Pulmonary Effects of Traumatic Brain Injury in Mice: A Gene Set Enrichment Analysis.

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.

Glutamine antagonist DRP-104 suppresses tumor growth and enhances response to checkpoint blockade in KEAP1 mutant lung cancer.

Loss-of-function mutations in KEAP1 frequently occur in lung cancer and are associated with poor prognosis and resistance to standard of care treatment, highlighting the need for the development of targeted therapies. We previously showed that KEAP1 mutant tumors consume glutamine to support the metabolic rewiring associated with NRF2-dependent antioxidant production. Here, using preclinical patient-derived xenograft models and antigenic orthotopic lung cancer models, we show that the glutamine antagonist prodrug DRP-104 impairs the growth of KEAP1 mutant tumors. We find that DRP-104 suppresses KEAP1 mutant tumors by inhibiting glutamine-dependent nucleotide synthesis and promoting antitumor T cell responses. Using multimodal single-cell sequencing and ex vivo functional assays, we demonstrate that DRP-104 reverses T cell exhaustion, decreases Tregs, and enhances the function of CD4 and CD8 T cells, culminating in an improved response to anti-PD1 therapy. Our preclinical findings provide compelling evidence that DRP-104, currently in clinical trials, offers a promising therapeutic approach for treating patients with KEAP1 mutant lung cancer.

Anatomical Anal Stenosis after PPH: Insights from a Retrospective Study and Rat Model.

High-grade hemorrhoids are usually recommended to receive operational treatments. However, these traditional surgeries are associated with severe postoperative pain. A procedure for prolapse and hemorrhoids (PPH), a circular staple device, has been developed to improve short-term outcomes, including reducing the severity of postoperative pain. PPH, compared to conventional surgery, has been associated with the incidence of anatomical anal stenosis. The causes of stenosis after PPH are not yet clear. We first analyzed the complications of our patients with PPH, and then developed a rat model to verify the tension force of PPH using Hematoxylin-eosin, Masson's trichrome, immunohistochemistry, and immunofluorescence staining. Our clinical data showed that PPH significantly improved postoperative pain, but that it resulted in higher incidences of complications, including anal stenosis, than hemorrhoidectomy. We simulated the status of PPH and developed a rat model to verify PPH's tension force, including the scarring area and the deposition of proinflammatory factors, angiogenic factors, and fibrotic factors. The tension wound histological data showed more extensive granulation tissue and inflammatory cell infiltration and a thicker epidermis than the control group on day 12 post-operation and tension treatment. In addition to IL-1ß and IL-10 cytokines on day 3 and IL-1ß, IL-6, and IL-10 cytokines on day 12 post-operation in the tension group, two angiogenic factors, CD31 and VEGF-A, were found to have a more significant expression on day 7 post-operation in the tension group. The mean scar area was larger and the distribution of fibrotic proteins (collagen 1, α-SMA, CTGF, and MMP2) in the tension group was significantly broader than in the control on day 12 post-operation and tension treatment. Based on the findings of our animal model, the development of a lesser tensile force for PPH to decrease the deposition of proinflammatory factors, angiogenic factors, and fibrotic factors is urgently required.

Identification and characterization of a novel CASR mutation causing familial hypocalciuric hypercalcemia.

Context: Although a monoallelic mutation in the calcium-sensing receptor (CASR) gene causes familial hypocalciuric hypercalcemia (FHH), the functional characterization of the identified CASR mutation linked to the clinical response to calcimimetics therapy is still limited. Objective: A 45-year-old male presenting with moderate hypercalcemia, hypocalciuria, and inappropriately high parathyroid hormone (PTH) had a good response to cinacalcet (total serum calcium (Ca2+) from 12.5 to 10.1 mg/dl). We identified the genetic mutation and characterized the functional and pathophysiological mechanisms, and then linked the mutation to calcimimetics treatment in vitro. Design: Sanger sequencing of the CASR, GNA11, and AP2S1 genes was performed in his family. The simulation model was used to predict the function of the identified mutant. In vitro studies, including immunoblotting, immunofluorescence, a cycloheximide chase study, Calbryte™ 520 Ca2+ detection, and half-maximal effective concentration (EC50), were examined. Results: This proband was found to carry a de novo heterozygous missense I554N in the cysteine-rich domain of CASR, which was pathogenic based on the different software prediction models and ACGME criteria. The simulation model showed that CASR I554N mutation decreased its binding energy with Ca2+. Human CASR I554N mutation attenuated the stability of CASR protein, reduced the expression of p-ERK 1/2, and blunted the intracellular Ca2+ response to gradient extracellular Ca2+ (eCa2+) concentration. The EC50 study also demonstrated the correctable effect of calcimimetics on the function of the CASR I554N mutation. Conclusion: This novel CASR I554N mutation causing FHH attenuates CASR stability, its binding affinity with Ca2+, and the response to eCa2+ corrected by therapeutic calcimimetics.

