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Patients with hemorrhagic stroke have high rates of morbidity and mortality, and drugs for prevention are very limited. Mendelian randomization (MR) analysis can increase the success rate of drug development by providing genetic evidence. Previous MR analyses only analyzed the role of individual drug target genes in hemorrhagic stroke; therefore, we used MR analysis to systematically explore the druggable genes for hemorrhagic stroke. We sequentially performed summary-data-based MR analysis and two-sample MR analysis to assess the associations of all genes within the database with intracranial aneurysm, intracerebral hemorrhage, and their subtypes. Validated genes were further analyzed by colocalization. Only genes that were positive in all three analyses and were druggable were considered desirable genes. We also explored the mediators of genes affecting hemorrhagic stroke incidence. Finally, the associations of druggable genes with other cardiovascular diseases were analyzed to assess potential side effects. We identified 56 genes that significantly affected hemorrhagic stroke incidence. Moreover, TNFSF12, SLC22A4, SPARC, KL, RELT, and ADORA3 were found to be druggable. The inhibition of TNFSF12, SLC22A4, and SPARC can reduce the risk of intracranial aneurysm, subarachnoid hemorrhage, and intracerebral hemorrhage. Gene-induced hypertension may be a potential mechanism by which these genes cause hemorrhagic stroke. We also found that blocking these genes may cause side effects, such as ischemic stroke and its subtypes. Our study revealed that six druggable genes were associated with hemorrhagic stroke, and the inhibition of TNFSF12, SLC22A4, and SPARC had preventive effects against hemorrhagic strokes., Competing Interests: Declarations. Ethics Approval: This study was a reanalysis of previously published data and did not require ethical consent. Informed consent and ethical approval for the study of data were obtained from all participants according to the protocol of the original GWAS data. Consent to Participate: Not applicable. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: The question of whether to treat patients with chronic hepatitis B (CHB) during the immune tolerance (IT) period is a matter of ongoing debate, as it is difficult to discern different levels of liver disease severity. We created and assessed a novel diagnostic model for identifying significant liver tissue damage in individuals with CHB in IT phase., Methods: From November 2018 to December 2022, a cross-sectional study of 311 patients with chronic hepatitis B virus infection (HBV DNA > 30 IU/mL) at Ningbo No. 2 Hospital, Ningbo, China, who underwent liver biopsy, including 44 patients in IT phase. Utilizing univariate regression analyses and logistics analysis, and model was developed and validated to predict the severity of hepatic inflammatory and fibrosis in CHB patients and in IT phase., Results: Chitinase 3-like Protein (CHI3L1), albumin (ALB), alanine transaminase (ALT) / aspartate aminotransferase (AST) were identified as independent predictors of liver lesion severity in CHB patients with IT. The three were combined to build the model (named as CAA index), which demonstrated good performance. The CAA index achieved an area under the receiver operating characteristic curve (AUC) of 0.916 (95 % CI, 0.820-1.000) and AUC of validation group was 0.875 (95 % CI, 0.683-1.000)., Conclusions: CHI3L1 serves as an independent measure of liver fibrosis and inflammation in CHB. This diagnostic model has some value in assessing the severity of the patient's liver lesion severity and may be a reliable non-invasive diagnostic model helping determine whether treatment is necessary among CHB patients in IT phase., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Owing to the absence of specific symptoms in early-stage gastric cancer, most patients are diagnosed at intermediate or advanced stages. As a result, treatment often shifts from surgery to other therapies, with chemotherapy and targeted therapies being the primary options for advanced gastric cancer treatment., Aim: To investigate both treatment efficacy and immune modulation., Methods: A total of 116 patients with advanced gastric cancer, admitted from January 2021 to December 2023, were selected and divided into two groups of 58 each using the random number table method. The control group received FOLFOX4 chemotherapy (oxaliplatin + calcium + folinate + 5-fluorouracil) combined with intravenous sindilizumab. The observation group received the same treatment as the control group, supplemented by oral administration of Senqi Shiyiwei granules. Both groups underwent treatment cycles of 3 weeks, with a minimum of two cycles. The therapeutic efficacy, immune mechanisms, and treatment-related toxicity and side effects were compared between the groups., Results: The objective remission rate in the observation group (55.17%) was higher than that of the control group (36.21%) ( P < 0.05). After two treatment cycle, CD3+, CD4+, and CD4+/CD8+ levels were higher in the observation group compared to the control group, while CD8+, regulatory T cells, and natural killer cells were lower ( P < 0.05). Additionally, the incidence of leukopenia, nausea, and vomiting was lower in observed group ( P < 0.05). No significant differences were observed in the incidence of other adverse reactions ( P > 0.05)., Conclusion: Adjuvant therapy with Shenqixian granules may enhance the efficacy of simudizumab combined with FOLFOX4 chemotherapy in advanced gastric cancer and the immune function by increasing immune cell counts, making it a valuable option in clinical treatment., Competing Interests: Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article., (©The Author(s) 2025. Published by Baishideng Publishing Group Inc. All rights reserved.)

Mitochondrial oxidative phosphorylation (OXPHOS) is a therapeutic vulnerability in glycolysis-deficient cancers. Here we show that inhibiting OXPHOS similarly suppresses the proliferation and tumorigenicity of glycolytically competent colorectal cancer (CRC) cells in vitro and in patient-derived CRC xenografts. While the increased glycolytic activity rapidly replenished the ATP pool, it did not restore the reduced production of aspartate upon OXPHOS inhibition. This shortage in aspartate, in turn, caused nucleotide deficiencies, leading to S phase cell cycle arrest, replication fork stalling, and enrichment of the p53 pathway, manifestations of replication stress. The addition of purine nucleobases adenine and guanine along with the pyrimidine nucleoside uridine restored replication fork progression and cell proliferation, whereas the supplementation of exogenous aspartate recovered the nucleotide pool, demonstrating a causal role of the aspartate shortage in OXPHOS inhibition-induced nucleotide deficiencies and consequently replication stress and reductions in proliferation. Moreover, we demonstrate that glutamic-oxaloacetic transaminase 1 (GOT1) is critical for maintaining the minimum aspartate pool when OXPHOS is inhibited, as knockdown of GOT1 further reduced aspartate levels and rendered CRC cells more sensitive to OXPHOS inhibition both in vitro and in vivo. These results propose GOT1 targeting as a potential avenue to sensitize cancer cells to OXPHOS inhibitors, thus lowering the necessary doses to efficiently inhibit cancer growth while alleviating their adverse effects., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Objective: Epilepsy is one of the most prevalent brain diseases. Approximately one third of patients consistently experience drug-resistant epilepsy (DRE), a condition where seizures persist despite the use of antiseizure medications. Exploration of new therapies for DRE is urgently needed. In this single-center, randomized, sham-controlled, crossover clinical trial (NCT05042726), we aimed to investigate the effectiveness and safety of transcranial magnetic continuous theta burst stimulation (cTBS) targeting the cerebellum to treat DRE., Methods: Patients with DRE for ≥2 years and a seizure frequency of ≥2 seizures per month were enrolled and randomized 1:1 to receive active stimulation followed by sham stimulation or vice versa. The bilateral cerebellum was targeted by navigated cTBS focusing on the cerebellar dentate nucleus, once daily on workdays for 2 weeks. The primary outcomes were the percentage of seizure reduction and 50% responder rate in the per-protocol population within 2 months after treatment., Results: Forty-four patients were enrolled and randomized; 18 patients in the active stimulation-first group and 20 in the sham stimulation-first group were included in the final analysis. Active cTBS significantly reduced seizures compared to sham stimulation (difference in percentage of seizure reduction between treatments = 25%, 95% confidence interval [CI] = 5%-46%, p = .018). The 50% responder rate after active stimulation was significantly higher than that after sham stimulation (difference in 50% responder rate between treatments = 24%, 95% CI = 11%-40%, p = .029). Adverse events occasionally occurred during active stimulation (moderate headache in 5% of patients, tinnitus in 3% of patients, dizziness in 3% of patients) but resolved spontaneously within days after treatment completion., Significance: This trial suggested that cTBS targeting the cerebellum was effective and well tolerated in the treatment of DRE. Further studies are warranted to confirm its effectiveness and mechanism., (© 2024 International League Against Epilepsy.)

The dual inhibition of histone lysine-specific demethylase 1 (LSD1) and histone deacetylase (HDAC) has emerged as a promising strategy for cancer therapy. In this study, we report the discovery of novel 5-cyano-3-phenylindole-based LSD1/HDAC dual inhibitors, evaluated through both in vitro and in vivo assays. Among these inhibitors, compound 20c was identified as particularly potent, exhibiting high inhibitory activity against LSD1 (IC 50 = 39.0 nM) and HDAC1/2/3/6/8 (IC 50 = 1.4, 1.0, 1.3, 2.9, and 16.0 nM, respectively). Compound 20c effectively modulated the expression of biomarkers associated with LSD1 and HDAC inhibition and demonstrated superior antiproliferative activity compared to SAHA and 4SC-202 across multiple colorectal cancer cell lines. Following pharmacokinetic studies, 20c was further assessed in HCT-116 and HT-29 xenograft mouse models. It demonstrated significantly enhanced antitumor efficacy compared to SAHA, without causing observable toxicity. These findings highlight the potential of LSD1/HDAC dual inhibitors for the treatment of malignant cancers.

