Homogeneous continuous flow nitration of O-methylisouronium sulfate and its optimization by kinetic modeling.

Nitration of O-methylisouronium sulfate under mixed acid conditions gives O-methyl-N-nitroisourea, a key intermediate of neonicotinoid insecticides with high application value. The reaction is a fast and highly exothermic process with a high mass transfer resistance, making its control difficult and risky. In this paper, a homogeneous continuous flow microreactor system was developed for the nitration of O-methylisouronium sulfate under high concentrations of mixed acids, with a homemade static mixer eliminating the mass transfer resistance. In addition, the kinetic modeling of this reaction was performed based on the theory of NO2 + attack, with the activation energy and pre-exponential factor determined. Finally, based on the response surface generated by the kinetic model, the reaction was optimized with a conversion of 87.4% under a sulfuric acid mass fraction of 94%, initial reactant concentration of 0.5 mol/L, reaction temperature of 40 °C, molar ratio of reactants at 4.4:1, and a residence time of 12.36 minutes.

A RANKL-UCHL1-sCD13 negative feedback loop limits osteoclastogenesis in subchondral bone to prevent osteoarthritis progression.

Abnormal subchondral bone remodeling plays a pivotal role in the progression of osteoarthritis (OA). Here, we analyzed subchondral bone samples from OA patients and observed a significant upregulation of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) specifically in subchondral bone osteoclasts. Notably, we found a strong correlation between UCHL1 expression and osteoclast activity in the subchondral bone during OA progression in both human and murine models. Conditional UCHL1 deletion in osteoclast precursors exacerbated OA progression, while its overexpression, mediated by adeno-associated virus 9, alleviated this process in male mice. Mechanistically, RANKL stimulates UCHL1 expression in osteoclast precursors, subsequently stabilizing CD13, augmenting soluble CD13 (sCD13) release, and triggering an autocrine inhibitory effect on the MAPK pathway, thereby suppressing osteoclast formation. These findings unveil a previously unidentified negative feedback loop, RANKL-UCHL1-sCD13, that modulates osteoclast formation and presents a potential therapeutic target for OA.

RNA N6-methyladenosine modification in arthritis: New insights into pathogenesis.

The commonest type of eukaryotic RNA modification, N6-methyladenosine (m6A), has drawn increased scrutiny in the context of pathological functioning as well as relevance in determination of RNA stability, splicing, transportation, localization, and translation efficiency. The m6A modification plays an important role in several types of arthritis, especially osteoarthritis and rheumatoid arthritis. Recent studies have reported that m6A modification regulates arthritis pathology in cells, such as chondrocytes and synoviocytes via immune responses and inflammatory responses through functional proteins classified as writers, erasers, and readers. The aim of this review was to highlight recent advances relevant to m6A modification in the context of arthritis pathogenesis and detail underlying molecular mechanisms, regulatory functions, clinical applications, and future perspectives of m6A in arthritis with the aim of providing a foundation for future research directions.

Binding structures of SERF1a with NT17-polyQ peptides of huntingtin exon 1 revealed by SEC-SWAXS, NMR and molecular simulation.

The aberrant fibrillization of huntingtin exon 1 (Httex1) characterized by an expanded polyglutamine (polyQ) tract is a defining feature of Huntington's disease, a neurodegenerative disorder. Recent investigations underscore the involvement of a small EDRK-rich factor 1a (SERF1a) in promoting Httex1 fibrillization through interactions with its N terminus. By establishing an integrated approach with size-exclusion-column-based small- and wide-angle X-ray scattering (SEC-SWAXS), NMR, and molecular simulations using Rosetta, the analysis here reveals a tight binding of two NT17 fragments of Httex1 (comprising the initial 17 amino acids at the N terminus) to the N-terminal region of SERF1a. In contrast, examination of the complex structure of SERF1a with a coiled NT17-polyQ peptide (33 amino acids in total) indicates sparse contacts of the NT17 and polyQ segments with the N-terminal side of SERF1a. Furthermore, the integrated SEC-SWAXS and molecular-simulation analysis suggests that the coiled NT17 segment can transform into a helical conformation when associated with a polyQ segment exhibiting high helical content. Intriguingly, NT17-polyQ peptides with enhanced secondary structures display diminished interactions with SERF1a. This insight into the conformation-dependent binding of NT17 provides clues to a catalytic association mechanism underlying SERF1a's facilitation of Httext1 fibrillization.

