Genome-Wide Methylation Analysis Reveals a KCNK3-Prominent Causal Cascade on Hypertension.

BACKGROUND: Despite advances in understanding hypertension's genetic structure, how noncoding genetic variants influence it remains unclear. Studying their interaction with DNA methylation is crucial to deciphering this complex disease's genetic mechanisms. METHODS: We investigated the genetic and epigenetic interplay in hypertension using whole-genome bisulfite sequencing. Methylation profiling in 918 males revealed allele-specific methylation and methylation quantitative trait loci. We engineered rs1275988T/C mutant mice using CRISPR/Cas9, bred them for homozygosity, and subjected them to a high-salt diet. Telemetry captured their cardiovascular metrics. Protein-DNA interactions were elucidated using DNA pull-downs, mass spectrometry, and Western blots. A wire myograph assessed vascular function, and analysis of the Kcnk3 gene methylation highlighted the mutation's role in hypertension. RESULTS: We discovered that DNA methylation-associated genetic effects, especially in non-CpG island and noncoding distal regulatory regions, significantly contribute to hypertension predisposition. We identified distinct methylation quantitative trait locus patterns in the hypertensive population and observed that the onset of hypertension is influenced by the transmission of genetic effects through the demethylation process. By evidence-driven prioritization and in vivo experiments, we unearthed rs1275988 in a cell type-specific enhancer as a notable hypertension causal variant, intensifying hypertension through the modulation of local DNA methylation and consequential alterations in Kcnk3 gene expression and vascular remodeling. When exposed to a high-salt diet, mice with the rs1275988C/C genotype exhibited exacerbated hypertension and significant vascular remodeling, underscored by increased aortic wall thickness. The C allele of rs1275988 was associated with elevated DNA methylation levels, driving down the expression of the Kcnk3 gene by attenuating Nr2f2 binding at the enhancer locus. CONCLUSIONS: Our research reveals new insights into the complex interplay between genetic variations and DNA methylation in hypertension. We underscore hypomethylation's potential in hypertension onset and identify rs1275988 as a causal variant in vascular remodeling. This work advances our understanding of hypertension's molecular mechanisms and encourages personalized health care strategies.

A versatile and convenient tool for regulation of DNA strand displacement and post-modification on pre-fabricated DNA nanodevices.

Toehold-mediated strand displacement and its regulatory tools are fundamental for DNA nanotechnology. However, current regulatory tools all need to change the original sequence of reactants, making the regulation inconvenient and cumbersome. More importantly, the booming development of DNA nanotechnology will soon promote the production of packaged and batched devices or circuits with specified functions. Regarding standardized, packaged DNA nanodevices, access to personalized post-modification will greatly help users, whereas none of the current regulatory tools can provide such access, which has greatly constrained DNA nanodevices from becoming more powerful and practical. Herein, we developed a novel regulation tool named Cap which has two basic functions of subtle regulation of the reaction rate and erasability. Based on these functions, we further developed three advanced functions. Through integration of all functions of Cap and its distinct advantage of working independently, we finally realized personalized tailor-made post-modification on pre-fabricated DNA circuits. A pre-fabricated dual-output DNA circuit was successfully transformed into an equal-output circuit, a signal-antagonist circuit and a covariant circuit according to our requirements. Taken together, Cap is easy to design and generalizable for all strand displacement-based DNA nanodevices. We believe the Cap tool will be widely used in regulating reaction networks and personalized tailor-made post-modification of DNA nanodevices.

Rational Design of Conjugated Acetylenic Polymers Enables a Two-Electron Water Oxidation Pathway for Enhanced Photosynthetic Hydrogen Peroxide Generation.

