Characterizing the Oligomers Distribution along the Aggregation Pathway of Amyloid Aß1-40 by NMR.

This study delves into the early aggregation process of the Aß1-40 amyloid peptide, elucidating the associated oligomers distribution. Motivated by the acknowledged role of small oligomers in the neurotoxic damage linked to Alzheimer's disease, we present an experimental protocol for preparing 26-O-acyl isoAß1-40, a modified Aß1-40 peptide facilitating rapid isomerization to the native amide form at neutral pH. This ensures seed-free solutions, minimizing experimental variability. Additionally, we demonstrate the efficacy of coupling NMR diffusion ordered spectroscopy (DOSY) with the Inverse Laplace Transform (ILT) reconstruction method, for effective characterization of early aggregation processes. This innovative approach efficiently maps oligomers distributions across a wide spectrum of initial peptide concentrations offering unique insights into the evolution of oligomers relative populations. As a proof of concept, we demonstrate the efficacy of our approach assessing the impact of Epigallocathechin gallate, a known remodeling agent of amyloid fibrils, on the oligomeric distributions of aggregated Aß1-40. The DOSY-ILT proposed approach stands as a robust and discriminating asset, providing a powerful strategy for rapidly gaining insight into potential inhibitors' impact on the aggregation process.

Facelift: Assessment of Total Platysma Muscle Transection to Prevent the Recurrence of Platysmal Bands.

INTRODUCTION: Determining which facelift technique yields the most effective long-term rejuvenation results and ensures optimal stability over time remains a significant question in cosmetic surgery: Does the most invasive surgery lead to the best long-term outcomes? This study aims to evaluate the authors' approach using total platysma muscle transection to prevent platysma band recurrence, and to provide anatomical observations supporting and justifying their procedure. MATERIAL AND METHODS: A preliminary study in anatomical basic sciences was conducted to establish the rationale for our method. A prospective single-blind study was conducted, involving eighty patients seeking facial rejuvenation with platysmal band correction. They underwent face and neck-lift procedures with total platysma transection by the same surgeon between May 2013 and May 2016. Cosmetic outcomes were assessed using the Face and Neck-Lift Objective Photo-Numerical Assessment Scale. Scores by three blind evaluators before surgery, at 1 and 5 years postoperatively, were compared using a matched T Test (p < 0.05). RESULTS: The preliminary anatomical study revealed a consistent anastomotic system between the cervical branch of the facial nerve and the branches of the cervical plexus. Incomplete platysma section during a facelift might contribute to platysma band recurrence. The clinical study demonstrated satisfactory outcomes, with significant overall appearance improvement (p < 0.00001) and no platysma band recurrence. Complication rate was low. CONCLUSION: The authors' technique achieved satisfactory long-term results with minimal complications. However, due to the lengthy operating time and steep learning curve, it should be reserved for highly motivated patients. LEVEL OF EVIDENCE II: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Molecular Characterization of the Recombinant Ig1 Axl Receptor Domain: An Intriguing Bait for Screening in Drug Discovery.

Axl receptor tyrosine kinase and its ligand Gas6 regulate several biological processes and are involved in both the onset and progression of tumor malignancies and autoimmune diseases. Based on its key role in these settings, Axl is considered a promising target for the development of molecules with therapeutic and diagnostic purposes. In this paper, we describe the molecular characterization of the recombinant Ig1 domain of Axl (Ig1 Axl) and its biochemical properties. For the first time, an exhaustive spectroscopic characterization of the recombinant protein through circular dichroism and fluorescence studies is also reported, as well as a binding analysis to its natural ligand Gas6, paving the way for the use of recombinant Ig1 Axl as a bait in drug discovery screening procedures aimed at the identification of novel and specific binders targeting the Axl receptor.

Determinants of increased nursing workload in the COVID-era: A retrospective analysis of prospectively collected data.

