Significance and amplification methods of the purine salvage pathway in human brain cells.

Previous studies suggest that uric acid or reactive oxygen species, products of xanthine oxidoreductase (XOR), may associate with neurodegenerative diseases. However, neither relationship has ever been firmly established. Here, we analyzed human brain samples, obtained under protocols approved by research ethics committees, and found no expression of XOR and only low levels of uric acid in various regions of the brain. In the absence of XOR, hypoxanthine will be preserved and available for incorporation into the purine salvage pathway. To clarify the importance of salvage in the brain, we tested using human-induced pluripotent stem cell-derived neuronal cells. Stable isotope analyses showed that the purine salvage pathway was more effective for ATP synthesis than purine de novo synthesis. Blood uric acid levels were related to the intracellular adenylate pool (ATP + ADP + AMP), and reduced levels of this pool result in lower uric acid levels. XOR inhibitors are related to extracellular hypoxanthine levels available for uptake into the purine salvage pathway by inhibiting the oxidation of hypoxanthine to xanthine and uric acid in various organs where XOR is present and can prevent further decreases in the intracellular adenylate pool under stress. Furthermore, adding precursors of the pentose phosphate pathway enhanced hypoxanthine uptake, indicating that purine salvage is activated by phosphoribosyl pyrophosphate replenishment. These findings resolve previous contradictions regarding XOR products and provide new insights into clinical studies. It is suggested that therapeutic strategies maximizing maintenance of intracellular adenylate levels may effectively treat pathological conditions associated with ischemia and energy depletion.

Characteristics and clinical outcomes of patients with combined burns and trauma in Japan: Analysis of a nationwide trauma registry database.

INTRODUCTION: Patients with combined burns and trauma are often seen in the United States. The combination of trauma with burns increases mortality. In contrast, the characteristics and outcomes of these cases remain unknown in Japan. This study investigated the characteristics and outcomes of trauma associated with burns in Japan. METHODS: This multicenter retrospective cohort study was conducted by utilizing data from the Japan Trauma Data Bank for the period between 2004 and 2017. We evaluated the characteristics of burn patients (n = 5783) divided into two groups: burns only (n = 5537) and combined burns and trauma (n = 246). Clinical characteristics, including patient background, severity of trauma, injury mechanism, total body surface area affected, injury location, treatments, and clinical outcomes, were examined. RESULTS: Most patients in both the groups were injured by flames. The number proportion of patients with 40-89% of the total body surface area affected was 1069/5537 (19.3%) in the burn-only group and 23/246 (9.3%) in the combined burn and trauma group. The in-hospital mortality was 1006/5537 (18.2%) in the burn-only group and 17/246 (6.9%) in the combined burn and trauma group. CONCLUSIONS: We demonstrated the characteristics of Japanese patients with burns only compared with those with combined burns and trauma. Flames were the main cause of burns, and in-hospital mortality was lower in the combined burn and trauma group associated with a smaller burn area.

The components of the AhR-molecular chaperone complex differ depending on whether the ligands are toxic or non-toxic.

The aryl hydrocarbon receptor (AhR) forms a complex with the HSP90-XAP2-p23 molecular chaperone when the cells are exposed to toxic compounds. Recently, 1,4-dihydroxy-2-naphthoic acid (DHNA) was reported to be an AhR ligand. Here, we investigated the components of the molecular chaperone complex when DHNA binds to AhR. Proteins eluted from the 3-Methylcolanthrene-affinity column were AhR-HSP90-XAP2-p23 complex. The AhR-molecular chaperone complex did not contain p23 in the eluents from the DHNA-affinity column. In 3-MC-treated cells, AhR formed a complex with HSP90-XAP2-p23 and nuclear translocation occurred within 30 min, while in DHNA-treated cells, AhR formed a complex with AhR-HSP90-XAP2, and translocation was slow from 60 min. Thus, the AhR activation mechanism may differ when DHNA is the ligand compared to toxic ligands.

