32 patients with 44 LN detected by US, 19 classified as inflammatory and 25 as suspicious. 15 of those 25 suspicious LN had high FNA-Tg (13 of the 15 had positive cytology and 2 indeterminate). All of these 15 LN (11 patients) were proven to be PTC metastasis by histopathology. All 19 inflammatory LN and those 10/25 suspicious LN, had cytology negative for malignancy and undetectable FNA-Tg. We conclude that fine needle aspiration biopsy and FNA-Tg combined with neck US are essential for detecting positive cervical lymph nodes due to its high sensitivity and specificity and it should be considered the standard for investigating locally recurrent disease in patients with PTC.Com a introdução da ultra-sonografia cervical (USC) no seguimento dos pacientes com carcinoma papilífero de tiróide (CPT), tornou-se freqüente o encontro de pequenos linfonodos (LNs) cervicais. Porém, apesar de a USC apresentar alta sensibilidade, o estudo citológico obtido por punção aspirativa (PAAF) e, nos últimos anos, a dosagem da tiroglobulina (Tg) no lavado da agulha da PAAF (Tg-PAAF) vêm assumindo papel importante no diagnóstico de LNs cervicais. O objetivo deste estudo é verificar a acurácia da combinação da USC, citologia e Tg-PAAF em LNs suspeitos. Estudamos 32 pacientes que apresentavam 44 LNs à USC, classificados como inflamatórios (19) ou suspeitos (25). Dos 25 LNs suspeitos, 15 apresentavam Tg-PAAF elevada (13 com citologia compatível com metástases e 2 com citologia não-diagnóstica). Esses 15 LNs (11 pacientes) foram confirmados como metástase de CP pelo exame histopatológico. Os 19 LNs inflamatórios e os 10/25 LNs suspeitos apresentaram citologia negativa e Tg-PAAF indetectável. Concluímos que a USC apresenta alta sensibilidade na detecção de linfonodos cervicais, porém citologia e dosagem de Tg-PAAF são fundamentais para o diagnóstico. A associação USC, citologia e Tg-PAAF pode ser considerada a abordagem mais sensível e específica na detecção de LNs metastáticos em pacientes com CPT.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de MedicinaLaboratório FleuryUNIFESP, EPM, Depto. de MedicinaSciEL

Direct enzymatic esterification of cotton and Avicel with wild-type and engineered cutinases

In this work, the surface of cellulose, either Avicel or cotton fabric, was modified using cutinases without any previous treatment to swell or to solubilise the polymer. Aiming further improvement of cutinase ester synthase activity on cellulose, an engineered cutinase was investigated. Wild-type cutinase from Fusarium solani and its fusion with the carbohydrate-binding module N1 from Cellulomonas fimi were able to esterify the hydroxyl groups of cellulose with distinct efficiencies depending on the acid substrate/solvent system used, as shown by titration and by ATR-FTIR. The carbonyl stretching peak area increased significantly after enzymatic treatment during 72 h at 30 °C. Cutinase treatment resulted in relative increases of 31 and 9 % when octanoic acid and vegetable oil were used as substrates, respectively. Cutinase-N1 treatment resulted in relative increases of 11 and 29 % in the peak area when octanoic acid and vegetable oil were used as substrates, respectively. The production and application of cutinase fused with the domain N1 as a cellulose ester synthase, here reported for the first time, is therefore an interesting strategy to pursuit.This work was co-funded by the European Social Fund through the management authority POPH and FCT, Postdoctoral fellowship reference: SFRH/BPD/47555/2008. The authors also want to thank Doctor Raul Machado for his valuable help on FTIR spectral data treatment

Quantitative structure-activity relationships (QSAR) of 4-amino-2,6-diarylpyrimidine-5-carbonitriles with anti-inflammatory activity

The experimental anti-inflammatory activities of eight 4-amino-2,6-diarylpyrimidine-5-carbonitriles were subjected to a QSAR analysis based on results from B3LYP/6-31G(d,p) and AM1 electronic structure calculations. Principal component analyses and regressions based on these data indicate that potentially more active compounds should have low dipole moment and partition coefficient values and also be affected by the values of the charges of the carbon atoms through which the two aromatic rings are bonded to the pyrimidinic ring. Two new molecules were predicted to be at least as active as those with the highest activities used in the model building stage. One of them, having a methoxy group attached to one of the aromatic rings, was predicted to have an anti-inflammatory activity value of 52.3%. This molecule was synthesized and its experimental activity was found to be 52.8%, in agreement with the AM1 theoretical prediction. This value is 5% higher than the largest value used for modeling