Mechanisms of social buffering of fear in zebrafish

The supplementary materials such the videos, are not present in the deposit due to incompatibilty of the extension of the files with the repository.Some humans thrive whereas others resign when exposed to threatening situations throughout life. Social support has been identified as an important modulator of these discrepancies in human behaviour, and other social animals also exhibit phenomena in which individuals recover better from aversive events when conspecifics are present - aka social buffering. Here we studied social buffering in zebrafish, by exposing focal fish to an aversive stimulus (alarm substance - AS) either in the absence or presence of conspecific cues. When exposed to AS in the presence of both olfactory (shoal water) and visual (sight of shoal) conspecific cues, focal fish exhibited a lower fear response than when tested alone, demonstrating social buffering in zebrafish. When separately testing each cue's effectiveness, we verified that the visual cue was more effective than the olfactory in reducing freezing in a persistent threat scenario. Finally, we verified that social buffering was independent of shoal size and coincided with a distinct pattern of co-activation of brain regions known to be involved in mammalian social buffering. Thus, this study suggests a shared evolutionary origin for social buffering in vertebrates, bringing new evidence on the behavioural, sensory and neural mechanisms underlying this phenomenon.info:eu-repo/semantics/publishedVersio

The importance of lipid conjugation on anti-fusion peptides against Nipah virus

© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).Nipah virus (NiV) is a recently emerging zoonotic virus that belongs to the Paramyxoviridae family and the Henipavirus genus. It causes a range of conditions, from asymptomatic infection to acute respiratory illness and fatal encephalitis. The high mortality rate of 40 to 90% ranks these viruses among the deadliest viruses known to infect humans. Currently, there is no antiviral drug available for Nipah virus disease and treatment is only supportive. Thus, there is an urgent demand for efficient antiviral therapies. NiV F protein, which catalyzes fusion between the viral and host membranes, is a potential target for antiviral drugs, as it is a key protein in the initial stages of infection. Fusion inhibitor peptides derived from the HRC-domain of the F protein are known to bind to their complementary domain in the protein's transient intermediate state, preventing the formation of a six-helix bundle (6HB) thought to be responsible for driving the fusion of the viral and cell membranes. Here, we evaluated the biophysical and structural properties of four different C-terminal lipid-tagged peptides. Different compositions of the lipid tags were tested to search for properties that might promote efficacy and broad-spectrum activity. Fluorescence spectroscopy was used to study the interaction of the peptides with biomembrane model systems and human blood cells. In order to understand the structural properties of the peptides, circular dichroism measurements and molecular dynamics simulations were performed. Our results indicate a peptide preference for cholesterol-enriched membranes and a lipid conjugation-driven stabilization of the peptide α-helical secondary structure. This work may contribute for the development of highly effective viral fusion against NiV inhibitors.This work was financially supported by Fundação para a Ciência e a Tecnologia—Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal), through projects PTDC/BBB-BQB/3494/2014, PTDC/QUI-BIQ/114774/2009, PTDC/CCI-BIO/28200/2017 and Pest-OE/EQB/LA0004/2011, and by National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), project R01AI114736, lead by Anne Moscona (Columbia University Medical Center, NY, USA). This work was also financially supported by Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020-Programa Operacional Competitividade e Internacionalização (POCI) and by national funds through FCT-MCTES. MCM, PMS and DL were supported by FCT-MCTES fellowships SFRH/BPD/118731/2016, SFRH/BD/118413/2016 and SFRH/BPD/92537/2013, respectively.info:eu-repo/semantics/publishedVersio

Understanding dengue virus capsid protein interaction with key biological targets