Herein, the design of conjugated acetylenic polymers (CAPs) featuring diverse spatial arrangements and intramolecular spacers of diacetylene moieties (─C≡C─C≡C─) for photocatalytic hydrogen peroxide (H2 O2 ) production from water and O2 , without the need for sacrificial agents, is presented. It is shown that the linear configuration of diacetylene moieties within conjugated acetylenic polymers (CAPs) induces a pronounced polarization of electron distribution, which imparts enhanced charge-carrier mobility when compared to CAPs' networks featuring cross-linked arrangements. Moreover, optimizing the intramolecular spacer between diacetylene moieties within the linear structure leads to the exceptional modulation of the band structures, specifically resulting in a downshifted valence band (VB) and rendering the two-electron water oxidation pathway thermodynamically feasible for H2 O2 production. Consequently, the optimized CAPs with a linear configuration (LCAP-2), featuring spatially separated reduction centers (benzene rings) and oxidation centers (diacetylene moieties), exhibit a remarkable H2 O2 yield rate of 920.1 µmol g-1 h-1 , superior than that of the linear LCAP-1 (593.2 µmol g-1 h-1 ) and the cross-linked CCAP (433.4 µmol g-1 h-1 ). The apparent quantum efficiency (AQE) and solar-to-chemical energy conversion (SCC) efficiency of LCAP-2 are calculated to be 9.1% (λ = 420 nm) and 0.59%, respectively, surpassing the performance of most previously reported conjugated polymers.

HtrAs are essential for the survival of the haloarchaeon Natrinema gari J7-2 in response to heat, high salinity, and toxic substances.

Bacterial and eukaryotic HtrAs can act as an extracytoplasmic protein quality control (PQC) system to help cells survive in stress conditions, but the functions of archaeal HtrAs remain unknown. Particularly, haloarchaea route most secretory proteins to the Tat pathway, enabling them to fold properly in well-controlled cytoplasm with cytosolic PQC systems before secretion. It is unclear whether HtrAs are required for haloarchaeal survival and stress response. The haloarchaeon Natrinema gari J7-2 encodes three Tat signal peptide-bearing HtrAs (NgHtrA, NgHtrB, and NgHtrC), and the signal peptides of NgHtrA and NgHtrC contain a lipobox. Here, the in vitro analysis reveals that the three HtrAs show different profiles of temperature-, salinity-, and metal ion-dependent proteolytic activities and could exhibit chaperone-like activities to prevent the aggregation of reduced lysozyme when their proteolytic activities are inhibited at low temperatures or the active site is disrupted. The gene deletion and complementation assays reveal that NgHtrA and NgHtrC are essential for the survival of strain J7-2 at elevated temperature and/or high salinity and contribute to the resistance of this haloarchaeon to zinc and inhibitory substances generated from tryptone. Mutational analysis shows that the lipobox mediates membrane anchoring of NgHtrA or NgHtrC, and both the membrane-anchored and free extracellular forms of the two enzymes are involved in the stress resistance of strain J7-2, depending on the stress conditions. Deletion of the gene encoding NgHtrB in strain J7-2 causes no obvious growth defect, but NgHtrB can functionally substitute for NgHtrA or NgHtrC under some conditions.IMPORTANCEHtrA-mediated protein quality control plays an important role in the removal of aberrant proteins in the extracytoplasmic space of living cells, and the action mechanisms of HtrAs have been extensively studied in bacteria and eukaryotes; however, information about the function of archaeal HtrAs is scarce. Our results demonstrate that three HtrAs of the haloarchaeon Natrinema gari J7-2 possess both proteolytic and chaperone-like activities, confirming that the bifunctional nature of HtrAs is conserved across all three domains of life. Moreover, we found that NgHtrA and NgHtrC are essential for the survival of strain J7-2 under stress conditions, while NgHtrB can serve as a substitute for the other two HtrAs under certain circumstances. This study provides the first biochemical and genetic evidence of the importance of HtrAs for the survival of haloarchaea in response to stresses.

A TrmBL2-like transcription factor mediates the growth phase-dependent expression of halolysin SptA in a concentration-dependent manner in Natrinema gari J7-2.

