TBC1 domain-containing proteins are frequently involved in triple-negative breast cancers in connection with the induction of a glycolytic phenotype.

Metabolic plasticity is a hallmark of cancer, and metabolic alterations represent a promising therapeutic target. Since cellular metabolism is controlled by membrane traffic at multiple levels, we investigated the involvement of TBC1 domain-containing proteins (TBC1Ds) in the regulation of cancer metabolism. These proteins are characterized by the presence of a RAB-GAP domain, the TBC1 domain, and typically function as attenuators of RABs, the master switches of membrane traffic.

Lipid A heterogeneity and its role in the host interactions with pathogenic and commensal bacteria.

Lipopolysaccharide (LPS) is for most but not all Gram-negative bacteria an essential component of the outer leaflet of the outer membrane. LPS contributes to the integrity of the outer membrane, which acts as an effective permeability barrier to antimicrobial agents and protects against complement-mediated lysis. In commensal and pathogenic bacteria LPS interacts with pattern recognition receptors (e.

Activation of Canine, Mouse and Human TLR2 and TLR4 by Inactivated Vaccine Strains.

Canine vaccines contain inactivated strains of pathogenic , the causative agents of leptospirosis. For an effective response to vaccination, activation of the innate immune system pattern recognition receptors such as TLRs is crucial. However, it is not known which TLRs are activated by in dogs.

Nanoflow LC-MS Method Allowing In-Depth Characterization of Natural Heterogeneity of Complex Bacterial Lipopolysaccharides.

The variable modification of the outer membrane lipopolysaccharide (LPS) in Gram-negative bacteria contributes to bacterial pathogenesis through various mechanisms, including the development of antibiotic resistance and evasion of the immune response of the host. Characterizing the natural structural repertoire of LPS is challenging due to the high heterogeneity, branched architecture, and strong amphipathic character of these glycolipids. To address this problem, we have developed a method enabling the separation and structural profiling of complex intact LPS mixtures by using nanoflow reversed-phase high-performance liquid chromatography (nLC) coupled to electrospray ionization Fourier transform mass spectrometry (ESI-FT-MS).

Naturally circulating pertactin-deficient strains induce distinct gene expression and inflammatory signatures in human dendritic cells.

Respiratory infections caused by are reemerging despite high pertussis vaccination coverage. Since the introduction of the acellular pertussis vaccine in the late twentieth century, circulating strains increasingly lack expression of the vaccine component pertactin (Prn). In some countries, up to 90% of the circulating strains are deficient in Prn.

AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity.

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability.

Shortening the Lipid A Acyl Chains of Enables Depletion of Lipopolysaccharide Endotoxic Activity.

Whooping cough, or pertussis, is an acute respiratory infectious disease caused by the Gram-negative bacterium Whole-cell vaccines, which were introduced in the fifties of the previous century and proved to be effective, showed considerable reactogenicity and were replaced by subunit vaccines around the turn of the century. However, there is a considerable increase in the number of cases in industrialized countries. A possible strategy to improve vaccine-induced protection is the development of new, non-toxic, whole-cell pertussis vaccines.

KRAS-Driven Metabolic Rewiring Reveals Novel Actionable Targets in Cancer.

Tumors driven by mutant KRAS are among the most aggressive and refractory to treatment. Unfortunately, despite the efforts, targeting alterations of this GTPase, either directly or by acting on the downstream signaling cascades, has been, so far, largely unsuccessful. However, recently, novel therapeutic opportunities are emerging based on the effect that this oncogenic lesion exerts in rewiring the cancer cell metabolism.

Selective autophagy maintains centrosome integrity and accurate mitosis by turnover of centriolar satellites.

The centrosome is the master orchestrator of mitotic spindle formation and chromosome segregation in animal cells. Centrosome abnormalities are frequently observed in cancer, but little is known of their origin and about pathways affecting centrosome homeostasis. Here we show that autophagy preserves centrosome organization and stability through selective turnover of centriolar satellite components, a process we termed doryphagy.

Substrate specificity of the pyrophosphohydrolase LpxH determines the asymmetry of lipid A.

