Tandem Mass Spectrometry Analysis Research Articles

Lemongrass (Cymbopogon citratus) oil nanoparticle synthesis, characteristic, and evaluation of antibacterial and antifungal effects and the influence on hardness of acrylic resin

Context: Acrylic resin is used in dentistry as a removable denture base. It can cause various pathologies when not properly cleaned. One of the pathologies is denture stomatitis caused by Candida albicans and Streptococcus mutans accumulation on the acrylic resin surface. Therefore, microbial agents such as denture cleansers are needed. Aims: To evaluate the characteristics of lemongrass (Cymbopogon citratus) nanoparticles as a better antibacterial and antifungal herbal ingredient and their relationship with acrylic hardness. Methods: C. citratus oil nanoparticles (LON) were synthesized and analyzed by transmission electron microscopy (TEM). Electrospray ionization tandem mass spectrometry (ESI-MS) analysis was used to analyze the characteristics of LON bioactive components. Minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) against C. albicans and S. mutans and mechanical hardness test of acrylic were performed. Results: The LON concentration of MIC and MBC against C. albicans and S. mutans was 25 and 100%, respectively. One-way ANOVA showed no significant difference between groups of LON with different concentrations (p=0.687). A paired t-test showed significant differences in acrylic resin hardness before and after treatment of LON with 100% (p=0.022) and 50% (p=0.021) concentration. There was no significant difference in hardness before and after treatment of other concentrations of LON and chlorhexidine as positive control. Conclusions: LON treatment on acrylic resin decreased the growth of C. albicans and S. mutans without altering the mechanical properties (hardness).

Simultaneous electrochemical removal of three microcystin congeners and sulfamethoxazole in natural water

Microcystins (MCs), frequently detected in freshwater ecosystems, have raised significant human health and ecological concerns. New approaches are being developed to control and remove MCs. In this study, we examined factors influencing the efficacy of electrochemical oxidation as a means of control. Anode material (Pt/Ti, Ta2O5-IrO2/Ti, SnO2-SbO2/Ti, boron-doped diamond (BDD)/Si), anode surface area ratios and solution volumes, initial pollutant concentrations, and the co-existing antibiotic sulfamethoxazole (SMX) were investigated. MCs and SMX were dissolved in filtered Taihu Lake water to simulate the natural aquatic environment. The results showed that non-active anodes, lower initial concentration of MC, larger surface area ratio of anode to cathode, and smaller ratio of reaction solution volume to anode surface area could promote the degradation target pollutants. Under optimal conditions in this study, the degradation rates of MC-LR, MC-YR, MC-RR, and SMX each reached more than 90% within 6 hours, and the removal efficiency of MC-YR was the highest among three congeners. The effect of SMX on the degradation of MC congeners depended mainly on their concentration differences, such that when the initial concentration of SMX was one to two orders of magnitude lower than microcystin, the presence of SMX would promote the degradation of MCs. In contrast, when the initial concentration of SMX was higher than that of microcystin by approximately an order of magnitude, sulfamethoxazole would inhibit the degradation of MCs by between 4.6% and 24.5%. Ultra-high-performance liquid chromatography tandem mass spectrometry analysis revealed that the three MC congeners were electrochemically degraded through aromatic ring oxidation, alkene oxidation, and bond cleavage on the ADDA (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid) side chain. Notably, the removal of MCs was accompanied by a decline in the hardness of the reaction water. This study provided insights into electrochemical degradation of microcystins and antibiotics in natural water, offering suggestions for its practical application.

S-nitrosylation of aconitase-2 facilitates coronary microembolization-induced cardiac injury by suppressing iron-sulfur cluster assembly

