Size-exclusion HPLC Research Articles

Does Fungal Chitosan Leave Noticeable Traces in Treated Wines?

Background (1): The use of fungal chitosan as an antiseptic in wine appears as a promising alternative to sulfur dioxide for the elimination of Brettanomyces bruxellensis sensitive strains. Nevertheless, its utilization raises the question, "how are the treated wines different from the untreated ones?" Methods (2): Chitosan treatment residues were sought in the oligosaccharide and polysaccharide fractions and among 224 low MW ions (<1800 g·mol-1) in several wines by using liquid chromatography (size exclusion HPLC or LC-MS) and GC-MS. Standard oenological parameters were also examined as well as possible sensory modifications by a panel of tasters composed of experts and non-experts. Results (3): None of these methods enabled the reproducible and reliable identification of a treated wine without comparing it to its untreated control. The fingerprints of treatment are not reliably detectable by the analytical methods used in this study. However, the treated wines seem permanently protected against the development of chitosan-sensitive strains of B. bruxellensis. Conclusions (4): If chitosan treatment modifies the wine, the associated changes were not identified by the liquid chromatography method mentioned above and they were not perceived by most people in our taster panel. However, the expected antimicrobial action of chitosan was observed on B. bruxellensis sensitive strains and persisted at least one year. Tolerant strains were less affected by these persistent effects.

Analysis of Therapeutic Monoclonal Antibodies Using a Platform Size Exclusion-HPLC Method

In biotherapeutics manufacturing, controlling size variants of therapeutic monoclonal antibodies (mAbs) is critical for ensuring product quality. Size-exclusion chromatography (SEC) is a leading method for analyzing size variants by separating high molecular weight and low molecular weight species from the monomer based on their hydrodynamic radius. We present a platform size exclusion-HPLC (SE–HPLC) method for analyzing a wide range of mAbs of different subclasses, demonstrating its feasibility through rigorous validation and evaluation. Our study highlights the method’s reproducibility, robustness, and ability to distinguish differences in size variants.

Impact on Quality during In-Use Preparation of an Antibody Drug Conjugate with Eight Different Closed System Transfer Device Brands.

The aim of this study was to investigate the in-use compatibility of eight commercially available closed system transfer device brands (CSTDs) with a formulated model antibody drug conjugate (ADC). Overall, in-use simulated dosing preparation applying the CSTD systems investigated raised concerns for several product quality attributes. The incompatibilities observed were mainly associated with increased visible and subvisible particles formation as well as significant changes in holdup volumes. Visible and subvisible particles contained heterogeneous mixtures of particle classes, with the majority of subvisible particles associated with silicone oil leaching from CSTD systems during simulated dose preparation upon contact with the ADC formulation. These observations demonstrate that CSTD use may adversely impact product quality and delivered dose which could potentially lead to safety and efficacy concerns during administration. Other product quality attributes measured including turbidity, color, ADC recovery, and purity by size exclusion HPLC, did not show relevant changes. It is therefore strongly recommended to test and screen the compatibility of CSTDs with the respective ADC, in a representative in-use simulated administration setting, during early CMC development, i.e., well before the start of clinical studies, to include information about compatibility and to ensure that the CSTD listed in the manuals of preparation for clinical handling has been thoroughly assessed before human use.

Contribution of a C-Terminal Extension to the Substrate Affinity and Oligomeric Stability of Aldehyde Dehydrogenase from Thermus thermophilus HB27.

