Identification and characterization of new isoforms of human fas apoptotic inhibitory molecule (FAIM)

Altres ajuts: La Marató de TV3 (201414-30); Fellowship BES-2014-069550Fas Apoptosis Inhibitory Molecule (FAIM) is an evolutionarily highly conserved death receptor antagonist, widely expressed and known to participate in physiological and pathological processes. Two FAIM transcript variants have been characterized to date, namely FAIM short (FAIM-S) and FAIM long (FAIM-L). FAIM-S is ubiquitously expressed and serves as an anti-apoptotic protein in the immune system. Furthermore, in neurons, this isoform promotes NGF-induced neurite outgrowth through NF-кB and ERK signaling. In contrast FAIM-L is found only in neurons, where it exerts anti-apoptotic activity against several stimuli. In addition to these two variants, in silico studies point to the existence of two additional isoforms, neither of which have been characterized to date. In this regard, here we confirm the presence of these two additional FAIM isoforms in human fetal brain, fetal and adult testes, and placenta tissues. We named them FAIM-S_2a and FAIM-L_2a since they have the same sequence as FAIM-S and FAIM-L, but include exon 2a. PCR and western blot revealed that FAIM-S_2a shows ubiquitous expression in all the tissues and cellular models tested, while FAIM-L_2a is expressed exclusively in tissues of the nervous system. In addition, we found that, when overexpressed in non-neuronal cells, the splicing factor nSR100 induces the expression of the neuronal isoforms, thus identifying it as responsible for the generation of FAIM-L and FAIM-L_2a. Functionally, FAIM-S_2a and FAIM-L_2a increased neurite outgrowth in response to NGF stimulation in a neuronal model. This observation thus, supports the notion that these two isoforms are involved in neuronal differentiation. Furthermore, subcellular fractionation experiments revealed that, in contrast to FAIM-S and FAIM-L, FAIM-S_2a and FAIM-L_2a are able to localize to the nucleus, where they may have additional functions. In summary, here we report on two novel FAIM isoforms that may have relevant roles in the physiology and pathology of the nervous system

CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3

ObjectivesCARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain.MethodsIn total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.ResultsIn total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA–48 (263/377), blaKPC–3 (62/377), blaVIM–1 (28/377), and blaNDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5).ConclusionThis study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3

