Haemodynamic Response to a Standard Meal: Consideration on A Case of Significant Blood Pressure Peaks in a Diabetic Hypertensive Patient Treated with ARB and Comparison with Normal Age and Sex Matched Subjects

A standard meal is a stimulus that produces a response that consists in the redistribution of blood flow to the splanchnic district, potentially, affecting systemic blood pressure, this phenomenon was studied in animal models and in critic patient. Here we report a case of a diabetic hypertensive-in-treatment woman where two significant blood pressure peaks were recorded, during lunch and dinner, over an optimal 24/h blood pressure control. In the absence of previous normal reference values in the literature, we retrieved a series of n=10 age and sex matched subjects diagnosed normotensive on the mean of 24/h Ambulatory Blood Pressure Monitoring. We finally present our considerations on the normal response to a standard meal compared to what was found in the literature and in the present case, where an impaired control of resistance is hypothesised, and on the possible mechanisms supporting

Advanced analytical strategies for recombinant therapeutic proteins

Therapeutic proteins produced using recombinant DNA technologies are generally complex, heterogeneous, and subject to a variety of enzymatic or chemical modifications during expression, purification, and long-term storage. Hence the analytical strategies for characterization, quantitation, purity assay and evaluation of the biological activity of recombinant proteins still represent a big challenge and a matter for debate. The aim of the proposed special issue is to point out the main applications as well as future potentialities of the most advanced analytical techniques in the different aspects of the quality assessment of therapeutic proteins and in particular for conformation analysis, aggregates and impurities detection and quantitation, intact protein characterization, post-translational modifications (PTMs) identification and biological activity assessment. The special issue contains six in depth reviews and two original papers. Protein conformation is a key aspect to be assessed, because a specific conformation is essential for the biological function of the protein. The paper by Bertucci et al. points out the growing role of circular dichroism (CD) as a valuable and reliable technique to obtain this information, representing a useful tool for the study of pharmaceutical peptides themselves, in new formulations, after new processes of derivatization, production and storage. The paper also contains examples on the use of CD spectroscopy in the structural characterization of free and formulated recombinant proteins, looking at the prediction of the secondary structure, propensity to conformational changes, stability, and tendency to aggregation. Characterization of protein conformation by high resolution mass spectrometry (direct ESI-MS and hydrogen/ deuterium exchange) is reviewed by Bobst & Kaltashov. The paper provides an overview of the MS techniques and current trends for the characterization of the higher order structure and dynamics of biopharmaceutical products. Recombinant proteins often fail to reach their native conformation and in such cases they form different kinds of aggregates which are unsuitable for the intended applications. This has pressed, on one hand, the exploration of different approaches to favour protein folding, and on the other hand, the development of analytical strategies aimed at detecting soluble and insoluble aggregates. In the review by Garcia- Fruitos et al. the biological aspects of protein folding and unfolding are addressed together with an overview of the most advanced analytical techniques suitable for the fast evaluation of conformational quality and aggregation of recombinant drugs, even if showing apparent solubility

Silkworm pupae as source of high-value edible proteins and of bioactive peptides

To characterize the high-value protein content and to discover new bioactive peptides, present in edible organisms, as silkworm pupae, semiquantitative analytical approach has been applied. The combination of appropriate protein extraction methods, semiquantitative high-resolution mass spectrometry analyses of peptides, in silico bioactivity and gene ontology analyses, allowed protein profiling of silkworm pupae (778 gene products) and the characterization of bioactive peptides. The semiquantitative analysis, based on the measurement of the emPAI, revealed the presence of high-abundance class of proteins, such as larval storage protein (LSP) class. This class of proteins, beside its nutrient reservoir activity, is of great pharmaceutical interest for their efficacy in cardiovascular diseases. Potential allergens were also characterized and quantified, such as arginine kinase, thiol peroxiredoxin, and Bom m 9. This powerful bioanalytical approach proved the potential industrial applications of Bombyx mori pupae, as source of high-value proteins in a green and \u201ccircular\u201d economy perspective

Development of a direct ESI-MS method for measuring the tannin precipitation effect of proline-rich peptides and in silico studies on the proline role in tannin-protein interactions

Tannins are a heterogeneous class of polyphenols that are present in several plants and foods. Their ability to interact and precipitate proline-rich proteins leads to different effects such as astringency or antidiarrheal activity. Thus, evaluation of the tannin content in plant extracts plays a key role in understanding their potential use as pharmaceuticals and nutraceuticals. Several methods have been proposed to study tannin-protein interactions but few of them are focused on quantification. The purpose of the present work is to set up a suitable and time efficient method able to quantify the extent of tannin protein precipitation. Bradykinin, chosen as a model, was incubated with increasing concentrations of 1,2,3,4,6-penta-O-galloyl-\u3b2-D-glucose and tannic acid selected as reference of tannic compounds. Bradykinin not precipitated was determined by a mass spectrometer TSQ Quantum Ultra Triple Quadrupole (direct infusion analysis). The results were expressed as PC 50 , which is the concentration able to precipitate 50% of the protein. The type of tannin-protein interaction was evaluated also after precipitate solubilisation. The involvement of proline residues in tannin-protein interactions was confirmed by repeating the experiment using a synthesized peptide (RR-9) characterized by the same bradykinin sequence, but having proline residues replaced by glycine residues: no interaction occurred between the peptide and the tannins. Moreover, modelling studies on PGG-BK and PGG-RR-9 were performed to deeply investigate the involvement of prolines: a balance of hydrophobic and H-bond contacts stabilizes the PGG-BK cluster and the proline residues exert a crucial role thus allowing the PGG molecules to elicit a sticking effect

