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Boron neutron capture therapy (BNCT) targets invasive, radioresistant cancers but requires a selective and high B-10 loading boron drug. This manuscript investigates boron-rich poly(ethylene glycol)- block -(poly(4-vinylphenyl boronate ester)) polymer micelles synthesized via atom transfer radical polymerization for their potential application in BNCT. Transmission electron microscopy (TEM) revealed spherical micelles with a uniform size of 43 ± 10 nm, ideal for drug delivery. Additionally, probe sonication proved effective in maintaining the micelles' size and morphology postlyophilization and reconstitution. In vitro studies with B16-F10 melanoma cells demonstrated a 38-fold increase in boron accumulation compared to the borophenylalanine drug for BNCT. In vivo studies in a B16-F10 tumor-bearing mouse model confirmed enhanced tumor selectivity and accumulation, with a tumor-to-blood (T/B) ratio of 2.5, surpassing BPA's T/B ratio of 1.8. As a result, mice treated with these micelles experienced a significant delay in tumor growth, highlighting their potential for BNCT and warranting further research.

Malignant cancers, known for their pronounced heterogeneity, pose substantial challenges to monotherapeutic strategies and contribute to the risk of metastasis. Addressing this, our study explores the synergistic potential of combining boron neutron capture therapy (BNCT) with immune checkpoint blockade to enhance cancer treatment efficacy. We synthesized boron-rich block copolymer micelles as a novel boron drug for BNCT. Characterization was conducted using nuclear magnetic resonance, gel-permeation chromatography, transmission electron microscopy, and dynamic light scattering. These micelles, with an optimal size of 91.3 nm and a polydispersity index of 0.18, are suitable for drug delivery applications. In vitro assessments on B16-F10 melanoma cells showed a 13-fold increase in boron uptake with the micelles compared to borophenyl alanine (BPA), the conventional boron drug for BNCT. This resulted in a substantial increase in BNCT efficacy, reducing cell viability to 77% post-irradiation in micelle-treated cells, in contrast to 90% in BPA-treated cells. In vivo, melanoma-bearing mice treated with these micelles exhibited an 8-fold increase in boron accumulation in tumor tissues versus those treated with BPA, leading to prolonged tumor growth delay (5.4 days with micelles versus 3.3 days with BPA). Moreover, combining BNCT with anti-PD-L1 immunotherapy further extended the tumor growth delay to 6.6 days, and enhanced T-cell infiltration and activation at tumor sites, thereby indicating a boosted immune response. This combination demonstrates a promising approach by enhancing cytotoxic T-cell priming and mitigating the immunosuppressive effects of melanoma tumors., Competing Interests: Competing interes ts: The authors declare that they have no competing interests., (Copyright © 2024 Yi-Lin Chiu et al.)

Fixed-dose combination (FDC) therapies can enhance patient convenience and adherence to prescribed treatment regimens. Elagolix is a novel oral gonadotropin-releasing hormone receptor antagonist approved for management of moderate to severe pain associated with endometriosis and heavy menstrual bleeding associated with uterine fibroids. Hormonal add-back therapy can attenuate the reversible hypoestrogenic effects of elagolix. An FDC formulation containing elagolix/estradiol (E2)/norethindrone acetate (NETA) 300/1/0.5 mg as the morning dose and an elagolix 300 mg capsule as the evening dose, were evaluated in 2 bioequivalence studies including the effects of food. Study 1 in premenopausal women assessed the bioavailability of the elagolix 300-mg capsule relative to the commercially available elagolix 300-mg tablet. Study 2 in postmenopausal women, elagolix/E2/NETA (300 mg/1 mg/0.5 mg) FDC capsule was assessed relative to the elagolix 300-mg tablet coadministered with E2/NETA 1-mg/0.5-mg tablet, the regimen that was studied in Phase 3 uterine fibroid studies. Under fasting conditions, the test elagolix 300-mg capsule was bioequivalent to the reference elagolix 300-mg tablet. Under fasting conditions, the elagolix/E2/NETA FDC capsule was bioequivalent to the coadministered elagolix 300-mg tablet and E2/NETA 1/0.5-mg tablet. Following administration of elagolix/E2/NETA FDC capsule after a high-fat breakfast, elagolix mean maximum concentration (C max ) and area under the plasma concentration-time curve (AUC) were 38% and 28% lower, relative to fasting conditions. NETA mean C max was 51% lower and AUC from time 0 to infinity was 20% higher, while baseline-adjusted total estrone mean C max and AUC were 46% and 14% lower, respectively. No safety concerns were identified. These results enabled bridging the elagolix/E2/NETA FDC capsule., (© 2024 AbbVie Inc. Clinical Pharmacology in Drug Development published by Wiley Periodicals LLC on behalf of American College of Clinical Pharmacology.)

Acute lung injury occurs in 20-25% of cases following traumatic brain injury (TBI). We investigated changes in lung transcriptome expression post-TBI using animal models and bioinformatics. Employing unilateral controlled cortical impact for TBI, we conducted microarray analysis after lung acquisition, followed by gene set enrichment analysis of differentially expressed genes. Our findings indicate significant upregulation of inflammation-related genes and downregulation of nervous system genes. There was enhanced infiltration of adaptive immune cells, evidenced by positive enrichment in Lung-Th1, CD4, and CD8 T cells. Analysis using the Tabula Sapiens database revealed enrichment in lung-adventitial cells, pericytes, myofibroblasts, and fibroblasts, indicating potential effects on lung vasculature and fibrosis. Gene set enrichment analysis linked TBI to lung diseases, notably idiopathic pulmonary hypertension. A Venn diagram overlap analysis identified a common set of 20 genes, with FOSL2 showing the most significant fold change. Additionally, we observed a significant increase in ADRA1A → IL6 production post-TBI using the L1000 library. Our study highlights the impact of brain trauma on lung injury, revealing crucial gene expression changes related to immune cell infiltration, cytokine production, and potential alterations in lung vasculature and fibrosis, along with a specific spectrum of disease influence.

Triple-negative breast cancer (TNBC) is characterized by highly proliferative cancer cells and is the only subtype of breast cancer that lacks a targeted therapy. Boron neutron capture therapy (BNCT) is an approach that combines chemotherapy with radiotherapy and can potentially offer beneficial targeted treatment for TNBC patients owing to its unique ability to eradicate cancer cells selectively while minimizing damage to the surrounding healthy cells. Since BNCT relies on specific delivery of a high loading of B10 to the tumor site, there is growing research interest to develop more potent boron-based drugs for BNCT that can overcome the limitations of small-molecule boron compounds. In this study, polyethylene-glycol-coated boron carbon oxynitride nanoparticles (PEG@BCNO) of size 134.2±23.6nm were prepared as a promising drug for BNCT owing to their high boron content and enhanced biocompatibility. The therapeutic efficiency of PEG@BCNO was compared with a state-of-the-art 10 BPA boron drug in mice bearing MDA-MB-231 tumor. In the orthotopic mouse model, PEG@BCNO showed higher B10 accumulation in the tumor tissues (6 μg 10 B/g tissue compared to 3 μg 10 B/g tissue in mice administered B10-enriched 10 BPA drug) despite using the naturally occurring 11 B/ 10 B boron precursor in the preparation of the BCNO nanoparticles. The in vivo biodistribution of PEG@BCNO in mice bearing MDA-MB-231 showed a tumor/blood ratio of ~3.5, which is comparable to that of the state-of-the-art 10 BPA-fructose drug. We further demonstrated that upon neutron irradiation, the mice bearing MDA-MB-231 tumor cells treated with PEG@BCNO and 10 BPA showed tumor growth delay times of 9 days and 1 day, respectively, compared to mice in the control group after BNCT. The doubling times (DTs) for mice treated with PEG@BCNO and 10 BPA as well as mice in the control group were calculated to be 31.5, 19.8, and 17.7 days, respectively. Immunohistochemical staining for the p53 and caspase-3 antibodies revealed that mice treated with PEG@BCNO showed lower probability of cancer recurrence and greater level of cellular apoptosis than mice treated with 10 BPA and mice in the control group. Our study thus demonstrates the potential of pegylated BCNO nanoparticles in effectively inhibiting the growth of TNBC tumors compared to the state-of-the-art boron drug 10 BPA., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Pei Yuin Keng reports financial support was provided by National Science and Technology Council., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Urothelial cancer, a common urinary system malignancy, often presents treatment challenges due to metastasis and chemotherapy side effects. Angiogenesis, crucial for tumor growth, has become a target for drug development. This study explores the expression, prognostic value, and clinical correlation of RHOJ in the TCGA BLCA, GSE31684, and GSE32894 datasets. We identify common differentially expressed genes across these databases and utilize g:Profiler and Cytoscape ClueGO for functional assessment. Further, we perform a gene set enrichment analysis (GSEA) using Hallmark gene sets and use the imsig package for immune cell infiltration analysis. Our analysis indicates that RHOJ expression levels significantly impact survival rates, tumor progression, and immune response in urothelial tumors. High RHOJ expression correlated with poor prognosis, advanced disease stages, and an increase in monocyte population within the tumor microenvironment. This aligns with current literature indicating a key role of immune infiltration in bladder cancer progression and treatment response. Moreover, the GSEA and imsig results further suggest a potential mechanistic link between RHOJ expression and immune-related pathways. Considering the increasing emphasis on immunotherapeutic strategies in bladder cancer management, our findings on RHOJ 's potential as a diagnostic biomarker and its association with immune response open new avenues for therapeutic interventions.