Imbalance of synaptic and extrasynaptic NMDA receptors induced by the deletion of CRMP1 accelerates age-related cognitive decline in mice.

Collapsin response mediator protein 1 (CRMP1) is involved in semaphorin 3A signaling pathway, promoting neurite extension and growth cone collapse. It is highly expressed in the nervous system, especially the hippocampus. The crmp1 knockout (KO) mice display impaired spatial learning and memory, and this phenomenon seemingly tends to deteriorate with age. Here we investigated whether CRMP1 is involved in age-related cognitive decline in WT and crmp1 KO mice at adult, middle-aged and older stages. The results revealed that cognitive dysfunction in the Morris water maze task became more severe and decreased glutamate and glutamine level in middle-aged crmp1 KO mice. Additionally, increasing levels of extrasynaptic NMDA receptors and phosphorylation of Tau were observed in middle-aged crmp1 KO mice, leading to synaptic and neuronal loss in the CA3 regions of hippocampus. These findings suggest that deletion of CRMP1 accelerates age-related cognitive decline by disrupting the balance between synaptic and extrasynaptic NMDA receptors, resulting in the loss of synapses and neurons.

Why does diseased parathyroid appear weak or heterogenous intensity during intraoperative near-infrared autofluorescence?

Background: During intraoperative autofluorescence, the imaging intensity of diseased parathyroid glands is often lower than that of normal parathyroid glands, and some diseased glands especially those in secondary hyperparathyroidism (HPT) show heterogeneous intensities. This study aimed to investigate the reasons for these findings. Methods: After formalin and paraffin fixation and bivalve cutting, 18 diseased glands from patients with primary HPT, 35 diseased parathyroid glands from patients with uremic HPT, and the surrounding thyroid and thymus tissues were measured using near-infrared autofluorescence with a Fluorobeam imaging system (Fluoptics, France). None of the tissues were stained with indocyanine green. Hematoxylin and eosin staining matched the intensity of the autofluorescence. Results: Using the bright white intensity of the adult normal parathyroid gland as a reference (index score of 2), the chief cells and oxyphilic cell tissues of the diseased parathyroid had the same intensity score of 2 as that of the normal parathyroid gland, and the clear water parathyroid cell had a weaker intensity score (1-1.5). Their glandular architecture, including the trabecular, follicular, or solid arrangements, did not affect the level of intensity. The thymus, thyroid, fat, fibrosis, and necrosis had very low intensities (scores of 0). The red blood cell-hemorrhage appeared dark black (intensity score -1). The thickness of the fibrotic capsule varied in the diseased parathyroid glands; however, only a very thin capsule was observed in the normal parathyroid glands. Conclusions: Various degrees of fibrotic capsules in the diseased parathyroid gland may be the main factor contributing to the lower intensity during autofluorescence, and different cell types, necrosis, fibrosis, and hemorrhage may explain the appearance of heterogeneous intensity in the diseased parathyroid glands.

Decoding the prognostic significance of integrator complex subunit 9 (INTS9) in glioma: links to TP53 mutations, E2F signaling, and inflammatory microenvironments.

