Top-down versus bottom-up oxidation of a neonicotinoid pesticide by OH radicals.

Emerging contaminants (EC) distributed on surfaces in the environment can be oxidized by gas phase species (top-down) or by oxidants generated by the underlying substrate (bottom-up). One class of EC is the neonicotinoid (NN) pesticides that are widely distributed in air, water, and on plant and soil surfaces as well as on airborne dust and building materials. This study investigates the OH oxidation of the systemic NN pesticide acetamiprid (ACM) at room temperature. ACM on particles and as thin films on solid substrates were oxidized by OH radicals either from the gas phase or from an underlying TiO2 or NaNO2 substrate, and for comparison, in the aqueous phase. The site of OH attack is both the secondary >CH2 group as well as the primary -CH3 group attached to the tertiary amine nitrogen, with the latter dominating. In the case of top-down oxidation of ACM by gas phase OH radicals, addition to the -CN group also occurs. Major products are carbonyls and alcohols, but in the presence of sufficient water, their hydrolyzed products dominate. Kinetics measurements show ACM is more reactive toward gas phase OH radicals than other NN nitroguanidines, with an atmospheric lifetime of a few days. Bottom-up oxidation of ACM on TiO2 exposed to sunlight outdoors (temperatures were above 30 °C) was also shown to occur and is likely to be competitive with top-down oxidation. These findings highlight the different potential oxidation processes for EC and provide key data for assessing their environmental fates and toxicologies.

Identification of two archaeal GDGT lipid-modifying proteins reveals diverse microbes capable of GMGT biosynthesis and modification.

Archaea produce unique membrane-spanning lipids (MSLs), termed glycerol dialkyl glycerol tetraethers (GDGTs), which aid in adaptive responses to various environmental challenges. GDGTs can be modified through cyclization, cross-linking, methylation, hydroxylation, and desaturation, resulting in structurally distinct GDGT lipids. Here, we report the identification of radical SAM proteins responsible for two of these modifications-a glycerol monoalkyl glycerol tetraether (GMGT) synthase (Gms), responsible for covalently cross-linking the two hydrocarbon tails of a GDGT to produce GMGTs, and a GMGT methylase (Gmm), capable of methylating the core hydrocarbon tail. Heterologous expression of Gms proteins from various archaea in Thermococcus kodakarensis results in the production of GMGTs in two isomeric forms. Further, coexpression of Gms and Gmm produces mono- and dimethylated GMGTs and minor amounts of trimethylated GMGTs with only trace GDGT methylation. Phylogenetic analyses reveal the presence of Gms homologs in diverse archaeal genomes spanning all four archaeal superphyla and in multiple bacterial phyla with the genetic potential to synthesize fatty acid-based MSLs, demonstrating that GMGT production may be more widespread than previously appreciated. We demonstrate GMGT production in three Gms-encoding archaea, identifying an increase in GMGTs in response to elevated temperature in two Archaeoglobus species and the production of GMGTs with up to six rings in Vulcanisaeta distributa. The occurrence of such highly cyclized GMGTs has been limited to environmental samples and their detection in culture demonstrates the utility of combining genetic, bioinformatic, and lipid analyses to identify producers of distinct archaeal membrane lipids.

A radical polymer membrane for simultaneous degradation of organic pollutants and water filtration.

Integrating reactive radicals into membranes that resemble biological membranes has always been a pursuit for simultaneous organics degradation and water filtration. In this research, we discovered that a radical polymer (RP) that can directly trigger the oxidative degradation of sulfamethozaxole (SMX). Mechanistic studies by experiment and density functional theory simulations revealed that peroxyl radicals are the reactive species, and the radicals could be regenerated in the presence of O2. Furthermore, an interpenetrating RP network membrane consisting of polyvinyl alcohol and the RP was fabricated to demonstrate the simultaneous filtration of large molecules in the model wastewater stream and the degradation of ~ 85% of SMX with a steady permeation flux. This study offers valuable insights into the mechanism of RP-triggered advanced oxidation processes and provides an energy-efficient solution for the degradation of organic compounds and water filtration in wastewater treatment.

Molecular mechanism of the metal-independent production of hydroxyl radicals by thiourea dioxide and H2O2.