Insights about cervical lymph nodes: Evaluating deep learning-based reconstruction for head and neck computed tomography scan.

Purpose: This study aimed to investigate differences in cervical lymph node image quality on dual-energy computed tomography (CT) scan with datasets reconstructed using filter back projection (FBP), hybrid iterative reconstruction (IR), and deep learning-based image reconstruction (DLIR) in patients with head and neck cancer. Method: Seventy patients with head and neck cancer underwent follow-up contrast-enhanced dual-energy CT examinations. All datasets were reconstructed using FBP, hybrid IR with 30 % adaptive statistical IR (ASiR-V), and DLIR with three selectable levels (low, medium, and high) at 2.5- and 0.625-mm slice thicknesses. Herein, signal, image noise, signal-to-noise ratio, and contrast-to-noise ratio of lymph nodes and overall image quality, artifact, and noise of selected regions of interest were evaluated by two radiologists. Next, cervical lymph node sharpness was evaluated using full width at half maximum. Results: DLIR exhibited significantly reduced noise, ranging from 3.8 % to 35.9 % with improved signal-to-noise ratio (11.5-105.6 %) and contrast-to-noise ratio (10.5-107.5 %) compared with FBP and ASiR-V, for cervical lymph nodes (p < 0.001). Further, 0.625-mm-thick images reconstructed using DLIR-medium and DLIR-high had a lower noise than 2.5-mm-thick images reconstructed using FBP and ASiR-V. The lymph node margins and vessels on DLIR-medium and DLIR-high were sharper than those on FBP and ASiR-V (p < 0.05). Both readers agreed that DLIR had a better image quality than the conventional reconstruction algorithms. Conclusion: DLIR-medium and -high provided superior cervical lymph node image quality in head and neck CT. Improved image quality affords thin-slice DLIR images for dose-reduction protocols in the future.

Shearing-induced formation of Au nanowires.

Shearing-induced nucleation is known in our daily lives, yet rarely discussed in nano-synthesis. Here, we demonstrate an unambiguous shearing-induced growth of Au nanowires. While in static solution Au would predominately deposit on pre-synthesized triangular nanoplates to form nano-bowls, the introduction of stirring or shaking gives rise to nanowires, where an initial nucleation could be inferred. Under specific growth conditions, CTAB is responsible for stabilizing the growth materials and the resulting oversaturation promotes shearing-induced nucleation. At the same time, all Au surfaces are passivated by ligands, so that the growth materials are diverted to relatively fresher sites. We propose that the different degrees of "focused growth" in active surface growth could be represented by watersheds of different slopes, so that the subtle differences between neighbouring sites would set course to opposite pathways, with some sites becoming ever more active and others ever more inhibited. The shearing-induced nuclei, with their initially ligand-deficient surface and higher accessibility to growth materials, win the dynamic inter-particle competition against other sites, explaining the dramatic diversion of growth materials from the seeds to the nanowires.

GAPS: a geometric attention-based network for peptide binding site identification by the transfer learning approach.

Protein-peptide interactions (PPepIs) are vital to understanding cellular functions, which can facilitate the design of novel drugs. As an essential component in forming a PPepI, protein-peptide binding sites are the basis for understanding the mechanisms involved in PPepIs. Therefore, accurately identifying protein-peptide binding sites becomes a critical task. The traditional experimental methods for researching these binding sites are labor-intensive and time-consuming, and some computational tools have been invented to supplement it. However, these computational tools have limitations in generality or accuracy due to the need for ligand information, complex feature construction, or their reliance on modeling based on amino acid residues. To deal with the drawbacks of these computational algorithms, we describe a geometric attention-based network for peptide binding site identification (GAPS) in this work. The proposed model utilizes geometric feature engineering to construct atom representations and incorporates multiple attention mechanisms to update relevant biological features. In addition, the transfer learning strategy is implemented for leveraging the protein-protein binding sites information to enhance the protein-peptide binding sites recognition capability, taking into account the common structure and biological bias between proteins and peptides. Consequently, GAPS demonstrates the state-of-the-art performance and excellent robustness in this task. Moreover, our model exhibits exceptional performance across several extended experiments including predicting the apo protein-peptide, protein-cyclic peptide and the AlphaFold-predicted protein-peptide binding sites. These results confirm that the GAPS model is a powerful, versatile, stable method suitable for diverse binding site predictions.