Mitochondrial dysfunction and immune cell infiltration play crucial yet incompletely understood roles in the pathogenesis of calcific aortic valve disease (CAVD). This study aimed to identify immune-related mitochondrial genes critical to the pathological process of CAVD using multiomics approaches. The CIBERSORT algorithm was employed to evaluate immune cell infiltration characteristics in CAVD patients. An integrative analysis combining weighted gene coexpression network analysis (WGCNA), machine learning, and summary data-based Mendelian randomization (SMR) was performed to identify key mitochondrial genes implicated in CAVD. Spearman's rank correlation analysis was also performed to assess the relationships between key mitochondrial genes and infiltrating immune cells. Compared with those in normal aortic valve tissue, an increased proportion of M0 macrophages and resting memory CD4 T cells, along with a decreased proportion of plasma cells and activated dendritic cells, were observed in CAVD patients. Additionally, eight key mitochondrial genes associated with CAVD, including PDK4, LDHB, SLC25A36, ALDH9A1, ECHDC2, AUH, ALDH2, and BNIP3, were identified through the integration of WGCNA and machine learning methods. Subsequent SMR analysis, incorporating multiomics data, such as expression quantitative trait loci (eQTLs) and methylation quantitative trait loci (mQTLs), revealed a significant causal relationship between ALDH9A1 expression and a reduced risk of CAVD. Moreover, ALDH9A1 expression was inversely correlated with M0 macrophages and positively correlated with M2 macrophages. These findings suggest that increased ALDH9A1 expression is significantly associated with a reduced risk of CAVD and that it may exert its protective effects by modulating mitochondrial function and immune cell infiltration. Specifically, ALDH9A1 may contribute to the shift from M0 macrophages to anti-inflammatory M2 macrophages, potentially mitigating the pathological progression of CAVD. In conclusion, ALDH9A1 represents a promising molecular target for the diagnosis and treatment of CAVD. However, further validation through in vivo and n vitro studies is necessary to confirm its role in CAVD pathogenesis and therapeutic potential., (© 2024. The Author(s).)

The complexity of test and fault information within electronic devices makes their integrated diagnosis a challenging problem when designing equipment reliability. Current integrated diagnosis is analyzed for test optimization and test resource optimization. However, this neglects the connection between them. This paper proposes a design strategy for integrated diagnosis optimization based on the spatial mapping principle to quantitatively describe the constraint relationship between them. The integrated diagnosis optimization model is established by constructing the logical mapping relationship between test space, resource space, and fault space, and the optimal test configuration and test resource configuration are sought based on the grey wolf optimization algorithm. Seven high-dimensional benchmark functions and an integrated diagnosis model of electronic equipment are used to verify the efficiency of the algorithm proposed in this paper. The proposed algorithm is compared with the other four in terms of the algorithm's optimization speed and accuracy. The results indicate that the electronic equipment after integrated diagnosis optimization has critical fault detection, fault detection, fault isolation, and false alarm rates of 100%, 99.99%, 98.99%, and 0.2993%, respectively. After the integrated diagnosis optimization, the number of tests of the equipment is reduced by 88.9%, and the test cost is saved by 89%. Compared with the other algorithms, grey wolf optimization achieves the best optimization results, reduces the number of tests by 42%-55%, and decreases the test cost by 77.63%-83.91%. This strategy not only considers the test optimization of equipment and test resources optimization but also dramatically reduces the test cost while improving the test efficiency., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objective: Acute pericoronitis (AP) is a prevalent cause of odontogenic toothache which can significantly impact brain function. Previous research has predominantly concentrated on localized brain activity. However, the synergistic changes between brain hemispheres induced by toothache and resulting abnormal functional connectivity across the brain have not been comprehensively studied., Methods: A total of 34 patients with AP and 34 healthy individuals, matched for age, sex, and education were recruited for this study. All participants underwent resting-state functional magnetic resonance imaging (rs-MRI) scans. The voxel mirror homotopic connectivity (VMHC) method was used to identify intergroup differences. Brain regions exhibiting statistically significant differences were selected as regions of interest for further functional connectivity analysis. The partial correlation method was utilized to assess the correlation between abnormal VMHC values in different regions and clinical parameters, with age and sex included as covariates., Results: Patients with AP exhibited reduced VMHC values in the thalamus and elevated VMHC values in the inferior frontal gyrus compared with healthy controls. Subsequent functional connectivity analyses revealed extensive changes in functional networks, predominantly affecting the default, frontoparietal, cerebellar, and pain networks., Conclusion: Changes in functional patterns across these brain networks offer novel insights into the neurophysiological mechanisms underlying pain information processing., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 by Mutaz B. Habal, MD.)

Skp1-CUL1-ROC1-F-box E3 ubiquitin ligases' main component S-phase kinase-associated protein 2 (Skp2) is responsible for specifically recognizing ubiquitination-modified substrates to be degraded such as p27 and p21 in the case of binding with adaptor protein Cks1. Pharmacological inhibition of Skp2 has exhibited promising antitumor activity. Herein, we present the design and optimization of a series of [1,2,4]triazolo[1,5- a ]pyrimidine-based small molecules targeting Skp2. Among them, E35 demonstrated excellent inhibitory activities against the binding of Skp2-Cks1. In addition, compound E35 significantly inhibited colony formation and migration, as well as arrested the cell cycle at the S-phase. Mechanistically, compound E35 markedly decreased the expression of Skp2, as well as increased the expression of its substrates p21 and p27. Furthermore, compound E35 showed an obvious inhibitory effect on MGC-803 xenograft mice without obvious toxicity. All of these results suggest that compound E35 might be a valuable lead compound for antitumor agents targeting Skp2.

Cholangiocarcinoma (CCA) is widely noted for its high degree of malignancy, rapid progression, and limited therapeutic options. This study was carried out on transcriptome data of 417 CCA samples from different anatomical locations. The effects of lipid metabolism related genes and immune related genes as CCA classifiers were compared. Key genes were derived from MVI subtypes and better molecular subtypes. Pathways such as epithelial mesenchymal transition (EMT) and cell cycle were significantly activated in MVI-positive group. CCA patients were classified into three (four) subtypes based on lipid metabolism (immune) related genes, with better prognosis observed in lipid metabolism-C1, immune-C2, and immune-C4. IPTW analysis found that the prognosis of lipid metabolism-C1 was significantly better than that of lipid metabolism-C2 + C3 before and after correction. KRT16 was finally selected as the key gene. And knockdown of KRT16 inhibited proliferation, migration and invasion of CCA cells., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Atherosclerotic cardiovascular disease is closely correlated with elevated low density lipoprotein-cholesterol. In feeding state, glucose and insulin activate mammalian target of rapamycin 1 that phosphorylates the deubiquitylase ubiquitin-specific peptidase 20 (USP20). USP20 then stabilizes HMG-CoA reductase, thereby increasing lipid biosynthesis. In this study, we applied clinically approved lipid nanoparticles to encapsulate the siRNA targeting Usp20. We demonstrated that silencing of hepatic Usp20 by siRNA decreased body weight, improved insulin sensitivity, and increased energy expenditure through elevating UCP1. In Ldlr -/- mice, silencing Usp20 by siRNA decreased lipid levels and prevented atherosclerosis. This study suggests that the RNAi-based therapy targeting hepatic Usp20 has a translational potential to treat metabolic disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Alkaline phosphatase (ALP) reflects changes in the condition of multiple myeloma (MM) patients to some extent. However, the relationship of ALP in MM remains uncertain. Our study aimed to determine the association between initial ALP levels and overall survival in newly diagnosed MM patients., Methods: Clinical data from 202 newly diagnosed MM patients at Beijing Chaoyang Hospital between 2012 and 2016 were collected. Baseline characteristics, disease progression staging, serum markers, and patient survival data were recorded. The cut-off value for ALP was calculated based on patient survival data, and patients were divided into groups. Differences in patients' 3- and 5-year survival rates, liver function, bone disease and other indicators among different groups were compared. Independent risk factors influencing newly diagnosed MM patients were identified using COX regression analysis., Results: Patients were categorized into three groups based on ALP cut-off points: Group 1 (ALP <70 U/L), Group 2 (ALP 70 to <120 U/L), and Group 3 (ALP ≥120 U/L). Significant differences were observed in lactate dehydrogenase, serum calcium, white blood cell count, hemoglobin, and liver function indicators (including alanine aminotransferase, aspartate aminotransferase, albumin, and γ-glutamyl transferase) among different ALP groups (P<0.05). ALP levels varied significantly among patients with different bone disease grades (P<0.05). Median survival times for Groups 1, 2, and 3 were 25, 52, and 31 months, respectively. Group 2 exhibited significantly higher 3-year survival compared to the other two groups (P=0.006), while no significant difference was observed in 5-year survival among the three groups (P=0.51). Age, International Staging System staging, aspartate aminotransferase, β2-microglobulin, ALP grading, and severe bone disease were identified as independent factors influencing survival in newly diagnosed patients (P<0.05)., Conclusions: ALP levels are correlated with the prognosis of MM patients, and an ALP range of 70 to <120 U/L reflects a better survival expectation., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-330/coif). The authors have no conflicts of interest to declare., (2024 Translational Cancer Research. All rights reserved.)