BACKGROUND: COVID-19 is associated with increased nursing workload, therefore a high nurse-to-patient ratio would be required. AIM: To analyse difference in nursing workload, as expressed with the Nursing Activities Score (NAS), between COVID-19 patients versus control patients without COVID-19 disease (NCOVID-19 group) in an Italian Extracorporeal Membrane Oxygenation (ECMO) centre. STUDY DESIGN: Retrospective analysis of prospectively collected data, enrolling consecutive patients admitted to a general Intensive Care Unit, between 1st May 2019 and 28th February 2021. A multivariate analysis was then performed to assess if COVID-19 disease was an independent predictor of higher NAS and to assess which other factors and procedures are independently associated with increased workload. RESULTS: We enrolled 574 patients, of which 135 (24%) were in the COVID-19 group and 439 (76%) in the NCOVID-19 group. The average NAS was higher in the COVID-19 group (79 ± 11 vs. 65 ± 15, T = -10.026; p < 0.001). Prone positioning, continuous renal replacement therapy (CRRT) and ECMO were used more frequently in the COVID-19 group. A higher fraction of patients in the COVID group showed colonization from multidrug resistant bacteria. COVID-19 group had a higher duration of mechanical ventilation and longer ICU stay. The COVID-19 diagnosis was independently associated with a higher NAS. Other independent predictors of higher NAS were the use of prone positioning and continuous renal replacement therapy (CRRT). Colonization from multidrug resistant bacteria and ECMO support were not independently associated with higher NAS. CONCLUSIONS: The higher nursing workload in COVID-19 patients is mainly due to specific procedures required to treat the most hypoxemic patients, such as prone positioning. Colonization with multidrug resistant bacteria and ECMO support were not independently associated with a higher NAS. RELEVANCE TO CLINICAL PRACTICE: Higher workload in COVID-19 patients was due to specific interventions, such as prone positioning and CRRT, with the related nursing activities, as continuous presence at patient's bed, mobilization, positioning and complex hygienic procedures.

A Chemical Strategy for the Preparation of Multimodified Peptide Imaging Probes.

Multimodality probes appear of great interest for innovative imaging applications in disease diagnosis. Herein, we present a chemical strategy enabling site-specific double-modification and cyclization of a peptide probe exploiting native chemical ligation (NCL) and thiol-maleimide addition. The synthetic strategy is straightforward and of general applicability for the development of double-labeled peptide multimodality probes.

Anisotropic Mechanical Response of Bovine Pericardium Membrane Through Bulge Test and In-Situ Confocal-Laser Scanning.

In this work, we present a new experimental setup for the assessment of the anisotropic properties of Bovine Pericardium (BP) membranes. The chemically fixed BP samples have been subjected to a bulge test with in situ confocal laser scanning at increasing applied pressure. The high resolution topography provided by the confocal laser scanning has allowed to obtain a quantitative measure of the bulge displacement; after polynomial fitting, principal curvatures have been obtained and a degree of anisotropy (DA) has been defined as the normalized difference between the maximum and minimum principal curvatures. The experiments performed on the BP membranes have allowed us to obtain pressure-displacement data which clearly exhibit distinct principal curvatures indicating an anisotropic response. A comparison with curvatures data obtained on isotropic Nitrile Buthadiene Rubber (NBR) samples has confirmed the effectiveness of the experimental setup for this specific purpose. Numerical simulations of the bulge tests have been performed with the purpose of identifying a range of constitutive parameters which well describes the obtained range of DA on the BP membranes. The DA values have been partially validated with biaxial tests available in literature and with suitably performed uni-axial tensile tests.

Temporins: Multifunctional Peptides from Frog Skin.

Temporins are short peptides secreted by frogs from all over the world. They exert antimicrobial activity, mainly against Gram-positive bacteria, including resistant pathogens; recent studies highlight other possible applications of these peptides as anticancer or antiviral agents. This review is meant to describe the main features of temporins produced by different ranid genera. Due to the abundance of published papers, we focus on the most widely investigated peptides. We report studies on their mechanism of action and three-dimensional structure in model systems mimicking bacterial membranes or in the presence of cells. The design and the antimicrobial activity of peptide analogues is also described, with the aim of highlighting elements that are crucial to improve the bioactivity of peptides while reducing their toxicity. Finally, a short section is dedicated to the studies aimed at applying these peptides as drugs, to produce new antimicrobial materials or in other technological uses.

Derivatives of GdAAZTA Conjugated to Amino Acids: A Multinuclear and Multifrequency NMR Study.