Transfer learning across different chemical domains: virtual screening of organic materials with deep learning models pretrained on small molecule and chemical reaction data.

Machine learning is becoming a preferred method for the virtual screening of organic materials due to its cost-effectiveness over traditional computationally demanding techniques. However, the scarcity of labeled data for organic materials poses a significant challenge for training advanced machine learning models. This study showcases the potential of utilizing databases of drug-like small molecules and chemical reactions to pretrain the BERT model, enhancing its performance in the virtual screening of organic materials. By fine-tuning the BERT models with data from five virtual screening tasks, the version pretrained with the USPTO-SMILES dataset achieved R2 scores exceeding 0.94 for three tasks and over 0.81 for two others. This performance surpasses that of models pretrained on the small molecule or organic materials databases and outperforms three traditional machine learning models trained directly on virtual screening data. The success of the USPTO-SMILES pretrained BERT model can be attributed to the diverse array of organic building blocks in the USPTO database, offering a broader exploration of the chemical space. The study further suggests that accessing a reaction database with a wider range of reactions than the USPTO could further enhance model performance. Overall, this research validates the feasibility of applying transfer learning across different chemical domains for the efficient virtual screening of organic materials.Scientific contributionThis study verifies the feasibility of applying transfer learning to large language models in different chemical fields to help organic materials perform virtual screening. Through the comparison of transfer learning from different chemical fields to a variety of organic material molecules, the high precision virtual screening of organic materials is realized.

Successful Extension of Vascularized Composite Allograft Perfusion Cold Storage to 24 h in a Rat Hindlimb Transplant Model.

Background: Vascularized composite allograft transplantation is a treatment option for complex tissue injuries; however, ischemia reperfusion injury and high acute rejection rates remain a challenge. Hypothermic machine perfusion using acellular storage perfusate is a potential solution. This study evaluated the University of Wisconsin Kidney Preservation Solution-1 (KPS-1) compared with normal saline (NS) for preservation of donor rat hindlimbs subjected to 24 h of ex vivo perfusion cold storage. Methods: Hindlimbs were subjected to 24-h perfusion cold storage with heparinized KPS-1 (n = 6) or heparinized NS (n = 6). Flow, resistance, and pH were measured continuously. At the end of the 24-h period, tissue was collected for histological analysis of edema and apoptosis. Results: KPS-1 perfused limbs showed significantly less edema than the NS group, as evidenced by lower limb weight gain (P < 0.001) and less interfascicular space (P < 0.001). KPS-perfused muscle had significantly less cell death than NS-perfused muscle based on terminal deoxynucleotidyl transferase dUTP nick-end labeling (P < 0.001) and cleaved caspase-3 staining (P = 0.045). During hypothermic machine perfusion, a significant decrease in pH over time was detected in both groups, with a significantly greater decline in pH in the KPS-1 group than in the NS group. There were no significant differences overall and over time in flow rate or vascular resistance between the KPS and NS groups. Conclusions: Perfusion with KPS-1 can successfully extend vascularized composite allograft perfusion cold storage for 24 h in a rat hindlimb model without significant edema or cell death.

Retinal transplant immunology and advancements.

Several gaps and barriers remain for transplanting stem cells into the eye to treat ocular disease, especially diseases of the retina. While the eye has historically been considered immune privileged, recent thinking has identified the immune system as both a barrier and an opportunity for eye stem cell transplantation. Recent approaches leveraging scaffolds or cloaking have been considered in other tissues beyond immune suppression. This perspective paper outlines approaches for transplantation and proposes opportunities to overcome barriers of the immune system in stem cell transplantation in the eye.