Various pathological alterations, including lipid-deposition-induced comparative cardiac lipotoxicity, contribute to cardiac aging in the failing heart. A decline in endogenous myogenin proteins can lead to the reversal of muscle cell differentiation and the creation of mononucleated muscle cells. Myogenin may be a specific regulator of adaptive responses to avoid pathological hypertrophy in the heart. Hence, it is important to understand the regulation of myogenin expression and functions in response to exposure to varied stresses. In this study, we first examined and verified the cytotoxic effect of palmitic acid on H9c2 cells. The reduction in myogenin mRNA and protein expression by palmitic acid was independent of the effect of glucose. Meanwhile, the induction of cyclooxygenase 2 and activating transcription factor 3 mRNAs and proteins by palmitic acid was dependent on the presence of glucose. In addition, palmitic acid failed to disrupt cell cycle progression when H9c2 cells were treated with no glucose. Next, we examined the functional role of myogenin in palmitic-acid-treated H9c2 cells and found that myogenin may be involved in palmitic-acid-induced mitochondrial and cytosolic ROS generation, cellular senescence, and mitochondrial membrane potential. Finally, the GSE150059 dataset was deposited in the Gene Expression Omnibus website and the dataset was further analyzed via the molecular microscope diagnostic system (MMDx), demonstrating that many heart transplant biopsies currently diagnosed as no rejection have mild molecular-antibody-mediated rejection-related changes. Our data show that the expression levels of myogenin were lower than the average level in the studied population. Combining these results, we uncover part of the functional role of myogenin in lipid- and glucose-induced cardiac cell stresses. This finding provides valuable insight into the differential role of fatty-acid-associated gene expression in cardiovascular tissues. Additionally, the question of whether this gene expression is regulated by myogenin also highlights the usefulness of a platform such as MMDx-Heart and can help elucidate the functional role of myogenin in heart transplantation.

Introduction: MicroRNAs may be implicated in the acquisition of drug resistance in chronic myeloid leukemia as they regulate the expression of not only BCR-ABL1 but also genes associated with the activation of drug transfer proteins or essential signaling pathways., Methods: To understand the impact of specifically expressed miRNAs in chronic myeloid leukemia and their target genes, we collected peripheral blood mononuclear cells (PBMC) from patients diagnosed with chronic myeloid leukemia (CML) and healthy donors to determine whole miRNA expression by small RNA sequencing and screened out 31 differentially expressed microRNAs (DE-miRNAs) with high expression. With the utilization of miRNA set enrichment analysis tools, we present here a comprehensive analysis of the relevance of DE-miRNAs to disease and biological function. Furthermore, the literature-based miRNA-target gene database was used to analyze the overall target genes of the DE-miRNAs and to define their associated biological responses. We further integrated DE-miRNA target genes to identify CML miRNA targeted gene signature singscore (CMTGSS) and used gene-set enrichment analysis (GSEA) to analyze the correlation between CMTGSS and Hallmark gene-sets in PBMC samples from clinical CML patients. Finally, the association of CMTGSS stratification with multiple CML cell lineage gene sets was validated in PBMC samples from CML patients using GSEA., Results: Although individual miRNAs have been reported to have varying degrees of impact on CML, overall, our results show that abnormally upregulated miRNAs are associated with apoptosis and aberrantly downregulated miRNAs are associated with cell cycle. The clinical database shows that our defined DE-miRNAs are associated with the prognosis of CML patients. CMTGSS-based stratification analysis presented a tendency for miRNAs to affect cell differentiation in the blood microenvironment., Conclusion: Collectively, this study defined differentially expressed miRNAs by miRNA sequencing from clinical samples and comprehensively analyzed the biological functions of the differential miRNAs in association with the target genes. The analysis of the enrichment of specific myeloid differentiated cells and immune cells also suggests the magnitude and potential targets of differentially expressed miRNAs in the clinical setting. It helps us to make links between the different results obtained from the multi-faceted studies to provide more potential research directions., 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., (Copyright © 2023 Wu, Lai, Lu, Lai, Chen, Chen, Ho, Wu and Chiu.)

Glioblastoma multiforme (GBM) is a grade IV human glioma. It is the most malignant primary central nervous system tumor in adults, accounting for around 15% of intracranial neoplasms and 40-50% of all primary malignant brain tumors. However, the median survival time of GBM patients is still less than 15 months, even after treatment with surgical resection, concurrent chemoradiotherapy, and adjuvant chemotherapy with temozolomide (TMZ). Telomere maintenance 2 (TELO2) mRNA is highly expressed in high-grade glioma patients, and its expression correlates with shorter survival outcomes. Hence, it is urgent to address the functional role of TELO2 in the tumorigenesis and TMZ treatment of GBM. In this study, we knocked down TELO2 mRNA in GBM8401 cells, a grade IV GBM, compared with TELO2 mRNA overexpression in human embryonic glial SVG p12 cells and normal human astrocyte (NHA) cells. We first analyzed the effect of TELO2 on the Elsevier pathway and Hallmark gene sets in GBM8401, SVG p12, and NHA via an mRNA array analysis. Later, we further examined and analyzed the relationship between TELO2 and fibroblast growth factor receptor 3, cell cycle progression, epithelial-mesenchymal transient (EMT), reactive oxygen species (ROS), apoptosis, and telomerase activity. Our data showed that TELO2 is involved in several functions of GBM cells, including cell cycle progression, EMT, ROS, apoptosis, and telomerase activity. Finally, we examined the crosstalk between TELO2 and the responsiveness of TMZ or curcumin mediated through the TELO2-TTI1-TTI2 complex, the p53-dependent complex, the mitochondrial-related complex, and signaling pathways in GBM8401 cells. In summary, our work provides new insight that TELO2 might modulate target proteins mediated through the complex of phosphatidylinositol 3-kinase-related kinases in its involvement in cell cycle progression, EMT, and drug response in GBM patients.

Global warming increases the incidence of heat stroke (HS) and HS causes the reduction of visceral blood flow during hyperthermia, leading to intestinal barrier disruption, microbial translocation, systemic inflammation and multiple organ failure. Cathelicidin LL-37 exhibits antimicrobial activities, helps innate immunity within the gut to maintain intestinal homeostasis, and augments intestinal wound healing and barrier function. Thus, we evaluated the effects and possible mechanisms of cathelicidin LL-37 on HS. Wistar rats were placed in a heating-chamber of 42 ̊C to induce HS. Changes in rectal temperature, hemodynamic parameters, and survival rate were measured during the experimental period. Blood samples and ilea were collected to analyze the effects of LL-37 on systemic inflammation, multiple organ dysfunction, and intestinal injury. Furthermore, LS174T and HT-29 cells were used to assess the underlying mechanisms. Our data showed cathelicidin LL-37 ameliorated the damage of intestinal cells induced by HS. Intestinal injury, systemic inflammation, and nitrosative stress (high nitric oxide level) caused by continuous hyperthermia were attenuated in HS rats treated with cathelicidin LL-37, and hence, improved multiple organ dysfunction, coagulopathy, and survival rate. These beneficial effects of cathelicidin LL-37 were attributed to the protection of intestinal goblet cells (by increasing transepithelial resistance, mucin-2 and Nrf2 expression) and the improvement of intestinal barrier function (less cyclooxygenase-2 expression and FITC-dextran translocation). Interestingly, high cathelicidin expression in the ileal samples of inflammatory bowel disease patients was associated with better clinical outcome. These results suggest that cathelicidin LL-37 could prevent heat stress-induced intestinal damage and heat-related illnesses., Competing Interests: Conflict of interest statement The authors have declared that no conflict of interest exists., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Aims: Heat stroke is a life-threatening disorder triggered by thermoregulatory failure. Hyperthermia-induced splanchnic hypoperfusion has been reported to induce intestinal barrier dysfunction and systemic immune response that ultimately cause multiple-organ failure and death. Intestinal goblet cells contribute greatly to the formation of mucus barrier, which hinders translocation of gut microorganisms. Studies have reported that misoprostol can not only alleviate ischemic injury but also protect GI mucosal layer. Therefore, we evaluated the effects of misoprostol on intestinal goblet cells after heat stress and on multiple-organ dysfunction in heat stroke rats., Main Methods: Heat stress was established in the heating chamber and followed by misoprostol treatment. Changes in hemodynamics, organ function indices, inflammation, oxidative stress, and survival rate were analyzed. Furthermore, ilea and LS174T cells were used to examine intestinal functions., Key Findings: Heat stress caused dysfunction of intestinal goblet cells and damage to ilea by increasing oxidative stress and apoptosis. Increased nitrosative stress and inflammation accompanied by hypotension, hypoperfusion, tachycardia, multiple-organ dysfunction, and death were observed in the heat stroke rat model. Treatment of LS174T cells with misoprostol not only decreased oxidative stress and apoptosis but also reduced cytotoxicity caused by heat stress. Moreover, misoprostol prevented disruption of the enteric barrier, multiple-organ injury, and death in rats with heat stroke., Significance: This study indicates that misoprostol could alleviate intestinal damage and organ injury caused by heat stress and be a potential therapy for heat-related illnesses., Competing Interests: Declaration of competing interest All authors have read the journal's policy on disclosure of potential conflicts of interest and have no conflicts of interest to declare., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose: In this study, we use animal models combined with bioinformatics strategies to investigate the potential changes in overall renal transcriptional expression after traumatic brain injury., Methods: Microarray analysis was performed after kidney acquisition using unilateral controlled cortical impact as the primary mouse TBI model. Multi-oriented gene set enrichment analysis was performed for differentially expressed genes., Results: The results showed that TBI affected the gene set associated with mitochondria function in kidney cells, and a negative enrichment of gene sets associated with immune cell migration and epidermal development was also observed. Analysis of the disease phenotype gene set revealed that differential expression of mitochondria-related genes was associated with lactate metabolism. Alternatively, activation and adhesion of immune cells associated with the complement system may promote autoinflammation in kidney tissue. The simulated immune cell infiltration analysis showed an increase in the proportion of activated memory CD4 T cells and a decrease in the proportion of resting memory CD4 T cells, suggesting that activated memory CD4 T cell infiltration may be involved in the inflammation of renal tissue and cause damage to renal cells, such as principal cells, mesangial cells and loops of Henle cells., Conclusion: This study is the first to reveal the effects of brain trauma on the kidney. TBI may affect the expression of mitochondria function-related gene sets in renal cells by increasing lactate. It may also affect renal mesangial cells by inducing increased infiltration of immune cells through mechanisms related to complement system activation or autoimmune antibodies., Competing Interests: The authors report no conflicts of interest in this work., (© 2022 Chan et al.)

