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The Journal of Nutrition May 2024Infant formulas (IFs), the only adequate substitute to human milk, are complex matrices that require numerous ingredients and processing steps that may impact protein...
BACKGROUND
Infant formulas (IFs), the only adequate substitute to human milk, are complex matrices that require numerous ingredients and processing steps that may impact protein digestion and subsequent amino acid (AA) absorption.
OBJECTIVES
The objective was to understand the impact of the protein ingredient quality within IFs on postprandial plasma AA profiles.
METHODS
Four isonitrogenous and isocaloric IFs were produced at a semi-industrial scale using whey proteins from different origins (cheese compared with ideal whey) and denaturation levels (IF-A, -B, -C), and caseins with different supramolecular organizations (IF-C, -D). Ten Yucatan minipiglets (12- to 27-d-old) were used as a human infant model and received each IF for 3 d according to a Williams Latin square followed by a 2-d wash-out period. Jugular plasma was regularly sampled from 10 min preprandial to 4 h postprandial on the third day to measure free AAs, urea, insulin, and glucose concentrations. Data were statistically analyzed using a mixed linear model with diet (IFs), time, and sex as fixed factors and piglet as random factor.
RESULTS
IFs made with cheese whey (IF-A and -B) elicited significantly higher plasma total and essential AA concentrations than IFs made with ideal whey (IF-C and -D), regardless of the pre- and postprandial times. Most of the differences observed postprandially were explained by AA homeostasis modifications. IFs based on cheese whey induced an increased plasma concentration of Thr due to both a higher Thr content in these IFs and a Thr-limiting degrading capability in piglets. The use of a nonmicellar casein ingredient led to reduced plasma content of AA catabolism markers (IF-D compared with IF-C).
CONCLUSIONS
Overall, our results highlight the importance of the protein ingredient quality (composition and structure) within IFs on neonatal plasma AA profiles, which may further impact infant protein metabolism.
PubMed: 38801861
DOI: 10.1016/j.tjnut.2024.05.009 -
Protein Science : a Publication of the... Jun 2024Thermal stability of proteins is a primary metric for evaluating their physical properties. Although researchers attempted to predict it using machine learning...
Thermal stability of proteins is a primary metric for evaluating their physical properties. Although researchers attempted to predict it using machine learning frameworks, their performance has been dependent on the quality and quantity of published data. This is due to the technical limitation that thermodynamic characterization of protein denaturation by fluorescence or calorimetry in a high-throughput manner has been challenging. Obtaining a melting curve that derives solely from the target protein requires laborious purification, making it far from practical to prepare a hundred or more samples in a single workflow. Here, we aimed to overcome this throughput limitation by leveraging the high protein secretion efficacy of Brevibacillus and consecutive treatment with plate-scale purification methodologies. By handling the entire process of expression, purification, and analysis on a per-plate basis, we enabled the direct observation of protein denaturation in 384 samples within 4 days. To demonstrate a practical application of the system, we conducted a comprehensive analysis of 186 single mutants of a single-chain variable fragment of nivolumab, harvesting the melting temperature (T) ranging from -9.3 up to +10.8°C compared to the wild-type sequence. Our findings will allow for data-driven stabilization in protein design and streamlining the rational approaches.
Topics: Protein Stability; Thermodynamics; Protein Denaturation; High-Throughput Screening Assays; Brevibacillus
PubMed: 38801228
DOI: 10.1002/pro.5029 -
International Journal of Molecular... May 2024CIGB-258, a 3 kDa peptide from heat shock protein 60, exhibits synergistic anti-inflammatory activity with apolipoprotein A-I (apoA-I) in reconstituted high-density...
Synergistic Anti-Inflammatory Activity of Lipid-Free Apolipoprotein (apo) A-I and CIGB-258 in Acute-Phase Zebrafish via Stabilization of the apoA-I Structure to Enhance Anti-Glycation and Antioxidant Activities.
