The insertion/deletion in the DNA-binding region allows the discrimination and subsequent identification of the glucocorticoid receptor 1 (gr1) and gr2 nucleotide sequences in gilthead sea bream (Sparus aurata): Standardizing the gr nomenclature for a better understanding of the stress response in teleost fish species

Cortisol carries out its physiological mechanism of action through the recognition by the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) 1 (GR1) and GR2. Previous studies reported that the main difference between gr1 and gr2 nucleotide sequences resides in a 27-nucleotide insertion/deletion in the DNA-binding region, respectively. However, in gilthead sea bream (Sparus aurata) the annotation for gr1 and gr2 seems contradictory. The gr2 sequence possesses the characteristic 27-nucleotide insertion that, in fact, is associated with the gr1 nucleotide sequence. Thus, this study aimed to elucidate the nucleotide sequences for the gr1 and gr2 in gilthead sea bream. The Clustal Omega alignment for different fish species corroborated the presence of such 27-nucleotide insertion/deletion in the DNA-binding region for gr1 and gr2, respectively. Then, we design specific primers set for the amplification of the gilthead sea bream gr1 by polymerase chain reaction (PCR). Importantly, the gr1 nucleotide partial sequence has a high similarity with other gr1 sequences already published for other fish species, being present in all of them the 27-nucleotide insertion in the DNA-binding region. We also detected that in European sea bass the gr1 and gr2 sequences had not been named according to the 27-nucleotide insertion/deletion criteria in the DNA-binding region. Thus, our study makes an urgent call to the scientific community to discuss the establishment of an updated agreement that allows homogenizing the criteria for the nomenclature defining the gr1 and gr2 nucleotide sequences for a better understanding of the stress response in teleost fish species.This study thanks to the AGL2016-76069-C2-2- R, PID2020-117557RB-C21, PID2020-117557RB-C22 grants (AEI-MINECO; Spain). EV-V thanks the support of Fondecyt iniciacion grant (project number 11221308; Agencia Nacional de Investigacion y Desarrollo de Chile, Government of Chile). AK was the recipient of a Ministry of Science, Research, and Technology (Iran) fellowship. MT thanks for the support of the post-doctoral fellowship "Ramon y Cajal" (ref. RYC2019-026841-I) (Ministerio de Ciencia e Innovacion, Spanish Government). FER-L thanks the support of Fondecyt regular grant (project number: 1211841; Agencia Nacional de Investigacion y Desarrollo de Chile, Government of Chile)

The gene expression profile of the glucocorticoid receptor 1 (gr1) but not gr2 is modulated in mucosal tissues of gilthead sea bream (Sparus aurata) exposed to acute air-exposure stress

The perception of an acute stressor (short-duration; high-intensity) induces a physiological response that activates the hypothalamic-pituitary-interrenal (HPI) axis and the subsequent release of cortisol. Cortisol carries out its effect at the molecular level through its recognition by the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). Recently, we unveiled the nucleotide sequence of the glucocorticoid receptor 1 (gr1) and gr2 in gilthead sea bream (Sparus aurata). Importantly, GR1 and GR2 respond to different levels of cortisol concentration in fish and, consequently, play a differential role in the stress response. To date, and despite their relevance, no data describes the modulation of these receptors in response to an acute stressor in gilthead sea bream (S. aurata). In this study, we evaluated the kinetics of modulation of cortisol receptors expression (gr1, gr2, mr), and its similarity with the expression pattern of selected genes associated with stress (hsp70; enolase) and immune response (lysozyme; c3; il-1 beta; tnf-alpha; il-10; tgf-beta 1) in gilthead sea bream mucosal tissues (skin; gills; anterior gut). To do it, fish were acutely stressed by three-minute air exposure, and the expression profile was evaluated at zero, 1 h, 6 h, and 24 h post-stress (hps). The cortisol level in plasma and skin mucus peaked at 1 hps. All the mucosal tissues showed a time-dependent and tissue-specific upregulation of gr1 and mr. The immune-related genes showed the upregulation of il-1 beta at 6 hps (gills; anterior gut), and tnf-alpha and c3 at 24 hps (anterior gut). Taking together, our study concludes that fish subjected to three-minute air exposure modulated the expression of gr1 but not gr2 in mucosal tissues (skin; gills; anterior gut). Furthermore, our data reinforce the idea of a stimulatory effect induced in genes associated with the innate immune response after acute stress but focused at the mucosal level and in a time- and tissue-dependent manner