Neuronal phenotype characterization of FAIM-KO mice /

El gen Fas apoptotic inhibitory molecule 1 (FAIM1) fue descubierto en las células B del sistema y está evolutivamente conservado. FAIM1 codifica al menos para cuatro isoformas, de entre ellas FAIM-S y FAIM-L son las más relevantes. FAIM-L está codificada por splicing alternativo, se expresa sólo en neuronas y presenta veintidós aminoácidos más que la isoforma descrita en células B, FAIM-S. Ambas proteínas fueron identificadas como antagonistas de los receptores de muerte en diferentes células, siendo FAIM-L la única isoforma capaz de ejecutar una acción protectora en neuronas. Recientemente se ha revelado que las isoformas de FAIM1 también protegen contra la muerte inducida por estrés oxidativo. Estás proteínas ejercen adicionalmente otras funciones no relacionadas con la apoptosis. FAIM-S regula distintas funciones en el sistema inmune, participa en el metabolismo lipídico y glucolítico, en la agregación proteica y en el crecimiento neurítico. FAIM-L modula la transmisión y la plasticidad sináptica y la neurodegeneración axonal. Además, las isoformas de FAIM1 son importantes en diferentes patologías como el mieloma múltiple, Alzheimer y la obesidad.Aunque diversas funciones de FAIM1 han sido definidas en el sistema nervioso, todavía quedan muchas preguntas por elucidar sobre su papel en el sistema nervioso. Por ello, decidimos estudiar el fenotipo neuronal del ratón FAIM-KO. Inesperadamente observamos que los ratones FAIM-KO desarrollaban convulsiones tras ser expuestos a estímulos sensoriales y/o estresantes y que esta predisposición se iniciaba en la edad adulta. Este hallazgo nos llevó a estudiar qué alteraciones podían favorecer el desarrollo de estas crisis convulsivas. Observamos que los ratones FAIM-KO que convulsionaban presentaban alteraciones en el hipocampo típicas de cerebro epilépticos tales como aumento de la neurogénesis, expresión ectópica del neuropéptido Y y aumento de la expresión de c-fos en el hipocampo. Sin embargo, estos cambios no aparecían en los ratones sin convulsiones. Aunque los ratones FAIM-KO no presentaban inflamación o muerte celular, su densidad de células gliales estaba ligeramente disminuida y la densidad de ciertas poblaciones de interneuronas también estaban alteradas en el hipocampo. Además, la expresión de algunas proteínas sinápticas (SNAP25 y vGLUT) y apoptóticas (Fas y XIAP) así como en el número de dendritas primarias de las neuronas granulares estaban alterados en el hipocampo de estos ratones. En cuanto a su comportamiento, estos ratones eran más activos y tenían una mayor predisposición a interaccionar con otros ratones, mientras que presentaban deficiencias en sus habilidades cognitivas y de construcción del nido. Estos resultados revelan interesantes nuevas funciones de FAIM1 en el cerebro, sin embargo, es necesario corroborar el papel de esta proteína en el desarrollo de convulsiones ya que los ratones FAIM-KO usados en este estudio tienen un fondo genético mixto.Fas apoptotic inhibitory molecule 1 (FAIM1) gene is highly conserved in the evolution and was firstly characterized in B cells in 1999. FAIM1 codifies for at least four isoforms, among which FAIM-L and FAIM-S are the most relevant. FAIM-L is formed by alternative splicing, is exclusively expressed in neurons and contains twenty-two amino acids more than the first isoform characterized in B cells, FAIM-S. The first described role of FAIM1 was this protective effect against death-receptor mediated cell death. FAIM-S and FAIM-L exert this function in different cells, and FAIM-L is the only FAIM1 isoform that protects neurons against induced apoptosis. Recently, a protective role of FAIM1 in cellular stress-induced cell death has been reported. FAIM1 have been also implicated in non-apoptotic functions. FAIM-S participates in different non-apoptotic functions in the immune system, in glucose and lipid metabolism, in protein aggregation and in neurite outgrowth. Otherwise, FAIM-L acts as a regulator in synaptic transmission, axonal degeneration and synaptic plasticity process of long-term depression. Besides, FAIM1 isoforms have relevant roles in different diseases. In that sense, FAIM-S is deregulated in multiple myeloma and obesity and FAIM-L is downregulated in Alzheimer's disease. Although some FAIM1 isoforms functions have been reported in nervous system, many questions about their actions in CNS are still a high exciting mystery. Therefore, we decided to characterize the neuronal phenotype of FAIM-KO mice to unravel FAIM1 functions in brain. Surprisingly, we observed age-dependent sensory-induced seizures in FAIM-KO mice. Owing to that finding, the work was focused to unravel mechanisms related with seizure susceptibility in FAIM-KO mice. FAIM-KO mice with seizure showed typical hippocampal cellular and molecular alterations reported in epilepsy models such ectopic neuropeptide Y expression, increase neurogenesis and increase c-fos expression in hippocampus. However, these effects have not been observed in non-convulsive FAIM-KO mice. Although, neuroinflammation and cell death were not apparent in FAIM-KO mice, these animals exhibit a decrease in glial density in hippocampus and alterations in parvalbumin- and calretinin-containing interneuron populations. These mice also exhibited slightly changes in the expression of synaptic proteins SNAP25 and vGLUT1, deregulation in mRNA levels of Fas and XIAP, and alteration in primary dendrites of granule cells in hippocampus. Interestingly, FAIM-KO mice were hyperactive, had impairment in cognitive tasks and in nest construction and exhibited an increase in social interactions. These results point to new exciting roles of FAIM1 in CNS. However, the use in this work of FAIM-KO mice derived of mixed background makes the replication of these experiments in other genetic background necessary for ensuring a role of FAIM1 in seizure susceptibility