Background: Benzo[a]pyrene (BaP), a toxic carcinogen, is associated with various adverse effects but is rarely discussed in muscle-related disorders. This study investigated in vitro and in vivo effects triggered by BaP exposure in muscles and hypothesized that exposure might induce conditions similar to sarcopenia due to the shared mechanism of oxidative stress. In vitro experiments used C2C12 mouse myoblasts to examine effects induced by BaP exposure in control (untreated) and BaP-treated (10 µM/ml) muscle cells. An established TNF-α-treated sarcopenia model was utilized to verify our results. In vivo experiments compared immunohistochemical staining of sarcopenia-related markers in rats exposed to clean air and polluted air., Results: In C2C12 cells, after 2-72 h of BaP exposure, elevated mRNA and protein expressions were observed in aryl hydrocarbon receptor (AhR) and cytochrome P450 1A1, subsequently in ROS (NOX2 and NOX4) production, inflammatory cytokines (IL-6, TNF-α, and NF-kB), and proteins mediating apoptotic cell death (caspase-3 and PARP). Two myokines also altered mRNA and protein expressions akin to changes in sarcopenia, namely decreased irisin levels and increased myostatin levels. In addition, N-acetylcysteine, a well-known antioxidant, led to decrease in oxidative markers induced by BaP. The validation by TNF-α-treated sarcopenia model revealed comparable biological responses in either TNF-α or BaP treated C2C12 cells. In vivo experiments with rats exposed to air pollution showed increased expression of BaP, AhR, 8-hydroxydeoxyguanosine, and myostatin and decreased irisin expression in immunohistochemical staining., Conclusions: Our results suggest that BaP exerts deleterious effects on the muscle, leading to conditions indicative of sarcopenia. Antioxidant supplementation may be a treatment option for BaP-induced sarcopenia, but further validation studies are needed., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) is the primary histological subtype of liver cancer, and its incidence rates increase with age. Recently, systemic therapies, such as immune checkpoint inhibitors, monoclonal antibodies, and tyrosine kinase inhibitors (TKIs), have been more beneficial than conventional therapies for treating HCC. Nonetheless, the prognosis of late-stage HCC remains dismal because of its high recurrence rates, even with substantial advances in current therapeutic strategies. A new treatment, such as a combination of current systemic therapies, is urgently required. Therefore, we adopted a repurposing strategy and tried to combine ascorbate with TKIs, including lenvatinib and regorafenib, in HepG2 and Hep3B cells. We investigated the potential functional impact of pharmacological concentrations of ascorbate on the cell-cycle profiles, mitochondrial membrane potential, oxidative response, synergistic effects of lenvatinib or regorafenib, and differential responsiveness between HepG2 and Hep3B cells. Our data suggest that the relative level of cell density is an important determinant for ascorbate cytotoxicity in HCC. Furthermore, the data also revealed that the cytotoxic effect of pharmacological concentrations of ascorbate might not be mediated via our proposed elevation of ROS generation. Ascorbate might be involved in redox homeostasis to enhance the efficacy of TKIs in HepG2 and Hep3B cells. The synergistic effects of ascorbate with TKIs (lenvatinib and regorafenib) support their potential as an adjuvant for HCC targeted TKI therapy. This research provides a cheap and new combinatory therapy for HCC treatment., 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., (Copyright © 2022 Fan, Liu, Chang, Chiu, Huang and Chen.)

Objective: To highlight the clinical characteristics of primary biliary cholangitis on the basis of gender in terms of the extent of liver injury and extra-liver autoimmune expressions., Methods: The retrospective study was conducted at the Tri-Service General Hospital, Taiwan, and comprised data of patients aged >20 years diagnosed with primary biliary cholangitis between January 2000 and December 2018. Patients in the control group were randomly selected from the health examination centre. Liver injury manifestations and susceptibilities were analysed along gender lines. The gene expression microarray data from the National Centre for Biotechnology Information Gene Expression Omnibus database was also used to explore the relationship between autoimmune-induced inflammation and androgen response expression. Statistical analysis was done using Graph-Pad Prism 7.0., Results: Of the 75 cases, 63(84%) were females with a mean age of 64.6±1.78 years, and 12(16%%) were males with a mean age of 46.6±5.6 years. Of the 66 controls, 55(83.3%) were females with a mean age of 51.67 years, and 11(16.6%) were males with a mean age of 45.9 years. There were no significant differences in terms of liver profiles related to gender in the control group (p>0.05). Among the cases, male patients showed fewer extrahepatic autoimmune disorders and more severe liver injuries before or after ursodeoxycholic acid treatment (p<0.05). There was a positive correlation between androgen receptor response and the extent of systemic inflammation (p<0.05). Conclusion: The association between androgen receptor responses and inflammation was linked to gender-related hepatic injuries, which may explain why liver inflammation in male patients is generally more severe compared to the female patients., Conclusion: The association between androgen receptor responses and inflammation was linked to gender-related hepatic injuries, which may explain why liver inflammation in male patients is generally more severe compared to the female patients.

Rupture of abdominal aortic aneurysms (AAAs) is one of the leading causes of sudden death in the elderly population. The osteogenic transcription factor runt-related gene (RUNX) encodes multifunctional mediators of intracellular signal transduction pathways in vascular remodeling and inflammation. We aimed to evaluate the roles of RUNX2 and its putative downstream target miR-424/322 in the modulation of several AAA progression-related key molecules, such as matrix metalloproteinases and vascular endothelial growth factor. In the GEO database, we found that male patients with AAAs had higher RUNX2 expression than did control patients. Several risk factors for aneurysm induced the overexpression of MMPs through RUNX2 transactivation, and this was dependent on Smad2/3 upregulation in human aortic smooth muscle cells. miR-424 was overexpressed through RUNX2 after angiotensin II (AngII) challenge. The administration of siRUNX2 and miR-424 mimics attenuated the activation of the Smad/RUNX2 axis and the overexpression of several AAA progression-related molecules in vitro . Compared to their littermates, miR-322 KO mice were susceptible to AngII-induced AAA, whereas the silencing of RUNX2 and the administration of exogenous miR-322 mimics ameliorated the AngII-induced AAA in ApoE KO mice. Overall, we established the roles of the Smad/RUNX2/miR-424/322 axis in AAA pathogenesis. We demonstrated the therapeutic potentials of miR-424/322 mimics and RUNX2 inhibitor for AAA progression., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Overexpression of the deubiquitinase USP2a leads to stabilization of fatty acid synthase (FAS), the levels of which are often elevated in aggressive human cancers. Consequently, there is an urgent need for inhibitors to suppress the deubiquitination activity of USP2a so as to upregulate FAS protein degradation. We first analyzed the relationship between the expression level of USP2a and survival using The Cancer Genome Atlas Head-Neck Squamous Cell Carcinoma (HNSC) data collection. Our results suggested survival rates were lower among HNSC patients expressing higher levels of USP2a. We then investigated two thiopurine drugs, 6-thioguanine (6-TG) and 6-mercaptopurine (6-MP), to determine whether they could potentially serve as inhibitors of USP2a. Western blot analysis showed that levels of two USP2a target proteins, FAS and Mdm2, were dose-dependently decreased in A253 submaxillary carcinoma cells treated with 6-TG or 6-MP. Responding to the degradation of Mdm2, levels of p53 were increased. We found that 6-TG and 6-MP also suppressed levels of both USP2a mRNA and protein, suggesting these two thiopurines do not act solely through direct inhibition of USP2a. The effects of 6-TG and 6-MP were not cell type-specific, as they elicited similar decreases in FAS protein in leukemia, prostate and cervical cancer cell lines. 6-TG and 6-MP had effects on several cell cycle proteins, including another USP2a target protein, cyclin D1. The populations of cells in subG1 and S phase were increased by 6-TG and 6-MP, which was accompanied by reductions in G1 phase cells. In untreated cells, USP2a transfection increased FAS and cyclin D1 levels compared to an enzyme-dead USP2a C276A mutant, which lacked deubiquitinating activity. However, USP2a transfection failed to reverse the suppressive effects of 6-TG and 6-MP on FAS levels. In summary, these findings suggest 6-TG and 6-MP reduce the stability of some USP2a targets, including FAS and Mdm2, by inhibiting USP2a-catalyzed deubiquitination in some cancer cells. Our work also provides repurposing evidence supporting 6-TG and 6-MP as target therapeutic drugs, such as USP2a/FAS in this study., 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., (Copyright © 2021 Cheng, Liu, Chiu, Huang and Ho.)

