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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 -
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi =... May 2024Objective To prepare monoclonal antibodies against the envelope protein extracellular domain (Eecto) of Zika virus (ZIKV) in mice. Methods A prokaryotic expression...
Objective To prepare monoclonal antibodies against the envelope protein extracellular domain (Eecto) of Zika virus (ZIKV) in mice. Methods A prokaryotic expression plasmid, pET28a-ZIKV-Eecto of ZIKV Eecto, was constructed, transformed into Escherichia coli BL21 and induced by isopropyl β-D-thiogalactoside (IPTG). The recombinant Eecto protein was expressed in the form of inclusion bodies, and purified proteins were obtained through denaturation, renaturation and ultrafiltration. After three rounds of immunization with the Eecto protein, the serum of BALB/c mice was obtained and the titer of polyclonal antibodies in serum was determined. The reactivity of polyclonal antibodies was analyzed with Western blotting and immunofluorescence assay in HEK293T cells expressing the ZIKV prME. Spleen cells from mice with higher antibody titers were prepared and fused with SP2/0 myeloma cells. The hybridoma cells secreting antibodies were screened through the limited dilution method, and the ascites containing antibody were harvested for titer measurement and subclass analysis. The Eecto from the envelope proteins of Japanese encephalitis virus (JEV), Yellow fever virus (YFV), Dengue virus (DENV1-4), and Tick borne encephalitis virus (TBEV) were coated and used to analyze the cross-reactivity of ZIKV monoclonal antibodies by ELISA. Further specificity analysis was conducted on antibodies with high titers and strong specificity. Results The plasmid pET28a-ZIKV-Eecto was successfully constructed. The purified Eecto protein was obtained with good immunogenicity. Four monoclonal antibodies were prepared and screened, namely 1D6, 4F11, 4H7, and 4F8. Among them, 1D6, 4H7, and 4F8 are IgG (K) type antibodies, and 4F11 is an IgM (K) antibody. The ascitic fluid titer of 1D6 was higher than 1:10. Antibodies 1D6 and 4H7 are ZIKV-specific and showed no cross-reactivity with other Flaviviruses. Conclusion The mice monoclonal antibodies against ZIKV-Eecto are produced successfully, which will provide experimental materials for the establishment of ZIKV detection methods and the study of its pathogenesis.
Topics: Animals; Zika Virus; Mice, Inbred BALB C; Antibodies, Monoclonal; Viral Envelope Proteins; Mice; Humans; HEK293 Cells; Female; Antibodies, Viral; Protein Domains; Enzyme-Linked Immunosorbent Assay
PubMed: 38790101
DOI: No ID Found -
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 -
Frontiers in Oncology 2024For liquid biopsy of cancer, the extraction of circulating cell-free DNA (cfDNA) from plasma is required. We evaluated the efficacy of use of magnetic submicron...
OBJECTIVE
For liquid biopsy of cancer, the extraction of circulating cell-free DNA (cfDNA) from plasma is required. We evaluated the efficacy of use of magnetic submicron particles coated with abundant small zwitterions (MSP-ZEWBs) for extracting short fragments of cfDNA.
METHODS
We developed and optimized an MSP-ZEWB-based cfDNA extraction method using ampholytic ion-exchange materials and compared its results with those using a control kit. We measured the cfDNA concentration by quantitative polymerase-chain-reaction and using the Qubit method and analyzed cfDNA fragmentation patterns using a bioanalyzer.
RESULTS
The fragment size of cfDNA isolated from glycine hydrochloric acid at a pH of 2.2 exhibited a better alignment with the DNA marker. The highest DNA intensity was observed at the final concentration of 0.8% polyethylene glycol 8000. The intensity of cfDNA decreased significantly when isolated from plasma with DNA marker using MSP-ZEWBs with an adsorption buffer containing guanidine hydrochloride or isothiocyanoguanidine. All fragments were successfully extracted using MSP-ZEWBs from both plasma and phosphate-buffered saline. Notably, the intensity of short cfDNA fragments isolated using MSP-ZEWBs remained consistent for recovery of long DNA fragments. indicating a potential selective of small fragments.
CONCLUSION
The extraction of plasma cfDNA with MSP-ZEWBs requires no protein denaturation, shows resistance to cells remaining in plasma, and demonstrates higher overall efficiency and better reproducibility than other extraction methods. Use of MSP-ZEWBs may greatly enhance liquid biopsy of cancers through the analysis of plasma cfDNA in clinical practice.
PubMed: 38779084
DOI: 10.3389/fonc.2024.1397680 -
The Journal of Applied Laboratory... Jul 2024The clinically significant Factor V Leiden (FVL) point mutation (1691 G/A) causes replacement of Arg with Gln (glutamine), preventing activated protein C from... (Comparative Study)
Comparative Study
BACKGROUND
The clinically significant Factor V Leiden (FVL) point mutation (1691 G/A) causes replacement of Arg with Gln (glutamine), preventing activated protein C from inactivating Factor V leading to a lengthened clotting process. Individuals with the Factor V Leiden mutations have an increased risk for venous thrombosis. The aim of this study is to compare an unlabeled probe high-resolution melting analysis (HRMA) assay for Factor V Leiden mutation to a TaqMan hydrolysis assay (fluorogenic 5' nuclease PCR hydrolysis assay). HRMA is a post-PCR, homogenous, closed-tube system for the detection of sequence variants. Post-PCR, the amplicons are heated gradually until the melting temperature is reached and the fluorescent dye unbinds from the amplicon and exhibits low fluorescence. A melt-curve analysis is generated that is characteristic of a particular sequence variant. Therefore, HRMA allows for comparison of one base changes in genetic sequences based on their differences in melting rate.
METHODS
Blood samples were collected in EDTA tubes and DNA extracted using the Roche MagNaPure. Reactions of both HRMA and TaqMan were carried out on 3 controls (1691 G/G, 1691 G/A, and 1691 G/G and G/A) and 20 samples.
RESULTS
The genotypes for 3 reference controls purchased from Coriell (F5 1691 G/G, FVL 1691 G/A, and Heterozygote 1691 G/G and G/A) were confirmed by both the HRMA and TaqMan FVL assays. All 20 samples were confirmed to be F5 1691 G/G by both HRMA and TaqMan assays.
CONCLUSIONS
Comparing the results of the unlabeled probe HRMA FVL assay with a real-time TaqMan probe end point genotyping assay resulted in 100% sensitivity and 100% specificity for both assays.
Topics: Factor V; Humans; Polymerase Chain Reaction; Fluorescent Dyes; Point Mutation; Nucleic Acid Denaturation; Hydrolysis; Transition Temperature
PubMed: 38775465
DOI: 10.1093/jalm/jfae039