Indeterminate thyroid cytology: Detecting malignancy using analysis of nuclear images

Background: Thyroid nodules diagnosed as 'atypia of undetermined significance/ follicular lesion of undetermined significance' (AUS/FLUS) or 'follicular neoplasm/ suspected follicular neoplasm' (FN/SFN), according to Bethesda’s classification, represena challenge in clinical practice. Computerized analysis of nuclear images (CANI) could be a useful tool for these cases. Our aim was to evaluate the ability of CANI to correctly classify AUS/FLUS and FN/SFN thyroid nodules for malignancy. Methods: We studied 101 nodules cytologically classified as AUS/FLUS (n = 68) or FN/SFN (n = 33) from 97 thyroidectomy patients. Slides with cytological material were submitted for manual selection and analysis of the follicular cell nuclei for morphometric and texture parameters using ImageJ software. The histologically benign and malignant lesions were compared for such parameters which were then evaluated for the capacity to predict malignancy using the classification and regression trees gini model. The intraclass coefficient of correlation was used to evaluate method reproducibility. Results: In AUS/FLUS nodule analysis, the benign and malignant nodules differed for entropy (P < 0.05), while the FN/SFN nodules differed for fractal analysis, coefficient of variation (CV) of roughness, and CV-entropy (P < 0.05). Considering the AUS/FLUS and FN/SFN nodules separately, it correctly classified 90.0 and 100.0% malignant nodules, with a correct global classification of 94.1 and 97%, respectively. We observed that reproducibility was substantially or nearly complete (0.61–0.93) in 10 of the 12 nuclear parameters evaluated. Conclusion: CANI demonstrated a high capacity for correctly classifying AUS/FLUS and FN/SFN thyroid nodules for malignancy. This could be a useful method to help increase diagnostic accuracy in the indeterminate thyroid cytology.This study received financial support from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; processes number 2016/14987-0 and number 2016/14988-6). Further funding through 'Fundação para a Ciência e Tecnologi' – FCT and FEDER 'Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020'; by Operacional Programme for Competitiveness and Internationalization 'POCI' (Grant no. POCI-01-0145-FEDER-007274); by the 'Advancing cancer research: from basic knowledge to application' (grant no. NORTE-01-0145-FEDER-000029); and by the 'Projetos Estruturados de I & D & I', funded by Norte 2020 – Programa Operacional Regional do Norte

Activity of the fungus Pleurotus ostreatus and of its proteases on Panagrellus sp. larvae

Biological control has been shown to be one of the possible biotechnological applications of fungi and their proteases. The objective of this study was to evaluate the nematicidal activity of the fungus Pleurotus ostreatus and its proteases on Panagrellus sp. larvae. Proteolytic activity of P. ostreatus (PLO 06) was measured and characterized at different pHs, temperatures and in the presence of a inhibitor (PMSF). Daily samples of culture medium were collected in order to determine the period of maximum enzyme production. A zymogram showed the profile of several proteases. Predatory activity of the fungus P. ostreatus (PLO 06) was evaluated on Panagrellus sp. larvae (assay A) as well as the nematicidal activity of PLO 06 proteases on the same larvae (assay B). At pH 9 and 60°C, the activity of the proteases reached the maximum. In the presence of inhibitor, there was no proteolytic activity. A sample collected on the fifth day of incubation showed the highest enzyme activity. P. ostreatus demonstrated capture activity on larvae Panagrellus sp. The values of the reduction of the larvae (Assay A) were: day 1 (65.6%); day 2 (77.4%); day 3 (95.2%). The reduction of the larvae (Assay B) was 42%. P. ostreatus (PLO 06) and its proteases were very effective against Panagrellus sp. larvae, demonstrating great potential for use in integrated biological control.Keywords: Pleurotus, protease, Panagrellus sp., biological control, nematicidal. Abbreviation: PMSF, Phenylmethylsulfonyl fluoride

A Multi-task Network to Detect Junctions in Retinal Vasculature

Junctions in the retinal vasculature are key points to be able to extract its topology, but they vary in appearance, depending on vessel density, width and branching/crossing angles. The complexity of junction patterns is usually accompanied by a scarcity of labels, which discourages the usage of very deep networks for their detection. We propose a multi-task network, generating labels for vessel interior, centerline, edges and junction patterns, to provide additional information to facilitate junction detection. After the initial detection of potential junctions in junction-selective probability maps, candidate locations are re-examined in centerline probability maps to verify if they connect at least 3 branches. The experiments on the DRIVE and IOSTAR showed that our method outperformed a recent study in which a popular deep network was trained as a classifier to find junctions. Moreover, the proposed approach is applicable to unseen datasets with the same degree of success, after training it only once.Comment: MICCAI 2018 Camera Ready Versio