Coronary artery diseases are major problems of the world. Coronary artery disease patients frequently suffer from peptic ulcers when they receive aspirin treatment. For diagnostic and therapeutic purposes, the implementation of panendoscopy (PES) with biopsy is necessary. Some biopsy samples are wasted after the assay is completed. In the present study, we established a protocol for human gastric fibroblast isolation and induced pluripotent stem cell (iPSC) generation from gastric fibroblasts via PES with biopsy. We showed that these iPSCs can be differentiated into functional cardiomyocytes in vitro. To our knowledge, this is the first study to generate iPSCs from gastric fibroblasts in vitro.

Background: The soluble receptor for advanced glycation end products (sRAGE) has been proposed to serve as a marker for disease severity, but its role in sarcopenia, an age-related progressive loss of muscle mass and function, remains elusive. This study examines the association between sRAGE and sarcopenia., Methods: A total of 314 community-dwelling elderly adults who had their health examination at Tri-Service General Hospital from 2017 to 2019 underwent protein analysis with enzyme-linked immunosorbent assay. The relationship with sarcopenia and its detailed information, including components and diagnosis status, were examined using linear and logistic regressions., Results: As for sarcopenia components, low muscle mass (β = 162.8, p = 0.012) and strength (β = 181.31, p = 0.011) were significantly correlated with sRAGE, but not low gait speed (p = 0.066). With regard to disease status, confirmed sarcopenia (β = 436.93, p < 0.001), but not probable (p = 0.448) or severe sarcopenia (p = 0.488), was significantly correlated with sRAGE. In addition, females revealed a stronger association with sRAGE level by showing significant correlations with low muscle mass (β = 221.72, p = 0.014) and low muscle strength (β = 208.68, p = 0.043)., Conclusions: sRAGE level showed a positive association with sarcopenia, illustrating its involvement in the evolution of sarcopenia. This association is more evident in female groups, which may be attributed to the loss of protection from estrogen in postmenopausal women. Utilizing sRAGE level as a prospective marker for sarcopenia deserves further investigation in future studies., (© 2021. The Author(s).)

Chronic myeloid leukemia (CML) is a myeloproliferative disorder associated with the Philadelphia chromosome, and the current standard of care is the use of tyrosine kinase inhibitors (TKI). However, some patients will not achieve a molecular response and may progress to blast crisis, and the underlying mechanisms remain to be clarified. In this study, next-generation sequencing was used to explore endogenous miRNAs in CML patients versus healthy volunteers, and miR-342-5p was identified as the primary target. We found that miR-342-5p was downregulated in CML patients and had a significant inhibitory effect on cell proliferation in CML. Through a luciferase reporter system, miR-342-5p was reported to target the 3'-UTR domain of CCND1 and downregulated its expression. Furthermore, overexpression of miR-342-5p enhanced imatinib-induced DNA double-strand breaks and apoptosis. Finally, by analyzing clinical databases, we further confirmed that miR-342-5p was associated with predicted molecular responses in CML patients. In conclusion, we found that both in vivo and in vitro experiments and database cohorts showed that miR-342-5p plays a key role in CML patients, indicating that miR-342-5p may be a potential target for future CML treatment or prognostic evaluation., (© 2021. The Author(s).)

Cilostazol was suggested to be beneficial to retard in-stent atherosclerosis and prevent stent thrombosis. However, the mechanisms responsible for the beneficial effects of cilostazol are not fully understood. In this study, we attempted to verify the mechanism of the antithrombotic effect of cilostazol. Human umbilical vein endothelial cells (HUVECs) were cultured with various concentrations of cilostazol to verify its impact on endothelial cells. KLF2, silent information regulator transcript-1 (SIRT1), endothelial nitric oxide synthase (eNOS), and endothelial thrombomodulin (TM) expression levels were examined. We found cilostazol significantly activated KLF2 expression and KLF2-related endothelial function, including eNOS activation, Nitric oxide (NO) production, and TM secretion. The activation was regulated by SIRT1, which was also stimulated by cilostazol. These findings suggest that cilostazol may be capable of an antithrombotic and vasculoprotective effect in endothelial cells.

Clinical bladder tumor histological analysis shows that high expression of S1PR1 is associated with poor patient prognosis. However, there are no studies that describe the underlying mechanism. To investigate the relative distribution and actual function of S1PR1 in bladder tumors, we analyzed multiple clinical databases in combination with tumor purity and immune cell infiltration simulations, as well as databases of well-defined histological phenotypes of bladder cancer, and single-cell sequencing of adjacent normal tissues and bladder tumors, and further compared them with bladder cancer cell lines. The results showed that S1PR1 expression was generally higher in normal tissues than in bladder cancer tissues, and its distribution was mainly in endothelial cells or immune cells. The association between high S1PR1 expression and poor prognosis may be due to tumor invasion of adjacent normal tissues, where highly expressed S1PR1 may affect prognostic interpretation. The effect of S1PR1 itself on cancer cells was associated with cell adhesion, and in bladder cancer cells, S1PR1 expression was negatively correlated with cell motility. Moreover, the use of FTY-720 will cause an increased metastatic ability of bladder cancer cells. In conclusion, we suggest that the use of S1PR1 -specific inhibition as a synergistic treatment requires more observation and consideration.

According to the International Conference on Harmonisation E14 Q&As R3, concentration-QTc analysis can serve as an alternative to the by-time-point analysis or intersection-union test as the primary basis for decisions to classify the QTc risk of a drug. In a recent scientific white paper on concentration-QTc analysis, a pre-specified linear mixed effect model was suggested to study a QTc prolongation effect. The model assumes a direct time-concordant relationship (direct effect) between QTc interval and drug-concentrations. In the case of lagged drug QTc effects, also called 'hysteresis', a linear direct effect model may yield a biased QTc estimate. In this work, we estimate the bias of QTc effects via simulations when hysteresis is not accounted for in the linear mixed effect model analysis. Simulations are performed to compare different methods of identifying hysteresis when two physiologically plausible QT drug mechanisms are considered. The focus of this paper is to provide hysteresis identification methods and assess the influence of hysteresis in estimating the QT prolongation for most commonly observed QT-Concentration profiles.

Background: Two pharmacokinetic/pharmacodynamic studies were conducted to evaluate the potential drug-drug interaction between elagolix, an oral gonadotropin-releasing hormone receptor antagonist, and an oral contraceptive (ethinylestradiol [EE] 0.035 mg and norgestimate 0.18/0.215/0.25 mg) or progestin-only contraceptive (norethindrone 0.35 mg) in healthy premenopausal women., Methods: These phase I studies used a two-period, sequential design, where period 1 included treatment with oral contraceptives, followed by period 2 with contraceptives coadministered with elagolix 150 mg once daily., Results: In study 1, pharmacokinetic exposures for EE in period 2 increased by 30% and the norgestimate metabolites decreased by approximately 15% when coadministered with elagolix. Mean hormone exposure appeared lower for follicle-stimulating hormone (FSH; 31%), luteinizing hormone (LH; 38%), and estradiol (E2; 16%). The percentage of women with consecutive progesterone (P) concentrations above 5 nmol/L was similar in both periods. Norethindrone pharmacokinetic exposures were comparable in both periods. The hormone exposure for LH and FSH was similar, and mean E2 exposure was 32% lower in period 2. The percentage of subjects with consecutive ovulatory P concentrations was also similar in both periods (study 2). Safety and tolerability profiles were unremarkable in both studies., Conclusions: Coadministration of elagolix 150 mg once daily with oral contraceptives containing EE and norgestimate, or norethindrone, resulted in small pharmacokinetic changes in the oral contraceptive components. Similar or lower FSH, LH, and E2 exposures were observed during coadministration, with ovulatory P concentrations also comparable in both periods. The pharmacodynamic profiles of the oral contraceptives were maintained when coadministered with elagolix., (© 2021. The Author(s).)