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/Supplementary information accompanies this paper at http://www.nature.com/srepDengue virus (DENV) causes over 500,000 hospitalizations and 20,000 deaths worldwide every year. Dengue epidemics now reach temperate regions due to globalization of trade and travel and climate changes. Currently, there are no successful therapeutic or preventive approaches. We previously developed a peptide drug lead, pep14-23, that inhibits the biologically relevant interaction of DENV capsid (C) protein with lipid droplets (LDs). Surprisingly, pep14-23 also inhibits DENV C interaction with very low-density lipoproteins (VLDL). We thus investigated the similarity between the proposed DENV C molecular targets in LDs and VLDL, respectively, the proteins perilipin 3 (PLIN3) and apolipoprotein E (APOE). APOE N-terminal and PLIN3 C-terminal regions are remarkably similar, namely APOE α -helix 4 (APOEα 4) and PLIN3 α -helix 5 (PLIN3α 5) sequences, which are also highly superimposable structurally. Interestingly, APOE α -helical N-terminal sequence and structure superimposes with DENV C α -helices α 1 and α 2. Moreover, the DENV C hydrophobic cleft can accommodate the structurally analogous APOEα 4 and PLIN3α 5 helical regions. Mirroring DENV C-LDs interaction (previously shown experimentally to require PLIN3), we experimentally demonstrated that DENV C-VLDL interaction requires APOE. Thus, the results fit well with previous data and suggest future drug development strategies targeting the above mentioned α –helical structures.We acknowledge the support of Fundação para a Ciência e Tecnologia – Ministério da Educação e Ciência (FCT-MEC, Portugal) project PTDC/SAU-ENB/117013/2010, and Calouste Gulbenkian Foundation (Portugal). AFF and ICM also acknowledge FCT-MEC fellowship SFRH/BD/77609/2011 and Investigador FCT Program research contract IF/00772/2013, respectively

Structural and functional properties of the capsid protein of Dengue and related Flavivirus

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Dengue, West Nile and Zika, closely related viruses of the Flaviviridae family, are an increasing global threat, due to the expansion of their mosquito vectors. They present a very similar viral particle with an outer lipid bilayer containing two viral proteins and, within it, the nucleocapsid core. This core is composed by the viral RNA complexed with multiple copies of the capsid protein, a crucial structural protein that mediates not only viral assembly, but also encapsidation, by interacting with host lipid systems. The capsid is a homodimeric protein that contains a disordered N-terminal region, an intermediate flexible fold section and a very stable conserved fold region. Since a better understanding of its structure can give light into its biological activity, here, first, we compared and analyzed relevant mosquito-borne Flavivirus capsid protein sequences and their predicted structures. Then, we studied the alternative conformations enabled by the N-terminal region. Finally, using dengue virus capsid protein as main model, we correlated the protein size, thermal stability and function with its structure/dynamics features. The findings suggest that the capsid protein interaction with host lipid systems leads to minor allosteric changes that may modulate the specific binding of the protein to the viral RNA. Such mechanism can be targeted in future drug development strategies, namely by using improved versions of pep14-23, a dengue virus capsid protein peptide inhibitor, previously developed by us. Such knowledge can yield promising advances against Zika, dengue and closely related Flavivirus.This work was supported by “Fundação para a Ciência e a Tecnologia–Ministério da Ciência, Tecnologia e Ensino Superior” (FCT-MCTES, Portugal) project PTDC/SAU-ENB/117013/2010, Calouste Gulbenkian Foundation (FCG, Portugal) project Science Frontiers Research Prize 2010. A.F.F., A.S.M. and J.C.R. also acknowledge FCT-MCTES fellowships SFRH/BD/77609/2011, PD/BD/113698/2015 and SFRH/BD/95856/2013, respectively. I.C.M. acknowledges FCT-MCTES Programs “Investigador FCT” (IF/00772/2013) and “Concurso de Estímulo ao Emprego Científico” (CEECIND/01670/2017). This work was also supported by UID/BIM/50005/2019, project funded by Fundação para a Ciência e a Tecnologia (FCT)/ Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through Fundos do Orçamento de Estado.info:eu-repo/semantics/publishedVersio

Alpha-thalassemia due to novel deletions and complex rearrangements in the subtelomeric region of chromosome 16p