To cope with a high-salinity environment, haloarchaea generally employ the twin-arginine translocation (Tat) pathway to transport secretory proteins across the cytoplasm membrane in a folded state, including Tat-dependent extracellular subtilases (halolysins) capable of autocatalytic activation. Some halolysins, such as SptA of Natrinema gari J7-2, are produced at late-log phase to prevent premature enzyme activation and proteolytic damage of cellular proteins in haloarchaea; however, the regulation mechanism for growth phase-dependent expression of halolysins remains largely unknown. In this study, a DNA-protein pull-down assay was performed to identify the proteins binding to the 5'-flanking sequence of sptA encoding halolysin SptA in strain J7-2, revealing a TrmBL2-like transcription factor (NgTrmBL2). The ΔtrmBL2 mutant of strain J7-2 showed a sharp decrease in the production of SptA, suggesting that NgTrmBL2 positively regulates sptA expression. The purified recombinant NgTrmBL2 mainly existed as a dimer although monomeric and higher-order oligomeric forms were detected by native-PAGE analysis. The results of electrophoretic mobility shift assays (EMSAs) showed that NgTrmBL2 binds to the 5'-flanking sequence of sptA in a non-specific and concentration-dependent manner and exhibits an increased DNA-binding affinity with the increase in KCl concentration. Moreover, we found that a distal cis-regulatory element embedded in the neighboring upstream gene negatively regulates trmBL2 expression and thus participates in the growth phase-dependent biosynthesis of halolysin SptA. IMPORTANCE: Extracellular proteases play important roles in nutrient metabolism, processing of functional proteins, and antagonism of haloarchaea, but no transcription factor involved in regulating the expression of haloaechaeal extracellular protease has been reported yet. Here we report that a TrmBL2-like transcription factor (NgTrmBL2) mediates the growth phase-dependent expression of an extracellular protease, halolysin SptA, of haloarchaeon Natrinema gari J7-2. In contrast to its hyperthermophilic archaeal homologs, which are generally considered to be global transcription repressors, NgTrmBL2 functions as a positive regulator for sptA expression. This study provides new clues about the transcriptional regulation mechanism of extracellular protease in haloarchaea and the functional diversity of archaeal TrmBL2.

Epigenetic regulation of high light-induced anthocyanin biosynthesis by histone demethylase IBM1 in Arabidopsis.

In plant species, anthocyanin accumulation is specifically regulated by light signaling. Although the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) complex is known to control anthocyanin biosynthesis in response to light, the precise mechanism underlying this process remains largely unknown. Here, we report that Increase in BONSAI Methylation 1 (IBM1), a JmjC domain-containing histone demethylase, participates in the regulation of light-induced anthocyanin biosynthesis in Arabidopsis. The expression of IBM1 was induced by high light (HL) stress, and loss-of-function mutations in IBM1 led to accelerated anthocyanin accumulation under HL conditions. We further identified that IBM1 is directly associated with SPA1/3/4 chromatin in vivo to establish a hypomethylation status on H3K9 and DNA non-CG at these loci under HL, thereby releasing their expression. Genetic analysis showed that quadruple mutants of IBM1 and SPA1/3/4 resemble spa134 mutants. Overexpression of SPA1 in ibm1 mutants complements the mutant phenotype. Our results elucidate the significance and mechanism of IBM1 histone demethylase in the epigenetic regulation of anthocyanin biosynthesis in Arabidopsis under HL conditions.

Photoionization and Dissociative Photoionization of Acetaldehyde in the 10.0-13.7†eV Range by Synchrotron Photoelectron Photoion Coincidence Spectroscopy.

Photoionization and dissociative photoionization of acetaldehyde (CH3CHO) in the 10.0-13.7 eV energy range are studied by using synchrotron radiation double imaging photoelectron photoion coincidence spectroscopy (i2PEPICO). The X2A' and A2A" electronic states of CH3CHO+ as well as the Franck-Condon gap region between these two states have been populated with several vibrational sequences and assigned in the high-resolution slow photoelectron spectrum (SPES). The adiabatic ionization energies (AIEs) of the X2A' and A2A" states are measured at 10.228±0.006 and 12.52±0.05 eV, respectively. The present results show that the X2A' state is a stable state while the A2A" state is fully dissociative to produce CH3CO+, CHO+ and CH4 + fragment ions. The 0 K appearance energies (AE0K) of CH3CO+ and CHO+ fragment ions are determined through the modeling of the breakdown diagram, i. e., AE0K(CH3CO+)=10.89±0.01 eV (including a reverse barrier of ~0.19 eV) and AE0K(CHO+)=11.54±0.05 eV. In addition, the dissociation mechanisms of CH3CHO+ including statistical dissociation, direct bond breaking and isomerization are discussed with the support of the calculated dissociation limits and transition state energies.

Nanoclay Drug-Delivery System Loading Potassium Iodide Promotes Endocytosis and Targeted Therapy in Anaplastic Thyroid Cancer.