Introduction: Planting without mulching can eliminate the residual film pollution caused by the long-term use of plastic film covers, but it will increase soil moisture evaporation and heat loss and severely reduce water use efficiency and cotton productivity in cotton ( Gossypium hirsutum L.) fields in arid regions. It is unclear whether the advantages of subsurface drip irrigation and nighttime irrigation can be leveraged to reduce the amount of irrigation applied in fields, improve the soil and leaf hydrothermal environments, and increase the synchronicity of yield and water use efficiency (WUE)., Methods: Therefore, in a two-year field experiment (2019-2020), cotton was grown under different irrigation treatments (I5, 3753 m 3 ha -1 ; I4, 3477 m 3 ha -1 ; I3, 3201 m 3 ha -1 ; I2, 2925 m 3 ha -1 ; and I1, 2649 m 3 ha -1 ). The soil volumetric moisture content, soil temperature, leaf relative water content (RWC), daily changes in gas exchange parameters, lint yield, and WUE were evaluated., Results and Discussion: The results showed that reducing irrigation can reduce the soil volumetric moisture content (0-40 cm soil layer), increase the soil temperature and soil temperature conductivity, and increase the leaf temperature, intercellular carbon dioxide concentration (Ci), and WUE; however, reducing irrigation is not conducive to increasing the leaf RWC, net photosynthetic rate (Pn), stomatal conductance (Gs), or transpiration rate (Tr). There was no significant difference in WUE between the I3 and I4 treatments from 8:00 to 20:00, but the lint yield in these treatments increased by 2.8-12.2% compared to that in the I5 treatment, with no significant difference between the I3 and I4 treatments. In addition, a related analysis revealed that the positive effects of the leaf hydrothermal environment on the Pn and soil temperature on the WUE occurs during the same period (10:00-16:00). Overall, an irrigation amount of 3201-3477 m 3 ha -1 applied with a subsurface nighttime irrigation system without mulching can enhance the soil moisture content and soil temperature, maintain a high photosynthetic capacity, and increase the lint yield and WUE. These results revealed that the negative impacts of plastic film contamination in arid areas can be alleviated., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AK declared a past co-authorship with the author H-hL to the handling editor., (Copyright © 2024 Li, Li, Shi, Shi, Tian, Wang, Hao, Luo and Wang.)

Autophagy plays a pivotal role in diverse biological processes, including the maintenance and differentiation of neural stem cells (NSCs). Interestingly, while complete deletion of Fip200 severely impairs NSC maintenance and differentiation, inhibiting canonical autophagy via deletion of core genes, such as Atg5 , Atg16l1 , and Atg7 , or blockade of canonical interactions between FIP200 and ATG13 (designated as FIP200-4A mutant or FIP200 KI) does not produce comparable detrimental effects. This highlights the likely critical involvement of the non-canonical functions of FIP200, the mechanisms of which have remained elusive. Here, utilizing genetic mouse models, we demonstrated that FIP200 mediates non-canonical autophagic degradation of p62/sequestome1, primarily via TAX1BP1 in NSCs. Conditional deletion of Tax1bp1 in fip200 hGFAP conditional knock-in (cKI) mice led to NSC deficiency, resembling the fip200 hGFAP conditional knockout (cKO) mouse phenotype. Notably, reintroducing wild-type TAX1BP1 not only restored the maintenance of NSCs derived from tax1bp1 -knockout fip200 hGFAP cKI mice but also led to a marked reduction in p62 aggregate accumulation. Conversely, a TAX1BP1 mutant incapable of binding to FIP200 or NBR1/p62 failed to achieve this restoration. Furthermore, conditional deletion of Tax1bp1 in fip200 hGFAP cKO mice exacerbated NSC deficiency and p62 aggregate accumulation compared to fip200 hGFAP cKO mice. Collectively, these findings illustrate the essential role of the FIP200-TAX1BP1 axis in mediating the non-canonical autophagic degradation of p62 aggregates towards NSC maintenance and function, presenting novel therapeutic targets for neurodegenerative diseases.

Background: To investigate the prognosis of patients with Multiple Myeloma (MM) after surgery, analyze the risk factors leading to adverse postoperative outcomes, and establish a nomogram., Methods: Clinical data from 154 patients with MM who underwent surgery at our institution between 2007 and 2019 were retrospectively analyzed. Assessing and comparing patients' pain levels, quality of life, and functional status before and after surgery (P < 0.05) were considered statistically significant. The Kaplan-Meier survival curve was used to estimate the median survival time. Adverse postoperative outcomes were defined as worsened symptoms, lesion recurrence, complication grade ≥ 2, or a postoperative survival period < 1 year. Logistic regression analysis was used to determine the prognostic factors. Based on the logistic regression results, a nomogram predictive model was developed and calibrated., Results: Postoperative pain was significantly alleviated in patients with MM, and there were significant improvements in the quality of life and functional status (P < 0.05). The median postoperative survival was 41 months. Forty-nine patients (31.8%) experienced adverse postoperative outcomes. Multivariate logistic regression analysis identified patient age, duration of MM, International Staging System, preoperative Karnofsky Performance Status, and Hb < 90 g/L as independent factors influencing patient prognosis. Based on these results, a nomogram was constructed, with a C-index of 0.812. The calibration curve demonstrated similarity between the predicted and actual survival curves. Decision curve analysis favored the predictive value of the model at high-risk thresholds from 10% to-69%., Conclusion: This study developed a nomogram risk prediction model to assist in providing quantifiable assessment indicators for preoperative evaluation of surgical risk., (© 2024. The Author(s).)

Migration is an initial step in tumor expansion and metastasis; suppressing cellular migration is beneficial to cancer therapy. Herein, we designed a novel biogated nanoagents that integrated the migration inhibitory factor into the mesoporous silica nanoparticle (MSN) drug delivery nanosystem to realize cell migratory inhibition and synergistic treatment. Antisense oligonucleotides (Anti) of microRNA-330-3p, which is positively related with cancer cell proliferation, migration, invasion, and angiogenesis, not only acted as the locker for blocking drugs but also acted as the inhibitory factor for suppressing migration via gene therapy. Synergistic with gene therapy, the biogated nanoagents (termed as MSNs-Gef-Anti) could achieve on-demand drug release based on the intracellular stimulus-recognition and effectively kill tumor cells. Experimental results synchronously demonstrated that the migration suppression ability of MSNs-Gef-Anti nanoagents (nearly 30%) significantly contributed to cancer therapy, and the lethality rate of the non-small-cell lung cancer was up to 70%. This strategy opens avenues for realizing efficacious cancer therapy and should provide an innovative way for pursuing the rational design of advanced nano-therapeutic platforms with the combination of cancer cell migratory inhibition., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Panax ginseng is reputed to be capable of replenishing healthy Qi and bolstering physical strength, and P. notoginseng can resolve blood stasis and alleviate pain. P. ginseng and P. notoginseng are frequently employed to treat ischemic heart diseases caused by blockages in the heart vessels. Mitochondrial dysfunction often coexists with abnormal mitochondrial morphology, and mitochondrial plasticity and dynamics play key roles in cardiovascular diseases. In this study, primary neonatal rat cardiomyocytes were exposed to 4 hours of hypoxia(H) followed by 2 hours of reoxygenation(R). MitoTracker Deep Red and Hoechst 33342 were used to label mitochondria and nuclei, respectively. Fluorescence images were then acquired using ImageXpress Micro Confocal. Automated image processing and parameter extraction/calculation were carried out using ImagePro Plus. Subsequently, representative parameters were selected as indicators to assess alterations in mitochondrial morphology and function. The active compounds of P. ginseng and P. notoginseng were screened out and identified based on the UPLC-Triple-TOF-MS results and mitochondrial morphometric parameters. The findings demonstrated that RS-2, RS-4, SQ-1, and SQ-4 significantly increased the values of three key morphometric parameters, including mitochondrial length, branching, and area, which might contribute to rescuing morphological features of myocardial cells damaged by H/R injury. Among the active components of the two medicinal herbs, 20(R)-ginsenoside Rg&#95;3, ginsenoside Re, and gypenoside ⅩⅦ exhibited the strongest protective effects on mitochondria in cardiomyocytes. Specifically, 20(R)-ginsenoside Rg&#95;3 might upregulate expression of optic atrophy 1(OPA1) and mitofusin 2(MFN2), and ginsenoside Re and gypenoside ⅩⅦ might selectively upregulate OPA1 expression. Collectively, they promoted mitochondrial membrane fusion and mitigated mitochondrial damage, thereby exerting protective effects on cardiomyocytes. This study provides experimental support for the discovery of novel therapeutic agents for myocardial ischemia-reperfusion injury from P. ginseng and P. notoginseng and offers a novel approach for large-scale screening of bioactive compounds with cardioprotective effects from traditional Chinese medicines.