Background: Emerging studies have proposed that cytokines secreted following macrophage polarization may contribute to skeletal muscle aging. The current study primarily aimed to determine whether these cytokines have an impact on the progression of sarcopenia in an elderly population., Methods: In total, 120 elderly adults aged 65 years and older who underwent health examinations from 2015 to 2019 were included in this retrospective study. Sarcopenia was based on the criteria proposed by the European Working Group on Sarcopenia in Older People in 2019. Macrophages and cytokines datasets were obtained from Gene Expression Omnibus (GEO) database. Comprehensive assessments were performed for muscle strength, muscle mass, gait speed, tumor growth factor-β (TGF-β), and interleukin-12 (IL-12). Thereafter, the association between sarcopenia and cytokines was analyzed using regression models., Results: Low muscle strength and low-speed gait were negatively associated with IL-12 [β: -8.96 (95% CI: -14.12, -3.79) and -7.16 (95% CI: -12.54, -1.78), respectively]. Participants with more sarcopenia components and more severe sarcopenia had lower IL-12 (P for trend < 0.001). Conversely, more amount of sarcopenia components were associated with increased TGF-β (P for trend < 0.05). A definite diagnosis of sarcopenia was associated with decreased IL-12 and increased TGF-β with β of -8.96 (95% CI: -14.12, -3.79) and 147.75 (95% CI: 36.27, 259.23). Furthermore, increased IL-12 levels were significantly associated with reduced occurrence of sarcopenia with and odd ratio (OR) of 0.36 (95% CI: 0.15-0.834)., Conclusion: Our findings on the relationship between cytokines and age-related muscle loss showed that IL-12 may be an early diagnosis indicator for sarcopenia in the elderly population., Competing Interests: The authors declared that they had no competing interests., (© 2021 Chen et al.)

Background: Xanthelasma palpebrarum (XP) is a sign of hyperlipidemia and is closely linked to atherosclerosis. Since fatty liver shares similar risk factors with atherosclerosis, we hypothesized that patients with XP are also at risk of non-alcoholic fatty liver disease (NAFLD)., Methods: In this retrospective cohort study, 37 patients with XP were compared with sex- and age-matched controls undergoing general health examination. Moreover, demographic information and lipid profiles were compared. The risk of NAFLD was evaluated using the hepatic steatosis and ZJU indices. In addition, we analyzed publicly available RNA sequencing data from the GSE48452 and GSE61260 datasets in the Gene Expression Omnibus database., Findings: Patients with XP had higher scores of hepatic steatosis index (37 ± 1.13 vs 32 ± 0.82, p=0.0006) and ZJU index (38.77 ± 1.0 vs 33.88 ± 0.74, p=0.0002). In addition, they had higher levels of lipid parameters, including total cholesterol, low-density lipoprotein (LDL), and fasting glucose. Among patients with fatty liver, individuals presenting with XP showed higher serum levels of total cholesterol (216 ± 10.4 vs 188.9 ± 7.6, p=0.04), fasting glucose (117.1 ± 6.4 vs 98.3 ± 2.4, p=0.002), and low-density lipoprotein (145.1 ± 8.7 vs 115.6 ± 6.4, p=0.009) than those without XP. In gene expression analysis, individuals presenting with non-alcoholic steatohepatitis showed higher Z scores of xanthelasma than those without non-alcoholic steatohepatitis., Conclusion: Our results suggest that individuals with XP have a higher risk of progression to NAFLD and develop a more severe dyslipidemia., Competing Interests: The authors report no conflicts of interest in this work., (© 2021 Chen et al.)

Background: Sarcopenia is a multifactorial pathophysiologic condition of skeletal muscle mass and muscle strength associated with aging. However, biomarkers for predicting the occurrence of sarcopenia are rarely discussed in recent studies. The aim of the study was to elucidate the relationship between sarcopenia and several pertinent biomarkers., Methods: Using the Gene Expression Omnibus (GEO) profiles of the National Center for Biotechnology Information, the associations between mRNA expression of biomarkers and sarcopenia were explored, including high temperature requirement serine protease A1 (HtrA1), procollagen type III N-terminal peptide (P3NP), apelin, and heat shock proteins 70 (Hsp72). We enrolled 408 community-dwelling adults aged 65 years and older with sarcopenia and nonsarcopenia based on the algorithm proposed by the Asian Working Group for Sarcopenia (AWGS). Muscle strength is identified by hand grip strength using an analogue isometric dynamometer. Muscle mass is estimated by skeletal mass index (SMI) using a bioelectrical impedance analysis. Physical performance is measured by gait speed using 6 m walking distance. The associations between these biomarkers and sarcopenia were determined using receiver operating characteristic (ROC) curve analysis and multivariate regression models., Results: From the GEO profiles, the sarcopenia gene set variation analysis score was correlated significantly with the mRNA expression of APLNR (p < 0.001) and HSPA2 (p < 0.001). In our study, apelin was significantly associated with decreased hand grip strength with β values of - 0.137 (95%CI: - 0.229, - 0.046) in men. P3NP and HtrA1 were significantly associated with increased SMI with β values of 0.081 (95%CI: 0.010, 0.153) and 0.005 (95%CI: 0.001, 0.009) in men, respectively. Apelin and HtrA1 were inversely associated with the presence of sarcopenia with an OR of 0.543 (95%CI: 0.397-0.743) and 0.003 (95%CI: 0.001-0.890) after full adjustment. The cutoff point of HtrA1 was associated with the presence of sarcopenia with an OR of 0.254 (95%CI: 0.083-0.778) in men. The cutoff point of apelin was negatively associated with the presence of sarcopenia with an OR of 0.254 (95%CI: 0.083-0.778)., Conclusion: Our study highlights that P3NP, HtrA, and apelin are useful for diagnosis of sarcopenia in the clinical setting.

Background: Vitamin D contributes to bone health and extra-skeletal effects. The mechanisms underlying vitamin D metabolism have not been extensively evaluated. The relationships between vitamin D and inflammatory cytokines are debated. Our objective was to investigate whether supplemental interferons are associated with longitudinal change of vitamin D status in humans., Methods: A total of 48 patients with 24 or 48 weeks of pegylated interferon-α plus ribavirin therapy were examined for serum 25-hydroxyvitamin D (25[OH]D) level before treatment, at the end of treatment, and 24 weeks after treatment. In addition, we analyzed publicly available RNA sequencing data from accession GSE42697 and GSE7123 in the Gene Expression Omnibus., Findings: The overall sustained virologic response (SVR) rate was 62.5%. There was no statistically significant association between baseline 25(OH)D concentrations and liver fibrosis. In patients with SVR, serum 25(OH)D increased markedly at end-of-treatment and decreased markedly by the end of the 24-week follow-up period. In the non-SVR group, this treatment-dependent change was lost. In gene expression analysis, the vitamin D biosynthesis process was activated in subjects with SVR, but not in patients without SVR. Furthermore, vitamin D receptor (VDR) signaling in peripheral blood mononuclear cells (PBMCs) was triggered in marked responders but not in poor responders., Conclusion: In the aggregate, these data suggest that interferons have a regulatory influence on vitamin D status that can contribute to VDR signaling in PBMCs., Competing Interests: The authors declare no conflict of interest., (© 2020 Chen et al.)

The integral membrane, Kunitz-type serine protease inhibitors HAI-1 and HAI-2, can suppress the proteolytic activity of the type 2 transmembrane serine protease matriptase with high specificity and potency. High levels of extracellular matriptase proteolytic activity have, however, been observed in some neoplastic B-cells with high levels of endogenous HAI-2, indicating that HAI-2 may be an ineffective matriptase inhibitor at the cellular level. The different effectiveness of the HAIs in the control of extracellular matriptase proteolytic activity is examined here. Upon inducing matriptase zymogen activation in the HAI Teton Daudi Burkitt lymphoma cells, which naturally express matriptase with very low levels of HAI-2 and no HAI-1, nascent active matriptase was rapidly inhibited or shed as an enzymatically active enzyme. With increasing HAI-1 expression, cellular matriptase-HAI-1 complex increased, and extracellular active matriptase decreased proportionally. Increasing HAI-2 expression, however, resulted in cellular matriptase-HAI-2 complex levels reaching a plateau, while extracellular active matriptase remained high. In contrast to this differential effect, both HAI-1 and HAI-2, even at very low levels, were shown to promote the expression and cell-surface translocation of endogenous matriptase. The difference in the suppression of extracellular active matriptase by the two closely related serine protease inhibitors could result from the primarily cell surface expression of HAI-1 compared to the mainly intracellular localization of HAI-2. The HAIs, therefore, resemble one another with respect to promoting matriptase expression and surface translocation but differ in their effectiveness in the control of extracellular matriptase enzymatic activity., (© 2020 Chongqing Medical University. Production and hosting by Elsevier B.V.)