It is well-known that highly reactive hydroxyl radicals (HO•) can be produced by the classic Fenton system and our recently discovered haloquinone/H2O2 system, but rarely from thiol-derivatives. Here, we found, unexpectedly, that HO• can be generated from H2O2 and thiourea dioxide (TUO2), a widely used and environmentally friendly bleaching agent. A carbon-centered radical and sulfite were detected and identified as the transient intermediates, and urea and sulfate as the final products, with the complementary application of electron spin-trapping, oxygen-18 isotope labeling coupled with HPLC/MS analysis. Density functional theory calculations were conducted to further elucidate the detailed pathways for HO• production. Taken together, we proposed that the molecular mechanism for HO• generation by TUO2/H2O2: TUO2 tautomerizes from sulfinic acid into ketone isomer (TUO2-K) through proton transfer, then a nucleophilic addition of H2O2 on the S atom of TUO2-K, forming a S-hydroperoxide intermediate TUO2-OOH, which dissociates homolytically to produce HO•. Our findings represent the first experimental and computational study on an unprecedented new molecular mechanism of HO• production from simple thiol-derived sulfinic acids, which may have broad chemical, environmental, and biomedical significance for future research on the application of the well-known bleaching agent and its analogs.

Protein engineering a PhotoRNR chimera based on a unifying evolutionary apparatus among the natural classes of ribonucleotide reductases.

Ribonucleotide reductases (RNRs) are essential enzymes that catalyze the de novo transformation of nucleoside 5'-di(tri)phosphates [ND(T)Ps, where N is A, U, C, or G] to their corresponding deoxynucleotides. Despite the diversity of factors required for function and the low sequence conservation across RNRs, a unifying apparatus consolidating RNR activity is explored. We combine aspects of the protein subunit simplicity of class II RNR with a modified version of Escherichia coli class la photoRNRs that initiate radical chemistry with light to engineer a mimic of a class II enzyme. The design of this RNR involves fusing a truncated form of the active site containing α subunit with the functionally important C-terminal tail of the radical-generating ß subunit to render a chimeric RNR. Inspired by a recent cryo-EM structure, a [Re] photooxidant is located adjacent to Y356[ß], which is an essential component of the radical transport pathway in class I RNRs. Combination of this RNR photochimera with cytidine diphosphate (CDP), adenosine triphosphate (ATP), and light resulted in the generation of Y356• along with production of deoxycytidine diphosphate (dCDP) and cytosine. The photoproducts reflect an active site chemistry consistent with both the consensus mechanism of RNR and chemistry observed when RNR is inactivated by mechanism-based inhibitors in the active site. The enzymatic activity of the RNR photochimera in the absence of any ß metallocofactor highlights the adaptability of the 10-stranded αß barrel finger loop to support deoxynucleotide formation and accommodate the design of engineered RNRs.

Lipid-derived electrophiles inhibit the function of membrane channels during ferroptosis.

The therapeutic targeting of ferroptosis requires full understanding of the molecular mechanism of this regulated cell death pathway. While lipid-derived electrophiles (LDEs), including 4-hydroxy-2-nonenal (4-HNE), are important biomarkers of ferroptosis, a functional role for these highly reactive species in ferroptotic cell death execution has not been established. Here, through mechanistic characterization of LDE-detoxification impairment, we demonstrate that LDEs mediate altered protein function during ferroptosis. Applying live cell fluorescence imaging, we first identified that export of glutathione-LDE-adducts through multidrug resistance-associated protein (MRP) channels is inhibited following exposure to a panel of ferroptosis inducers (FINs) with different modes of action (type I-IV FINs erastin, RSL3, FIN56, and FINO2). This channel inhibition was recreated by both initiation of lipid peroxidation and treatment with 4-HNE. Importantly, treatment with radical-trapping antioxidants prevented impaired LDE-adduct export when working with both FINs and lipid peroxidation initiators but not 4-HNE, pinpointing LDEs as the cause of this inhibited MRP activity observed during ferroptosis. Our findings, when combined with reports of widespread LDE alkylation of key proteins following ferroptosis induction, including MRP1, set a precedent for LDEs as critical mediators of ferroptotic cell damage. Lipid hydroperoxide breakdown to form truncated phospholipids and LDEs may fully explain membrane permeabilization and modified protein function downstream of lipid peroxidation, offering a unified explanation of the molecular cell death mechanism of ferroptosis.

Bacterial stigmasterol degradation involving radical flavin delta-24 desaturase and molybdenum-dependent C26 hydroxylase.