It remains a challenge to obtain biocompatible afterglow materials with long emission wavelengths, durable lifetimes, and good water solubility. Herein we develop a photooxidation strategy to construct near-infrared afterglow carbon nanodots with an extra-long lifetime of up to 5.9 h, comparable to that of the well-known rare-earth or organic long-persistent luminescent materials. Intriguingly, size-dependent afterglow lifetime evolution from 3.4 to 5.9 h has been observed from the carbon nanodots systems in aqueous solution. With structural/ultrafast dynamics analysis and density functional theory simulations, we reveal that the persistent luminescence in carbon nanodots is activated by a photooxidation-induced dioxetane intermediate, which can slowly release and convert energy into luminous emission via the steric hindrance effect of nanoparticles. With the persistent near-infrared luminescence, tissue penetration depth of 20 mm can be achieved. Thanks to the high signal-to-background ratio, biological safety and cancer-specific targeting ability of carbon nanodots, ultralong-afterglow guided surgery has been successfully performed on mice model to remove tumor tissues accurately, demonstrating potential clinical applications. These results may facilitate the development of long-lasting luminescent materials for precision tumor resection., (© 2024. The Author(s).)

In this study, we sought to quantify the effect of planting structure change on fertilizer input and environmental cost in Chongqing and develop scientific and rational strategies for chemical fertilizer reduction. Based on the crop fertilizer quota standard and large sample farmer survey data under the medium productivity level in Chongqing, we evaluated and analyzed the application reduction potential and environmental benefits of fertilizer with the difference method and life cycle assessment. The results showed that:① since Chongqing became a municipality directly under the central government (1997), Chongqing crop planting structure had greatly changed, and the proportion of food crop (rice, corn, wheat, bean, and potato) decreased by 21%. The area of fruits and vegetables increased from 3.36×10 5 hm 2 to 1.05×10 6 hm 2 , and their proportion increased by 20%. ② Nearly 55% of fertilizers had been consumed by vegetable (37%) and citrus production systems, and 11%, 12%, and 12% of fertilizers were consumed by rice, corn, and potato, respectively. ③ The total fertilizer reduction of the Chongqing planting industry could reach up to 1.69×10 5 tons during the period of "the 14th Five-Year Plan," with a fertilizer reduction potential of 18.6%. The fertilizer reduction potential (reduction amount) of rice, corn, citrus, and vegetables would reach 0.3% (2.9×10 2 tons), 12% (1.45×10 4 tons), 21% (3.65×10 4 tons), and 30% (1.18×10 5 tons), respectively. On the other hand, the rape system was insufficient in phosphorus potassium fertilizers, and the corn tended to be insufficient in potash fertilizer. ④ The current production level was low, and the nitrogen loss, greenhouse gas emissions, and eutrophication potential in the planting industry of Chongqing reached 1.81×10 5 tons (N), 1.43×10 7 tons (CO 2 -eq), and 1.74×10 5 tons (PO 4 -eq). With the increase in the realization degree of the crop quota standard (60%-100%), the reactive nitrogen loss, greenhouse gas emissions, and eutrophication potential decreased by 14.9%-24.9%, 10.1%-16.7%, and 13.8%-23%, respectively. The structure of the planting industry in Chongqing significantly changed, the total fertilizer consumption in Chongqing tended to decline gradually, and the fertilization intensity of commercial crops stayed at a high level. The agricultural fertilizer reduction potential and the reactive nitrogen and greenhouse gas emission reduction potential were large, especially for citrus and vegetable production systems. However, it is also necessary to pay attention to insufficient corn potash fertilizer and rape phosphorus potassium fertilizer investment and carry out collaborative promotion of fertilizer reduction.

Ethnopharmacological Relevance: The Mongolian medicine Nuangong Qiwei Pill (NGQW) is a folk prescription with a long history of use by the Mongolian people. NGQW comprises seven Mongolian medicines, which have the effects of regulating and nourishing blood, warming the uterus, dispelling cold and relieving pain. For a long time, it has been used as a good remedy for gynecological diseases, with remarkable curative effects, favored by the majority of patients and recommended by doctors. Polycystic ovary syndrome (PCOS) is a common gynecological endocrine disorder that can lead to menstrual disorders or infertility. In the gynecological classification of Mongolian medicine, polycystic ovary syndrome has not been distinguished in detail, and the mechanism of NGQW in the treatment of polycystic ovary syndrome has not been scientifically studied and standardized., Aim of the Study: The aim of this study was to clarify the mechanism of action of NGQW and macelignan in the treatment of PCOS and to provide a reference for the clinical application of these drugs., Materials and Methods: The effect of intragastric administration of NGQW and macelignan on PCOS model mice was observed. The mental status of mice was examined behaviorally, and serum hormone levels and oxidative stress parameters were measured by ELISA. Giemsa staining was used to detect the reproductive cycle, and HE staining was used to observe the ovarian status. Immunofluorescence staining was performed to observe the proliferation and apoptosis of ovarian granulosa cells. qRT‒PCR was conducted to measure the expression of IL-6, BAX, BCL-2, and estrogen synthesis-related genes in ovarian tissue and particle cells., Results: In the dehydroepiandrosterone (DHEA)-induced PCOS model mice, both NGQW and macelignan improved the estrous cycle; increased the estradiol (E2) content; lowered testosterone (T), progesterone (P) and luteinizing hormone (LH) levels; reduced the number of polycystic follicles; promoted granulosa cell proliferation; reduced granulosa cell apoptosis; and alleviated depression and anxiety. In addition, Nuangong Qiwei Pill and macelignan reduced the mRNA levels of the ovarian inflammatory factor IL-6; improved the disordered levels of the antioxidant indicators GSH, MDA, and SOD; and activated the TGF-β3 signaling pathway to increase the transcription of Cyp19a1, which increases estrogen secretion., Conclusion: NGQW and macelignan can treat PCOS through the TGF-β3/Smad/Cyp19a1 signaling pathway to regulate the secretion ability of ovarian granulosa cells. Our research justifies the traditional use of NGQW to treat PCOS and enriches the scope of action of macelignan., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: The hypoxic environment during bone healing is important in regulating the differentiation of periosteal stem cells (PSCs) into osteoblasts or chondrocytes; however, the underlying mechanisms remain unclear., Aim: To determine the effect of hypoxia on PSCs, and the expression of microRNA-584-5p (miR-584-5p) and RUNX family transcription factor 2 (RUNX2) in PSCs was modulated to explore the impact of the miR-584-5p/RUNX2 axis on hypoxia-induced osteogenic differentiation of PSCs., Methods: In this study, we isolated primary mouse PSCs and stimulated them with hypoxia, and the characteristics and functional genes related to PSC osteogenic differentiation were assessed. Constructs expressing miR-584-5p and RUNX2 were established to determine PSC osteogenic differentiation., Results: Hypoxic stimulation induced PSC osteogenic differentiation and significantly increased calcified nodules, intracellular calcium ion levels, and alkaline phosphatase (ALP) activity in PSCs. Osteogenic differentiation-related factors such as RUNX2, bone morphogenetic protein 2, hypoxia-inducible factor 1-alpha, and ALP were upregulated; in contrast, miR-584-5p was downregulated in these cells. Furthermore, upregulation of miR-584-5p significantly inhibited RUNX2 expression and hypoxia-induced PSC osteogenic differentiation. RUNX2 was the target gene of miR-584-5p , antagonizing miR-584-5p inhibition in hypoxia-induced PSC osteogenic differentiation., Conclusion: Our study showed that the interaction of miR-584-5p and RUNX2 could mediate PSC osteogenic differentiation induced by hypoxia., Competing Interests: Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article., (©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.)

