Development of a Potency Assay for Nous-209, a Multivalent Neoantigens-Based Genetic Cancer Vaccine.

Quality control testing of vaccines, including potency assessment, is critical to ensure equivalence of clinical lots. We developed a potency assay to support the clinical advancement of Nous-209, a cancer vaccine based on heterologous prime/boost administration of two multivalent viral vector products: GAd-209 and MVA-209. These consist of a mix of four Adeno (Great Ape Adenovirus; GAd) and four Modified Vaccinia Ankara (MVA) vectors respectively, each containing a different transgene encoding a synthetic polypeptide composed of antigenic peptide fragments joined one after the other. The potency assay employs quantitative Reverse Transcription PCR (RT-Q-PCR) to quantitatively measure the transcripts from the four transgenes encoded by each product in in vitro infected cells, enabling simultaneous detection. Results showcase the assay's robustness and biological relevance, as it effectively detects potency loss in one component of the mixture comparably to in vivo immunogenicity testing. This report details the assay's setup and validation, offering valuable insights for the clinical development of similar genetic vaccines, particularly those encoding synthetic polypeptides.

Antimicrobial Peptides: A New Hope in Biomedical and Pharmaceutical Fields.

Antibiotics are essential drugs used to treat pathogenic bacteria, but their prolonged use contributes to the development and spread of drug-resistant microorganisms. Antibiotic resistance is a serious challenge and has led to the need for new alternative molecules less prone to bacterial resistance. Antimicrobial peptides (AMPs) have aroused great interest as potential next-generation antibiotics, since they are bioactive small proteins, naturally produced by all living organisms, and representing the first line of defense against fungi, viruses and bacteria. AMPs are commonly classified according to their sources, which are represented by microorganisms, plants and animals, as well as to their secondary structure, their biosynthesis and their mechanism of action. They find application in different fields such as agriculture, food industry and medicine, on which we focused our attention in this review. Particularly, we examined AMP potential applicability in wound healing, skin infections and metabolic syndrome, considering their ability to act as potential Angiotensin-Converting Enzyme I and pancreatic lipase inhibitory peptides as well as antioxidant peptides. Moreover, we argued about the pharmacokinetic and pharmacodynamic approaches to develop new antibiotics, the drug development strategies and the formulation approaches which need to be taken into account in developing clinically suitable AMP applications.

New Pieces in the Puzzle of uPAR Role in Cell Migration Mechanisms.

The urokinase (uPA) receptor (uPAR) plays a key role in cell migration. We previously showed that uPAR-negative HEK-293 cells efficiently migrate toward serum but, after uPAR ectopic expression, migrate only in a uPAR-dependent manner. In fact, migration of uPAR-transfected HEK-293 (uPAR-293) cells is impaired by anti-uPAR antibodies, without recovery of the uPAR-independent migration mechanisms formerly active. Prostate carcinoma PC3 cells, which express high endogenous uPAR levels, migrated only through a uPAR-dependent mechanism; in fact, the silencing of uPAR expression inhibited their migration. We hypothesize a crucial role of the uPAR glycosyl-phosphatidyl-inositol (GPI) tail, which promotes uPAR partitioning to lipid rafts, in uPAR-controlled cell migration. Here, we show that removal of the uPAR GPI-tail, or lipid rafts disruption by methyl-beta-cyclodextrin impairs migration of PC3 cells, incapable of uPAR-independent migration, whereas it restores uPAR-independent migration in uPAR-293 cells. We then show that, in PC3 cells, both uPAR signaling partners, ß1 integrins and receptors for formylated peptides (FPRs), partly associate with lipid rafts. Inhibition of their interaction with uPAR impairs this association and impairs cell migration. Interestingly, blocking uPAR association with FPRs also impairs ß1 integrin partitioning to lipid rafts, whereas blocking its association with ß1 integrins has no effect on FPRs partitioning. On these bases, we propose that uPAR controls cell migration by connecting ß1 integrins and FPRs and, through its GPI tail, by driving them into lipid rafts, thus promoting pro-migratory signals. uPAR-mediated partitioning of integrins to lipid rafts is strictly dependent on uPAR association with FPRs.

Type I and II PRMTs regulate catabolic as well as detoxifying processes in Aspergillus nidulans.

In filamentous fungi, arginine methylation has been implicated in morphogenesis, mycotoxin biosynthesis, pathogenicity, and stress response although the exact role of this posttranslational modification in these processes remains obscure. Here, we present the first genome-wide transcriptome analysis in filamentous fungi that compared expression levels of genes regulated by type I and type II protein arginine methyltransferases (PRMTs). In Aspergillus nidulans, three conserved type I and II PRMTs are present that catalyze asymmetric or symmetric dimethylation of arginines. We generated a double type I mutant (ΔrmtA/rmtB) and a combined type I and type II mutant (ΔrmtB/rmtC) to perform genome-wide comparison of their effects on gene expression, but also to monitor putative overlapping activities and reciprocal regulations of type I and type II PRMTs in Aspergillus. Our study demonstrates, that rmtA and rmtC as type I and type II representatives act together as repressors of proteins that are secreted into the extracellular region as the majority of up-regulated genes are mainly involved in catabolic pathways that constitute the secretome of Aspergillus. In addition to a strong up-regulation of secretory genes we found a significant enrichment of down-regulated genes involved in processes related to oxidation-reduction, transmembrane transport and secondary metabolite biosynthesis. Strikingly, nearly 50% of down-regulated genes in both double mutants correspond to redox reaction/oxidoreductase processes, a remarkable finding in light of our recently observed oxidative stress phenotypes of ΔrmtA and ΔrmtC. Finally, analysis of nuclear and cytoplasmic extracts for mono-methylated proteins revealed the presence of both, common and specific substrates of RmtA and RmtC. Thus, our data indicate that type I and II PRMTs in Aspergillus seem to co-regulate the same biological processes but also specifically affect other pathways in a non-redundant fashion.