Sterols are ubiquitous membrane constituents that persist to a large extent in the environment due to their water insolubility and chemical inertness. Recently, an oxygenase-independent sterol degradation pathway was discovered in a cholesterol-grown denitrifying bacterium Sterolibacterium (S.) denitrificans. It achieves hydroxylation of the unactivated primary C26 of the isoprenoid side chain to an allylic alcohol via a phosphorylated intermediate in a four-step ATP-dependent enzyme cascade. However, this pathway is incompatible with the degradation of widely distributed steroids containing a double bond at C22 in the isoprenoid side chain such as the plant sterol stigmasterol. Here, we have enriched a prototypical delta-24 desaturase from S. denitrificans, which catalyzes the electron acceptor-dependent oxidation of the intermediate stigmast-1,4-diene-3-one to a conjugated (22,24)-diene. We suggest an α4ß4 architecture of the 440 kDa enzyme, with each subunit covalently binding an flavin mononucleotide cofactor to a histidyl residue. As isolated, both flavins are present as red semiquinone radicals, which can be reduced by stigmast-1,4-diene-3-one but cannot be oxidized even with strong oxidizing agents. We propose a mechanism involving an allylic radical intermediate in which two flavin semiquinones each abstract one hydrogen atom from the substrate. The conjugated delta-22,24 moiety formed allows for the subsequent hydroxylation of the terminal C26 with water by a heterologously produced molybdenum-dependent steroid C26 dehydrogenase 2. In conclusion, the pathway elucidated for delta-22 steroids achieves oxygen-independent hydroxylation of the isoprenoid side chain by bypassing the ATP-dependent formation of a phosphorylated intermediate.

Small molecule MarR modulators potentiate metronidazole antibiotic activity in aerobic E. coli by inducing activation by the nitroreductase NfsA.

Antibiotic-resistant Enterobacterales pose a major threat to healthcare systems worldwide, necessitating the development of novel strategies to fight such hard-to-kill bacteria. One potential approach is to develop molecules that force bacteria to hyper-activate prodrug antibiotics, thus rendering them more effective. In the present work, we aimed to obtain proof-of-concept data to support that small molecules targeting transcriptional regulators can potentiate the antibiotic activity of the prodrug metronidazole (MTZ) against Escherichia coli under aerobic conditions. By screening a chemical library of small molecules, a series of structurally related molecules were identified that had little inherent antibiotic activity but showed substantial activity in combination with ineffective concentrations of MTZ. Transcriptome analyses, functional genetics, thermal shift assays, and electrophoretic mobility shift assays were then used to demonstrate that these MTZ boosters target the transcriptional repressor MarR, resulting in the upregulation of the marRAB operon and its downstream MarA regulon. The associated upregulation of the flavin-containing nitroreductase, NfsA, was then shown to be critical for the booster-mediated potentiation of MTZ antibiotic activity. Transcriptomic studies, biochemical assays, and electron paramagnetic resonance measurements were then used to show that under aerobic conditions, NfsA catalyzed 1-electron reduction of MTZ to the MTZ radical anion which in turn induced lethal DNA damage in E. coli. This work reports the first example of prodrug boosting in Enterobacterales by transcriptional modulators and highlights that MTZ antibiotic activity can be chemically induced under anaerobic growth conditions.

Antioxidants are ineffective at quenching reactive oxygen species inside bacteria and should not be used to diagnose oxidative stress.

A wide variety of stresses have been proposed to exert killing effects upon bacteria by stimulating the intracellular formation of reactive oxygen species (ROS). A key part of the supporting evidence has often been the ability of antioxidant compounds to protect the cells. In this study, some of the most-used antioxidants-thiourea, glutathione, N-acetylcysteine, and ascorbate-have been examined. Their ability to quench superoxide and hydrogen peroxide was verified in vitro, but the rate constants were orders of magnitude too slow for them to have an impact upon superoxide and peroxide concentrations in vivo, where these species are already scavenged by highly active enzymes. Indeed, the antioxidants were unable to protect the growth and ROS-sensitive enzymes of E. coli strains experiencing authentic oxidative stress. Similar logic posits that antioxidants cannot substantially quench hydroxyl radicals inside cells, which contain abundant biomolecules that react with them at diffusion-limited rates. Indeed, antioxidants were able to protect cells from DNA damage only if they were applied at concentrations that slow metabolism and growth. This protective effect was apparent even under anoxic conditions, when ROS could not possibly be involved, and it was replicated when growth was similarly slowed by other means. Experimenters should discard the use of antioxidants as a way of detecting intracellular oxidative stress and should revisit conclusions that have been based upon such experiments. The notable exception is that these compounds can effectively degrade hydrogen peroxide from environmental sources before it enters cells.

Morc2a variants cause hydroxyl radical-mediated neuropathy and are rescued by restoring GHKL ATPase.

