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.

Effects of Hst3p inhibition in Candida albicans: a genome-wide H3K56 acetylation analysis.

Candida spp. represent the third most frequent worldwide cause of infection in Intensive Care Units with a mortality rate of almost 40%. The classes of antifungals currently available include azoles, polyenes, echinocandins, pyrimidine derivatives, and allylamines. However, the therapeutical options for the treatment of candidiasis are drastically reduced by the increasing antifungal resistance. The growing need for a more targeted antifungal therapy is limited by the concern of finding molecules that specifically recognize the microbial cell without damaging the host. Epigenetic writers and erasers have emerged as promising targets in different contexts, including the treatment of fungal infections. In C. albicans, Hst3p, a sirtuin that deacetylates H3K56ac, represents an attractive antifungal target as it is essential for the fungus viability and virulence. Although the relevance of such epigenetic regulator is documented for the development of new antifungal therapies, the molecular mechanism behind Hst3p-mediated epigenetic regulation remains unrevealed. Here, we provide the first genome-wide profiling of H3K56ac in C. albicans resulting in H3K56ac enriched regions associated with Candida sp. pathogenicity. Upon Hst3p inhibition, 447 regions gain H3K56ac. Importantly, these genomic areas contain genes encoding for adhesin proteins, degradative enzymes, and white-opaque switching. Moreover, our RNA-seq analysis revealed 1330 upregulated and 1081 downregulated transcripts upon Hst3p inhibition, and among them, we identified 87 genes whose transcriptional increase well correlates with the enrichment of H3K56 acetylation on their promoters, including some well-known regulators of phenotypic switching and virulence. Based on our evidence, Hst3p is an appealing target for the development of new potential antifungal drugs.

Selective crossectomy combined with mechanochemical ablation in the treatment of great saphenous vein insufficiency: Early results of a single center experience.

BACKGROUND: Selective crossectomy and mechanochemical ablation (MOCA) of great saphenous vein (GSV) have been used, for years, individually in the treatment of chronic venous insufficiency. In this paper, we focus on the advantages of a combination of the two techniques, in order to prevent complications and recurrence. METHODS: A preoperative clinical and instrumental screening phase was conducted for the purpose of dividing patients into three groups: "Saph+Cross" group (51/139 patients) underwent saphenectomy and crossectomy; "MOCA" group (44/139 patients) underwent MOCA of GSV with Flebogrif® device; "MOCA + Cross" group (44/139 patients) subjected to both MOCA and crossectomy procedures.Recurrence rate, defined as total recanalization of GSV and/or onset of neosaphena and/or new varicose veins, was used as a primary outcome. Secondary outcomes were procedural time and intra- and post-procedural complications. RESULTS: We conducted a 1-, 6-, and 12-month follow-up with Duplex scan. The recurrence rates were 3.9%, 21.8%, and 4.5% for "Saph+Cross," "MOCA," and "MOCA+Cross," respectively, with a significant difference for the comparison between "MOCA" and "Saph+Cross" (MOCA vs Saph+Cross: OR 5.35, CI95% [0.98; 54.6], p-value .040).The sub-analysis of primary outcome highlighted a lower recanalization rate of GSV when combining the crossectomy with MOCA procedure (2.2% MOCA+Cross vs 15.9% MOCA; 0.12 OR, [0.002; 1.02] CI95%, p-value .029).Among the secondary outcomes, "MOCA" showed a shorter procedural time than the other groups (Saph+Cross: 51.3 ± 11.4; MOCA: 45.1 ± 7.5; MOCA+Cross: 50.4 ± 10; p-value .027). No significant differences were noted in terms of intra- and post-procedural complications. CONCLUSIONS: The results showed that patients treated with saphenectomy and crossectomy have a lower recurrence rate compared to MOCA alone and MOCA + crossectomy procedures.The association of crossectomy with MOCA significantly reduces the recanalization rate of GSV, and it is also characterized by a higher free survival from recurrence (SSF) than with MOCA alone.

