Inhibiting DNA methyltransferase DNMT3B confers protection against ferroptosis in nucleus pulposus and ameliorates intervertebral disc degeneration via upregulating SLC40A1.

Epigenetic changes are important considerations for degenerative diseases. DNA methylation regulates crucial genes by epigenetic mechanism, impacting cell function and fate. DNA presents hypermethylation in degenerated nucleus pulposus (NP) tissue, but its role in intervertebral disc degeneration (IVDD) remains elusive. This study aimed to demonstrate that methyltransferase mediated hypermethylation was responsible for IVDD by integrative bioinformatics and experimental verification. Methyltransferase DNMT3B was highly expressed in severely degenerated NP tissue (involving human and rats) and in-vitro degenerated human NP cells (NPCs). Bioinformatics elucidated that hypermethylated genes were enriched in oxidative stress and ferroptosis, and the ferroptosis suppressor gene SLC40A1 was identified with lower expression and higher methylation in severely degenerated human NP tissue. Cell culture using human NPCs showed that DNMT3B induced ferroptosis and oxidative stress in NPCs by downregulating SLC40A1, promoting a degenerative cell phenotype. An in-vivo rat IVDD model showed that DNA methyltransferase inhibitor 5-AZA alleviated puncture-induced IVDD. Taken together, DNA methyltransferase DNMT3B aggravates ferroptosis and oxidative stress in NPCs via regulating SLC40A1. Epigenetic mechanism within DNA methylation is a promising therapeutic biomarker for IVDD.

RARRES2 is involved in the "lock-and-key" interactions between osteosarcoma stem cells and tumor-associated macrophages.

Osteosarcoma (OS) is a type of tumor. Osteosarcoma stem cells (OSCs) are responsible for drug resistance, recurrence, and immunosuppression in OS. We aimed to determine the heterogeneity of OSCs and the immunosuppression mechanisms underlying the interactions between OSCs and tumor-associated macrophages (TAMs). The cell components, trajectory changes, and cell communication profiles of OS cells were analyzed by transcriptomics at the single-cell level. The intercellular communication patterns of OSCs were verified, and the role of the cell hub genes was revealed. Hub geneS are genes that play important roles in regulating certain biological processes; they are often defined as the genes with the strongest regulatory effect on differentially expressed gene sets. Moreover, various cellular components of the OS microenvironment were identified. Malignant cells were grouped, and OSCs were identified. Further regrouping and communication analysis revealed that the genes in the stemness maintenance and differentiation subgroups were involved in communication with macrophages. Key receptor-ligand pairs and target gene sets for cell communication were obtained. Transcriptome data analysis revealed the key gene RARRES2, which is involved in intercellular communication between OSCs and TAMs. In vitro studies confirmed that macrophages promote RARRES2-mediated stemness maintenance in OSCs via the TAM-secreted cytokine insulin-like growth factor 1. Patient studies confirmed that RARRES2 could be a biomarker of OS. OSCs are highly heterogeneous, and different subgroups are responsible for proliferation and communication with other cells. The IGF-RARRES2 axis plays a key role in maintaining OSC stemness through communication with TAMs.

Decomposition and humification process of domestic biodegradable waste by black soldier fly (Hermetia illucens L.) larvae from the perspective of dissolved organic matter.

Black soldier fly larvae (Hermitia illucens L.) (BSFL) bioconversion is a promising technology for domestic biodegradable waste (DBW) management and resource recovery. However, little is known about the DBW biodegradation during the BSFL bioconversion from the perspective of dissolved organic matter (DOM). In the current study, field tests were conducted on a full-scale BSFL bioconversion facility with treatment capacity of 15 tons DBW/day. Composition of DOM in DBW were investigated by spectral analysis (UV-vis, fluorescence, and Fourier Transform Infrared spectroscopy (FT-IR)), coupled with enzyme activity analysis. After BSFL bioconversion, DOM concentrations, total carbon and total nitrogen in residues decreased by 51.5%, 18.3% and 19.9%, respectively. Meanwhile, enzymes like catalase, lipase, protease, sucrase, urease and cellulase significantly increased (9.28%-56.3%). The specific UV absorbance at 254 nm and 280 nm (SUVA254, SUVA280), the area at 226-400 nm (A226-400) and slope in the 280-400 nm region (S280-400) of DOM increased by 230%, 186%, 143% and 398%, respectively. Moreover, the characteristic peaks at 1636, 1077 and 1045 cm-1 in FT-IR increased continuously, with a significant decrease in peak at 1124 and 1572 cm-1. DOM spectral data show that BSFL decomposed the carboxylic, cellulose and aliphatic components, resulting in the increase of oxygen-containing functional groups (e.g., hydroxyl, carboxyl, carbonyl) and aromatic compounds. Furthermore, fluorescence profiles show that Region Ⅰ, Ⅱ (aromatic proteins) and Ⅳ (soluble microbial byproducts) decreased while Region Ⅴ (humic-like substances) increased significantly. Humification index increased by 122% while biological index decreased by 18.0%, indicating a significant increase in degree of humification and stabilization of the residues. The current evidence provides a theoretical basis for technical re-innovation and improving economic potential of BSFL technology.