Aldehyde dehydrogenase enzymes (ALDHs) are widely studied for their roles in disease propagation and cell metabolism. Their use in biocatalysis applications, for the conversion of aldehydes to carboxylic acids, has also been recognized. Understanding the structural features and functions of both prokaryotic and eukaryotic ALDHs is key to uncovering novel applications of the enzyme and probing its role in disease propagation. The thermostable enzyme ALDHTt originating fromThermus thermophilus, strain HB27, possesses a unique extension of its C-terminus, which has been evolutionarily excluded from mesophilic counterparts and other thermophilic enzymes in the same genus. In this work, the thermophilic adaptation is studied by the expression and optimized purification of mutant ALDHTt-508, with a 22-amino acid truncation of the C-terminus. The mutant shows increased activity throughout production compared to native ALDHTt, indicating an opening of the active site upon C-terminus truncation and giving rationale into the evolutionary exclusion of the C-terminal extension from similar thermophilic and mesophilic ALDH proteins. Additionally, the C-terminus is shown to play a role in controlling substrate specificity of native ALDH, particularly in excluding catalysis of certain large and certain aromatic ortho-substituted aldehydes, as well as modulating the protein's pH tolerance by increasing surface charge. Dynamic light scattering and size-exclusion HPLC methods are used to show the role of the C-terminus in ALDHTt oligomeric stability at the cost of catalytic efficiency. Studying the aggregation rate of ALDHTt with and without a C-terminal extension leads to the conclusion that ALDHTt follows a monomolecular reaction aggregation mechanism.

Optimizing expression, purification, structural and functional assessments of a novel dimeric incretin (GLP-1cpGLP-1)

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that reduces postprandial glycemic excursions by enhancing insulin secretion. In this study, a new dimeric GLP-1 analogue (GLP-1cpGLP-1) was designed by inserting human insulin C-peptide (CP) in the middle of a dimer of [Gly8] GLP-1 (7–36). Then, the dimeric incretin (GLP-1cpGLP-1) was ligated to human αB-crystallin (αB-Cry) to create a hybrid protein, abbreviated as αB-GLP-1cpGLP-1. The constructed gene was well expressed in the bacterial host system. After specific chemical release from the hybrid protein, the dimeric incretin was purified by size exclusion chromatography (SEC). Finally, the RP-HPLC analysis indicated a purity of >99 % for the dimeric incretin. The secondary structure assessments by various spectroscopic methods, and in silico analysis suggested that the dimeric incretin has α-helical rich structure. The dynamic light scattering (DLS) analysis indicates that our dimeric incretin forms large oligomeric structures. This incretin analogue significantly reduced blood glucose levels in both healthy and diabetic mice while effectively triggering insulin release. The size exclusion HPLC also indicates the interaction of the new incretin analogue with human serum albumin, the main carrier protein in the bloodstream. Consistent with the results obtained from the biological activity assessments, this significant interaction indicates its potential as a viable therapeutic agent with a long-lasting effect. The results of our research represent a significant breakthrough in the successful design of an active incretin dimer capable of effectively controlling blood sugar levels and inducing insulin secretion in the realm of diabetes treatment.

B-304 Performance of Alternative Proficiency Testing Program for Bayesian Forecasting Tool of Infliximab Exposure in Clinical Pharmacokinetic Laboratory

Abstract Background We have developed a Bayesian (PK) forecasting and dosing tool for Infliximab (IFX), a monoclonal antibody targeting Tumor necrosis factor α. This novel dosing tool employs machine learning and proficiency of the algorithm must be established. Our objective was to implement an alternative proficiency testing program (APT) in the routine clinical (PK) laboratory. Methods The test (PredictrPK® IFX) is validated and measures serum IFX and antibodies to IFX (ATI) concentrations using a homogenous mobility shift assay by size exclusion HPLC and albumin (ALB) by nephelometry. Determinations are inputted with weight, dose, and interdose interval provided on test requisition in the Bayesian forecasting PK tool which uses non-linear mixed effect to calculate the individual parameter estimates, Clearance and predicted trough concentrations. APT is conducted using five previously tested patient samples, blinded to testing personnel and retested in the same manner as the routine method of analysis every six months. APT consists of two components, a component evaluating standalone IFX, ATI, and ALB analytes, and a second component evaluating the Bayesian forecasting PK tool in estimating Clearance and trough concentrations. Criteria for acceptable performance is set at target value ± 20% for standalone analytes and forecasted PK estimates. Acceptable ATP score corresponds to 4/5 samples with passing grade. Results The results from two consecutive APT results with six months intervals is presented in Fig. 1. Comparing the results to the original assayed data at the time of collection, the APT results showed IFX concentration, ATI and ALB within 20% of target value. Clearance and estimated trough concentrations calculated using the Bayesian forecasting tool also met acceptability criteria (100% passing grade). Conclusion We have implemented an APT for Bayesian forecasting tool in the clinical PK laboratory.

