Estudio de la contribución de una mutación puntual en el gen atpE en la resistencia a bedaquilina de Mycobacterium abscessus

Mycobacterium abscessus se está convirtiendo en uno de los patógenos más preocupantes en hospitales y centros de fibrosis quística de todo el mundo. Se trata de una micobacteria no tuberculosa de crecimiento rápido causante de enfermedades respiratorias, más frecuentes en pacientes inmunocomprometidos. La relevancia de esta micobacteria reside en su resistencia frente a los antituberculosos convencionales así como a la mayoría de antibióticos que se disponen actualmente para uso clínico y que, por tanto, dificulta el tratamiento de los pacientes que la presentan (Cortes, Nessar, & Singh, 2010).La aprobación en 2012 de un medicamento para tratar la tuberculosis multirresistente denominado Bedaquilina y la comprobación de que también presenta actividad antimicrobiana contra M. abscessus supuso una alternativa en el tratamiento de las enfermedades causadas por este microorganismo (Mahajan, 2013). Sin embargo, la identificación de un aislado clínico que presenta una mutación en el gen diana de la Bedaquilina (atpE) que codifica la subunidad c de la ATP sintasa, puede ver comprometida la eficacia del medicamento.En este trabajo, se pretende obtener una cepa de M. abscessus que presente un alelo del gen atpE con la mutación encontrada haciendo uso de técnicas de ingeniería genética y caracterizar su resistencia a Bedaquilina en comparación con la cepa silvestre. Para ello, se clonó el gen atpE con la mutación en un vector apropiado utilizando distintas técnicas de clonaje así como utilizando una técnica novedosa, el recombineering. Aunque no se pudo secuenciar, hay evidencias de que dicha mutación confiere resistencia a Bedaquilina.Este trabajo proporciona una estrategia de caracterización de las cepas bacterianas a través de estudios genotípicos que permite optimizar cualquier régimen terapéutico para evitar tratamientos ineficaces al estudiar la resistencia a antimicrobianos y puede ser trasladada a otros microorganismos.<br /

Identificación mediante la huella del pabellón auricular

Desarrollo del actual protocolo para el tratamiento de huellas de oreja y análisis tanto de la técnica como de la validez jurídica actual de la identificación por este métodoUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Dealing with the ubiquity of phthalates in the laboratory when determining plasticizers by gas chromatography/mass spectrometry and PARAFAC

Determining plasticizers and other additives migrated from plastic materials becomes a hard task when these substances are already present in the laboratory environment. This work dealt with this drawback in the multiresidue determination of four plasticizers (2,6-di-tert-butyl-4-methyl-phenol (BHT), diisobutyl phthalate (DiBP), bis(2-ethylhexyl) adipate (DEHA) and diisononyl phthalate (DiNP)) and a UV stabilizer (benzophenone (BP)) by gas chromatography/mass spectrometry (GC/MS) using DiBP-d4 as internal standard. The ubiquity of DiBP by a non-constant leaching process in the laboratory was detected, which could not guarantee the achievement of a trustworthy quantification. To handle this, the assessment of the level of DiBP in solvent blanks having fixed the probabilities of false non-compliance (α) and false compliance (β) at 0.01 was performed. On the other hand, another special case was that of DiNP, in whose chromatogram finger peaks appear because of an array of possible C9 isomers. PARAFAC, used for the identification and quantification of all the substances, is a useful chemometric tool that enabled a more reliable determination of this analyte since no peak areas were considered but chromatographic and spectral loadings. Since phthalates may migrate from rubber latex items, an evaluation of the existence of matrix effects on the determination of the five analytes was conducted prior to an extraction with hexane from a dummy for infants. As matrix effects were present, the quantification of the compounds under study was performed following the standard addition method using PARAFAC sample loadings as response variable. As a result, the presence of BHT was confirmed, being its concentration equal to 37.87 μg L−1. Calibrations based on PARAFAC yielded the following values for the decision limit (CCα): 1.16 μg L−1 for BHT, 1.34 μg L−1 for BP, 1.84 μg L−1 for DEHA and 51.42 μg L−1 for DiNP(for α = 0.05 and two replicates).Ministerio de Economía y Competitividad (CTQ2014-53157-R).M.L

