ABSTRACT
Nitric oxide synthases (NOSs), a family of flavo-hemoproteins with relatively rigid domains linked by flexible regions, require optimal FMN domain docking to the heme domain for efficient interdomain electron transfer (IET). To probe the FMN-heme interdomain docking, the magnetic dipole interactions between the FMN semiquinone radical (FMNHâ¢) and the low-spin ferric heme centers in oxygenase/FMN (oxyFMN) constructs of neuronal and inducible NOS (nNOS and iNOS, respectively) were measured using the relaxation-induced dipolar modulation enhancement (RIDME) technique. The FMNH⢠RIDME data were analyzed using the mesoscale Monte Carlo calculations of conformational distributions of NOS, which were improved to account for the native degrees of freedom of the amino acid residues constituting the flexible interdomain tethers. This combined computational and experimental analysis allowed for the estimation of the stabilization energies and populations of the docking complexes of calmodulin (CaM) and the FMN domain with the heme domain. Moreover, combining the five-pulse and scaled four-pulse RIDME data into a single trace has significantly reduced the uncertainty in the estimated docking probabilities. The obtained FMN-heme domain docking energies for nNOS and iNOS were similar (-3.8 kcal/mol), in agreement with the high degree of conservation of the FMN-heme domain docking interface between the NOS isoforms. In spite of the similar energetics, the FMN-heme domain docking probabilities in nNOS and iNOS oxyFMN were noticeably different (~ 0.19 and 0.23, respectively), likely due to differences in the lengths of the FMN-heme interdomain tethers and the docking interface topographies. The analysis based on the IET theory and RIDME experiments indicates that the variations in conformational dynamics may account for half of the difference in the FMN-heme IET rates between the two NOS isoforms.
ABSTRACT
NF-κB (Nuclear factor-kappa B) family proteins are versatile transcription factors that play crucial regulatory roles in cell development, growth, apoptosis, inflammation, and immune response. However, there is limited research on the function of these key genes in echinoderms. In this study, an NF-κB family gene (SiRel) was identified in sea urchin Strongylocentrotus intermedius. The gene has an open reading frame length of 1809 bp and encodes for 602 amino acids. Domain prediction results revealed that the N-terminal of SiRel protein encodes a conserved Rel homology domain (RHD), including the RHD-DNA binding domain and the RHD-dimerization domain. Multiple sequence comparison results showed that the protein sequences of these two domains were conserved. Phylogenetic analysis indicated that SiRel clustered with Strongylocentrotus purpuratus p65 protein and Rel protein of other echinoderms. Results from quantitative real-time PCR demonstrated detectable SiRel mRNA expression in all tested sea urchin tissues, with the highest expression level found in the gills. And SiRel mRNA expression levels were significantly induced after LPS (Lipopolysaccharide) and poly(I:C) (Polyinosinic:polycytidylic acid) stimulation. In addition, SiRel protein expression can be found in cytoplasm and nucleus of HEK293T cells. Co-immunoprecipitation results showed that SiRel could interact with sea urchin IκB (Inhibitor of NF-κB) protein. Western blotting and dual-luciferase reporter gene assay results indicated that overexpression of SiRel in HEK293T cells could impact the phosphorylation levels of JNK (c-Jun N-terminal kinase) and Erk1/2 (Extracellular signal-regulated kinases1/2) and activate interleukin-6 (IL-6), activating protein 1 (AP-1), interferon (IFN)α/ß/γ, and signal transducer and activator of transcription 3 (STAT3) reporter genes in HEK293T cells. In conclusion, this study reveals that SiRel plays an important role in the innate immune response of sea urchins and enriches our understanding of comparative immunology theory.