Background: Studies have shown that the release of endogenous glutamate (Glu) participates in lung injury by activating N-methyl-D-aspartate receptor (NMDAR), but the mechanism is still unclear. This study was to investigate the effects and related mechanisms of Glu on the lipid synthesis of pulmonary surfactant (PS) in isolated rat lung tissues., Methods: The cultured lung tissues of adult SD rats were treated with Glu. The amount of [3H]-choline incorporation into phosphatidylcholine (PC) was detected. RT-PCR and Western blot were used to detect the changes of mRNA and protein expression of cytidine triphosphate: phosphocholine cytidylyltransferase alpha (CCTα), a key regulatory enzyme in PC biosynthesis. Western blot was used to detect the expression of NMDAR1, which is a functional subunit of NMDAR. Specific protein 1 (Sp1) expression plasmids were used. After transfected with Sp1 expression plasmids, the mRNA and protein levels of CCTα were detected by RT-PCR and Western blot in A549 cells. After treated with NMDA and MK-801, the mRNA and protein levels of Sp1 were detected by RT-PCR and Western blot in A549 cells., Results: Glu decreased the incorporation of [3H]-choline into PC in a concentration- and time- dependent manner. Glu treatment significantly reduced the mRNA and protein levels of CCTα in lungs. Glu treatment up-regulated NMDAR1 protein expression, and the NMDAR blocker MK-801 could partially reverse the reduction of [3H]-choline incorporation induced by Glu (10-4 mol/L) in lungs. After transfected with Sp1 plasmid for 30 h, the mRNA and protein expression levels of CCTα were increased and the protein expression of Sp1 was also up-regulated. After A549 cells were treated with NMDA, the level of Sp1 mRNA did not change significantly, but the expression of nucleus protein in Sp1 was significantly decreased, while the expression of cytoplasmic protein was significantly increased. However, MK-801could reverse these changes., Conclusions: Glu reduced the biosynthesis of the main lipid PC in PS and inhibited CCTα expression by activating NMDAR, which were mediated by the inhibition of the nuclear translocation of Sp1 and the promoter activity of CCTα. In conclusion, NMDAR-mediated Glu toxicity leading to impaired PS synthesis may be a potential pathogenesis of lung injury., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Recent studies have shown that microglia/macrophages and astrocytes can mediate synaptic phagocytosis through the MER proto-oncokinase in developmental or stroke models, but it is unclear whether the same mechanism is also active in traumatic brain injury. In this study, we established a mouse model of traumatic brain injury and found that both microglia/macrophages and astrocytes phagocytosed synapses and expression of the MER proto-oncokinase increased 14 days after injury. Specific knockout of MER in microglia/macrophages or astrocytes markedly reduced injury volume and greatly improved neurobehavioral function. In addition, in both microglia/macrophages-specific and astrocytes-specific MER knock-out mice, the number of microglia/macrophage and astrocyte phagocytosing synapses was markedly decreased, and the total number of dendritic spines was increased. Our study suggested that MER proto-oncokinase expression in microglia/macrophages and astrocytes may play an important role in synaptic phagocytosis, and inhibiting this process could be a new strategy for treating traumatic brain injury., Competing Interests: None

Memory performance evaluation has generally been based on behavioral tests in the past decades. However, its neural correlates remain largely unknown, particularly in humans. Here we addressed this question using intracranial electroencephalography in patients with refractory epilepsy, performing an episodic memory test. We used the presurgical Wechsler Memory Scale (WMS) test to assess the memory performance of each patient. We found that hippocampal ripples significantly exhibited a transient increase during visual stimulation or before verbal recall. This increase in hippocampal ripples positively correlated with memory performance. By contrast, memory performance is negatively correlated with hippocampal interictal epileptic discharges (IEDs) or epileptic ripples in the memory task. However, these correlations were not present during quiet wakefulness. Thus, our findings uncover the neural correlates of memory performance in addition to traditional behavioral tests., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The aim of the present study was to explore the diagnostic value of a deep convolutional neural network (DCNN) model for the diagnosis of pulmonary nodules in adolescent and young adult patients with osteosarcoma. For the present study, 675 chest CT images were retrospectively collected from 109 patients with clinically confirmed osteosarcoma who underwent chest CT examination at Hangzhou Third People's Hospital (Hangzhou, China) from March 2011 to February 2022. CT images were then evaluated using the DCNN and manual models. Subsequently, pulmonary nodules of osteosarcoma were divided into calcified nodules, solid nodules, partially solid nodules and ground glass nodules using the DCNN model. Those patients with osteosarcoma who were diagnosed and treated were followed up to observe dynamic changes in the pulmonary nodules. A total of 3,087 nodules were detected, while 278 nodules were missed compared with those determined using the reference standard given by the consensus of three Experienced radiologists., which was analyzed by two diagnostic radiologists. In the manual model group, 2,442 nodules were detected, while 657 nodules were missed. The DCNN model showed significantly higher sensitivity and specificity compared with the manual model (sensitivity, 0.923 vs. 0.908; specificity, 0.552 vs. 0.351; P<0.05). In addition, the DCNN model yielded an area under the curve (AUC) value of 0.795 [95% confidence interval (CI), 0.743-0.846], outperforming that of the manual model (AUC, 0.687; 95% CI, 0.629-0.732; P<0.05). The film reading time of the DCNN model was also significantly shorter compared with that of the manual model [mean ± standard deviation (SD); 173.25±24.10 vs. 328.32±22.72 sec; P<0.05)]. The AUC of calcified nodules, solid nodules, partially solid nodules and ground glass nodules was calculated to be 0.766, 0.771, 0.761 and 0.796, respectively, using the DCNN model. Using this model, the majority of the pulmonary nodules were detected in patients with osteosarcoma at the initial diagnosis (69/109, 62.3%), and the majority of these were found with multiple pulmonary nodules instead of a single nodule (71/109, 65.1% vs. 38/109, 34.9%). These data suggest that, compared with the manual model, the DCNN model proved to be beneficial for the detection of pulmonary nodules in adolescent and young adult patients with osteosarcoma, which may reduce the time of artificial radiograph reading. In conclusion, the proposed DCNN model, developed using data from 675 chest CT images retrospectively collected from 109 patients with clinically confirmed osteosarcoma, may be used as an effective tool to evaluate pulmonary nodules in patients with osteosarcoma., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Ni et al.)

F-box protein S-phase kinase-associated protein 2 (Skp2) is a component of cullin-RING ligases, which is responsible for recruiting and ubiquitinating substrates and subsequently plays its proteolytic and non-proteolytic role. High expression of Skp2 is frequently observed in multiple aggressive tumor tissues and associated with poor prognosis. Several of the Skp2 inhibitors have been reported in the last decades; however, few of them have shown detailed structure-activity relationship (SAR) and potent bioactivity. Herein, based on the hit compound 11a from our in-house library, we optimize and synthesize a series of new 2,3-diphenylpyrazine-based inhibitors targeting the Skp2-Cks1 interaction and further systematically study the SAR. Among them, compound 14i shows potent activity against the Skp2-Cks1 interaction with an IC 50 value of 2.8 μM and against PC-3 and MGC-803 cells with IC 50 values of 4.8 and 7.0 μM, respectively. Most importantly, compound 14i exhibited effectively anticancer effects on PC-3 and MGC-803 xenograft mice models without obvious toxicity.

Histone acetyltransferase human males absent on the first (hMOF) is a member of MYST family which participates in posttranslational chromatin modification by controlling the acetylation level of histone H4K16. Abnormal activity of hMOF occurs in multiple cancers and biological alteration of hMOF expression can affect diverse cellular functions including cell proliferation, cell cycle progression and embryonic stem cells (ESCs) self-renewal. The relationship between hMOF and cisplatin resistance was investigated in The Cancer Genome Atlas (TCGA) and Genomics of Drug Sensitivity in Cancer (GDSC) database. Lentiviral-mediated hMOF-overexpressed cells or hMOF-knockdown cells were established to investigate its role on cisplatin-based chemotherapy resistance in vitro ovarian cancer cells and animal models. Furthermore, a whole transcriptome analysis with RNA sequencing was used to explore the underlying molecular mechanism of hMOF affecting cisplatin-resistance in ovarian cancer. The data from TCGA analysis and IHC identification demonstrated that hMOF expression was closely associated with cisplatin-resistance in ovarian cancer. The expression of hMOF and cell stemness characteristics increased significantly in cisplatin-resistant OVCAR3/DDP cells. In the low hMOF expressing ovarian cancer OVCAR3 cells, overexpression of hMOF improved the stemness characteristics, inhibited cisplatin-induced apoptosis and mitochondrial membrane potential impairment, as well as reduced the sensitivity of OVCAR3 cells to cisplatin treatment. Moreover, overexpression of hMOF diminished tumor sensitivity to cisplatin in a mouse xenograft tumor model, accompanied by decrease in the proportion of cisplatin-induced apoptosis and alteration of mitochondrial apoptosis proteins. In addition, opposite phenotype and protein alterations were observed when knockdown of hMOF in the high hMOF expressing ovarian cancer A2780 cells. Transcriptomic profiling analysis and biological experimental verification orientated that MDM2-p53 apoptosis pathway was related to hMOF-modulated cisplatin resistance of OVCAR3 cells. Furthermore, hMOF reduced cisplatin-induced p53 accumulation by stabilizing MDM2 expression. Mechanistically, the increased stability of MDM2 was due to the inhibition of ubiquitinated degradation, which resulted by increased of MDM2 acetylation levels by its direct interaction with hMOF. Finally, genetic inhibition MDM2 could reverse hMOF-mediated cisplatin resistance in OVCAR3 cells with up-regulated hMOF expression. Meanwhile, treatment with adenovirus expressing shRNA of hMOF improved OVCAR3/DDP cell xenograft sensitivity to cisplatin in mouse. Collectively, the results of the study confirm that MDM2 as a novel non-histone substrate of hMOF, participates in promoting hMOF-modulated cisplatin chemoresistance in ovarian cancer cells. hMOF/MDM2 axis might be a potential target for the treatment of chemotherapy-resistant ovarian cancer., (© 2023. The Author(s).)