Mitochondria are the main source and one of the targets of Pb (lead)-induced oxidative stress in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model organism for studying lead (Pb) toxicity. Yeast cells of a laboratory S. cerevisiae strain (WT strain) were incubated with Pb concentrations up to 1,000 μmol/l for 3 h. Cells exposed to Pb lost proliferation capacity without damage to the cell membrane, and they accumulated intracellular superoxide anion (O2 .−) and hydrogen peroxide (H2O2). The involvement of the mitochondrial electron transport chain (ETC) in the generation of reactive oxygen species (ROS) induced by Pb was evaluated. For this purpose, an isogenic derivative ρ0 strain, lacking mitochondrial DNA, was used. The ρ0 strain, without respiratory competence, displayed a lower intracellular ROS accumulation and a higher resistance to Pb compared to the WT strain. The kinetic study of ROS generation in yeast cells exposed to Pb showed that the production of O2 .− precedes the accumulation of H2O2, which is compatible with the leakage of electrons from the mitochondrial ETC. Yeast cells exposed to Pb displayed mutations at the mitochondrial DNA level. This is most likely a consequence of oxidative stress. In conclusion, mitochondria are an important source of Pb-induced ROS and, simultaneously, one of the targets of its toxicity.The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013

LOCUS (LOng Covid–Understanding Symptoms, events and use of services in Portugal): A three-component study protocol

Approximately 10% of patients experience symptoms of Post COVID-19 Condition (PCC) after a SARS-CoV-2 infection. Akin acute COVID-19, PCC may impact a multitude of organs and systems, such as the cardiovascular, respiratory, musculoskeletal, and neurological systems. The frequency and associated risk factors of PCC are still unclear among both community and hospital settings in individuals with a history of COVID-19. The LOCUS study was designed to clarify the PCC's burden and associated risk factors. LOCUS is a multi-component study that encompasses three complementary building blocks. The "Cardiovascular and respiratory events following COVID-19" component is set to estimate the incidence of cardiovascular and respiratory events after COVID-19 in eight Portuguese hospitals via electronic health records consultation. The "Physical and mental symptoms following COVID-19" component aims to address the community prevalence of self-reported PCC symptoms through a questionnaire-based approach. Finally, the "Treating and living with Post COVID-19 Condition" component will employ semi-structured interviews and focus groups to characterise reported experiences of using or working in healthcare and community services for the treatment of PCC symptoms. This multi-component study represents an innovative approach to exploring the health consequences of PCC. Its results are expected to provide a key contribution to the optimisation of healthcare services design.info:eu-repo/semantics/publishedVersio

Evaluation of the role of glutathione in the lead-induced toxicity in Saccharomyces cerevisiae

The effect of intracellular reduced glutathione (GSH) in the lead stress response of Saccharomyces cerevisiae was investigated. Yeast cells exposed to Pb, for 3 h, lost the cell proliferation capacity (viability) and decreased intracellular GSH level. The Pb-induced loss of cell viability was compared among yeast cells deficient in GSH1 (∆gsh1) or GSH2 (∆gsh2) genes and wild-type (WT) cells. When exposed to Pb, ∆gsh1 and ∆gsh2 cells did not display an increased loss of viability, compared with WT cells. However, the depletion of cellular thiols, including GSH, by treatment of WT cells with iodoacetamide (an alkylating agent, which binds covalently to thiol group), increased the loss of viability in Pb-treated cells. In contrast, GSH enrichment, due to the incubation of WT cells with amino acids mixture constituting GSH (l-glutamic acid, l-cysteine and glycine), reduced the Pb-induced loss of proliferation capacity. The obtained results suggest that intracellular GSH is involved in the defence against the Pb-induced toxicity; however, at physiological concentration, GSH seems not to be sufficient to prevent the Pb-induced loss of cell viability

Phototriggered release of tetrapeptide AAPV from coumarinyl and pyrenyl cages

Ala-Ala-Pro-Val (AAPV) is a bioactive tetrapeptide that inhibits human neutrophil elastase (HNE), an enzyme involved in skin chronic inflammatory diseases like psoriasis. Caged derivatives of this peptide were prepared by proper N- and C-terminal derivatisation through a carbamate or ester linkage, respectively, with two photoactive moieties, namely 7-methoxycoumarin-2-ylmethyl and pyren-2-ylmethyl groups. These groups were chosen to assess the influence of the photosensitive group and the type of linkage in the controlled photorelease of the active molecule. The caged peptides were irradiated at selected wavelengths of irradiation (254, 300, and 350 nm), and the photolytic process was monitored by HPLC-UV. The results established the applicability of the tested photoactive groups for the release of AAPV, especially for the derivative bearing the carbamate-linked pyrenylmethyl group, which displayed the shortest irradiation times for the release at the various wavelengths of irradiation (ca. 4 min at 254 nm, 8 min at 300 nm and 46 min at 350 nm).Thanks are due to the Fundação para a Ciência e Tecnologia (FCT, Portugal) for financial support to the portuguese NMR network (PTNMR, Bruker Avance III 400- Univ. Minho), FCT and FEDER (European Fund for Regional Development)- COMPETE-QREN-EU for financial support through the Chemistry Research Centre of the University of Minho (Ref. UID/QUI/00686/2013 and UID/QUI/0686/2016). A PhD grant to A.M.S. (SFRH/BD/80813/2011) is also acknowledged.info:eu-repo/semantics/publishedVersio

