Feasibility and outcomes with subclavian vein access for crescent jugular dual lumen catheter for venovenous extracorporeal membrane oxygenation in COVID-19 related acute respiratory distress syndrome.

INTRODUCTION: Femoral-femoral Veno-Venous ExtraCorporeal Life Support (V-V ECLS) has been associated with higher infections rates, vascular site bleeding complications, and restricted patient mobility. Jugular or bicaval dual lumen V-V ECLS conceptually overcomes some of these adverse factors, but experience has shown that jugular vein cannulation still limits mobility and has increased bleeding complications. Technique and outcomes of subclavian vein single-cannulation with Crescent jugular dual-lumen V-V ECLS is described. METHOD: five patients with COVID-19 related acute respiratory distress syndrome (ARDS) underwent right subclavian vein V-V ECLS placement with the Crescent 32 French jugular dual-lumen V-V ECLS catheter. A standardized percutaneous technique was developed that allowed efficient insertion without need for any specialized imaging (i.e. transesophageal echocardiogram) and outcomes assessed. RESULTS: Mean age of the five patients was 41.2 years, all obese with an average basal mass index of 45.2 kg/m2 and mean days to decannulation of 24.2 days. Outcomes discovered included; improved patient mobility allowing physical rehabilitation, no vascular access site related complications requiring surgery or endovascular intervention, and none had evidence of superior vena cava syndrome. One patient had subclavian/axillary vein thrombosis with resolution after 3 months of direct-acting oral anticoagulants, and one patient had blood cultures positive at day 37, nearing decannulation. CONCLUSION: Subclavian vein access for crescent jugular dual lumen V-V ECLS catheter appears to be safe and feasible with added benefits of decreased bleeding and increased mobility over jugular or femoral-femoral access site for long term V-V ECLS support in COVID-19 related ARDS patients.

The PSI Domain of the MET Oncogene Encodes a Functional Disulfide Isomerase Essential for the Maturation of the Receptor Precursor.

The tyrosine kinase receptor encoded by the MET oncogene has been extensively studied. Surprisingly, one extracellular domain, PSI, evolutionary conserved between plexins, semaphorins, and integrins, has no established function. The MET PSI sequence contains two CXXC motifs, usually found in protein disulfide isomerases (PDI). Using a scrambled oxidized RNAse enzymatic activity assay in vitro, we show, for the first time, that the MET extracellular domain displays disulfide isomerase activity, abolished by PSI domain antibodies. PSI domain deletion or mutations of CXXC sites to AXXA or SXXS result in a significant impairment of the cleavage of the MET 175 kDa precursor protein, abolishing the maturation of α and ß chains, of, respectively, 50 kDa and 145 kDa, disulfide-linked. The uncleaved precursor is stuck in the Golgi apparatus and, interestingly, is constitutively phosphorylated. However, no signal transduction is observed as measured by AKT and MAPK phosphorylation. Consequently, biological responses to the MET ligand-hepatocyte growth factor (HGF)-such as growth and epithelial to mesenchymal transition, are hampered. These data show that the MET PSI domain is functional and is required for the maturation, surface expression, and biological functions of the MET oncogenic protein.

The Long-Lasting Protective Effect of HGF in Cardiomyoblasts Exposed to Doxorubicin Requires a Positive Feed-Forward Loop Mediated by Erk1,2-Timp1-Stat3.

Previous studies showed that the hepatocyte growth factor (HGF)-Met receptor axis plays long-lasting cardioprotection against doxorubicin anti-cancer therapy. Here, we explored the mechanism(s) underlying the HGF protective effect. DNA damage was monitored by histone H2AX phosphorylation and apoptosis by proteolytic cleavage of caspase 3. In doxorubicin-treated H9c2 cardiomyoblasts, the long-lasting cardioprotection is mediated by activation of the Ras/Raf/Mek/Erk (extracellular signal-regulated kinase 1,2) signaling pathway and requires Stat3 (signal transducer and activator of transcription 3) activation. The HGF protection was abrogated by the Erk1,2 inhibitor, PD98059. This translated into reduced Y705 phosphorylation and impaired nuclear translocation of Stat3, showing crosstalk between Erk1,2 and Stat3 signaling. An array of 29 cytokines, known to activate Stat3, was interrogated to identify the molecule(s) linking the two pathways. The analysis showed a selective increase in expression of the tissue inhibitor of metalloproteinases-1 (Timp1). Consistently, inhibition in cardiomyoblasts of Timp1 translation by siRNAs blunted both Stat3 activation and the cardioprotective effect of HGF. Thus, Timp1 is responsible for the generation of a feed-forward loop of Stat3 activation and helps cardiomyocytes to survive during the genotoxic stress induced by anthracyclines.