Lipopolysaccharides are anchored to the outer membrane of Gram-negative bacteria by a hydrophobic moiety known as lipid A, which potently activates the host innate immune response. Lipid A of , the causative agent of whooping cough, displays unusual structural asymmetry with respect to the length of the acyl chains at the 3 and 3' positions, which are 3OH-C10 and 3OH-C14 chains, respectively. Both chains are attached by the acyltransferase LpxA, the first enzyme in the lipid A biosynthesis pathway, which, in , has limited chain length specificity.

Kinesin-2 Controls the Motility of RAB5 Endosomes and Their Association with the Spindle in Mitosis.

RAB5 is a small GTPase that belongs to the wide family of Rab proteins and localizes on early endosomes. In its active GTP-bound form, RAB5 recruits downstream effectors that, in turn, are responsible for distinct aspects of early endosome function, including their movement along microtubules. We previously reported that, at the onset of mitosis, RAB5positive vesicles cluster around the spindle poles and, during metaphase, move along spindle microtubules.

High USP6NL Levels in Breast Cancer Sustain Chronic AKT Phosphorylation and GLUT1 Stability Fueling Aerobic Glycolysis.

USP6NL, also named RN-tre, is a GTPase-activating protein involved in control of endocytosis and signal transduction. Here we report that USP6NL is overexpressed in breast cancer, mainly of the basal-like/integrative cluster 10 subtype. Increased USP6NL levels were accompanied by gene amplification and were associated with worse prognosis in the METABRIC dataset, retaining prognostic value in multivariable analysis.

Sema4C/PlexinB2 signaling controls breast cancer cell growth, hormonal dependence and tumorigenic potential.

Semaphorin 4C (Sema4C) expression in human breast cancers correlates with poor disease outcome. Surprisingly, upon knock-down of Sema4C or its receptor PlexinB2 in diverse mammary carcinoma cells (but not their normal counterparts), we observed dramatic growth inhibition associated with impairment of G2/M phase transition, cytokinesis defects and the onset of cell senescence. Mechanistically, we demonstrated a Sema4C/PlexinB2/LARG-dependent signaling cascade that is required to maintain critical RhoA-GTP levels in cancer cells.

Th17-Mediated Cross Protection against Pneumococcal Carriage by Vaccination with a Variable Antigen.

Serotype-specific protection against is an important limitation of the current polysaccharide-based vaccines. To prevent serotype replacement, reduce transmission, and limit the emergence of new variants, it is essential to induce broad protection and restrict pneumococcal colonization. In this study, we used a prototype vaccine formulation consisting of lipopolysaccharide (LPS)-detoxified outer membrane vesicles (OMVs) from serovar Typhimurium displaying the variable N terminus of PspA (α1α2) for intranasal vaccination, which induced strong Th17 immunity associated with a substantial reduction of pneumococcal colonization.

Modulating endotoxin activity by combinatorial bioengineering of meningococcal lipopolysaccharide.

Neisseria meningitidis contains a very potent hexa-acylated LPS that is too toxic for therapeutic applications. We used systematic molecular bioengineering of meningococcal LPS through deletion of biosynthetic enzymes in combination with induction of LPS modifying enzymes to yield a variety of novel LPS mutants with changes in both lipid A acylation and phosphorylation. Mass spectrometry was used for detailed compositional determination of the LPS molecular species, and stimulation of immune cells was done to correlate this with endotoxic activity.

Vaccine-Mediated Activation of Human TLR4 Is Affected by Modulation of Culture Conditions during Whole-Cell Pertussis Vaccine Preparation.

The potency of whole-cell pertussis (wP) vaccines is still determined by an intracerebral mouse protection test. To allow development of suitable in vitro alternatives to this test, insight into relevant parameters to monitor the consistency of vaccine quality is essential. To this end, a panel of experimental wP vaccines of varying quality was prepared by sulfate-mediated suppression of the BvgASR master virulence regulatory system of Bordetella pertussis during cultivation.

Rebound Effects Caused by Withdrawal of MET Kinase Inhibitor Are Quenched by a MET Therapeutic Antibody.

MET oncogene amplification is emerging as a major mechanism of acquired resistance to EGFR-directed therapy in lung and colorectal cancers. Furthermore, MET amplification predicts responsiveness to MET inhibitors currently in clinical trials. Among the anti-MET drugs available, ATP-competitive small-molecule kinase inhibitors abrogate receptor autophosphorylation and downstream activation of ERK1/2 and AKT, resulting in cell-cycle arrest.

