Elucidation of the Molecular Mechanisms Underlying the Ability of <i>C. Elegans</i> Pharynx to Recognize as Toxicant the Soluble, Pre-Fibrillar Assemblies of the Amyloidogenic Proteins

Immunoglobulin light chain amyloidosis (AL) is the most common form of systemic amyloidosis in which a plasma-cell dyscrasia results in the overproduction of amyloidogenic immunoglobulin light chains (LC). Although LC organize in extracellular deposits in different target organs, approximately 75% of patients manifest heart involvement at presentation, with a median survival of only 6 months if chemotherapy fails to stop LC production. Although an active role of radical oxygen species (ROS) has already been envisaged, the actual mechanisms behind their generation remain elusive. In this study, it was observed that amyloidogenic LC purified from patients with cardiac involvement intrinsically generated high levels of ROS and, when administered to C. elegans resulted in ROS production. These species can directly target the pharyngeal cells, causing remarkable damage particularly at the mitochondrial level, similar to that observed in amyloid-affected hearts from AL patients. Tetracycline counteracted all these ROS-mediated effects and, when added to standard chemotherapy, they reduced early deaths in patients with cardiac AL. To limit and repair the stress-induced damage, different intracellular signalling events are activated. In particular, the nuclear translocation of the FOXO/DAF-16 transcription factor triggers the transcription of stress-responsive genes, including heat-shock protein (hsp)-16.2 and manganese superoxide dismutase (sod)-3, and controlling stress resistance and survival. All these effects were entirely dependent on the presence of metal ions, particularly copper. Metal chelators or metal-binding compounds, particularly the new drug PBT2, block ROS production and interrupt the vicious cycle of oxidative stress. In C. elegans, low doses of PBT2 in combination with tetracycline result in a synergistic beneficial effect, highlighting the potential application of this pharmacological strategy for AL patients. These findings indicate that metal-induced oxidative stress, already reported to be linked with some neurological disorders, is also a key element in cardiac AL amyloidosis

Work-Related Stress Mediates the Impact of Safety Climate on Safety Outcomes

WORK-RELATED STRESS MEDIATES THE IMPACT OF SAFETY CLIMATE ON SAFETY OUTCOMES The risk of work related injuries and accidents is still one of the main issues in the world of work in Italy. In 2011 there were 726,000 accidents in the workplace, 930 of which were fatal. These figures have been steadily declining since 2007, although this trend seems to be more related to the economic crisis than to a real improvement in working conditions. During the last few years the approach to prevention of injury has focused more on the human factor, in particular highlighting the important role that work-related stress, safety climate and safety culture can play in preventing work accidents. In this context, this research aims to investigate the relationship between safety climate in the work environment and safety performance and also to analyse how work-related stress may affect this interaction. Safety climate has been defined as shared perceptions regarding the policies, procedures and practices. These provide a series of regulations regarding behaviour in the work environment and mean that it is possible to determine performance in terms of safety of workers. This in turn leads to a positive climate which enhances safer behaviour, whereas in a negative climate unsafe behaviour increases. There is therefore a close connection between safety climate and the occurrence of accidents and injuries. Psychological distress directly affects individual psychophysical well-being and behaviour and can affect performance in terms of safety procedures (Melià 2008; Zohar 2005). The presence of a safety climate, safety performance and the risk of work-related stress were assessed in 175 metal and mechanical workers employed in two different companies in the north-east of Italy. The workers were all men, divided into 6 age groups: 4 workers (2.3%) were aged 19 to 25 years, 36 (20.6%) were from 26 to 35 years, 79 (45.1%) were from 36 to 45 years, 45 (25.7%) were from 46 to 55 years and 8 (4.6%) were more than 55 years. 3 subjects (1.7%) did not provide information about their demographic status. The validated Integrated Organizational Safety Climate Questionnaire was used to assess three safety climate measures (Organizational, Supervisor and Co-worker) (Brondino, Pasini and Silva 2011). Each item has a 7-point scale that ranges from 1 = never to 7 = always. Organizational safety climate (OSC) is measured with a 12-item scale, in which the worker is asked to judge the safety climate of the entire organization. Supervisor's safety climate (SSC) is measured with a 10-item scale, in which the workers had to judge the real importance given to safety by their direct supervisor in the work-group. Co-workers' safety climate (CSC) is measured with a 12-item scale in which the workers rate the degree to which safety is a real priority of their colleagues. Safety performance are measured with a 4-item scale which refers to individual performance of safety compliance. The scale is an adjusted version of Griffin & Neal scale about safety behaviour (2000). Responses were given on a 7-point Likert scale, from 1 = “not at all” to 7 = “very much”. The GHQ-12 questionnaire, which is widely used to assess levels of psychological distress, was also employed. The Italian version of the HSE Management Standards Indicator Tool was used to asses work related stress. It consists of 35 items that identify six organizational dimensions. These are: demands (including issues such as workload, work patterns and the work environment), control (how much say the person has in the way they do their work), support (including the encouragement, sponsorship and resources provided by the organisation, line management and colleagues), relationships (whether the organisation promotes positive working to avoid conflict and deals with unacceptable behaviour), role (whether people understand their role within the organisation and whether the organisation ensures that they do not have conflicting roles) and change (how organisational change is managed and communicated in the organisation). Data were analysed using the AMOS package, a software package designed to create structural equation modelling (SEM). The results of the study seem to confirm that work related stress, connected with psychological distress, mediate the relationship between safety climate and safety performance