Mutations in the Microrchidia CW-type zinc finger 2 (MORC2) GHKL ATPase module cause a broad range of neuropathies, such as Charcot-Marie-Tooth disease type 2Z; however, the aetiology and therapeutic strategy are not fully understood. Previously, we reported that the Morc2a p.S87L mouse model exhibited neuropathy and muscular dysfunction through DNA damage accumulation. In the present study, we analysed the gene expression of Morc2a p.S87L mice and designated the primary causing factor. We investigated the pathological pathway using Morc2a p.S87L mouse embryonic fibroblasts and human fibroblasts harbouring MORC2 p.R252W. We subsequently assessed the therapeutic effect of gene therapy administered to Morc2a p.S87L mice. This study revealed that Morc2a p.S87L causes a protein synthesis defect, resulting in the loss of function of Morc2a and high cellular apoptosis induced by high hydroxyl radical levels. We considered the Morc2a GHKL ATPase domain as a therapeutic target because it simultaneously complements hydroxyl radical scavenging and ATPase activity. We used the adeno-associated virus (AAV)-PHP.eB serotype, which has a high CNS transduction efficiency, to express Morc2a or Morc2a GHKL ATPase domain protein in vivo. Notably, AAV gene therapy ameliorated neuropathy and muscular dysfunction with a single treatment. Loss-of-function characteristics due to protein synthesis defects in Morc2a p.S87L were also noted in human MORC2 p.S87L or p.R252W variants, indicating the correlation between mouse and human pathogenesis. In summary, CMT2Z is known as an incurable genetic disorder, but the present study demonstrated its mechanisms and treatments based on established animal models. This study demonstrates that the Morc2a p.S87L variant causes hydroxyl radical-mediated neuropathy, which can be rescued through AAV-based gene therapy.

Chaotropic Effect-Induced Sol-Gel Transition and Radical Stabilization for Bacterially Sensitive Near-Infrared Photothermal Therapy.

Deep-seated bacterial infections (DBIs) are stubborn and deeply penetrate tissues. Eliminating deep-seated bacteria and promoting tissue regeneration remain great challenges. Here, a novel radical-containing hydrogel (SFT-B Gel) cross-linked by a chaotropic effect was designed for the sensing of DBIs and near-infrared photothermal therapy (NIR-II PTT). A silk fibroin solution stained with 4,4',4″-(1,3,5-triazine-2,4,6-triyl)tris(1-methylpyridin-1-ium) (TPT3+) was employed as the backbone, which could be cross-linked by a closo-dodecaborate cluster (B12H122-) through a chaotropic effect to form the SFT-B Gel. More interestingly, the SFT-B Gel exhibited the ability to sense DBIs, which could generate a TPT2+• radical with obvious color changes in the presence of bacteria. The radical-containing SFT-B Gel (SFT-B★ Gel) possessed strong NIR-II absorption and a remarkable photothermal effect, thus demonstrating excellent NIR-II PTT antibacterial activity for the treatment of DBIs. This work provides a new approach for the construction of intelligent hydrogels with unique properties using a chaotropic effect.

Prognostic Value of P63 Expression in Muscle-Invasive Bladder Cancer and Association with Molecular Subtypes-Preliminary Report.

There is an ongoing need for biomarkers that could reliably predict the outcome of BC and that could guide the management of this disease. In this setting, we aimed to explore the prognostic value of the transcription factor P63 in patients with muscle-invasive bladder cancer (MIBC) having undergone radical cystectomy. The correlation between P63 expression and clinicopathological features (tumor stage, nodes involvement, patterns of muscularis propria invasion, papillary architecture, anaplasia, concomitant carcinoma in situ, lymphovascular invasion, perineural invasion, necrosis) and molecular subtyping (basal and luminal type tumors) was tested in 65 radical cystectomy specimens and matched with cancer-specific survival (CSS) and overall survival (OS). P63-negative tumors displayed significantly higher rates of pattern 2 of muscularis propria invasion (50% vs. 14%, p = 0.002) and variant histology (45% vs. 19%, p = 0.022) compared to P63-positive ones. According to the combined expression of CK5/6 and CK20 (Algorithm #1), P63-positive and P63-negative tumors were mostly basal-like and double-negative, respectively (p = 0.004). Using Algorithm #2, based on the combined expression of CK5/6 and GATA3, the vast majority of tumors were luminal overall and in each group (p = 0.003). There was no significant difference in CSS and OS between P63-positive and P63-negative tumors, but the former featured a trend towards longer OS. Though associated with pathological features harboring negative prognostic potential, P63 status as such failed to predict CSS and OS. That said, it may contribute to better molecular subtyping of MIBC.

Clinical and histopathological parameters in transrectal ultrasound-guided biopsies associated with tumor upgrading after radical prostatectomy: A comparative analysis of risk groups.