The use of the Amplatzer Vascular Plug in the prevention of endoleaks during abdominal endovascular aneurysm repair: A systematic literature review on current applications.

OBJECTIVES: The Amplatzer Vascular Plug (AVP) is a vascular occlusion device designed to provide optimal embolization in several fields of the endovascular surgery. A full literature review was conducted to analyze AVPs in comparison with coils for the prevention of endoleaks during endovascular abdominal aortic aneurysm repair. METHODS: A systematic review was designed under PRISMA statement guidelines for systematic reviews and meta-analyses. The results were updated with a subsequent electronic search using Medline and Scopus databases up to December 2019. RESULTS: Eighteen articles making this comparison were found. In 79.7% of the cases, the target vessel was the internal iliac artery; in 1.6%, the common iliac artery; and in 16.7%, the inferior mesenteric artery. Risk of complications (buttock claudication, groin hematoma, endoleaks, and erectile dysfunction) after AVP was low. A cost comparison revealed that the mean cost for coils was around US$2262, while the average cost for the AVP was US$310. CONCLUSIONS: The AVP is an effective and safe device for occluding peripheral vessels, proved to have lower complications rates. Compared with coil embolization, the AVP technique is potentially associated with lower procedural costs.

Efficacy and Safety of Jotec E-Ventus BX Stent Graft for Iliac Branch Device Procedure: A Retrospective Clinical Study.

BACKGROUND: The endovascular aneurysm repair (EVAR) is a successful treatment for aorto-iliac aneurysms. The success of EVAR is enhanced by the use of devices that maintain the patency of targeted arteries namely the iliac branch device (IBD) With this study we aimed to evaluate the association between the use of Jotec E-ventus during EVAR with IBD and prognosis in patients with aorto-iliac aneurysms. METHODS: This is a retrospective, multicentric study enrolling patients referred to our Vascular Surgery Units from January 2015 to January 2020. All patients underwent EVAR with IBD using Jotec E-ventus as bridging stent. Primary endpoint was the development of types I and III endoleaks. Secondary endpoint was the onset of device occlusion with loss of vascular patency. RESULTS: We studied 32 patients (mean age 71.7±4.5y). Of these, 25 patients were treated with standard EVAR procedure whereas 7 were treated with isolated IBD due to extension of disease involving iliac bifurcation. Median follow-up lasted 15[IQR11-27] months. During follow-up, incidence rates for endoleaks and occlusion were 3.98(95%CI 0.48-14.41) and 1.99(95%CI 0.05-11.12) per 100 pts/year. CONCLUSIONS: Jotec E-ventus during EVAR is associated with a low rate of severe complications in a small cohort of patients with aorto-iliac aneurysms.

Position Paper on Young Vascular Surgeons Training of the Mediterranean Federation for the Advancing of Vascular Surgery (MeFAVS): State of the Art and Perspectives.

The Mediterranean Federation for the Advancing of Vascular Surgery (MeFAVS) was founded in 2018, with the aim to promote cooperation among vascular professionals within Mediterranean countries. Due to its prominent social and economic impact on national health systems, diabetic peripheral artery was selected as the very first topic to be investigated by the federation. In this second paper, different experiences from delegates of participating countries were shared to define common strategies to harmonize, standardize, and optimize education and training in the Vascular Surgery specialty.

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.

Integer sequence discovery from small graphs.

We have exhaustively enumerated all simple, connected graphs of a finite order and have computed a selection of invariants over this set. Integer sequences were constructed from these invariants and checked against the Online Encyclopedia of Integer Sequences (OEIS). 141 new sequences were added and six sequences were extended. From the graph database, we were able to programmatically suggest relationships among the invariants. It will be shown that we can readily visualize any sequence of graphs with a given criteria. The code has been released as an open-source framework for further analysis and the database was constructed to be extensible to invariants not considered in this work.

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.