Evaluation of the Accuracy of Cr and BUN Using the ABL90 FLEX PLUS Blood Gas Analyzer and the Equivalence of Candidate Specimens for Assessment of Renal Function.

BACKGROUND: The ABL90 FLEX PLUS (Radiometer) is a blood gas analyzer that also provides creatinine (Cr) and blood urea nitrogen (BUN) results. We assessed the accuracy of the ABL90 FLEX PLUS to measure Cr and BUN and find suitable candidate specimens against primary specimens (heparinized whole-blood (H-WB)). METHODS: Paired H-WB, serum, and sodium-citrated whole-blood (C-WB) samples (105) were collected. The Cr and BUN levels in the H-WB using the ABL90 FLEX PLUS were compared with those of the serum using four automated chemistry analyzers. The suitability of the candidate specimens was assessed at each medical decision level according to the CLSI guideline EP35-ED1. RESULTS: The respective mean differences of the ABL90 FLEX PLUS for the Cr and BUN were below -0.10 and -3.51 mg/dL compared to the other analyzers. The systematic differences between the serum and the H-WB at the low, medium, and high medical decision levels were all 0% for Cr, but those of the C-WB were -12.96%, -11.81%, and -11.30%, respectively. Regarding imprecision, the SDserum/SDH-WB ratios at each level were 0.14, 1.41, and 0.68, whereas the SDC-WB/SDH-WB ratios were 0.35, 2.00, and 0.73, respectively. CONCLUSIONS: The ABL90 FLEX PLUS provided Cr and BUN results comparable with the four widely used analyzers. Among the candidates, the serum was suitable for Cr testing using the ABL90 FLEX PLUS, while the C-WB did not satisfy the acceptance criteria.

Neutrophil Recruitment in Arterial Thrombus and Characteristics of Stroke Patients with Neutrophil-Rich Thrombus.

PURPOSE: Neutrophils contribute to thrombosis. However, there is limited information on the temporal course of neutrophil recruitment in thrombosis, the contribution of neutrophils to thrombus growth, and the characteristics of stroke patients with neutrophil-rich thrombi. MATERIALS AND METHODS: After inducing carotid artery thrombosis in Institute of Cancer Research mice using ferric chloride, aged thrombi were produced by ligating the distal portion of the carotid artery in mice for 0.5, 1, 2, 3, 6, or 24 h. For thrombus analysis in stroke patients, we used registry data and thrombi that were obtained during intra-arterial thrombectomy. Immunohistochemistry was performed to determine thrombus composition. RESULTS: In the thrombi of 70 mice, Ly6G positive cell counts (neutrophils) and histone H3-positive cell counts increased in a time-dependent manner (both p<0.001). Ly6G-positive cell count was strongly correlated with histone H3-positive cell counts (r=0.910, p<0.001), but not with thrombus size (p=0.320). In 75 stroke patients, atrial fibrillation and cardioembolism were more frequent in the higher neutrophil group (32/37, 86.5%) than in the lower neutrophil group (19/38, 50%) (p=0.002). The median erythrocyte fraction was higher [52.0 (interquartile range 39.9-57.8)] in the higher neutrophil group than in the lower neutrophil group [40.3 (interquartile range 23.5-53.2)]. The fraction of neutrophils was positively correlated with that of erythrocytes (R=0.35, p=0.002). CONCLUSION: Neutrophils were recruited and increased in arterial thrombosis in a time-dependent manner; however, they were not associated with the growth of formed thrombi. Neutrophil fractions in the thrombi of stroke patients appeared to be associated with atrial fibrillation and erythrocyte fraction.

Effect of leukopenia induced by cyclophosphamide on the initial stage of arterial thrombosis in mice.

INTRODUCTION: Leukocytes are found in organizing thrombi and are associated with thrombus growth. However, their role in the initial stage of thrombus formation is not well known. We investigated the role of leukocytes in the early stage of arterial thrombosis by inducing leukopenia. METHODS: In this double-blind, randomized, placebo-controlled study, 72 Institute of Cancer Research mice were randomly treated with intraperitoneal 100 mg/kg cyclophosphamide or normal saline. The primary outcome was time to occlusion after FeCl3 treatment. We also compared thrombus size, histological composition, and association with peripheral blood cell counts between cyclophosphamide and control groups. RESULTS: Cyclophosphamide treatment significantly decreased leukocyte counts by 82.8% compared to placebo (P < 0.001). The time to occlusion was significantly longer in the cyclophosphamide group (3.31 ± 1.59 min) than in the control group (2.30 ± 1.14 min; P = 0.003). The immunoreactivity for Ly6G-positive cells, intracellular histone H3, and released histone H3 in thrombi was significantly reduced in the cyclophosphamide group by 92.8%, 50.2%, and 34.3%, respectively. Time to occlusion had a moderate negative correlation with leukocyte count in peripheral blood (r = -0.326, P = 0.022) in the entire group. CONCLUSIONS: Cyclophosphamide-induced leukopenia attenuated thrombus formation during the early stage of arterial thrombosis. Our findings suggest the potential role of leukocytes in the initial stage of arterial thrombosis.