The GdAAZTA (AAZTA = 6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid) complex represents a platform of great interest for the design of innovative MRI probes due to its remarkable magnetic properties, thermodynamic stability, kinetic inertness, and high chemical versatility. Here, we detail the synthesis and characterization of new derivatives functionalized with four amino acids with different molecular weights and charges: l-serine, l-cysteine, l-lysine, and l-glutamic acid. The main reason for conjugating these moieties to the ligand AAZTA is the in-depth study of the chemical properties in aqueous solution of model compounds that mimic complex structures based on polypeptide fragments used in molecular imaging applications. The analysis of the 1H NMR spectra of the corresponding Eu(III)-complexes indicates the presence of a single isomeric species in solution, and measurements of the luminescence lifetimes show that functionalization with amino acid residues maintains the hydration state of the parent complex unaltered (q = 2). The relaxometric properties of the Gd(III) chelates were analyzed by multinuclear and multifrequency NMR techniques to evaluate the molecular parameters that determine their performance as MRI probes. The relaxivity values of all of the novel chelates are higher than that of GdAAZTA over the entire range of applied magnetic fields because of the slower rotational dynamics. Data obtained in reconstituted human serum indicate the occurrence of weak interactions with the proteins, which result in larger relaxivity values at the typical imaging fields. Finally, all of the new complexes are characterized by excellent chemical stability in biological matrices over time, by the absence of transmetallation processes, or the formation of ternary complexes with oxyanions of biological relevance. In particular, the kinetic stability of the new complexes, measured by monitoring the release of Gd3+ in the presence of a large excess of Zn2+, is ca. two orders of magnitude higher than that of the clinical MRI contrast agent GdDTPA.

Switching the N-Capping Region from all-L to all-D Amino Acids in a VEGF Mimetic Helical Peptide.

The N-capping region of an α-helix is a short N-terminal amino acid stretch that contributes to nucleate and stabilize the helical structure. In the VEGF mimetic helical peptide QK, the N-capping region was previously demonstrated to be a key factor of QK helical folding. In this paper, we explored the effect of the chiral inversion of the N-capping sequence on QK folding, performing conformational analysis in solution by circular dichroism and NMR spectroscopy. The effect of such a modification on QK stability in serum and the proliferative effect were also evaluated.

Probing the helical stability in a VEGF-mimetic peptide.

The analysis of the forces governing helix formation and stability in peptides and proteins has attracted considerable interest in order to shed light on folding mechanism. We analyzed the role of hydrophobic interaction, steric hindrance and chain length on i, i + 3 position in QK peptide, a VEGF mimetic helical peptide. We focused on position 10 of QK, occupied by a leucine, as previous studies highlighted the key role of the Leu7-Leu10 interaction in modulating the helix formation and inducing an unusual thermodynamic stability. Leu10 has been replaced by hydrophobic amino acids with different side-chain length, hydrophobicity and steric hindrance. Ten peptides were, hence, synthesized and analyzed combining circular dichroism, calorimetry and NMR spectroscopy. We found that helical content and thermal stability of peptide QK changed when Leu10 was replaced. Interestingly, we observed that the changes in the helical content and thermal stability were not always correlated and they depend on the type of interaction (strength and geometry) that could be established between Leu7 and the residue in position 10.

Exploiting Protein N-Terminus for Site-Specific Bioconjugation.

Although a plethora of chemistries have been developed to selectively decorate protein molecules, novel strategies continue to be reported with the final aim of improving selectivity and mildness of the reaction conditions, preserve protein integrity, and fulfill all the increasing requirements of the modern applications of protein conjugates. The targeting of the protein N-terminal alpha-amine group appears a convenient solution to the issue, emerging as a useful and unique reactive site universally present in each protein molecule. Herein, we provide an updated overview of the methodologies developed until today to afford the selective modification of proteins through the targeting of the N-terminal alpha-amine. Chemical and enzymatic strategies enabling the selective labeling of the protein N-terminal alpha-amine group are described.

Intraocular pressure reduction with once-a-day application of a new prostaglandin eye drop: a pilot placebo-controlled study in 12 patients.

PURPOSE: To assess the ocular hypotensive effect of 15-keto fluprostenol, the oxidized metabolite of travoprost, on glaucoma patients, through a randomized double-masked placebo-controlled study. METHODS: Twelve patients with ocular normal tension glaucoma (NTG) (intraocular pressure [IOP] < 22 mmHg) were enrolled. In order to ensure patient compliance to treatment, all study subjects were hospitalized. In each patient, the eye to be submitted to the treatments was randomly chosen. After hospital admission (day 1), those patients received for 5 days at 8 P.M. either one drop of 15-keto fluprostenol (35 µg/ml) or one drop of placebo. IOP evaluation was performed within 8 A.M. and 8 P.M. for 6 days. Furthermore, we performed a determination of cardiovascular parameters before and after the treatments. RESULTS: Starting with the first IOP measurement after the first treatment (8 A.M. on day 2), IOP was reduced of about 14% in the eyes treated 15-keto fluprostenol, in comparison with baseline IOP values of 15-keto fluprostenol-treated patients. The IOP reduction in the 15-keto fluprostenol-treated group was significantly compared to placebo group (p < 0.05) starting from day 3 till day 6 of the study. Except for mild hyperemia in one 15-keto fluprostenol-treated eye, no other side effects were observed or reported by the enrolled patients. CONCLUSIONS: The travoprost metabolite 15-keto fluprostenol was effective in decrease IOP and maintained IOP reduction along 5 days of treatment. The 15-keto fluprostenol can be developed as a good candidate for once-a-day NTG patients' treatment.

