-
Central-European Journal of Immunology 2024Fever is an adaptive host-defense response to infection and nowadays is rightly considered to be an expression of a healthy body and a well-functioning immune system....
Fever is an adaptive host-defense response to infection and nowadays is rightly considered to be an expression of a healthy body and a well-functioning immune system. The condition is that it must be tightly regulated. Therefore, in individual cases, fever may be detrimental and should be treated. Specific excessive febrile reaction to pathogens which occurs after aseptic injuries is one among such cases. We previously found that among necrotic products, high mobility group box protein 1 (HMGB1) released from the site of aseptic injury affects immune effectors (cells) to mediate higher fever in response to further contact with bacterial lipopolysaccharide (LPS). Here we observed that intraperitoneal (i.p.) pre-injection of recombinant HMGB1 (5 µg/rat i.p.) provoked an increase in plasma levels of prostaglandin E2 (PGE2) in rats and augmented release of interleukin (IL)-1β and IL-6 after LPS administration at a dose of 50 µg/kg i.p. compared to rats pre-injected with saline or heat-denatured HMGB1. Furthermore, peripheral blood mononuclear cells (PBMCs) isolated from rats injected with HMGB1 were more sensitized to produce enhanced levels of IL-1β and PGE2 when stimulated with LPS in vitro (1 µg/ml/10 cells for 4 h) compared to control animals injected with saline or heat-denatured HMGB1. We also noted a significant increase in activation of nuclear factor κB (NF-κB) in cells isolated from rats injected with HMGB1. Altogether, the obtained results suggest that HMGB1 participates in priming of immune cells to further contact with pathogens.
PubMed: 38812604
DOI: 10.5114/ceji.2024.138600 -
MBio May 2024Pathogenic bacteria rely on secreted virulence factors to cause disease in susceptible hosts. However, in Gram-positive bacteria, the mechanisms underlying secreted...
UNLABELLED
Pathogenic bacteria rely on secreted virulence factors to cause disease in susceptible hosts. However, in Gram-positive bacteria, the mechanisms underlying secreted protein activation and regulation post-membrane translocation remain largely unknown. Using proteomics, we identified several proteins that are dependent on the secreted chaperone PrsA2. We followed with phenotypic, biochemical, and biophysical assays and computational analyses to examine the regulation of a detected key secreted virulence factor, listeriolysin O (LLO), and its interaction with PrsA2 from the bacterial pathogen (). Critical to virulence is internalization by host cells and the subsequent action of the cholesterol-dependent pore-forming toxin, LLO, which enables bacterial escape from the host cell phagosome. Since is a Gram-positive organism, the space between the cell membrane and wall is solvent exposed. Therefore, we hypothesized that the drop from neutral to acidic pH as the pathogen is internalized into a phagosome is critical to regulating the interaction of PrsA2 with LLO. Here, we demonstrate that PrsA2 directly interacts with LLO in a pH-dependent manner. We show that PrsA2 protects and sequesters LLO under neutral pH conditions where LLO can be observed to aggregate. In addition, we identify molecular features of PrsA2 that are required for interaction and ultimately the folding and activity of LLO. Moreover, protein-complex modeling suggests that PrsA2 interacts with LLO via its cholesterol-binding domain. These findings highlight a mechanism by which a Gram-positive secretion chaperone regulates the secretion, stability, and folding of a pore-forming toxin under conditions relevant to host cell infection.
IMPORTANCE
is a ubiquitous food-borne pathogen that can cause severe disease to vulnerable populations. During infection, relies on a wide repertoire of secreted virulence factors including the LLO that enables the bacterium to invade the host and spread from cell to cell. After membrane translocation, secreted factors must become active in the challenging bacterial cell membrane-wall interface. However, the mechanisms required for secreted protein folding and function are largely unknown. encodes a chaperone, PrsA2, that is critical for the activity of secreted factors. Here, we show that PrsA2 directly associates and protects the major virulence factor, LLO, under conditions corresponding to the host cytosol, where LLO undergoes irreversible denaturation. Additionally, we identify molecular features of PrsA2 that enable its interaction with LLO. Together, our results suggest that and perhaps other Gram-positive bacteria utilize secreted chaperones to regulate the activity of pore-forming toxins during infection.