2º Dia do Jovem Investigador do Instituto Nacional de Saúde Doutor Ricardo Jorge, INSA, 8 maio 2017Introduction: Inherited deletions removing the α-globin genes and/or their upstream regulatory elements (MCSs) give rise to alpha-thalassemia, one of the most common genetic recessive disorders worldwide. The pathology is characterized by microcytic hypochromic anemia due to reduction of the α-globin chain synthesis, which are essential for hemoglobin tetramerization. Material and Methods: In order to clarify the suggestive α-thalassemia phenotype in eleven patients, we performed Multiplex Ligation-dependent Probe Amplification with commercial and synthetic engineered probes, gap-PCR, and Sanger sequencing to search for deletions in the subtelomeric region of chromosome 16p. Results: We have identified five distinct large deletions, two of them novel, and one indel. The deletions range from approximately 3.3 to 323 kb, and i) remove the whole α-globin cluster; or ii) remove exclusively the upstream regulatory elements leaving the α-globin genes structurally intact. The indel consists in the loss of MCS-R2 (HS-40), which is the most important distal regulatory element for the α-globin gene expression, and the insertion of 39 bp, seemingly resulting from a complex rearrangement involving two DNA segments (probably from chromosome 3q) bridging the deletion breakpoints with a CC-bp orphan sequence in between. Finally, in one patient no α-globin deletion or point mutation were found. This patient revealed to be a very unusual case of acquired alpha-thalassemia associated with a myelodysplastic syndrome. Conclusions: Our study widens the spectrum of molecular lesions by which α-thalassemia may occur and emphasizes the importance of diagnosing large α-zero-deletions to provide patients with appropriate genetic counseling.info:eu-repo/semantics/publishedVersio

The Portuguese Society of Rheumatology position paper on the use of biosimilars

Biotechnological drugs have become a fundamental resource for the treatment of rheumatic patients. Patent expiry of some of these drugs created the opportunity for biopharmaceutical manufacturers to develop biosimilar drugs intended to be as efficacious as the originator product but with a lower cost to healthcare systems. Due to the complex manufacturing process and highly intricate structure of biologicals, a biosimilar can never be an exact copy of its reference product. Consequently, regulatory authorities issued strict preclinical and clinical guidelines to ensure safety and efficacy equivalence and, in September 2013, the biosimilar of infliximab was the first biosimilar monoclonal antibody to be authorized for use in the European Union. The current document is a position statement of the "Sociedade Portuguesa de Reumatologia" (Portuguese Society of Rheumatology) on the use of biosimilar drugs in rheumatic diseases. Two systematic literature reviews were performed, one concerning clinical trials and the other one concerning international position papers on biosimilars. The results were presented and discussed in a national meeting and a final position document was discussed, written and approved by Portuguese rheumatologists. Briefly, this position statement is contrary to automatic substitution of the originator by the biosimilar, defends either a different INN or the prescription by brand name, supports that switching between biosimilars and the originator molecule should be done after at least 6 months of treatment and based on the attending physician decision and after adequate patient information, recommends the registration of all biosimilar treated patients in Reuma.pt for efficacy, safety and immunogenicity surveillance, following the strategy already ongoing for originators, and opposes to extrapolation of indications approved to the originator to completely different diseases and/or age groups without adequate pre-clinical, safety or efficacy data.info:eu-repo/semantics/publishedVersio

Development of a detector (ALFA) to measure the absolute LHC luminosity at ATLAS

The ATLAS collaboration plans to determine the absolute luminosity of the CERN LHC at Interaction Point 1 by measuring the trajectory of protons elastically scattered at very small angles ($\mu rad$). A scintillating fibre tracker system called ALFA (Absolute Luminosity For ATLAS) is proposed for this measurement. Detector modules will be placed above and below the LHC beam axis in roman pot units at a distance of 240 m on each side of the ATLAS interaction point. They allow the detectors to approach the beam axis to millimeter distance. Overlap detectors also based on the scintillating fibre technology, will measure the precise relative position of the two detector modules. Results obtained during beam tests at DESY and at CERN validate the detectors design and demonstrate the achievable resolution. We also report about radiation hardness studies of the scintillating fibres to estimate the lifetime of the ALFA system at different operating conditions of the LHC