Fas apoptosis inhibitory molecules: more than death-receptor antagonists in the nervous system

The importance of death receptor (DR) signaling in embryonic development and physiological homeostasis is well established, as is the existence of several molecules that modulate DRs function, among them Fas Apoptotis Inhibitory Molecules. Although FAIM 1, FAIM 2, and FAIM 3 inhibit Fas‐induced cell death, they are not structurally related, nor do they share expression patterns. Moreover, they inhibit apoptosis through completely different mechanisms. FAIM 1 and FAIM 2 protect neurons from DR ‐induced apoptosis and are involved in neurite outgrowth and neuronal plasticity. FAIM 1 inhibits Fas ligand‐ and tumor necrosis factor alpha‐induced apoptosis by direct interaction with Fas receptor and through the stabilization of levels of X‐linked inhibitor of apoptosis protein, a potent anti‐apoptotic protein that inhibits caspases. Low FAIM 1 levels are found in Alzheimer's disease, thus sensitizing neurons to tumor necrosis factor alpha and prompting neuronal loss. FAIM 2 protects from Fas by direct interaction with Fas receptor, as well as by modulating calcium release at the endoplasmic reticulum through interaction with Bcl‐xL . Several studies prove the role of FAIM 2 in diseases of the nervous system, such as ischemia, bacterial meningitis, and neuroblastoma. The less characterized member of the FAIM family is FAIM 3, which is expressed in tissues of the digestive and urinary tracts, bone marrow and testes, and restricted to the cerebellum in the nervous system. FAIM 3 protects against DR ‐induced apoptosis by inducing the expression of other DR ‐antagonists such as CFLAR or through the interaction with the DR ‐adaptor protein Fas‐associated via death domain. FAIM 3 null mouse models reveal this protein as an important mediator of inflammatory autoimmune responses such as those triggered in autoimmune encephalomyelitis. Given the differences between FAIM s and the variety of processes in which they are involved, here we sought to provide a concise review about these molecules and their roles in the physiology and pathology of the nervous system

Identification and characterization of new isoforms of human fas apoptotic inhibitory molecule (FAIM)

Altres ajuts: La Marató de TV3 (201414-30); Fellowship BES-2014-069550Fas Apoptosis Inhibitory Molecule (FAIM) is an evolutionarily highly conserved death receptor antagonist, widely expressed and known to participate in physiological and pathological processes. Two FAIM transcript variants have been characterized to date, namely FAIM short (FAIM-S) and FAIM long (FAIM-L). FAIM-S is ubiquitously expressed and serves as an anti-apoptotic protein in the immune system. Furthermore, in neurons, this isoform promotes NGF-induced neurite outgrowth through NF-кB and ERK signaling. In contrast FAIM-L is found only in neurons, where it exerts anti-apoptotic activity against several stimuli. In addition to these two variants, in silico studies point to the existence of two additional isoforms, neither of which have been characterized to date. In this regard, here we confirm the presence of these two additional FAIM isoforms in human fetal brain, fetal and adult testes, and placenta tissues. We named them FAIM-S_2a and FAIM-L_2a since they have the same sequence as FAIM-S and FAIM-L, but include exon 2a. PCR and western blot revealed that FAIM-S_2a shows ubiquitous expression in all the tissues and cellular models tested, while FAIM-L_2a is expressed exclusively in tissues of the nervous system. In addition, we found that, when overexpressed in non-neuronal cells, the splicing factor nSR100 induces the expression of the neuronal isoforms, thus identifying it as responsible for the generation of FAIM-L and FAIM-L_2a. Functionally, FAIM-S_2a and FAIM-L_2a increased neurite outgrowth in response to NGF stimulation in a neuronal model. This observation thus, supports the notion that these two isoforms are involved in neuronal differentiation. Furthermore, subcellular fractionation experiments revealed that, in contrast to FAIM-S and FAIM-L, FAIM-S_2a and FAIM-L_2a are able to localize to the nucleus, where they may have additional functions. In summary, here we report on two novel FAIM isoforms that may have relevant roles in the physiology and pathology of the nervous system

mRNA secondary structure.