CIGB-258, a 3 kDa peptide from heat shock protein 60, exhibits synergistic anti-inflammatory activity with apolipoprotein A-I (apoA-I) in reconstituted high-density lipoproteins (rHDLs) via stabilization of the rHDL structure. This study explored the interactions between CIGB-258 and apoA-I in the lipid-free state to assess their synergistic effects in the structural and functional enhancement of apoA-I and HDL. A co-treatment of lipid-free apoA-I and CIGB-258 inhibited the cupric ion-mediated oxidation of low-density lipoprotein (LDL) and a lowering of oxidized species in the dose-responsive manner of CIGB-258. The co-presence of CIGB-258 caused a blue shift in the wavelength of maximum fluorescence (WMF) of apoA-I with protection from proteolytic degradation. The addition of apoA-I:CIGB-258, with a molar ratio of 1:0.1, 1:0.5, and 1:1, to HDL and HDL remarkably enhanced the antioxidant ability against LDL oxidation up to two-fold higher than HDL alone. HDL-associated paraoxonase activities were elevated up to 28% by the co-addition of apoA-I and CIGB-258, which is linked to the suppression of Cu-mediated HDL oxidation with the slowest electromobility. Isothermal denaturation by a urea treatment showed that the co-presence of CIGB-258 attenuated the exposure of intrinsic tryptophan (Trp) and increased the mid-points of denaturation from 2.33 M for apoA-I alone to 2.57 M for an apoA-I:CIGB-258 mixture with a molar ratio of 1:0.5. The addition of CIGB-258 to apoA-I protected the carboxymethyllysine (CML)-facilitated glycation of apoA-I with the prevention of Trp exposure. A co-treatment of apoA-I and CIGB-258 synergistically safeguarded zebrafish embryos from acute death by CML-toxicity, suppressing oxidative stress and apoptosis. In adult zebrafish, the co-treatment of apoA-I+CIGB-258 exerted the highest anti-inflammatory activity with a higher recovery of swimming ability and survivability than apoA-I alone or CIGB-258 alone. A co-injection of apoA-I and CIGB-258 led to the lowest infiltration of neutrophils and interleukin (IL)-6 generation in hepatic tissue, with the lowest serum triglyceride, aspartate transaminase, and alanine transaminase levels in plasma. In conclusion, the co-presence of CIGB-258 ameliorated the beneficial functionalities of apoA-I, such as antioxidant and anti-glycation activities, by enhancing the structural stabilization and protection of apoA-I. The combination of apoA-I and CIGB-258 synergistically enforced the anti-inflammatory effect against CML toxicity in embryos and adult zebrafish.
Topics: Apolipoprotein A-I; Animals; Zebrafish; Antioxidants; Anti-Inflammatory Agents; Lipoproteins, HDL; Lipoproteins, LDL; Oxidation-Reduction; Drug Synergism
PubMed: 38791598
DOI: 10.3390/ijms25105560 -
International Journal of Molecular... May 2024Glutathione -transferase omega 1 (GstO1) catalyzes deglutathionylation and plays an important role in the protein glutathionylation cycle in cells. GstO1 contains four...
Glutathione -transferase omega 1 (GstO1) catalyzes deglutathionylation and plays an important role in the protein glutathionylation cycle in cells. GstO1 contains four conserved cysteine residues (C32, C90, C191, C236) found to be mutated in patients with associated diseases. In this study, we investigated the effects of cysteine mutations on the structure and function of GstO1 under different redox conditions. Wild-type GstO1 (WT) was highly sensitive to hydrogen peroxide (HO), which caused precipitation and denaturation at a physiological temperature. However, glutathione efficiently inhibited the HO-induced denaturation of GstO1. Cysteine mutants C32A and C236A exhibited redox-dependent stabilities and enzyme activities significantly different from those of WT. These results indicate that C32 and C236 play critical roles in GstO1 regulation by sensing redox environments and explain the pathological effect of cysteine mutations found in patients with associated diseases.
Topics: Cysteine; Oxidation-Reduction; Glutathione Transferase; Humans; Glutathione; Hydrogen Peroxide; Mutation
PubMed: 38791319
DOI: 10.3390/ijms25105279 -
International Journal of Molecular... May 2024High-resolution melting (HRM) is a cost-efficient tool for targeted DNA methylation analysis. HRM yields the average methylation status across all CpGs in PCR products....
High-resolution melting (HRM) is a cost-efficient tool for targeted DNA methylation analysis. HRM yields the average methylation status across all CpGs in PCR products. Moreover, it provides information on the methylation pattern, e.g., the occurrence of monoallelic methylation. HRM assays have to be calibrated by analyzing DNA methylation standards of known methylation status and mixtures thereof. In general, DNA methylation levels determined by the classical calibration approach, including the whole temperature range in between normalization intervals, are in good agreement with the mean of the DNA methylation status of individual CpGs determined by pyrosequencing (PSQ), the gold standard of targeted DNA methylation analysis. However, the classical calibration approach leads to highly inaccurate results for samples with heterogeneous DNA methylation since they result in more complex melt curves, differing in their shape compared to those of DNA standards and mixtures thereof. Here, we present a novel calibration approach, i.e., temperature-wise calibration. By temperature-wise calibration, methylation profiles over temperature are obtained, which help in finding the optimal calibration range and thus increase the accuracy of HRM data, particularly for heterogeneous DNA methylation. For explaining the principle and demonstrating the potential of the novel calibration approach, we selected the promoter and two enhancers of , a gene encoding the repair protein MGMT.