Bleb characteristics assessed by ultrasound biomicroscopy correlate with intraocular pressure after Ahmed glaucoma valve surgery in dogs.

OBJECTIVE: To assess the characteristics of blebs formed after Ahmed glaucoma valve (AGV) surgery in dogs using ultrasound biomicroscopy (UBM) and to analyze their correlation with postoperative intraocular pressure (IOP). ANIMALS: 16 eyes (13 dogs) were diagnosed with primary angle-closure glaucoma and were followed up after AGV surgery from June 2021 to September 2023. METHODS: In this prospective study, UBM examinations were performed to assess bleb characteristics, including bleb wall thickness and reflectivity. IOP at the time of UBM imaging and the duration from AGV surgery to UBM imaging were recorded. Histological examination of an enucleated eye removed due to uncontrolled IOP leading to blindness was also conducted. RESULTS: A significant correlation was observed between IOP and relative reflectivity (Pearson r = 0.60; P = .01), and a negative correlation was observed between bleb wall thickness and relative reflectivity (Pearson r = -0.72; P = .002). No significant correlation was observed between the duration from AGV surgery to UBM imaging and either bleb wall thickness or relative reflectivity, respectively. Histological examination of the enucleated eye revealed collagen-rich fibrous encapsulation of the bleb wall, including myofibroblasts that exhibited positive α-smooth muscle actin immunostaining. CLINICAL RELEVANCE: In dogs that underwent AGV surgery, less dense, thick-walled blebs on UBM tended to maintain IOP within the normal range. However, denser, thinner-walled blebs showed IOP levels above the normal range despite the use of antiglaucoma medications. UBM is a useful tool for evaluating bleb characteristics and their influence on IOP regulation after AGV surgery in dogs.

Clinical significance of an elevated on-admission beta-hydroxybutyrate in acutely ill adult patients without diabetes.

OBJECTIVE: To determine the relationship between point-of-care ß-hydroxybutyrate (BHB) concentration and outcomes in adult patients without diabetes admitted through ED. METHODS: This was a prospective study from 10 March to 2 July 2021. Admitted patients without diabetes had capillary BHB sampled in ED. Outcomes of length-of-stay (LOS), composite mortality/ICU admission rates and clinical severity scores (Quick Sepsis Organ Failure Assessment score/National Early Warning Score [qSOFA/NEWS]) were measured. BHB was assessed as a continuous variable and between those with BHB above and equal to 1.0 mmol/L and those below 1.0 mmol/L. RESULTS: A total of 311 patients were included from 2377 admissions. Median length-of-stay was 4.1 days (IQR 2.1-9.8), 18 (5.8%) died and 37 (11.8%) were admitted to ICU. Median BHB was 0.2 mmol/L (IQR 0.1-0.4). Twenty-five patients had BHB ≥1.0 mmol/L and five were >3.0 mmol/L. There was no significant difference in median LOS for patients with BHB ≥1.0 mmol/L compared to non-ketotic patients, 5.3 days (IQR 2.2-7.5) versus 4.1 days, respectively (IQR 2.0-9.8) (P = 0.69). BHB did not correlate with LOS (Spearman ρ = 0.116, 95% confidence interval: 0.006-0.223). qSOFA and NEWS also did not differ between these cohorts. For those 25 patients with BHB ≥1.0 mmol/L, an infective/inflammatory diagnosis was present in 11 (44%), at least 2 days of fasting in 10 (40%) and ethanol intake >40 g within 48 h in 4 (16%). CONCLUSIONS: Routine BHB measurement in patients without diabetes does not add to clinical bedside assessment and use should be limited to when required to confirm a clinical impression.

Recurrent Tuberous Sclerosis Complex/Mammalian Target of Rapamycin Mutations Define Primary Renal Hemangioblastoma as a Unique Entity Distinct From Its Central Nervous System Counterpart.