Clinical landscape of LAG-3-targeted therapy

Lymphocyte-activated gene 3 (LAG-3) is a cell surface inhibitory receptor and a key regulator of immune homeostasis with multiple biological activities related to T-cell functions. LAG-3 is considered a next-generation immune checkpoint of clinical importance, right next to programmed cell death protein 1 (PD-1) and cytotoxic T-cell lymphocyte antigen-4 (CTLA-4). Indeed, it is the third inhibitory receptor to be exploited in human anticancer immunotherapies. Several LAG-3-antagonistic immunotherapies are being evaluated at various stages of preclinical and clinical development. In addition, combination therapies blocking LAG-3 together with other immune checkpoints are also being evaluated at preclinical and clinical levels. Indeed, the co-blockade of LAG-3 with PD-1 is demonstrating encouraging results. A new generation of bispecific PD-1/LAG-3-blocking agents have also shown strong capacities to specifically target PD-1+ LAG-3+ highly dysfunctional T cells and enhance their proliferation and effector activities. Here we identify and classify preclinical and clinical trials conducted involving LAG-3 as a target through an extensive bibliographic research. The current understanding of LAG-3 clinical applications is summarized, and most of the publically available data up to date regarding LAG-3-targeted therapy preclinical and clinical research and development are reviewed and discussed.The OncoImmunology group is funded by the Spanish Association against Cancer ( AECC ) [grant number PROYE16001ESCO ]; Instituto de Salud Carlos III (ISCIII)-FEDER project grants [grant numbers FIS PI17/02119, FIS PI20/00010, COV20/00000, TRANSPOCART ICI19/00069]; a Biomedicine Project grant from the Department of Health of the Government of Navarre [grant number BMED 050-2019 ]; strategic projects from the Department of Industry, Government of Navarre (AGATA, Ref. 0011-1411-2020-000013; LINTERNA, Ref. 0011-1411-2020-000033; DESCARTHES, 0011-1411-2019-000058); European Project Horizon 2020 Improved Vaccination for Older Adults (ISOLDA; ID: 848166); Crescendo Biologics Ltd. supported the OncoImmunology group for the development and testing of PD-1 and LAG-3 bispecifics

Sudden cessation of fluoxetine before alcohol drinking reinstatement alters microglial morphology and TLR4/inflammatory neuroadaptation in the rat brain

Preclinical studies on the efects of abrupt cessation of selective serotonin reuptake inhibitors (SSRIs), a medication often prescribed in alcohol use disorder (AUD) patients with depression, results in alcohol consumption escalation after resuming drinking. However, a potential neuroinfammatory component on this escalation remains unexplored despite the immunomodulatory role of serotonin. Here, we utilized a rat model of 14-daily administration of the SSRI fuoxetine (10 mg/kg/day) along alcohol self-administration deprivation to study the efects of fuoxetine cessation on neuroinfammation after resuming alcohol drinking. Microglial morphology and infammatory gene expression were analyzed in prelimbic cortex, striatum, basolateral amygdala and dorsal hippocampus. Results indicated that alcohol drinking reinstatement increased microglial IBA1 immunoreactivity and altered morphometric features of activated microglia (fractal dimension, lacunarity, density, roughness, and cell area, perimeter and circularity). Despite alcohol reinstatement, fuoxetine cessation modifed microglial morphology in a brain region-specifc manner, resulting in hyper-ramifed (spatial complexity of branching), reactive (lower heterogeneity and circularity)-like microglia. We also found that microglial cell area correlated with changes in mRNA expression of chemokines (Cx3cl1/fractalkine, Cxcl12/SDF1α, Ccl2/MCP1), cytokines (IL1β, IL6, IL10) and the innate immune toll-like receptor 4 (TLR4) in dorsal hippocampus. Specifcally, TLR4 correlated with microglial spatial complexity assessed by fractal dimension in striatum, suggesting a role in process branching. (...)Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Funding for open access charge: Universidad de Málaga/CBUA. RETICS Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISCIII), Ministerio de Ciencia e Innovación and European Regional Development Funds-European Union (ERDF-EU) (Grant No. RD16/0017/0001); ISCIII, ERDF-EU (Grant No. PI17/02026, Grant No. PI19/01577); Ministerio de Sanidad, Delegación de Gobierno para el Plan Nacional sobre Drogas (Grant No. PND 2020/048, Grant No. PND 2019/040, Grant No. PND 2018/044, Grant No. PND 2018/033); and Consejería de Salud y Familia, Junta de Andalucía (Neuro-RECA, Grant No. RIC-0111–2019). JS (Grant No. CPII17/00024), FJP (Grant No. CPII19/00022) and AS (Grant No. CPII19/00031) hold “Miguel Servet II” research contracts from the National System of Health, ISCIII, ERDF-EU. FJP also holds a “Nicolas Monardes” contract from Servicio Andaluz de Salud, Consejería de Salud y Familia, Junta de Andalucía (Grant No. C1-0049–2019). PR (Grant No. CP19/00068) hold “Miguel Servet I” research contracts from the National System of Health, ISCIII, ERDF-EU. The funding sources had no further role in study design; in the collection, analysis and interpretation of data; in writing of the report; and in the decision to submit the paper for publication

Plasma endocannabinoid alterations as a link in the comorbidity of Alzheimer's disease and type 2 diabetes mellitus.

