Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway

Background. Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with intracellular mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC.  Methods. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed.  Results. Macrophages infected with intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca 2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca 2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells.  Conclusion. These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies

Neutrophils Promote Mycobacterial Trehalose Dimycolate-Induced Lung Inflammation via the Mincle Pathway

Trehalose 6,6′-dimycolate (TDM), a cord factor of Mycobacterium tuberculosis (Mtb), is an important regulator of immune responses during Mtb infections. Macrophages recognize TDM through the Mincle receptor and initiate TDM-induced inflammatory responses, leading to lung granuloma formation. Although various immune cells are recruited to lung granulomas, the roles of other immune cells, especially during the initial process of TDM-induced inflammation, are not clear. In this study, Mincle signaling on neutrophils played an important role in TDM-induced lung inflammation by promoting adhesion and innate immune responses. Neutrophils were recruited during the early stage of lung inflammation following TDM-induced granuloma formation. Mincle expression on neutrophils was required for infiltration of TDM-challenged sites in a granuloma model induced by TDM-coated-beads. TDM-induced Mincle signaling on neutrophils increased cell adherence by enhancing F-actin polymerization and CD11b/CD18 surface expression. The TDM-induced effects were dependent on Src, Syk, and MAPK/ERK kinases (MEK). Moreover, coactivation of the Mincle and TLR2 pathways by TDM and Pam3CSK4 treatment synergistically induced CD11b/CD18 surface expression, reactive oxygen species, and TNFα production by neutrophils. These synergistically-enhanced immune responses correlated with the degree of Mincle expression on neutrophil surfaces. The physiological relevance of the Mincle-mediated anti-TDM immune response was confirmed by defective immune responses in Mincle−/− mice upon aerosol infections with Mtb. Mincle-mutant mice had higher inflammation levels and mycobacterial loads than WT mice. Neutrophil depletion with anti-Ly6G antibody caused a reduction in IL-6 and monocyte chemotactic protein-1 expression upon TDM treatment, and reduced levels of immune cell recruitment during the initial stage of infection. These findings suggest a new role of Mincle signaling on neutrophils during anti-mycobacterial responses

Phthiocerol Dimycocerosates of M. tuberculosis Participate in Macrophage Invasion by Inducing Changes in the Organization of Plasma Membrane Lipids

Phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), in particular during the early step of infection when bacilli encounter their host macrophages. However, their cellular and molecular mechanisms of action remain unknown. Using Mtb mutants deleted for genes involved in DIM biosynthesis, we demonstrated that DIM participate both in the receptor-dependent phagocytosis of Mtb and the prevention of phagosomal acidification. The effects of DIM required a state of the membrane fluidity as demonstrated by experiments conducted with cholesterol-depleting drugs that abolished the differences in phagocytosis efficiency and phagosome acidification observed between wild-type and mutant strains. The insertion of a new cholesterol-pyrene probe in living cells demonstrated that the polarity of the membrane hydrophobic core changed upon contact with Mtb whereas the lateral diffusion of cholesterol was unaffected. This effect was dependent on DIM and was consistent with the effect observed following DIM insertion in model membrane. Therefore, we propose that DIM control the invasion of macrophages by Mtb by targeting lipid organisation in the host membrane, thereby modifying its biophysical properties. The DIM-induced changes in lipid ordering favour the efficiency of receptor-mediated phagocytosis of Mtb and contribute to the control of phagosomal pH driving bacilli in a protective niche

Organocatalytic trifluoromethylation of imines using phase-transfer catalysis with phenoxides. A general platform for catalytic additions of organosilanes to imines

A new approach to additions of silicon nucleophiles to imines was developed. The method is based on the phase-transfer of phenoxides by ammonium catalysts, overcoming the inability of amide adducts in promoting the reactions

Prognostic role of non-sustained ventricular tachycardia detected with remote interrogation in a pacemaker population