The rapid proliferative biological behavior of primary foci of anaplastic thyroid cancer (ATC) makes it a lethal tumor. According to the specific iodine uptake capacity of thyroid cells and enhanced endocytosis of ATC cells, we designed a kind of nanoclay drug-loading system and showed a promising treatment strategy for ATC. Introducing potassium iodide (KI) improves the homoaggregation of clay nanoparticles and then affects the distribution of nanoparticles in vivo, which makes KI@DOX-KaolinMeOH enriched almost exclusively in thyroid tissue. Simultaneously, the improvement of dispersibility of KI@DOX-KaolinMeOH changes the target uptake of ATC cells by improving the endocytosis and nanoparticle-induced autophagy, which regulate the production of autolysosomes and autophagy-enhanced chemotherapy, eventually contributing to a tumor inhibition rate of more than 90% in the primary foci of ATC. Therefore, this facile strategy to improve the homoaggregation of nanoclay by introducing KI has the potential to become an advanced drug delivery vehicle in ATC treatment.

SlCK2α as a novel substrate for CRL4 E3 ligase regulates fruit size through maintenance of cell division homeostasis in tomato.

MAIN CONCLUSION: This study unravels a novel regulatory module (CRL4-CK2α-CDK2) involving fruit size control by mediating cell division homeostasis (SlCK2α and SlCDK2) in tomato. Fruit size is one of the crucial agronomical traits for crop production. UV-damaged DNA binding protein 1 (DDB1), a core component of Cullin4-RING E3 ubiquitin ligase complex (CRL4), has been identified as a negative regulator of fruit size in tomato (Solanum lycopersicum). However, the underlying molecular mechanism remains largely unclear. Here, we report the identification and characterization of a SlDDB1-interacting protein putatively involving fruit size control through regulating cell proliferation in tomato. It is a tomato homolog SlCK2α, the catalytic subunit of the casein kinase 2 (CK2), identified by yeast two-hybrid (Y2H) assays. The interaction between SlDDB1 and SlCK2α was demonstrated by bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP). RNA interference (RNAi) and CRISPR/Cas9-based mutant analyses showed that lack of SlCK2α resulted in reduction of fruit size with reduced cell number, suggesting it is a positive regulator on fruit size by promoting cell proliferation. We also showed SlDDB1 is required to ubiquitinate SlCK2α and negatively regulate its stability through 26S proteasome-mediated degradation. Furthermore, we found that a tomato homolog of cell division protein kinase 2 (SlCDK2) could interact with and specifically be phosphorylated by SlCK2α, resulting in an increase of SlCDK2 protein stability. CRISPR/Cas9-based genetic evidence showed that SlCDK2 is also a positive regulator of fruit size by influencing cell division in tomato. Taken together, our findings, thus, unravel a novel regulatory module CRL4-CK2α-CDK2 in finely modulating cell division homeostasis and the consequences on fruit size.

The transcription factor MYB43 antagonizes with ICE1 to regulate freezing tolerance in Arabidopsis.

Previous discovering meticulously illustrates the post-translational modifications and protein stability regulation of ICE1 and their role in cold stress. However, the studies on the interaction of ICE1 with other transcription factors, and their function in modulation cold stress tolerance, as well as in the transition between cold stress and growth are largely insufficient. In this work, we found that maltose binding protein (MBP) 43 directly binds to the promoters of CBF genes to repress their expression, thereby negatively regulating freezing tolerance. Biochemical and genetic analyses showed that MYB43 interacts and antagonizes with ICE1 to regulate the expression of CBF genes and plant's freezing stress tolerance. PLEIOTROPIC REGULATORY LOCUS 1 (PRL1) accumulates under cold stress and promotes MYB43 protein degradation; however, when cold stress disappears, PRL1 restores normal protein levels, causing MYB43 protein to re-accumulate to normal levels. Furthermore, PRL1 positively regulates freezing tolerance by promoting degradation of MYB43 to attenuate its repression of CBF genes and antagonism with ICE1. Thus, our study reveals that MYB43 inhibits CBF genes expression under normal growth condition, while PRL1 promotes MYB43 protein degradation to attenuate its repression of CBF genes and antagonism with ICE1, and thereby to the precise modulation of plant cold stress responses.

Regulatory module WRKY33-ATL31-IRT1 mediates cadmium tolerance in Arabidopsis.