Self-Esteem and Risk Behaviours in Adolescents: A Systematic Review.

Adolescence is recognised as a notoriously vulnerable period in the human life cycle. Influenced by a complex interplay of biological, psychological and social factors, adolescents show a marked propensity to engage in risk behaviours. A systematic review was conducted of studies published in the Web of Science, PsycInfo and MEDLINE databases over the last decade, with the aim of collecting studies on the relationship between self-esteem and risk behaviour in individuals aged 12-18 years. The aim was to confirm the role of high self-esteem as a consistent protective factor against risk behaviour. The results show that self-esteem is negatively related to risk behaviour. Our results also reflect the need for further research on how sociodemographic factors, among others, affect the relationship between self-esteem and risk behaviours. This review highlights the relevance of implementing specific educational interventions to strengthen self-esteem in adolescents, with the aim of preventing various risk behaviours that may emerge during adolescence and persist throughout life if not addressed early.

Protocol for in vitro establishment of heterogeneous stem-like cultures derived from whole human glioblastoma tumors.

Cultures enriched in glioblastoma stem-like cells (GSCs) are prominent in vitro models to investigate molecular determinants and therapeutic targets of glioblastoma; however, conventional GSC derivation protocols fail to preserve GSC heterogeneity. Here, we present a protocol for the propagation of heterogeneous GSC cultures starting from cell resuspensions containing the entire tumor mass. We describe steps for isolation of GSCs and their maintenance and expansion in culture. We then detail procedures for preliminary analysis to be performed on freshly isolated material. For complete details on the use and execution of this protocol, please refer to De Bacco et al.1.

Use of Impella Devices for Acute Cardiogenic Shock in the Perioperative Period of Cardiac Surgery.

INTRODUCTION: The Impella ventricular support system is a device that can be inserted percutaneously or directly across the aortic valve to unload the left ventricle. The purpose of this study is to determine the role of Impella devices in patients with acute cardiogenic shock in the perioperative period of cardiac surgery. METHODS: A retrospective single-surgeon review of 11 consecutive patients who underwent placement of Impella devices in the perioperative period of cardiac surgery was performed. Patient records were evaluated for demographics, indications for placement, and postoperative outcomes. RESULTS: Impella devices were placed for refractory cardiogenic shock preoperatively in 6 patients, intraoperatively in 4 patients, and postoperatively as a rescue in 1 patient. Seven patients received Impella CP, 1 Impella RP, 1 Impella CP and RP, and 2 Impella 5.0. Additionally, 3 patients required preoperative venovenous extracorporeal membrane oxygenation (VV-ECMO), and 1 patient required intraoperative venoarterial extracorporeal membrane oxygenation (VA-ECMO). All Impella devices were removed 1 to 28 days after implantation. Length of stay in the intensive care unit stay ranged from 2 to 53 days (average 23.9±14.6). The 30-day and 1-year mortality were 0%. Ten of 11 patients were alive at 2 years. Also, 1 patient died 18 months after surgery from complications of coronavirus disease (Covid-19). Device-related complications included varying degrees> of hemolysis in 8 patients (73%) and device malfunction in 1 patient (9%). CONCLUSIONS: The Impella ventricular support system can be combined with other mechanical support devices for additional hemodynamic support. All patients demonstrated myocardial recovery with no deaths in the perioperative period and in 1-year of follow-up. Larger studies are necessary to validate these findings.

Coexisting cancer stem cells with heterogeneous gene amplifications, transcriptional profiles, and malignancy are isolated from single glioblastomas.

Glioblastoma (GBM) is known as an intractable, highly heterogeneous tumor encompassing multiple subclones, each supported by a distinct glioblastoma stem cell (GSC). The contribution of GSC genetic and transcriptional heterogeneity to tumor subclonal properties is debated. In this study, we describe the systematic derivation, propagation, and characterization of multiple distinct GSCs from single, treatment-naive GBMs (GSC families). The tumorigenic potential of each GSC better correlates with its transcriptional profile than its genetic make-up, with classical GSCs being inherently more aggressive and mesenchymal more dependent on exogenous growth factors across multiple GBMs. These GSCs can segregate and recapitulate different histopathological aspects of the same GBM, as shown in a paradigmatic tumor with two histopathologically distinct components, including a conventional GBM and a more aggressive primitive neuronal component. This study provides a resource for investigating how GSCs with distinct genetic and/or phenotypic features contribute to individual GBM heterogeneity and malignant escalation.