Over the last several years, studies have suggested a role of endocannabinoids such as 2-AG and 2-OG in the impairment of β-cell function and insulin secretion, as well as in the control of lipid and glucose metabolism in the periphery. Besides, alterations in the endocannabidiome are associated with the development of dementia. Since type 2 diabetes mellitus (T2DM) is an established risk factor for late-life cognitive decline, we sought to evaluate the possible link between the alterations in plasma endocannabinoids as potential biomarkers of cognitive decline in elderly patients with T2DM. In the present study, we evaluated the plasma levels of endocannabinoids in a cohort of elder controls and patients suffering from T2DM, with either mild cognitive impairment (MCI) or Alzheimer’s disease (AD). The cognitive performance of these patients was evaluated at the beginning of the study and their regional brain metabolic activity was assessed by PET-18FDG. We found that T2DM patients showed decreased levels of brain metabolic activity determined by PET-18FDG in the inferior parietal lobe, caudate, and thalamus, which were decreased and related to poor cognitive performance shown by both BLESSED and MMSE tests. Segregation of patients according to their cognitive status (MCI or AD) showed lower basal metabolism in the aforementioned regions, which was exacerbated in patients with AD and T2DM comorbidity. Correlation analysis showed plasma levels of the endocannabinoids 2-AG, 2-LG, and 2-OG were inversely related to brain metabolism in these areas, as well as to worse BLESSED and MMSE scores. Our results depict that plasma endocannabinoids are potential biomarkers linking the development of cognitive decline to the occurrence of T2DM.Consejería de Universidad, Investigación e Innovación, Junta de Andalucía, grant number PI21/00291. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Cutting-edge: preclinical and clinical development of the first approved LAG-3 inhibitor

Immune checkpoint inhibitors (ICIs) have revolutionized medical practice in oncology since the FDA approval of the first ICI 11 years ago. In light of this, Lymphocyte-Activation Gene 3 (LAG-3) is one of the most important next-generation immune checkpoint molecules, playing a similar role as Programmed cell Death protein 1 (PD-1) and Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4). 19 LAG-3 targeting molecules are being evaluated at 108 clinical trials which are demonstrating positive results, including promising bispecific molecules targeting LAG-3 simultaneously with other ICIs. Recently, a new dual anti-PD-1 (Nivolumab) and anti-LAG-3 (Relatimab) treatment developed by Bristol Myers Squibb (Opdualag), was approved by the Food and Drug Administration (FDA) as the first LAG-3 blocking antibody combination for unresectable or metastatic melanoma. This novel immunotherapy combination more than doubled median progression-free survival (PFS) when compared to nivolumab monotherapy (10.1 months versus 4.6 months). Here, we analyze the large clinical trial responsible for this historical approval (RELATIVITY-047), and discuss the preclinical and clinical developments that led to its jump into clinical practice. We will also summarize results achieved by other LAG-3 targeting molecules with promising anti-tumor activities currently under clinical development in phases I, I/II, II, and III. Opdualag will boost the entry of more LAG-3 targeting molecules into clinical practice, supporting the accumulating evidence highlighting the pivotal role of LAG-3 in cancer.The OncoImmunology group is funded by the Spanish Association against Cancer (AECC) [grant number PROYE16001ESCO]; Instituto de Salud Carlos III (ISCIII)-FEDER project grants [grant numbers FIS PI17/02119, FIS PI20/00010, COV20/00000, TRANSPOCART ICI19/00069]; a Biomedicine Project grant from the Department of Health of the Government of Navarre [grant number BMED 050-2019]; strategic projects from the Department of Industry, Government of Navarre (AGATA, Ref. 0011-1411-2020-000013; LINTERNA, Ref. 0011-1411-2020-000033; DESCARTHES, 0011-1411-2019-000058); European Project Horizon 2020 Improved Vaccination for Older Adults (ISOLDA; ID: 848166); Crescendo Biologics Ltd. supported the OncoImmunology group for the development and testing of PD-1 and LAG-3 bispecifics

Structure and function of the N-terminal extension of the formin INF2

In INF2—a formin linked to inherited renal and neurological disease in humans—the DID is preceded by a short N-terminal extension of unknown structure and function. INF2 activation is achieved by Ca2+-dependent association of calmodulin (CaM). Here, we show that the N-terminal extension of INF2 is organized into two α-helices, the first of which is necessary to maintain the perinuclear F-actin ring and normal cytosolic F-actin content. Biochemical assays indicated that this helix interacts directly with CaM and contains the sole CaM-binding site (CaMBS) detected in INF2. The residues W11, L14 and L18 of INF2, arranged as a 1-4-8 motif, were identified as the most important residues for the binding, W11 being the most critical of the three. This motif is conserved in vertebrate INF2 and in the human population. NMR and biochemical analyses revealed that CaM interacts directly through its C-terminal lobe with the INF2 CaMBS. Unlike control cells, INF2 KO cells lacked the perinuclear F-actin ring, had little cytosolic F-actin content, did not respond to increased Ca2+ concentrations by making more F-actin, and maintained the transcriptional cofactor MRTF predominantly in the cytoplasm. Whereas expression of intact INF2 restored all these defects, INF2 with inactivated CaMBS did not. Our study reveals the structure of the N-terminal extension, its interaction with Ca2+/CaM, and its function in INF2 activatio