BACKGROUND: Thanks to technological advances, prostate cancer (PCa) can be diagnosed at a younger age. It is known that most of these patients are in the low-intermediate risk group, and the histological grade of the tumor increases in half of those undergoing radical prostatectomy (Rp) compared to their diagnostic biopsies. This is especially important in terms of active surveillance (AS) and/or the timely evaluation of curative treatment options in patients diagnosed at an early age. Our aim was to investigate clinical and histopathological parameters that may be associated with an increase in the histological grade of the tumor in patients with acinar adenocarcinoma who were diagnosed by transrectal ultrasound-guided biopsy (TRUS-Bx) and underwent Rp. METHODS: A total of 205 patients with classical acinar adenocarcinoma diagnosed by TRUS-Bx without metastasis and who underwent Rp were grouped according to the D'Amico risk classification. Age at diagnosis, serum prostate-specific antigen (PSA), PSA density, prostate volume, Prostate Imaging Reporting and Data System (PI-RADS) score, clinical stage, Gleason Grade Group (GGG), high-grade intraepithelial neoplasia in tumor-free cores (HGPIN) (single and ≥2 cores), perineural invasion (PNI), and lymphovascular invasion (LVI) was obtained. Additionally, GGG, pathological stage, lymph node metastasis, surgical margin positivity, and tumor volume obtained from Rp were evaluated. Comparisons were made between the case groups in which the tumor grade increased and remained the same, in terms of age, serum PSA, PSA density, HGPIN in tumor-free cores (single and ≥2 cores), PNI, and LVI in all biopsies (with or without tumors), as well as risk groups. In addition, the relationships of HGPIN in tumor-free cores (single and ≥2 cores), PNI, and LVI on TRUS-Bx with age, serum PSA and PSA density, tumor volume, surgical margin positivity, pathological stage, lymph node metastasis, and risk groups were examined separately. RESULTS: Of the patients, 72 (35.1%) were in the low-risk group, 95 (46.3%) in the intermediate-risk group, and 38 (18.5%) in the high-risk group. Most of the patients with an increased histological grade (n = 38, 48.1%) were in the low-risk group (p < 0.05) and had an advanced median age. HGPIN in single and ≥2 tumor-free cores and PNI were more common in these patients (p < 0.01, p < 0.001, and p < 0.05, respectively). According to the multivariable analysis, advanced age (odds ratio [OR]: 1.087, 95% confidence interval [CI]: 1.029-1.148, p < 0.05), high serum PSA (OR: 1.047, 95% CI: 1.006-1.090, p < 0.05), HGPIN in ≥2 tumor-free cores (OR: 6.346, 95% CI: 3.136-12.912, p < 0.001), and PNI (OR: 3.138, 95% CI: 1.179-8.356, p < 0.05) were independent risk factors for a tumor upgrade. Furthermore, being in the low-risk group was an independent risk factor when compared to the intermediate- and high-risk groups (OR: 0.187, 95% CI: 0.080-0.437, p < 0.001 and OR: 0.054, 95% CI: 0.013-0.230, p < 0.001, respectively). The HGPIN diagnosis was more common in the low- and intermediate-risk groups. Advanced age at diagnosis, high serum PSA and PSA density values were associated with PNI on TRUS-Bx. High serum PSA and PSA density values were associated with LVI on TRUS-Bx. Surgical margin positivity was higher in cases with PNI and LVI detected by TRUS-Bx. HGPIN in ≥2 tumor-free cores, PNI, and LVI on TRUS-Bx were associated with a higher rate of lymph node metastases. CONCLUSIONS: In patients diagnosed with acinar adenocarcinoma, the presence of HGPIN even in a single tumor-free core on TRUS-Bx was found to be significant in terms of showing an increase in the histological tumor grade in Rp. The diagnosis of HGPIN in ≥2 tumor-free cores on TRUS-Bx was determined as an independent risk factor for an increased Gleason score after Rp. Furthermore, an advanced age, a high serum PSA value, being in the low-risk group, and the presence of PNI were associated with a tumor upgrade. HGPIN in ≥2 tumor-free cores, PNI, and LVI were also associated with lymph node metastasis. Therefore, the diagnosis of HGPIN should be signed out on pathological reports.

Overall and metastasis-free survival of Afro-Caribbean patients with biochemical recurrence after radical prostatectomy.