Polymorphism of G-quadruplexes formed by short oligonucleotides containing a 3′-3′ inversion of polarity: From G:C:G:C tetrads to π–π stacked G-wires

G-wires are supramolecular DNA structures based on the G-quadruplex (G4) structural motif obtained by the self-assembly of interlocked slipped G-rich oligonucleotide (ON) strands, or by end-to-end stacking of G4 units. Despite the increasing interest towards G-wires due to their potential applications in DNA nanotechnologies, the self-assembly process to obtain G-wires having a predefined length and stability is still neither completely understood nor controlled. In our previous studies, we demonstrated that the d(5′CG2-3′-3′-G2C5′) ON, characterized by the presence of a 3′-3′-inversion of polarity site self-assembles into a G-wire structure when annealed in the presence of K+ ions. Herein, by using CD, PAGE, HPLC size exclusion chromatography, and NMR investigations we studied the propensity of shorter analogues having sequences 5′CGn-3′-3′-GmC5′ (with n = 1 and 1 ≤ m ≤ 3) to form the corresponding G-quadruplexes and stacked G-wires. The results revealed that the formation of G-wires starting from d(5′CGn-3′-3′-GmC5′) ONs is possible only for the sequences having n and m > 1 in which both guanosines flanking the 5′-ending cytosines are not involved into the 3′-3′ phosphodiester bond.

The Role of Ion Pairing Agents in Liquid Chromatography (LC) Separations

Ion pairing agents are frequently used in liquid chromatography (LC), especially in reversed-phase liquid chromatography (RPLC), to increase the separation and retention of charged analytes. However, their use in other modes of HPLC, such as sizeexclusion HPLC (SE–HPLC) and ion exchange chromatography (IEC) has not been extensively explored. In RPLC, the target analytes may have charged functional groups, which makes it difficult to maintain them on the hydrophobic stationary phase. This difficulty is often overcome by the inclusion of an ion pairing agent, which introduces ionic contacts and strengthens the hydrophobic connections between the analytes and stationary phase. The separation mechanisms in both SE–HPLC and IEC rely on size and charge differences, respectively, between analytes. Thus, they are naturally constructed to separate analytes without the requiring extra ion pairing agents. In this study, we introduced an ion pairing agent (&lt;0.1% formic acid) to the protein sample, and as a result, an alteration in the elution profile (separation efficiency, selectivity, retention of analytes, and resolution) has been achieved. When samples were introduced with an ion-pairing agent in SE–HPLC, there was a considerable improvement in low molecular weight species (LMWs) separation with four extra peaks with higher resolution (up to 2%), without significant alteration in total area percentage. Similarly, a minor new acidic variant peak was resolved in the weak cation-exchange LC (WCX–LC) analysis of the formic acid-enhanced sample, with the resolution being increased by 3%. The results show that using ion pairing agents should be explored in other modes of HPLC as well as for applications where they may offer enhanced chromatographic separations.

Inhibition of ROS-Scavenging Enzyme System Is a Key Event in Tomato Genetic Resistance against Root-Knot Nematodes.