Abstract Background Coronary microembolization (CME) is a common reason for periprocedural myocardial infarction after coronary interventions. S-nitrosylation (SNO), a prototypic redox-based posttranslational modification, is involved in the pathogenesis of several cardiovascular diseases. Purpose To explore the role of SNO of aconitase-2 (ACO2) in CME-induced cardiac injury, as well as the mechanism by which SNO-ACO2 modulates myocardial injury in response to CME. Methods CME models were established in C57BL/6J mice by injecting 300,000 polyethylene microspheres (diameter 9 µm) into the left ventricle chamber with the occlusion of the ascending aorta. Cardiac function was evaluated by echocardiography. Histological and serum analysis was performed to evaluate myocardial injury. Myocardial samples were scanned for S-nitrosylated proteins using biotin-switch procedures and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. SNO sites of ACO2 were further identified through LC-MS/MS. De-nitrosylation of ACO2 was achieved by the mutation of SNO site or overexpression of the de-nitrosylation enzyme thioredoxin 1 (TRX1). Interacting effectors of SNO- ACO2 were screened through LC-MS/MS and confirmed by coimmunoprecipitation. Neonatal rat ventricular myocytes (NRVMs) were used for in vitro study. The cellular ACO2 activity was analysed using an ACO2 enzyme activity assay kit. Mitochondrial morphology and function were assessed by fluorescent staining and mitochondrial stress testing. Results Cardiac injury was exacerbated by CME as demonstrated by impaired cardiac contractile function, morphological changes, and increased fibrosis, microinfarct size and serum troponin I level. We identified 789 S-nitrosylated proteins in CME mouse hearts, and ACO2 is one of the highly S-nitrosylated proteins. The increased level of SNO-ACO2 was verified by immunoblot assay in CME mouse hearts and hypoxia-stimulated NRVMs. SNO site of ACO2 at cysteine 126 was identified by LC-MS/MS and confirmed in NRVMs. De-nitrosylation of ACO2 by the mutation of cysteine 126 or overexpression of TRX1 both greatly improved the activity of ACO2 by 39.7% and 31.4% respectively, furthermore restored the mitochondrial function, and attenuated CME-induced cardiac injury. Mechanistically, SNO-ACO2 at cysteine 126 suppressed the interaction between ACO2 and NFU1, an iron-sulfur (Fe-S) cluster scaffold protein. The dissociation from NFU1 deprived ACO2 of the Fe-S cluster and the enzyme activity, thereby promoting the disruption of the mitochondrial tricarboxylic acid cycle and cardiac injury. Conclusions Our data defined a previously unrecognized role of SNO-ACO2 in CME-induced cardiac injury. We demonstrated that SNO-ACO2 at cysteine 126 disrupted ACO2/NFU1 interaction to exacerbate myocardial injury after CME. Our results suggested that de-nitrosylation of ACO2 may provide a new therapeutic target for the treatment of CME.

Macrophage MST4 exacerbates cardiac microvascular ischaemia/reperfusion injury by phosphorylating ACLY

Abstract Background Cardiac microvascular dysfunction is a contributing factor in the development of cardiac ischaemia/reperfusion (I/R) injury. The STE20-type kinases play important role in cardiovascular diseases. However, the impact of mammalian Ste20-like kinase 4 (MST4) on cardiac microvascular I/R injury remains unknown. Purpose To decipher the role of MST4 in cardiac microvascular I/R injury and the underlying mechanisms. Methods Single-cell RNA sequencing of I/R mouse heart was performed to explore the expression changes of MST4 in different cell types. MST4 flox (MST4 f/Y) mice was crossed with myeloid-specific LysM-Cre mice to generate myeloid-specific MST4 knockout mice (MST4 CKO). Bone marrow transplantation was used to testify the role of myeloid-derived MST4 in cardiac microvascular I/R injury. Bone-marrow-derived macrophage (BMDM) and cardiac microvascular endothelial cell (CMEC) were used to dissect molecular mechanisms. Adeno-associated virus and adenovirus were constructed to overexpress ATP Citrate Lyase (ACLY) with mutation of serine 455 (S455A) in vivo and in vitro, respectively. Phosphoproteomics, coimmunoprecipitation coupled with liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis and metabolomics were performed to decipher the downstream of MST4. Results MST4 is dominantly upregulated in CCR2+ monocyte-derived macrophages. Myeloid-specific deficiency of MST4 improves cardiac performance in I/R mouse model. Wild type (WT) mice transplanted with BMDM from MST4 CKO mice also exhibited improved cardiac function after I/R, however, MST4 CKO mice transplanted with BMDM from WT mice showed deteriorated cardiac performance. Metabolomics showed that primary bile acid biosynthesis pathway was upregulated in BMDM overexpressing MST4. The level of 7α-hydroxyl3-oxo-4-cholestenoic acid (7-HOCA), an intermediate of bile acid, was dramatically increased in I/R mice or BMDM overexpressing MST4, which can be reversed by knocking down MST4. Phosphoproteomics and LC-MS/MS analysis discovered that ACLY was one of the most important substrates of MST4 in the process of I/R injury. ACLY was directly phosphorylated by MST4 at serine 455, which increased ACLY enzyme activity. Mechanistically, ACLY promoted cholesterol catabolism through the bile acid synthesis pathway, leading to the accumulation of 7-HOCA in the macrophage-endothelial cell microenvironment. High level of 7-HOCA was able to promote endothelial cell mtDNA release, leading to endothelial cell pyroptosis through inflammasome pathway. However, mutation of serine 455 of ACLY blocked the role of MST4. Conclusions Our data defined a previously unrecognized role of MST4/ACLY pathway in cardiac microvascular I/R injury. MST4 led to the accumulation of 7-HOCA by phosphorylating ACLY, furthermore, 7-HOCA promoted endothelial cell injury. Targeting MST4/ACLY pathway ameliorated microvascular I/R injury, which may provide a novel therapeutic strategy for I/R injury.