Subject(s)
Amino Acid Sequence , Gene Expression Regulation , Immunity, Innate , Lipopolysaccharides , Phylogeny , Poly I-C , Sequence Alignment , Strongylocentrotus , Animals , Immunity, Innate/genetics , Poly I-C/pharmacology , Lipopolysaccharides/pharmacology , Strongylocentrotus/genetics , Strongylocentrotus/immunology , Sequence Alignment/veterinary , Gene Expression Regulation/immunology , Cloning, Molecular , Gene Expression Profiling/veterinary , Humans , NF-kappa B/genetics , NF-kappa B/metabolism , NF-kappa B/immunology , Base Sequence , Proto-Oncogene Proteins c-rel/genetics , Proto-Oncogene Proteins c-rel/metabolism , HEK293 CellsABSTRACT
Nitric oxide synthase (NOS) in mammals is a family of multidomain proteins in which interdomain electron transfer (IET) is controlled by domain-domain interactions. Calmodulin (CaM) binds to the canonical CaM-binding site in the linker region between the FMN and heme domains of NOS and allows tethered FMN domain motions, enabling an intersubunit FMN-heme IET in the output state for NO production. Our previous cross-linking mass spectrometric (XL MS) results demonstrated site-specific protein dynamics in the CaM-responsive regions of rat neuronal NOS (nNOS) reductase construct, a monomeric protein [Jiang et al., Biochemistry, 2023, 62, 2232-2237]. In this work, we have extended our combined approach of XL MS structural mapping and AlphaFold structural prediction to examine the homodimeric nNOS oxygenase/FMN (oxyFMN) construct, an established model of the NOS output state. We employed parallel reaction monitoring (PRM) based quantitative XL MS (qXL MS) to assess the CaM-induced changes in interdomain dynamics and interactions. Intersubunit cross-links were identified by mapping the cross-links onto top AlphaFold structural models, which was complemented by comparing their relative abundances in the cross-linked dimeric and monomeric bands. Furthermore, contrasting the CaM-free and CaM-bound nNOS samples shows that CaM enables the formation of the intersubunit FMN-heme docking complex and that CaM binding induces extensive, allosteric conformational changes across the NOS regions. Moreover, the observed cross-links sites specifically respond to changes in ionic strength. This indicates that interdomain salt bridges are responsible for stabilizing and orienting the output state for efficient FMN-heme IET. Taken together, our targeted qXL MS results have revealed that CaM and ionic strength modulate specific dynamic changes in the CaM/FMN/heme complexes, particularly in the context of intersubunit interdomain FMN-heme interactions.
Subject(s)
Calmodulin , Flavin Mononucleotide , Heme , Mass Spectrometry , Nitric Oxide Synthase Type I , Nitric Oxide Synthase Type I/metabolism , Nitric Oxide Synthase Type I/chemistry , Flavin Mononucleotide/metabolism , Flavin Mononucleotide/chemistry , Heme/metabolism , Heme/chemistry , Animals , Rats , Calmodulin/metabolism , Calmodulin/chemistry , Mass Spectrometry/methods , Protein Domains , Models, Molecular , Binding Sites , Cross-Linking Reagents/chemistry , Protein BindingABSTRACT
Calmodulin (CaM) binds to a linker between the oxygenase and reductase domains of nitric oxide synthase (NOS) to regulate the functional conformational dynamics. Specific residues on the interdomain interface guide the domain-domain docking to facilitate the electron transfer in NOS. Notably, the docking interface between CaM and the heme-containing oxygenase domain of NOS is isoform specific, which is only beginning to be investigated. Toward advancing understanding of the distinct CaM-NOS docking interactions by infrared spectroscopy, we introduced a cyano-group as frequency-resolved vibrational probe into CaM individually and when associated with full-length and a bi-domain oxygenase/FMN construct of the inducible NOS isoform (iNOS). Site-specific, selective labeling with p-cyano-L-phenylalanine (CNF) by amber suppression of CaM bound to the iNOS has been accomplished by protein coexpression due to the instability of recombinant iNOS protein alone. We introduced CNF at residue 108, which is at the putative CaM-heme (NOS) docking interface. CNF was also introduced at residue 29, which is distant from the docking interface. FT IR data show that the 108 site is sensitive to CaM-NOS complex formation, while insensitivity to its association with the iNOS protein or peptide was observed for the 29 site. Moreover, narrowing of the IR bands at residue 108 suggests the C≡N probe experiences a more limited distribution of environments, indicating side chain restriction apparent for the complex with iNOS. This initial work sets the stage for residue-specific characterizations of structural dynamics of the docked states of NOS proteins.