COVID-19, caused by SARS-CoV-2, has rapidly spread to more than 160 countries worldwide since 2020. Despite tremendous efforts and resources spent worldwide trying to explore antiviral drugs, there is still no effective clinical treatment for COVID-19 to date. Approximately 15% of COVID-19 cases progress to pneumonia, and patients with severe pneumonia may die from acute respiratory distress syndrome (ARDS). It is believed that pulmonary fibrosis from SARS-CoV-2 infection further leads to ARDS, often resulting in irreversible impairment of lung function. If the mechanisms by which SARS-CoV-2 infection primarily causes an immune response or immune cell infiltration can be identified, it may be possible to mitigate excessive immune responses by modulating the infiltration and activation of specific targets, thereby reducing or preventing severe lung damage. However, the extent to which immune cell subsets are significantly altered in the lung tissues of COVID-19 patients remains to be elucidated. This study applied the CIBERSORT-X method to comprehensively evaluate the transcriptional estimated immune infiltration landscape in the lung tissues of COVID-19 patients and further compare it with the lung tissues of patients with idiopathic pulmonary fibrosis (IPF). We found a variety of immune cell subtypes in the COVID-19 group, especially naïve B cells were highly infiltrated. Comparison of functional transcriptomic analyses revealed that non-differentiated naïve B cells may be the main cause of the over-active humoral immune response. Using several publicly available single-cell RNA sequencing data to validate the genetic differences in B-cell populations, it was found that the B-cells collected from COVID-19 patients were inclined towards naïve B-cells, whereas those collected from IPF patients were inclined towards memory B-cells. Further differentiation of B cells between COVID-19 mild and severe patients showed that B cells from severe patients tended to be antibody-secreting cells, and gene expression showed that B cells from severe patients were similar to DN2 B cells that trigger extrafollicular response. Moreover, a higher percentage of B-cell infiltration seems associated with poorer clinical outcome. Finally, a comparison of several specific COVID-19 cases treated with targeted B-cell therapy suggests that appropriate suppression of naïve B cells might potentially be a novel strategy to alleviate the severe symptoms of COVID-19., Competing Interests: The authors have declared that no competing interests exist.

The pathophysiological functions of matriptase, a type 2 transmembrane serine protease, rely primarily on its enzymatic activity, which is under tight control through multiple mechanisms. Among those regulatory mechanisms, the control of zymogen activation is arguably the most important. Matriptase zymogen activation not only generates the mature active enzyme but also initiates suppressive mechanisms, such as rapid inhibition by HAI-1, and matriptase shedding. These tightly coupled events allow the potent matriptase tryptic activity to fulfill its biological functions at the same time as limiting undesired hazards. Matriptase is converted to the active enzyme via a process of autoactivation, in which the activational cleavage is thought to rely on the interactions of matriptase zymogen molecules and other as yet identified proteins. Matriptase autoactivation can occur spontaneously and is rapidly followed by the formation and then shedding of matriptase-HAI-1 complexes, resulting in the presence of relatively low levels of the complex on cells. Activation can also be induced by several non-protease factors, such as the exposure of cells to a mildly acidic buffer, which rapidly causes high-level matriptase zymogen activation in almost all cell lines tested. In the current study, the structural requirements for this acid-induced zymogen activation are compared with those required for spontaneous activation through a systematic analysis of the impact of 18 different mutations in various structural domains and motifs on matriptase zymogen activation. Our study reveals that both acid-induced matriptase activation and spontaneous activation depend on the maintenance of the structural integrity of the serine protease domain, non-catalytic domains, and posttranslational modifications. The common requirements of both modes of activation suggest that acid-induced matriptase activation may function as a physiological mechanism to induce pericellular proteolysis by accelerating matriptase autoactivation.

The zinc-finger protein which regulates apoptosis and cell cycle arrest 1 (Zac1), encoded by Plagl1 gene, is a seven-zinc-finger containing transcription factor belonging to the imprinted genome and is expressed in diverse types of embryonic and adult human tissues. Zac1 is postulated to be a tumor suppressor by inducing cell cycle arrest and apoptosis through interacting and modulating transcriptional activity of p53 as it was named. Correspondingly, the reduction or loss of Zac1 expression is associated with the incidence and progression of several human tumors, including cervical cancer, breast cancer, ovarian cancer, pituitary tumors, and basal cell carcinoma, implying the rationality of utilizing Zac1 expression as novel a biomarker for the evaluation of cervical cancer prognosis. However, to date, it has not been elucidated whether Zac1 expression is related to the prognosis of patients in clinical cervical cancer tumor samples. To address the questions outlined above, we report here a comprehensive investigation of Zac1 expression in biopsies of clinical cervical carcinoma. By analyzing Zac1 expression in various gene expression profiling of cervical cancer databases, we show the association between high Zac1 expression and poor prognosis of cervical cancer. Functional enrichment analysis showed that high Zac1 expression was associated with epithelial-mesenchymal transition (EMT), which was further observed in clinical characteristics and metastatic carcinoma samples using immunohistochemical staining. Correspondingly, hypomethylation of CpG island on Zac1 promoter was observed in samples with high Zac1 expression in cervical carcinoma. Finally, overexpression of Zac1 in a variety of cervical cancer cell lines increase their mesenchymal biomarker expression and migration, strengthening the correlation between cervical cancers with high Zac1 expression and metastasis in clinical. In summary, this research firstly revealed that identifying Zac1 expression or the methylation status of CpG site on Zac1 promoter may provide us with novel indicators for the evaluation of cervical cancer metastasis.

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is an enzyme that detoxifies aldehydes by converting them to carboxylic acids. ALDH2 deficiency is known to increase oxidative stress. Increased oxidative stress plays a pivotal role in abdominal aortic aneurysm (AAA) pathogenesis. Reactive oxygen species (ROS) promote degradation of the extracellular matrix (ECM) and vascular smooth muscle cell (VSMC) apoptosis. Reducing oxidative stress by an ALDH2 activator could have therapeutic potential for limiting AAA development. We hypothesized that ALDH2 deficiency could increase the risk for AAA by decreasing ROS elimination and that an ALDH2 activator could provide an alternative option for AAA treatment. The National Center for Biotechnology (NCBI) Gene Expression Omnibus (GEO) database was used. Human aortic smooth muscle cells (HASMCs) were used for the in vitro experiments. Gene-targeted ALDH2*2 KI knock-in mice on a C57BL/6J background and apolipoprotein E knockout (ApoE KO) mice were obtained. An animal model of AAA was constructed using osmotic minipumps to deliver 1000 ng/kg/min angiotensin II (AngII) for 28 days. Patients with AAA had significantly lower ALDH2 expression levels than normal subjects. ALDH2*2 KI mice were susceptible to AngII administration, exhibiting significantly increased AAA incidence rates and increased aortic diameters. Alda-1, an ALDH2 activator, reduced AngII-induced ROS production, NF-kB activation, and apoptosis in HASMCs. Alda-1 attenuated AngII-induced aneurysm formation and decreased aortic expansion in ApoE KO mice. We concluded that ALDH2 deficiency is associated with the development of AAAs in humans and a murine disease model. ALDH2 deficiency increases susceptibility to AngII-induced AAA formation by attenuating anti-ROS effects and increasing VSMC apoptosis and vascular inflammation. Alda-1 was shown to attenuate the progression of experimental AAA in a murine model., (© 2020 Federation of American Societies for Experimental Biology.)

Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne flaviviruses that cause severe illness after infection. Currently, there are no specific or effective treatments against DENV and ZIKV. Previous studies have shown that tyrosine kinase activities and signal transduction are involved in flavivirus replication, suggesting a potential therapeutic strategy for DENV and ZIKV. In this study, we found that compound L3 can significantly reduce viral protein expression and viral titers in HEK-293, MCF-7, HepG2, and Huh-7 cells and exhibits superior therapeutic efficacy against flaviviral infection compared to other tyrosine kinase inhibitors. In addition, compound L3 can decrease endogenous HER2 activation and inhibit the phosphorylation of the HER2 downstream signaling molecules Src and ERK1/2, the levels of which have been associated with viral protein expression in MCF-7 cells. Moreover, silencing HER2 diminished DENV-2 and ZIKV expression in MCF-7 cells, which suggests that HER2 activity is involved in flavivirus replication. Furthermore, in DENV-2-infected AG129 mice, treatment with compound L3 increased the survival rates and reduced the viremia levels. Overall, compound L3 demonstrates therapeutic efficacy both in vitro and in vivo and could be developed as a promising antiviral drug against emerging flaviviruses or for concurrent DENV and ZIKV outbreaks.

Context: Elagolix is an oral gonadotropin-releasing hormone (GnRH) antagonist recently approved for the treatment of endometriosis-associated pain and being developed for heavy menstrual bleeding associated with uterine fibroids., Objective: The objective was to evaluate the effects of elagolix on ovulation and ovarian sex hormones., Design and Setting: This was a randomized, open-label, multicenter study., Participants: Participants were healthy ovulatory women aged 18 to 40 years., Interventions: Elagolix was administered orally for 3 continuous 28-day dosing intervals at 100 to 200 mg once daily (QD), 100 to 300 mg twice daily (BID), and 300 mg BID plus estradiol/norethindrone acetate (E2/NETA) 1/0.5 mg QD., Main Outcome Measures: The main outcomes measures were ovulation rates measured by transvaginal ultrasound, progesterone concentrations, and hormone suppression., Results: Elagolix suppressed ovulation in a dose-dependent manner. The percentage of women who ovulated was highest at 100 mg QD (78%), intermediate at 150 and 200 mg QD and 100 mg BID (47%-57%), and lowest at 200 and 300 mg BID (32% and 27%, respectively). Addition of E2/NETA to elagolix 300 mg BID further suppressed the ovulation rate to 10%. Elagolix also suppressed luteinizing hormone and follicle stimulating hormone in a dose-dependent manner, leading to dose-dependent suppression of estradiol and progesterone. Elagolix had no effect on serum biomarker of ovarian reserve, and reduced endometrial thickness compared to the screening cycle., Conclusion: Women being treated with elagolix may ovulate and should use effective methods of contraception. The rate of ovulation was lowest with elagolix 300 mg BID plus E2/NETA 1/0.5 mg QD., (© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The effects of thrombo-prevention, such as antiplatelet and anticoagulant activity, have been reported with the usage of Ginkgo biloba extract (GbE); however, the detailed mechanism has not yet been fully investigated, especially the role of Krüppel-like factor 2 (KLF2). This study aimed to investigate whether GbE can activate KLF2 and then induce thrombomodulin (TM) and tissue-type plasminogen activator (t-PA) secretion to enhance the effects of thrombo-prevention. Different concentrations of GbE were incubated with human umbilical vein endothelial cells (HUVECs) to evaluate its effect on endothelial cells. We found that KLF2 expression is correlated to the risk of atherosclerosis and venous thromboembolism in clinical practice. In the HUVEC cell model, GbE stimulated the expression of KLF2 in a dose-dependent manner. Moreover, TM and t-PA secretion increased when the cells were cultured with GbE. Both the expressions and activities of TM and t-PA in the GbE-treated cells declined after KLF2 was blocked by shKLF2. In sum, with GbE treatment, KLF2 expression in human endothelial cells was significantly activated, which in turn induced an increase in the protein expression and activity of TM and t-PA. After shRNA inhibited the KLF2 expression, GbE stopped inducing the expression and activity of TM and t-PA. These findings suggest that GbE exerts an antithrombotic effect on endothelial cells by increasing the TM expression and t-PA secretion; further, KLF2 is a key factor in this mechanism.