INTRODUCTION: Gliomas, a type of brain neoplasm, are prevalent and often fatal. Molecular diagnostics have improved understanding, but treatment options are limited. This study investigates the role of INTS9 in processing small nuclear RNA (snRNA), which is crucial to generating mature messenger RNA (mRNA). We aim to employ advanced bioinformatics analyses with large-scale databases and conduct functional experiments to elucidate its potential role in glioma therapeutics. MATERIALS AND METHODS: We collected genomic, proteomic, and Whole-Exon-Sequencing data from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) for bioinformatic analyses. Then, we validated INTS9 protein expression through immunohistochemistry and assessed its correlation with P53 and KI67 protein expression. Gene Set Enrichment Analysis (GSEA) was performed to identify altered signaling pathways, and functional experiments were conducted on three cell lines treated with siINTS9. Then, we also investigate the impacts of tumor heterogeneity on INTS9 expression by integrating single-cell sequencing, 12-cell state prediction, and CIBERSORT analyses. Finally, we also observed longitudinal changes in INTS9 using the Glioma Longitudinal Analysis (GLASS) dataset. RESULTS: Our findings showed increased INTS9 levels in tumor tissue compared to non-neoplastic components, correlating with high tumor grading and proliferation index. TP53 mutation was the most notable factor associated with upregulated INTS9, along with other potential contributors, such as combined chromosome 7 gain/10 loss, TERT promoter mutation, and increased Tumor Mutational Burden (TMB). In GSEA analyses, we also linked INTS9 with enhanced cell proliferation and inflammation signaling. Downregulating INTS9 impacted cellular proliferation and cell cycle regulation during the function validation. In the context of the 12 cell states, INTS9 correlated with tumor-stem and tumor-proliferative-stem cells. CIBERSORT analyses revealed increased INTS9 associated with increased macrophage M0 and M2 but depletion of monocytes. Longitudinally, we also noticed that the INTS9 expression declined during recurrence in IDH wildtype. CONCLUSION: This study assessed the role of INTS9 protein in glioma development and its potential as a therapeutic target. Results indicated elevated INTS9 levels were linked to increased proliferation capacity, higher tumor grading, and poorer prognosis, potentially resulting from TP53 mutations. This research highlights the potential of INTS9 as a promising target for glioma treatment.

Glutamine antagonist DRP-104 suppresses tumor growth and enhances response to checkpoint blockade in KEAP1 mutant lung cancer.

Loss-of-function mutations in KEAP1 frequently occur in lung cancer and are associated with resistance to standard of care treatment, highlighting the need for the development of targeted therapies. We have previously shown that KEAP1 mutant tumors have increased glutamine consumption to support the metabolic rewiring associated with NRF2 activation. Here, using patient-derived xenograft models and antigenic orthotopic lung cancer models, we show that the novel glutamine antagonist DRP-104 impairs the growth of KEAP1 mutant tumors. We find that DRP-104 suppresses KEAP1 mutant tumor growth by inhibiting glutamine-dependent nucleotide synthesis and promoting anti-tumor CD4 and CD8 T cell responses. Using multimodal single-cell sequencing and ex vivo functional assays, we discover that DRP-104 reverses T cell exhaustion and enhances the function of CD4 and CD8 T cells culminating in an improved response to anti-PD1 therapy. Our pre-clinical findings provide compelling evidence that DRP-104, currently in phase 1 clinical trials, offers a promising therapeutic approach for treating patients with KEAP1 mutant lung cancer. Furthermore, we demonstrate that by combining DRP-104 with checkpoint inhibition, we can achieve suppression of tumor intrinsic metabolism and augmentation of anti-tumor T cell responses.

Tumor-intrinsic LKB1-LIF signaling axis establishes a myeloid niche to promote immune evasion and tumor growth.

Tumor mutations can influence the surrounding microenvironment leading to suppression of anti-tumor immune responses and thereby contributing to tumor progression and failure of cancer therapies. Here we use genetically engineered lung cancer mouse models and patient samples to dissect how LKB1 mutations accelerate tumor growth by reshaping the immune microenvironment. Comprehensive immune profiling of LKB1 -mutant vs wildtype tumors revealed dramatic changes in myeloid cells, specifically enrichment of Arg1 + interstitial macrophages and SiglecF Hi neutrophils. We discovered a novel mechanism whereby autocrine LIF signaling in Lkb1 -mutant tumors drives tumorigenesis by reprogramming myeloid cells in the immune microenvironment. Inhibiting LIF signaling in Lkb1 -mutant tumors, via gene targeting or with a neutralizing antibody, resulted in a striking reduction in Arg1 + interstitial macrophages and SiglecF Hi neutrophils, expansion of antigen specific T cells, and inhibition of tumor progression. Thus, targeting LIF signaling provides a new therapeutic approach to reverse the immunosuppressive microenvironment of LKB1 -mutant tumors.

