Changes in the biophysical properties of the cell membrane are involved in the response of neurospora crassa to staurosporine

Neurospora crassa is a non-pathogenic filamentous fungus widely used as a multicellular eukaryotic model. Recently, the biophysical properties of the plasma membrane of N. crassa conidia were thoroughly characterized. They evolve during conidial germination at a speed that depends on culture conditions, suggesting an important association between membrane remodeling and the intense membrane biogenesis that takes place during the germinative process. Staurosporine (STS) is a drug used to induce programmed cell death in various organisms. In N. crassa, STS up-regulates the expression of the ABC transporter ABC-3, which localizes at the plasma membrane and pumps STS out. To understand the role of plasma membrane biophysical properties in the fungal drug response, N. crassa was subjected to STS treatment during early and late conidial development stages. Following 1 h treatment with STS, there is an increase in the abundance of the more ordered, sphingolipid-enriched, domains in the plasmamembrane of conidia. This leads to higher fluidity in othermembrane regions. The global order of the membrane remains thus practically unchanged. Significant changes in sphingolipid-enriched domains were also observed after 15min challenge with STS, but they were essentially opposite to those verified for the 1 h treatment, suggesting different types of drug responses. STS effects on membrane properties that are more dependent on ergosterol levels also depend on the developmental stage. There were no alterations on 2 h-grown cells, clearly contrasting to what happens at longer growth times. In this case, the differences were more marked for longer STS treatment, and rationalized considering that the drug prevents the increase in the ergosterol/glycerophospholipid ratio that normally takes place at the late conidial stage/transition to the mycelial stage. This could be perceived as a drug-induced development arrest after 5 h growth, involving ergosterol, and pointing to a role of lipid rafts possibly related with an up-regulated expression of the ABC-3 transporter. Overall, our results suggest the involvement of membrane ordered domains in the response mechanisms to STS in N. crassa. Neurospora crassa is a non-pathogenic filamentous fungus widely used as a multicellular eukaryotic model. Recently, the biophysical properties of the plasma membrane of N. crassa conidia were thoroughly characterized. They evolve during conidial germination at a speed that depends on culture conditions, suggesting an important association between membrane remodeling and the intense membrane biogenesis that takes place during the germinative process. Staurosporine (STS) is a drug used to induce programmed cell death in various organisms. In N. crassa, STS up-regulates the expression of the ABC transporter ABC-3, which localizes at the plasma membrane and pumps STS out. To understand the role of plasma membrane biophysical properties in the fungal drug response, N. crassa was subjected to STS treatment during early and late conidial development stages. Following 1 h treatment with STS, there is an increase in the abundance of the more ordered, sphingolipid-enriched, domains in the plasmamembrane of conidia. This leads to higher fluidity in othermembrane regions. The global order of the membrane remains thus practically unchanged. Significant changes in sphingolipid-enriched domains were also observed after 15min challenge with STS, but they were essentially opposite to those verified for the 1 h treatment, suggesting different types of drug responses. STS effects on membrane properties that are more dependent on ergosterol levels also depend on the developmental stage. There were no alterations on 2 h-grown cells, clearly contrasting to what happens at longer growth times. In this case, the differences were more marked for longer STS treatment, and rationalized considering that the drug prevents the increase in the ergosterol/glycerophospholipid ratio that normally takes place at the late conidial stage/transition to the mycelial stage. This could be perceived as a drug-induced development arrest after 5 h growth, involving ergosterol, and pointing to a role of lipid rafts possibly related with an up-regulated expression of the ABC-3 transporter. Overall, our results suggest the involvement of membrane ordered domains in the response mechanisms to STS in N. crassa.Fundação para a Ciência e a Tecnologia (FCT), Portugal, is acknowledged for grants PTDC/BBB-BQB/6071/2014, UID/Multi/00612/2013, IF/00317/2012, and PT2020 referring to research unit 4293. FS acknowledges Ph.D. scholarship SFRH/BD/108031/2015, also from FCT

