RB1 gene mutation up-date, a meta-analysis based on 932 reported mutations available in a searchable database

BACKGROUND: Retinoblastoma, a prototype of hereditary cancer, is the most common intraocular tumour in children and potential cause of blindness from therapeutic eye ablation, second tumours in germ line carrier's survivors, and even death when left untreated. The molecular scanning of RB1 in search of germ line mutations lead to the publication of more than 900 mutations whose knowledge is important for genetic counselling and the characterization of phenotypic-genotypic relationships. RESULTS: A searchable database (RBGMdb) has been constructed with 932 published RB1 mutations. The spectrum of these mutations has been analyzed with the following results: 1) the retinoblastoma protein is frequently inactivated by deletions and nonsense mutations while missense mutations are the main inactivating event in most genetic diseases. 2) Near 40% of RB1 gene mutations are recurrent and gather in sixteen hot points, including twelve nonsense, two missense and three splicing mutations. The remainder mutations are scattered along RB1, being most frequent in exons 9, 10, 14, 17, 18, 20, and 23. 3) The analysis of RB1 mutations by country of origin of the patients identifies two groups in which the incidence of nonsense and splicing mutations show differences extremely significant, and suggest the involvement of predisposing ethnic backgrounds. 4) A significant association between late age at diagnosis and splicing mutations in bilateral retinoblastoma patients suggests the occurrence of a delayed-onset genotype. 5) Most of the reported mutations in low-penetrance families fall in three groups: a) Mutations in regulatory sequences at the promoter resulting in low expression of a normal Rb; b) Missense and in-frame deletions affecting non-essential sequence motifs which result in a partial inactivation of Rb functions; c) Splicing mutations leading to the reduction of normal mRNA splicing or to alternative splicing involving either true oncogenic or defective (weak) alleles. CONCLUSION: The analysis of RB1 gene mutations logged in the RBGMdb has shown relevant phenotype-genotype relationships and provided working hypothesis to ascertain mechanisms linking certain mutations to ethnicity, delayed onset of the disease and low-penetrance. Gene profiling of tumors will help to clarify the genetic background linked to ethnicity and variable expressivity or delayed onset phenotypes

Cauliflower mosaic virus is preferentially acquired from the phloem by its aphid vectors

Cauliflower mosaic virus (CaMV) is transmitted in a non-circulative manner by aphids following the helper strategy. Helper proteins P2 and P3 act as a bridge between virions and the aphid cuticle. Electronic monitoring of aphid stylet activities (EPG technique), transmission tests and electron microscopy showed that CaMV is preferentially acquired from the phloem by its most common aphid vectors, Brevycorine brassicae and Myzus persicae. We also found that CaMV is semipersistently transmitted and that the rate of acquisition does not follow a typical bimodal curve. Instead, the virus could be acquired from non-phloem tissues at a low and fairly constant rate after one or more intracellular punctures within a few minutes, but the probability of acquisition rose significantly when aphids reached the phase of committed ingestion from the phloem. The acquisition rate of CaMV did not increase with increasing number of intracellular punctures, but the total duration of intracellular puncture was one of the variables selected by the stepwise logistic regression model used to fit the data that best explained acquisition of CaMV. Furthermore, aphids reaching the phloem faster had a higher probability of acquiring the virus. Our results support the hypothesis that multiple intracellular punctures of epidermal and mesophyll cells result in loading aphids with the CaMV-encoded aphid transmission factor (P2), and that aphids, in most cases, subsequently acquire CaMV particles during phloem sap ingestion. Consistently, immunoelectron microscopy showed that P3–virions are frequently found in the sieve element lumen, whereas P2 could not be detected.We are indebted to the Spanish Ministry of Science and Technology (Research Grant, no. AGL:2000-2006) for funding this work. Also, we acknowledge the Comunidad Autónoma de Madrid (Spain) and CAPES (Brazil) for funding the fellowships awarded to I.P. and S.L., respectively. M.D. was awarded a fellowship from the ACI-JC of the French Ministry for Research. We thank Takii Ltd seed company for kindly providing turnip ‘Just Right’ seedsPeer reviewe