INTRODUCTION: Early salvage radiotherapy is indicated for patients with biochemical recurrence after radical prostatectomy. However, for various reasons, certain patients do not benefit from this treatment (OBS) or only at a late stage (LSR). There are few studies on this subject and none on a "high-risk" population, such as patients of African descent. Our objective was to estimate the metastasis-free (MFS) and overall survival (OS) of patients who did not receive salvage radiotherapy, and to identify risk factors of disease progression. PATIENTS AND METHODS: This was a single-center retrospective study that included 154 patients, 99 in the OBS group and 55 in the LSR group. All were treated by total prostatectomy for localized prostate cancer between January 2000 and December 2020 and none received early salvage radiotherapy after biochemical recurrence. RESULTS: Baseline characteristics were similar between groups, except for the time to biochemical recurrence. The median follow-up was 10.0 and 11.8 years for the OBS and LSR groups, respectively. The median time from surgery to LSR was 5.1 years. The two groups did not show a significant difference in MFS: 90.6% at 10 years for the OBS group and 93.3% for the LSR group. The median MFS was 19.8 and 19.6 years for the OBS and LSR groups respectively. OS for the OBS group was significantly higher than that for the LSR group (HR: 2.14 [1.07-4.29]; p = 0.03), with 10-year OS of 95.9% for the OBS group and 76.1% for the LSR group. Median OS was 16 and 15.6 years for the OBS and LSR groups, respectively. CONCLUSION: In this study, we observed satisfactory metastasis-free and OS rates relative to those reported in the scientific literature. The challenge is not to question the benefit of early salvage radiotherapy, but to improve the identification of patients at risk of progression through the development of molecular and genomic tests for more highly personalized medicine.

Distribution of neurovascular structures within the prostate gland and their relationship to complications after radical prostatectomy.

BACKGROUND: Radical prostatectomy remains the main choice of treatment for prostate cancer. However, despite improvements in surgical techniques and neurovascular sparing procedures, rates of erectile dysfunction, and urinary incontinence remain variable. This is due, at least in part, to an incomplete understanding of neurovascular structures associated with the prostate. The objective of this study was to provide a comprehensive, detailed histological overview of the distribution of nerves and blood vessels within the prostate, facilitating subsequent correlation of prostatic neurovascular structures with patients' clinical outcomes after radical prostatectomy. METHODS: Neurovascular structures within the prostate were investigated in a total of 309 slides obtained from 15 patients who underwent non-nerve-sparing radical prostatectomy. Immunohistochemical staining was performed to identify and distinguish between parasympathetic and sympathetic nerves, whereas hematoxylin and eosin staining was used to identify blood vessels. The total number, density, and relative position of nerves and blood vessels were established using quantitative morphometry and illustrated using visualization approaches. Patient-specific outcome data were then used to establish whether the internal distribution of nerves and blood vessels within the prostate correlated with the nature and extent of complications after surgery. One-way analysis of variance tests and unpaired t tests were applied to establish statistically significant differences across the measured variables. RESULTS: Nerves and blood vessels were present across all prostatic levels and regions. However, their number and density varied considerably between regions. Assessment of the precise positioning of neurovascular structures revealed that the majority of nerve fibers were located within the dorsal and peripheral aspects of the gland. In contrast, blood vessels were predominantly located within ventral and dorsal midline regions. The number of intraprostatic nerves was found to be significantly lower in patients who recovered their continence within 12 months of surgery, compared to those whose recovery took 12 months or longer. CONCLUSION: We report an unexpected disconnect between the localization and positioning of nerve fibers and blood vessels within the prostate. Moreover, individual variability in the density of intraprostatic neurovascular structures appears to correlate with the successful recovery of urinary continence after radical prostatectomy, suggesting that differences in intrinsic neurovascular arrangements of the prostate influence postoperative outcomes.

Serum (-2)proPSA/freePSAratio, (-2)proPSA/freePSA density, prostate health index, and prostate health index density as clues to reveal postoperative clinically significant prostate cancer in men with prostate-specific antigen 2-10 ng/mL.