Antibiotics and Antimicrobial Resistance in the COVID-19 Era: Perspective from Resource-Limited Settings

The dissemination of COVID-19 around the globe has been followed by an increased consumption of antibiotics. This is related to the concern for bacterial superinfection in COVID-19 patients. The identification of bacterial pathogens is challenging in low and middle income countries (LMIC), as there are no readily-available and cost-effective clinical or biological markers that can effectively discriminate between bacterial and viral infections. Fortunately, faced with the threat of COVID-19 spread, there has been a growing awareness of the importance of antimicrobial stewardship programs, as well as infection prevention and control measures that could help reduce the microbial load and hence circulation of pathogens, with a reduction in dissemination of antimicrobial resistance. These measures should be improved particularly in developing countries. Studies need to be conducted to evaluate the worldwide evolution of antimicrobial resistance during the COVID-19 pandemic, because pathogens do not respect borders. This issue takes on even greater importance in developing countries, where data on resistance patterns are scarce, conditions for infectious pathogen transmission are optimal, and treatment resources are suboptimal

Antimicrobial functionalized genetically engineered spider silk

Genetically engineered fusion proteins offer potential as multifunctional biomaterials for medical use. Fusion or chimeric proteins can be formed using recombinant DNA technology by combining nucleotide sequences encoding different peptides or proteins that are otherwise not found together in nature. In the present study, three new fusion proteins were designed, cloned and expressed and assessed for function, by combining the consensus sequence of dragline spider silk with three different antimicrobial peptides. The human antimicrobial peptides human neutrophil defensin 2 (HNP-2), human neutrophil defensins 4 (HNP-4) and hepcidin were fused to spider silk through bioengineering. The spider silk domain maintained its self-assembly features, a key aspect of these new polymeric protein biomaterials, allowing the formation of b-sheets to lock in structures via physical interactions without the need for chemical crosslinking. These new functional silk proteins were assessed for antimicrobial activity against Gram e Escherichia coli and Gram þ Staphylococcus aureus and microbicidal activity was demonstrated. Dynamic light scattering was used to assess protein aggregation to clarify the antimicrobial patterns observed. Attenuated-total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and circular dichroism (CD) were used to assess the secondary structure of the new recombinant proteins. In vitro cell studies with a human osteosarcoma cell line (SaOs-2) demonstrated the compatibility of these new proteins with mammalian cells.Fundação para a Ciência e a Tecnologia (FCT) - Bolsa de doutoramento (SFRH/BD/28603/2006); Chimera project (PTDC/EBB-EBI/109093/2008); NIH and Tissue Engineering Resource Center EB003210, P41 EB002520, DE017207

AFM study of morphology and mechanical properties of a chimeric 2 spider silk and bone sialoprotein protein for bone regeneration

Atomic force microscopy (AFM) was used to assess a new chimeric protein consisting of a fusion protein of the consensus repeat for Nephila clavipes spider dragline protein and bone sialoprotein (6merþBSP). The elastic modulus of this protein in film form was assessed through force curves, and film surface roughness was also determined. The results showed a significant difference among the elastic modulus of the chimeric silk protein, 6merþBSP, and control films consisting of only the silk component (6mer). The behavior of the 6merþBSP and 6mer proteins in aqueous solution in the presence of calcium (Ca) ions was also assessed to determine interactions between the inorganic and organic components related to bone interactions, anchoring, and biomaterial network formation. The results demonstrated the formation of protein networks in the presence of Ca2þ ions, characteristics that may be important in the context of controlling materials assembly and properties related to bone formation with this new chimeric silk-BSP protein.Silvia Games thanks the Foundation for Science and Technology (FCT) for supporting her Ph.D. grant, SFRH/BD/28603/2006. This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283), the Chimera project (PTDC/EBB-EBI/109093/2008) funded by the FCT agency, the NIH (P41 EB002520) Tissue Engineering Resource Center, and the NIH (EB003210 and DE017207)