Phytochemistry of compounds isolated from the leaf-surface extract of Psiadia punctulata (DC.) Vatke growing in Saudi Arabia.

The surface extract of an accession of Psiadia punctulata (DC.) Vatke (Asteraceae) growing in Saudi Arabia was investigated for its phytochemical composition. A bio-guided investigation of the extract led to the isolation of thirteen ent-kaurane and trachylobane diterpenes and seventeen compounds previously described, including nine flavonoids and eight diterpenes. Three flavonoids and one ent-kaurane diterpene showed antimicrobial activity with MIC100 values ranging from 25 to 150 µg/ml. The extract showed antibacterial activity against Staphylococcus aureus (MIC100 = 180 µg/ml) and antifungal activity against Candida albicans (MIC0 = 130 µg/ml). The isolated 3',4',5,7-tetramethoxyflavone, at a concentration of 40 µg/ml, displayed the ability to reduce biofilm formation of S. aureus and C. albicans by 50% and 90% respectively.

Structure Modification of an Active Azo-Compound as a Route to New Antimicrobial Compounds.

Some novel (phenyl-diazenyl)phenols 3a-g were designed and synthesized to be evaluated for their antimicrobial activity. A previously synthesized molecule, active against bacteria and fungi, was used as lead for modifications and optimization of the structure, by introduction/removal or displacement of hydroxyl groups on the azobenzene rings. The aim of this work was to evaluate the consequent changes of the antimicrobial activity and to validate the hypothesis that, for these compounds, a plausible mechanism could involve an interaction with protein receptors, rather than an interaction with membrane. All newly synthesized compounds were analyzed by ¹H-NMR, DSC thermal analysis and UV-Vis spectroscopy. The in vitro minimal inhibitory concentrations (MIC) of each compound was determined against Gram-positive and Gram-negative bacteria and Candida albicans. Compounds 3b and 3g showed the highest activity against S. aureus and C. albicans, with remarkable MIC values of 10 µg/mL and 3 µg/mL, respectively. Structure-activity relationship studies were capable to rationalize the effect of different substitutions on the phenyl ring of the azobenzene on antimicrobial activity.

Design and expression of peptides with antimicrobial activity against Salmonella typhimurium.

We showed previously that insertion of Synechocystis Δ12 -desaturase in salmonella's membrane alters membrane physical state (MPS), followed by the expression of stress genes causing inability to survive within murine macrophages (MΦ). Recently, we showed that expression of one membrane lipid domain (MLD) of Δ12 -desaturase (ORF200) interferes with salmonella MPS, causing loss of virulence in mice and immunoprotection. Here, we postulate that an α-antimicrobial peptide (α-AMP) intercalates within membrane lipids, and depending on its amino acid sequence, it does so within specific key sensors of MLD. In this study, we choose as target for a putative synthetic AMP, PhoP/PhoQ, a sensor that responds to low Mg2+ concentration. We synthesised a modified DNA fragment coding for an amino acid sequence (NUF) similar to that fragment and expressed it in salmonella typhimurium. We showed that the pattern of gene expression controlled by PhoP/PhoQ highlights dysregulation of pathways involving phospholipids biosynthesis, stress proteins and genes coding for antigens. RNA-Seq of strain expressing ORF200 showed that the pattern of those genes is also altered here. Accumulation of NUF conferred temporary immunoprotection. This represents a powerful procedure to address synthetic α-AMPs to a specific MLD generating live non-virulent bacterial strains.

HRMS Profile of a Hazelnut Skin Proanthocyanidin-rich Fraction with Antioxidant and Anti-Candida albicans Activities.

Roasted hazelnut skins (RHS) represent a byproduct of kernel industrial processing. In this research, a RHS extract (RHS-M) and its fraction RHS-M-F3 enriched in proanthocyanidins (PAs), with antioxidant activity, were characterized in terms of total phenolic compound and PA contents. RHS-M and RHS-M-F3 showed antifungal properties against Candida albicans SC5314 (MIC2 = 3.00 and 0.10 µg/mL and MIC0 = 5.00 and 0.50 µg/mL, respectively), determined by the microbroth dilution method and Candida albicans morphological analysis. No cytotoxic effect on HEKa and HDFa cell lines was exhibited by RHS-M and RHS-M-F3. The metabolite profiling of RHS-M and RHS-M-F3 was performed by thiolysis followed by HPLC-UV-HRMS analysis and a combination of HRMS-FIA and HPLC-HRMS(n). Extract and fraction contain oligomeric PAs (mDP of 7.3 and 6.0, respectively, and DP up to 10) mainly constituted by B-type oligomers of (epi)-catechin. Also, (epi)-gallocatechin and gallate derivatives were identified as monomer units, and A-type PAs were detected as minor compounds.