Targeting the VEGFR2 signaling pathway for angiogenesis and fibrosis regulation in neovascular age-related macular degeneration

Neovascular age-related macular degeneration (nAMD) is characterized by abnormal blood vessel growth from the choroid, leading to complications and eventual blindness. Despite anti-VEGF therapy, subretinal fibrosis remains a major concern, as VEGF/VEGF receptor-2 (VEGFR2) signaling can contribute to both angiogenesis and fibrosis. For the identification of the aqueous humor proteome, we performed liquid chromatography with tandem mass spectrometry analysis. To investigate the potential therapeutic effects of targeting the VEGF signaling pathway using apatinib, a highly selective VEGFR2 tyrosine kinase inhibitor, this study employed in vitro (THP-1 conditioned media-treated ARPE-19 cells) and in vivo (laser-induced choroidal neovascularization mouse) models of nAMD. This study revealed elevated VEGFR2 protein levels in the aqueous humor of nAMD patients, suggesting a potential target to mitigate neovascularization and fibrosis in nAMD. Apatinib effectively reduced VEGFA and αSMA levels in both in vitro and in vivo models. Moreover, apatinib showed improvement in laser-induced subretinal hyper-reflective lesions. The action mechanism was linked to the inhibition of VEGFR2 activation, leading to the suppression of both angiogenesis and fibrosis through the downregulation of STAT3 phosphorylation. Therefore, the VEGFR2 signaling pathway appears to play a central role in the development of nAMD by regulating both angiogenesis and fibrosis.

Novel Serum Markers that Distinguish Behcet’s Disease from Idiopathic Recurrent Aphthous Stomatitis

ABSTRACT Background Behcet’s disease (BD) is a rare and recurrent autoinflammatory disorder characterized by systemic vasculitis, frequently manifested as recurrent aphthous stomatitis (RAS). We aim to identify specific serum proteins to discriminate between BD and idiopathicRAS. Method Peripheral blood was collected from 12 BD patients, 12 idiopathic RAS patients, and 21 healthy volunteers. The serum samples underwent Tandem Mass Tag-based mass spectrometry analysis. Differentially expressed proteins (DEPs) were identified for KEGG pathway enrichment, Gene Ontology (GO), and protein-protein interaction (PPI) analyses. ELISA was utilized to verify two BD-specific DEPs in another cohort consisting of 18 BD patients, 18 idiopathic RAS patients, and 18 controls. Results Compared with RAS serum, BD serum showed 242 DEPs. 49 proteins were differentially expressed in BD but not RAS serum compared to healthy controls. KEGG pathway and GO analyses revealed that DEPs in BD and RAS have similar biological functions and cellular distributions, featuring a significant association with pathways regulating blood coagulation and immune response. When comparing DEPs between BD and RAS, several keratins emerged as markers that distinguish RAS from BD. We also identified multiple DEPs in BD but not RAS patients. PPI analysis uncovered that lipoprotein metabolism regulators serve as hub proteins, indicating their potentially essential roles in BD pathology. In addition, ELISA results confirmed the elevated LRG1 and SOD3 levels in BD, but not RAS patients, compared to healthy donors. Conclusion Our data uncovered novel serum proteins that distinguish BD from RAS, which may potentially be useful in BD diagnosis and treatment.