Refolding, Crystallization, and Crystal Structure Analysis of a Scavenger Receptor Cysteine-Rich Domain of Human Salivary Agglutinin Expressed in Escherichia coli.

Scavenger receptors are a protein superfamily that typically consists of one or more repeats of the scavenger receptor cysteine-rich structural domain (SRCRD), which is an ancient and highly conserved protein module. The expression and purification of eukaryotic proteins containing multiple disulfide bonds has always been challenging. The expression systems that are commonly used to express SRCRD proteins mainly consist of eukaryotic protein expression systems. Herein, we established a high-level expression strategy of a Type B SRCRD unit from human salivary agglutinin using the Escherichia coli expression system, followed by a refolding and purification process. The untagged recombinant SRCRD was expressed in E. coli using the pET-32a vector, which was followed by a refolding process using the GSH/GSSG redox system. The SRCRD expressed in E. coli SHuffle T7 showed better solubility after refolding than that expressed in E. coli BL21(DE3), suggesting the importance of the disulfide bond content prior to refolding. The quality of the refolded protein was finally assessed using crystallization and crystal structure analysis. As proteins refolded from inclusion bodies exhibit a high crystal quality and reproducibility, this method is considered a reliable strategy for SRCRD protein expression and purification. To further confirm the structural integrity of the refolded SRCRD protein, the purified protein was subjected to crystallization using sitting-drop vapor diffusion method. The obtained crystals of SRCRD diffracted X-rays to a resolution of 1.47 Å. The solved crystal structure appeared to be highly conserved, with four disulfide bonds appropriately formed. The surface charge distribution of homologous SRCRD proteins indicates that the negatively charged region at the surface is associated with their calcium-dependent ligand recognition. These results suggest that a high-quality SRCRD protein expressed by E. coli SHuffle T7 can be successfully folded and purified, providing new options for the expression of members of the scavenger receptor superfamily.

"KAIZEN" method realizing implementation of deep-learning models for COVID-19 CT diagnosis in real world hospitals.

Numerous COVID-19 diagnostic imaging Artificial Intelligence (AI) studies exist. However, none of their models were of potential clinical use, primarily owing to methodological defects and the lack of implementation considerations for inference. In this study, all development processes of the deep-learning models are performed based on strict criteria of the "KAIZEN checklist", which is proposed based on previous AI development guidelines to overcome the deficiencies mentioned above. We develop and evaluate two binary-classification deep-learning models to triage COVID-19: a slice model examining a Computed Tomography (CT) slice to find COVID-19 lesions; a series model examining a series of CT images to find an infected patient. We collected 2,400,200 CT slices from twelve emergency centers in Japan. Area Under Curve (AUC) and accuracy were calculated for classification performance. The inference time of the system that includes these two models were measured. For validation data, the slice and series models recognized COVID-19 with AUCs and accuracies of 0.989 and 0.982, 95.9% and 93.0% respectively. For test data, the models' AUCs and accuracies were 0.958 and 0.953, 90.0% and 91.4% respectively. The average inference time per case was 2.83 s. Our deep-learning system realizes accuracy and inference speed high enough for practical use. The systems have already been implemented in four hospitals and eight are under progression. We released an application software and implementation code for free in a highly usable state to allow its use in Japan and globally.

Evaluation of the therapeutic efficiency and efficacy of blood purification in the treatment of severe acute pancreatitis.