PubMed: 38809022
DOI: 10.1128/mbio.00743-24 -
RSC Advances May 2024Deep eutectic solvents (DESs), characterized by their low volatility, non-toxicity, and biodegradability, have gained attention as green solvents due to their minimal...
Deep eutectic solvents (DESs), characterized by their low volatility, non-toxicity, and biodegradability, have gained attention as green solvents due to their minimal environmental impact and sustainability. The choline chloride/glucose DES, composed solely of biomass, is notable for its high biocompatibility and ability to be prepared at low cost. However, it is also known for its low thermal stability and tendency to denature when heated. In this study, we approached the choline chloride/glucose DES, with its thermal denaturation properties, as a unique chemical conversion medium entirely constituted from biomass. We investigated the thermal denaturation and reaction behaviors of the DES when subjected to prolonged heating. It was found that the choline chloride/glucose DES was relatively thermally stable at around 100 °C, but underwent thermal denaturation at 130 °C, enabling the production of 5-HMF and seven types of rare sugars derived from glucose. The yield of disaccharides containing seven types of rare sugars and 5-HMF relative to the weight of glucose was as high as approximately 70% and 5%, respectively. This study thus reveals that simply heating a liquid composed exclusively of biomass under mild conditions can generate a range of high-value compounds.
PubMed: 38808234
DOI: 10.1039/d4ra02546f -
PloS One 2024As imaging techniques rapidly evolve to probe nanoscale genome organization at higher resolution, it is critical to consider how the reagents and procedures involved in...
As imaging techniques rapidly evolve to probe nanoscale genome organization at higher resolution, it is critical to consider how the reagents and procedures involved in sample preparation affect chromatin at the relevant length scales. Here, we investigate the effects of fluorescent labeling of DNA sequences within chromatin using the gold standard technique of three-dimensional fluorescence in situ hybridization (3D FISH). The chemical reagents involved in the 3D FISH protocol, specifically formamide, cause significant alterations to the sub-200 nm (sub-Mbp) chromatin structure. Alternatively, two labeling methods that do not rely on formamide denaturation, resolution after single-strand exonuclease resection (RASER)-FISH and clustered regularly interspaced short palindromic repeats (CRISPR)-Sirius, had minimal impact on the three-dimensional organization of chromatin. We present a polymer physics-based analysis of these protocols with guidelines for their interpretation when assessing chromatin structure using currently available techniques.
Topics: Formamides; In Situ Hybridization, Fluorescence; DNA; Chromatin; Nucleic Acid Denaturation; Animals
PubMed: 38805476
DOI: 10.1371/journal.pone.0301000 -
Journal of Pharmaceutical Sciences May 2024Protein denaturation and aggregation resulting from the effects of interfacial stress, often enhanced by flow and shear stress, pose significant challenges in the...
Protein denaturation and aggregation resulting from the effects of interfacial stress, often enhanced by flow and shear stress, pose significant challenges in the production of therapeutic proteins and monoclonal antibodies. The influence of flow on protein stability is closely intertwined with interfacial effects. In this study, we have developed a microfluidic device capable of exposing low volume (< 320 µL) protein solutions to highly uniform shear. To disentangle the synergistic impact of flow and interfaces on protein aggregation, we fabricated two devices composed of different materials, namely poly(methyl methacrylate) (PMMA) and stainless steel. Upon application of shear, we observed formation of protein particles in the micron-size range. Notably, The number of particles generated in the steel devices was ∼ 3.5 fold lower than in the PMMA device, hinting at an interface-mediated effect. With increasing the protein concentration from 1 to 50 mg/mL we observed a saturation in the amount of aggregates, further confirming the key role of solid-liquid interfaces in inducing particle formation. Introduction of non-ionic surfactants prevented protein aggregation, even at the highest tested protein concentration and low surfactant concentrations of 0.05 mg/mL. Overall, our findings corroborate the synergistic impact of shear and interface effects on protein aggregation. The device developed in this study offers a small-scale platform for assessing the stability of antibody formulations throughout various stages of the development and manufacturing process.
PubMed: 38801973
DOI: 10.1016/j.xphs.2024.05.024 -
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 -
Frontiers in Microbiology 2024Rapid identification of infected individuals through viral RNA or antigen detection followed by effective personal isolation is usually the most effective way to prevent...