Topics: DNA Methylation; Calibration; Humans; Nucleic Acid Denaturation; Promoter Regions, Genetic; DNA Modification Methylases; Tumor Suppressor Proteins; Temperature; DNA Repair Enzymes; CpG Islands; Sequence Analysis, DNA; DNA
PubMed: 38791122
DOI: 10.3390/ijms25105082 -
Biomedicines Apr 2024Cystathione beta-synthase (CBS) T236N is a novel mutation associated with pyridoxine non-responsiveness, which presents a significant difficulty in the medical treatment...
BACKGROUND
Cystathione beta-synthase (CBS) T236N is a novel mutation associated with pyridoxine non-responsiveness, which presents a significant difficulty in the medical treatment of homocystinuria. Reported severe phenotypes in homocystinuria patients highlight the urgent requirement to comprehend the molecular mechanisms underlying mutation pathogenicity for the advancement of the disease.
METHODOLOGY
In this study, we used a multidisciplinary approach to investigate the molecular properties of bacterially expressed and purified recombinant CBS protein, which we directly compared to those of the wild-type (CBS) protein.
RESULTS
Our data revealed a profound impact of the p.T236N mutation on CBS enzymatic activity, with a dramatic reduction of ~96% compared to the CBS protein. Circular dichroism (CD) experiments indicated that the p.T236N mutation did not significantly alter the secondary structure of the protein. However, CD spectra unveiled distinct differences in the thermal stability of CBS and CBS mutant protein species. In addition, chemical denaturation experiments further highlighted that the CBS protein exhibited greater thermodynamic stability than the CBS mutant, suggesting a destabilizing effect of this mutation.
CONCLUSIONS
Our findings provide an explanation of the pathogenicity of the p.T236N mutation, shedding light on its role in severe homocystinuria phenotypes. This study contributes to a deeper understanding of CBS deficiency and may improve the development of targeted therapeutic strategies for affected individuals.
PubMed: 38790892
DOI: 10.3390/biomedicines12050929 -
Foods (Basel, Switzerland) May 2024Various drying temperatures impact the texture of pasta and cause different drying defects. These by-products could reflect techno-functional characteristics which are...
Various drying temperatures impact the texture of pasta and cause different drying defects. These by-products could reflect techno-functional characteristics which are suitable for cereal products. This research addresses the influence of low (LT) and high (HT) drying pasta defects with two granulations on the theoretical and functional characteristics of hard dough biscuits. By shifting from a LT to HT drying temperature, a higher onset and peak temperature was found due to the higher mobility of starch molecules with increasing crystalline stability. The lowest transition enthalpy of biscuit formulation was also observed for higher incorporation of fine HT pasta regrinds. The algebraic model of dough with consistography determined the poor-extensible gluten and a high resistance with a greater value of P/L and P indices for LT regrinds. Scanning electron microscopy revealed a heavy and dense texture with immersed starch granules for additional fine regrinds while coarse samples caused swell granules with greater diameter. Moreover, fine HT regrinds reflected the lowest L* value for biscuit due to heat gradient tension with the hard milling process which leads to protein denaturation with decreasing nitrogenous.
PubMed: 38790787
DOI: 10.3390/foods13101487 -
Foods (Basel, Switzerland) May 2024Bee products are considered true wonders of nature, used since ancient times, and studied even today for their various biological activities. In this study, we...
Bee products are considered true wonders of nature, used since ancient times, and studied even today for their various biological activities. In this study, we hypothesise that Romanian bee products from different origins (micro apiary products, lyophilised forms, commercial) exhibit distinct chemical compositions, influencing their biological activities. An LC-MS analysis revealed varied polyphenolic content patterns, with cumaric acid, ferulic acid, rosmarinic acid, and quercitine identified in significant amounts across all samples. Primary anti-inflammatory evaluation phases, including the inhibition of haemolysis values and protein denaturation, unveiled a range of protective effects on red blood cells (RBC) and blood proteins, contingent upon the sample concentration. Antimicrobial activity assessments against 12 ATCC strains and 6 pathogenic isolates demonstrated varying efficacy, with propolis samples showing low efficacy, royal jelly forms displaying moderate effectiveness, and apilarnin forms exhibiting good inhibitory activity, mostly against Gram-positive bacteria. Notably, the lyophilised form emerged as the most promising sample, yielding the best results across the biological activities assessed. Furthermore, molecular docking was employed to elucidate the inhibitory potential of compounds identified from these bee products by targeting putative bacterial and fungal proteins. Results from the docking analysis showed rosmarinic and rutin exhibited strong binding energies and interactions with the putative antimicrobial proteins of bacteria (-9.7 kcal/mol to -7.6 kcal/mol) and fungi (-9.5 kcal/mol to -8.1 kcal/mol). The findings in this study support the use of bee products for antimicrobial purposes in a biologically active and eco-friendly proportion while providing valuable insights into their mechanism of action.