Short PlGF-derived peptides bind VEGFR-1 and VEGFR-2 in vitro and on the surface of endothelial cells.

The placental growth factor (PlGF), a member of VEGF family, plays a crucial role in pathological angiogenesis, especially ischemia, inflammation, and cancer. This activity is mediated by its selective binding to VEGF receptor 1 (VEGFR-1), which occurs predominantly through receptor domains 2 and 3. The PlGF ß-hairpin region spanning residues Q87 to V100 is one of the key binding elements on the protein side. We have undertaken a study on the design, preparation, and functional characterization of the peptide reproducing this region and of a set of analogues where glycine 94, occurring at the corner of the hairpin in the native protein, is replaced by charged as well as hydrophobic residues. Also, some peptides with arginine 96 replaced by other residues have been studied. We find that the parent peptide weakly binds VEGFR-1, but replacement of G94 with residues bearing H-bond donating residues significantly improves the affinity. Replacement of R96 instead blocks the interaction between the peptide and the domain. The strongest affinity is observed with the G94H (peptide PlGF-2) and G94W (peptide PlGF-10) mutants, while the peptide PlGF-8, bearing the R96G mutation, is totally inactive. The PlGF-1 and PlGF-2 peptides also bind the VEGFR-2 receptors, though with a reduced affinity, and are able to interfere with the VEGF-induced receptor signaling on endothelial cells. The peptides also bind VEGFR-2 on the surface of cells, while PlGF-8 is inactive. Data suggest that these peptides have potential applications as PlGF/VEGF mimic in various experimental settings.

Labeling of VEGFR1D2 through oxime ligation.

We reported an useful protocol for the labeling of the second domain of the Vascular Endothelial Growth Factor Receptor 1 (VEGFR1D2), a small protein ligand able to bind VEGF, the main regulator of angiogenesis. We developed a bioconjugation strategy based on the use of oxime-ligation reaction conjugating an aldehyde derivative of the VEGFR1D2 to a molecular probe harboring an alkoxyamine functional group. We applied the synthetic protocol to prepare a biotinylated conjugate of VEGFR1D2 and we demonstrate that the bioconjugate retains its ability to specifically bind its natural ligand, VEGF, with high affinity. The biotinylated VEGFR1D2 could be useful to detect and quantify VEGF for diagnostic purposes as well as a tool for the screening of new molecules targeting VEGFRs for therapeutic applications. The labeling protocol is versatile and can be extended to different molecular probes, such as fluorophores, chelators or multimeric scaffolds, affording a biomedical platform for VEGF targeting.

Self-Made Rapid Prototyping Technique for Orbital Floor Reconstruction: Showcases for Technical Description.

BACKGROUND: Restoring the orbital cavity integrity in orbital floor defects is a challenging issue due to the anatomical complexity of the floor's surface. This is a showcase for technical description of a novel "in house" rapid prototyping protocol aimed to customize implant for orbital floor reconstruction. METHODS: The authors present 4 cases to show our Computer-aided-design and Computer-aided-manufacturing digital workflow. The system was based on a 3D-printed press that; through a virtually designed mold, was used to conform a patient specific titanium mesh for orbital floor reconstruction. RESULTS: The merging procedure analysis by iPlan Cranial 3.0 (Brainlab, Munich, Germany) highlighted a 0.71 ±â€Š0.23 mm (P <0.05) discrepancy in a point-to-point superimposition between the digital planned reconstruction and the real in vivo result. CONCLUSIONS: The authors expect that this technique will reduce operative time and cost however further study and larger series may better define the applicability in everyday surgical practice.

VEGFR Recognition Interface of a Proangiogenic VEGF-Mimetic Peptide Determined In Vitro and in the Presence of Endothelial Cells by NMR Spectroscopy.