Taxonomias para os argumentos e contra-argumentos no debate sobre o princípio de identidade dos indiscerníveis

Este artigo visa apresentar uma taxonomia original dos argumentos mais difundidos contra o Princípio de Identidade dos Indiscerníveis ao longo da história da Filosofia, mas focando em versões defendidas no século XX e XXI; bem como uma taxonomia das respostas mais efetivas para esses argumentos usados no início do século XXI com uma breve avaliação sobre quais são as mais efetivas para cada argumento de ataque. O leitor também encontrará uma bibliografia atualizada sobre os debates envolvendo esses argumentos e contra-argumentos nas seções correspondentes. Palavras-chave: Virtudes epistêmicas. História da Filosofia. Louis Rougier. Liberalismo

Strategies for defending the Principle of Identity of Indiscernibles: a critical survey and a new approach

The Principle of Identity of Indiscernibles (PII) is the focus of much controversy in the history of Metaphysics and in contemporary Physics. Many questions rover the debate about its truth or falsehood, for example, to which objects the principle applies? Which properties can be counted as discerning properties? Is the principle necessary? In other words, which version of the principle is the correct and is this version true? This thesis aims to answer this questions in order to show that PII is a necessarily true principle of metaphysics. To accomplish this task, the reader will find, in this thesis, an encyclopaedical introduction to the history of PII and to the reasons it matters so much, followed by a presentation of the most famous arguments against it and the defences used against these arguments. Then, the reader finds in-depth discussion of the minutiae involved in postulating the principle as to make clear what is in fact being attacked and defended. With these preliminaries solved, a deeper analysis of these defences is presented aiming to discover which is the most appropriate example to use against the attacks to the principle. This analysis allowed a classification of these defences in four families with different strategies within them. Finally, with these defensive strategies at hand we are able to confront alleged counterexamples to PII in Mathematics with the intention to test these defences

Regulatory Cells, Cytokine Pattern and Clinical Risk Factors for Asthma in Infants and Young Children with Recurrent Wheeze

Several risk factors for asthma have been identified in infants and young children with recurrent wheeze. However, published literature has reported contradictory findings regarding the underlying immunological mechanisms. OBJECTIVES: This study was designed to assess and compare the immunological status during the first 2 years in steroid-naive young children with >or= three episodes of physician-confirmed wheeze (n=50), with and without clinical risk factors for developing subsequent asthma (i.e. parental asthma or a personal history of eczema and/or two of the following: wheezing without colds, a personal history of allergic rhinitis and peripheral blood eosinophilia >4%), with age-matched healthy controls (n=30). METHODS: Peripheral blood CD4(+)CD25(+) and CD4(+)CD25(high) T cells and their cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), GITR and Foxp3 expression were analysed by flow cytometry. Cytokine (IFN-gamma, TGF-beta and IL-10), CTLA-4 and Foxp3 mRNA expression were evaluated (real-time PCR) after peripheral blood mononuclear cell stimulation with phorbol 12-myristate 13-acetate (PMA) (24 h) and house dust mite (HDM) extracts (7th day). RESULTS: Flow cytometry results showed a significant reduction in the absolute number of CD4(+)CD25(high) and the absolute and percentage numbers of CD4(+)CD25(+)CTLA-4(+) in wheezy children compared with healthy controls. Wheezy children at a high risk of developing asthma had a significantly lower absolute number of CD4(+)CD25(+) (P=0.01) and CD4(+)CD25(high) (P=0.04), compared with those at a low risk. After PMA stimulation, CTLA-4 (P=0.03) and Foxp3 (P=0.02) expression was diminished in wheezy children compared with the healthy children. After HDM stimulation, CTLA-4 (P=0.03) and IFN-gamma (P=0.04) expression was diminished in wheezy children compared with healthy children. High-risk children had lower expression of IFN-gamma (P=0.03) compared with low-risk and healthy children and lower expression of CTLA-4 (P=0.01) compared with healthy children. CONCLUSIONS: Although our findings suggest that some immunological parameters are impaired in children with recurrent wheeze, particularly with a high risk for asthma, further studies are needed in order to assess their potential as surrogate predictor factors for asthma in early life