Cadmium (Cd) is one of the most dangerous environmental pollutants among heavy metals, and threatens food safety and human health by accumulating in plant sink tissues. Here, we report a novel regulatory cascade that profoundly influences Cd tolerance in Arabidopsis. Phenotypic analysis showed that an insertional knockdown mutation at the Arabidopsis Tóxicos en Levadura 31 (ATL31) locus resulted in hypersensitivity to Cd stress, most likely due to a significant increase in Cd accumulation. Consistently, ATL31-overexpressing lines exhibited enhanced Cd stress tolerance and reduced Cd accumulation. Further, IRON-REGULATED TRANSPORTER 1 (IRT1) was identified, and yeast two-hybrid, co-immunoprecipitation and bimolecular fluorescence complementation assays demonstrated its interaction with ATL31. Biochemical, molecular, and genetic analyses showed that IRT1 is targeted by ATL31 for ubiquitin-conjugated degradation in response to Cd stress. Intriguingly, transcription of ATL31 was strongly induced by Cd stress. In addition, transgenic and molecular analyses showed that WRKY33 directly activated the transcription of ATL31 in response to Cd stress and positively regulated Cd tolerance. Genetic analysis indicated that ATL31 acts upstream of IRT1 and downstream of WRKY33 to regulate Cd tolerance. Our study revealed that the WRKY33-ATL31-IRT1 module plays a crucial role in timely blocking Cd absorption to prevent metal toxicity in Arabidopsis.

Beam smoothing by introducing spatial dispersion for high-peak-power laser pulse compression.

Post-compression can effectively further improve the peak power of laser pulses by shortening the pulse duration. Which has been investigated in various ranges of energy and central wavelength. However, the spatial intensity profile of high-peak-power laser pulses is generally inhomogeneous due to pump lasers, imperfect optical components, and dust in the optical layout. In post-compression, the B-integral is proportional to intensity, and wavefront distortions are induced in the spectral broadening stage, leading to a decrease in focusing intensity. Moreover, the beam intensity may be strongly modulated and beam inhomogeneity will be intensified in this process, causing damage to optical components and limiting the achievement of high peak power enhancement. In this study, to address these challenges, the laser pulse is first smoothed by introducing spatial dispersion using prism pairs or asymmetric four-grating compressors, and then the smoothed pulse is used for post-compression. The simulation results indicate that this method can effectively remove hot spots from laser pulses and maintain high peak power enhancement in post-compression.

Using ultrasound radiomics analysis to diagnose cervical lymph node metastasis in patients with nasopharyngeal carcinoma.

OBJECTIVE: This study aimed to explore the clinical value of ultrasound radiomics analysis in the diagnosis of cervical lymph node metastasis (CLNM) in patients with nasopharyngeal carcinoma (NPC). METHODS: A total of 205 cases of NPC CLNM and 284 cases of benign lymphadenopathy with pathologic diagnosis were retrospectively included. Grayscale ultrasound (US) images of the largest section of every lymph node underwent feature extraction. Feature selection was done by maximum relevance minimum redundancy (mRMR) algorithm and multivariate logistic least absolute shrinkage and selection operator (LASSO) regression. Logistic regression models were developed based on clinical features, radiomics features, and the combination of those features. The AUCs of models were analyzed by DeLong's test. RESULTS: In the clinical model, lymph nodes in the upper neck, larger long axis, and unclear hilus were significant factors for CLNM (p < 0.001). MRMR and LASSO regression selected 7 significant features for the radiomics model from the 386 radiomics features extracted. In the validation dataset, the AUC value was 0.838 (0.776-0.901) in the clinical model, 0.810 (0.739-0.881) in the radiomics model, and 0.880 (0.826-0.933) in the combined model. There was not a significant difference between the AUCs of clinical models and radiomics models in both datasets. DeLong's test revealed a significantly larger AUC in the combined model than in the clinical model in both training (p = 0.049) and validation datasets (p = 0.027). CONCLUSION: Ultrasound radiomics analysis has potential value in screening meaningful ultrasound features and improving the diagnostic efficiency of ultrasound in CLNM of patients with NPC. KEY POINTS: • Radiomics analysis of gray-scale ultrasound images can be used to develop an effective radiomics model for the diagnosis of cervical lymph node metastasis in nasopharyngeal carcinoma patients. • Radiomics model combined with general ultrasound features performed better than the clinical model in differentiating cervical lymph node metastases from benign lymphadenopathy.