Role of ferroptosis-associated genes in ankylosing spondylitis and immune cell infiltration.

Ankylosing spondylitis (AS) is a chronic progressive autoimmune disease with insidious onset, high rates of disability among patients, unknown pathogenesis, and no effective treatment. Ferroptosis is a novel type of regulated cell death that is associated with various cancers and diseases. However, its relation to AS is not clear. In the present study, we identified two potential therapeutic targets for AS based on genes associated with ferroptosis and explored their association with immune cells and immune cell infiltration (ICI). We studied gene expression profiles of two cohorts of patients with AS (GSE25101 and GSE41038) derived from the gene expression omnibus database, and ferroptosis-associated genes (FRGs) were obtained from the FerrDb database. LASSO regression analysis was performed to build predictive models for AS based on FRGs, and the ferroptosis level in each sample was assessed via single-sample gene set enrichment analysis. Weighted gene co-expression network and protein-protein interaction network analyses were performed for screening; two key genes, DDIT3 and HSPB1, were identified in patients with AS. The relationship between key genes and ICI levels was assessed using the CIBERSORT algorithm, followed by gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. Finally, DDIT3 and HSPB1 were identified as diagnostic markers and potential therapeutic targets for AS. DDIT3 was highly positively correlated with the infiltration levels of various immune cells, while HSPB1 was negatively correlated with the infiltration levels of several different types of immune cells. In conclusion, DDIT3 and HSPB1 may induce ferroptosis in the cells of patients with AS via changes in the inflammatory response in the immune microenvironment, and these genes could serve as molecular targets for AS therapy.

Theaflavin-3,3'-Digallate Plays a ROS-Mediated Dual Role in Ferroptosis and Apoptosis via the MAPK Pathway in Human Osteosarcoma Cell Lines and Xenografts.

Globally, osteosarcoma (OS) is the most prevalent form of primary bone cancer in children and adolescents. Traditional neoadjuvant chemotherapy regimens have reached a bottleneck; thus, OS survivors have unsatisfactory outcomes. Theaflavin-3,3'-digallate (TF3) exhibits potent anticancer properties against many human cancers. Nevertheless, the biological effects and the underlying molecular mechanism of TF3 in human OS remain unclear. The objective of this study was to investigate the effects of TF3 on human OS cell lines and mouse xenograft models. The results showed that TF3 reduced cell viability, suppressed cell proliferation, and caused G0/G1 cell cycle arrest in both MG63 and HOS cell lines in a concentration-dependent manner. TF3 also altered the homeostatic mechanisms for iron storage in the examined cell lines, resulting in an excess of labile iron. Unsurprisingly, TF3 caused oxidative stress through reduced glutathione (GSH) exhaustion, reactive oxygen species (ROS) accumulation, and the Fenton reaction, which triggered ferroptosis and apoptosis in the cells. TF3 also induced MAPK signalling pathways, including the ERK, JNK, and p38 MAPK pathways. Furthermore, oxidative stress was shown to be the primary reason for TF3-induced proliferation inhibition, programmed cell death, and MAPK pathway activation in vitro. Moreover, TF3 exhibited markedly strong antitumour efficacy in vivo in mouse models. In summary, this study demonstrates that TF3 concomitantly plays dual roles in apoptotic and ferroptotic cell death by triggering the ROS and MAPK signalling pathways in both in vitro and in vivo models.

Targeting Ferroptosis Holds Potential for Intervertebral Disc Degeneration Therapy.

Intervertebral disc degeneration (IVDD) is a common pathological condition responsible for lower back pain, which can significantly increase economic and social burdens. Although considerable efforts have been made to identify potential mechanisms of disc degeneration, the treatment of IVDD is not satisfactory. Ferroptosis, a recently reported form of regulated cell death (RCD), is characterized by iron-dependent lipid peroxidation and has been demonstrated to be responsible for a variety of degenerative diseases. Accumulating evidence suggests that ferroptosis is implicated in IVDD by decreasing viability and increasing extracellular matrix degradation of nucleus pulposus cells, annulus fibrosus cells, or endplate chondrocytes. In this review, we summarize the literature regarding ferroptosis of intervertebral disc cells and discuss its molecular pathways and biomarkers for treating IVDD. Importantly, ferroptosis is verified as a promising therapeutic target for IVDD.