Subject(s)
Calmodulin , Spectrophotometry, Infrared , Calmodulin/chemistry , Calmodulin/metabolism , Nitric Oxide Synthase Type II/chemistry , Nitric Oxide Synthase Type II/metabolism , Protein Binding , Molecular Docking SimulationABSTRACT
Selenium (Se) and tellurium (Te) contaminations in soils and water bodies have been widely reported in recent years. Se(IV) and Te(IV) were regarded as their most dangerous forms. Microbial treatments of Se(IV)- and Te(IV)-containing wastes are promising approaches because of their environmentally friendly and sustainable advantages. However, the salt-tolerant microbial resources that can be used for selenium/tellurium pollution control are still limited since industrial wastewaters usually contain a large number of salts. In this study, a marine Shewanella sp. FDA-1 (FDA-1) was reported for efficient Se(IV) and Te(IV) reduction under saline conditions. Process and product analyses were performed to investigate the bioreduction processes of Se(IV) and Te(IV). The results showed that FDA-1 can effectively reduce Se(IV) and Te(IV) to Se0 and Te0 Se(IV)/Te(IV) to Se0/Te0 in 72 h, which were further confirmed by XRD and XPS analyses. In addition, enzymatic and RT‒qPCR assays showed that flavin-related proteins, reductases, dehydrogenases, etc., could be involved in the bioreduction of Se(IV)/Te(IV). Overall, our results demonstrate the ability of FDA-1 to reduce high concentrations of Se(IV)/or Te(IV) to Se0/or Te0 under saline conditions and thus provide efficient microbial candidate for controlling Se and Te pollution.(AU)
Subject(s)
Humans , Bacteria , Metals/toxicity , Selenious Acid/metabolism , Selenium/metabolism , Tellurium/metabolism , Microbiology , Microbiological Techniques , Soil Microbiology , Water MicrobiologyABSTRACT
The coastal ocean systems play paramount role in the nutrient biogeochemistry because of its interconnected environment. To gain a novel insight into coupling relationships between bacterial community, functioning properties and nutrient metabolism, we conducted analysis on the patterns and driving factors of planktonic bacterial functional community across subsurface water of marine ranching near the Yellow Sea in both summer and winter. Illumina HiSeq Sequencing and a corresponding set of biogeochemical data were used to assess distribution patterns of taxa, adaptive mechanism and metabolic function. Results demonstrated that Proteobacteria, Cyanobacteria, Actinobacteriota and Bacteroidota were dominant phyla both in summer and winter. Taxonomic profiles related to nutrient variation were found to be highly correlated with Dissolved Oxygen (DO) and Chlorophyll fluorescence (FLUO), and distinct diversity differences were also found between summer and winter samples. Functional activity in summer associated with the relative abundance of phototrophy and photoautotrophy were the highest in the subsurface water, while in winter the dominant functional properties were mainly include chemoheterotrophy and aerobic_ chemoheterotrophy. A significant difference related to functional activity between summer and winter, mainly representing ligninolysis and iron_respiration. In general, our study provides a framework for understanding the relative importance of environmental factors, temperature variation and nutrient availability in shaping the metabolic processes of aquatic microorganisms, particularly in ocean mariculture systems.
Subject(s)
Cyanobacteria , Seawater , Seawater/chemistry , Biodiversity , Plankton , WaterABSTRACT
Microplastics (MPs) aging in natural ecosystems are caused by solar irradiation. Photo-aged MPs in aquatic systems are a major threat to molluscs. In this study, polystyrene (PS) photo-aging was simulated using a sunlight simulator. After exposure of Crassostrea gigas to photo-aged PS, a decreased gonadosomatic index, coupled with histological alterations, suggested an inhibitory effect on the gonadal development of bivalves. As the concentration of aged PS increased, the inhibitory effects on gonadal development became more severe. The sex hormone (testosterone and estradiol) and energy metabolism (glycogen, lipid, and protein content) differences between C. gigas males and females suggested a disruption of sex hormonal homeostasis and a shift in energy allocation strategy, which may have affected reproduction, especially female oysters. In addition, the substantial downregulation of SOX-8, SOX-E, Piwi1, and TGF-ß genes may be contributing factors causing the inhibitory effect of aged PS on the gonadal development of C. gigas. This study provides an essential reference for evaluating the reproductive health risks posed by aged MPs and offers novel insights and perspectives for exploring the impact of MPs under natural conditions.