Atomically Dispersed Fe-N4 Site as a Conductive Bridge Enables Efficient and Stable Activation of Peroxymonosulfate: Active Site Renewal, Anti-Oxidative Capacity, and Pathway Alternation Mechanism.

Atomically dispersed metal sites anchored on nitrogen-doped carbonaceous substrates (M-NCs) have emerged as promising alternatives to conventional peroxymonosulfate (PMS) activators; however, the exact contribution of each site still remains elusive. Herein, isolated Fe-N4 active site-decorated three-dimensional NC substrates (FeSA-NC) via a micropore confinement strategy are fabricated to initiate PMS oxidation reaction, achieving a specific activity of 5.16 × 103 L·min-1·g-1 for the degradation of bisphenol A (BPA), which outperforms most of the state-of-the-art single-atom (SA) catalysts. Mechanism inquiry reveals enhanced chemisorption and electron transfer between PMS and FeSA-NC, enabling an inner electron shuttle mechanism in which Fe-N4 serves as a conductive bridge. The Fe-N4 sites reduce the energy barrier for the formation of SO5* and H*, thereby transforming the reaction pathway from directly adjacent electron transfer into reactive oxygen species (ROS)-dominated oxidation. Theoretical calculations and dynamic simulations reveal that the Fe-N4 sites induce facilitated desorption of reaction intermediates (PMS*/BPA*), which collectively contribute to the renewal of active sites and eventually enhance the catalytic durability. This work offers a reasonable interpretation for the important role of the Fe-N4 moiety in altering the activation mechanism and enhancing the antioxidative capacity of NC materials, which fundamentally furnishes theoretical support for SA material design.

Rate constant and branching ratio of the reaction of ethyl peroxy radicals with methyl peroxy radicals.

The cross-reaction of ethyl peroxy radicals (C2H5O2) with methyl peroxy radicals (CH3O2) (R1) has been studied using laser photolysis coupled to time resolved detection of the two different peroxy radicals by continuous wave cavity ring down spectroscopy (cw-CRDS) in their AÃ-X̃ electronic transition in the near-infrared region, C2H5O2 at 7602.25 cm-1, and CH3O2 at 7488.13 cm-1. This detection scheme is not completely selective for both radicals, but it is demonstrated that it has great advantages compared to the widely used, but unselective UV absorption spectroscopy. Peroxy radicals were generated from the reaction of Cl-atoms with the appropriate hydrocarbon (CH4 and C2H6) in the presence of O2, whereby Cl-atoms were generated by 351 nm photolysis of Cl2. For different reasons detailed in the manuscript, all experiments were carried out under excess of C2H5O2 over CH3O2. The experimental results were best reproduced by an appropriate chemical model with a rate constant for the cross-reaction of k = (3.8 ± 1.0) × 10-13 cm3 s-1 and a yield for the radical channel, leading to CH3O and C2H5O, of (ϕ1a = 0.40 ± 0.20).

A liquid chromatography-tandem mass spectrometry method for comprehensive determination of metabolites in the purine pathway of rat plasma and its application in anti-gout effects of Lycium ruthenicum Murr.

It has been proved that purine metabolites are implicated in various biological syndromes and disorders. Therefore, the realization of panoramic detection of purine metabolites will be of great significance to the pathogenesis of purine metabolic disorders. In the present study, an ultra-high performance liquid chromatography with tandem mass spectrometry method was developed for the comprehensive quantification of purine metabolites in rat plasma. The 17 purine metabolites were separated and quantified in the short running time of 15 min. The proposed method was strictly validated by applying SeraSub solution as a matrix and proved to be linear (R2 ≥ 0.9944), accurate (the recoveries of all analytes ranged from 85.3% to 103.0%, with relative standard deviation values ≤ 9.3%), and precise (the intra- and inter-day precisions were less than 10.8% and 12.4%, respectively). The method was then successfully applied to the qualification of the endogenous purine metabolites in acute gouty arthritis rats, as well as colchicine and anthocyanin-intervened rats. Results showed that uric acid, xanthine, hypoxanthine, and xanthine were considered the key factors of acute gouty arthritis. The established method and measurement of purines in rat plasma might help the investigation of the action mechanisms between purine disorders and related diseases.