Background: Chronic hepatitis B virus (HBV) infection remains a major global public health problem. Chronic hepatitis B (CHB) patients can be divided into treatment indication and non-treatment indication individuals according to alanine transaminase (ALT), HBV DNA, serum hepatitis B e antigen status, disease status [liver cirrhosis, hepatocellular carcinoma (HCC), or liver failure], liver necroinflammation or fibrosis, patients' age, and family history of HCC or cirrhosis. For example, normal ALT patients in 'immune-tolerant' phase with HBV DNA higher than 10 7 or 2 × 10 7 IU/mL, and those in 'inactive-carrier' phase with HBV DNA lower than 2 × 10 3 IU/mL do not require antiviral therapy. However, is it reasonable to set the defined values of HBV DNA as the fundamental basis to estimate the disease state and to determine whether to start treatment? In fact, we should pay more attention to those who do not match the treatment indications (gray-zone patients both in the indeterminate phase and in the 'inactive-carrier' phase)., Aim: To analyze the correlation of HBV DNA level and liver histopathological severity, and to explore the significance of HBV DNA for CHB with normal ALT., Methods: From January 2017 to December 2021, a retrospective cross-sectional set of 1299 patients with chronic HBV infection (HBV DNA > 30 IU/mL) who underwent liver biopsy from four hospitals, including 634 with ALT less than 40 U/L. None of the patients had received anti-HBV treatment. The degrees of liver necroinflammatory activity and liver fibrosis were evaluated according to the Metavir system. On the basis of the HBV DNA level, patients were divided into two groups: Low/moderate replication group, HBV DNA ≤ 10 7 IU/mL [7.00 Log IU/mL, the European Association for the Study of the Liver (EASL) guidelines] or ≤ 2 × 10 7 IU/mL [7.30 Log IU/mL, the Chinese Medical Association (CMA) guidelines]; high replication group, HBV DNA > 10 7 IU/mL or > 2 × 10 7 IU/mL. Relevant factors (demographic characteristics, laboratory parameters and noninvasive models) for liver histopathological severity were analyzed by univariate analysis, logistics analysis and propensity score-matched analysis., Results: At entry, there were 21.45%, 24.29%, and 30.28% of the patients had liver histopathological severities with ≥ A2, ≥ F2, and ≥ A2 or/and ≥ F2, respectively. HBV DNA level (negative correlation) and noninvasive model liver fibrosis 5 value (positive correlation) were independent risk factors for liver histopathological severities (liver necroinflammation, liver fibrosis, and treatment indication). The AUROCs of the prediction probabilities (PRE&#95;) of the models mentioned above (< A2 vs ≥ A2, < F2 vs ≥ F2, < A2 and < F2 vs ≥ A2 or/and ≥ F2) were 0.814 (95%CI: 0.770-0.859), 0.824 (95%CI: 0.785-0.863), and 0.799 (95%CI: 0.760-0.838), respectively. HBV DNA level (negative correlation) was still an independent risk factor when diagnostic models were excluded, the P values (< A2 vs ≥ A2, < F2 vs ≥ F2, < A2 and < F2 vs ≥ A2 or/and ≥ F2) were 0.011, 0.000, and 0.000, respectively. For the propensity score-matched pairs, whether based on EASL guidelines or CMA guidelines, the group with significant liver histology damage (≥ A2 or/and ≥ F2) showed much lower HBV DNA level than the group with non- significant liver histology damage (< A2 and < F2). Patients in the moderate replication group (with indeterminate phase) had the most serious liver disease pathologically and hematologically, followed by patients in the low replication group (with 'inactive-carrier' phase) and then the high replication group (with 'immune-tolerant' phase)., Conclusion: HBV DNA level is a negative risk factor for liver disease progression. The phase definition of CHB may be revised by whether the level of HBV DNA exceeds the detection low limit value. Patients who are in the indeterminate phase or 'inactive carriers' should receive antiviral therapy., Competing Interests: Conflict-of-interest statement: We have no financial relationships to disclose., (©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.)

MicroRNAs (miRNAs) exhibit a crucial role in the pathogenesis and progress of neurodegenerative disorders. Recent studies have shown abnormal levels of miRNA expression in patients with amyotrophic lateral sclerosis (ALS). Clinical data also confirmed that miRNAs in these patients are inconsistent across studies. A comprehensive systematic review and meta-analysis of current studies can help recognize the important roles of miRNAs during ALS development. Therefore, we initially aimed to perform a systematic literature review on the muscle or serum miRNAs in patients with ALS and healthy individuals. Subsequently, we quantitatively summarized the clinical data of muscle or serum miRNA of patients with ALS and healthy individuals using a meta-analytical technique. 11 studies comprising 281 patients with ALS and 244 healthy control (HC) controls were identified from PubMed and Web of Science for meta-analysis. A systematic review revealed that miRNA levels are closely associated with the occurrence of ALS disease. The expression levels of the most relevant miRNAs were either increased or decreased. The random-effects meta-analysis indicated that the levels of miR-206, miR-133b, and miR-338-3p were significantly elevated in patients with ALS than in HC subjects. By contrast, there was no significant differences in the miR-133a levels between patients with ALS and HC subjects. Collectively, our outcomes demonstrated that serum miR-206, miR-133b, and miR-338-3p were significantly increased in patients with ALS. We speculated that the increased expression levels of miR-206, miR-133b and miR-338-3p are potential promising biomarkers for ALS., Competing Interests: Competing interests Not applicable in this study., (Copyright © 2023. Published by Elsevier Inc.)

Hedgehog (Hh) signaling pathway plays a pivotal role in embryonic development. Hh binding to Patched1 (PTCH1) derepresses Smoothened (SMO), thereby activating the downstream signal transduction. Covalent SMO modification by cholesterol in its cysteine-rich domain (CRD) is essential for SMO function. SMO cholesterylation is a calcium-accelerated autoprocessing reaction, and STIM1-ORAI1-mediated store-operated calcium entry promotes cholesterylation and activation of endosome-localized SMO. However, it is unknown whether the Hh-PTCH1 interplay regulates the activity of the endoplasmic reticulum (ER)-localized SMO. Here, we found that PTCH1 inhibited the COPII-dependent export of SMO from the ER, whereas Hh promoted this process. The RRxWxR amino acid motif in the cytosolic tail of SMO was essential for COPII recognition, ciliary localization, and signal transduction activity. Hh and PTCH1 regulated cholesterol modification of the ER-localized SMO, and SMO cholesterylation accelerated its exit from ER. The GRAMD1/ASTER sterol transport proteins facilitated cholesterol transfer to ER from PM, resulting in increased SMO cholesterylation and enhanced Hh signaling. Collectively, we reveal a regulatory role of GRAMD-mediated cholesterol transport in ER-resident SMO maturation and Hh signaling., (© 2022 The Authors.)

Purpose: To evaluate the long-term survival and functional outcomes of patients with prolonged disorders of consciousness (pDoC) 1-8 years after brain injuries., Methods: Retrospective study to assess the long-term survival and functional outcomes of patients with pDoC was conducted. We performed Cox regression and multivariate logistic regression to calculate hazard ratios (HRs) for the outcome of survival and to identify risk factors of the functional outcome., Results: We recruited 154 patients with pDoC. The duration of follow-up from disease onset was 1-8 years. The median age was 46 years (IQR, 32-59), and 65.6% ( n = 101) of them were men. During the follow-up period, one hundred and ten patients (71.4%) survived; among them, 52 patients had a good outcome. From the overall survival curve, the 1-, 3-, and 8-year survival rates of patients were about 80.5%, 72.0%, and 69.7%, respectively. Cox regression analysis revealed a significant association between the lower APACHE II score ( p = 0.005) (cut-off score ≥ 18) and the presence of sleep spindles ( p = 0.001) with survival. Logistic regression analysis demonstrated a higher CRS-R score (cut-off score ≥ 7), and presence of sleep spindles were related to a favorable outcome among patients with pDoC., Conclusions: Sleep spindles are correlated with both long-term survival and long-term functional outcome in pDoC patients.

Background: Hematogenous metastasis is essential for the progression of advanced hepatocellular carcinoma (HCC) and can occur even after patients receive multidisciplinary therapies, including immunotherapy and hepatectomy; circulating tumor cells (CTCs) are one of the dominant components of the metastatic cascade. However, the CTC capture efficiency for HCC is low due to the low sensitivity of the detection method. In this study, epithelial cell adhesion molecule (EpCAM)/vimentin/Glypican-3 (GPC3) antibody-modified lipid magnetic spheres (LMS) were used to capture tumor cells with epithelial phenotype, mesenchymal phenotype and GPC3 phenotype, respectively, in order to capture more CTCs with a more comprehensive phenotype for monitoring tumor metastasis., Results: The novel CTC detection system of Ep-LMS/Vi-LMS/GPC3-LMS was characterized by low toxicity, strong specificity (96.94%), high sensitivity (98.12%) and high capture efficiency (98.64%) in vitro. A sudden increase in CTC counts accompanied by the occurrence of lung metastasis was found in vivo, which was further validated by a clinical study. During follow-up, the rapid increase in CTCs predicted tumor progression in HCC patients. Additionally, genetic testing results showed common genetic alterations in primary tumors, CTCs and metastatic tissues. The proportion of patients predicted to benefit from immunotherapy with the CTC detection method was higher than that for the tissue detection method (76.47% vs. 41.18%, P = 0.037), guiding the application of clinical individualized therapy., Conclusions: The Ep-LMS/Vi-LMS/GPC3-LMS sequential CTC capture system is convenient and feasible for the clinical prediction of HCC progression. CTCs captured by this system could be used as a suitable alternative to HCC tissue detection in guiding immunotherapy, supporting the clinical application of CTC liquid biopsy., (© 2023. The Author(s).)