Tankyrase inhibition impairs directional migration and invasion of lung cancer cells by affecting microtubule dynamics and polarity signals.

Background: Tankyrases are poly(adenosine diphosphate)-ribose polymerases that contribute to biological processes as diverse as modulation of Wnt signaling, telomere maintenance, vesicle trafficking, and microtubule-dependent spindle pole assembly during mitosis. At interphase, polarized reshaping of the microtubule network fosters oriented cell migration. This is attained by association of adenomatous polyposis coli with the plus end of microtubules at the cortex of cell membrane protrusions and microtubule-based centrosome reorientation towards the migrating front.

Lipopolysaccharide engineering in Neisseria meningitidis: structural analysis of different pentaacyl lipid A mutants and comparison of their modified agonist properties.

Engineering the lipopolysaccharide (LPS) biosynthetic pathway offers the potential to obtain modified derivatives with optimized adjuvant properties. Neisseria meningitidis strain H44/76 was modified by expression of the pagL gene encoding lipid A 3-O-deacylase from Bordetella bronchiseptica and by inactivation of the lgtB gene encoding the terminal oligosaccharide galactosyltransferase. Mass spectrometry analysis of purified mutant LPS was used for detailed compositional analysis of all present molecular species.

Intact rough- and smooth-form lipopolysaccharides from Escherichia coli separated by preparative gel electrophoresis exhibit differential biologic activity in human macrophages.

We established a new preparative separation procedure, based on DOC/PAGE, to isolate intact lipopolysaccharide (LPS) fractions from natural LPS preparations of Escherichia coli. Analysis of the chemical integrity of LPS fractions by MS showed that no significant chemical modifications were introduced by the procedure. Contamination with toll-like receptor 2 (TLR2)-reactive cell-wall components present in the natural LPS mixture was effectively removed by the procedure, as determined by the absence of reactivity of the purified fractions in a HEK293-TLR2 cell line.

Hydrogen sulfide promotes calcium signals and migration in tumor-derived endothelial cells.

Hydrogen sulfide (H(2)S) is a gasotransmitter that plays several roles in various tissues, including the cardiovascular system. Because it has been recently proposed to act as a mediator of angiogenesis progression, here we investigate the effects of H(2)S in a well-established model of tumor angiogenesis: endothelial cells obtained from human breast carcinoma (B-TECs). Ca(2+) imaging and patch-clamp experiments reveal that acute perfusion with NaHS, a widely employed H(2)S donor, activates cytosolic calcium (Ca(c)) increase, as well as potassium and nonselective cationic currents, in B-TECs.

Isolation of smooth-type lipopolysaccharides to electrophoretic homogeneity.

Polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate or sodium deoxycholate (SDS- or DOC-slab-PAGE) is a powerful technique for the separation of smooth(S)-type bacterial lipopolysaccharides (LPS). In order to recover the individual LPS species from the polyacrylamide gel for subsequent analyses, a sensitive, nondestructive reverse staining of slab-PAGE-separated LPS has been developed. The individual reverse-stained LPS bands can be rapidly and efficiently recovered into an aqueous 5% triethylamine solution when they are extruded to produce fine gel microparticles.

Hydrogen sulfide regulates intracellular Ca2+ concentration in endothelial cells from excised rat aorta.

Hydrogen sulphide (H2S) is a recently discovered gasotransmitter that may regulate a growing number of endothelial functions, including nitric oxide (NO) release, proliferation, adhesion and migration, which are the key steps of angiogenesis. The mechanism whereby H2S impacts on endothelial physiology is still unclear: however, the aforementioned processes are driven by an increase in intracellular Ca2+ concentration ([Ca2+]i). In the present study, we exploited the excised rat aorta to gain insights into the regulation of [Ca2+]i by H2S within in situ endothelial cells (ECs).

Multiple roles of protein kinase a in arachidonic acid-mediated Ca2+ entry and tumor-derived human endothelial cell migration.

We recently showed that arachidonic acid (AA) triggers calcium signals in endothelial cells derived from human breast carcinoma (B-TEC). In particular, AA-dependent Ca(2+) entry is involved in the early steps of tumor angiogenesis in vitro. Here, we investigated the multiple roles of the nitric oxide (NO) and cyclic AMP/protein kinase A (PKA) pathways in AA-mediated Ca(2+) signaling in the same cells.