Subject(s)
Crassostrea , Water Pollutants, Chemical , Animals , Male , Female , Microplastics , Polystyrenes , Plastics , Ecosystem , Crassostrea/physiology , Sunlight , Water Pollutants, Chemical/toxicity , Water Pollutants, Chemical/metabolismABSTRACT
Selenium (Se) and tellurium (Te) contaminations in soils and water bodies have been widely reported in recent years. Se(IV) and Te(IV) were regarded as their most dangerous forms. Microbial treatments of Se(IV)- and Te(IV)-containing wastes are promising approaches because of their environmentally friendly and sustainable advantages. However, the salt-tolerant microbial resources that can be used for selenium/tellurium pollution control are still limited since industrial wastewaters usually contain a large number of salts. In this study, a marine Shewanella sp. FDA-1 (FDA-1) was reported for efficient Se(IV) and Te(IV) reduction under saline conditions. Process and product analyses were performed to investigate the bioreduction processes of Se(IV) and Te(IV). The results showed that FDA-1 can effectively reduce Se(IV) and Te(IV) to Se0 and Te0 Se(IV)/Te(IV) to Se0/Te0 in 72 h, which were further confirmed by XRD and XPS analyses. In addition, enzymatic and RTâqPCR assays showed that flavin-related proteins, reductases, dehydrogenases, etc., could be involved in the bioreduction of Se(IV)/Te(IV). Overall, our results demonstrate the ability of FDA-1 to reduce high concentrations of Se(IV)/or Te(IV) to Se0/or Te0 under saline conditions and thus provide efficient microbial candidate for controlling Se and Te pollution.
Subject(s)
Selenious Acid , Selenium , Selenious Acid/metabolism , Selenium/metabolism , Tellurium/metabolism , MetalsABSTRACT
Neuronal nitric oxide synthase (nNOS) is regulated by phosphorylation in vivo, yet the underlying biochemical mechanisms remain unclear, primarily due to difficulty in obtaining milligram quantities of phosphorylated nNOS protein; detailed spectroscopic and rapid kinetics investigations require purified protein samples at a concentration in the range of hundreds microM. Moreover, the functional diversity of the nNOS isoform is linked to its splice variants. Also of note is that determination of protein phosphorylation stoichiometry remains as a challenge. To address these issues, this study first expanded a recent genetic code expansion approach to produce phosphorylated rat nNOSµ and nNOSα holoproteins through site-specific incorporation of phosphoserine (pSer) at residues 1446 and 1412, respectively; this site is at the C-terminal tail region, a NOS-unique regulatory element. A quantitative mass spectrometric approach was then developed in-house to analyze unphosphorylated peptides in phosphatase-treated and -untreated phospho-nNOS proteins. The observed pSer-incorporation efficiency consistently exceeded 80%, showing high pSer-incorporation efficiency. Notably, EPR spin trapping results demonstrate that under l-arginine-depleted conditions, pSer1412 nNOSα presented a significant reduction in superoxide generation, whereas pSer1446 nNOSµ exhibited the opposite effect, compared to their unphosphorylated counterparts. This suggests that phosphorylation at the C-terminal tail has a regulatory effect on nNOS uncoupling that may differ between variant forms. Furthermore, the methodologies for incorporating pSer into large, complex protein and quantifying the percentage of phosphorylation in recombinant purified protein should be applicable to other protein systems.
Subject(s)
Nitric Oxide Synthase Type I , Nitric Oxide , Superoxides , Animals , Rats , Nitric Oxide/metabolism , Nitric Oxide Synthase Type I/genetics , Phosphorylation , Phosphoserine/metabolism , Recombinant Proteins/metabolism , Superoxides/metabolismABSTRACT
Infrared sensors capture infrared rays radiated by objects to form thermal images. They have a steady ability to penetrate smoke and fog, and are widely used in security monitoring, military, etc. However, civilian infrared detectors with lower resolution cannot compare with megapixel RGB camera sensors. In this paper, we propose a dynamic attention mechanism-based thermal image super-resolution network for infrared sensors. Specifically, the dynamic attention modules adaptively reweight the outputs of the attention and non-attention branches according to features at different depths of the network. The attention branch, which consists of channel- and pixel-wise attention blocks, is responsible for extracting the most informative features, while the non-attention branch is adopted as a supplement to extract the remaining ignored features. The dynamic weights block operates with 1D convolution instead of the full multi-layer perceptron on the global average pooled features, reducing parameters and enhancing information interaction between channels, and the same structure is adopted in the channel attention block. Qualitative and quantitative results on three testing datasets demonstrate that the proposed network can superior restore high-frequency details while improving the resolution of thermal images. And the lightweight structure of the proposed network with lower computing cost can be practically deployed on edge devices, effectively improving the imaging perception quality of infrared sensors.