BACKGROUND: There is a strong clinical need to fill the gap of identifying clinically significant prostate cancer (csPCa) in men with prostate-specific antigen (PSA) gray zone values. Promising, but not definitive results have been obtained using PSA derivatives such as prostate health index (PHI) and PHI density (PHID) and the percentage (-2)proPSA/free PSA (%p2PSA/fPSA). Thus, this study aimed to compare the diagnostic value of PHI, PHID, %proPSA/fPSA, and (-2)proPSA/freePSA density (-2pPSA/fPSAD) for csPCa in the patients with PSA within 2-10 ng/mL. METHODS: Serum samples and clinicopathological features were prospectively collected from 142 patients who underwent robot-assisted radical prostatectomy  between September 2021 and December 2023. According to the inclusion criteria, the patients with total PSA  within 2 and 10 ng/mL and negative or suspicious digital rectal examination  were enrolled. We used two different classifications for csPCa: 1) patients with Gleason score (GS) ≥ 7(4 + 3) and 2) patients with GS ≥ 7(3 + 4). The receiver operating characteristic curves and the area under the curve (AUC) values were used to assess the diagnostic performance. RESULTS: Of the 142 men included, 116 (82%) patients were diagnosed with csPCa as GS ≥ 3 + 4 and 107 (75%) defined as csPCa as GS ≥ 7(4 + 3), respectively. We found that p2PSA/fPSA, p2PSA/fPSAD, PHI, and PHID were significantly higher in csPCa classified as GS ≥ 7(3 + 4) as well as GS ≥ 7(4 + 3), with p-values 0.027, 0.054, 0.0016, and 0.0027, respectively. AUCs of the analyzed variables were higher when used to predict csPCa as GS ≥ 6 compared to csPCa as GS ≥7(4 + 3), with an AUC equal, respectively, to 0.679 (95% CI: 0.571-0.786), 0.685 (95% CI: 0.571-0.799), 0.737 (95% CI: 0.639-0.836), and 0.736 (95% CI: 0.630-0.841) in the first subgroup and with an AUC equal, respectively, to 0.653 (95% CI: 0.552-0.754), 0.665 (95% CI: 0.560-0.770), 0.668 (95% CI: 0.568-0.769), and 0.670 (95% CI: 0.567-0.773) in the second, respectively. Both PHID and p2PSA/fPSAD allowed improvement in the diagnostic accuracy with respect to PHI and p2PSA/fPSA ratio, however the differences were not statistically significant (p = 0.409, 0.180 for csPCa as G ≥ Gleason grade (GG) 2 and 0.558 and 0.087 for csPCa as G ≥ GG3, respectively). We found that PHI, PHID, p2PSA/fPSA ratio, and p2PSA/fPSAD showed higher sensitivity, specificity, and positive predictive value when used to predict csPCa as GG ≥ 2, whereas negative predictive value of all four parameters was higher when used to predict GG ≥ 3. CONCLUSIONS: In men with a PSA level between 2 and 10 ng/mL, PHI and PHID, p2PSA/fPSA, and p2PSA/fPSAD showed good diagnostic performance for postoperative csPCa. However, PHID and p2PSA/fPSAD had a small advantage over PHI which needs to be further investigated for the reduction of unnecessary surgical interventions. This finding suggests that it could be a promising biomarker for making the treatment-decision strategy.

Postradical prostatectomy prostate-specific antigen outcomes after 6 versus 18 months of perioperative androgen-deprivation therapy in men with localized, unfavorable intermediate-risk or high-risk prostate cancer: Results of part 2 of a randomized phase 2 trial.

BACKGROUND: Patients with localized, unfavorable intermediate-risk and high-risk prostate cancer have an increased risk of relapse after radical prostatectomy (RP). The authors previously reported on part 1 of this phase 2 trial testing neoadjuvant apalutamide, abiraterone, prednisone, plus leuprolide (AAPL) or abiraterone, prednisone, and leuprolide (APL) for 6 months followed by RP. The results demonstrated favorable pathologic responses (tumor <5 mm) in 20.3% of patients (n = 24 of 118). Herein, the authors report the results of part 2. METHODS: For part 2, patients were randomized 1:1 to receive either AAPL for 12 months (arm 2A) or observation (arm 2B), stratified by neoadjuvant therapy and pathologic tumor classification. The primary end point was 3-year biochemical progression-free survival. Secondary end points included safety and testosterone recovery (>200 ng/dL). RESULTS: Overall, 82 of 118 patients (69%) enrolled in part 1 were randomized to part 2. A higher proportion of patients who were not randomized to adjuvant therapy had a favorable prostatectomy pathologic response (32.3% in nonrandomized patients compared with 17.1% in randomized patients). In the intent-to-treat analysis, the 3-year biochemical progression-free survival rate was 81% for arm 2A and 72% for arm 2B (hazard ratio, 0.81; 90% confidence interval, 0.43-1.49). Of the randomized patients, 81% had testosterone recovery in the AAPL group compared with 95% in the observation group, with a median time to recovery of <12 months in both arms. CONCLUSIONS: In this study, because 30% of patients declined adjuvant treatment, part B was underpowered to detect differences between arms. Future perioperative studies should be biomarker-directed and include strategies for investigator and patient engagement to ensure compliance with protocol procedures.

Decoding the neural impact of radical complexity in Chinese characters during working memory task.