Engineering, Characterization, and Biological Evaluation of an Antibody Targeting the HGF Receptor.

The Hepatocyte growth factor (HGF) and its receptor (MET) promote several physiological activities such as tissue regeneration and protection from cell injury of epithelial, endothelial, neuronal and muscle cells. The therapeutic potential of MET activation has been scrutinized in the treatment of acute tissue injury, chronic inflammation, such as renal fibrosis and multiple sclerosis (MS), cardiovascular and neurodegenerative diseases. On the other hand, the HGF-MET signaling pathway may be caught by cancer cells and turned to work for invasion, metastasis, and drug resistance in the tumor microenvironment. Here, we engineered a recombinant antibody (RDO24) and two derived fragments, binding the extracellular domain (ECD) of the MET protein. The antibody binds with high affinity (8 nM) to MET ECD and does not cross-react with the closely related receptors RON nor with Semaphorin 4D. Deletion mapping studies and computational modeling show that RDO24 binds to the structure bent on the Plexin-Semaphorin-Integrin (PSI) domain, implicating the PSI domain in its binding to MET. The intact RDO24 antibody and the bivalent Fab2, but not the monovalent Fab induce MET auto-phosphorylation, mimicking the mechanism of action of HGF that activates the receptor by dimerization. Accordingly, the bivalent recombinant molecules induce HGF biological responses, such as cell migration and wound healing, behaving as MET agonists of therapeutic interest in regenerative medicine. In vivo administration of RDO24 in the murine model of MS, represented by experimental autoimmune encephalomyelitis (EAE), delays the EAE onset, mitigates the early clinical symptoms, and reduces inflammatory infiltrates. Altogether, these results suggest that engineered RDO24 antibody may be beneficial in multiple sclerosis and possibly other types of inflammatory disorders.

ERBB3 overexpression due to miR-205 inactivation confers sensitivity to FGF, metabolic activation, and liability to ERBB3 targeting in glioblastoma.

In glioblastoma (GBM), the most frequent and lethal brain tumor, therapies suppressing recurrently altered signaling pathways failed to extend survival. However, in patient subsets, specific genetic lesions can confer sensitivity to targeted agents. By exploiting an integrated model based on patient-derived stem-like cells, faithfully recapitulating the original GBMs in vitro and in vivo, here, we identify a human GBM subset (∼9% of all GBMs) characterized by ERBB3 overexpression and nuclear accumulation. ERBB3 overexpression is driven by inheritable promoter methylation or post-transcriptional silencing of the oncosuppressor miR-205 and sustains the malignant phenotype. Overexpressed ERBB3 behaves as a specific signaling platform for fibroblast growth factor receptor (FGFR), driving PI3K/AKT/mTOR pathway hyperactivation, and overall metabolic upregulation. As a result, ERBB3 inhibition by specific antibodies is lethal for GBM stem-like cells and xenotransplants. These findings highlight a subset of patients eligible for ERBB3-targeted therapy.

Intramolecular cation-pi interactions as the driving force to restrict the conformation of certain nucleosides.

Despite the well-established importance of intermolecular cation-pi interactions in molecular recognition, intramolecular cation-pi interactions have been less studied. Here we describe how the simultaneous presence of an aromatic ring at the 5'-position of an inosine derivative and a positively charged imidazolium ring in the purine base drive the conformation of the nucleoside toward a very major conformer in solution that is stabilized by an intramolecular cation-pi interaction. Therefore, the cation-pi interaction between imidazolium ions and aromatic rings can also be proposed in the design of small molecules where this type of interaction is desirable. The imidazolium ion can be obtained by a simple acidification of the pH of the media. So a simple change in pH can shift the conformational equilibrium from a random to a restricted conformation stabilized by an intramolecular cation-pi interaction. Thus the here described nucleosides can be considered as a new class of pH-dependent conformationally switchable molecules.

Activation of the MET receptor attenuates doxorubicin-induced cardiotoxicity in vivo and in vitro.