QK peptide is a vascular endothelial growth factor (VEGF)-mimetic molecule with significant proangiogenic activity. In particular, QK is able to bind and activate VEGF receptors (VEGFRs) to stimulate a functional response in endothelial cells. To characterize the peptide bioactivity and its molecular recognition properties, a detailed picture of the interaction between peptide QK and VEGF receptors is reported. By combining NMR spectroscopy studies in solution on the purified receptor and in the presence of intact endothelial cells, a molecular description of the binding interaction between peptide QK and VEGFR2 in the cellular context is obtained. These results reveal useful insights into the peptide biological mechanism, which opens the way to further optimization of this class of VEGF-mimicking peptides.

Pro-angiogenic peptides in biomedicine.

Pro-angiogenic therapy provides a promising new perspective in tackling of many common and severe pathological conditions, such as central and peripheral vascular diseases. Pro-angiogenic therapy also finds interesting applications in the regenerative medicine for the treatment of chronic wounds and in tissue engineering. However, clinical studies on therapeutic angiogenesis, mainly performed by administrating growth factors, have not led to convincing results until now, mainly due to the unfavorable pharmacokinetic and to safety concerns. Thus, the research of new pro-angiogenic molecules endowed of improved pharmacological profile is strongly encouraged. This review focuses on Vascular Endothelial Growth Factor (VEGF) mimetic peptides exerting a pro-angiogenic activity, which are considered among the most promising alternatives to the VEGF based therapy. Peptides show a great potential in drug discovery, as they feature straightforward development approaches, robust and cheap synthetic methodologies for their preparation and functionalization, improved safety and efficacy profiles. Thus, pro-angiogenic peptides represent a valuable alternative to traditional drugs for biomedical applications in cardiovascular diseases and regenerative medicine.

Conformational stabilization of a ß-hairpin through a triazole-tryptophan interaction.

Molecular tools to stabilize the ß-hairpin conformation are needed as ß-hairpin peptides are useful molecules for pharmaceutical, biological and materials applications. We explored the use of a "triazole bridge", a covalent link between two ß-hairpin strands obtained through Cu-catalyzed alkyne-azide cycloaddition, combined with an aromatic-aromatic interaction. Highly conformationally stable peptides were identified by NMR screening of a small collection of cyclic peptides based on the Trpzip2 scaffold. The characteristic Trp-Trp interaction of Trpzip2 was replaced by a diagonal triazole bridge of variable length. NMR and CD analyses showed that triazole and indole rings could favorably interact to stabilize a ß-hairpin conformation. The conformational stabilization depends on the length of the triazole bridge and the reciprocal position between the aromatic rings. Combining aromatic interactions and the covalent inter-strand triazole bridge is a useful strategy to obtain peptides with a high ß-hairpin content.

Venoplant Effect in the Management of the Post-operative Oedema in Plastic Surgery: Results of a Randomized and Controlled Clinical Trial.

BACKGROUND: Post-operative oedema and ecchymosis represent early post-operative complications, impacting negatively on the final aesthetic outcome of each surgical procedure. In particular, such complications are very frustrating for patients and-sometimes-are difficult to be managed by surgeons. Several strategies are available for managing oedema, although some side effects have been reported. A new promising compound for the management of oedema is Venoplant, and this study aims to assess its effectiveness in decreasing post-operative oedema. METHODS: Patients were randomly allocated for receiving three different treatments: (1) Venoplant tablets and Venoplant gel; (2) only Venoplant tablets; and (3) not treated with Venoplant. The aesthetical outcome has been evaluated using the Global Aesthetic Improvement Scale (GAIS), compiled by both patient and clinician. The GAIS scale was administered several times: the day following the surgical procedure (T0) after 3 days (T1), after 7 days (T2), after 15 days (T3) and after 1 month (T4). RESULTS: Forty-three patients participated in the study. According to patient's evaluations, at T0 in Group 1 and in Group 2 a significant statistical difference was found compared to the control group (p < 0.001 and p < 0.05, respectively). Over time, a significant reduction in swelling and ecchymosis was reported by patients treated with Venoplant (tablets alone or in combination with gel) compared to the control group. According to the physician's assessment, during the different time points of evaluation, a significant reduction in post-operative oedema in Group 1 and in Group 2 compared to the control group was found. CONCLUSION: Venoplant represents a valid therapeutic strategy for the management of post-operative oedema, guaranteeing a good level of patient satisfaction, in the absence of common side effects which are often associated with other therapies. LEVEL OF EVIDENCE I: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .