Vassilios Tsikaris

Brain tumor therapy remains limited by the blood–brain barrier (BBB), which restricts drug access. BBB‐penetrating peptides offer a promising strategy for delivering therapeutic and diagnostic payloads. Angiopep‐2 is a well‐established vector, yet novel radioconjugates based on this vector remain of interest. We report the synthesis and evaluation of DOTA‐Angiopep‐2 for radiolabeling with Lutetium‐177 ( ¹⁷⁷ Lu) and Terbium‐161 ( ¹⁶¹ Tb). Notably, ¹⁷⁷ Lu serves as a β‐ and γ‐emitter, whereas ¹⁶¹ Tb is an Auger and β‐emitter; both are utilized in therapy and SPECT imaging. Peptides were synthesized via solid‐phase peptide synthesis. Cytotoxicity assays in T98 glioblastoma cells showed that Angiopep‐2 is well‐tolerated, maintaining ~100% viability at 20 μM and a moderate decline up to 100 μM. Radiolabeling achieved yields > 95% with excellent radiochemical stability at room temperature for up to 10 days and moderate stability in the presence of human serum. Biodistribution in healthy CFW mice showed a brain‐associated radioactivity of 0.24% ± 0.05% IA/g at 5 min p.i. and a 12‐fold increase in the brain‐to‐blood ratio (0.028–0.339) by 60 min p.i. These results support DOTA‐Angiopep‐2 as a versatile platform for radionuclide delivery and a potential candidate for future glioma‐targeted studies. Further studies in tumor‐bearing models are ongoing to evaluate therapeutic efficacy and translational potential.

Identifying blood components influenced by rearing systems that serve as biomarkers to distinguish free-range from conventional broilers can improve animal health, welfare, and productivity. The current study aimed to evaluate specific blood parameters related to immune function and tissue stress, as biomarkers to differentiate free-range, slow-growing Sasso broilers from conventionally raised fast-growing Ross 308 broilers. For this purpose, serum IgM Natural Antibodies (NAbs) targeting actin and lipopolysaccharides (LPS) as key immunological parameters of natural immunity, along with creatine phosphokinase (CPK) and other significant stress and tissue-related biochemical parameters, were measured in a total of 300 broilers (150 per group) raised under industrial scale rearing systems, by standard methodology. Our results showed significantly higher IgM-NAb levels to both antigens and lower CPK and Aspartate Aminotransferase levels in Sasso broilers compared to Ross 308 broilers (p < 0.001). Combination of anti-LPS IgM-NAbs and CPK levels, as the most potent biomarker candidates, achieved 90% sensitivity and 84% specificity in distinguishing Ross 308 from Sasso broilers. In conclusion, the combined evaluation of these blood biomarkers can effectively discriminate conventional from free-range broilers analyzed herein, with potent novel applications in the poultry industry making it a promising, cost effective and easy-to-use, diagnostic tool.

Advances in synthetic peptide methodologies have enabled the development of macromolecules mimicking protein properties and have found applications in various fields, particularly in immunology. Furthermore, Sequential Oligopeptide Carriers (SOC_n and CPSOC) have been designed as multi-functional core molecules, to which multiple bio-cargos can be anchored, allowing the construction of high molecular weight molecules (>3000 Da) capable of inducing a strong immune response. This study presents the design and synthesis of the Ac-[K-Aib-C(3,9-Acm; 6,12-SSNC_(Acm)QENSDK)]₄-NH₂ peptide conjugate of branched architecture. The peptide epitope S¹²⁸SNCQENSDK¹³⁷ belongs to the V. berus basic phospholipase A₂, a member of the European viper species’ most lethal protein families. The peptide epitope was synthesized according to the SPPS Fmoc/tBu strategy and characterized by HR-ESI-MS and NMR experiments, while the conjugate was purified by RP-HPLC and characterized by HR-ESI-MS.

Chemoselective reactions have played a crucial role in the development of high-molecular-weight (>3000 Da) macromolecules with a branched architecture, particularly as peptides and epitope-based vaccines have emerged as promising tools for drug development. Based on this, in this study, we designed and synthesized the peptide macromolecule CH₃CO-[K-Aib-C(3,9-CH₂NHCOCH₃; 6,12-DFLC(CH₂NHCOCH₃)KKESEK)]₄-NH₂ [Ac-K-Aib-C(3,9-Acm); 6,12-epitope)]₄-NH₂] using chemoselective thioether bond formation between the peptide carrier CPSOC (3,9 Acm) and the IAc-DFLC_(Acm)KKESEK-NH₂ peptide epitope. The conjugate was purified via RP-HPLC and characterized via HR-ESI-MS. The epitope D¹²⁸FLCKKESEK¹³⁷ derives from the lethal toxin, ammodytoxin A, from the V. ammodytes snake species, and it was synthesized using the SPPS Fmoc/tBu methodology and characterized via HR-ESI-MS and NMR techniques.