BACKGROUND AND PURPOSE: Doxorubicin anti-cancer therapy is associated with cardiotoxicity, resulting from DNA damage response (DDR). Hepatocyte growth factor (HGF) protects cardiomyocytes from injury, but its effective use is compromised by low biodistribution. In this study, we have investigated whether the activation of the HGF receptor-encoded by the Met gene-by an agonist monoclonal antibody (mAb) could protect against doxorubicin-induced cardiotoxicity. EXPERIMENTAL APPROACH: The mAb (5 mg·kg-1 ) was injected in vivo into C57BL/6J mice, before doxorubicin (three doses of 7 mg·kg-1 ). Cardiac functions were evaluated through MRI after treatment termination. Heart histological staining and mRNA levels of genes associated with heart failure (Acta1 and Nppa), inflammation (IL-6), and fibrosis (Ctgf, Col1a2, Timp1, and Mmp9) were assessed. MAb (100 nM) was administered in vitro to H9c2 cardiomyoblasts before addition of doxorubicin (25 µM). DDR and apoptosis markers were evaluated by quantitative western blotting, flow cytometry, and immunofluorescence. Stattic was used for pharmacological inactivation of STAT3. KEY RESULTS: In vivo, administration of the mAb alleviated doxorubicin-induced cardiac dysfunction and fibrosis. In vitro, mAb mimicked the response to HGF by (a) inhibiting histone H2AX phosphorylation at S139, (b) quenching the expression of the DNA repair enzyme PARP1, and (c) reducing the proteolytic activation of caspase 3. The MET-driven cardioprotection involved, at least in vitro, the phosphorylation of STAT3. CONCLUSION AND IMPLICATIONS: The MET agonist mAb provides a new tool for cardioprotection against anthracycline cardiotoxicity.

Baseline cortisol levels, total proteins, and eosinophil count as predictors of hemodynamic response to steroid treatment in septic shock.

BACKGROUND: Our aim was to study if baseline serum cortisol is related to the hemodynamic response to steroid treatment in septic shock patients and if the measurement of total proteins and eosinophil count improves its accuracy. METHODS: A retrospective analysis was performed in 66 consecutive surgical septic shock patients receiving steroid treatment. Four criteria were chosen to define hemodynamic improvement based on the combination of noradrenaline (NA) withdrawal (at 24 and 48 hour) and an increase of the hemodynamic index (HI = mean arterial pressure/NA dose) of 150% at 24 hour and of 350% at 48 hour. The accuracy of the serum cortisol to predict the hemodynamic response to steroid treatment following the four criteria was determined by receiver operating characteristic curve analysis. RESULTS: The largest area under curve was found for the NA withdrawal or an increase of the hemodynamic index >350% at 48 hour after starting the steroid treatment (area under curve, 0.686; 95% CI, 0.553-0.819; p = 0.01). This criteria was met by 35 patients (53%) and was associated with a lower mortality (25.7% vs. 67.7%, p = 0.001). However, no clear serum cortisol cutoff value for the diagnosis of adrenal insufficiency based on the hemodynamic response could be found. Neither the baseline proteins nor the eosinophils improved the accuracy of cortisol to predict hemodynamic improvement. CONCLUSION: The measurement of serum cortisol in surgical septic shock patients does not accurately predict the hemodynamic response to steroids. No clear cutoff value for cortisol, alone or in combination with total protein and/or eosinophil count, can be defined to indicate steroid treatment.

Use of Impella Devices for Acute Cardiogenic Shock in the Perioperative Period of Cardiac Surgery

ABSTRACT Introduction: The Impella ventricular support system is a device that can be inserted percutaneously or directly across the aortic valve to unload the left ventricle. The purpose of this study is to determine the role of Impella devices in patients with acute cardiogenic shock in the perioperative period of cardiac surgery. Methods: A retrospective single-surgeon review of 11 consecutive patients who underwent placement of Impella devices in the perioperative period of cardiac surgery was performed. Patient records were evaluated for demographics, indications for placement, and postoperative outcomes. Results: Impella devices were placed for refractory cardiogenic shock preoperatively in 6 patients, intraoperatively in 4 patients, and postoperatively as a rescue in 1 patient. Seven patients received Impella CP, 1 Impella RP, 1 Impella CP and RP, and 2 Impella 5.0. Additionally, 3 patients required preoperative venovenous extracorporeal membrane oxygenation (VV-ECMO), and 1 patient required intraoperative venoarterial extracorporeal membrane oxygenation (VA-ECMO). All Impella devices were removed 1 to 28 days after implantation. Length of stay in the intensive care unit stay ranged from 2 to 53 days (average 23.9±14.6). The 30-day and 1-year mortality were 0%. Ten of 11 patients were alive at 2 years. Also, 1 patient died 18 months after surgery from complications of coronavirus disease (Covid-19). Device-related complications included varying degrees> of hemolysis in 8 patients (73%) and device malfunction in 1 patient (9%). Conclusions: The Impella ventricular support system can be combined with other mechanical support devices for additional hemodynamic support. All patients demonstrated myocardial recovery with no deaths in the perioperative period and in 1-year of follow-up. Larger studies are necessary to validate these findings.