<p>5´UTR sequences of FAIM isoforms structures as shown by the output of the RNAStructure web server (<a href="http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html" target="_blank">http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html</a>). The optimal secondary prediction for all the sequences was obtained in dot-bracket notation with the lowest free energy structure for the input sequence. Colour annotation of the structures provides information about the confidence in the prediction of a specific pair (base paired or unpaired nucleotides). The highest probabilities are red and the lowest are purple.</p

Isoforms expression in cell lines.

<p><b>A:</b> SH-SY5Y cells were transfected with the pCDNA3-FLAG-FAIM-S, pCDNA3-FLAG-FAIM-S_2a, pCDNA3-FLAG-FAIM-L or pCDNA3-FLAG-FAIM-L_2a vector. At a range of time points, cells were harvested and protein expression was assessed by western blot using an anti-FLAG antibody (dilution 1:20000). <b>B</b>: PC12 cells were transfected with the isoform vectors (above mentioned) and treated with MG-132 (2.5 μM). Cell extracts were then resolved by western blot analysis, and FAIM expression was measured using an anti-FLAG antibody (dilution 1:20000). <b>C:</b> HEK293T cells transfected with pcDNA3-FLAG-FAIM-L, pcDNA3-FLAG-FAIM-S, pcDNA3-FLAG-FAIM-L-2a or pcDNA3-FLAG-FAIM-S-2a vector were lysed, and protein extracts were analyzed by western blot. An anti-FAIM-L (anti-2b FAIM, specific for neuronal exon 2b) and anti-FAIM (that recognizes the common part of the isoforms) were used. Anti-tubulin was used as a loading control. Two different exposures of the film are shown in order to facilitate observation of the bands of all isoforms. DIV: days <i>in vitro</i> (n = 3).</p

FAIM-S_2a and FAIM-L_2a are localized in the cytoplasm and nucleus.

<p><b>A:</b> Western blot analysis using anti-FLAG to detect the presence of FAIM-S, FAIM-L, FAIM-S_2a and FAIM-L_2a in the distinct cellular compartments. Anti-calnexin was used as a marker for the membrane fraction, anti-actin as a marker of the cytosolic fraction, and anti-Tri-Methyl-Histone H3 as a marker of the nucleus. <b>B:</b> Immunofluorescence in Vero cells 24 h after transfection with pcDNA3-GFP containing the extra-long isoforms. Anti-calnexin (reticular protein), Mitotracker (mitochondrial marker) and Hoechst (nuclei staining) were used to examine the co-localization of FAIM isoforms. Scale bars 10 <b>μ</b>m.</p

Treatment with Actinomycin D (ActD).

<p>SH-SY5Y cells were treated with ActD for a range of times (3, 6, 9 and 12 h). <b>A:</b> The half-life of mRNA was measured by treating cells with ActD (5 μg/ml) and collecting total RNA at the times shown, whereupon the levels of <i>FAIM</i> mRNA and <i>18S mRNA</i> (a stable, housekeeping control mRNA) were measured by RT–qPCR analysis. mRNA half-life was calculated as the time needed to reduce transcript levels to half (50%, discontinuous line) of their initial abundance at time 0. <b>B:</b> Number of cycles needed to detect similar size product of FAIM-S, FAIM-L, FAIM-S_2a and FAIM-L_2a by qPCR (SybrGreen) using the following pairs of primers: (1bF2/3R for FAIM-S; 2bF/3R for FAIM-L; 2aF/2a3R for FAIM-S_2a and 2aF/2bR for FAIM-L_2a).</p

Thermodynamic stability of the secondary structure of the 5´UTR.

<p>Thermodynamic stability of the secondary structure of the 5´UTR.</p