Genetic resistance in plants against incompatible pests is expressed by the activation of an immune system; however, the molecular mechanisms of pest recognition and expression of immunity, although long the object of investigation, are far from being fully understood. The immune response triggered by the infection of soil-borne parasites, such as root-knot nematodes (RKNs), to incompatible resistant tomato plants was studied and compared to the compatible response that occurred when RKNs attacked susceptible plants. In compatible interactions, the invading nematode juveniles were allowed to fully develop and reproduce, whilst that was impeded in incompatible interactions. In crude root extracts, a first assay of reactive oxygen species (ROS)-scavenging enzymatic activity was carried out at the earliest stages of tomato-RKN incompatible interaction. Membrane-bound and soluble CAT, which is the most active enzyme in hydrogen peroxide (H2O2) scavenging, was found to be specifically inhibited in roots of inoculated resistant plants until 5 days after inoculation, with respect to uninoculated plants. The expression of genes encoding for antioxidant enzymes, such as CAT and glutathione peroxidase (GPX), was not always inhibited in roots of nematode-infected resistant tomato. Therefore, the biochemical mechanisms of CAT inhibition were further investigated. Two CAT isozymes were characterized by size exclusion HPLC as a tetrameric form with a molecular weight of 220,000 dalton and its subunits (55,000 dalton). Fractions containing such isozymes were tested by their sensitivity to both salicylic acid (SA) and H2O2. It was evidenced that elevated concentrations of both chemicals led to a partial inactivation of CAT. Elevated concentrations of H2O2 in incompatible interactions have been suggested to be produced by membrane-bound superoxide anion generating, SOD, and isoperoxidase-enhanced activities. Such partial inactivation of CAT has been depicted as one of the earliest key metabolic events, which is specifically associated with tomato immunity to RKNs. Enhanced ROS production and the inhibition of ROS-scavenging systems have been considered to trigger all the metabolic events leading to cell death and tissue necrosis developed around the head of the invading juveniles by which this special type of plant resistance is exerted.

Impact of chitosan and/or transglutaminase treatment on the colloidal stability and air-water interfacial properties of gliadin based nanoparticles

Wheat gliadin nanoparticles (GNPs) hold great potential as food ingredients. However, they have limited stability in aqueous systems as they precipitate rapidly in the presence of salt or at certain pH values. GNP modification strategies can increase their applicability in food systems. In this work, GNPs and hybrid gliadin-chitosan nanoparticles (GCNPs) were produced by liquid anti-solvent precipitation. For the first time ever, the impact of chitosan addition, both as such and in combination with microbial transglutaminase (TG) treatment to covalently conjugate protein and chitosan, on gliadin based NP stability and foaming properties was investigated. GCNPs had excellent colloidal stability over the course of 17 days of storage and in the presence of 1.5 M NaCl. This was attributed to the high chitosan-induced surface charge of GCNPs. Size exclusion HPLC revealed that TG treatment resulted in gliadin-chitosan conjugation, although no additional positive impact on GCNP stability was noted. At pH 4.5, the addition of charged chitosan resulted in worse GNP foaming properties, and TG had only a limited positive impact on the functionality. The worse foaming properties were attributed to the chitosan charged groups reducing mutual interactions of nanoparticle constituents resulting in a weaker adsorbed interfacial film. Even though GCNPs possessed poor foaming properties, they still hold great potential due to their excellent colloidal stability under food-system relevant conditions.

Mechanistic insights into the 2-step dimerization process FO the hepatitis C virus RNA genome.

The 3ʹX RNA of the Hepatitis C Virus (HCV) is a highly conserved, 98-nt sequence located at the 3ʹ terminus of the HCV genome. This region of the genome can engage in several viral RNA-RNA binding interactions that have previously been shown to be critical for regulating translation, replication, and viral particle assembly. Within the 3ʹX RNA there is a stretch of 16 nucleotides that is responsible for two different RNA-RNA binding interactions: one heterotypic and the other homotypic. Notably, the nucleotide sequence for the heterotypic interaction is fully nested within the homotypic interaction and therefore formation of one interaction precludes formation of the other. Although these two interactions have important regulatory roles, it is not known what determines which interaction is formed at any given time and how they might interconvert. Using single-molecule FRET (smFRET) and size exclusion HPLC (SE-HPLC) techniques to monitor the conformational status of the 3ʹX RNA under a variety of conditions, we are beginning to resolve these unanswered questions. First, in the absence of either binding partner, we observe two distinct conformations of monomeric 3ʹX with their relative abundance dictated by either solution conditions (e.g., [Mg2+]) or nucleotide deletions. Additionally, we a observe large conformational change in the 3ʹX RNA when exposed to its homotypic binding partner, resulting in the formation of a homodimeric extended duplex of the 3ʹX RNA. These findings support an emerging hypothesis that the conformation changes associated with the 3ʹX RNA function as riboregulatory switches that halt protein and RNA synthesis, ultimately giving way for virus particles to begin to assemble.