Exploring the Functional Roles of Telomere Maintenance 2 in the Tumorigenesis of Glioblastoma Multiforme and Drug Responsiveness to Temozolomide.

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.

Ancient ubiquitous protein 1 (AUP1) is a prognostic biomarker connected with TP53 mutation and the inflamed microenvironments in glioma.

INTRODUCTION: Glioblastoma (GBM) is the most common and lethal brain tumor. The current treatment is surgical removal combined with radiotherapy and chemotherapy, Temozolomide (TMZ). However, tumors tend to develop TMZ resistance which leads to therapeutic failure. Ancient ubiquitous protein 1 (AUP1) is a protein associated with lipid metabolism, which is widely expressed on the surface of ER and Lipid droplets, involved in the degradation of misfolded proteins through autophagy. It has recently been described as a prognostic marker in renal tumors. Here, we aim to use sophisticated bioinformatics and experimental validation to characterize the AUP1's role in glioma. MATERIAL AND METHODS: We collected the mRNA, proteomics, and Whole-Exon-Sequencing from The Cancer Genome Atlas (TCGA) for bioinformatics analyses. The analyses included the expression difference, Kaplan-Meier-survival, COX-survival, and correlation to the clinical factors (tumor mutation burden, microsatellite instability, and driven mutant genes). Next, we validated the AUP1 protein expression using immunohistochemical staining on the 78 clinical cases and correlated them with P53 and KI67. Then, we applied GSEA analyses to identify the altered signalings and set functional experiments (including Western Blot, qPCR, BrdU, migration, cell-cycle, and RNAseq) on cell lines when supplemented with small interfering RNA targeting the AUP1 gene (siAUP1) for further validation. We integrated the single-cell sequencing and CIBERSORT analyses at the Chinese Glioma Genome Atlas (CGGA) and Glioma Longitudinal AnalySiS (GLASS) dataset to rationale the role of AUP1 in glioma. RESULTS: Firstly, the AUP1 is a prognostic marker, increased in the tumor component, and correlated with tumor grade in both transcriptomes and protein levels. Secondly, we found higher AUP1 associated with TP53 status, Tumor mutation burden, and increased proliferation. In the function validation, downregulated AUP1 expression merely impacted the U87MG cells' proliferation instead of altering the lipophagy activity. From the single-cell sequencing and CIBERSORT analyses at CGGA and GLASS data, we understood the AUP1 expression was affected by the tumor proliferation, stromal, and inflammation compositions, particularly the myeloid and T cells. In the longitudinal data, the AUP1 significantly dropped in the recurrent IDH wildtype astrocytoma, which might result from increased AUP1-cold components, including oligodendrocytes, endothelial cells, and pericytes. CONCLUSION: According to the literature, AUP1 regulates lipophagy by stabilizing the ubiquitination of lipid droplets. However, we found no direct link between AUP1 suppression and altered autophagy activity in the functional validation. Instead, we noticed AUP1 expression associated with tumor proliferation and inflammatory status, contributed by myeloid cells and T cells. In addition, the TP53 mutations seem to play an important role here and initiate inflamed microenvironments. At the same time, EGFR amplification and Chromosome 7 gain combined 10 loss are associated with increased tumor growth related to AUP1 levels. This study taught us that AUP1 is a poorer predictive biomarker associated with tumor proliferation and could report inflamed status, potentially impacting the clinical application.

Oxidative Status Determines the Cytotoxicity of Ascorbic Acid in Human Oral Normal and Cancer Cells.

