The present study aims to investigate coelomocytes, immune mediators cells in the echinoderm Holothuria tubulosa, as an unusual source of antimicrobial and antibiofilm agents. The activity of the 5kDa peptide fraction of the cytosol from H. tubulosa coelomocytes (5-HCC) was tested against a reference group of Gram-negative and Gram-positive human pathogens. Minimal inhibitory concentrations (MICs) ranging from 125 to 500 mg/ml were determined against tested strains. The observed biological activity of 5-HCC could be due to two novel peptides, identified by capillary RP-HPLC/nESI-MS/MS, which present the common chemical-physical characteristics of antimicrobial peptides. Such peptides were chemically synthesized and their antimicrobial activity was tested. The synthetic peptides showed broad-spectrum activity at 12.5 mg/ml against the majority of the tested Gram-positive and Gram-negative strains, and they were also able to inhibit biofilm formation in a significant percentage at a concentration of 3.1 mg/ml against staphylococcal and Pseudomonas aeruginosa strains.

VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications

VDAC (voltage-dependent anion channel) is the pore-forming protein located in the outer mitochondrial membrane. In higher eukaryotes, three genes encode VDAC. Nevertheless, the knowledge of VDAC isoforms is mainly restricted to VDAC1, the only isoform that has been characterized from living tissues to date. We have highly enriched the isoform VDAC2 using as starting material bovine spermatozoa. VDAC2 was obtained in the hydroxyapatite/celite pass-through of sperm proteins solubilized with Triton X-100. This fraction showed in SDS/PAGE two major bands and one faint band in the molecular mass range of 30–35 kDa. Two-dimensional electrophoresis resolved these bands in ten spots with various Coomassie Blue staining intensities. Western-blot analysis with antibodies monospecific for each isoform and MS peptide sequencing showed that the main protein resolved in electrophoresis was VDAC2 with minor contaminations of the other isoforms. Proteomic analysis of the higher molecular mass VDAC2 protein allowed the coverage of the whole protein with the exception of the tripeptide A₂₄AR₂₆. In the same material, the presence of two possible amino acid substitutions (T₈₈ to L₈₈ and A₉₇ to Q₉₇) was revealed. Reconstitution of VDAC2 pores in planar lipid bilayers showed typical features of mitochondrial porins. Stepwise increases in membrane conductance were observed with a predominant conductance of approx. 3.5 nS (nanoSiemens) in 1 M KCl. Very often, small short-lived fluctuations were observed with single-channel conductance of approx. 1.5 nS. Bovine spermatozoa VDAC2 was anion selective and showed voltage dependence. The present study is the first work to report the purification and characterization of VDAC2 from a mammalian tissue.

Mass spectrometry-based approaches have been successfully applied for identifying ancient proteins in bones and other tissues. On the contrary, there are relatively few examples of the successful recovery and identification of archeological protein residues from ceramic artifacts; this is because ceramics contain much lower levels of proteins which are extensively degraded by diagenetic effects. In this paper, we report the results of the characterization of proteins extracted from pottery of the Maltese site of Baħrija, the guide-site for the Baħrija period (half of 9th–second half of eighth century BCE), recently identified as the final part of the Borġ in-Nadur culture. Proteomic data here reported confirm that one of the major issue of these kind of studies is represented by contamination of animal and human agents that may complicate endogenous protein identification and authentication. The samples tested included a small group of ceramic forms, namely three tableware and six coarse ware thought to have been used in food preparation and/or storage. In this context, the limited availability of paleobotanical and archeozoological analyses may be compensated by the outcomes of the first proteomics profiling which, even if obtained on a limited selection of vessels, revealed the centrality of wheat in the diet of the ancient community of Baħrija. The data have been deposited to the ProteomeXchange with identifier < PXD022848 > .