Role of PFKFB3-driven glycolysis in vessel sprouting.

Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. Here, we show that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator PFKFB3 in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalizing with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the pro-stalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching.

A Cholesterol-Based Allostery Model of T Cell Receptor Phosphorylation.

Signaling through the T cell receptor (TCR) controls adaptive immune responses. Antigen binding to TCRαß transmits signals through the plasma membrane to induce phosphorylation of the CD3 cytoplasmic tails by incompletely understood mechanisms. Here we show that cholesterol bound to the TCRß transmembrane region keeps the TCR in a resting, inactive conformation that cannot be phosphorylated by active kinases. Only TCRs that spontaneously detached from cholesterol could switch to the active conformation (termed primed TCRs) and then be phosphorylated. Indeed, by modulating cholesterol binding genetically or enzymatically, we could switch the TCR between the resting and primed states. The active conformation was stabilized by binding to peptide-MHC, which thus controlled TCR signaling. These data are explained by a model of reciprocal allosteric regulation of TCR phosphorylation by cholesterol and ligand binding. Our results provide both a molecular mechanism and a conceptual framework for how lipid-receptor interactions regulate signal transduction.

CCR5 deficiency impairs CD4+ T-cell memory responses and antigenic sensitivity through increased ceramide synthesis.

CCR5 is not only a coreceptor for HIV-1 infection in CD4+ T cells, but also contributes to their functional fitness. Here, we show that by limiting transcription of specific ceramide synthases, CCR5 signaling reduces ceramide levels and thereby increases T-cell antigen receptor (TCR) nanoclustering in antigen-experienced mouse and human CD4+ T cells. This activity is CCR5-specific and independent of CCR5 co-stimulatory activity. CCR5-deficient mice showed reduced production of high-affinity class-switched antibodies, but only after antigen rechallenge, which implies an impaired memory CD4+ T-cell response. This study identifies a CCR5 function in the generation of CD4+ T-cell memory responses and establishes an antigen-independent mechanism that regulates TCR nanoclustering by altering specific lipid species.

Suicidal behaviour: emergency department attendance for suicidal recurrence and other psychiatric or medical reasons in the 6 months following the index episode.

The prevention of a complex phenomenon, such as suicide, requires an interdisciplinary approach that provides a comprehensive response to the care needs of people with suicidal behavior (SB). The aim of this study is to investigate the clinical and healthcare features of people presenting thoughts and/or attempts of suicide to define risk factors for recurrence.

Immuno-priming durvalumab with bevacizumab in HER2-negative advanced breast cancer: a pilot clinical trial.

BACKGROUND: Preclinical research suggests that the efficacy of immune checkpoint inhibitors in breast cancer can be enhanced by combining them with antiangiogenics, particularly in a sequential fashion. We sought to explore the efficacy and biomarkers of combining the anti-PD-L1 durvalumab plus the antiangiogenic bevacizumab after bevacizumab monotherapy for advanced HER2-negative breast cancer. METHODS: Patients had advanced HER2-negative disease that progressed while receiving single-agent bevacizumab maintenance as a part of a previous chemotherapy plus bevacizumab regimen. Treatment consisted of bi-weekly durvalumab plus bevacizumab (10 mg/kg each i.v.). Peripheral-blood mononuclear cells (PBMCs) were obtained before the first durvalumab dose and every 4 weeks and immunophenotyped by flow-cytometry. A fresh pre-durvalumab tumor biopsy was obtained; gene-expression studies and immunohistochemical staining to assess vascular normalization and characterize the immune infiltrate were conducted. Patients were classified as "non-progressors" if they had clinical benefit (SD/PR/CR) at 4 months. The co-primary endpoints were the changes in the percentage T cell subpopulations in PBMCs in progressors versus non-progressors, and PFS/OS time. RESULTS: Twenty-six patients were accrued. Median PFS and OS were 3.5 and 11 months; a trend for a longer OS was detected for the hormone-positive subset (19.8 versus 7.4 months in triple-negatives; P = 0.11). Clinical benefit rate at 2 and 4 months was 60% and 44%, respectively, without significant differences between hormone-positive and triple-negative (P = 0.73). Non-progressors' tumors displayed vascular normalization features as a result of previous bevacizumab, compared with generally abnormal patterns observed in progressors. Non-progressors also showed increased T-effector and T-memory signatures and decreased TREG signatures in gene expression studies in baseline-post-bevacizumab-tumors compared with progressors. Notably, analysis of PBMC populations before durvalumab treatment was concordant with the findings in tumor samples and showed a decreased percentage of circulating TREGs in non-progressors. CONCLUSIONS: This study reporting on sequential bevacizumab+durvalumab in breast cancer showed encouraging activity in a heavily pre-treated cohort. The correlative studies agree with the preclinical rationale supporting an immunopriming effect exerted by antiangiogenic treatment, probably by reducing TREGs cells both systemically and in tumor tissue. The magnitude of this benefit should be addressed in a randomized setting. TRIAL REGISTRATION: (www.clinicaltrials.gov): NCT02802098 . Registered on June 16, 2020.