ABSTRACT
The biological reduction of selenite (Se(iv)) or tellurite (Te(iv)) to Se0 or Te0 has received increasing attention, as related studies have favored the development of Se/Te pollution control methods. In the presence of the electron donor, the microbes acquired energy and transferred electrons to Se(iv) or Te(iv) to achieve their detoxication. However, the microbial electron transfer pathways involved in this process are still not fully understood. In this study, we reported that marine Shewanella sp. FDL-2 (FDL-2) was capable of reducing Se(iv) and Te(iv) through a novel riboflavin-involved pathway. The results showed that FDL-2 can effectively reduce 10 mM Se(iv) and 5 mM Te(iv) to Se0 and Te0, which was further confirmed by XPS and XRD analyses. RT-qPCR results indicate the upregulation of genes coding flavin-related proteins, and the production of flavin-related substances by strain FDL-2 during Se(iv)/Te(iv) bioreduction was proven by fluorescence chromatography analysis. In addition, the presence of riboflavin enhanced the electron transfer efficiency, indicating its promoting effect on the bioreduction of Se(iv)/Te(iv). Overall, our results highlight a riboflavin-involved electron transfer pathway during Se(iv)/Te(iv) bioreduction and thus deepen our understanding of the corresponding mechanism.
ABSTRACT
As a toxic element of global concern, the elevated concentration of antimony (Sb) in the environment has attracted increasing attention. Microorganisms have been reported as important driving forces for Sb transformation. Iron (Fe) is the most important metal associated element of Sb, however, how Fe-bearing minerals affect the biological transformation of Sb is still unclear. In this study, the effects of Fe-bearing minerals on biological Sb(V) reduction were investigated by employing a marine Shewanella sp. CNZ-1 (CNZ-1). Our results showed that the presence of hematite, magnetite and ferrihydrite (1 g/L) resulted in a decrease in Sb(III) concentration of ~19-31 % compared to the Fe(III)-minerals free system. The calculated Sb(V) reduction rates are 0.0256 (R2 0.71), 0.0389 (R2 0.87), 0.0299 (R2 0.96) and 0.0428 (R2 0.95) h-1 in the hematite-, magnetite-, ferrihydrite-supplemented and Fe(III)-minerals free systems, respectively. The cube-shaped Sb2O3 was characterized as a reductive product by using XRD, XPS, FTIR, TG and SEM approaches. Differential proteomic analysis showed that flagellar protein, cytochrome c, electron transfer flavoprotein, nitrate reductase and polysulfide reductase (up-regulation >1.5-fold, p value <0.05) were supposed to be included in the electron transport pathway of Sb(V) reduction by strain CNZ-1, and the key role of nitrate reductases was further highlighted during this reaction process based on the RT-qPCR and confirmatory experiments. Overall, these findings are beneficial to understand the environmental fate of Sb in the presence of Fe-bearing minerals and provide guidance in developing the bacteria/enzyme-mediated control strategy for Sb pollution.
Subject(s)
Ferric Compounds , Iron , Ferric Compounds/metabolism , Iron/metabolism , Nitrate Reductase/metabolism , Ferrosoferric Oxide , Proteomics , Oxidation-Reduction , Minerals/metabolism , Antimony/analysisABSTRACT
DNA methylation (DNAme) alterations are known to initiate from the precancerous stage of tumorigenesis. Herein, we investigated the global and local patterns of DNAme perturbations in tumorigenesis by analysing the genome-wide DNAme profiles of the cervix, colorectum, stomach, prostate, and liver at precancerous and cancer stages. We observed global hypomethylation in tissues of both two stages, except for the cervix, whose global DNAme level in normal tissue was lower than that of the other four tumour types. For alterations shared by both stages, there were common hyper-methylation (sHyperMethyl) and hypo-methylation (sHypoMethyl) changes, of which the latter type was more frequently identified in all tissues. Biological pathways interrupted by sHyperMethyl and sHypoMethyl alterations demonstrated significant tissue specificity. DNAme bidirectional chaos indicated by the enrichment of both sHyperMethyl and sHypoMethyl changes in the same pathway was observed in most tissues and was a common phenomenon, particularly in liver lesions. Moreover, for the same enriched pathways, different tissues may be affected by distinct DNAme types. For the PI3K-Akt signalling pathway, sHyperMethyl enrichment was observed in the prostate dataset, but sHypoMethyl enrichment was observed in the colorectum and liver datasets. Nevertheless, they did not show an increased possibility in survival prediction of patients in comparison with other DNAme types. Additionally, our study demonstrated that gene-body DNAme changes of tumour suppressor genes and oncogenes may persist from precancerous lesions to the tumour. Overall, we demonstrate the tissue specificity and commonality of cross-stage alterations in DNA methylation profiles in multi-tissue tumorigenesis.