Background: Exosomal proteins from cancer cells are becoming new biomarkers for cancer monitoring and efficacy evaluation. However, their biological function and molecular mechanism underlying tumor metastasis are largely unknown., Methods: Bioinformatic methods such as bulk gene expression analysis, single-cell RNA sequencing data analysis, and gene set enrichment analysis were employed to identify metastasis-associated proteins. The in vitro and in vivo experiments were used to investigate the function of RAB13 in HCC metastasis., Results: We identified RAB13 as one of the critical regulators of metastasis in HCC-derived exosomes for the first time. In vitro, the invasiveness of HCC cell lines could be attenuated by RAB13 silence. In vivo, tumor size and proportion of high-grade lung metastatic nodule could be reduced in the mice with orthotopic transplantation of tumors and intravenously injected with exosomes derived from MHCC97H cell with RAB13 silence (si-RAB13-Exo), as compared with those without RAB13 silence (si-NC-Exo). Moreover, in si-RAB13-Exo group, circulating tumor cell counts were decreased at the third, fourth, and fifth weeks after orthotopic transplantation of tumors, and MMP2 (matrix metalloproteinase 2)/TIMP2 (tissue inhibitor of metalloproteinases 2) ratio was also significantly decreased. In addition, RAB13 expression was also associated with VEGF levels, microvessel density, and tube formation of vascular endothelial cells by both in vitro and in vivo models, indicating that RAB13 was associated with angiogenesis in HCC., Conclusions: We have demonstrated exosomal RAB13 as a potential regulator of metastasis for HCC by in silico, in vitro, and in vivo methods, which greatly improve our understanding of the functional impact of exosomal proteins on HCC metastasis., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc on behalf of the American Association for the Study of Liver Diseases.)

Background: Schizophrenia（SCZ）is a common clinically chronic psychiatric disease, and there have no effective specific therapeutic drugs in clinical practice currently. Studies have shown that the expression level of brain-derived neurotrophic factor (BDNF) in schizophrenics has decreased, so the expression level of BDNF has always been one of the evaluation indicators of SCZ. The neurotrophic factor hypothesis believes that increase or decrease of the expression level of BDNF may be one of the pathophysiological basis of SCZ., Methods: In this study, schizophrenic mice model with MK-801-induced glutamate dysfunction was established, and two doses of BDNF were administered to schizophrenic mice by intranasal administration. The four groups of mice: Control group, Model group, BDNF-20, BDNF-100 performed a series of behavioral tests to explore the effects of BDNF on sensory motor gating, anxiety, depression, social interaction, spontaneous activity, and memory in schizophrenic mice. Transcriptome sequencing of the BDNF high group and Model group in prefrontal cortex and hippocampus, using Metascape for gene function annotation and enrichment pathway analysis, to obtain BDNF transcription regulation information, understand the molecular mechanism of BDNF in SCZ further. Subsequently，immunofluorescence detected the effects of BDNF on neurons and glial cells in the prefrontal cortex and hippocampus., Conclusion: The results show that BDNF can improve the behavior of SCZ by regulating the construction of the nervous system, affecting the growth and distribution of neurons and glial cells, and changing inflammation and apoptosis in the brain., Competing Interests: Declarations of competing interest The authors have no potential or actual conflicts of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Through field investigation, UPLC-MS/MS technology and MaxEnt model were performed to predict the suitable distribution area for red huajiao ( Zanthoxylum bungeanum maxim.) in China from 2021 s to 2060 s, and evaluate the effects of climate factors on the quality of red huajiao. The results demonstrated that mean temperature of the coldest quarter and min temperature of the coldest month were the most important environmental variables influencing red huajiao distribution. Suitable habitats for red huajiao were located mainly in dry and hot valley zone in the Qinba Mountains and the semi-humid and semi-arid areas of the Loess Plateau. The amides contents were higher in high suitability areas, while it was decreased in medium and low suitability areas, and temperature, wind speed and precipitation played a key role in their accumulation. This investigation was of great significance for the planting area optimization, quality control, benefit improvement and industrial development of red huajiao., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Background: Pancreatic ductal adenocarcinoma (PDAC) is a fatal malignancy due to the lack of early detection method, therapeutic drug and target. We noticed that the expression of Protein Tyrosine Phosphatase Mitochondria1(PTPMT1) is upregulated in PDAC. However, its role in pancreatic cancer remains unknown., Methods: We first analyzed the expression of PTPMT1 from 50 PDAC patients. Secondly, the survival proportions of different PTPMT1-expressed patients were analyzed. Then, the role and mechanism of PTPMT1 in PDAC were studied by lentivirus transduction system., Results: PTPMT1 was upregulated in PDAC and patients with high PTPMT1 expression displayed lower overall survival rate. Knockdown of PTPMT1 increased the sensitivity to erastin or RSL3 induced ferroptosis. Mechanically, knockdown of PTPMT1 resulted in upregulated Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) and downregulated Solute Carrier Family 7 Member 11 (SLC7A11). In addition, SLC7A11 was upregulated in PDAC tumor tissue and correlated positively with the expression of PTPMT1. However, the expression of ACSL4 was downregulated in PDAC and negatively correlated with the expression of PTPMT1., Conclusion: Our study demonstrates that PTPMT1 is upregulated in PDAC and PTPMT1 inhibits ferroptosis by suppressing the expression of ACSL4 and upregulating SLC7A11 in Panc-1 cells, suggesting PTPMT1 might be a potential prognosis biomarker and therapeutic target in PDAC., (Copyright © 2022 Asian Surgical Association and Taiwan Robotic Surgery Association. Published by Elsevier B.V. All rights reserved.)

Objective: To compare and analyze the clinicopathological features and significance for indications of different types of antiviral therapy in chronic hepatitis B (CHB). Methods: Clinical data of 861 CHB cases who received liver biopsy, had hepatitis B virus (HBV) DNA-positive (> 30 IU/ml) and met the indications for antiviral therapy from January 2014 to December 2019 were included. Liver pathological changes and their correlation with clinical characteristics were compared and analyzed. According to different data, t -test, analysis of variance, nonparametric test, χ 2 test, Ridit and logistic regression analysis were used for statistical analysis. Results: Most of the cases (72.24%) had remarkable pathological damage. The degree of liver fibrosis was higher in the normal than the abnormal group ( P <0.001). 17.54% cases had hepatic steatosis. The vast majority of cases (97.33%) had positive hepatitis B surface antigen (HBsAg), while only 50.87% had positive hepatitis B core antigen (HBcAg). The positive correlation factors affecting the severity of liver histopathology were alkaline phosphatase level, while the negative correlation factors were positive HBcAg staining, albumin and platelet level. The degree of liver inflammation and fibrosis had statistically significant differences with different HBcAg staining levels ( χ 2 =44.142 and 102.386, respectively; P <0.001), and the severity was more apparent in the negative group. Conclusion: There exist differences in clinicopathological features for indications of different types of antiviral therapy in patients with CHB. Liver function test range is inconsistent with degrees of hepatic histological severity. The positive and intensity of liver tissue HBcAg staining, and albumin and alanine aminotransferase levels have negative correlation with disease severity.

Few reports have investigated the predictive value of urinary cadmium (UCd) and telomere length on renal function impairment. Therefore, we constructed nomogram models, using a cross-sectional survey to analyze the potential function of UCd and telomere length in renal function impairment risk. We randomly selected two community populations in Shanxi, China, and general information of the subjects was collected through face-to-face questionnaire surveys. Venous blood of subjects was collected to detect absolute telomere length (ATL) by real-time quantitative chain reaction (RT-PCR). Collecting urinary samples detected UCd and urinary N-acetyl-β-d-glucosaminidase (UNAG). Estimated glomerular filtration rate (eGFR) was obtained based on serum creatinine (SCr). Nomogram models on risk prediction analysis of renal function impairment was constructed. After adjusting for other confounding factors, UCd (β = 0.853, 95% confidence interval (CI): 0.739 ~ 0.986) and ATL (β = 1.803, 95%CI: 1.017 ~ 1.154) were independent risk influencing factors for increased UNAG levels, and the risk factors for eGFR reduction were UCd (β = 1.011, 95%CI: 1.187 ~ 1.471), age (β = 1.630, 95%CI: 1.303 ~ 2.038), and sex (β = 0.181, 95%CI: 0.105 ~ 0.310). Using UCd, ATL, sex, and age to construct the nomogram, and the C-statistics 0.584 (95%CI: 0.536 ~ 0.632) and 0.816 (95%CI: 0.781 ~ 0.851) were obtained by internal verification of the calibration curve, C-statistics revealed nomogram model validation was good and using decision curve analysis (DCA) confirmed a good predictive value of the nomogram models. In a nomogram model, ATL, UCd, sex, and age were detected as independent risk factors for renal function impairment, with UCd being the strongest predictor., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) is a non-receptor protein tyrosine phosphatase ubiquitously expressed mainly in the cytoplasm of several tissues. SHP2 modulates diverse cell signaling events that control metabolism, cell growth, differentiation, cell migration, transcription and oncogenic transformation. It interacts with diverse molecules in the cell, and regulates key signaling events including RAS/ERK, PI3K/AKT, JAK/STAT and PD-1 pathways downstream of several receptor tyrosine kinases (RTKs) upon stimulation by growth factors and cytokines. SHP2 acts as both a phosphatase and a scaffold, and plays prominently oncogenic functions but can be tumor suppressor in a context-dependent manner. It typically acts as a positive regulator of RTKs signaling with some inhibitory functions reported as well. SHP2 expression and activity is regulated by such factors as allosteric autoinhibition, microRNAs, ubiquitination and SUMOylation. Dysregulation of SHP2 expression or activity causes many developmental diseases, and hematological and solid tumors. Moreover, upregulated SHP2 expression or activity also decreases sensitivity of cancer cells to anticancer drugs. SHP2 is now considered as a compelling anticancer drug target and several classes of SHP2 inhibitors with different mode of action are developed with some already in clinical trial phases. Moreover, novel SHP2 substrates and functions are rapidly growing both in cell and cancer. In view of this, we comprehensively and thoroughly reviewed literatures about SHP2 regulatory mechanisms, substrates and binding partners, biological functions, roles in human cancers, and different classes of small molecule inhibitors target this oncoprotein in cancer., (© 2022. Springer Nature Switzerland AG.)