Mutations in Wnt2 Alter Presynaptic Motor Neuron Morphology and Presynaptic Protein Localization at the Drosophila Neuromuscular Junction

Wnt proteins are secreted proteins involved in a number of developmental processes including neural development and synaptogenesis. We sought to determine the role of the Drosophila Wnt7b ortholog, Wnt2, using the neuromuscular junction (NMJ). Mutations in wnt2 produce an increase in the number of presynaptic branches and a reduction in immunolabeling of the active zone proteins, Bruchpilot and synaptobrevin, at the NMJ. There was no change, however, in immunolabeling for the presynaptic proteins cysteine-string protein (CSP) and synaptotagmin, nor the postsynaptic proteins GluRIIA and DLG at the NMJ. Consistent with the presynaptic defects, wnt2 mutants exhibit approximately a 50% reduction in evoked excitatory junctional currents. Rescue, RNAi, and tissue-specific qRT-PCR experiments indicate that Wnt2 is expressed by the postsynaptic cell where it may serve as a retrograde signal that regulates presynaptic morphology and the localization of presynaptic proteins

Un examen actualizado de la percepción de las barreras para la implementación de la farmacogenómica y la utilidad de los pares fármaco/gen en América Latina y el Caribe

La farmacogenómica (PGx) se considera un campo emergente en los países en desarrollo. La investigación sobre PGx en la región de América Latina y el Caribe (ALC) sigue siendo escasa, con información limitada en algunas poblaciones. Por lo tanto, las extrapolaciones son complicadas, especialmente en poblaciones mixtas. En este trabajo, revisamos y analizamos el conocimiento farmacogenómico entre la comunidad científica y clínica de ALC y examinamos las barreras para la aplicación clínica. Realizamos una búsqueda de publicaciones y ensayos clínicos en este campo en todo el mundo y evaluamos la contribución de ALC. A continuación, realizamos una encuesta regional estructurada que evaluó una lista de 14 barreras potenciales para la aplicación clínica de biomarcadores en función de su importancia. Además, se analizó una lista emparejada de 54 genes/fármacos para determinar una asociación entre los biomarcadores y la respuesta a la medicina genómica. Esta encuesta se comparó con una encuesta anterior realizada en 2014 para evaluar el progreso en la región. Los resultados de la búsqueda indicaron que los países de América Latina y el Caribe han contribuido con el 3,44% del total de publicaciones y el 2,45% de los ensayos clínicos relacionados con PGx en todo el mundo hasta el momento. Un total de 106 profesionales de 17 países respondieron a la encuesta. Se identificaron seis grandes grupos de obstáculos. A pesar de los continuos esfuerzos de la región en la última década, la principal barrera para la implementación de PGx en ALC sigue siendo la misma, la "necesidad de directrices, procesos y protocolos para la aplicación clínica de la farmacogenética/farmacogenómica". Las cuestiones de coste-eficacia se consideran factores críticos en la región. Los puntos relacionados con la reticencia de los clínicos son actualmente menos relevantes. Según los resultados de la encuesta, los pares gen/fármaco mejor clasificados (96%-99%) y percibidos como importantes fueron CYP2D6/tamoxifeno, CYP3A5/tacrolimus, CYP2D6/opioides, DPYD/fluoropirimidinas, TMPT/tiopurinas, CYP2D6/antidepresivos tricíclicos, CYP2C19/antidepresivos tricíclicos, NUDT15/tiopurinas, CYP2B6/efavirenz y CYP2C19/clopidogrel. En conclusión, aunque la contribución global de los países de ALC sigue siendo baja en el campo del PGx, se ha observado una mejora relevante en la región. La percepción de la utilidad de las pruebas PGx en la comunidad biomédica ha cambiado drásticamente, aumentando la concienciación entre los médicos, lo que sugiere un futuro prometedor en las aplicaciones clínicas de PGx en ALC.Pharmacogenomics (PGx) is considered an emergent field in developing countries. Research on PGx in the Latin American and the Caribbean (LAC) region remains scarce, with limited information in some populations. Thus, extrapolations are complicated, especially in mixed populations. In this paper, we reviewed and analyzed pharmacogenomic knowledge among the LAC scientific and clinical community and examined barriers to clinical application. We performed a search for publications and clinical trials in the field worldwide and evaluated the contribution of LAC. Next, we conducted a regional structured survey that evaluated a list of 14 potential barriers to the clinical implementation of biomarkers based on their importance. In addition, a paired list of 54 genes/drugs was analyzed to determine an association between biomarkers and response to genomic medicine. This survey was compared to a previous survey performed in 2014 to assess progress in the region. The search results indicated that Latin American and Caribbean countries have contributed 3.44% of the total publications and 2.45% of the PGx-related clinical trials worldwide thus far. A total of 106 professionals from 17 countries answered the survey. Six major groups of barriers were identified. Despite the region’s continuous efforts in the last decade, the primary barrier to PGx implementation in LAC remains the same, the “need for guidelines, processes, and protocols for the clinical application of pharmacogenetics/pharmacogenomics”. Cost-effectiveness issues are considered critical factors in the region. Items related to the reluctance of clinicians are currently less relevant. Based on the survey results, the highest ranked (96%–99%) gene/drug pairs perceived as important were CYP2D6/tamoxifen, CYP3A5/tacrolimus, CYP2D6/opioids, DPYD/fluoropyrimidines, TMPT/thiopurines, CYP2D6/tricyclic antidepressants, CYP2C19/tricyclic antidepressants, NUDT15/thiopurines, CYP2B6/efavirenz, and CYP2C19/clopidogrel. In conclusion, although the global contribution of LAC countries remains low in the PGx field, a relevant improvement has been observed in the region. The perception of the usefulness of PGx tests in biomedical community has drastically changed, raising awareness among physicians, which suggests a promising future in the clinical applications of PGx in LAC