Oral squamous cell carcinoma (OSCC) can arise anywhere in the oral cavity. OSCC's molecular pathogenesis is complex, resulting from a wide range of events that involve the interplay between genetic mutations and altered levels of transcripts, proteins, and metabolites. Platinum-based drugs are the first-line treatment for OSCC; however, severe side-effects and resistance are challenging issues. Thus, there is an urgent clinical need to develop novel and/or combinatory therapeutics. In this study, we investigated the cytotoxic effects of pharmacological concentrations of ascorbate on two human oral cell lines, the oral epidermoid carcinoma meng-1 (OECM-1) cell and the Smulow-Glickman (SG) human normal gingival epithelial cell. Our study examined the potential functional impact of pharmacological concentrations of ascorbates on the cell-cycle profiles, mitochondrial-membrane potential, oxidative response, the synergistic effect of cisplatin, and the differential responsiveness between OECM-1 and SG cells. Two forms of ascorbate, free and sodium forms, were applied to examine the cytotoxic effect and it was found that both forms had a similar higher sensitivity to OECM-1 cells than to SG cells. In addition, our study data suggest that the determinant factor of cell density is important for ascorbate-induced cytotoxicity in OECM-1 and SG cells. Our findings further revealed that the cytotoxic effect might be mediated through the induction of mitochondrial reactive oxygen species (ROS) generation and the reduction in cytosolic ROS generation. The combination index supported the agonistic effect between sodium ascorbate and cisplatin in OECM-1 cells, but not in SG cells. In summary, our current findings provide supporting evidence for ascorbate to serve as a sensitizer for platinum-based treatment of OSCC. Hence, our work provides not only repurposing of the drug, ascorbate, but also an opportunity to decrease the side-effects of, and risk of resistance to, platinum-based treatment for OSCC.

Sedoanalgesia with intravenous midazolam and fentanyl for angioplasty of dysfunctional arteriovenous access: A retrospective single-center analysis.

BACKGROUND: Endovascular therapy, such as percutaneous transluminal angioplasty (PTA), stenting, or embolization, is a well-established form of treatment to prolong the lifespan of arteriovenous access. These procedures, especially PTA, can be accompanied with severe pain. We reviewed and analyzed the efficacy, safety, and outcome of sedoanalgesia using intravenous midazolam and fentanyl, for pain relief during these procedures. METHODS: Two hundred and two consecutive patients with dysfunctional dialysis access that had undergone endovascular procedure in our institute between July and November 2017 were included in this study. The dialysis access profile, procedure complication, and 10-point Visual Analog Scale (VAS) were collected. One-year clinical follow-up record was also collected to evaluate arteriovenous access patency and long-term complications. RESULTS: Among the 202 patients, the mean age was (mean ± SD) 67.0 ± 12.08 years. Dialysis access profile of these patients were 119 (58.9%) native arteriovenous fistula and 162 (74.2%) forearm access. The number of lesions treated were 1.63 ± 0.802.Immediate complications included 11 (5.44%) nausea/vomiting, 24 (11.88%) desaturation (SpO2 < 90%, resolved after pillow removal or jaw trust), 16 (7.9%) hypotension (SBP < 90 mmHg, resolved without inotropic agents). There was a low average pain score (1.16 ± 1.594) during the procedure with 136 (67.3%) no pain (VAS 0-1) and 56 (27.7%) mild pain (VAS 2-4). Higher VAS score correlated with overweight patients, longer PTA time and pain after procedure. Six-month primary patency rate was 49.17% and primary assisted patency rate was 93.04%. CONCLUSIONS: Sedoanalgesia with intravenous midazolam and fentanyl is an easy, safe, and effective method for surgeons.

Apoptosis, Proliferation, and Autophagy Are Involved in Local Anesthetic-Induced Cytotoxicity of Human Breast Cancer Cells.

Breast cancer accounts for almost one quarter of all female cancers worldwide, and more than 90% of those who are diagnosed with breast cancer undergo mastectomy or breast conservation surgery. Local anesthetics effectively inhibit the invasion of cancer cells at concentrations that are used in surgical procedures. The limited treatment options for triple-negative breast cancer (TNBC) demonstrate unmet clinical needs. In this study, four local anesthetics, lidocaine, levobupivacaine, bupivacaine, and ropivacaine, were applied to two breast tumor cell types, TNBC MDA-MB-231 cells and triple-positive breast cancer BT-474 cells. In addition to the induction of apoptosis and the suppression of the cellular proliferation rate, the four local anesthetics decreased the levels of reactive oxygen species and increased the autophagy elongation indicator in both cell types. Our combination index analysis with doxorubicin showed that ropivacaine had a synergistic effect on the two cell types, and lidocaine had a synergistic effect only in MDA-MB-231 cells; the others had no synergistic effects on doxorubicin. Lidocaine contributed significantly to the formation of autophagolysosomes in a dose-dependent manner in MDA-MB-231 cells but not in BT-474 cells. Our study demonstrated that the four local anesthetics can reduce tumor growth and proliferation and promote apoptosis and autophagy.

