Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules

Background: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement

Genetic Diversity among Enterococcus faecalis

Enterococcus faecalis, a ubiquitous member of mammalian gastrointestinal flora, is a leading cause of nosocomial infections and a growing public health concern. The enterococci responsible for these infections are often resistant to multiple antibiotics and have become notorious for their ability to acquire and disseminate antibiotic resistances. In the current study, we examined genetic relationships among 106 strains of E. faecalis isolated over the past 100 years, including strains identified for their diversity and used historically for serotyping, strains that have been adapted for laboratory use, and isolates from previously described E. faecalis infection outbreaks. This collection also includes isolates first characterized as having novel plasmids, virulence traits, antibiotic resistances, and pathogenicity island (PAI) components. We evaluated variation in factors contributing to pathogenicity, including toxin production, antibiotic resistance, polymorphism in the capsule (cps) operon, pathogenicity island (PAI) gene content, and other accessory factors. This information was correlated with multi-locus sequence typing (MLST) data, which was used to define genetic lineages. Our findings show that virulence and antibiotic resistance traits can be found within many diverse lineages of E. faecalis. However, lineages have emerged that have caused infection outbreaks globally, in which several new antibiotic resistances have entered the species, and in which virulence traits have converged. Comparing genomic hybridization profiles, using a microarray, of strains identified by MLST as spanning the diversity of the species, allowed us to identify the core E. faecalis genome as consisting of an estimated 2057 unique genes

In vitro activity of daptomycin, linezolid and rifampicin on Staphylococcus epidermidis biofilms

Owing to their massive use, Staphylococcus epidermidis has recently developed significant resistance to several antibiotics, and became one of the leading causes of hospital-acquired infections. Current antibiotics are typically ineffective in the eradication of bacteria in biofilmassociated persistent infections. Accordingly, the paucity of effective treatment against cells in this mode of growth is a key factor that potentiates the need for new agents active in the prevention or eradication of biofilms. Daptomycin and linezolid belong to the novel antibiotic therapies that are active against gram-positive cocci. On the other hand, rifampicin has been shown to be one of the most potent, prevalent antibiotics against S. epidermidis biofilms. Therefore, the main aim of this study was to study the susceptibility of S. epidermidis biofilm cells to the two newer antimicrobial agents previously mentioned, and compare the results obtained with the antimicrobial effect of rifampicin, widely used in the prevention/treatment of indwelling medical device infections. To this end the in vitro activities of daptomycin, linezolid, and rifampicin on S. epidermidis biofilms were accessed, using these antibiotics at MIC and peak serum concentrations. The results demonstrated that at MIC concentration, rifampicin was the most effective antibiotic tested. At peak serum concentration, both strains demonstrated similar susceptibility to rifampicin and daptomycin, with colony-forming units (CFUs) reductions of approximately 3–4 log10, with a slightly lower response to linezolid, which was also more strain dependent. However, considering all the parameters studied, daptomycin was considered the most effective antibiotic tested, demonstrating an excellent in vitro activity against S. epidermidis biofilm cells. In conclusion, this antibiotic can be strongly considered as an acceptable therapeutic option for S. epidermidis biofilm-associated infections and can represent a potential alternative to rifampicin in serious infections where rifampicin resistance becomes prevalent.Bruna Leite acknowledges the financial support from ISAC/Program Erasmus Munds External Cooperation and the IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus of Gualtar. Fernanda Gomes and Pilar Teixeira fully acknowledge the financial support from Fundacao para a Ciencia e Tecnologia (FCT) through the grants SFRH/BD/32126/2006 and SFRH/BPD/26803/2006, respectively

A Call for Action: The Application of the International Health Regulations to the Global Threat of Antimicrobial Resistance

Stephen Harbarth and colleagues argue that the International Health Regulations (IHR) should be applied to the global health threat of antimicrobial resistance

Structural Basis for Cyclic Py-Im Polyamide Allosteric Inhibition of Nuclear Receptor Binding

Pyrrole-imidazole polyamides are a class of small molecules that can be programmed to bind a broad repertoire of DNA sequences, disrupt transcription factor−DNA interfaces, and modulate gene expression pathways in cell culture experiments. In this paper we describe a high-resolution X-ray crystal structure of a β-amino turn-linked eight-ring cyclic Py-Im polyamide bound to the central six base pairs of the sequence d(5′-CCAGTACTGG-3′)_2, revealing significant modulation of DNA shape. We compare the DNA structural perturbations induced by DNA-binding transcripton factors, androgen receptor and glucocorticoid receptor, in the major groove to those induced by cyclic polyamide binding in the minor groove. The cyclic polyamide is an allosteric modulator that perturbs the DNA structure in such a way that nuclear receptor protein binding is no longer compatible. This allosteric perturbation of the DNA helix provides a molecular basis for disruption of transcription factor−DNA interfaces by small molecules, a minimum step in chemical control of gene networks

Acquisition of Chemoresistance in Gliomas Is Associated with Increased Mitochondrial Coupling and Decreased ROS Production