Continuous hemofiltration in hyperthermic septic shock patients.

BACKGROUND: Severe hyperthermia commonly accompanies septic shock. High body temperature in absence of infection activates the inflammatory response and is associated with a high mortality. Three years ago, our hypothesis that sustained fever is harmful in septic shock led us to the development of a protocol aiming at decreasing hyperthermia (>/=39.5 degrees C) by means of hemofiltration when the patients did not respond to antipyretics. We present a report of temperature and hemodynamic changes and the outcome of 19 consecutive hyperthermic septic shock patients with multiorgan system failure and compare them with a historical similar group of patients in whom hyperthermia was not treated with hemofiltration. METHODS: Depending on renal function, patients were treated with continuous low-flow hemofiltration (n = 8) or hemodiafiltration, (n = 11). Core temperature was registered every hour. A hemodynamic index (HI) was defined (mean arterial pressure to noradrenaline dose) and used during the first 24 hours to describe the patients' hemodynamic profile by means of its percent variation starting 6 hours before instituting the hemofiltration. RESULTS: The patients' temperature decreased linearly from 39.8 degrees C +/- 0.5 degrees C before hemofiltration to 37 degrees C +/- 1.2 degrees C after 24 hours of treatment (p < 0.001). The HI decreased significantly from -6 hours to the onset of hemofiltration (p = 0.002) and increased significantly after 24 hours (p = 0.008). Twenty-eight-day mortality was 32% (6 of 19) when compared with 100% (11 of 11) in the historical group (p < 0.001). CONCLUSIONS: Continuous low-flow hemofiltration decreased body temperature and vasopressor requirements in hyperthermic septic shock patients. The mortality was unexpectedly low.

7,5'-O-dibenzylinosines: synthesis and studies on their conformational properties.

Reaction of 2',3'-O-isopropylidene inosine with benzyl bromide (1 h, rt) led to the 1,5'-O-dibenzylderivative 4, but by increasing the reaction time or the temperature, compound 4 is further transformed into the 1,7,5'-O-tribenzylinosine derivative 5. Similarly, the 7-methyl-1,5'-O-dibenzylderivative 6 has been synthesized from 4. The 1H-NMR spectra of 5 and 6 showed peculiar chemical shifts for geminal protons (H5' and H5'' of the ribose, and the CH2 of the benzyl groups). Preliminary NMR studies have been performed, including NOESY experiments that point toward the predominant existence of conformers that are stabilized by an electrostatic interaction between the positively charged imidazole of the base moiety and the high electron density of the 5'-benzyl substituent.

5'-O-tritylinosine and analogues as allosteric inhibitors of human thymidine phosphorylase.

On the basis of our previous findings that 5'-O-tritylinosine (KIN59) behaves as an allosteric inhibitor of the angiogenic enzyme thymidine phosphorylase (TPase), we have undertaken the synthesis and enzymatic evaluation of a novel series of nucleoside analogues modified at positions 1, 2, or 6 of the purine ring and at the 5'-position of the ribose moiety of the lead compound KIN59. SAR studies indicate that quite large structural variations can be performed on KIN59 without compromising TPase inhibition. Thus, incorporation of a cyclopropylmethyl or a cyclohexylmethyl group at position N(1) of 5'-O-tritylinosine increases the inhibitory activity against TPase 10-fold compared to KIN59. Moreover, the trityl group at the 5'-position of the ribose seems to be crucial for TPase inhibition. The here reported results further substantiate that 5'-O-trityl nucleosides represent a new class of TPase inhibitors that should be further explored in those biological systems where TPase plays an instrumental role (i.e. angiogenesis).