Uncovering the biophysical mechanisms of riboregulatory interactions involving the hepatitis C virus 3'X RNA using single-molecule FRET.

The last 98 nucleotides of the hepatitis C virus genome form what is called the 3ʹX RNA, which is a structured and highly conserved non-coding RNA element that has been implicated in the regulation of several different RNA-dependent viral processes like replication, translation, and particle assembly. Current research suggests that this riboregulatory behavior arises from the ability of 3ʹX to form multiple mutually exclusive interactions with highly conserved RNA elements across the HCV genome. Here, we use time-resolved single-molecule fluorescence spectroscopy in conjunction with analytical size exclusion HPLC to characterize the structural, energetic, and kinetic principles governing these RNA-RNA interactions. In the absence of any interaction partners, we see that 3ʹX adopts two distinct conformations. Furthermore, these two conformations have different preferences for various inter- and intragenomic RNA-RNA interactions, with the interactions themselves depending on various external factors (e.g., [Mg2+]). These findings have made it possible for us to predict which of these mutually exclusive interactions will be formed under specific conditions and ultimately how the formation and disruption of them regulates various RNA-dependent viral processes.

Valorization of shrimp (Heterocarpus reedi) processing waste via enzymatic hydrolysis: Protein extractions, hydrolysates and antioxidant peptide fractions

Shrimp waste valorization offers a solution to environmental pollution and increases the sustainability of fishery products that, in most cases, are discarded. This study aimed to recover protein from shrimp waste and obtain antioxidant peptides through enzymatic hydrolysis. First, two different protein recovery strategies such as aqueous extraction and alkaline extraction were evaluated. Then the use of two commercial proteases (Alcalase and Flavourzyme) was assessed to obtain antioxidant hydrolysates (by ORAC and DPPH assay). The higher protein yield was obtained by alkaline extraction, and the highest antioxidant activity was obtained using Flavourzyme (31.7 μmol TE g⁻1 and 52.4% of DPPH). In addition, a response surface methodology (RSM) was used to optimize the hydrolysis conditions (time and enzyme/substrate ratio). Finally, the hydrolysate was sequentially fractionated by ultrafiltration using different molecular weight cut-offs (10 and 3 kDa). The most antioxidant fraction was <3 kDa reaching 0.91 ± 0.04 μmol TE mg−1 protein. This fraction was separated by Size-Exclusion and Reverse-Phase HPLC into four peptides of 3558, 2568, 1747, and 718 kDa, where the prevalence of hydrophobic and aromatic amino acids could contribute positively to the antioxidant activity. These results established sustainable strategies for obtaining peptide fractions with a high potential to be used as natural alternatives to antioxidants.

Comparative study on structural, biological and functional activities of hydrolysates from Adzuki bean (Vigna angularis) and mung bean (Vigna radiata) protein concentrates using Alcalase and Flavourzyme