The cDNA of the neutrophil antibiotic Bac5 predicts a pro-sequence homologous to a cysteine proteinase inhibitor that is common to other neutrophil antibiotics.

Bac5 is a 5-kDa proline- and arginine-rich antibiotic, stored as inactive precursor (proBac5) in the large granules of bovine neutrophils. A full-length cDNA encoding the precursor form of Bac5 has been cloned. The encoded protein (pre-proBac5) has a calculated mass of 20,031 Da and a pI of 9.21. This comprises a putative signal peptide of 29 amino acid residues and a 101-residue pro-sequence that precede the mature antibiotic. The pro-sequence is acidic and may neutralize the highly cationic Bac5, thus accounting for the inactivation of the antibiotic activity observed in in vitro experiments. The structure of mature Bac5 agrees closely with the amino acid sequence previously determined, with an additional tripeptide tail predicting carboxyl-terminal amidation. A valyl residue is deduced at the cleavage site for the proteolytic maturation of proBac5, consistent with a previous observation showing elastase as the enzyme involved in this processing step. The region upstream of Bac5 reveals high identity to corresponding regions of two neutrophil antimicrobial polypeptides, CAP18 from rabbit and bovine indolicidin. The COOH-terminal sequences of these antibiotics are completely unrelated. The proregion also exhibits remarkable similarity to pig cathelin, an inhibitor of cathepsin L, indicating a common evolutionary origin

MBOAT7 is anchored to endomembranes by six transmembrane domains.

Abstract Membrane bound O-acyltransferase domain- containing 7 (MBOAT7, also known as LPIAT1) is a protein involved in the acyl chain remodeling of phospholipids via the Lands' cycle. The MBOAT7 is a susceptibility risk genetic locus for non-alcoholic fatty liver disease (NAFLD) and mental retardation. Although it has been shown that MBOAT7 is associated to membranes, the MBOAT7 topology remains unknown. To solve the topological organization of MBOAT7, we performed: A) solubilization of the total membrane fraction of cells overexpressing the recombinant MBOAT7-V5, which revealed MBOAT7 is an integral protein strongly attached to endomembranes; B) in silico analysis by using 22 computational methods, which predicted the number and localization of transmembrane domains of MBOAT7 with a range between 5 and 12; C) in vitro analysis of living cells transfected with GFP-tagged MBOAT7 full length and truncated forms, using a combination of Western Blotting, co-immunofluorescence and Fluorescence Protease Protection (FPP) assay; D) in vitro analysis of living cells transfected with FLAG-tagged MBOAT7 full length forms, using a combination of Western Blotting, selective membrane permeabilization followed by indirect immunofluorescence. All together, these data revealed that MBOAT7 is a multispanning transmembrane protein with six transmembrane domains. Based on our model, the predicted catalytic dyad of the protein, composed of the conserved asparagine in position 321 (Asn-321) and the preserved histidine in position 356 (His-356), has a lumenal localization. These data are compatible with the role of MBOAT7 in remodeling the acyl chain composition of endomembranes

Bovine Neutrophil Antibiotic Peptides and Their Precursors: Structure and Role in Innate Immunity

Four peptides were characterized in extracts of bovine neutrophil granules: an Arg-rich dodecapeptide, maintained in a cyclic structure by a disulfide bridge; a Trp-rich tridecapeptide named indo- licidin; and two 43- and 59 amino acids long peptides, named Bac5 and Bac7, with frequent repeats of the triplets Arg-Pro-Pro and Pro-Arg-Pro, respectively. The full length cDNA of the first three of these peptides was characterized recently. Sequence analysis showed that the prosequences of the predicted precursors of all the three peptides are highly identical and exhibited also a remarkable similarity to cathelin, a porcine inhibitor of cathepsin L. Purified proBacö actually proved in in vitro assays to inhibit cathepsin L, but not other cysteine proteinases such as cathepsin B. Unlike proBacö, proBac7 is selectively chemotactic to monocytes. Several fragments of Bac5 and Bac7 (from 6 to 35 residues) were synthesized by the Fmoc method. The results of antibacterial assays show that the N-terminal portion, the most cationic one in both Bac5 and Bac7, is essential for the antimicrobial activity and that the minimal length necessary to arrest the growth of susceptible bacteria is 18-20 residues