Decoding human cardiac stem cells regenerative potential in acute myocardial infarction

Acute Myocardial Infarction (AMI) remains a leading cause of death worldwide. After AMI, clinical restauration of blood flow aggravates tissue damage (Ischemia/Reperfusion, I/R injury), critically decreasing the number of viable cardiomyocytes (CMs). Human myocardium harbors a population of endogenous cardiac stem/progenitor cells (CSCs) that is activated upon I/R injury, contributing to myocardial repair through the establishment of an auto/paracrine molecular crosstalk between CSCs and CMs in stress. Transplantation of CSCs is currently being tested in several clinical trials, and although some improvements have been reported regarding decrease of the infarcted area, it is still not enough to show benefit over pharmacological standard-of-care. Our work aims at combining the development of relevant I/R in vitro human cell models with implementation of advanced mass spectrometry (MS)-based proteomic tools to further characterize hCSC and unveil associated regenerative mechanisms upon AMI. hCSCs employed in the phase I/II clinical trial CARE-MI (NCT02439398) were used (allogeneic therapy). Different strategies were explored to recapitulate both phases of I/R injury in the human adult heart, including: the use of human adult/mature cells, 3D culture system and stirred-tank bioreactor technology. Firstly, we developed a transwell co-culture cell based I/R model, with human CSCs and human induced pluripotent stem cell derived CMs (hiPSC-CMs). Following this work, and aiming at further improving the relevance of the I/R injury in vitro setup, 3D hiPSC-CM aggregate cultures and bioreactors were combined, allowing the control/monitoring of environmental parameters such as pH and dissolved oxygen, critical in the context of I/R physiology. Important features of I/R injury were successfully captured in the two models, including hiPSC-CM death upon reperfusion, disruption of cell ultra-structure organization, as well as increased release of angiogenic and inflammatory cytokines, consistent with the described pathophysiology of AMI. hCSCs response to I/R was further probed using whole proteome analysis (including quantitative SWATH methodology), allowing us to propose new pathways in the hCSCs-mediated regenerative process along the different phases of I/R injury through the identification of more than 3800 proteins and quantification of 714 proteins. Our data shows that our AMI-setup up-regulates hCSC proteins associated with several pro-migratory, proliferation and stress response-related pathways. Moreover, our results reinforce the idea that paracrine-mediated mechanisms are a central response in hCSC activation, with the enrichment of several paracrine signaling and pro-angiogenic pathways. We also show for the first time increased CXCL6 secretion by hCSCs upon injury, suggesting a relevant role of this angiogenic cytokine in hCSC mediated myocardial regeneration. Overall, multiple strategies were used to develop novel and robust I/R injury in vitro models, recapitulating several features of the human adult myocardium. The systems established allowed to better characterize hCSC mechanisms of action in response to AMI contexts. The knowledge generated has the potential to be used in the development of novel strategies excelling endogenous and transplanted hCSCs regenerative potential

Unveiling human Cardiac Stem Cells regenerative potential in Ishemia/Reperfusion Injury