Temozolomide (TMZ) is an alkylating agent used for treating gliomas. Chemoresistance is a severe limitation to TMZ therapy; there is a critical need to understand the underlying mechanisms that determine tumor response to TMZ. We recently reported that chemoresistance to TMZ is related to a remodeling of the entire electron transport chain, with significant increases in the activity of complexes II/III and cytochrome c oxidase (CcO). Moreover, pharmacologic and genetic manipulation of CcO reverses chemoresistance. Therefore, to test the hypothesis that TMZ-resistance arises from tighter mitochondrial coupling and decreased production of reactive oxygen species (ROS), we have assessed mitochondrial function in TMZ-sensitive and -resistant glioma cells, and in TMZ-resistant glioblastoma multiform (GBM) xenograft lines (xenolines). Maximum ADP-stimulated (state 3) rates of mitochondrial oxygen consumption were greater in TMZ-resistant cells and xenolines, and basal respiration (state 2), proton leak (state 4), and mitochondrial ROS production were significantly lower in TMZ-resistant cells. Furthermore, TMZ-resistant cells consumed less glucose and produced less lactic acid. Chemoresistant cells were insensitive to the oxidative stress induced by TMZ and hydrogen peroxide challenges, but treatment with the oxidant L-buthionine-S,R-sulfoximine increased TMZ-dependent ROS generation and reversed chemoresistance. Importantly, treatment with the antioxidant N-acetyl-cysteine inhibited TMZ-dependent ROS generation in chemosensitive cells, preventing TMZ toxicity. Finally, we found that mitochondrial DNA-depleted cells (ρ°) were resistant to TMZ and had lower intracellular ROS levels after TMZ exposure compared with parental cells. Repopulation of ρ° cells with mitochondria restored ROS production and sensitivity to TMZ. Taken together, our results indicate that chemoresistance to TMZ is linked to tighter mitochondrial coupling and low ROS production, and suggest a novel mitochondrial ROS-dependent mechanism underlying TMZ-chemoresistance in glioma. Thus, perturbation of mitochondrial functions and changes in redox status might constitute a novel strategy for sensitizing glioma cells to therapeutic approaches

Whole Genome Characterization of the Mechanisms of Daptomycin Resistance in Clinical and Laboratory Derived Isolates of Staphylococcus aureus

Background: Daptomycin remains one of our last-line anti-staphylococcal agents. This study aims to characterize the genetic evolution to daptomycin resistance in S. aureus. Methods: Whole genome sequencing was performed on a unique collection of isogenic, clinical (21 strains) and laboratory (12 strains) derived strains that had been exposed to daptomycin and developed daptomycin-nonsusceptibility. Electron microscopy (EM) and lipid membrane studies were performed on selected isolates. Results: On average, six coding region mutations were observed across the genome in the clinical daptomycin exposed strains, whereas only two mutations on average were seen in the laboratory exposed pairs. All daptomycin-nonsusceptible strains had a mutation in a phospholipid biosynthesis gene. This included mutations in the previously described mprF gene, but also in other phospholipid biosynthesis genes, including cardiolipin synthase (cls2) and CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase (pgsA). EM and lipid membrane composition analyses on two clinical pairs showed that the daptomycin-nonsusceptible strains had a thicker cell wall and an increase in membrane lysyl-phosphatidylglycerol. Conclusion: Point mutations in genes coding for membrane phospholipids are associated with the development of reduced susceptibility to daptomycin in S. aureus. Mutations in cls2 and pgsA appear to be new genetic mechanisms affecting daptomycin susceptibility in S. aureus

NOTCH1 Signaling Promotes Human T-Cell Acute Lymphoblastic Leukemia Initiating Cell Regeneration in Supportive Niches

Leukemia initiating cells (LIC) contribute to therapeutic resistance through acquisition of mutations in signaling pathways, such as NOTCH1, that promote self-renewal and survival within supportive niches. Activating mutations in NOTCH1 occur commonly in T cell acute lymphoblastic leukemia (T-ALL) and have been implicated in therapeutic resistance. However, the cell type and context specific consequences of NOTCH1 activation, its role in human LIC regeneration, and sensitivity to NOTCH1 inhibition in hematopoietic microenvironments had not been elucidated.We established humanized bioluminescent T-ALL LIC mouse models transplanted with pediatric T-ALL samples that were sequenced for NOTCH1 and other common T-ALL mutations. In this study, CD34(+) cells from NOTCH1(Mutated) T-ALL samples had higher leukemic engraftment and serial transplantation capacity than NOTCH1(Wild-type) CD34(+) cells in hematopoietic niches, suggesting that self-renewing LIC were enriched within the NOTCH1(Mutated) CD34(+) fraction. Humanized NOTCH1 monoclonal antibody treatment reduced LIC survival and self-renewal in NOTCH1(Mutated) T-ALL LIC-engrafted mice and resulted in depletion of CD34(+)CD2(+)CD7(+) cells that harbor serial transplantation capacity.These results reveal a functional hierarchy within the LIC population based on NOTCH1 activation, which renders LIC susceptible to targeted NOTCH1 inhibition and highlights the utility of NOTCH1 antibody targeting as a key component of malignant stem cell eradication strategies