An Updated Examination of the Perception of Barriers for Pharmacogenomics Implementation and the Usefulness of Drug/Gene Pairs in Latin America and the Caribbean

Pharmacogenomics (PGx) is considered an emergent field in developing countries. Research on PGx in the Latin American and the Caribbean (LAC) region remains scarce, with limited information in some populations. Thus, extrapolations are complicated, especially in mixed populations. In this paper, we reviewed and analyzed pharmacogenomic knowledge among the LAC scientific and clinical community and examined barriers to clinical application. We performed a search for publications and clinical trials in the field worldwide and evaluated the contribution of LAC. Next, we conducted a regional structured survey that evaluated a list of 14 potential barriers to the clinical implementation of biomarkers based on their importance. In addition, a paired list of 54 genes/drugs was analyzed to determine an association between biomarkers and response to genomic medicine. This survey was compared to a previous survey performed in 2014 to assess progress in the region. The search results indicated that Latin American and Caribbean countries have contributed 3.44% of the total publications and 2.45% of the PGx-related clinical trials worldwide thus far. A total of 106 professionals from 17 countries answered the survey. Six major groups of barriers were identified. Despite the region’s continuous efforts in the last decade, the primary barrier to PGx implementation in LAC remains the same, the “need for guidelines, processes, and protocols for the clinical application of pharmacogenetics/pharmacogenomics”. Cost-effectiveness issues are considered critical factors in the region. Items related to the reluctance of clinicians are currently less relevant. Based on the survey results, the highest ranked (96%–99%) gene/drug pairs perceived as important were CYP2D6/tamoxifen, CYP3A5/tacrolimus, CYP2D6/opioids, DPYD/fluoropyrimidines, TMPT/thiopurines, CYP2D6/tricyclic antidepressants, CYP2C19/tricyclic antidepressants, NUDT15/thiopurines, CYP2B6/efavirenz, and CYP2C19/clopidogrel. In conclusion, although the global contribution of LAC countries remains low in the PGx field, a relevant improvement has been observed in the region. The perception of the usefulness of PGx tests in biomedical community has drastically changed, raising awareness among physicians, which suggests a promising future in the clinical applications of PGx in LAC