Diagnostic Validation of a Clinical Laboratory-Oriented Targeted RNA Sequencing System for Detecting Gene Fusions in Hematologic Malignancies.

Targeted RNA sequencing (RNA-seq) is a highly accurate method for sequencing transcripts of interest with a high resolution and throughput. However, RNA-seq has not been widely performed in clinical molecular laboratories because of the complexity of data processing and interpretation. We developed and validated a customized RNA-seq panel and data processing protocol for fusion detection using 4 analytical validation samples and 51 clinical samples, covering seven types of hematologic malignancies. Analytical validation showed that the results for target gene coverage and between- and within-run precision and linearity tests were reliable. Using clinical samples, RNA-seq based on filtering and prioritization strategies detected all 25 known fusions previously found by multiplex reverse transcriptase-PCR and fluorescence in situ hybridization. It also detected nine novel fusions. Known fusions detected by RNA-seq included two IGH rearrangements supported by expression analysis. Novel fusions included six that targeted just one partner gene. In addition, 18 disease- and drug resistance-associated transcript variants in ABL1, GATA2, IKZF1, JAK2, RUNX1, and WT1 were designated simultaneously. Expression analysis showed distinct clustering according to subtype and lineage. In conclusion, this study showed that our customized RNA-seq system had a reliable and stable performance for fusion detection, with enhanced diagnostic yield for hematologic malignancies in a clinical diagnostic setting.

The HLA-A*02:01:175 allele newly identified in a Korean hematopoietic stem cell donor by next-generation sequencing.

HLA-A*02:01:175 has a single synonymous nucleotide polymorphism when compared with HLA-A*02:01:01:01 [c165.G>C].

Changing the frequency and spectra of chromosomal aberrations in Korean patients with acute leukemia in a tertiary care hospital.

BACKGROUND: Chromosomal analysis is essential for the diagnosis and risk stratification of all leukemia patients. Not surprisingly, racial differences in chromosomal aberrations (CA) in hematological malignancies could be found, and CA incidence in leukemia might change over time, possibly due to environmental and lifestyle changes. Thus, we compared the frequency and range of CA in patients with acute leukemia (AL) during two time periods (2006‒2009 vs. 2010‒2015) and compared them with other prior studies. METHODS: We enrolled 717 patients with AL during a six-year period (2010‒2015). We compared the results to those of our earlier study (2006‒2009) [1]. Conventional cytogenetics, a multiplex reverse transcriptase (RT)-PCR system, and fluorescence in situ hybridization were employed to assess bone marrow specimens or peripheral blood at the diagnostic stage in AL patients to detect CA. RESULTS: The incidence of CA changed in the leukemia subgroups during the two time periods. Notably, the most frequent CA of childhood acute myeloid leukemia (AML) was PML/RARA, and was followed by RUNX1/RUNX1T1 in the current study. In contrast, the most common CA was RUNX1/RUNX1T1 in a previous study [1] and was followed by PML/RARA. In this study, the most frequent CA of the mixed phenotype AL was BCR/ABL1, which was followed by KMT2A/MLLT3. In a previous report, [1] the most frequent CA was BCR/ABL1, which was followed by KMT2A/ELL. CONCLUSION: The distribution of CA in Korean AL patients changed over time in a single institute. This change might be due to environmental and lifestyle changes.

ATP Production Relies on Fatty Acid Oxidation Rather than Glycolysis in Pancreatic Ductal Adenocarcinoma.

Glycolysis is known as the main pathway for ATP production in cancer cells. However, in cancer cells, glucose deprivation for 24 h does not reduce ATP levels, whereas it does suppress lactate production. In this study, metabolic pathways were blocked to identify the main pathway of ATP production in pancreatic ductal adenocarcinoma (PDAC). Blocking fatty acid oxidation (FAO) decreased ATP production by 40% in cancer cells with no effect on normal cells. The effects of calorie balanced high- or low-fat diets were tested to determine whether cancer growth is modulated by fatty acids instead of calories. A low-fat diet caused a 70% decrease in pancreatic preneoplastic lesions compared with the control, whereas a high-fat diet caused a two-fold increase in preneoplastic lesions accompanied with increase of ATP production in the Kras (G12D)/Pdx1-cre PDAC model. The present results suggest that ATP production in cancer cells is dependent on FAO rather than on glycolysis, which can be a therapeutic approach by targeting cancer energy metabolism.

PDX models of human lung squamous cell carcinoma: consideration of factors in preclinical and co-clinical applications.

BACKGROUND: Treatment of human lung squamous cell carcinoma (LUSC) using current targeted therapies is limited because of their diverse somatic mutations without any specific dominant driver mutations. These mutational diversities preventing the use of common targeted therapies or the combination of available therapeutic modalities would require a preclinical animal model of this tumor to acquire improved clinical responses. Patient-derived xenograft (PDX) models have been recognized as a potentially useful preclinical model for personalized precision medicine. However, whether the use of LUSC PDX models would be appropriate enough for clinical application is still controversial. METHODS: In the process of developing PDX models from Korean patients with LUSC, the authors investigated the factors influencing the successful initial engraftment of tumors in NOD scid gamma mice and the retainability of the pathological and genomic characteristics of the parental patient tumors in PDX tumors. CONCLUSIONS: The authors have developed 62 LUSC PDX models that retained the pathological and genomic features of parental patient tumors, which could be used in preclinical and co-clinical studies. Trial registration Tumor samples were obtained from 139 patients with LUSC between November 2014 and January 2019. All the patients provided signed informed consents. This study was approved by the institutional review board (IRB) of Samsung Medical Center (2018-03-110).