The global demand for improvement in the welfare conditions of broilers has generated the necessity to implement alternative rearing systems as well as less intensive growth hybrids. The majority of the data on alternative farming methods, notwithstanding their abundance, are the result of small-scale experiments. The present extended field study examined the effect of two different industrial farming systems on broilers’ antioxidant status, performance, and meat quality, including 13 replicates of each industrial breeding system (intensive conventional; free range) and two different chicken genotypes (fast growth; slow growth). The duration of the study was 51 months, and the total number of broilers was 260.000 for the conventional and 78.000 for the free-range system. The results showed that fast-growth chicks demonstrated a more satisfactory performance (in terms of body weight gain (BWG) and feed conversion ratio (FCR) with p ≤ 0.001), reduced serum lipid oxidation (p ≤ 0.05), and more tender meat. Contrarily, slow-growth chickens presented significantly higher total antioxidant capacity (TAC) in serum and thigh muscle (p ≤ 0.001), significantly lower (p ≤ 0.05) thigh muscle oxidation (in terms of thiobarbituric acid reactive substances, TBARS), increased protein and decreased fat content (p ≤ 0.05), and better smell, taste, color, and texture. In conclusion, the free-range farming system for slow-growth chickens may result in an overall higher nutritional value, sensory score, and serum and thigh muscle antioxidant profile than the conventional farming system for fast-growth broilers. However, fast-growth broilers exhibit better performance and might undergo less stress.

Naturally occurring antibodies (NAbs), which are major components of innate immunity, exist in circulation under healthy conditions without prior antigenic stimulation and are able to recognize both self- and non-self-constituents. The present study aimed at identifying potential immunological differences between commercial fast- and slow-growth broilers (n = 555) raised in conventional and free-range systems, respectively, through the use of the specificity, isotypes and levels of circulating NAbs. The possible beneficial effect of oregano-based dietary supplementation was also evaluated. To this end, serum IgM and IgY NAbs against self- (actin and DNA) and non-self- antigens (trinitrophenol and lipopolysaccharide) were measured by ELISA and further correlated with genotype, season and performance. Significantly higher levels of IgM NAbs against all antigens were found in slow-growth compared to fast-growth broilers. IgM NAb levels were also significantly increased in dietarily supplemented slow-growth broilers versus those consuming standard feed. Moreover, significantly elevated levels of anti-DNA IgY NAbs were found in fast-growth compared to slow-growth broilers, whereas the opposite was observed for anti-LPS IgY NAbs. Multivariate linear regression analysis confirmed multiple interactions between NAb levels, genotype, season and performance. Overall, serum NAbs have proven to be valuable innovative immunotools in the poultry industry, efficiently differentiating fast-growing versus slow-growing broilers, and dietary supplementation of plant extracts can enhance natural immunity.

The generation of cell‐penetrating peptides as cargo‐delivery systems has produced an immense number of studies owing to the importance of these systems as tools to deliver molecules into the cells, as well as due to the interest to shed light into a yet unclear mechanism of the entrance of these peptides into the cells. However, many cell‐penetrating peptides might present drawbacks due to causing cellular toxicity, or due to being entrapped in endosomes, or as a result of their degradation before they meet their target. In this work, a cargo transporting molecule, the Cell Penetrating Sequential Oligopeptide Carrier (CPSOC), formed by the repetitive ‐Lys‐Aib‐Cys‐ moiety, was tested for its ability to penetrate the cell membrane and transport the conjugated peptides into the cells. The cysteine residue anchors bioactive molecules through a stable thioether bond. The lysine supplies the positive charge to the construct, whereas the α‐amino isobutyric acid is well known to induce helicoid conformation to the peptide backbone and protects from enzymatic degradation. The present study demonstrates that CPSOC penetrates the membrane transporting the conjugated cargo into the cell. When we tested CPSOC‐conjugated peptides carrying critical domains of Cdc42, a small GTPase implicated in exocytosis, the internalized peptides were found to be functional because they inhibited exocytosis of von Willebrand factor from endothelial Weibel–Palade bodies a trafficking event depending on the Cdc42 protein. The data suggest that the carrier can deliver efficiently functional peptides into the cells, and thus, it can be used as a multiple‐cargo transporting molecule.