ABSTRACT: Renal hemangioblastoma (HB) is a rare subset of HBs arising outside of the central nervous system (CNS), with its molecular drivers remaining entirely unknown. There were no significant alterations detected in previous studies, including von Hippel-Lindau gene alterations, which are commonly associated with CNS-HB. This study aimed to determine the real molecular identity of renal HB and better understand its relationship with CNS-HB. A cohort of 10 renal HBs was submitted for next-generation sequencing technology. As a control, 5 classic CNS-HBs were similarly analyzed. Based on the molecular results, glycoprotein nonmetastatic B (GPNMB) immunohistochemistry was further performed in the cases of renal HB and CNS-HB. Mutational analysis demonstrated that all 10 renal HBs harbored somatic mutations in tuberous sclerosis complex 1 ( TSC1 , 5 cases), TSC2 (3 cases), and mammalian target of rapamycin (2 cases), with the majority classified as pathogenic or likely pathogenic. The CNS-HB cohort uniformly demonstrated somatic mutations in the von Hippel-Lindau gene. GPNMB was strong and diffuse in all 10 renal HBs and completely negative in CNS-HBs, reinforcing the molecular findings. Our study reveals a specific molecular hallmark in renal HB, characterized by recurrent TSC/mammalian target of rapamycin mutations, which defines it as a unique entity distinct from CNS-HB. This molecular finding potentially expands the therapeutic options for patients with renal HB. GPNMB can be considered for inclusion in immunohistochemical panels to improve renal HB identification.

Spermidine ameliorates osteoarthritis via altering macrophage polarization.

OBJECTIVE: Spermidine (SPD) is an anti-aging natural substance, and it exerts effects through anti-apoptosis and anti-inflammation. However, the specific protective mechanism of SPD in osteoarthritis (OA) remains unclear. Here, we explored the role of SPD on the articular cartilage and the synovial tissue, and tested whether the drug would regulate the polarization of synovial macrophages by in vivo and in vitro experiments. METHODS: By constructing an OA model in mice, we preliminarily explored the protective effect of SPD on the articular cartilage and the synovial tissue. Meanwhile, we isolated and cultured human primary chondrocytes and bone marrow-derived macrophages (BMDMs), and prepared a conditioned medium (CM) to explore the specific protective effect of SPD in vitro. RESULTS: We found that SPD alleviated cartilage degeneration and synovitis, increased M2 polarization and decreased M1 polarization in synovial macrophages. In vitro experiments, SPD inhibited ERK MAPK and p65/NF-κB signaling in macrophages, and transformed macrophages from M1 to M2 subtypes. Interestingly, SPD had no direct protective effect on chondrocytes in vitro; however, the conditioned medium (CM) from M1 macrophages treated with SPD promoted the anabolism and inhibited the catabolism of chondrocytes. Moreover, this CM markedly suppressed IL-1ß-induced p38/JNK MAPK signaling pathway activation in chondrocytes. CONCLUSIONS: This work provides new perspectives on the role of SPD in OA. SPD does not directly target chondrocytes, but can ameliorate the degradation of articular cartilage through regulating M1/M2 polarization of synovial macrophages. Hence, SPD is expected to be the potential therapy for OA.

Temporomandibular joint degenerative changes following mandibular fracture: a computed tomography-based study on the role of condylar involvement.

OBJECTIVES: This study assessed the incidence of postfracture radiological temporomandibular joint (TMJ) degeneration in patients with different types of mandibular fractures, focusing on the impact of condylar fractures. METHODS: This retrospective review included patients diagnosed as having mandibular fractures from 2016 to 2020 who had undergone initial computed tomography (CT) and a follow-up CT scan at least 1-month postfracture. Patient demographics, fracture details, treatment methods, and radiological signs of TMJ degeneration on CT were analyzed to identify risk factors for postfracture TMJ degeneration, with a focus on condylar head fracture and non-head (condylar neck or base) fractures. RESULTS: The study included 85 patients (mean age: 38.95 ± 17.64 years). The per-patient analysis indicated that the incidence of new radiologic TMJ degeneration on CT was significantly the highest (p < 0.001) in patients with condylar head fractures (90.91%), followed by those with non-head condylar fractures (57.14%), and those without condylar involvement (24.49%). The per-joint analysis indicated nearly inevitable degeneration (93.94%) in 33 TMJs with ipsilateral condylar head fractures. For the remaining 137 TMJs, multivariate logistic regression revealed that other patterns (ipsilateral non-head, contralateral, or both) of condylar fractures (odds ratio (OR) = 3.811, p = 0.007) and the need for open reduction and internal fixation (OR = 5.804, p = 0.005) significantly increased the risk of TMJ degeneration. CONCLUSIONS: Ipsilateral non-head condylar fractures and contralateral condylar fractures are associated with a high risk of postfracture TMJ degeneration. Indirect trauma plays a vital role in postfracture TMJ degeneration.