Readers of Chinese characters need to recognize how they are formed in order to identify them correctly. However, our understanding of the cognitive processing of characters in working memory is limited. In Experiment 1, using the character N-back task paradigm, electrophysiological data were recorded from 26 participants to investigate the effects of the visual feature of radicals on neural activity during the character recognition, updating and maintenance in the N-back task. Results showed that compound characters required longer response times than single-component characters. For the event-related potentials (ERPs), the compound character condition had more negative N2pc and lower P300 amplitudes than the single-component character condition. In Experiment 2, data from 26 participants were used to analyse the effect of the phonological feature of radicals on neural activity during the character recognition, updating and maintenance in the N-back task. Results showed that there was a larger P200 in the irregular character condition than in the regular character condition, but there was no difference between the regular and the irregular characters in the N2pc, P300 and slow wave (SW) components. The visual feature and the phonological feature of the radicals may have different effects on the character processing. This study reveals the neural effects of Chinese character radicals on cognitive processing in a working memory task and provides behavioural and electrophysiological evidence for a theoretical model of verbal working memory subprocesses.

Cisplatin eligibility in the neoadjuvant setting of patients with muscle-invasive bladder cancer undergoing radical cystectomy.

BACKGROUND: To examine the agreement of different calculated estimated glomerular filtration rate (eGFR) formulas and measured creatinine clearance (CrCI) at the primary diagnosis of muscle-invasive bladder cancer (MIBC). MATERIALS AND METHODS: We performed a multicenter analysis of patients with MIBC, treated with cisplatin-based neoadjuvant chemotherapy (NAC) and radical cystectomy (RC), or with RC alone, between 2011 and 2021. Baseline eGFR was computed using 4 calculated serum equations including Cockcroft-Gault (CG), MDRD, CKD-EPI 2009, and race-free CKD-EPI 2021. To examine the association between calculated eGFR and measured CrCI, subgroup analyses were performed among patients in whom measured 24-hour urine CrCl was determined. Cisplatin-ineligibility was defined as CrCI and/or eGFR < 60 mL/minute per 1.73 m2. RESULTS: Of 956 patients, 30.0%, 33.3%, 31.9%, and 27.7% were found to be cisplatin-ineligible by the CG, MDRD, CKD-EPI, and race-free CKD-EPI equations (P = .052). The concordance between calculated eGFR formulas was rated substantial (Cohen's kappa (k): 0.66-0.95). Among the subgroup (n = 245) with measured CrCl, 37 (15.1%) patients had a CrCI less than 60 mL/minute. Concordance between measured CrCl and calculated eGFR was poor (ĸ: 0.29-0.40). All calculated eGFR formulas markedly underestimated the measured CrCI. Specifically, 78%-87.5% of patients with a calculated eGFR between 40 and 59 mL/minute exhibited a measured CrCI ≥ 60 mL/minute. CONCLUSIONS: Comparing calculated eGFR formulas, similar percentages of patients with MIBC were deemed cisplatin-ineligible. However, a significant number of patients could be upgraded by being cisplatin-fit based on measured CrCI, particularly when the calculated eGFR was falling within the gray range of 40-59 mL/minute.

Underlying mechanism of Dendrobium huoshanense resistance to lead stress using the quantitative proteomics method.

Lead affects photosynthesis and growth and has serious toxic effects on plants. Here, the differential expressed proteins (DEPs) in D. huoshanense were investigated under different applications of lead acetate solutions. Using label-free quantitative proteomics methods, more than 12,000 peptides and 2,449 proteins were identified. GO and KEGG functional annotations show that these differential proteins mainly participate in carbohydrate metabolism, energy metabolism, amino acid metabolism, translation, protein folding, sorting, and degradation, as well as oxidation and reduction processes. A total of 636 DEPs were identified, and lead could induce the expression of most proteins. KEGG enrichment analysis suggested that proteins involved in processes such as homologous recombination, vitamin B6 metabolism, flavonoid biosynthesis, cellular component organisation or biogenesis, and biological regulation were significantly enriched. Nearly 40 proteins are involved in DNA replication and repair, RNA synthesis, transport, and splicing. The effect of lead stress on D. huoshanense may be achieved through photosynthesis, oxidative phosphorylation, and the production of excess antioxidant substances. The expression of 9 photosynthesis-related proteins and 12 oxidative phosphorylation-related proteins was up-regulated after lead stress. Furthermore, a total of 3 SOD, 12 POD, 3 CAT, and 7 ascorbate-related metabolic enzymes were identified. Under lead stress, almost all key enzymes involved in the synthesis of antioxidant substances are up-regulated, which may facilitate the scavenging of oxygen-free radical scavenging. The expression levels of some key enzymes involved in sugar and glycoside synthesis, the phenylpropanoid synthesis pathway, and the terpene synthesis pathway also increased. More than 30 proteins involved in heavy metal transport were also identified. Expression profiling revealed a significant rise in the expression of the ABC-type multidrug resistance transporter, copper chaperone, and P-type ATPase with exposure to lead stress. Our findings lay the basis for research on the response and resistance of D. huoshanense to heavy metal stress.