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Nucleic Acids Research Jun 2024Whole-genome bisulfite sequencing (BS-Seq) measures cytosine methylation changes at single-base resolution and can be used to profile cell-free DNA (cfDNA). In plasma,...
Whole-genome bisulfite sequencing (BS-Seq) measures cytosine methylation changes at single-base resolution and can be used to profile cell-free DNA (cfDNA). In plasma, ultrashort single-stranded cfDNA (uscfDNA, ∼50 nt) has been identified together with 167 bp double-stranded mononucleosomal cell-free DNA (mncfDNA). However, the methylation profile of uscfDNA has not been described. Conventional BS-Seq workflows may not be helpful because bisulfite conversion degrades larger DNA into smaller fragments, leading to erroneous categorization as uscfDNA. We describe the '5mCAdpBS-Seq' workflow in which pre-methylated 5mC (5-methylcytosine) single-stranded adapters are ligated to heat-denatured cfDNA before bisulfite conversion. This method retains only DNA fragments that are unaltered by bisulfite treatment, resulting in less biased uscfDNA methylation analysis. Using 5mCAdpBS-Seq, uscfDNA had lower levels of DNA methylation (∼15%) compared to mncfDNA and was enriched in promoters and CpG islands. Hypomethylated uscfDNA fragments were enriched in upstream transcription start sites (TSSs), and the intensity of enrichment was correlated with expressed genes of hemopoietic cells. Using tissue-of-origin deconvolution, we inferred that uscfDNA is derived primarily from eosinophils, neutrophils, and monocytes. As proof-of-principle, we show that characteristics of the methylation profile of uscfDNA can distinguish non-small cell lung carcinoma from non-cancer samples. The 5mCAdpBS-Seq workflow is recommended for any cfDNA methylation-based investigations.
Topics: DNA Methylation; Humans; Cell-Free Nucleic Acids; CpG Islands; DNA, Single-Stranded; 5-Methylcytosine; Lung Neoplasms; Sulfites; Promoter Regions, Genetic; Sequence Analysis, DNA; Whole Genome Sequencing
PubMed: 38797520
DOI: 10.1093/nar/gkae276 -
Micromachines Apr 2024The thermal stability of DNA immobilized on a solid surface is one of the factors that affects the efficiency of solid-phase amplification (SP-PCR). Although variable...
The thermal stability of DNA immobilized on a solid surface is one of the factors that affects the efficiency of solid-phase amplification (SP-PCR). Although variable temperature amplification ensures high specificity of the reaction by precisely controlling temperature changes, excessively high temperatures during denaturation can negatively affect DNA stability. Formamide (FA) enables DNA denaturation at lower temperatures, showing potential for SP-PCR. Research on FA's impacts on DNA microarrays is still limited, necessitating further optimization in exploring the characteristics of FA in SP-PCR according to particular application needs. We immobilized DNA on a chip using a crosslinker and generated DNA microarrays through bridge amplification based on FA denaturation on our automated reaction device. We optimized the denaturation and hybridization parameters of FA, achieving a maximum cluster density of 2.83 × 10 colonies/mm. Compared to high-temperature denaturation, FA denaturation required a lower template concentration and milder reaction conditions and produced higher cluster density, demonstrating that FA effectively improves hybridization rates on surfaces. Regarding the immobilized DNA stability, the FA group exhibited a 45% loss of DNA, resulting in a 15% higher DNA retention rate compared to the high-temperature group, indicating that FA can better maintain DNA stability. Our study suggests that using FA improves the immobilized DNA stability and amplification efficiency in SP-PCR.
PubMed: 38793138
DOI: 10.3390/mi15050565 -
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 2024The aim of this study is to produce a biodegradable food packaging material that reduces environmental pollution and protects food safety. The effects of total solids...
The aim of this study is to produce a biodegradable food packaging material that reduces environmental pollution and protects food safety. The effects of total solids content, substrate ratio, polyphenol content, and magnetic stirring time on bovine bone gelatin/sodium carboxymethylcellulose nanoemulsion (BBG/SCMC-NE) were investigated using particle size, PDI, turbidity, rheological properties, and zeta potential as evaluation indexes. The micro, structural, antioxidant, encapsulation, and release properties were characterized after deriving its optimal preparation process. The results showed that the nanoemulsion was optimally prepared with a total solids content of 2%, a substrate ratio of 9:1, a polyphenol content of 0.2%, and a magnetic stirring time of 60 min. SEM showed that the nanoemulsion showed a dense and uniform reticulated structure. FTIR and XRD results showed that covalent cross-linking of proteins and polysaccharides altered the structure of gelatin molecular chains to a more compact form but did not change its semi-crystalline structure. DSC showed that the 9:1 BBG/SCMC-NE had a higher thermal denaturation temperature and greater thermal stability, and its DPPH scavenging rate could reach 79.25% and encapsulation rate up to 90.88%, with excellent slow-release performance. The results of the study provide basic guidance for the preparation of stable active food packaging with excellent properties.
PubMed: 38790806
DOI: 10.3390/foods13101506 -
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 -
Antioxidants (Basel, Switzerland) May 2024Alcohol is toxic to neurons and can trigger alcohol-related brain damage, neuronal loss, and cognitive decline. Neuronal cells may be vulnerable to alcohol toxicity and...
Alcohol is toxic to neurons and can trigger alcohol-related brain damage, neuronal loss, and cognitive decline. Neuronal cells may be vulnerable to alcohol toxicity and damage from oxidative stress after differentiation. To consider this further, the toxicity of alcohol to undifferentiated SH-SY5Y cells was compared with that of cells that had been acutely differentiated. Cells were exposed to alcohol over a concentration range of 0-200 mM for up to 24 h and alcohol effects on cell viability were evaluated via MTT and LDH assays. Effects on mitochondrial morphology were examined via transmission electron microscopy, and mitochondrial functionality was examined using measurements of ATP and the production of reactive oxygen species (ROS). Alcohol reduced cell viability and depleted ATP levels in a concentration- and exposure duration-dependent manner, with undifferentiated cells more vulnerable to toxicity. Alcohol exposure resulted in neurite retraction, altered mitochondrial morphology, and increased the levels of ROS in proportion to alcohol concentration; these peaked after 3 and 6 h exposures and were significantly higher in differentiated cells. Protein carbonyl content (PCC) lagged behind ROS production and peaked after 12 and 24 h, increasing in proportion to alcohol concentration, with higher levels in differentiated cells. Carbonylated proteins were characterised by their denatured molecular weights and overlapped with those from adult post-mortem brain tissue, with levels of PCC higher in alcoholic subjects than matched controls. Hence, alcohol can potentially trigger cell and tissue damage from oxidative stress and the accumulation of oxidatively damaged proteins.
PubMed: 38790685
DOI: 10.3390/antiox13050580