Antioxidant and Anti-Inflammaging Ability of Prune (Prunus Spinosa L.) Extract Result in Improved Wound Healing Efficacy

none12sìPrunus spinosa L. fruit (PSF) ethanol extract, showing a peculiar content of biologically active molecules (polyphenols), was investigated for its wound healing capacity, a typical feature that declines during aging and is negatively affected by the persistence of inflammation and oxidative stress. To this aim, first, PSF anti-inflammatory properties were tested on young and senescent LPS-treated human umbilical vein endothelial cells (HUVECs). As a result, PSF treatment increased miR-146a and decreased IRAK-1 and IL-6 expression levels. In addition, the PSF antioxidant effect was validated in vitro with DPPH assay and confirmed by in vivo treatments in C. elegans. Our findings showed beneficial effects on worms’ lifespan and healthspan with positive outcomes on longevity markers (i.e., miR-124 upregulation and miR-39 downregulation) as well. The PSF effect on wound healing was tested using the same cells and experimental conditions employed to investigate PSF antioxidant and anti-inflammaging ability. PSF treatment resulted in a significant improvement of wound healing closure (ca. 70%), through cell migration, both in young and older cells, associated to a downregulation of inflammation markers. In conclusion, PSF extract antioxidant and antiinflammaging abilities result in improved wound healing capacity, thus suggesting that PSF might be helpful to improve the quality of life for its beneficial health effects.openSofia Coppari, Mariastella Colomba, Daniele Fraternale, Vanessa Brinkmann, Margherita Romeo, Marco Bruno Luigi Rocchi, Barbara Di Giacomo, Michele Mari, Loretta Guidi, Seeram Ramakrishna, Natascia Ventura, Maria Cristina AlbertiniCoppari, Sofia; Colomba, Mariastella; Fraternale, Daniele; Brinkmann, Vanessa; Romeo, Margherita; Rocchi, MARCO BRUNO LUIGI; DI GIACOMO, Barbara; Mari, Michele; Guidi, Loretta; Ramakrishna, Seeram; Ventura, Natascia; Albertini, MARIA CRISTIN

Multigram synthesis and in vivo efficacy studies of a novel multitarget anti-Alzheimer's compound

We describe the multigram synthesis and in vivo efficacy studies of a donepezil‒huprine hybrid that has been found to display a promising in vitro multitarget profile of interest for the treatment of Alzheimer's disease (AD). Its synthesis features as the key step a novel multigram preparative chromatographic resolution of intermediate racemic huprine Y by chiral HPLC. Administration of this compound to transgenic CL4176 and CL2006 Caenorhabditis elegans strains expressing human Aβ42, here used as simplified animal models of AD, led to a significant protection from the toxicity induced by Aβ42. However, this protective effect was not accompanied, in CL2006 worms, by a reduction of amyloid deposits. Oral administration for 3 months to transgenic APPSL mice, a well-established animal model of AD, improved short-term memory, but did not alter brain levels of Aβ peptides nor cortical and hippocampal amyloid plaque load. Despite the clear protective and cognitive effects of AVCRI104P4, the lack of Aβ lowering effect in vivo might be related to its lower in vitro potency toward Aβ aggregation and formation as compared with its higher anticholinesterase activities. Further lead optimization in this series should thus focus on improving the anti-amyloid/anticholinesterase activity ratio

Exome-Wide Association Study on Alanine Aminotransferase Identifies Sequence Variants in the GPAM and APOE Associated With Fatty Liver Disease

BACKGROUND & AIMS: Fatty liver disease (FLD) is a growing epidemic that is expected to be the leading cause of end-stage liver disease within the next decade. Both environmental and genetic factors contribute to the susceptibility of FLD. Several genetic variants contributing to FLD have been identified in exome-wide association studies. However, there is still a missing hereditability indicating that other genetic variants are yet to be discovered. METHODS: To find genes involved in FLD, we first examined the association of missense and nonsense variants with alanine amino transferase at an exome-wide level in 425,671 participants from the UK Biobank. We then validated genetic variants with liver fat content in 8930 participants in whom liver fat measurement was available, and replicated 2 genetic variants in 3 independent cohorts comprising 2621 individuals with available liver biopsy. RESULTS: We identified 190 genetic variants independently associated with alanine aminotransferase after correcting for multiple testing with Bonferroni method. The majority of these variants were not previously associated with this trait. Among those associated, there was a striking enrichment of genetic variants influencing lipid metabolism. We identified the variants rs2792751 in GPAM/GPAT1, the gene encoding glycerol-3phosphate acyltransferase, mitochondrial, and rs429358 in APOE, the gene encoding apolipoprotein E, as robustly associated with liver fat content and liver disease after adjusting for multiple testing. Both genes affect lipid metabolism in the liver. CONCLUSIONS: We identified 2 novel genetic variants in GPAM and APOE that are robustly associated with steatosis and liver damage. These findings may help to better elucidate the genetic susceptibility to FLD onset and progression.Peer reviewe