International meeting on Wolf-Hirschhorn syndrome: Update on the nosology and new insights on the pathogenic mechanisms for seizures and growth delay.

"An International Meeting on Wolf-Hirschhorn Syndrome (WHS)" was held at The University Hospital La Paz in Madrid, Spain (October 13-14, 2017). One hundred and twenty-five people, including physicians, scientists and affected families, attended the meeting. Parent and patient advocates from the Spanish Association of WHS opened the meeting with a panel discussion to set the stage regarding their hopes and expectations for therapeutic advances. In keeping with the theme on therapeutic development, the sessions followed a progression from description of the phenotype and definition of therapeutic endpoints, to definition of genomic changes. These proceedings will review the major points of discussion.

Lack of CCM1 induces hypersprouting and impairs response to flow.

Cerebral cavernous malformation (CCM) is a disease of vascular malformations known to be caused by mutations in one of three genes: CCM1, CCM2 or CCM3. Despite several studies, the mechanism of CCM lesion onset remains unclear. Using a Ccm1 knockout mouse model, we studied the morphogenesis of early lesion formation in the retina in order to provide insight into potential mechanisms. We demonstrate that lesions develop in a stereotypic location and pattern, preceded by endothelial hypersprouting as confirmed in a zebrafish model of disease. The vascular defects seen with loss of Ccm1 suggest a defect in endothelial flow response. Taken together, these results suggest new mechanisms of early CCM disease pathogenesis and provide a framework for further study.

SRF selectively controls tip cell invasive behavior in angiogenesis.

Efficient angiogenic sprouting is essential for embryonic, postnatal and tumor development. Serum response factor (SRF) is known to be important for embryonic vascular development. Here, we studied the effect of inducible endothelial-specific deletion of Srf in postnatal and adult mice. We find that endothelial SRF activity is vital for postnatal growth and survival, and is equally required for developmental and pathological angiogenesis, including during tumor growth. Our results demonstrate that SRF is selectively required for endothelial filopodia formation and cell contractility during sprouting angiogenesis, but seems dispensable for vascular remodeling. At the molecular level, we observe that vascular endothelial growth factor A induces nuclear accumulation of myocardin-related transcription factors (MRTFs) and regulates MRTF/SRF-dependent target genes including Myl9, which is important for endothelial cell migration in vitro. We conclude that SRF has a unique function in regulating migratory tip cell behavior during sprouting angiogenesis. We hypothesize that targeting the SRF pathway could provide an opportunity to selectively target tip cell filopodia-driven angiogenesis to restrict tumor growth.

Relevance of Nck-CD3 epsilon interaction for T cell activation in vivo.

On TCR ligation, the adaptor Nck is recruited through its src homology 3.1 domain to a proline-rich sequence (PRS) in CD3ε. We have studied the relevance of this interaction for T cell activation in vitro and in vivo by targeting the interaction sites in both partners. The first approach consisted of studying a knockin (KI) mouse line (KI-PRS) bearing a conservative mutation in the PRS that makes the TCR incompetent to recruit Nck. This deficiency prevents T cell activation by Ag in vitro and inhibited very early TCR signaling events including the tyrosine phosphorylation of CD3ζ. Most important, KI-PRS mice are partly protected against the development of neurological symptoms in an experimental autoimmune encephalitis model, and show a deficient antitumoral response after vaccination. The second approach consisted of using a high-affinity peptide that specifically binds the src homology 3.1 domain and prevents the interaction of Nck with CD3ε. This peptide inhibits T cell proliferation in vitro and in vivo. These data suggest that Nck recruitment to the TCR is fundamental to mount an efficient T cell response in vivo, and that the Nck-CD3ε interaction may represent a target for pharmacological modulation of the immune response.

A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity.