B-150 Chromatographic Separation and Quantitation of Human Insulin and Lispro Analog in Serum Using Liquid Chromatography High-Resolution Mass Spectrometry

Abstract Background Factitious hypoglycemia, a deliberate attempt to induce low blood glucose levels occurring secondary to the surreptitious administration of exogenous insulin, can be difficult to diagnose due to a variety of factors including the increased use of synthetic insulin analogs in clinical practice. Traditional insulin immunoassays demonstrate variable cross-reactivity with commonly prescribed insulin analogues and their metabolites, making it difficult to measure specific analogs. The development of liquid chromatography high resolution accurate mass (LC-HRAM)-mass spectrometry (MS) assays has largely circumvented the limitations of traditional immunoassays for detection and quantitation of insulin analogs. Analytical challenges remain, however, specifically in differentiating lispro, an isomeric human insulin analog formed by the transposition of proline and lysine near the C-terminal end of the B chain, from human insulin in patient samples due to the equivalent mass-to-charge ratios of their precursor ions. Methods Immunoaffinity purification of insulin from calibrators and quality control material prepared by spiking insulin-depleted serum with pharmaceutical insulin preparations, as well as patient samples, was performed using tips coupled with anti-insulin monoclonal antibody. An automated liquid handler was programmed to perform aspiration and dispense cycles. Eluate from the purification protocol was submitted for analysis using a TLX-2 multiplex LC system paired with a Thermo Scientific Q Exactive Plus mass spectrometer. Various HPLC column chemistries were evaluated, a column passivation procedure was established, and column heater temperature was optimized to identify a combination of parameters yielding the best chromatographic separation of human insulin and lispro. Additionally, parallel reaction monitoring (PRM) targeted tandem mass spectrometry (MS/MS) analysis was utilized in combination with the full scan data to increase specificity through the monitoring of fragment ions produced from human insulin and each insulin analog. Results We found that a 1000 Å Diphenyl, 2.7 µm, 2.1 x 100 mm analytical column in a column heater maintained at a temperature of 80°C allowed for human insulin and lispro to be differentiated as two chromatographically separated extracted ion chromatographic peaks (XICs) and quantified down to 2.5 mcIU/mL with near-baseline separation. Before use, the column required passivation with a high protein buffer (0.05% bovine serum albumin in 20% acetonitrile [ACN] + 0.2% acetic acid) followed by increasingly organic buffers (20% ACN + 0.2% acetic acid and 50% ACN + 0.2% acetic acid). MS/MS was used to confirm the presence of analogs detected. Conclusions The LC-HRAM-MS method described here, coupled with optimized column chemistry, column passivation, and proper column temperature control allows for chromatographic separation of human insulin and lispro. Incorporating a PRM scan into the mass spectrometry full scan method provides confirmation of the insulin detected in the XICs. The results obtained from preliminary studies suggest this method is appropriate for use to facilitate the clinical investigation of suspected factitious hypoglycemia with the ability to differentiate isomeric compounds, such as human insulin and lispro.

CT1812 biomarker signature from a meta-analysis of CSF proteomic findings from two Phase 2 clinical trials in Alzheimer's disease.

CT1812 is in clinical development for the treatment of Alzheimer's disease (AD). Cerebrospinal fluid (CSF) exploratory proteomics was employed to identify pharmacodynamic biomarkers of CT1812 in mild to moderate AD from two independent clinical trials. Unbiased analysis of tandem-mass tag mass spectrometry (TMT-MS) quantitative proteomics, pathway analysis and correlation analyses with volumetric magnetic resonance imaging (vMRI) were performed for the SPARC cohort (NCT03493282). Comparative analyses and a meta-analysis with the interim SHINE cohort (NCT03507790; SHINE-A) followed by network analysis (weighted gene co-expression network analysis [WGCNA]) were used to understand the biological impact of CT1812. CT1812 pharmacodynamic biomarkers and biological pathways were identified that replicate across two clinical cohorts. The meta-analysis revealed novel candidate biomarkers linked to S2R biology and AD, and network analysis revealed treatment-associated networks driven by S2R. DISCUSSION: Early clinical validation of CT1812 candidate biomarkers replicating in independent cohorts strengthens the understanding of the biological impact of CT1812 in patients with AD, and supports CT1812's synaptoprotective mechanism of action and its continued clinical development. This exploratory proteomics study identified candidate biomarkers of CT1812 in SPARC (NCT03493282) Comparative analyses identified biomarkers replicating across trials/cohorts Two independent Ph2 trial cohorts (SPARC and interim SHINE [NCT03507790; SHINE-A]) were used in a meta-analysis Amyloid beta (Aβ)& synaptic biology impacted by CT1812 and volumetric magnetic resonance imaging (vMRI) treatment-related correlates emerge Network analyses revealed sigma-2 receptor (S2R)-interacting proteins that may be "drivers" of changes.