Subject(s)
DNA Methylation , Precancerous Conditions , Male , Female , Humans , Organ Specificity , Phosphatidylinositol 3-Kinases/genetics , Precancerous Conditions/genetics , Carcinogenesis/geneticsABSTRACT
Stable picosecond dissipative soliton pulses were observed in an ytterbium-doped fiber laser employing a high-quality mixture of BP/SnSe2-PVA saturable absorber (SA). The modulation depth, saturation intensity, and non-saturable loss of the mixture of BP/SnSe2-PVA SA were measured with values of 5.98%, 18.37 MW/cm2, and 33%, respectively. Within the pump power range of 150-270 mW, stable dissipative soliton pulses were obtained with an output power of 1.68-4 mW. When the minimum pulse duration is 1.28 ps, a repetition rate of 0.903 MHz, center wavelength of 1064.38 nm and 3 dB bandwidth of 2 nm were obtained. The maximum pulse energy of 4.43 nJ and the signal-to-noise ratio up to 72 dB were achieved at pump power of 270 mW. The results suggest that the BP/SnSe2-PVA mixture SA has outstanding nonlinear saturable absorption characteristics and broad ultrafast laser applications.
ABSTRACT
Nitric oxide synthase (NOS) is responsible for the biosynthesis of nitric oxide (NO), an important signaling molecule controlling diverse physiological processes such as neurotransmission and vasodilation. Neuronal NOS (nNOS) is a calmodulin (CaM)-controlled enzyme. In the absence of CaM, several intrinsic control elements, along with NADP+ binding, suppress electron transfer across the NOS domains. CaM binding relieves the inhibitory factors to promote the electron transport required for NO production. The regulatory dynamics of nNOS control elements are critical to governing NO signaling, yet mechanistic questions remain, because the intrinsic dynamics of NOS thwart traditional structural biology approaches. Here, we have employed cross-linking mass spectrometry (XL MS) to probe regulatory dynamics in nNOS, focusing on the CaM-responsive control elements. Quantitative XL MS revealed conformational changes differentiating the nNOS reductase (nNOSred) alone, nNOSred with NADP+, nNOS-CaM, and nNOS-CaM with NADP+. We observed distinct effects of CaM vs NADP+ on cross-linking patterns in nNOSred. CaM induces striking global changes, while the impact of NADP+ is primarily localized to the NADPH-binding subdomain. Moreover, CaM increases the abundance of intra-nNOS cross-links that are related to the formation of the inter-CaM-nNOS cross-links. Taken together, these XL MS results demonstrate that CaM and NADP+ site-specifically alter the nNOS conformational landscape.
ABSTRACT
We propose a method for imaging through a turbid medium by using a single-shot decoherence polarization gate (DPG). The DPG is made up of a polarizer, an analyzer, and a weakly scattering medium. Contrary to intuition, we discover that the preferential utilization of sparsely scattered photons by introducing weakly scattering mediums can lead to better image quality. The experimental results show that the visibilities of the images acquired from the DPG imaging method are obviously improved. The contrast of the bar can be increased by 50% by the DPG imaging technique. Furthermore, we study the effect of the volume concentration of the weakly scattering medium on the speckle suppression and the enhancement of the visibilities of the images. The variances of the contrasts of the image show that there exists an optimum optical depth (â¼0.8) of the weakly scattering medium for DPG imaging through a specific turbid medium.