The reprogramming of differentiated cells to pluripotent cells (induced pluripotent stem (iPS) cells) is known to be an inefficient process. We recently reported that cells with short telomeres cannot be reprogrammed to iPS cells despite their normal proliferation rates, probably reflecting the existence of 'reprogramming barriers' that abort the reprogramming of cells with uncapped telomeres. Here we show that p53 (also known as Trp53 in mice and TP53 in humans) is critically involved in preventing the reprogramming of cells carrying various types of DNA damage, including short telomeres, DNA repair deficiencies, or exogenously inflicted DNA damage. Reprogramming in the presence of pre-existing, but tolerated, DNA damage is aborted by the activation of a DNA damage response and p53-dependent apoptosis. Abrogation of p53 allows efficient reprogramming in the face of DNA damage and the generation of iPS cells carrying persistent DNA damage and chromosomal aberrations. These observations indicate that during reprogramming cells increase their intolerance to different types of DNA damage and that p53 is critical in preventing the generation of human and mouse pluripotent cells from suboptimal parental cells.

[EVALUATION OF THE EFFICIENCY OF CLEANING THE AIR BETWEEN TWO METHODS OF MAKING IN PATIENTS WITH AEROSOL CHRONIC OBSTRUCTIVE PULMONARY DISEASE]. / Evaluación de la eficacia en la limpieza de la vía aérea entre dos métodos de toma de aerosolterapia en pacientes con enfermedad pulmonar obstructiva croníca.

INTRODUCTION: Chronic Obstructive Pulmonary Disease (COPD) is a condition which requires, among others, the administration of bronchodilators and anti-inflammatory drugs to control the disease. They help to keep the airways clear and prevent the buildup of fluid and mucus. Inhalation is the most widely used form of administrating the medication because of its local and rapid action which normally is done by aerosol therapy. The objective of this study is to evaluate the effectiveness of two aerosol methods in clearing the airways of COPD patients, admitted with an exacerbation due to their disease. It also aims to evaluate its effects on the length of stay, oxygen saturation, dyspnea, autonomy and complications. METHODS: Randomized clinical trial. Traditional method (control group) and positive expiratory vibrating device (intervention group): two ways to deliver aerosol were compared. The following outcome variables were considered: length of stay, days of fever, oxygen saturation, need for NIV or VM, the basic activities of daily living index autonomy (Barthel), dyspnea (Borg scale) and peak-flow. RESULTS: 39 patients were included. Regarding hospital stay, patients in the intervention group spend an average of one day less in hospital. Also in this group there were fewer readmissions. No statistically significant differences were found in the remaining variables. CONCLUSIONS: The inhalation treatment with a vibrating device with positive exhalation, appears to reduce the length of stay and prevent readmissions. It is important to continue research on non-pharmacological interventions as to achieve the prevention of relapses.

XPF nuclease-dependent telomere loss and increased DNA damage in mice overexpressing TRF2 result in premature aging and cancer.

TRF2 is a telomere-binding protein that has a role in telomere protection. We generated mice that overexpress TRF2 in the skin. These mice had a severe phenotype in the skin in response to light, consisting of premature skin deterioration, hyperpigmentation and increased skin cancer, which resembles the human syndrome xeroderma pigmentosum. Keratinocytes from these mice were hypersensitive to ultraviolet irradiation and DNA crosslinking agents. The skin cells of these mice had marked telomere shortening, loss of the telomeric G-strand overhang and increased chromosomal instability. Telomere loss in these mice was mediated by XPF, a structure-specific nuclease involved in ultraviolet-induced damage repair and mutated in individuals with xeroderma pigmentosum. These findings suggest that TRF2 provides a crucial link between telomere function and ultraviolet-induced damage repair, whose alteration underlies genomic instability, cancer and aging. Finally, we show that a number of human skin tumors have increased expression of TRF2, further highlighting a role for TRF2 in skin cancer.

Preoperative respiratory therapy in patients undergoing surgery for lung cancer: A randomized controlled trial.