Liquid Chromatography Tandem Mass Spectrometry Analysis of Lipidomic Differences between Renal Cortex and Medulla in db/db Mice

Liquid Chromatography Tandem Mass Spectrometry Analysis of Lipidomic Differences between Renal Cortex and Medulla in db/db Mice

Proteomic analysis revealing mechanisms of κ-carrageenan modulating the gelling properties of heat-induced microbial transglutaminase cross-linked myofibrillar protein gels

The effects of κ-carrageenan (KC, 0.2 %, w/w) on the gelling properties and microstructures of heat-treated myofibrillar protein (MP) gels mediated by various levels (0, 0.1, 0.3, and 0.5 %, w/w) of microbial transglutaminase (TG) were investigated. The results revealed that regardless of TG level, the existence of KC significantly promoted the forming of disulphide bonds but limited the generation of large-sized protein polymers in MP gel treated with TG alone, as well as subsequently enhanced the water retention ability and gel strength of mixed MP gel, especially in the sample adding 0.3% TG and 0.2% KC, which was clearly verified by the forming of more denser and continuous gel network. Moreover, TG-induced changes in protein structure led to the alteration in the protein profiles of different MP gels, and an obvious down-regulation trend of myosin (Q9TV63, Q9TV62, P79293, and Q9TV61) was identified in the TG-0.3 group. Furthermore, liquid chromatography–tandem mass spectrometry analysis demonstrated that TG mainly induces protein crosslinking in the light meromyosin domain of the myosin rod. Meanwhile, in the presence of KC, the crosslinking-site percentage of the TG-0.3 group decreased from 37.7% to 36.2%, indicating that KC potentially reduces TG-induced crosslinking in MP gels. Thus, our findings illustrate the critical mechanisms underlying the modulation of KC on the gelling properties of TG-mediated MP gels at the molecular level, which providing potential guideline for the combined applications of TG and KC in the meat industry.

The lung proteome in HIV-associated obstructive lung disease (OLD)

RationaleObstructive lung disease is increasingly common among persons living with HIV (PLWH). There are currently no validated biomarkers that identify individuals at risk of developing obstructive lung disease (OLD) and specific mechanisms contributing to HIV-associated OLD remain elusive, independent of smoking.ObjectivesWe sought to identify biomarkers and biological pathways associated with OLD using a broad proteomic approach.MethodsWe performed tandem mass tagging and mass spectrometry (MS) analysis on bronchoalveolar lavage fluid (BALF) samples from persons living with HIV with OLD (n=26) and without OLD (n=26). We combined untargeted MS with a targeted SomaScan aptamer-based approach. We used Pearson correlation tests to identify associations between each protein and lung function (FEV1pp). We adjusted for multiple comparisons using a false discovery rate (FDR) adjustment. Significant proteins were entered into a pathway overrepresentation analysis. Protein-driven endotypes were constructed using K-means clustering.Measurements and main resultsWe identified over 3800 proteins by MS and identified 254 proteins that correlated with FEV1pp when we combined the MS and SomaScan proteomes when adjusting for smoking status. Pathway analysis revealed cell adhesion molecules as significant.ConclusionsProtein expression differs in the lung of PWH and decreased lung function (FEV1pp). Pathway analysis reveals cell adhesion molecules having potentially important roles in this process.

Comparative Study on Growth and Metabolomic Profiles of Six Lactobacilli Strains by Sodium Selenite

Selenium (Se) has garnered increasing attention in the field of nutrition, as it is essential for both humans and animals. Certain microorganisms can enrich inorganic selenium and convert it into organic selenium. The growth and metabolomic profiles of six lactobacilli strains exposed to 50 μg/mL of sodium selenite were performed using gas chromatography tandem time-off light mass spectrometry (GC-TOF-MS) analysis. The addition of selenium significantly increased both the population and weight of the Lacticaseibacillus rhamnosus PS5, Lbs. rhamnosus RT-B, Limosilactobacillus reuteri 3630, and Lmb. reuteri 1663 strains, while those of the other two strains decreased. A total of 271 metabolites were determined, with their concentrations ranked from highest to lowest as follows: organic acids and derivatives, oxygen compounds, lipids and lipid-like molecules, and benzenoids. In certain groups, the concentrations of serine, aspartic acid, trehalose, palmitic acid, methylthreonine, and melibiose increased significantly, whereas glucuronic acid, ribose, ornithine, and methionine were downregulated. The metabolic pathways were significantly associated with ABC transporters, glycine, serine, threonine metabolism, and aminobenzoate degradation and other pathways. Based on these findings, we concluded that the transport, absorption, assimilation, and stress response to selenium by lactobacilli in metabolomic changed. Furthermore, the metabolomic alterations among different types of lactobacilli varied primarily due to their distinct properties.