Advanced quantitative proteomics to evaluate molecular effects of low-molecular-weight hyaluronic acid in human dermal fibroblasts

Hyaluronic acid (HA) is physiologically synthesized by several human cells types but it is also a widespread ingredient of commercial products, from pharmaceuticals to cosmetics. Despite its extended use, the precise intra- and extra-cellular effects of HA at low-molecular-weight (LWM-HA) are currently unclear. At this regard, the aim of this study is to in-depth identify and quantify proteome's changes in normal human dermal fibroblasts after 24 h treatment with 0.125, 0.25 and 0.50 % LMW-HA (20 1250 kDa) respectively, vs controls. To do this, a label-free quantitative proteomic approach based on high-resolution mass spectrometry was used. Overall, 2328 proteins were identified of which 39 significantly altered by 0.125 %, 149 by 0.25 % and 496 by 0.50 % LMW-HA. Protein networking studies indicated that the biological effects involve the enhancement of intracellular activity at all concentrations, as well as the extracellular matrix reorganization, proteoglycans and collagen biosynthesis. Moreover, the cell's wellness was confirmed, although mild inflammatory and immune responses were induced at the highest concentration. The more complete comprehension of intra- and extra-cellular effects of LMW-HA here provided by an advanced analytical approach and protein networking will be useful to further exploit its features and improve current formulations

Insights into the effects of N-glycosylation on the characteristics of the VC1 domain of the human receptor for advanced glycation end products (RAGE) secreted by Pichia pastoris

Advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs), resulting from non-enzymatic modifications of proteins, are potentially harmful to human health. They directly act on proteins, affecting structure and function, or through receptor-mediated mechanisms. RAGE, a type I transmembrane glycoprotein, was identified as a receptor for AGEs. RAGE is involved in chronic inflammation, oxidative stress-based diseases and ageing. The majority of RAGE ligands bind to the VC1 domain. This domain was successfully expressed and secreted by Pichia pastoris. Out of two N-glycosylation sites, one (Asn25) was fully occupied while the other (Asn81) was under-glycosylated, generating two VC1 variants, named p36 and p34. Analysis of N-glycans and of their influence on VC1 properties were here investigated. The highly sensitive procainamide labeling method coupled to ES-MS was used for N-glycan profiling. N-glycans released from VC1 ranged from Man9GlcNAc2- to Man15GlcNAc2- with major Man10GlcNAc2- and Man11GlcNAc2- species for p36 and p34, respectively. Circular dichroism spectra indicated that VC1 maintains the same conformation also after removal of N-glycans. Thermal denaturation curves showed that the carbohydrate moiety has a small stabilizing effect on VC1 protein conformation. The removal of the glycan moiety did not affect the binding of VC1 to sugar-derived AGE- or malondialdehyde-derived ALE-human serum albumin. Given the crucial role of RAGE in human pathologies, the features of VC1 from P. pastoris will prove useful in designing strategies for the enrichment of AGEs/ALEs from plasma, urine or tissues, and in characterizing the nature of the interaction

Colostrum from cows immunized with a veterinary vaccine against bovine rotavirus displays enhanced in vitro anti-human rotavirus activity

Human rotaviruses represent a major cause of severe diarrheal disease in infants and young children. The limited impact of oral vaccines on global estimates of rotavirus mortality and the suboptimal use of oral rehydration justify the need for alternative prophylactic and therapeutic strategies, especially for immunocompromised hosts. The protective effects of colostrum\u2014the first milk produced during the initial 24 to 48 h after parturition\u2014are well documented in the literature. In particular, the ingestion of hyperimmune bovine colostrum has been proposed as an alternative preventive approach against human rotavirus gastroenteritis. Although the immunization of pregnant cows with human rotavirus boosts the release of specific immunoglobulin G in bovine colostrum, it raises regulatory and safety issues. In this study, we demonstrated that the conventional bovine rotavirus vaccine is sufficient to enhance the anti-human rotavirus protective efficacy of bovine colostrum, thus providing a conservative approach to produce hyperimmune bovine colostrum, making it exploitable as a functional food

Selective Protein Conjugation of Poly(glycerol monomethacrylate) and Poly(polyethylene glycol methacrylate) with Tunable Topology via Reductive Amination with Multifunctional ATRP Initiators for Activity Preservation

In this study, we compare poly(glycerol monomethacrylate) (PGMA) of different chain lengths and architectures (linear and two-arm) with poly(poly(ethylene glycol) methyl ether methacrylate) (PPEGMA) as an alternative polymer platform for the synthesis of a new generation of protein-polymer conjugates. Mono-and two-arm functional atom-transfer radical polymerization (ATRP) initiators were designed and selectively attached to lysozyme at the N-terminus via reductive amination. Site-specific, grafting from activator regenerated by electron transfer (ARGET) ATRP was carried out in phosphate buffer, and the reaction parameters were optimized to obtain polymer conjugates with predetermined molar mass and topology. The activity preservation under proteolytic and high-temperature conditions showed a clear dependence on the structure of the repeating unit and on the macromolecular architecture. These results highlighted the potential of PGMA as a poly(ethylene glycol) (PEG) alternative for the half-life extension of biotherapeutics. Moreover, this synthetic approach may inspire the design of a new class of protein-polymer conjugates through an optimal combination of macromolecular composition and topology

PP271—Novel therapeutic strategy to prevent chemotherapy-induced persistent sensory neuropathy by TRPA1 blockade

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