After an Acute Myocardial Infarction (AMI), Ischemia-Reperfusion (I/R) injury is responsible for a critical decrease in the number of viable cardiomyocytes (hCMs). Human myocardium harbors a population of endogenous cardiac stem cells (hCSCs) that is activated upon I/R injury, contributing to myocardial repair through the establishment of an auto/paracrine molecular crosstalk between hCSCs and hCMs in stress. Clinical trials involving transplantation of hCSCs into the infarcted myocardium have demonstrated the potential of these cells. Although some improvements have been reported regarding increase in viable myocardium and improved tissue contractility, extensive data indicates that transplanted cells do not survive in the myocardium and this has led to the postulation of a paracrine mechanism for the observed beneficial effects. Using the same cells currently employed in the allogenous hCSCs transplantation clinical trial CARE-MI (EUDRA 2013-001358-81), our work aims at setting up the first in vitro human I/R injury model in order to better decipher the mechanisms of action of hCSCs upon AMI using proteomic tools. Mono-cultures of donor derived hCSCs, hCMs and co-cultures with the two cell types were established using human donor derived CSCs and cardiomyocytes derived from human induced pluripotent stem cells at different maturation stages (hiPSC-CMs). Ischemia was mimicked by substituting growth media by Ischemia Mimetic Solution and placing the cells at 0% O2 for 5 hours. In the reperfusion step, cells were placed back in their physiological culture conditions (3% O2). The effect of I/R injury in hCSCs was accessed by total proteome analysis (using LC-MS) at different time points. Growth factor secretion, cells’ viability, as well as hCSCs proliferation was also monitored in both mono- and co-culture systems. More than 2000 proteins were identified in hCSCs exposed to injury including proteins associated with mitochondrial dysfunction and oxidative stress. Important features of I/R injury were successfully captured, namely hCSCs proliferation activation upon insult, increase in key growth factors secretion, and the protective effect of hCSCs on hiPSC-CMs. This system will allow further understanding on the molecular landscape of the myocardium during AMI, namely regarding hCSCs regenerative response and hCMs survival. The knowledge generated in this work will hopefully potentiate the development of novel molecular and cell-based therapies for myocardium regeneration. The authors acknowledge the projects CARE-MI (HEALTH-2009-242038), CARDIOSTEM (MITP-TB/ECE/0013/2013, the Portuguese Foundation for Science and Technology- FCT (PTDC/BBB-BIO/1414) and iNOVA4Health (UID/Multi/04462/2013) for financial support. MJCS is a recipient of the FCT fellowship SFRH/BPD/52339/2013

Quality of live and quality of work life in organic versus conventional farmers

Quality of life (QOL) and quality of work life (QOWL) play a key role in the overall concept of sustainability. In this paper we analyze QOL and QOWL variables in relation to the type of livestock farm (organic/conventional) and the use of some quality label (PDO Protected Designation of Origin, PGI Protected Geographical Indication). Data were collected through regular visits in 2011 to 70 small ruminant farms in Spain using five-point Likert items with 1-5 range. Of all the 70 farms, 6 are organics, 39 have a quality label and 17 use traditional manufacturing methods. The possession of quality labels in the small ruminant farms analyzed seems not to be related with the quality of life and work. Farmers with traditional production show higher valuation of quality of life but not of quality of work. Organic farms provide quality of life and quality of work significantly better than conventional ones

Advancing the knowledge on immunomodulatory properties of human cardiac stem cells

Transplantation of allogeneic human cardiac/stem progenitor cells (hCSC) is currently being tested in several phase I/II clinical trials as a novel and promising therapy for restauration of myocardial tissue function in acute myocardial infarction (AMI) patients. Previous findings demonstrate that these cells have an immune suppressive profile, interacting with different populations from the immune system, resulting in overall attenuation of myocardium inflammation. However, transplanted hCSCs are still recognized and cleared from the injured site impairing long retention times in the tissue that could be translated into a higher clinical benefit. In this work, different models of allogeneic hCSC/ T-lymphocyte interaction in vitro were explored, using the same hCSCs employed in the allogenous hCSCs transplantation phase I/II clinical trial CARE-MI, NCT02439398. T lymphocytes were cultured either in direct contact with hCSCs, or using transwell inserts or with hCSC conditioned medium. In our results, we show that IFN-γ activation is correlated with an increase in hCSC indoleamine 2,3-dioxygenase (IDO) enzyme expression. We also show a significant inhibition of T lymphocyte inhibition when cultivating human peripheral blood mononuclear cells (hPBMCs) in direct cell-cell contact, using transwells or with activated hCSC conditioned medium, combined with tryptophan depletion and kyurenine (a tryptophan metabolite) accumulation in activated hCSCs conditioned medium. These findings provide evidence, that although playing a role in the process, PDL-1 cell contact dependent T-regulatory cell modulation is not the exclusive neither the central mechanism involved in T-lymphocyte proliferation inhibition. This finding further supports the prominent paracrine-based beneficial CSC activities in the host tissue. Our results demonstrate for the first time that hCSCs exert an immune-suppressive effect on T lymphocyte proliferation through a paracrine mechanism associated with IDO enzyme mediated tryptophan metabolism. The knowledge generated contributes not only to a better understanding on hCSC immunomodulatory mechanisms, but also open new avenues in the development of new hCSC transplantation strategies in allogeneic settings

Synergism interaction between genetic polymorphisms in drug metabolizing enzymes and NSAIDs on upper gastrointestinal haemorrhage: a multicenter case-control study

Genetic variation; Non-steroidal anti-inflammatory drugs; Upper gastrointestinal haemorrhageVariació genètica; Fàrmacs antiinflamatoris no esteroides; Hemorràgia gastrointestinal superiorVariación genética; Medicamentos antiinflamatorios no esteroideos; Hemorragia gastrointestinal superiorBackground Interindividual genetic variations contribute to differences in patients’ response to drugs as well as to the development of certain disorders. Patients who use non-steroidal anti-inflammatory drugs (NSAIDs) may develop serious gastrointestinal disorders, mainly upper gastrointestinal haemorrhage (UGIH). Studies about the interaction between NSAIDs and genetic variations on the risk of UGIH are scarce. Therefore, we investigated the effect of 16 single nucleotide polymorphisms (SNPs) involved in drug metabolism on the risk of NSAIDs-induced UGIH. Materials and methods We conducted a multicenter case-control study of 326 cases and 748 controls. Participants were sub-grouped into four categories according to NSAID exposure and genetic profile. We estimated odds ratios (ORs) and their 95% confidence intervals (CI) using generalized linear mixed models for dependent binomial variables and then calculated the measures of interaction, synergism index (S), and relative excess risk due to interaction (RERI). We undertook stratified analyses by the type of NSAID (aspirin, non-aspirin). Results We observed an excess risk of UGIH due to an interaction between any NSAID, non-aspirin NSAIDs or aspirin and carrying certain SNPs. The greatest excess risk was observed for carriers of: rs2180314:C>G [any NSAID: S = 3.30 (95%CI: 1.24–8.80), RERI = 4.39 (95%CI: 0.70–8.07); non-aspirin NSAIDs: S = 3.42 (95%CI: 1.12–10.47), RERI = 3.97 (95%CI: 0.44–7.50)], and rs4809957:A>G [any NSAID: S = 2.11 (95%CI: 0.90–4.97), RERI = 3.46 (95%CI: −0.40–7.31)]. Aspirin use by carriers of rs6664:C>T is also associated with increased risk of UGIH [ORaspirin(+),wild-type: 2.22 (95%CI: 0.69–7.17) vs. ORaspirin(+),genetic-variation: 7.72 (95%CI: 2.75–21.68)], yet larger sample size is needed to confirm this observation. Conclusions The joint effect of the SNPs s2180314:C>G and rs4809957:A>G and NSAIDs are more than three times higher than the sum of their individual effects. Personalized prescriptions based on genotyping would permit a better weighing of risks and benefits from NSAID consumption.This study was supported by grants from: Carlos III Health Institute (P I12/02414, of the P E I+D+I 2012-2016); Fondo Europeo de Desarrollo Regional (FEDER); the Novartis, Pfizer and Dr Esteve pharmaceutical companies; the Health Research Fund/Fondo de Investigaciońn Sanitaria (P I021512, P I021364, P I020661, and P I021572); Ministry of Health & Consumer Affairs, Spain (SAF2002-04057); Galician Regional Authority, Spain (P GIDIT03P XIC20806P N); Department of Health of the Basque Country (03/11092 and 11/111103); Fundacion Vasca de innovacion e investigacion sanitarias (OSIBG19/002 and OSIBG18/105). The genotyping service was carried out at CEGEN-P RB3-ISCIII; Carlos III Health Institute and ERDF (P T17/0019, of the P E I+D+I 2013-2016). The funding sources do not have any role in the study design; data collection, analysis and interpretation; writing the manuscript; and in the decision to submit the article for publication

A multicenter case–control study of the effect of e-nos VNTR polymorphism on upper gastrointestinal hemorrhage in NSAID users

Gastroenterology; PharmacogeneticsGastroenterologia; FarmacogenèticaGastroenterología; FarmacogenéticaBleeding in non-steroidal anti-inflammatory drug (NSAID) users limited their prescription. This first multicenter full case–control study (325 cases and 744 controls), explored the association of e-NOS intron 4 variable number tandem repeat (VNTR) polymorphism with upper gastrointestinal hemorrhage (UGIH) in NSAID exposed and unexposed populations and assessed any interaction between this polymorphism and NSAIDs. NSAID users carrying e-NOS intron 4 wild type genotype or VNTR polymorphism have higher odds of UGIH than those unexposed to NSAIDs [Odds Ratio (OR): 6.62 (95% Confidence Interval (CI): 4.24, 10.36) and OR: 5.41 (95% CI 2.62, 11.51), respectively], with no effect modification from VNTR polymorphism-NSAIDs interaction [Relative Excess Risk due to Interaction (RERI): −1.35 (95% CI −5.73, 3.03); Synergism Index (S): 0.77 (95% CI 0.31, 1.94)]. Similar findings were obtained for aspirin exposure. Non-aspirin NSAID users who carry e-NOS intron 4 VNTR polymorphism have lower odds of UGIH [OR: 4.02 (95% CI 1.85, 8.75) than those users with wild type genotype [OR: 6.52 (95% CI 4.09, 10.38)]; though the interaction estimates are not statistically significant [RERI: −2.68 (95% CI −6.67, 1.31); S: 0.53 (95% CI 0.18, 1.55)]. This exploratory study suggests that the odds of UGIH in NSAID or aspirin users does not modify according to patient´s e-NOS intron 4 genotype.This work was supported by a grant from Instituto de Salud Carlos III [PI12/02414]/Plan Estatal de I + D + I 2012–2016; Fondo Europeo de Desarrollo Regional (FEDER); the Novartis, Pfizer and Dr Esteve pharmaceutical companies; the Health Research Fund/Fondo de Investigación Sanitaria [PI021512, PI021364, PI020661, PI021572]; Ministry of Health & Consumer Affairs, Spain [SAF2002-04057]; Galician Regional Authority, Spain [PGIDIT03PXIC20806PN]; Department of Health of the Basque Country [03/11092 and 11/111103]; and Fundacion vasca de innovacin e investigacin sanitarias [OSIBG19/002 and OSIBG18/105]. The genotyping service was carried out at CEGEN-PRB3-ISCIII; Instituto de Salud Carlos III and ERDF [PT17/0019, of the PE I + D + I 2013–2016]

Mitochondrial complex I dysfunction alters the balance of soluble and membrane-bound TNF during chronic experimental colitis.

Inflammatory bowel disease (IBD) is a complex, chronic, relapsing and heterogeneous disease induced by environmental, genomic, microbial and immunological factors. MCJ is a mitochondrial protein that regulates the metabolic status of macrophages and their response to translocated bacteria. Previously, an acute murine model of DSS-induced colitis showed increased disease severity due to MCJ deficiency. Unexpectedly, we now show that MCJ-deficient mice have augmented tumor necrosis factor alpha converting enzyme (TACE) activity in the context of chronic inflammation. This adaptative change likely affects the balance between soluble and transmembrane TNF and supports the association of the soluble form and a milder phenotype. Interestingly, the general shifts in microbial composition previously observed during acute inflammation were absent in the chronic model of inflammation in MCJ-deficient mice. However, the lack of the mitochondrial protein resulted in increased alpha diversity and the reduction in critical microbial members associated with inflammation, such as Ruminococcus gnavus, which could be associated with TACE activity. These results provide evidence of the dynamic metabolic adaptation of the colon tissue to chronic inflammatory changes mediated by the control of mitochondrial function