Computational Approaches Drive Developments in Immune-Oncology Therapies for PD-1/PD-L1 Immune Checkpoint Inhibitors

Funding Information: This research was funded by Fundação para a Ciência e Tecnologia (FCT) Portugal, grant number UIDB/50006/2020 (LAQV-REQUIMTE), UIDP/04378/2020 and UIDB/04378/2020 (UCIBIO) and LA/P/0140/2020 (i4HB), the European Commission GLYCOTwinning (GA 101079417), the EJPRD ProDGNE (EJPRD/0001/2020 EU 825575) and SI I&DT, DCMatters (AVISO Nº 17/SI/2019) REF 47212. F.P. gratefully acknowledges FCT for an Assistant Research Position (CEECIND/01649/2021). Publisher Copyright: © 2023 by the authors.Computational approaches in immune-oncology therapies focus on using data-driven methods to identify potential immune targets and develop novel drug candidates. In particular, the search for PD-1/PD-L1 immune checkpoint inhibitors (ICIs) has enlivened the field, leveraging the use of cheminformatics and bioinformatics tools to analyze large datasets of molecules, gene expression and protein–protein interactions. Up to now, there is still an unmet clinical need for improved ICIs and reliable predictive biomarkers. In this review, we highlight the computational methodologies applied to discovering and developing PD-1/PD-L1 ICIs for improved cancer immunotherapies with a greater focus in the last five years. The use of computer-aided drug design structure- and ligand-based virtual screening processes, molecular docking, homology modeling and molecular dynamics simulations methodologies essential for successful drug discovery campaigns focusing on antibodies, peptides or small-molecule ICIs are addressed. A list of recent databases and web tools used in the context of cancer and immunotherapy has been compilated and made available, namely regarding a general scope, cancer and immunology. In summary, computational approaches have become valuable tools for discovering and developing ICIs. Despite significant progress, there is still a need for improved ICIs and biomarkers, and recent databases and web tools have been compiled to aid in this pursuit.publishersversionpublishe

Toxicological evaluation of new tacrine analogues from 4-amino-1H-pyrrole-3-carbonitrile

Foundation for Science and Technology (FCT), FEDER and COMPET

Reconstruction of a Long Defect of the Ulnar Artery and Nerve with an Arterialized Neurovenous Free Flap in a Teenager: A Case Report and Literature Review

There is evidence that nerve flaps are superior to nerve grafts for bridging long nerve defects. Moreover, arterialized neurovenous flaps (ANVFs) have multiple potential advantages over traditional nerve flaps in this context. This paper describes a case of reconstruction of a long defect of the ulnar artery and nerve with an arterialized neurovenous free flap and presents a literature review on this subject. A 16-year-old boy sustained a stab wound injury to the medial aspect of the distal third of his right forearm. The patient was initially observed and treated at another institution where the patient was diagnosed with a flexor carpis ulnaris muscle and an ulnar artery section. The artery was ligated and the muscle was sutured. Four months later, the patient was referred to our institution with complaints of ulnar nerve damage, as well as hand pain and cold intolerance. Physical examination and ancillary tests supported the diagnosis of ulnar artery and nerve complete section. Surgery revealed an 8 cm hiatus of the ulnar artery and a 5 cm defect of the ulnar nerve. These gaps were bridged with a flow through ANVF containing the sural nerve and the lesser saphenous vein. The postoperative course was uneventful. Two years postoperatively, the patient had regained normal trophism and M5 strength in all previously paralyzed muscles according to the Medical Research Council Scale. Thermography revealed good perfusion in the right ulnar angiosome. The ANVF may be an expedite, safe and efficient option to reconstruct a long ulnar nerve and artery defect.info:eu-repo/semantics/publishedVersio

