Targeting nicotinamide N-methyltransferase decreased aggressiveness of osteosarcoma cells.

BACKGROUND: Osteosarcoma (OS) is a primary bone malignancy that mostly affects young people, characterized by high metastatic potential, and a marked chemoresistance that is responsible for disease relapse in most patients. Therefore, it is necessary to identify novel molecules to setup targeted strategies to improve the clinical outcome. The enzyme nicotinamide N-methyltransferase (NNMT) catalyses the N-methylation of nicotinamide and other analogs, playing a crucial role in the biotransformation of drugs and xenobiotics. NNMT overexpression was reported in a wide variety of cancers, and several studies demonstrated that is able to promote cell proliferation, migration and resistance to chemotherapy. The aim of this study was to explore the potential involvement of NNMT in OS. METHODS: Immunohistochemical analyses have been performed to evaluate NNMT expression in selected OS and healthy bone tissue samples. Subsequently, OS cell lines have been transfected with vectors targeting NNMT mRNA (shRNAs) and the impact of this downregulation on migration, cell proliferation, and response to chemotherapeutic treatment was also analysed by wound healing, MTT, SRB and Trypan blue assays, respectively. RESULTS: Results showed that OS samples display a significantly higher NNMT expression compared with healthy tissue. Preliminary results suggest that NNMT silencing in OS cell lines is associated to a decrease of cell proliferation and migration, as well as to enhanced sensitivity to chemotherapy. Data obtained showed that NNMT may represent an interesting marker for OS detection and a promising target for effective anti-cancer therapy.

Publisher Correction: Complement-dependent outer membrane perturbation sensitizes Gram-negative bacteria to Gram-positive specific antibiotics.

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Complement-dependent outer membrane perturbation sensitizes Gram-negative bacteria to Gram-positive specific antibiotics.

Gram-negative bacteria are refractory to the action of many antibiotics due to their impermeable outer membrane. An important player of the immune system is the complement system, a protein network in serum that directly kills Gram-negative bacteria through pore-formation by the Membrane Attack Complexes (MAC). We here show that the MAC rapidly perforates the outer membrane but that inner membrane damage, which is essential for killing, is relatively slow. Importantly, we demonstrate that MAC-induced outer membrane damage sensitizes Gram-negative bacteria to otherwise ineffective, Gram-positive-specific, antimicrobials. Synergy between serum and nisin was observed for 22 out of 53 tested Gram-negative clinical isolates and for multi-drug resistant (MDR) blood isolates. The in vivo relevance of this process is further highlighted by the fact that blood sensitizes a MDR K. pneumoniae strain to vancomycin. Altogether, these data imply that antibiotics that are considered ineffective to treat infections with Gram-negatives may have different functional outcomes in patients, due to the presence of the complement system.

Strategies for the Activation and Release of the Membranolytic Peptide Melittin from Liposomes Using Endosomal pH as a Trigger.

Endosomolytic peptides are often coupled to drug delivery systems to enhance endosomal escape, which is crucial for the delivery of macromolecular drugs that are vulnerable to degradation in the endolysosomal pathway. Melittin is a 26 amino acid peptide derived from bee venom that has a very high membranolytic activity. However, such lytic peptides also impose a significant safety risk when applied in vivo as they often have similar activity against red blood cells and other nontarget cell membranes. Our aim is to control the membrane-disrupting capacity of these peptides in time and space by physically constraining them to a nanocarrier surface in such a way that they only become activated when delivered inside acidic endosomes. To this end, a variety of chemical approaches for the coupling of lytic peptides to liposomes via functionalized PEG-lipids were explored, including maleimide-thiol chemistry, click-chemistry, and aldehyde-hydrazide chemistry. The latter enables reversible conjugation via a hydrazone bond, allowing for release of the peptide under endosomal conditions. By carefully choosing the conjugation site and by using a pH activated analog of the melittin peptide, lytic activity toward a model membrane is completely inhibited at physiological pH. At endosomal pH the activity is restored by hydrolysis of the acid-labile hydrazone bond, releasing the peptide in its most active, free form. Furthermore, using an analogue containing a nonhydrolyzable bond as a control, it was shown that the activity observed can be completely attributed to release of the peptide, validating dynamic covalent conjugation as a suitable strategy to maintain safety during circulation.

Analysis of the nutritional management practices in intensive care: Identification of needs for improvement.

OBJECTIVES: To analyze the nutritional management practices in Intensive Care (ICU) to detect the need for improvement actions. Re-evaluate the process after implementation of improvement actions. DESIGN: Prospective observational study in 3 phases: 1) observation; 2) analysis, proposal development and dissemination; 3) analysis of the implementation. SETTING: ICU of a hospital of high complexity. PARTICIPANTS: Adult ICU forecast more than 48h of artificial nutrition. PRIMARY ENDPOINTS: Parenteral nutrition (PN), enteral nutrition (EN) (type, average effective volume, complications) and average nutritional ratio. RESULTS: A total of 229 patients (phase 1: 110, phase 3: 119). After analyzing the initial results, were proposed: increased use and precocity of EN, increased protein intake, nutritional monitoring effectiveness and increased supplementary indication NP. The measures were broadcast at specific meetings. During phase 3 more patients received EN (55.5 vs. 78.2%, P=.001), with no significant difference in the start time (1.66 vs. 2.33 days), duration (6.82 vs. 10,12 days) or complications (37,7 vs. 47,3%).Use of hyperproteic diets was higher in phase 3 (0 vs. 13.01%, P<.05). The use of NP was similar (48.2 vs. 48,7%) with a tendency to a later onset in phase 3 (1.25±1.25 vs. 2.45±3.22 days). There were no significant differences in the average nutritional ratio (0.56±0.28 vs. 0.61±0.27, P=.56). CONCLUSIONS: The use of EN and the protein intake increased, without appreciating effects on other improvement measures. Other methods appear to be necessary for the proper implementation of improvement measures.

Virilizing adrenal adenoma in an adult with the Beckwith-Wiedemann syndrome: paradoxical response to dexamethasone.

An adult woman with Beckwith-Wiedemann syndrome, hemihypertrophy and an androgen-secreting adrenal adenoma is described. She presented with a 7-year history of progressive virilization and was found to have high plasma levels of testosterone and dehydroepiandrosterone (DHEA) sulphate and elevated levels of urinary metabolites of testosterone and its precursors. Administration of dexamethasone was associated with progressive rises in plasma 17 alpha OH progesterone, 11 beta-desoxycortisol, DHEA sulphate, androstenedione and testosterone, together with increased urinary excretion of androsterone, 11 beta OH androsterone, etiocholanolone, DHEA, and 16 alpha OH DHEA. Hormone levels fell to normal following removal of the tumour.