Prediction of Flavor Potential of Ocimum basilicum L. Side-Stream Phytoconstituents, Using Liquid Chromatography–Tandem Mass Spectrometry Analysis and In Silico Techniques

Although post-distillation side-streams of basil (Ocimum basilicum L.) pose significant economic and environmental challenges, they also bring forth new opportunities in the flavor industry. Thus, the objective of the current study was to assess the phenolic profile of basil side-stream extracts to identify key compounds and to evaluate their taste properties, using liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis, flavor prediction tools and molecular docking. In particular, 52 phytoconstituents, mainly phenolic acids, salvianolic acids, flavonoids and fatty acids derivatives, were elucidated in the side-streams of two different basil varieties (Minimum and Genovese) harvested and distilled in early and late autumn, highlighting the effect of pre-harvest factors on basil’s phenolic fingerprint. Furthermore, the results of tests undertaken using taste prediction tools showed that most of the identified compounds were very likely to taste bitter, while six of them (caffeoylferuloyltartaric acid, isoquercetin, lithospermic acid A, sagerinic acid, salvianolic acids C and F) presented a high bitterant capacity (70–90%). Moreover, according to molecular docking studies, these compounds exhibited a stronger binding affinity to the hTAS2R46 bitter receptor compared to its known agonist, strychnine. This outcome and consequently their bitterness were mainly attributed to interactions with Glu265, Thr180 and/or Trp88 through the formation of direct hydrogen bonds. Therefore, the present results provide insights into the taste profiles of basil side-streams, leading to more sustainable and innovative uses of aromatic herbs residues.

Bisphenols in daily clothes from conventional and recycled material: evaluation of dermal exposure to potentially toxic substances

Given the increasing concern about chemical exposure from textiles, our study examines the risks of dermal exposure to bisphenol A (BPA), bisphenol S (BPS), bisphenol B (BPB) and bisphenol F (BPF) from conventional and recycled textiles for adults, aiming to obtain new data, assess exposure, and evaluate the impact of washing on bisphenol levels. A total of 57 textile samples (33 from recycled and 24 from conventional material) were subjected to ultrasound-assisted extraction (UAE) followed by ultra-high performance liquid chromatography with tandem mass spectrometry analysis (UHPLC-MS/MS). The BPA and BPS concentrations varied widely (BPA: < 0.050 to 625 ng/g, BPS: 0.277–2,474 ng/g). The median BPA content in recycled textiles (13.5 ng/g) was almost twice as high as that of 7.66 ng/g in conventional textiles. BPS showed a median of 1.85 ng/g in recycled textiles and 3.42 ng/g in conventional textiles, indicating a shift from BPA to BPS in manufacturing practices. Simulated laundry experiments showed an overall reduction in bisphenols concentrations after washing. The study also assessed potential health implications via dermal exposure to dry and sweat-wet textiles compared to a tolerable daily intake (TDI) of 0.2 ng/kg bw/day for BPA set by the European Food Safety Authority (EFSA). Exposure from dry textiles remained below this threshold, while exposure from wet textiles often exceeded it, indicating an increased risk under conditions that simulate sweating or humidity. By finding the widespread presence of bisphenols in textiles, our study emphasises the importance of being aware of the potential risks associated with recycling materials as well as the benefits.

Detection of 6-PPD and 6-PPDQ in airborne particulates and assessment of their toxicity in lung cells