The physicochemical features of mung bean protein (MBP) and adzuki bean protein (ABP) hydrolysates derived from Alcalase (MBPHA, ABPHA) and Flavourzyme (MBPHF, ABPHF) were assessed using FTIR, hydrophobicity, emulsion activity, zeta potential, and health-promoting activities. The results proved that the choice of peptidase and substrate both have a significant effect on the hydrolysates in different physicochemical, structural and functional properties. Size exclusion-HPLC was used to fractionate the MBP and ABP hydrolysates. The results demonstrated that Alcalase hydrolysates included smaller peptides than Flavourzyme hydrolysates, and the chromatogram patterns of the two peptidases were similar. The peptides with the most potent antioxidant and ACE-inhibitory properties were identified using MALDI-TOF-MS. The fraction (F4) of MBPHA exhibited the highest levels of metal chelating activity. The Flavourzyme hydrolysates fraction (F2) and the ABPHA fraction (F2) showed the highest ABTS radical scavenging activity and ACE-inhibitory activity, respectively. Pro-Pro was identified in peptide sequences with ABTS radical scavenging activity as an active component while Pro-Gln was identified in peptide sequences with ACE-inhibitory activity. As a result, Pro-Pro and Pro-Gln, respectively, are likely-one of the characteristics of antioxidant and ACE-inhibitory peptides from MBP and ABP. Compared to mung bean and adzuki bean protein as substrate, Alcalase and Flavourzyme as peptidases significant impacted the development of distinct functionalities and biological activities.

Cytosine-rich oligonucleotides incorporating a non-nucleotide loop: A further step towards the obtainment of physiologically stable i-motif DNA

i-Motifs, also known as i-tetraplexes, are secondary structures of DNA occurring in cytosine-rich oligonucleotides (CROs) that recall increasing interest in the scientific community for their relevance in various biological processes and DNA nanotechnology. This study reports the design of new structurally modified CROs, named Double-Ended-Linker-CROs (DEL-CROs), capable of forming stable i-motif structures. Here, two C-rich strands having sequences d(AC4A) and d(C6) have been attached, in a parallel fashion, to the two linker's edges by their 3′ or 5′ ends. The resulting DEL-CROs have been investigated for their capability to form i-motif structures by circular dichroism, poly-acrylamide gel electrophoresis, HPLC–size-exclusion chromatography, and NMR studies. This investigation established that DEL-CROs could form more stable i-motif structures than the corresponding unmodified CROs. In particular, the i-motif formed by DEL-5′-d(C6)2 resulted stable enough to be detected even at near physiological conditions (37 °C, pH 7.0). The results open the way to developing pH-switchable nanocarriers and aptamers based on suitably functionalized DEL-CROs.

Abstract 2467: SPECT-imaging guided development and evaluation of targeted alpha therapy (TAT) for colorectal liver metastasis