Elevated intraspinal pressure drives edema progression after acute compression spinal cord injury in rabbits.

Elevated intraspinal pressure (ISP) following traumatic spinal cord injury (tSCI) can be an important factor for secondary SCI that may result in greater tissue damage and functional deficits. Our present study aimed to investigate the dynamic changes in ISP after different degrees of acute compression SCI in rabbits with closed canals and explore its influence on spinal cord pathophysiology. Closed balloon compression injuries were induced with different inflated volumes (40 µl, 50 µl or no inflation) at the T7/8 level in rabbits. ISP was monitored by a SOPHYSA probe at the epicenter within 7 days post-SCI. Edema progression, spinal cord perfusion and damage severity were evaluated by serial multisequence MRI scans, somatosensory evoked potentials (SEPs) and behavioral scores. Histological and blood spinal cord barrier (BSCB) permeability results were subsequently analyzed. The results showed that the ISP waveforms comprised three peaks, significantly increased after tSCI, peaked at 72 h (21.86 ± 3.13 mmHg) in the moderate group or 48 h (31.71 ± 6.02 mmHg) in the severe group and exhibited "slow elevated and fast decreased" or "fast elevated and slow decreased" dynamic changes in both injured groups. Elevated ISP after injury was correlated with spinal cord perfusion and edema progression, leading to secondary lesion enlargement. The secondary damage aggravation can be visualized by diffusion tensor tractography (DTT). Moreover, the BSCB permeability was significantly increased at the epicenter and rostrocaudal segments at 72 h after SCI; by 14 days, notable permeability was still observed at the caudal segment in the severely injured rabbits. Our results suggest that the ISP of rabbits with closed canals increased after acute compression SCI and exhibited different dynamic change patterns in moderately and severely injured rabbits. Elevated ISP exacerbated spinal cord perfusion, drove edema progression and led to secondary lesion enlargement that was strongly associated with BSCB disruption. For severe tSCI, early intervention targeting elevated ISP may be an indispensable choice to rescue spinal cord function.

Black soldier fly larvae vermicompost alters soil biochemistry and bacterial community composition.

Black soldier fly larvae (Hermetia illucens L. BSFL) bioconversion is a promising biotechnology for food waste recycling, yet little is known about how BSFL vermicompost affects soil health in terms of element availability and related microbial response. In this work, a field soil experiment for luffa (Luffa cylindrica (L.) Roem.) growth was conducted to examine the impacts of BSFL vermicompost (BV, 9750 kg ha-1, equal to total N input rate of chemically treated soil (CK)) on soil biochemistry and bacterial communities. Relative to CK, application of BV significantly increased total soil carbon by 149% and enhanced catalase and urease activity by 59.2% and 16.2%, respectively. BV increased the degree of aromaticity and humification in dissolved organic matter (DOM) in soil by 28.6% and 27.3%, respectively, compared to CK treatment. Among bacterial communities in soil, Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria were the phyla that showed the most substantial alteration in response to BV. Redundancy analysis further revealed that the bacterial community structure was affected by DOM and total phosphorus. Functional analyses indicated that BV enhanced xylanolysis (55.4%) and nitrogen fixation (46.3%), but inhibited nitrification (59.8%). BSFL vermicompost input might effectively prevent the harm of soil borne pathogens (e.g., wilt). Moreover, these function groups strongly correlated with Clostridiales, Actinomycetales, and Nitrospirales. Our study reveals that BSFL vermicompost promoted soil nutrient availability, microbial community succession, and biochemical function optimization, which is conducive to the popularization and application of BSFL vermicompost in the field of soil health. KEY POINTS: • Vermicompost enhanced catalase and urease levels while increased DOM aromaticity. • Vermicompost enriched Bacteroidetes and Firmicutes and improved soil health.

Insights on dissolved organic matter and bacterial community succession during secondary composting in residue after black soldier fly larvae (Hermetia illucens L.) bioconversion for food waste treatment.