Saccharomyces cerevisiae mutants affected in vacuole assembly or vacuolar H+-ATPase are hypersensitive to lead (Pb) toxicity

Lead is an important environmental pollutant. The role of vacuole, in Pb detoxification, was studied using a vacuolar protein sorting mutant strain (vps16Δ), belonging to class C mutants. Cells disrupted in VPS16 gene, did not display a detectable vacuolar-like structure. Based on the loss of cell proliferation capacity, it was found that cells from vps16Δ mutant exhibited a hypersensitivity to Pb-induced toxicity, compared to wild type (WT) strain. The function of vacuolar H+-ATPase (V-ATPase), in Pb detoxification, was evaluated using mutants with structurally normal vacuoles but defective in subunits of catalytic (vma1Δ or vma2Δ) or membrane domain (vph1Δ or vma3Δ) of V-ATPase. All mutants tested, lacking a functional V-ATPase, displayed an increased susceptibility to Pb, comparatively to cells from WT strain. Modification of vacuolar morphology, in Pb-exposed cells, was visualized using a Vma2p-GFP strain. The treatment of yeast cells with Pb originated the fusion of the medium size vacuolar lobes into one enlarged vacuole. In conclusion, it was found that vacuole plays an important role in the detoxification of Pb in Saccharomyces cerevisiae; in addition, a functional V-ATPase was required for Pb compartmentalization.The authors thank the Fundacao para a Ciencia e a Tecnologia (FCT) through the Portuguese Government for their financial support of this work through the grant PEST-OE/EQB/LA0023/2011 to IBB

Five-year review of an international clinical research-training program

The exponential increase in clinical research has profoundly changed medical sciences. Evidence that has accumulated in the past three decades from clinical trials has led to the proposal that clinical care should not be based solely on clinical expertise and patient values, and should integrate robust data from systematic research. As a consequence, clinical research has become more complex and methods have become more rigorous, and evidence is usually not easily translated into clinical practice. Therefore, the instruction of clinical research methods for scientists and clinicians must adapt to this new reality. To address this challenge, a global distance-learning clinical research-training program was developed, based on collaborative learning, the pedagogical goal of which was to develop critical thinking skills in clinical research. We describe and analyze the challenges and possible solutions of this course after 5 years of experience (2008-2012) with this program. Through evaluation by students and faculty, we identified and reviewed the following challenges of our program: 1) student engagement and motivation, 2) impact of heterogeneous audience on learning, 3) learning in large groups, 4) enhancing group learning, 5) enhancing social presence, 6) dropouts, 7) quality control, and 8) course management. We discuss these issues and potential alternatives with regard to our research and background

Correlation between nucleotide composition and folding energy of coding sequences with special attention to wobble bases

Background: The secondary structure and complexity of mRNA influences its accessibility to regulatory molecules (proteins, micro-RNAs), its stability and its level of expression. The mobile elements of the RNA sequence, the wobble bases, are expected to regulate the formation of structures encompassing coding sequences. Results: The sequence/folding energy (FE) relationship was studied by statistical, bioinformatic methods in 90 CDS containing 26,370 codons. I found that the FE (dG) associated with coding sequences is significant and negative (407 kcal/1000 bases, mean +/- S.E.M.) indicating that these sequences are able to form structures. However, the FE has only a small free component, less than 10% of the total. The contribution of the 1st and 3rd codon bases to the FE is larger than the contribution of the 2nd (central) bases. It is possible to achieve a ~ 4-fold change in FE by altering the wobble bases in synonymous codons. The sequence/FE relationship can be described with a simple algorithm, and the total FE can be predicted solely from the sequence composition of the nucleic acid. The contributions of different synonymous codons to the FE are additive and one codon cannot replace another. The accumulated contributions of synonymous codons of an amino acid to the total folding energy of an mRNA is strongly correlated to the relative amount of that amino acid in the translated protein. Conclusion: Synonymous codons are not interchangable with regard to their role in determining the mRNA FE and the relative amounts of amino acids in the translated protein, even if they are indistinguishable in respect of amino acid coding.Comment: 14 pages including 6 figures and 1 tabl