Abstract BACKGROUND: Only a small fraction of patients with colorectal liver metastases (CLM) is eligible for curative resection, and novel treatment options are highly needed. The high linear-energy transfer of short-ranged alpha particles can induce complex double-stranded DNA breaks, leading to cell death. With no known resistance mechanism, alpha-particle emitting isotopes represent a promising tool in cancer management. In targeted alpha therapy (TAT), an alpha-particle emitting isotope is attached to a tumor-targeting antibody (hereafter TAT antibody), ensuring specific delivery to the tumor. By substituting the alpha-particle emitting isotope (Actinium-225, 225Ac) with a diagnostic isotope (Zirconium-89, 89Zr), the in-vivo distribution of the TAT antibody can be monitored using PET/SPECT/CT. The aim of this work was to set up robust labeling and PET/SPECT protocols to evaluate TAT in CLM using novel experimental models. METHODS: For SPECT-imaging of 225Ac daughter isotopes, the combined gamma emission spectra were gated at 218 keV ± 20% and 440 keV ± 20% for Francium-221 and Bismuth-213, respectively. 89Zr was labeled to a desferrioxamine* (DFO*) conjugated TAT antibody, which recognizes both the murine and human antigen. Indium-111 (111In) labeling was performed through diethylenetriaminepentaacetic acid (DTPA) conjugated to an isotype antibody. Radiochemical purity and protein integrity were assessed by size exclusion HPLC, iTLC and SDS-PAGE. For orthotopic CLM model development, 106 cells of human colorectal cancer cell lines HT-55 or LS1034 were injected in the spleen of female Rj:NMRI-Foxn1nu mice, followed by splenectomy. Tumor growth was monitored by T2-weighted MR-imaging. Non-tumor bearing mice (n = 12, 3 per group) were injected with 4 MBq of 10, 20, 50 or 100 µg 89Zr-TAT antibody to evaluate biodistribution and the mice scanned on day 2, 4 and 6 after iv injection. RESULTS: Francium-221 and Bismuth-213 could be separately visualized after free Actinium-225 administration (i.v.). 89Zr and 111In were routinely labeled to their respective antibody with up to 500 MBq/mg with &amp;gt;95% radiochemical purity. 89Zr-DFO*-TAT antibody showed &amp;gt;95% radiochemical purity and &amp;gt;95% protein integrity after 7 days stability test in serum, shown by iTLC and SDS-PAGE. SPECT/CT of 89Zr-DFO*-TAT antibody in non-tumor bearing mice showed little uptake in healthy tissues apart from the liver due to hepatic antibody clearance. Orthotopic models of CLM show reproducible development of metastases for HT-55 and LS1034 within 3 weeks. CONCLUSION: Robust labeling methods, PET/SPECT imaging protocols and suitable animal models of CLM were established. Results of our dual-isotope SPECT/CT imaging of our 89Zr-DFO*-TAT antibody in comparison with an 111In-labeled isotype antibody, together with efficacy data of the TAT, will be presented at the meeting. Citation Format: Frans V. Suurs, Alexander Kristian, Gebregziabher Petros, Yuan Zeng Feng, Karianne G. Fleten, Roger M. Bjerke, Alan Cuthbertson, Kjersti Flatmark. SPECT-imaging guided development and evaluation of targeted alpha therapy (TAT) for colorectal liver metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2467.

Quantitative NMR for the structural analysis of novel bivalent glycoconjugates as vaccine candidates

Novel unimolecular bivalent glycoconjugates were assembled combining several functionalized capsular polysaccharides of Streptococcus pneumoniae and Neisseria meningitidis to a carrier protein by using an effective strategy based on the Ugi 4-component reaction. The development of multivalent glycoconjugates opens new opportunities in the field of vaccine design, but their high structural complexity involves new analytical challenges. Nuclear Magnetic Resonance has found wide applications in the characterization and impurity profiling of carbohydrate-based vaccines. Eight bivalent conjugates were studied by quantitative NMR analyzing the structural identity, the content of each capsular polysaccharide, the ratios between polysaccharides, the polysaccharide to protein ratios and undesirable contaminants. The qNMR technique involves experiments with several modified parameters for obtaining spectra with quantifiable signals. In addition, the achieved NMR results were combined with the results of colorimetric assay and Size Exclusion HPLC for assessing the protein content and free protein percentage, respectively. The application of quantitative NMR showed to be efficient to clear up the new structural complexities while allowing the quantitative assessment of the components.

Metal-binding biomolecules in the liver of northern pike (Esox lucius Linnaeus, 1758): The first data for the family Esocidae

Metal-handling strategies of various fish species are known to vary significantly in association with their intracellular metal behaviour. Thus, to better understand the possible consequences of increased metal exposure in fish it is important to perform comparative studies on metal-binding biomolecules in organs of different species. This study was the first of this kind on a liver of an esocid fish (northern pike, Esox lucius), and the gathered information were compared to fish belonging to three other families, Leuciscidae, Cyprinidae and Salmonidae. Distributions of ten elements among cytosolic biomolecules of different molecular masses were studied by size exclusion HPLC combined offline with high resolution ICP-MS. The results indicated predominant association of Co, Fe and Mo to high molecular mass biomolecules (>100 kDa), of Zn and Bi to both high and medium molecular mass biomolecules (>30 kDa), of Mn and Se to medium molecular mass biomolecules (30–100 kDa), and Ag, Cd and Cu to low molecular mass biomolecules (10–30 kDa), presumably metallothioneins. Evident binding to metallothioneins was also detected for Zn and Bi. For several metals, distinct differences were observed when cytosolic metal distributions of northern pike were compared to leuciscids, salmonids and cyprinids. More pronounced Zn binding to metallothioneins was recorded in leuciscids and cyprinids than both esocids and salmonids, whereas cytosolic Mn and Se distributions clearly differed between all studied fish families. Accordingly, in assessment of metal pollution it is vital to consider the exposed species, which requires prior comprehensive comparative research on numerous aquatic organisms.