Background: Non-sustained ventricular tachycardia (NSVT) can occur asymptomatically and can be incidentally detected in the internal records of pacemakers (PM). The clinical value of NSVT in the population of PM patients is still uncertain. Our aim was to assess the prevalence of NSVT detected by remote PM control, to describe the clinical and demographic characteristics of patients with NSVT, and to assess the prognostic significance of NSVT in terms of both overall and cardiovascular mortality. Methods: Consecutive patients followed with PM remote interrogations from September 2010 to December 2015 were included. The transmissions pertaining to the first 12 months of remote control were analysed and the patients were divided by those presenting NSVT and those without NSVT. The two groups were compared in terms of total mortality and cardiovascular mortality based on the administrative data provided by the regional administration of the Italian National Health System. Results: The prevalence of NSVT in 408 patients (62% males, mean age 75.6; SD 10.6 years old) was 21% in a year. During a mean follow-up duration of 44 months, NSVT did not emerge as independently associated with overall mortality, but was associated with cardiovascular mortality in a competing risk regression model with older age, male gender, diabetes, chronic renal insufficiency, ischemic cardiomyopathy and chronic obstructive pulmonary disease. Conclusions: We show that NSVT episodes recorded by remote control in a PM population are independently associated with cardiovascular mortality with possible implications for risk stratification and therapeutic options

TBL1XR1 ensures balanced neural development through NCOR complex-mediated regulation of the MAPK pathway.

TBL1XR1 gene is associated with multiple developmental disorders presenting several neurological aspects. The relative protein is involved in the modulation of important cellular pathways and master regulators of transcriptional output, including nuclear receptor repressors, Wnt signaling, and MECP2 protein. However, TBL1XR1 mutations (including complete loss of its functions) have not been experimentally studied in a neurological context, leaving a knowledge gap in the mechanisms at the basis of the diseases. Here, we show that Tbl1xr1 knock-out mice exhibit behavioral and neuronal abnormalities. Either the absence of TBL1XR1 or its point mutations interfering with stability/regulation of NCOR complex induced decreased proliferation and increased differentiation in neural progenitors. We suggest that this developmental unbalance is due to a failure in the regulation of the MAPK cascade. Taken together, our results broaden the molecular and functional aftermath of TBL1XR1 deficiency associated with human disorders

Remote Handling System for the DTT Fusion Reactor: A System Engineering Approach for Preliminary Conceptual Design of the Main Robotic Equipment

The aim of the Divertor Tokamak Test (DTT) facility, whose construction is starting at ENEA premises in Frascati, Italy, is to test different concepts of divertors under integrated physics and technical conditions that can reliably be extrapolated to DEMO experimental plant. The replacement of the divertor inside the Vacuum Vessel is, therefore, not simply a chance in case of failure of a divertor cassette, but a scheduled event that will occur several times in the lifetime of the machine. This operation, as well as replacement of First Wall modules (in case of failure), is therefore necessary and must be carried out through a Remote Handling (RH) System, being humans not allowed to enter inside the Vacuum Vessel, due to the high radiation levels inside it, and to stay in contact with activated dusts. The compact dimension of the DTT machine and, at the same time, the great dimensions of coils needed to get the requested performance, constrain port dimensions and, consequently, add additional issues to the design of the RH System. Starting from RH strategy already conceived by CREATE Consortium, and moving from a continuously growing list of technical and functional requirements, this paper describes the iterative process based on a system engineering approach which is driving the conceptual design of the main RH equipment for Divertor and First Wall modules. Compliance of the conceived concepts with requirements has been confirmed by first simulations on static force analysis, workspace and dexterity evaluation

Inhibition of Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1) signaling in the striatum reverts motor symptoms associated with l-dopa–induced dyskinesia

l-dopa–induced dyskinesia (LID) is a common debilitating complication of dopamine replacement therapy in Parkinson's disease. Recent evidence suggests that LID may be linked causally to a hyperactivation of the Ras–ERK signaling cascade in the basal ganglia. We set out to determine whether specific targeting of Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1), a brain-specific activator of the Ras–ERK pathway, may provide a therapy for LID. On the rodent abnormal involuntary movements scale, Ras-GRF1–deficient mice were significantly resistant to the development of dyskinesia during chronic l-dopa treatment. Furthermore, in a nonhuman primate model of LID, lentiviral vectors expressing dominant negative forms of Ras-GRF1 caused a dramatic reversion of dyskinesia severity leaving intact the therapeutic effect of l-dopa. These data reveal the central role of Ras-GRF1 in governing striatal adaptations to dopamine replacement therapy and validate a viable treatment for LID based on intracellular signaling modulation