BACKGROUND: A preoperative respiratory therapy treatment was performed to analyze the effectiveness, with respect to postoperative air leak and pain, in patients undergoing surgery for lung cancer. OBJECTIVES: To analyze air leakage and postoperative pain. MATERIAL AND METHODS: Seventy one patients were studied, with a mean age of 62.58 years. Descriptive variables of gender, carcinogenic pathology, type of surgical incision and lung resection, use of glue and endostapler, and presence of adhesions were analyzed. Likewise, analysis of the quantitative variables of age, body mass index and forced expiratory volume in 1 s Two homogeneous groups resulted. Differentiated, experimental group (EG) that performed preoperative respiratory therapy and control group (CG). RESULTS: There were statistically significant differences in favor of the EG with respect to postoperative air leak on days 1-2 during the performance of physiotherapy techniques, the food and during the performance of the exercises autonomously. Furthermore, differences in air leakage were observed on days 2-4 during gait. The number of patients decreased to a greater extent in the EG. Regarding pain, there were statistically significant differences in the sample on days 1-4, with greater intensity of pain in the CG and after doing physiotherapy every day except the second. CONCLUSIONS: Preoperative respiratory therapy in patients undergoing surgery for lung cancer was effective in reducing the number of patients who presented postoperative air leak and reducing pain in the EG.

Telomere shortening relaxes X chromosome inactivation and forces global transcriptome alterations.

Telomeres are heterochromatic structures at chromosome ends essential for chromosomal stability. Telomere shortening and the accumulation of dysfunctional telomeres are associated with organismal aging. Using telomerase-deficient TRF2-overexpressing mice (K5TRF2/Terc(-/-)) as a model for accelerated aging, we show that telomere shortening is paralleled by a gradual deregulation of the mammalian transcriptome leading to cumulative changes in a defined set of genes, including up-regulation of the mTOR and Akt survival pathways and down-regulation of cell cycle and DNA repair pathways. Increased DNA damage from dysfunctional telomeres leads to reduced deposition of H3K27me3 onto the inactive X chromosome (Xi), impaired association of the Xi with telomeric transcript accumulations (Tacs), and reactivation of an X chromosome-linked K5TRF2 transgene that is subjected to X-chromosome inactivation in female mice with sufficiently long telomeres. Exogenously induced DNA damage also disrupts Xi-Tacs, suggesting DNA damage at the origin of these alterations. Collectively, these findings suggest that critically short telomeres activate a persistent DNA damage response that alters gene expression programs in a nonstochastic manner toward cell cycle arrest and activation of survival pathways, as well as impacts the maintenance of epigenetic memory and nuclear organization, thereby contributing to organismal aging.

Molecular epidemiology of Panton-Valentine leukocidin-positive Staphylococcus aureus in Spain: emergence of the USA300 clone in an autochthonous population.

We characterized all of the Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus isolates collected between 2005 and 2008 in the Bilbao, Spain, area. For the first time, the USA300 clone is reported as predominant among PVL-positive clones in a European autochthonous population, requiring active monitoring of the incidence of USA300 in Spain and throughout Europe.

Comparing the effect of Cu-based fungicides and pure Cu salts on microbial biomass, microbial community structure and bacterial community tolerance to Cu.

The effect of Cu on three different microbial endpoints was studied using different Cu sources, in order to check the usefulness of pure Cu salts to estimate the toxicity of commercial Cu fungicides on soil microbes. Cu additions caused similar dose-response curves of substrate induced respiration (SIR) decreases regardless of Cu source, i.e. the use of pure Cu salts to estimate the effect of Cu fungicides on microbial biomass using SIR may be useful. Phospholipid fatty acid (PLFA) analysis showed that the Cu source was more important for the microbial community structure than Cu concentration. Thus, the use of Cu salts to infer the effects of Cu fungicides on microbial community structure using PLFA analysis is not recommended, since effects of Cu concentration will be confounded with Cu source. Analyzing pollution induced community tolerance (PICT) to Cu showed that the use of pure Cu salts may overestimate Cu effects if Cu salt additions modified the soil pH. The highest doses of Cu salts increased bacterial community tolerance to Cu between 300 and 600 times, while commercial Cu fungicide increases were between 20 and 160 times. Therefore, the use of pure Cu salts to estimate the Cu fungicides effects on soil microbes is not recommended for PLFAs analyses, not suitable for PICT at high Cu concentrations, while useful for SIR.

The Chemokine Receptor CCR5 Links Memory CD4+ T Cell Metabolism to T Cell Antigen Receptor Nanoclustering.