The extensive use of synthetic antioxidants, notably N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6-PPD), in rubber-related products, particularly in tire manufacturing, has induced concerns regarding their environmental impact and potential health hazards. Despite the identification of 6-PPD and its derivative, 6-PPD quinone (6-PPDQ), in various water samples and their lethal effects on certain aquatic species (e.g., coho salmon, rainbow trout and brook trout), the levels of airborne 6-PPD/6-PPDQ and their respiratory toxicity remain relatively unexplored. In this study, we aimed to evaluate the respiratory toxicity potential of 6-PPD and its derivatives, with a specific focus on detecting these compounds in airborne particulates and assessing their toxic effects on lung cells. Characterization of four airborne fine particulate (FP) samples revealed spherical morphologies with diameters ranging from 17.7 to 225.7 nm, displaying slight agglomeration and negative surface charge. methanol/acetonitrile extraction followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis confirmed the presence of both 6-PPD and 6-PPDQ on the surfaces of FPs, with significant variations (0.26–1.05 μg g−1) in loading capacity observed among the samples. Subsequent exposure of lung cells (THP-1, BEAS-2B, and A549) to 6-PPD and 6-PPDQ revealed dose-dependent declines in mitochondrial metabolic activity induced by 6-PPD, along with severe membrane damage, ATP depletion, and pro-inflammatory cytokine release. Conversely, 6-PPDQ exhibited negligible toxicity in all tested parameters. These findings underscore the potential health risks associated with airborne 6-PPD exposure and emphasize the importance of further research into the respiratory toxicity of 6-PPD derivatives.

Proteomic profiling of Arabidopsis nuclei reveals distinct protein accumulation kinetics upon heat stress

Heat stress (HS) impacts the nuclear proteome and, subsequently, protein activities in different nuclear compartments. In Arabidopsis thaliana, a short exposure to 37 °C leads to loss of the standard tripartite architecture of the nucleolus, the most prominent nuclear substructure, and, consequently, affects the assembly of ribosomes. Here, we report a quantitative label-free LC‒MS/MS (Liquid Chromatography coupled to tandem Mass Spectrometry) analysis to determine the nuclear proteome of Arabidopsis at 22 °C, HS (37 °C for 4 and 24 h), and a recovery phase. This analysis identified ten distinct groups of proteins based on relative abundance changes in the nucleus before, during and after HS: Early, Late, Transient, Early Persistent, Late Persistent, Recovery, Early-Like, Late-Like, Transient-Like and Continuous Groups (EG, LG, TG, EPG, LPG, RG, ELG, LLG, TLG and CG, respectively). Interestingly, the RNA polymerase I subunit NRPA3 and other main nucleolar proteins, including NUCLEOLIN 1 and FIBRILLARIN 1 and 2, were detected in RG and CG, suggesting that plants require increased nucleolar activity and likely ribosome assembly to restore protein synthesis after HS.

Interrogating the Presence of RNA Phosphorothioate in Nature by a Highly Selective and Sensitive Fluorescence Method.

In nature, DNA phosphorothioate (PT) is found in the genomic materials of some prokaryotes. In contrast, whether there is natural RNA PT is still a question under debate. A groundbreaking study reported the discovery of RNA PT in cellular RNA samples from both prokaryotes and eukaryotes at contents of >100 PT per million nucleotides (PPM-nt) according to liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. However, this finding was challenged by a later work showing that other RNA modifications, such as 2'-O-methylation, could give almost the same LC-MS/MS signal patterns as RNA PT. As the LC-MS/MS technique led to contradicting conclusions, another independent method is thus needed to interrogate the presence of RNA PT in nature. In this work, we have developed a highly selective and sensitive fluorescence method for RNA PT quantification based on a new RNA PT-specific conversion reaction. It can detect as low as 2.8 PPM-nt in RNA without interference from RNA thiobases or protein cysteines. We measured the total RNA samples from some bacteria and human cells using this method. None of these samples gave any RNA PT signal above the detection limit (2.8 PPM-nt), suggesting that the widespread presence of natural RNA PT at the 100 PPM-nt level or above is highly unlikely. Nevertheless, due to the limited number of cell species tested in this work, the possible existence of natural RNA PT cannot be excluded. The fluorescence method reported here is simple and low-cost; therefore, it should be an ideal assay for broadly screening various types of cells to search for the clue of RNA PT in nature.