Heavy metal contamination is one of the main factors causing ecological and environmental degradation. Soil contamination by heavy metals decreases soil quality, reduces agricultural productivity and quality, and even threatens human health. Therefore, optimizing remediation strategies for soils polluted with heavy metals is of great significance for high-yield, good-quality, and sustainable agriculture. Numerous domestic and foreign scholars have carried out a large number of studies on the phytoremediation of heavy metal contaminated soils. However, the remediation efficiency may be restricted by soil and climatic/environmental conditions. The synergistic remediation of microorganisms and plants is considered an effective means to improve metal remediation efficiency under environmental stresses. Metal-resistant plant growth-promoting bacteria (PGPB) not only promote plant growth and its resistance to biotic (e.g., phytopathogens, etc.) and abiotic (e.g., drought, salinity, extreme temperatures, heavy metals, etc.) stresses but also alter meal bioavailability in soils and metal toxicity in plants, thereby improving phytoremediation efficiency. In this paper, the mechanisms involved in promoting plant growth and its stress tolerance, and affecting metal bioavailability by metal-resistant PGPB, were systematically summarized. Furthermore, research progress on the application of PGPB in ecological restoration in recent years was extensively reviewed.

Objective: To investigate the effect of whole-process case management based on service process design on patients undergoing total knee arthroplasty (TKA) in areas including pain, function, satisfaction, and complications., Methods: A total of 204 patients who underwent unilateral TKA between April 2021 and March 2022 at the Department of Orthopedics, West China Hospital, Sichuan University were enrolled. By using a random number table, the patients were randomly assigned to two groups, 102 in the general case management group (group G) and 102 in the whole-process case management group (group W). Patients in group G received traditional perioperative case management, while those in the whole-process case management group received integrated case management optimized on the basis of the service process design. The two groups of patients were studied through comparison of their general data, Visual Analogue Scale (VAS) pain score, knee flexion and range of motion, Hospital for Special Surgery (HSS) knee score, the 18-item Patient Satisfaction Questionnaire Short Form (PSQ-18), ability to climb stairs, and complications at 3 days and 3, 8, and 12 weeks after TKA., Results: There was no significant difference between the two groups in patient general information or baseline data collected at the time of enrollment ( P >0.05). There was no significant difference in HSS score, joint range of motion, and VAS pain score between the two groups before the surgery and 3 days after the surgery ( P >0.05). However, the HSS score, joint range of motion, and VAS pain scores of group W were significantly superior to those of group G at 3, 8 and 12 weeks after the surgery (all P <0.05). In addition, group W demonstrated significantly better ability to climb up and down stairs than that of group G at 12 weeks after the surgery ( P < 0.001). In terms of satisfaction, patients in group W were significantly more satisfied than those in group G at 3 days, and 3, 8, and 12 weeks after the surgery ( P <0.001)., Conclusion: Whole-process case management based on service process design has a positive effect of relieving pain, increasing range of motion, improving function, increasing satisfaction, and reducing complications in patients undergoing TKA., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).)

Background: Glycolysis caused by hypoxia-induced abnormal activation of hypoxia inducible factor-1α (HIF-1α) in the immune microenvironment promotes the progression of hepatocellular carcinoma (HCC), leading to enhanced drug resistance in cancer cells. Therefore, altering the immunosuppressive microenvironment by imp-roving the hypoxic state is a new goal in improving cancer treatment., Aim: To analyse the role of HIF-1α, which is closely related to tumour proliferation, invasion, metastasis, and angiogenesis, in the proliferation and invasion of liver cancer, and to explore the HIF-1α pathway-mediated anti-cancer mechanism of sirolimus (SRL) combined with Huai Er., Methods: Previous studies on HCC tissues identified the importance of HIF-1α, glucose transporter 1 (GLUT1), and lactate dehydrogenase A (LDHA) expression. In this study, HepG2 and Huh7 cell lines were treated, under hypoxic and normoxic conditions, with a combination of SRL and Huai Er. The effects on proliferation, invasion, cell cycle, and apoptosis were analysed. Proteomics and genomics techniques were used to analyze the HIF-1α-related signalling pathway during SRL combined with Huai Er treatment and its inhibition of the proliferation of HCC cells., Results: High levels of HIF-1α, LDHA, and GLUT-1 were found in poorly differentiated HCC, with lower patient survival rates. Hypoxia promoted the proliferation of HepG2 and Huh7 cells and weakened the apoptosis and cell cycle blocking effects of the SRL/Huai Er treatment. This was achieved by activation of HIF-1α and glycolysis in HCC, leading to the upregulation of LDHA, GLUT-1, Akt/mammalian target of rapamycin (mTOR), vascular endothelial growth factor (VEGF), and Forkhead box P3 and downregulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and p27. The hypoxia-induced activation of HIF-1α showed the greatest attenuation in the SRL/Huai Er (S50 + H8) group compared to the drug treatments alone ( P < 0.001). The S50 + H8 treatment significantly downregulated the expression of mTOR and HIF-1α, and significantly reduced the expression of VEGF mRNA. Meanwhile, the combined blocking of mTOR and HIF-1α enhanced the downregulation of Akt/mTOR , HIF-1α , LDHA , and GLUT-1 mRNA and resulted in the downregulation of PTEN, p27 , and VEGF mRNA ( P < 0.001)., Conclusion: SRL increases the anti-cancer effect of Huai Er, which reduces the promotion of hypoxia-induced HIF-1α on the Warburg effect by inhibition of the PI3K/Akt/mTOR-HIF-1α and HIF-1α-PTEN signalling pathways in HCC., Competing Interests: Conflict-of-interest statement: The authors have no conflicts of interest to declare., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

Click chemistry is a concept wherein modular synthesis is used for rapid functional discovery. To this end, continuous discovery of clickable chemical transformations is the pillar to support the development of this field. This report details the development of a clickable C3-H selenylation of indole that is suitable for on-plate parallel and DNA-encoded library (SeDEL) synthesis via bioinspired LUMO activation strategy. This reaction is modular, robust and highly site-selective, and it features a simple and mild reaction system (catalyzed by nonmetallic B(C 6 F 5 ) 3 at room temperature), high yields and excellent functional group compatibility. Using this method, a library of 1350 indole-selenides was parallel synthesized in an efficient and practical manner, enabling the rapid identification of 3 ai as a promising compound with nanomolar antiproliferative activity in cancer cells via in situ phenotypic screening. These results indicate the great potential of this new clickable selenylation reaction in high-throughput medicinal chemistry and chemical biology., (© 2022 Wiley-VCH GmbH.)

Objective: Liver fibrosis is a frequently occurring liver injury which lacks of effective treatment clinically. Here, we investigated the protective effects of a novel compound Gorse isoflavone alkaloid (GIA) against liver fibrosis., Methods: Totally forty rats were randomly divided into four groups. Then we established a model of liver fibrosis induced by the intragastric administration of carbon tetrachloride (CCl 4 ). This treated group was followed by the intragastric administration of GIA and colchicine. Then the liver index and spleen index, and liver function indexes were detected by kit. Western blotting assay was performed to estimate the expression of Transforming Growth Factor-β1 (TGF-β1) and related proteins. Tissue fibrosis was observed by Masson staining., Results: Our results suggested that GIA reduced the deposition of collagen fibres and the fibrosis index hydroxyproline (Hyp) of liver tissue. Furthermore, we found that GIA significantly decreased the expression of Transforming Growth Factor-β1 (TGF-β1) and the ratio of p-smad2/3 to smad2/3, enhanced the level of superoxide dismutase (SOD), and decreased the concentration of malonic dialdehyde (MDA) in the liver., Conclusions: Our findings revealed that GIA has a beneficial effect to resist the liver fibrosis, and could be ideal for potential use in antifibrotic drugs for the liver., Competing Interests: None., (AJTR Copyright © 2022.)