Diagnosis and treatment of biliary mucinous cystic neoplasms: A single-center experience.

BACKGROUND: Biliary mucinous cystic neoplasms (BMCNs) are rare hepatobiliary cystic tumors, which can be divided into noninvasive and invasive types. This study aimed to investigate the diagnosis, treatment, and prognosis of BMCNs in a large single center. METHODS: We analyzed 49 patients with BMCNs confirmed by postoperative pathology at the First Affiliated Hospital, Zhejiang University School of Medicine between January 2007 and December 2021. RESULTS: Among the 49 patients, 37 were female (75.5%), and the average age was 57.04 years. Common symptoms included abdominal discomfort, jaundice and fever, while 22 patients (44.9%) had no symptoms. Serum carbohydrate antigen (CA) 19-9 and CA125 concentrations were elevated in 34.8% and 19.6% of patients, respectively. Forty-eight patients had tumors in the intrahepatic bile ducts and only one had a tumor in the extrahepatic bile duct. Forty-eight patients with noninvasive intrahepatic BMCNs were further analyzed in terms of pathological features: 34 (70.8%) had low-grade intraepithelial neoplasms (LGINs), and 14 (29.2%) had high-grade intraepithelial neoplasms (HGINs). The potential immunohistochemical markers of BMCNs were cytokeratin (CK) 19, CK7, estrogen receptor and progesterone receptor. Follow-up data for 37 patients with intrahepatic BMCNs were obtained. The median overall survival (OS) of BMCNs was not reached. The longest survival time was 137 months.The 5- and 10-year OS rates were 100% and 85.4%, respectively. The 5- and 10-year recurrence-free survival (RFS) rates were 93.9% and 80.2%, respectively. CONCLUSIONS: BMCNs are rare cystic neoplasms that commonly occur in middle-aged females. BMCNs can only be diagnosed and classified by postoperative pathology, as there are no specific clinical presentations, serological indicators or imaging modalities for preoperative diagnosis. Complete surgical resection is necessary for BMCNs, and the postoperative prognosis is favorable.

Dimeric Product of Peroxy Radical Self-Reaction Probed with VUV Photoionization Mass Spectrometry and Theoretical Calculations: The Case of C2H5OOC2H5.

Organic peroxy radicals (RO2) as key intermediates in tropospheric chemistry exert a controlling influence on the cycling of atmospheric reactive radicals and the production of secondary pollutants, such as ozone and secondary organic aerosols (SOA). Herein, we present a comprehensive study of the self-reaction of ethyl peroxy radicals (C2H5O2) by using advanced vacuum ultraviolet (VUV) photoionization mass spectrometry in combination with theoretical calculations. A VUV discharge lamp in Hefei and synchrotron radiation at the Swiss Light Source (SLS) are employed as the photoionization light sources, combined with a microwave discharge fast flow reactor in Hefei and a laser photolysis reactor at the SLS. The dimeric product, C2H5OOC2H5, as well as other products, CH3CHO, C2H5OH and C2H5O, formed from the self-reaction of C2H5O2 are clearly observed in the photoionization mass spectra. Two kinds of kinetic experiments have been performed in Hefei by either changing the reaction time or the initial concentration of C2H5O2 radicals to confirm the origins of the products and to validate the reaction mechanisms. Based on the fitting of the kinetic data with the theoretically calculated results and the peak area ratios in the photoionization mass spectra, a branching ratio of 10 ± 5% for the pathway leading to the dimeric product C2H5OOC2H5 is measured. In addition, the adiabatic ionization energy (AIE) of C2H5OOC2H5 is determined at 8.75 ± 0.05 eV in the photoionization spectrum with the aid of Franck-Condon calculations and its structure is revealed here for the first time. The potential energy surface of the C2H5O2 self-reaction has also been theoretically calculated with a high-level of theory to understand the reaction processes in detail. This study provides a new insight into the direct measurement of the elusive dimeric product ROOR and demonstrates its non-negligible branching ratio in the self-reaction of small RO2 radicals.

Maturation Process and Characterization of a Novel Thermostable and Halotolerant Subtilisin-Like Protease with High Collagenolytic Activity but Low Gelatinolytic Activity.