Identification of Agents That Ameliorate Hyperphosphatemia-Suppressed Myogenin Expression Involved in the Nrf2/p62 Pathway in C2C12 Skeletal Muscle Cells.

Hyperphosphatemia can occur as a result of reduced phosphate (Pi) excretion in cases of kidney dysfunction, which can induce muscle wasting and suppress myogenic differentiation. Higher Pi suppresses myogenic differentiation and promotes muscle atrophy through canonical (oxidative stress-mediated) and noncanonical (p62-mediated) activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. However, the crosstalk between myogenin and Nrf2/p62 and potential drug(s) for the regulation of myogenin expression needed to be addressed. In this study, we further identified that myogenin may negatively regulate Nrf2 and p62 protein levels in the mouse C2C12 muscle cell line. In the drug screening analysis, we identified N-acetylcysteine, metformin, phenformin, berberine, 4-chloro-3-ethylphenol, cilostazol, and cilomilast as ameliorating the induction of Nrf2 and p62 expression and reduction in myogenin expression that occur due to high Pi. We further elucidated that doxorubicin and hydrogen peroxide reduced the amount of myogenin protein mediated through the Kelch-like ECH-associated protein 1/Nrf2 pathway, differently from the mechanism of high Pi. The dual functional roles of L-ascorbic acid (L-AA) were found to be dependent on the working concentration, where concentrations below 1 mM L-AA reversed the effect of high Pi on myogenin and those above 1 mM L-AA had a similar effect of high Pi on myogenin when used alone. L-AA exacerbated the effect of hydrogen peroxide on myogenin protein and had no further effect of doxorubicin on myogenin protein. In summary, our results further our understanding of the crosstalk between myogenin and Nrf2, with the identification and verification of several potential drugs that can be applied in rescuing the decline of myogenin due to high Pi in muscle cells.

Adjuvant Chemoradiotherapy Associated with Improved Overall Survival in Resected Esophageal Squamous Cell Carcinoma after Neoadjuvant Chemoradiotherapy in Intensity-Modulated Radiotherapy Era.

BACKGROUND: The prognosis of patients with resected esophageal squamous cell carcinoma after neoadjuvant chemoradiotherapy is particularly poor in those who were staged as ypT3/T4 and/or ypN+. This study investigated whether adjuvant chemoradiotherapy was associated with improved clinical outcomes in these patients. METHODS: we identified patients with esophageal squamous cell carcinoma who were staged as ypT3/T4 and/or ypN+ after being treated with neoadjuvant chemoradiotherapy followed by esophagectomy between the years 2013 and 2019. Patients were divided into two groups based on whether they received adjuvant chemoradiotherapy. The Kaplan-Meier method and Cox regression modeling were performed for survival analyses and multivariable analysis, respectively. RESULTS: 76 eligible patients were included in the analyses. The median follow-up for the study cohort was 43.4 months. On Kaplan-Meier analyses of the overall population, adjuvant chemoradiotherapy was associated with significantly improved median overall survival (31.7 months vs. 16.3 months, p = 0.036). On Kaplan-Meier analyses of the 35 matched pairs generated by propensity score matching, adjuvant chemoradiotherapy was associated with significantly longer median overall survival (31.7 months vs. 14.3 months; p = 0.004) and median recurrence-free survival (18.9 months vs. 11.7 months; p = 0.020). In multivariable analysis, adjuvant chemoradiotherapy was independently associated with a 60% reduction in mortality (p = 0.003) and a 48% reduction in risk of recurrence (p = 0.035) after adjusting for putative confounders. In addition, microscopic positive resection margin and Mandard tumor regression grade 3-4 were independently associated with increased mortality and risk of recurrence. While a greater number of lymph nodes dissected was independently associated with significantly improved overall survival, the number of positive lymph nodes was independently associated with significantly worse overall survival and a trend (p = 0.058) towards worse recurrence-free survival. CONCLUSIONS: This study demonstrated that adjuvant CRT was independently associated with a significantly improved survival and lower risk of recurrence than observation in esophageal squamous cell carcinoma patients staged as ypT3 and/or ypN+ after receiving neoadjuvant chemoradiotherapy and radical surgery. The results of this study have implications for the design of future clinical trials and may improve treatment outcomes of patients in this setting who cannot afford or are without access to adjuvant nivolumab.