ABSTRACT
Programmed cell death (PCD) resistance is a key driver of cancer occurrence and development. The prognostic relevance of PCD-related genes in hepatocellular carcinoma (HCC) has attracted considerable attention in recent years. However, there is still a lack of efforts to compare the methylation status of different types of PCD genes in HCC and their roles in its surveillance. The methylation status of genes related to pyroptosis, apoptosis, autophagy, necroptosis, ferroptosis, and cuproptosis was analyzed in tumor and non-tumor tissues from TCGA. Whole-genome bisulfite sequencing (WGBS) data of paired tumor tissue and buffy coat samples were used to filter the potential interference of blood leukocytes in cell-free DNA (cfDNA). The WGBS data of healthy individuals' and early-stage HCC patients' cfDNA were analyzed to evaluate the distinguishing ability. The average gene body methylation (gbDNAme) of pyroptosis-related genes (PRGs) was significantly altered in HCC tissues relative to normal tissues, and their distinguishing ability was higher compared to the other types of PCD-related genes. The gbDNAme of NLRP7, NLRP2, and NLRP3 was reflective of the hypomethylation in HCC tissues, and methylation levels of NLRP3 correlated positively with its expression level (r=0.51). The candidate hypomethylated PRGs could discriminate between early HCC patients and healthy controls in cfDNA analysis with high accuracy (area under the receiver operation curve, AUC=0.94). Furthermore, the hypomethylation of PRGs was associated with poor prognosis of HCC. Gene body hypomethylation of PRGs is a promising biomarker for early HCC detection, monitoring of tumor recurrence, and prognosis prediction.
Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis Regulatory Proteins , Carcinoma, Hepatocellular , Liver Neoplasms , NLR Family, Pyrin Domain-Containing 3 Protein , Humans , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell-Free Nucleic Acids , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Pyroptosis/geneticsABSTRACT
Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease. However, a detailed DNA methylation (DNAme) landscape has not yet been elucidated. Our study combined DNAme and transcriptome profiles for HCM myocardium and identify aberrant DNAme associated with altered myocardial function in HCM. The transcription of methylation-related genes did not significantly differ between HCM and normal myocardium. Nevertheless, the former had an altered DNAme profile compared with the latter. The hypermethylated and hypomethylated sites in HCM tissues had chromosomal distributions and functional enrichment of correlated genes differing from those of their normal tissue counterparts. The GO analysis of network underlying the genes correlated with DNAme alteration and differentially expressed genes (DEGs) shows functional clusters centred on immune cell function and muscle system processes. In KEGG analysis, only the calcium signalling pathway was enriched either by the genes correlated with changes in DNAme or DEGs. The protein-protein interactions (PPI) underlying the genes altered at both the DNAme and transcriptional highlighted two important functional clusters. One of these was related to the immune response and had the estrogen receptor-encoding ESR1 gene as its node. The other cluster comprised cardiac electrophysiology-related genes. Intelliectin-1 (ITLN1), a component of the innate immune system, was transcriptionally downregulated in HCM and had a hypermethylated site within 1500 bp upstream of the ITLN1 transcription start site. Estimates of immune infiltration demonstrated a relative decline in immune cell population diversity in HCM. A combination of DNAme and transcriptome profiles may help identify and develop new therapeutic targets for HCM.
Subject(s)
Cardiomyopathy, Hypertrophic , Epigenome , Humans , DNA Methylation , Gene Expression Profiling , Cardiomyopathy, Hypertrophic/genetics , Cardiomyopathy, Hypertrophic/metabolism , Transcriptome , ElectrophysiologyABSTRACT
Chronic arsenic exposures via the consumption of contaminated drinking water are clearly associated with many deleterious health outcomes, including anemia. Following exposure, trivalent inorganic arsenic (AsIII) is methylated through a series of arsenic (+III oxidation state) methyltransferase (As3MT)-dependent reactions, resulting in the production of several intermediates with greater toxicity than the parent inorganic arsenicals. The extent to which inorganic vs. methylated arsenicals contribute to AsIII-induced hematotoxicity remains unknown. In this study, the contribution of As3MT-dependent biotransformation to the development of anemia was evaluated in male As3mt-knockout (KO) and wild-type, C57BL/6J, mice following 60-day drinking water exposures to 1 mg/L (ppm) AsIII. The evaluation of hematological indicators of anemia revealed significant reductions in red blood cell counts, hemoglobin levels, and hematocrit in AsIII-exposed wild-type mice as compared to unexposed controls. No such changes in the blood of As3mt-KO mice were detected. Compared with unexposed controls, the percentages of mature RBCs in the bone marrow and spleen (measured by flow cytometry) were significantly reduced in the bone marrow of AsIII-exposed wild-type, but not As3mt-KO mice. This was accompanied by increased levels of mature RBCS in the spleen and elevated levels of circulating erythropoietin in the serum of AsIII-exposed wild-type, but not As3mt-KO mice. Taken together, the findings from the present study suggest that As3MT-dependent biotransformation has an essential role in mediating the hematotoxicity of AsIII following drinking water exposures.