The inhibition of anabolic pathways, such as aerobic glycolysis, is a metabolic cornerstone of memory T cell differentiation and function. However, the signals that hamper these anabolic pathways are not completely known. Recent evidence pinpoints the chemokine receptor CCR5 as an important player in CD4+ T cell memory responses by regulating T cell antigen receptor (TCR) nanoclustering in an antigen-independent manner. This paper reports that CCR5 specifically restrains aerobic glycolysis in memory-like CD4+ T cells, but not in effector CD4+ T cells. CCR5-deficient memory CD4+ T cells thus show an abnormally high glycolytic/oxidative metabolism ratio. No CCR5-dependent change in glucose uptake nor in the expression of the main glucose transporters was detected in any of the examined cell types, although CCR5-deficient memory cells did show increased expression of the hexokinase 2 and pyruvate kinase M2 isoforms, plus the concomitant downregulation of Bcl-6, a transcriptional repressor of these key glycolytic enzymes. Further, the TCR nanoclustering defects observed in CCR5-deficient antigen-experienced CD4+ T cells were partially reversed by incubation with 2-deoxyglucose (2-DG), suggesting a link between inhibition of the glycolytic pathway and TCR nanoscopic organization. Indeed, the treatment of CCR5-deficient lymphoblasts with 2-DG enhanced IL-2 production after antigen re-stimulation. These results identify CCR5 as an important regulator of the metabolic fitness of memory CD4+ T cells, and reveal an unexpected link between T cell metabolism and TCR organization with potential influence on the response of memory T cells upon antigen re-encounter.

Hemoperitoneum secondary to haemocholecyst due to acute cholecystitis.

The haemocholecyst is a rare disease with a low index of suspiction, which is even lower if the patient does not present risk factors. Likewise, the course of this pathology with a haemoperitoneum without vesicular perforation is equally infrequent. We present an unusual case in which the diagnosis of the patient was carried out intraoperatively. We consider that communicating these unusual cases in clinical practice helps to increase clinical suspiction and prompt diagnosis.

El hemocolecisto es una patología poco frecuente y con un índice bajo de sospecha, que todavía es menor si el paciente no presenta factores de riesgo. Asimismo, su curso con clínica de hemoperitoneo sin perforación vesicular es igualmente infrecuente. Presentamos un caso poco habitual en el cual el diagnóstico de la paciente se llevó a cabo intraoperatoriamente. Consideramos que comunicar estos casos poco habituales en la práctica clínica colabora a aumentar la sospecha clínica y el diagnóstico precoz.

Comparison of Cu salts and commercial Cu based fungicides on toxicity towards microorganisms in soil.

Microbial responses to Cu pollution as a function of Cu sources (Cu salts and commercial Cu fungicides) were assessed in a soil using basal soil respiration, and bacterial and fungal community growth, as endpoints. The soil was amended with different concentrations (0-32 mmol Cu kg-1) of Cu nitrate, Cu sulfate, Bordeaux mixture and 3 types of Cu oxychloride. Cu salts decreased soil pH, while this was not found with the other Cu sources. This difference in soil pH effects caused differences in the respiration, bacterial growth and fungal growth response. Basal soil respiration was negatively affected by Cu addition when the soil was spiked with Cu salts, but almost unaffected by commercial Cu fungicides. Bacterial growth was significantly and negatively affected by Cu addition for all the Cu sources, but Cu toxicity was higher for Cu salts than for commercial Cu fungicides. Fungal growth response was also different for Cu salts and commercial Cu fungicides, but only in the long-term. High Cu amendments using Cu salts stimulated fungal growth, whereas for commercial Cu fungicides, these concentrations inhibited fungal growth. Thus, the use of products similar to those used in commercial fungicides is a recommended practice for Cu risk assessments in soil.

Coping with Wolf-Hirschhorn syndrome: quality of life and psychosocial features of family carers.

BACKGROUND: Wolf-Hirschhorn Syndrome (WHS) is a rare, congenital disease characterized by a distinctive facial phenotype, seizures, intellectual disability and developmental delay, and pre and postnatal growth requiring lifelong care. The psychosocial status of the family caregivers of children diagnosed with WHS is unknown. This study aims to characterize the sociodemographic and psychosocial profile of WHS caregivers and analyze how these variables impact their quality of life (QoL) and well-being. RESULTS: The sociodemographic and clinical profile of 22 Spanish caregivers of children with WHS and the characteristics of those affected have been described. Significant relationships were found between sociodemographic and psychosocial characteristics among caregivers. The impact on the parents' QoL and negative relationship with the symptomatology were assessed. The use of engagement strategies such as problem focused coping was associated with improved psychological QoL and social support. CONCLUSIONS: WHS caregivers share similarities in their profile and needs with caregivers of children with other rare diseases. Pychosocial support groups involving parents caring for children with the same disease could improve caregivers' well-being and QoL by strengthening their social support network and using positive coping styles.