PubMed: 38790755
DOI: 10.3390/foods13101455 -
Scientific Reports May 2024Oral disorders can exert systemic ramifications beyond their localized effects on dental tissues, implicating a wide array of physiological conditions. The utilization...
Oral disorders can exert systemic ramifications beyond their localized effects on dental tissues, implicating a wide array of physiological conditions. The utilization of essential oils (EOs) for protection of oral health represents a longstanding practice. Consequently, in this investigation, essential oil derived from Nigella sativa seeds (NSEO) underwent isolation via the hydro-distillation process, followed by a comprehensive evaluation of its antioxidant, anti-inflammatory, anti-fungal, antibacterial activities, and cytocompatibility. The isolated NSEO manifested as a pale-yellow substance and was found to harbor a diverse spectrum of bioactive constituents, including steroids, triterpenoids, flavonoids, phenols, proteins, alkaloids, tannin, sesquiterpenoid hydrocarbons, monoterpenoid alcohol, and monoterpenoid ketone (thymoquinone). Notably, the total phenolic content (TPC) and total flavonoid content (TFC) of NSEO were quantified at 641.23 μg GAE/gm and 442.25 μg QE/g, respectively. Furthermore, NSEO exhibited concentration-dependent inhibition of protein denaturation, HRBC membrane stabilization, and hemolysis inhibition. Comparative analysis revealed that NSEO and chlorhexidine (CHX) 0.2% displayed substantial inhibition of hemolysis compared to aspirin. While NSEO and CHX 0.2% demonstrated analogous antibacterial activity against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, NSEO showcased heightened efficacy against Lactobacillus acidophilus and Candida albicans. Additionally, NSEO exhibited pronounced effects against periodontal pathogens such as Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia. Importantly, no cytotoxicity was observed on human gingival fibroblast cell lines. These findings underscore the potential of NSEO as a potent antibacterial and antifungal agent in the management of oral microbial pathogens, thereby offering avenues for the development of innovative therapies targeting diverse oral inflammatory conditions. Nevertheless, further investigations are imperative to unlock its full therapeutic repertoire.
Topics: Oils, Volatile; Antioxidants; Nigella sativa; Anti-Inflammatory Agents; Humans; Microbial Sensitivity Tests; Anti-Bacterial Agents; Seeds; Anti-Infective Agents
PubMed: 38789533
DOI: 10.1038/s41598-024-62915-1 -
Gels (Basel, Switzerland) May 2024This study aimed to investigate the gelling behavior of faba bean (FB) and chickpea (CP) flour between 10 and 20% () concentration at pH 3.0, 5.0, and 7.0. Both sources...
This study aimed to investigate the gelling behavior of faba bean (FB) and chickpea (CP) flour between 10 and 20% () concentration at pH 3.0, 5.0, and 7.0. Both sources formed at pH 3.0 and 5.0 self-standing gels with 12% () of flour, while 16% () of flour was required to obtain a gel at pH 7.0. During gelling between 40 and 70 °C, a sharp increase of the elastic modulus G' was observed in both flours, mainly due to water absorption and swelling of the starch, one of the major constituents in the ingredients. Increasing the temperature at 95 °C, G' increased due to the denaturation of globulins and therefore the exposure of their internal part, which allowed more hydrophobic interactions and the formation of the gel. After cooling, both FB and CP gels displayed a solid-like behavior (tan δ ranging between 0.11 and 0.18) with G' values at pH 3.0 and 5.0 significantly ( < 0.05) higher than those at pH 7.0, due to the lower electrostatic repulsions at pHs far from the isoelectric point. The rheological properties were supported by the water binding capacity values, confirming the better gels' strength described by rheological analysis. These results will enhance our understanding of the role of legume flours in formulating innovative and sustainable food products as alternatives to animal ones.
PubMed: 38786226
DOI: 10.3390/gels10050309