Black soldier fly larvae (Hermetia illucens L. BSFL) bioconversion is a promising biotechnology for food waste treatment. However, the separated residues still do not meet criteria for use as land application biofertilizers. In this work, we investigated a full-scale BSFL bioconversion project to explore features of dissolved organic matter (DOM) and its associated responses of bacterial community succession in residue during secondary composting. Data showed that the concentrations of total nitrogen and ammonium nitrogen decreased by 11.8% and 22.6% during the secondary composting, respectively, while the nitrate nitrogen concentration increased 18.7 times. The DOM concentration decreased by 69.1%, in which protein-like, alcohol-phenol, and biodegradable aliphatic substances were metabolized by bacteria during the thermophilic phase together with the accumulation of humus-like substances, resulting in an increase in the relative concentration of aromatic compounds. The structure of the bacterial community varied at different stages of the bioprocess, in which Bacteroidetes, Actinobacteria, Proteobacteria, and Firmicutes were the dominant bacterial phyla. Lysinibacillus, Pusillimonas, and Caldicoprobacter were found to be key contributors in the degradation and formation of DOM. The DOM concentration (33.4%) and temperature (17.7%) were the prime environmental factors that promoted succession of the bacterial community. Through bacterial metabolism, the structural stability of DOM components was improved during the composting process, and the degrees of humification and aromaticity were also increased. This study depicted the dynamic features of DOM and the associated bacterial community succession in residue during secondary composting, which is conducive with the reuse of BSFL residue as biofertilizer for agriculture.

PM2.5-bound elements in Hebei Province, China: Pollution levels, source apportionment and health risks.

Particle-bound elements have attracted increasing attentions due to their health effects and atmospheric catalytic reactivity. However, elements in atmospheric fine particulate matter (PM2.5) have not been well investigated even in some highly polluted area. In this study, 22 elements in PM2.5 were measured by a multi-metal monitor in ten prefecture-level and county-level cities in Hebei province, one of the most polluted provinces in China, during the heating and non-heating seasons. Source apportionment of PM2.5-bound elements were conducted, and health risks of individual elements and different sources were assessed. The results showed that, total elements (TEs) measured contributed to 2%-7% of the PM2.5 mass, with potassium (K), calcium (Ca), iron (Fe), and zinc (Zn) as the most abundant elements, accounting for about 71%- 87% of TEs mass. Concentrations of chromium (Cr), arsenic (As), and cadmium (Cd) were more likely to exceed the World Health Organization (WHO) limits. Source apportionment results indicated that PM2.5-bound elements were primarily from coal combustion, dust, traffic, ferrous metal smelting and oil combustion, and other industrial related sources. Therein, ferrous metal smelting and oil combustion, coal combustion and industry were the predominant source of Cr, As and Cd, respectively. Health risk assessment indicated that the carcinogenic and non-carcinogenic risks of As for children could exceed the precautionary criteria, and coal combustion source had the highest carcinogenic and non-carcinogenic risks. This study suggested that attentions should be paid not only on PM2.5 mass but also PM2.5-bound compounds especially heavy metals and metalloids to reduce health risks in the future.

Correction: Cadmium-Induced Oxidative Stress and Apoptotic Changes in the Testis of Freshwater Crab, Sinopotamon henanense.

[This corrects the article DOI: 10.1371/journal.pone.0027853.].

Ruthenium(II) complex-based fluorescent sensor for peroxynitrite.

We have developed a new ruthenium complex, Ru(bpy)(2)[4-(2,2'-bipyridin-4-yloxy)phenol]Cl(2) (RuL), as a fluorescent sensor to detect peroxynitrite (ONOO(-)). The results showed that the addition of ONOO(-) to the aqueous solution of RuL would result in distinct fluorescence quenching at 600nm. RuL exhibits a good selectivity for ONOO(-) over other reactive oxygen species (ROS) and reactive nitrite species (RNS), and the reaction time is less than 1.5s. The sensing mechanism is proposed as the oxidative O-dealkylation reaction.

Synthesis and properties of fluorescence dyes: tetracyclic pyrazolo[3,4-b]pyridine-based coumarin chromophores with intramolecular charge transfer character.

Two series of new tetracyclic pyrazolo[3,4-b]pyridine-based coumarin chromophores were synthesized through a facile reaction between 3-aldehyde-7-diethylaminocoumarin (5) or 3-acetyl-7-diethylaminocoumarin (6) and 5-aminopyrazole derivatives (7) in a one-pot procedure. Different condensed products were obtained from compounds 5 and 6, and the potential reaction mechanism was studied using the reaction of 5 with 5-amino-1-phenylpyrazole (7a). The molecular structures were characterized by NMR and HRMS and confirmed by X-ray diffraction. The photophysical, electrochemical, and thermal properties of these compounds were investigated by absorption spectroscopy, fluorescence spectroscopy, single photon counting technique, cyclic voltammetry, thermogravimetric analysis, etc. Results show that the compounds exhibited high fluorescence quantum yields and good electrochemical, thermal, and photochemical stabilities. In addition, the application of these highly fluorescent compounds in living cell imaging was also explored by laser scanning confocal microscopy.