Clean-label breadmaking: Size exclusion HPLC analysis of proteins in dough supplemented with additives vs hard red spring wheat flour

Currently, there is a challenge for clean-label bread formulations to find replacements for dough improvers. For pan bread traditionally made from hard winter wheat, the addition of hard spring wheat flour may be an option to replace some dough improvers. Protein composition and properties of hard red spring wheat dough that would improve the quality of winter wheat dough for the purpose of replacing dough improvers need to be investigated. Therefore, this research aimed to compare molecular weight distribution (MWD) of unreduced proteins in dough samples that were made of a base hard red winter wheat flour supplemented with hard red spring (HRS) wheat flour or common additives such as potassium bromate, azodicarbonamide, ascorbic acid, sodium stearoyl lactylate, vital wheat gluten, non-fat dry milk, and others. The incorporation of the artificial additives significantly (p < .05) increased the quantity of total high molecular weight protein polymers (HMW-PP) in base-flour dough samples, which led to the improvement of breadmaking quality. Nonetheless, the HRS wheat flour naturally contained better protein quantity and quality, which sequentially contributed to forming more HMW-PP and a stronger gluten network in the dough than the base flour dough containing artificial additives. This research reported novel information on the variation of protein MWD in dough samples, especially those supplemented with artificial additives. The results support that the HRS wheat flour is an adequate choice as a blending flour to produce a clean-label bread substituting artificial additives.

Design, Synthesis and Preclinical Assessment of 99mTc-iFAP for In Vivo Fibroblast Activation Protein (FAP) Imaging.

Fibroblast activation protein (FAP) is expressed in the microenvironment of most human epithelial tumors. 68Ga-labeled FAP inhibitors based on the cyanopyrrolidine structure (FAPI) are currently used for the detection of the tumor microenvironment by PET imaging. This research aimed to design, synthesize and preclinically evaluate a new FAP inhibitor radiopharmaceutical based on the 99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (99mTc-iFAP) structure for SPECT imaging. Molecular docking for affinity calculations was performed using the AutoDock software. The chemical synthesis was based on a series of coupling reactions of 6-hidrazinylnicotinic acid (HYNIC) and D-alanine to a boronic acid derivative. The iFAP was prepared as a lyophilized formulation based on EDDA/SnCl2 for labeling with 99mTc. The radiochemical purity (R.P.) was verified via ITLC-SG and reversed-phase radio-HPLC. The stability in human serum was evaluated by size-exclusion HPLC. In vitro cell uptake was assessed using N30 stromal endometrial cells (FAP positive) and human fibroblasts (FAP negative). Biodistribution and tumor uptake were determined in Hep-G2 tumor-bearing nude mice, from which images were acquired using a micro-SPECT/CT. The iFAP ligand (Ki = 0.536 nm, AutoDock affinity), characterized by UV-Vis, FT-IR, 1H–NMR and UPLC-mass spectroscopies, was synthesized with a chemical purity of 92%. The 99mTc-iFAP was obtained with a R.P. >98%. In vitro and in vivo studies indicated high radiotracer stability in human serum (>95% at 24 h), specific recognition for FAP, high tumor uptake (7.05 ± 1.13% ID/g at 30 min) and fast kidney elimination. The results found in this research justify additional dosimetric and clinical studies to establish the sensitivity and specificity of the 99mTc-iFAP.