Characterization of the Temperature-Sensitive Mutations un-7 and png-1 in Neurospora crassa

The model filamentous fungus Neurospora crassa has been studied for over fifty years and many temperature-sensitive mutants have been generated. While most of these have been mapped genetically, many remain anonymous. The mutation in the N. crassa temperature-sensitive lethal mutant un-7 was identified by a complementation based approach as being in the open reading frame designated NCU00651 on linkage group I. Other mutations in this gene have been identified that lead to a temperature-sensitive morphological phenotype called png-1. The mutations underlying un-7 result in a serine to phenylalanine change at position 273 and an isoleucine to valine change at position 390, while the mutation in png-1 was found to result in a serine to leucine change at position 279 although there were other conservative changes in this allele. The overall morphology of the strain carrying the un-7 mutation is compared to strains carrying the png-1 mutation and these mutations are evaluated in the context of other temperature-sensitive mutants in Neurospora

The adult heart requires baseline expression of the transcription factor Hand2 to withstand RV pressure overload

AIMS: Research on the pathophysiology of right ventricular (RV) failure has, in spite of the associated high mortality and morbidity, lagged behind compared to the left ventricle (LV).Previous work from our lab revealed that the embryonic basic helix-loop-helix transcription factor heart and neural crest derivatives expressed-2 (Hand2) is re-expressed in the adult heart and activates a 'fetal gene program' contributing to pathological cardiac remodeling under conditions of LV pressure overload. As such, ablation of cardiac expression of Hand2 conferred protection to cardiac stress and abrogated the maladaptive effects that were observed upon increased expression levels. In this study, we aimed to understand the contribution of Hand2 to RV remodeling in response to pressure overload induced by pulmonary artery banding (PAB). METHODS AND RESULTS: In the present study, Hand2F/F and MCM- Hand2F/F mice were treated with tamoxifen (control and knockout, respectively) and subjected to six weeks of RV pressure overload induced by PAB. Echocardiographic- and MRI-derived hemodynamic parameters as well as molecular remodeling were assessed for all experimental groups and compared to sham-operated controls. Six weeks after PAB, levels of Hand2 expression increased in the control banded animals but, as expected, remained absent in the knockout hearts. Despite the dramatic differences in Hand2 expression, pressure overload resulted in impaired cardiac function independently of the genotype. In fact, Hand2 depletion seems to sensitize the RV to pressure overload as these mice develop more hypertrophy and more severe cardiac dysfunction. Higher expression levels of HAND2 were also observed in RV samples of human hearts from patients with pulmonary hypertension. In turn, the LV of RV-pressure overloaded hearts was also dramatically affected as reflected by changes in shape, decreased LV mass and impaired cardiac function. RNA sequencing revealed a distinct set of genes that are dysregulated in the pressure-overloaded RV, compared to the previously described pressure-overloaded LV. CONCLUSIONS: Cardiac-specific depletion of Hand2 is associated with severe cardiac dysfunction in conditions of RV pressure overload. While inhibiting Hand2 expression can prevent cardiac dysfunction in conditions of LV pressure overload, the same does not hold true for conditions of RV pressure overload. This study highlights the need to better understand the molecular mechanisms driving pathological remodeling of the RV in contrast to the LV, in order to better diagnose and treat patients with RV or LV failure. TRANSLATIONAL PERSPECTIVE: RV failure associated with pulmonary hypertension reduces long-term survival rate to 55% within 3 years, suggesting that 3 years after diagnosis almost half of the patients will die. To revert these numbers an adequate RV-specific and, therefore, more efficient treatment is needed. Our work suggests that current therapies and potential mechanisms underlying LV failure may not be suitable for RV failure. While Hand2 deletion is favorable in LV response to stress, it is particularly detrimental in the RV under similar conditions, and thus, highlighting potential severe consequences of not differentiating therapeutic targets or treatment for RV or LV failure