This study aimed to evaluate the therapeutic efficacy and effect of blood purification (BP) therapy on severe acute pancreatitis (SAP). Information on 305 patients (BP group 68, control group 237) diagnosed with SAP was retrieved from the Medical Information Mart for Intensive Care IV (MIMIC IV) database. Firstly, the influence of BP treatment was preliminarily evaluated by comparing the outcome indicators of the two groups. Secondly, multiple regression analysis was used to screen the mortality risk factors to verify the impact of BP on the survival outcome of patients. Then, the effect of BP treatment was re-validated with baseline data. Finally, cox regression was used to make the survival curve after matching to confirm whether BP could affect the death outcome. The results indicated that the BP group had a lower incidence of shock (p = 0.012), but a higher incidence of acute kidney injury (AKI) (p < 0.001), with no differences observed in other outcome indicators when compared to the control group. It was also found that the 28-day survival curve of patients between the two groups was significantly overlapped (p = 0.133), indicating that BP treatment had no significant effect on the survival outcome of patients with SAP. Although BP is beneficial in stabilizing hemodynamics, it has no effect on short- and long-term mortality of patients. The application of this technology in the treatment of SAP should be done with caution until appropriate BP treatment methods are developed, particularly for patients who are not able to adapt to renal replacement therapy.

Molecular mechanism of Fe3+ binding inhibition to Vibrio metschnikovii ferric ion-binding protein, FbpA, by rosmarinic acid and its hydrolysate, danshensu.

Global warming has increased the growth of pathogenic Vibrio bacteria, which can cause foodborne illnesses and death. Vibrio bacteria require iron for growth and survival. They utilize a ferric ion-binding protein (FbpA) to bind and transport Fe3+ into the cell. FbpA from Vibrio metschnikovii (Vm) is a potential target for inhibiting its growth. Rosmarinic acid (RA) can block the binding of VmFbpA to Fe3+ ; however, the molecular mechanism of Fe3+ binding and RA inhibition to VmFbpA is unclear. In this study, we used x-ray crystallography to determine the Fe3+ -binding mode of VmFbpA and the mechanism of RA inhibition. The structures revealed that in the Fe3+ bound form, Fe3+ was coordinated to VmFbpA by two Tyr residues, two HCO3 - ions, and two water molecules in a six-coordinated geometry. In contrast, in the inhibitor bound form, RA was initially bound to VmFbpA following gel filtration purification, but it was hydrolyzed to danshensu (DSS), which occupied the binding site as shown in an electron density map and reverse phase chromatography (RPC) analysis. Both RA and DSS exhibited a stronger binding affinity to VmFbpA, higher Fe3+ reduction capacity, and more potent bacteriostatic effect on V. metschnikovii compared with caffeic acid (CA), another hydrolysis product of RA. These results provide insight into the mechanism of iron acquisition by V. metschnikovii and inhibition by RA and DSS. Our findings offer clues on the development of effective strategies to prevent Vibrio infections.

A Machine Learning Prediction Model for Non-cardiogenic Out-of-hospital Cardiac Arrest with Initial Non-shockable Rhythm.

Objectives: The purpose of this study was to develop and validate a machine learning prediction model for the prognosis of non-cardiogenic out-of-hospital cardiac arrest (OHCA) with an initial non-shockable rhythm. Design: Data were obtained from a nationwide OHCA registry in Japan. Overall, 222,056 patients with OHCA and an initial non-shockable rhythm were identified from the registry in 2016 and 2017. Patients aged <18 years and OHCA caused by cardiogenic origin, cancer, and external factors were excluded. Finally, 58,854 participants were included. Methods: Patients were classified into the training dataset (n=29,304, data from 2016) and the test dataset (n=29,550, data from 2017). The training dataset was used to train and develop the machine learning model, and the test dataset was used for internal validation. We selected XGBoost as the machine learning classifier. The primary outcome was the poor prognosis defined as cerebral performance category of 3-5 at 1 month. Eleven prehospital variables were selected as outcome predictors. Results: In validation, the machine learning model predicted the primary outcome with an accuracy of 90.8% [95% confidence interval (CI): 90.5-91.2], a sensitivity of 91.4% [CI: 90.7-91.4], a specificity of 74.1% [CI: 69.2-78.6], and an area under the receiver operating characteristic value of 0.89 [0.87-0.92]. The important features for model development were the prehospital return of spontaneous circulation, prehospital adrenaline administration, and initial electrical rhythm. Conclusions: We developed a favorable machine learning model to predict the prognosis of non-cardiogenic OHCA with an initial non-shockable rhythm in the early stage of resuscitation.