INTRODUCTION
Rapid identification of infected individuals through viral RNA or antigen detection followed by effective personal isolation is usually the most effective way to prevent the spread of a newly emerging virus. Large-scale detection involves mass specimen collection and transportation. For biosafety reasons, denaturing viral transport medium has been extensively used during the SARS-CoV-2 pandemic. However, the high concentrations of guanidinium isothiocyanate (GITC) in such media have raised issues around sufficient GITC supply and laboratory safety. Moreover, there is a lack of denaturing transport media compatible with SARS-CoV-2 RNA and antigen detection.
METHODS
Here, we tested whether supplementing media containing low concentrations of GITC with ammonium sulfate (AS) would affect the throat-swab detection of SARS-CoV-2 or a viral inactivation assay targeting coronavirus and other enveloped and non-enveloped viruses. The effect of adding AS to the media on RNA stability and its compatibility with SARS-CoV-2 antigen detection were also tested.
RESULTS AND DISCUSSION
We found that adding AS to the denaturing transport media reduced the need for high levels of GITC, improved SARS-COV-2 RNA detection without compromising virus inactivation, and enabled the denaturing transport media compatible with SARS-CoV-2 antigen detection.
PubMed: 38800755
DOI: 10.3389/fmicb.2024.1384991 -
Journal of Applied Glycoscience 2024Trehalose is known to protect enzymes from denaturation. In the present study, we observed promotion of apple polyphenol oxidase (PPO) inactivation in a trehalose...
Trehalose is known to protect enzymes from denaturation. In the present study, we observed promotion of apple polyphenol oxidase (PPO) inactivation in a trehalose solution with thermal treatment. Crude PPO from Fuji apple was mixed with either sucrose or trehalose solutions, then the samples treated at 25 or 65 °C. In the presence of trehalose, PPO activities were markedly decreased upon treatment at 65 °C with increasing trehalose concentration. Furthermore, the reduction in PPO activity in the presence of trehalose was proportional to storage time after thermal treatment and thermal treatment time. Comparing PPO activities between treatment time 0 and 90 min at 65 °C, activities decreased 89 % for trehalose concentration of 0.2 M. These results indicates that trehalose acts not only as inhibitor but as promoter of inactivation of PPO. The Lineweaver-Burk plot indicated that trehalose acts on PPO as a non-competitive inhibitor during the 65 °C treatment. Two mechanisms of PPO inactivation in the presence of trehalose were suggested; one is the suppression of PPO activation cause by a thermal treatment, and another is the conformational change to inactivation form of PPO in conjunction with trehalose and a thermal treatment. Additionally, apple juice including 0.2 or 0.5 M trehalose with 65 °C treatment indicated slow browning than the juice with 0.2 or 0.5 M sucrose or without sugars. This result demonstrates that the preventing of browning with trehalose is a viable industrial food process.
PubMed: 38799413
DOI: 10.5458/jag.jag.JAG-2023_0009 -
Scientific Reports May 2024The present work investigates the quality and the chemical effects of dehydration, using a novel dehydration system based on an electromagnetic induction and low... (Comparative Study)
Comparative Study
The present work investigates the quality and the chemical effects of dehydration, using a novel dehydration system based on an electromagnetic induction and low pressures technique, comparing it with the thermo-solar drying system. High oleic sunflower seeds, which are an important oil seed crop, were used due to the fact that they have a special place in the food industry. The seed samples were exposed to electromagnetic induction and low pressures by 0.5 and 1 h, then several chemical characterizations were carried out, in the electrophoresis study, it was found that most proteins in the hull were degraded or denatured, some of them were lost during the time in the thermosolar dryer while in kernel keeps 94.9% of the concentration in control proteins. Otherwise, the electromagnetic induction dryer did not lose the most of proteins in the kernel keeping 99.1% in 0.5 h and 98.4% in 1 h, just degrading its concentration. Germination viability results did not show changes after 0.5 h in the electromagnetic fields, but they decreased in 1 h from 66 to 40% until the thermosolar method fell to 24% in 4 h, both analysis results change proportionally with the treatment time and moisture content and the amount of the oxygen.
Topics: Helianthus; Seeds; Germination; Plant Proteins; Desiccation; Water; Dehydration
PubMed: 38797730
DOI: 10.1038/s41598-024-62822-5