Enzymatic degradation of collagen is of great industrial and environmental significance; however, little is known about thermophile-derived collagenolytic proteases. Here, we report a novel collagenolytic protease (TSS) from thermophilic Brevibacillus sp. WF146. The TSS precursor comprises a signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain, a ß-jelly roll (ßJR) domain, and a prepeptidase C-terminal (PPC) domain. The maturation of TSS involves a stepwise autoprocessing of the N-terminal propeptide and the PPC domain, and the ßJR rather than the PPC domain is necessary for correct folding of the enzyme. Purified mature TSS displayed optimal activity at 70°C and pH 9.0, a half-life of 1.5 h at 75°C, and an increased thermostability as the NaCl concentration increased up to 4 M. TSS possesses an increased number of surface acidic residues and ion pairs, as well as four Ca2+-binding sites, which contribute to its high thermostability and halotolerance. At high temperatures, TSS exhibited high activity toward insoluble type I collagen and azocoll but showed a low gelatinolytic activity, with a strong preference for Arg and Gly at the P1 and P1' positions, respectively. Both the ßJR and PPC domains could bind but not swell collagen, and thus facilitate TSS-mediated collagenolysis via improving the accessibility of the enzyme to the substrate. Additionally, TSS has the ability to efficiently degrade fish scale collagen at high temperatures. IMPORTANCE Proteolytic degradation of collagen at high temperatures has the advantages of increasing degradation efficiency and minimizing the risk of microbial contamination. Reports on thermostable collagenolytic proteases are limited, and their maturation and catalytic mechanisms remain to be elucidated. Our results demonstrate that the thermophile-derived TSS matures in an autocatalytic manner and represents one of the most thermostable collagenolytic proteases reported so far. At elevated temperatures, TSS prefers hydrolyzing insoluble heat-denatured collagen rather than gelatin, providing new insight into the mechanism of collagen degradation by thermostable collagenolytic proteases. Moreover, TSS has the potential to be used in recycling collagen-rich wastes such as fish scales.

Auto- and Hetero-Catalytic Processing of the N-Terminal Propeptide Promotes the C-Terminal Fibronectin Type III Domain-Mediated Dimerization of a Thermostable Vpr-like Protease.

Microbial Vpr-like proteases are extracellular multidomain subtilases with diverse functions and can form oligomers, but their maturation and oligomerization mechanisms remain to be elucidated. Here, we report a novel Vpr-like protease (BTV) from thermophilic bacterium Brevibacillus sp. WF146. The BTV precursor comprises a signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain with an inserted protease-associated (PA) domain, two tandem fibronectin type III domains (Fn1 and Fn2), and a C-terminal propeptide. The BTV proform (pro-BTV) could be autoprocessed into the mature form (mBTV) via two intermediates lacking the N- or C-terminal propeptide, respectively, and the C-terminal propeptide delays the autocatalytic maturation of the enzyme. By comparison, pro-BTV is more efficiently processed into mBTV by protease TSS from strain WF146. Purified mBTV is a Ca2+-dependent thermostable protease, showing optimal activity at 60°C and retaining more than 60% of activity after incubation at 60°C for 8 h. The PA domain is important for enzyme stability and contributes to the substrate specificity of BTV by restricting the access of protein substrates to the active site. The proform and mature form of BTV exist as a monomer and a homodimer, respectively, and the dimerization is mediated by the Fn1 and Fn2 domains. The N-terminal propeptide of BTV not only acts as intramolecular chaperone and enzymatic inhibitor but also inhibits the homodimerization of the enzyme. The removal of the N-terminal propeptide leads to a structural adjustment of the enzyme and thus promotes enzyme dimerization. IMPORTANCE Vpr-like proteases are widely distributed in bacteria and fungi and are involved in processing lantibiotics, degrading collagen, keratin, and fibrin, and pathogenesis of microbes. The dissection of the roles of individual domains in enzyme maturation and oligomerization is crucial for understanding the action mechanisms of these multidomain proteases. Our results demonstrate that hetero-catalytic maturation of the extracellular Vpr-like protease BTV of Brevibacillus sp. WF146 is more efficient than autocatalytic maturation of the enzyme. Moreover, we found that the C-terminal tandem fibronectin type III domains rather than the PA domain mediate the dimerization of mature BTV, while the N-terminal propeptide inhibits the dimerization of the BTV proform. This study provides new insight into the activation and oligomerization mechanisms of Vpr-like proteases.