Integrated multi-omics analyses reveal microbial community resilience to fluctuating low oxygen in the East China sea

Climate change and eutrophication are accelerating ocean deoxygenation, leading to a global decline in oxygen levels. The East China Sea, frequently experiencing deoxygenation events, harbors diverse microbial communities. However, the response of these communities to the changing deoxygenation dynamics remains poorly understood. Here, we explored the composition and function of microbial communities inhabiting seawaters of the Changjiang Estuary and offshore areas. Our findings suggested that neutral processes significantly influenced the assembly of these communities. The overall bacterial composition demonstrated remarkable high stability across the oxygen gradient. Salinity exhibited a significantly stronger correlation with bacterial community structure than dissolved oxygen. Both metagenomics and metaproteomics revealed that all of the samples exhibited similar functional community structures. Heterotrophic metabolism dominated these sites, as evidenced by a diverse array of transporters and metabolic enzymes for organic matter uptake and utilization, which constituted a significant portion of the expressed proteins. O2 was the primary electron acceptor in bacteria even under hypoxic conditions, evidenced by expression of low- and high-affinity cytochrome oxidases. Proteins associated with anaerobic processes, such as dissimilatory sulfite reductases, were virtually undetectable. Untargeted liquid chromatography with tandem mass spectrometry analysis of seawater samples revealed a diverse range of dissolved organic matter (DOM) components in amino acids, lipids, organic acids, peptides, and carbohydrates, potentially fueling dominant taxa growth. Despite fluctuations in the abundance of specific genera, the remarkable similarity in community structure, function, and DOM suggests that this ecosystem possesses robust adaptive mechanisms that buffer against abrupt changes, even below the well-defined hypoxic threshold in marine ecosystem.

Efficacy of BRAF/MEK-inhibitor therapy for epithelioid glioblastoma with a novel BRAFV600 mutation

Epithelioid glioblastoma (eGB), a very aggressive and rare brain tumour, is associated with a dismal median overall survival. Effective therapies for patients with eGB, particularly with leptomeningeal dissemination, are still lacking. Here, we describe a case of a 25-year-old male diagnosed with an intramedullary cervical tumour with subsequent leptomeningeal disease. Histopathology identified a highly necrotising, epithelioid-type tumour with high cell density, most compatible with the diagnosis of an eGB. DNA analysis revealed an unprecedented B-Raf protooncogene, serine/threonine kinase (BRAF) gene variant in exon 15 (ENST00000288602.6, c.1799_1810delinsATG, p.(V600_W604delinsDG)), triggering activation of the mitogen-activated protein kinase (MAPK) pathway. Consequently, we initiated MAPK inhibitor (MAPKi) therapy, utilizing a combination of BRAF and mitogen-activated protein kinase kinase (MEK) inhibitors. Liquid chromatography–tandem mass spectrometry analysis confirmed the drugs’ presence in the patient’s cerebrospinal fluid, indicating their capacity to cross the blood-brain barrier. Remarkably, the patient responded very well to therapy and transitioned from a near-comatose state to significantly improved health, sustained for over three months. This study highlights that MAPKi, particularly targeted towards novel BRAFV600 mutations, might offer promising advancements in eGB treatment strategies.

Identification of the Biotransformation Pathways of a Potential Oral Male Contraceptive, 11β-Methyl-19-Nortestosterone (11β-MNT) and Its Prodrugs: An In Vitro Study Highlights the Contribution of Polymorphic Intestinal UGT2B17.

11β-Methyl-19-nortestosterone dodecylcarbonate (11β-MNTDC) is a prodrug of 11β-MNT and is being considered as a promising male oral contraceptive candidate in clinical development. However, the oral administration of 11β-MNTDC exhibits an ~200-fold lower serum concentration of 11β-MNT compared to 11β-MNTDC, resulting in the poor bioavailability of 11β-MNT. To elucidate the role of the first-pass metabolism of 11β-MNT in its poor bioavailability, we determined the biotransformation products of 11β-MNT and its prodrugs in human in vitro models. 11β-MNT and its two prodrugs 11β-MNTDC and 11β-MNT undecanoate (11β-MNTU) were incubated in cryopreserved human hepatocytes (HHs) and subjected to liquid chromatography-high resolution tandem mass spectrometry analysis, which identified ten 11β-MNT biotransformation products with dehydrogenated and glucuronidation (11β-MNTG) metabolites being the major metabolites. However, 11β-MNTG formation is highly variable and prevalent in human intestinal S9 fractions. A reaction phenotyping study of 11β-MNT using thirteen recombinant UDP-glucuronosyltransferase (UGT) enzymes confirmed the major role of UGT2B17 in 11β-MNTG formation. This was further supported by a strong correlation (R2 > 0.78) between 11β-MNTG and UGT2B17 abundance in human intestinal microsomes, human liver microsomes, and HH systems. These results suggest that 11β-MNT and its prodrugs are rapidly metabolized to 11β-MNTG by the highly polymorphic intestinal UGT2B17, which may explain the poor and variable bioavailability of the drug.