Matriptase is ectopically expressed in neoplastic B-cells, in which matriptase activity is enhanced by negligible expression of its endogenous inhibitor, hepatocyte growth factor activator inhibitor (HAI)-1. HAI-1, however, is also involved in matriptase synthesis and intracellular trafficking. The lack of HAI-1 indicates that other related inhibitor, such as HAI-2, might be expressed. Here, we show that HAI-2 is commonly co-expressed in matriptase-expressing neoplastic B-cells. The level of active matriptase shed after induction of matriptase zymogen activation in 7 different neoplastic B-cells was next determined and characterised. Our data reveal that active matriptase can only be generated and shed by those cells able to activate matriptase and in a rough correlation with the levels of matriptase protein. While HAI-2 can potently inhibit matriptase, the levels of active matriptase are not proportionally suppressed in those cells with high HAI-2. Our survey suggests that matriptase proteolysis might aberrantly remain high in neoplastic B-cells regardless of the levels of HAI-2.

This work characterized the time-course, circadian rhythm, and inherent variability in key cardiovascular variables (heart rate, corrected QT interval, and systolic and diastolic blood pressure) that are routinely collected as part of safety monitoring in phase I trials. Longitudinal data from 1,035 healthy volunteers who received placebo in 65 single-dose and multiple-dose phase I trials conducted by AbbVie were compiled and analyzed using nonlinear mixed-effects modeling. An independent nonlinear mixed-effects model was developed for each variable, and combinations of cosine functions were used to capture circadian oscillations. Gender, race, age, and body weight were significant covariates for variability in baseline measures, and the contributions of these covariates were quantitatively characterized. Based on the extensive data set analyzed, the developed models represent valuable tools to help contextualize and differentiate inherent variability that can be expected in a typical phase I setting from true drug-related cardiovascular safety signals. In addition, these placebo models can be used to support exposure-response analyses that estimate treatment-related effects on the evaluated cardiovascular measures., (© 2019 AbbVie Inc. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

[This corrects the article DOI: 10.1371/journal.pone.0192632.].

The membrane-bound serine proteases prostasin and matriptase and the Kunitz-type protease inhibitors HAI-1 and HAI-2 are all expressed in human skin and may form a tightly regulated proteolysis network, contributing to skin pathophysiology. Evidence from other systems, however, suggests that the relationship between matriptase and prostasin and between the proteases and the inhibitors can be context-dependent. In this study the in vivo zymogen activation and protease inhibition status of matriptase and prostasin were investigated in the human skin. Immunohistochemistry detected high levels of activated prostasin in the granular layer, but only low levels of activated matriptase restricted to the basal layer. Immunoblot analysis of foreskin lysates confirmed this in vivo zymogen activation status and further revealed that HAI-1 but not HAI-2 is the prominent inhibitor for prostasin and matriptase in skin. The zymogen activation status and location of the proteases does not support a close functional relation between matriptase and prostasin in the human skin. The limited role for HAI-2 in the inhibition of matriptase and prostasin is the result of its primarily intracellular localization in basal and spinous layer keratinocytes, which probably prevents the Kunitz inhibitor from interacting with active prostasin or matriptase. In contrast, the cell surface expression of HAI-1 in all viable epidermal layers renders it an effective regulator for matriptase and prostasin. Collectively, our study suggests the importance of tissue distribution and subcellular localization in the functional relationship between proteases and protease inhibitors.

ABT-493 is a hepatitis C virus nonstructural protein 3/4A protease inhibitor with pangenotypic antiviral activity. This study investigated the pharmacokinetics, safety, and tolerability of single and multiple ascending doses of ABT-493 and the effect of food and ritonavir coadministration on ABT-493 pharmacokinetics in healthy adults. In the blinded, randomized, placebo-controlled phase 1 single- and multiple-dose portions of the study, ABT-493 25-800 mg were evaluated as single doses, and 200, 400, and 800 mg were evaluated as multiple doses. The effect of food and ritonavir was assessed in a crossover unblinded fashion. ABT-493 pharmacokinetic parameters were estimated using noncompartmental methods. ABT-493 25-800 mg showed a greater than dose-proportional increase in exposures. Minimal accumulation (≤15%) was observed after ABT-493 200- and 400-mg multiple dosing; higher accumulations (approximately 80%) were observed after the 800-mg dose. ABT-493 harmonic mean half-life was 6-9 hours. Food had a minimal effect on ABT-493 exposures. All adverse events were assessed by the investigator as mild to moderate in severity, no serious adverse events were reported, and no subjects discontinued from the study. No clinically significant laboratory tests, vital signs, or electrocardiogram values were reported. A maximum tolerated dose was not reached., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The membrane-associated serine proteases matriptase and prostasin are believed to function in close partnership. Their zymogen activation has been reported to be tightly coupled, either as a matriptase-initiated proteolytic cascade or through a mutually dependent mechanism involving the formation of a reciprocal zymogen activation complex. Here we show that this putative relationship may not apply in the context of human matriptase and prostasin. First, the tightly coupled proteolytic cascade between matriptase and prostasin might not occur when modest matriptase activation is induced by sphingosine 1-phospahte in human mammary epithelial cells. Second, prostasin is not required and/or involved in matriptase autoactivation because matriptase can undergo zymogen activation in cells that do not endogenously express prostasin. Third, matriptase is not required for and/or involved in prostasin activation, since activated prostasin can be detected in cells expressing no endogenous matriptase. Finally, matriptase and prostasin both undergo zymogen activation through an apparently un-coupled mechanism in cells endogenously expressing both proteases, such as in Caco-2 cells. In these human enterocytes, matriptase is detected primarily in the zymogen form and prostasin predominantly as the activated form, either in complexes with protease inhibitors or as the free active form. The negligible levels of prostasin zymogen with high levels of matriptase zymogen suggests that the reciprocal zymogen activation complex is likely not the mechanism for matriptase zymogen activation. Furthermore, high level prostasin activation still occurs in Caco-2 variants with reduced or absent matriptase expression, indicating that matriptase is not required and/or involved in prostasin zymogen activation. Collectively, these data suggest that any functional relationship between natural endogenous human matriptase and prostasin does not occur at the level of zymogen activation., Competing Interests: C.-Y.L is an inventor on US patents #6,077,938 (Title: Monoclonal antibody to an 80-kDa protease) and #6,677,377 (Title: Structure based discovery of inhibitors of matriptase for the cancer diagnosis and therapy by detection and inhibition of matriptase activity), and M.D.J and C.-Y.L are inventors on US patent #7,355,015 (Title: Matriptase, a serine protease and its applications). This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Background and Aims: The three direct-acting antiviral regimen of ombitasvir/paritaprevir/ritonavir and dasabuvir (3D regimen) is approved for treatment of hepatitis C virus (HCV) genotype 1 infection. Drug-drug interaction (DDI) studies of the 3D regimen and commonly used medications were conducted in healthy volunteers to provide information on coadministering these medications with or without dose adjustments., Methods: Three phase I studies evaluated DDIs between the 3D regimen (ombitasvir/paritaprevir/ritonavir 25/150/100 mg once daily + dasabuvir 250 mg twice daily) and hydrocodone bitartrate/acetaminophen (5/300 mg), metformin hydrochloride (500 mg), diazepam (2 mg), cyclobenzaprine hydrochloride (5 mg), carisoprodol (250 mg), or sulfamethoxazole/trimethoprim (SMZ/TMP) (800/160 mg twice daily), all administered orally. DDI magnitude was determined using geometric mean ratios and 90 % confidence intervals for the maximum plasma concentration (C max) and area under the plasma concentration-time curve (AUC)., Results: Changes in exposures (C max and AUC geometric mean ratios) of acetaminophen, metformin, sulfamethoxazole, trimethoprim, and diazepam were ≤25 % upon coadministration with the 3D regimen. The C max and AUC of nordiazepam, an active metabolite of diazepam, increased by 10 % and decreased by 44 %, respectively. Exposures of cyclobenzaprine and carisoprodol decreased by ≤40 and ≤46 %, respectively, whereas exposures of hydrocodone increased up to 90 %. Ombitasvir, paritaprevir, ritonavir, and dasabuvir exposures changed by ≤25 %, except for a 37 % decrease in paritaprevir C max with metformin and a 33 % increase in dasabuvir AUC with SMZ/TMP., Conclusions: Acetaminophen, metformin, sulfamethoxazole, and trimethoprim can be coadministered with the 3D regimen without dose adjustment. Higher doses may be needed for diazepam, cyclobenzaprine, and carisoprodol based on clinical monitoring. A 50 % lower dose and/or clinical monitoring should be considered for hydrocodone. No dose adjustment is necessary for the 3D regimen.