Characteristics of the Cytotoxicity of Taraxacum mongolicum and Taraxacum formosanum in Human Breast Cancer Cells.

Breast cancer is a highly heterogeneous disease that has been clinically divided into three main subtypes: estrogen receptor (ER)- and progesterone receptor (PR)-positive, human epidermal growth factor receptor 2 (HER 2)-positive, and triple-negative breast cancer (TNBC). With its high metastatic potential and resistance to endocrine therapy, HER 2-targeted therapy, and chemotherapy, TNBC represents an enormous clinical challenge. The genus Taraxacum is used to treat breast cancer in traditional medicine. Here, we applied aqueous extracts from two Taraxacum species, T. mongolicum and T. formosanum, to compare their potential antitumor effects against three human breast cancer cell lines: MDA-MB-231 (ER-, PR-, and HER2-), ZR-75-1 (ER+, PR+/-, and HER2-), and MCF-7 (ER+, PR+, and HER2-). Our results show that T. mongolicum exerted cytotoxic effects against MDA-MB-231 cells, including the induction of apoptosis, the reduction of cell proliferation, the disruption of the mitochondrial membrane potential, and/or the downregulation of the oxygen consumption rate. Both T. mongolicum and T. formosanum decreased cell migration and colony formation in the three cell-lines and exerted suppressive effects on MCF-7 cell proliferation based on metabolic activity and BrdU incorporation, but an enhanced proliferation of ZR-75-1 cells based on BrdU incorporation. T. formosanum induced ribotoxic stress in MDA-MB-231and ZR-75-1 cells; T. mongolicum did not. In summary, these findings suggest that T. mongolicum showed greater cytotoxicity against all three tested breast cancer cell lines, especially the TNBC MDA-MB-231 cell line.

Tea consumption affects the absorption of levothyroxine.

Levothyroxine (LT4) is a convenient treatment for hypothyroidism. Coffee, however, prevents the body from efficaciously absorbing LT4. It is unknown whether the intake of tea interferes with LT4 absorption. Thirty-seven hypothyroidism patients with the following types of consumption were recruited: 1) only tea, 2) tea and coffee, and 3) only coffee. The concentrations of thyroid-stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4) were recorded before and 3 months after changing the consumption. The patients had reductions in the concentration of TSH to varying degrees after 3 months (p < 0.001 or p < 0.05). The natural logarithms of the differences between the concentrations of TSH before and after the change in the consumption (Δln-TSH) of tea and coffee, only coffee, and only tea were 1.94, 2.00, and 2.18, respectively. Long-term consumption of tea, like coffee, interfered with the absorption of LT4. We believe that avoiding tea when taking LT4 will reduce malabsorption.

ß-cell deletion of the PKm1 and PKm2 isoforms of pyruvate kinase in mice reveals their essential role as nutrient sensors for the KATP channel.

Pyruvate kinase (PK) and the phosphoenolpyruvate (PEP) cycle play key roles in nutrient-stimulated KATP channel closure and insulin secretion. To identify the PK isoforms involved, we generated mice lacking ß-cell PKm1, PKm2, and mitochondrial PEP carboxykinase (PCK2) that generates mitochondrial PEP. Glucose metabolism was found to generate both glycolytic and mitochondrially derived PEP, which triggers KATP closure through local PKm1 and PKm2 signaling at the plasma membrane. Amino acids, which generate mitochondrial PEP without producing glycolytic fructose 1,6-bisphosphate to allosterically activate PKm2, signal through PKm1 to raise ATP/ADP, close KATP channels, and stimulate insulin secretion. Raising cytosolic ATP/ADP with amino acids is insufficient to close KATP channels in the absence of PK activity or PCK2, indicating that KATP channels are primarily regulated by PEP that provides ATP via plasma membrane-associated PK, rather than mitochondrially derived ATP. Following membrane depolarization, the PEP cycle is involved in an 'off-switch' that facilitates KATP channel reopening and Ca2+ extrusion, as shown by PK activation experiments and ß-cell PCK2 deletion, which prolongs Ca2+ oscillations and increases insulin secretion. In conclusion, the differential response of PKm1 and PKm2 to the glycolytic and mitochondrial sources of PEP influences the ß-cell nutrient response, and controls the oscillatory cycle regulating insulin secretion.