Genome-Wide Association between Branch Point Properties and Alternative Splicing

The branch point (BP) is one of the three obligatory signals required for pre-mRNA splicing. In mammals, the degeneracy of the motif combined with the lack of a large set of experimentally verified BPs complicates the task of modeling it in silico, and therefore of predicting the location of natural BPs. Consequently, BPs have been disregarded in a considerable fraction of the genome-wide studies on the regulation of splicing in mammals. We present a new computational approach for mammalian BP prediction. Using sequence conservation and positional bias we obtained a set of motifs with good agreement with U2 snRNA binding stability. Using a Support Vector Machine algorithm, we created a model complemented with polypyrimidine tract features, which considerably improves the prediction accuracy over previously published methods. Applying our algorithm to human introns, we show that BP position is highly dependent on the presence of AG dinucleotides in the 3′ end of introns, with distance to the 3′ splice site and BP strength strongly correlating with alternative splicing. Furthermore, experimental BP mapping for five exons preceded by long AG-dinucleotide exclusion zones revealed that, for a given intron, more than one BP can be chosen throughout the course of splicing. Finally, the comparison between exons of different evolutionary ages and pseudo exons suggests a key role of the BP in the pathway of exon creation in human. Our computational and experimental analyses suggest that BP recognition is more flexible than previously assumed, and it appears highly dependent on the presence of downstream polypyrimidine tracts. The reported association between BP features and the splicing outcome suggests that this, so far disregarded but yet crucial, element buries information that can complement current acceptor site models

West Nile virus capsid protein interacts with biologically relevant host lipid systems

Copyright © 2019 Martins, Carvalho, Faustino, Martins and Santos. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.West Nile and dengue viruses are closely related flaviviruses, originating mosquito-borne viral infections for which there are no effective and specific treatments. Their capsid proteins sequence and structure are particularly similar, forming highly superimposable α-helical homodimers. Measuring protein-ligand interactions at the single-molecule level yields detailed information of biological and biomedical relevance. In this work, such an approach was successfully applied on the characterization of the West Nile virus capsid protein interaction with host lipid systems, namely intracellular lipid droplets (an essential step for dengue virus replication) and blood plasma lipoproteins. Dynamic light scattering measurements show that West Nile virus capsid protein binds very low-density lipoproteins, but not low-density lipoproteins, and this interaction is dependent of potassium ions. Zeta potential experiments show that the interaction with lipid droplets is also dependent of potassium ions as well as surface proteins. The forces involved on the binding of the capsid protein with lipid droplets and lipoproteins were determined using atomic force microscopy-based force spectroscopy, proving that these interactions are K+-dependent rather than a general dependence of ionic strength. The capsid protein interaction with host lipid systems may be targeted in future therapeutic strategies against different flaviviruses. The biophysical and nanotechnology approaches employed in this study may be applied to characterize the interactions of other important proteins from different viruses, in order to understand their life cycles, as well as to find new strategies to inhibit them.This work was supported by Fundação para a Ciência e a Tecnologia–Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal) project PTDC/SAUENB/117013/2010. AF and AM also acknowledge FCT-MCTES fellowships SFRH/BD/77609/2011 and PD/BD/113698/2015, respectively. IM acknowledges consecutive funding from the FCT-MCTES fellowship SFRH/BPD/74287/2010 and the Program Investigador FCT (Research Contract IF/00772/2013). This work was also supported by UID/BIM/50005/2019, project funded by FCT-MCTES through Fundos do Orçamento do Estado.info:eu-repo/semantics/publishedVersio