Toxicology, pharmacokinetics, and immunogenicity studies of CCR4-IL2 bispecific immunotoxin in rats and minipigs.

We have developed a diphtheria toxin-based recombinant human CCR4-IL2 bispecific immunotoxin (CCR4-IL2-IT) for targeted therapy of cutaneous T-cell lymphoma (CTCL). CCR4-IL2-IT demonstrated superior efficacy in an immunodeficient mouse CTCL model. Recently, we have compared the in vivo efficacy of CCR4-IL2-IT versus Brentuximab (FDA approved leading drug in CTCL market) in the same immunodeficient mouse CTCL model. The comparison demonstrated that CCR4-IL2-IT was significantly more effective than Brentuximab. In this study, we have performed non-GLP (Good Laboratory Practice) toxicology, pharmacokinetics, immunogenicity studies of CCR4-IL2-IT in both rats and minipigs. CCR4-IL2-IT demonstrated excellent safety profiles in both rats and minipigs. The maximum tolerated dose of CCR4-IL2-IT was determined as 0.4 mg/kg in both rats and minipigs. Complete blood count and chemistry analysis did not show significant difference for all measured parameters between the blood samples of pre-injection versus post-injection from the five-day toxicology studies of CCT4-IL2-IT in both rats and minipigs. Histology analysis did not show difference between the PBS treatment group versus CCR4-IL2-IT treatment group at 50 µg/kg in both rats and minipigs. The half-life of CCR4-IL2-IT was determined as about 45 min in rats and 30 min in minipigs. The antibodies against CCR4-IL2-IT were detected in about two weeks after CCR4-IL2-IT treatment. CCR4-IL2-IT did not induce cytokine release syndrome in a peripheral blood mononuclear cell derived humanized mouse model. The depletion of CCR4+ cell and CD25+ cell (two target cell populations of CCR4-IL2-IT) was observed in minipigs. The excellent safety profile promoted us to further develop CCR4-IL2-IT towards clinical trials.

Rutin promotes M2 phenotype microglia polarization by suppressing the JAK/STAT3 signaling to protect against retinal ischemia-reperfusion injury.

We aimed to investigate the neuroprotective effect of rutin on retinal ganglion cells (RGCs) under ischemia-reperfusion (I/R) conditions and the underlying mechanisms involving microglia polarization and JAK/STAT3 signaling. RGCs isolated from C57/Bl6 mice were co-cultured with BV2 microglial cells under normal or in vitro oxygen-glucose deprivation and reoxygenation (OGD/R) conditions. Rutin's effects were evaluated by assessing cell viability, apoptosis rates, cytokine levels, microglial polarization markers and JAK/STAT3 phosphorylation levels. The specific target is confirmed through the inhibitory effect of rutin on the respectively activated signaling factors. Furthermore, molecular docking analyses elucidated rutin-JAK1 interactions. OGD/R conditions significantly reduced RGC viability, exacerbated by BV2 co-culture. However, both 1 µM and 5 µM rutin treatment dose-dependently enhanced RGC viability, reduced apoptosis, and suppressed pro-inflammatory cytokine levels. Western blot analysis indicated that rutin promoted the M2 microglial phenotype and suppressed JAK/STAT3 signaling. Notably, rutin selectively inhibited JAK1 phosphorylation without affecting STAT3. Molecular docking highlighted potential interaction sites between rutin and specific JAK1 pseudokinase domain. Rutin exerts neuroprotective effects against retinal I/R injury by promoting M2 microglial polarization, potentially through the selective inhibition of JAK1 phosphorylation within the JAK/STAT3 signaling pathway. These findings provide a foundation for the therapeutic potential of rutin in retinal I/R injuries.