Cadmium-induced oxidative stress and apoptotic changes in the testis of freshwater crab, Sinopotamon henanense.

Cadmium (Cd), one of the most toxic environmental and industrial pollutants, is known to exert gonadotoxic and spermiotoxic effects. In the present study, we examined the toxic effect of Cd on the testis of freshwater crab, Sinopotamon henanense. Crabs were exposed to different Cd concentrations (from 0 to 116.00 mg·L(-1)) for 7 d. Oxidative stress and apoptotic changes in the testes were detected. The activities of SOD, GPx and CAT initially increased and subsequently decreased with increasing Cd concentrations, which was accompanied with the increase in malondialdehyde (MDA) and H(2)O(2) content in a concentration-dependent manner. Typical morphological characteristic and physiological changes of apoptosis were observed using a variety of methods (HE staining, AO/EB double fluorescent staining, Transmission Electron Microscope observation and DNA fragmentation analysis), and the activities of caspase-3 and caspase-9 were increased in a concentration-dependent manner after Cd exposure. These results led to the conclusion that Cd could induced oxidative damage as well as apoptosis in the testis, and the apoptotic processes may be mediated via mitochondria-dependent apoptosis pathway by regulating the activities of caspase-3 and caspase-9.

Reversible fluorescent probe for highly selective and sensitive detection of mercapto biomolecules.

A coumarin-derived complex, Hg(2)L(2), was reported as a highly sensitive and selective probe for the detection of mercapto biomolecules in aqueous solution. The addition of Cys to a 99% aqueous solution of Hg(2)L(2) resulted in rapid and remarkable fluorescence OFF-ON (emission at 525 nm) due to the ligand-exchange reaction of Cys with L coordinated to Hg(2+). The increased fluorescence can be completely quenched by Hg(2+) and recovered again by the subsequent addition of Cys. Such a fluorescence OFF-ON circle can be repeated at least 10 times by the alterative addition of Cys and Hg(2+) to the solution of Hg(2)L(2), indicating that it can be used as a convertible and reversible probe for the detection of Cys. The interconversion of Hg(2)L(2) and L via the decomplexation/complexation by the modulation of Cys/Hg(2+) was definitely verified from their crystal structures. Other competitive amino acids without a thiol group cannot induce any fluorescence changes, implying that Hg(2)L(2) can selectively determine mercapto biomolecules. Using confocal fluorescence imaging, L/Hg(2)L(2) as a pair of reversible probes can be further applied to track and monitor the self-detoxification process of Hg(2+) ions in SYS5 cells.

Enzyme sensing based on a controllable oxidation reaction.

We developed a new method for glutathione reductase (GR) enzyme sensing via a metal-controlled spontaneous oxidation reaction. A new complex HgL, composed of 3-benzothiazoliny-7-N,N-diethylaminocoumarin (L) and HgCl2, was used as an example for illustration. It was found that ligand L was released from complex HgL by a ligand exchange process in the presence of GSH, which was enzymatic reduced from GSSG in the presence of NADH. Subsequently, free ligand L was spontaneously oxidated to a laser dye (coumarin-6) with both fluorescence enhancement and color change. A good linear relationship between fluorescent intensity and GSH concentrations (in the range of 10-40 µM) elicits the potential use of HgL in GSH detection. Based on this result, complex HgL was applied to identify GR from other proteins/enzymes with good selectivity and sensitivity. HgL can also determine GR in the concentration range from 0.5 to 10 U/mL with a linear relationship. Furthermore, this novel sensing method was also expected to be a useful example for the design of other biosensing systems.

Colorimetric test kit for Cu2+ detection.

A coumarin-based colorimetric chemosensor 1 was designed and synthesized. It exhibits good sensitivity and selectivity for the copper cation over other cations such as Zn(2+), Cd(2+), Pb(2+), Co(2+), Fe(2+), Ni(2+), Ag(+), and alkali and alkaline earth metal cations both in aqueous solution and on paper-made test kits. The change in color is very easily observed by the naked eye in the presence of Cu(2+) cation, whereas other metal cations do not induce such a change. The quantitative detection of Cu(2+) was preliminarily examined.