The two direct-acting antiviral (2D) regimen of ombitasvir and paritaprevir (administered with low-dose ritonavir) is being developed for treatment of genotype subtype 1b and genotypes 2 and 4 chronic hepatitis C virus (HCV) infection. Drug-drug interactions were evaluated in healthy volunteers to develop dosing recommendations for HCV-infected subjects. Mechanism-based interactions were evaluated for ketoconazole, pravastatin, rosuvastatin, digoxin, warfarin, and omeprazole. Interactions were also evaluated for duloxetine, escitalopram, methadone, and buprenorphine-naloxone. Ratios of geometric means with 90% confidence intervals for the maximum plasma concentration and the area under the plasma concentration-time curve were estimated to assess the magnitude of the interactions. For most medications, coadministration with the 2D regimen resulted in a <50% change in exposures. Ketoconazole, digoxin, pravastatin, and rosuvastatin exposures increased by up to 105%, 58%, 76%, and 161%, respectively, and omeprazole exposures decreased by approximately 50%. Clinically meaningful changes in ombitasvir, paritaprevir, or ritonavir exposures were not observed. In summary, all 11 medications evaluated can be coadministered with the 2D regimen, with most medications requiring no dose adjustment. Ketoconazole, digoxin, pravastatin, and rosuvastatin require lower doses, and omeprazole may require a higher dose. No dose adjustment is required for the 2D regimen., (Copyright © 2015 Badri et al.)

Background: A novel ritonavir oral powder formulation has been developed to eliminate the alcohol and propylene glycol contents in the current ritonavir oral solution for paediatric use. Two clinical studies were conducted to assess the bioequivalence of the powder formulation to the marketed oral solution and to evaluate the effect of food and vehicles on bioavailability., Methods: Study 1 was a randomized, partial-crossover, 4-period study in 48 subjects. Regimens included: oral solution under moderate-fat conditions, powder formulation in water under fasting, moderate-fat or high-fat conditions, and powder formulation in chocolate milk or pudding under moderate-fat conditions. Study 2 was a randomized, crossover, 4-period study in 24 subjects. Subjects were randomized to a sequence of the oral solution and powder formulation in water, infant formula and apple sauce, all under moderate-fat conditions. Bioavailability comparisons were assessed by the 90% CIs for the geometric least-squares mean ratios., Results: Ritonavir powder formulation in water was found to be bioequivalent to the marketed oral solution. Ritonavir powder formulation administered in chocolate milk, pudding, infant formula or apple sauce was bioequivalent to the powder formulation administered in water. Compared with fasting conditions, moderate-fat and high-fat meals were associated with approximately 25-40% and 35-50% reduction in ritonavir concentrations, respectively., Conclusions: The novel ritonavir powder formulation is bioequivalent to marketed ritonavir oral solution under moderate-fat conditions with a similar effect of meals. None of the vehicles tested negatively affected the bioavailability, which suggests the potential for use of a broad range of vehicles for dose preparation.

Purpose: A phase 1 study was conducted to evaluate the bioavailability and food effect of a new veliparib formulation in subjects with solid tumors., Methods: Subjects (planned: Stage I, N = 20; Stage II, N = 16) received four regimens of a single oral dose of veliparib utilizing a group-sequential design. Subjects were administered single doses of 40 mg veliparib supplied as four 10 mg current formulation, four 10 mg new formulation and one 40 mg new formulation under fasting conditions and under non-fasting conditions. Serial blood samples were collected for the determination of veliparib pharmacokinetics. At the end of Stage I, the relative bioavailability between each pair of regimens was assessed by a two one-sided tests procedure from the analyses of the natural logarithms of C(max) and AUC. A 92.7 % confidence interval within the 0.80-1.25 range between each regimen pair determined bioequivalence., Results: Four 10 mg current formulation capsules, four 10 mg new formulation and one 40 mg new formulation were bioequivalent with respect to C(max) and AUC under fasting conditions. The administration of a high-fat meal did not have a significant effect on AUC and only caused a slight decrease in veliparib C(max) (17 %) and a delay of approximately 1 h in T(max)., Conclusions: The 40 mg new capsule was bioequivalent to currently used formulation. Food had no effect on the extent of veliparib absorption and only a small (17 %) decrease in peak exposure of veliparib.

Background and Objective: A pharmacokinetic substudy was conducted within a phase 3 clinical trial that evaluated the efficacy and safety of two leuprolide acetate 3-month depot formulations in children with central precocious puberty (CPP), where the pharmacokinetics of leuprolide and the exposure-response relationship between leuprolide concentration and the probability of luteinizing hormone (LH) suppression were assessed., Methods: Children diagnosed with CPP (N = 42 in each dosing cohort), who were treatment naïve or previously treated, received a total of two intramuscular injections of either leuprolide acetate depot 11.25 or 30 mg formulations administered 3 months apart. Serial blood samples were collected for leuprolide concentration determination in a subset of subjects (N = 24 in each cohort). One-way analysis of covariance was used to assess dose proportionality. The probability of LH suppression (peak-stimulated LH concentrations <4 mIU/mL) exposure-response relationship was modelled using repeated measures logistic regression. The predicted probability of LH suppression and the corresponding 95 % confidence interval at the mean leuprolide concentration of each dose group and at each time of measurement were computed., Results: Mean leuprolide concentrations between weeks 4 and 12 for 11.25 and 30 mg doses were relatively constant and dose proportional, with no accumulation of leuprolide upon repeated administration. Body weight and age were not found to be significant covariates on leuprolide pharmacokinetics. Higher leuprolide concentrations were associated with higher probability of LH suppression and both doses provided LH suppression levels <4 mIU/mL., Conclusion: Leuprolide pharmacokinetics were characterized for 11.25 and 30 mg 3-month depot injections. An exposure-response model was developed to link leuprolide concentrations and probability of peak-stimulated LH suppression.

Purpose: The primary objectives of this study were to evaluate the effect of food on the oral bioavailability and to evaluate the effect of diurnal variation on the pharmacokinetics of linifanib, a novel tyrosine kinase (TK) inhibitor selective for vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptors, in patients with advanced solid tumors. Adverse events were monitored., Methods: This was a phase 1, open-label, randomized, crossover study. Thirty-four patients received dosing regimens to evaluate linifanib pharmacokinetic parameters under fasting and non-fasting conditions and with morning or evening dosing. Adverse events (AEs) were assessed according to National Cancer Institute Common Terminology Criteria for AEs (Version 3.0)., Results: The administration with food had a negligible effect on the AUC∞ of linifanib, but the Cmax of linifanib was decreased by 40 % compared to the fasting condition. Evening dosing after a 2-h fast had a negligible effect on AUC₂₄; however, the dose-normalized Cmax of linifanib after evening dosing was 64 % of that after morning dosing following a 10-h fast. Common Grade 3/4 AEs were fatigue (24 %), hypertension (21 %), and palmar-plantar erythrodysaesthesia syndrome (15 %)., Conclusions: Dosing with food or in the evening has a significant effect on the oral bioavailability of linifanib that should be taken into consideration when designing future clinical studies. The pattern of adverse advents reported in this study is similar to that seen in other studies of linifanib and other agents in the VEGF/PDGF TK receptor inhibitor class.

Purpose: Linifanib is a selective inhibitor of the vascular endothelial growth factor and platelet-derived growth factor family of tyrosine kinase inhibitors. The purpose of this high-precision QT study was to evaluate the effects of linifanib on cardiac repolarization in patients with advanced metastatic tumors., Methods: Enrolled patients (n = 24) had measurable disease refractory to standard therapies, ECOG performance status of 0-1, and adequate organ function. Patients were randomized in a 2-sequence, 2-period crossover design. Serial ECG measurements and pharmacokinetic samples were collected for each crossover period. An intersection-union test was performed for time-matched baseline-adjusted QTcF intervals. An exposure-response analysis was explored to correlate the plasma concentration and QTcF., Results: The maximum 95 % upper confidence bound for the baseline-adjusted QTcF was 4.3 ms at hour 3 at the maximum tolerated linifanib dose of 0.25 mg/kg. Linifanib did not meet the regulatory threshold (10 ms) for QT prolongation. Exposure-response modeling showed that the QTcF change was not significant at the maximum plasma concentration., Conclusions: Linifanib does not significantly affect cardiac repolarization in patients with advanced solid tumors.