MET inhibition overcomes radiation resistance of glioblastoma stem-like cells.

Glioblastoma (GBM) contains stem-like cells (GSCs) known to be resistant to ionizing radiation and thus responsible for therapeutic failure and rapidly lethal tumor recurrence. It is known that GSC radioresistance relies on efficient activation of the DNA damage response, but the mechanisms linking this response with the stem status are still unclear. Here, we show that the MET receptor kinase, a functional marker of GSCs, is specifically expressed in a subset of radioresistant GSCs and overexpressed in human GBM recurring after radiotherapy. We elucidate that MET promotes GSC radioresistance through a novel mechanism, relying on AKT activity and leading to (i) sustained activation of Aurora kinase A, ATM kinase, and the downstream effectors of DNA repair, and (ii) phosphorylation and cytoplasmic retention of p21, which is associated with anti-apoptotic functions. We show that MET pharmacological inhibition causes DNA damage accumulation in irradiated GSCs and their depletion in vitro and in GBMs generated by GSC xenotransplantation. Preclinical evidence is thus provided that MET inhibitors can radiosensitize tumors and convert GSC-positive selection, induced by radiotherapy, into GSC eradication.

TNF-α promotes invasive growth through the MET signaling pathway.

The inflammatory cytokine Tumor Necrosis Factor Alpha (TNF-α) is known to trigger invasive growth, a physiological property for tissue healing, turning into a hallmark of progression in cancer. However, the invasive response to TNF-α relies on poorly understood molecular mechanisms. We thus investigated whether it involves the MET oncogene, which regulates the invasive growth program by encoding the tyrosine kinase receptor for Hepatocyte Growth Factor (HGF). Here we show that the TNF-α pro-invasive activity requires MET function, as it is fully inhibited by MET-specific inhibitors (small-molecules, antibodies, and siRNAs). Mechanistically, we show that TNF-α induces MET transcription via NF-κB, and exploits MET to sustain MEK/ERK activation and Snail accumulation, leading to E-cadherin downregulation. We then show that TNF-α not only induces MET expression in cancer cells, but also HGF secretion by fibroblasts. Consistently, we found that, in human colorectal cancer tissues, high levels of TNF-α correlates with increased expression of both MET and HGF. These findings suggest that TNF-α fosters a HGF/MET pro-invasive paracrine loop in tumors. Targeting this ligand/receptor pair would contribute to prevent cancer progression associated with inflammation.

Anti-HIV-1 activity of a tripodal receptor that recognizes mannose oligomers.

The glycoprotein gp120 of the HIV-1 viral envelope has a high content in mannose residues, particularly α-1,2-mannose oligomers. Compounds that interact with these high-mannose type glycans may disturb the interaction between gp120 and its (co)receptors and are considered potential anti-HIV agents. Previously, we demonstrated that a tripodal receptor (1), with a central scaffold of 1,3,5-triethylbenzene substituted with three 2,3,4-trihydroxybenzoyl groups, selectively recognizes α-1,2-mannose polysaccharides. Here we present additional studies to determine the anti-HIV-1 activity and the mechanism of antiviral activity of this compound. Our studies indicate that 1 shows anti-HIV-1 activity in the low micromolar range and has pronounced gp120 binding and HIV-1 integrase inhibitory capacity. However, gp120 binding rather than integrase inhibition seems to be the primary mechanism of antiviral activity of 1.

Leishmania infantum EndoG is an endo/exo-nuclease essential for parasite survival.

EndoG, a member of the DNA/RNA non-specific ßßα-metal family of nucleases, has been demonstrated to be present in many organisms, including Trypanosomatids. This nuclease participates in the apoptotic program in these parasites by migrating from the mitochondrion to the nucleus, where it takes part in the degradation of genomic DNA that characterizes this process. We now demonstrate that Leishmania infantum EndoG (LiEndoG) is an endo-exonuclease that has a preferential 5' exonuclease activity on linear DNA. Regardless of its role during apoptotic cell death, this enzyme seems to be necessary during normal development of the parasites as indicated by the reduced growth rates observed in LiEndoG hemi-knockouts and their poor infectivity in differentiated THP-1 cells. The pro-life role of this protein is also corroborated by the higher survival rates of parasites that over-express this protein after treatment with the LiEndoG inhibitor Lei49. Taken together, our results demonstrate that this enzyme plays essential roles in both survival and death of Leishmania parasites.