Washable Colorimetric Nanofiber Nonwoven for Ammonia Gas Detection.

The colorimetric sensor is a facile, cost-effective, and non-power-operated green energy material for gas detection. In this study, the colorimetric sensing property of a meta-aramid/dye 3 nanofiber sensor for ammonia (NH3) gas detection was investigated. This colorimetric sensor was prepared using various dye 3 concentrations via electrospinning. Morphological, thermal, structural, and mechanical analyses of the sensor were carried out by field-emission scanning electron microscopy, thermogravimetric analysis, Fourier-transform infrared spectroscopy, and a universal testing machine, respectively. A homemade computer color matching machine connected with a gas flow device characterized the response of the meta-aramid/dye 3 nanofiber colorimetric sensor to various exposure levels of NH3 gas. From the results, we confirmed that this colorimetric green energy sensor could detect ammonia gas in the concentration of 1-10 ppm with a sensing response time of 10 s at room temperature. After washing with laundry detergent for 30 min, the colorimetric sensors still exhibited sensing property and reversibility.

Transglutaminase 2-Mediated p53 Depletion Promotes Angiogenesis by Increasing HIF-1α-p300 Binding in Renal Cell Carcinoma.

Angiogenesis and the expression of vascular endothelial growth factor (VEGF) are increased in renal cell carcinoma (RCC). Transglutaminase 2 (TGase 2), which promotes angiogenesis in endothelial cells during wound healing, is upregulated in RCC. Tumor angiogenesis involves three domains: cancer cells, the extracellular matrix, and endothelial cells. TGase 2 stabilizes VEGF in the extracellular matrix and promotes VEGFR-2 nuclear translocation in endothelial cells. However, the role of TGase 2 in angiogenesis in the cancer cell domain remains unclear. Hypoxia-inducible factor (HIF)-1α-mediated VEGF production underlies the induction of angiogenesis in cancer cells. In this study, we show that p53 downregulated HIF-1α in RCC, and p53 overexpression decreased VEGF production. Increased TGase 2 promoted angiogenesis by inducing p53 degradation, leading to the activation of HIF-1α. The interaction of HIF-1α and p53 with the cofactor p300 is required for stable transcriptional activation. We found that TGase 2-mediated p53 depletion increased the availability of p300 for HIF-1α-p300 binding. A preclinical xenograft model suggested that TGase 2 inhibition can reverse angiogenesis in RCC.

Inhibition of Transglutaminase 2 but Not of MDM2 Has a Significant Therapeutic Effect on Renal Cell Carcinoma.

More than 50% of human cancers harbor TP53 mutations and increased expression of Mouse double minute 2 homolog(MDM2), which contribute to cancer progression and drug resistance. Renal cell carcinoma (RCC) has an unusually high incidence of wild-type p53, with a mutation rate of less than 4%. MDM2 is master regulator of apoptosis in cancer cells, which is triggered through proteasomal degradation of wild-type p53. Recently, we found that p53 protein levels in RCC are regulated by autophagic degradation. Transglutaminase 2 (TGase 2) was responsible for p53 degradation through this pathway. Knocking down TGase 2 increased p53-mediated apoptosis in RCC. Therefore, we asked whether depleting p53 from RCC cells occurs via MDM2-mediated proteasomal degradation or via TGase 2-mediated autophagic degradation. In vitro gene knockdown experiments revealed that stability of p53 in RCC was inversely related to levels of both MDM2 and TGase 2 protein. Therefore, we examined the therapeutic efficacy of inhibitors of TGase 2 and MDM2 in an in vivo model of RCC. The results showed that inhibiting TGase 2 but not MDM2 had efficient anticancer effects.

Fc-saxatilin inhibits VEGF-induced permeability by regulating claudin-5 expression in human brain microvascular endothelial cells.

The disruption of the blood-brain barrier influences the degree of brain damage and prognosis in cerebral ischemia or other brain diseases accompanied by inflammation. Vascular endothelial growth factor (VEGF) released during brain ischemia or inflammation has been implicated in the breakdown of the blood-brain barrier by increasing endothelial permeability. Saxatilin, a disintegrin-containing RGD motif, has been reported to disaggregate platelets via interactions with platelet integrins and to have a thrombolysis effect. Additionally, the Fc-saxatilin fusion protein reduces vascular leakage in cerebral ischemia in mice. In this study, we show that Fc-saxatilin prevents VEGF-induced permeability in human brain microvascular endothelial cells (HBMECs). The activation of Src and Fak, downstream signaling proteins of VEGF in the induction of endothelial permeability, was inhibited by Fc-saxatilin in HBMECs. The downregulation of a tight junction protein, claudin-5, at the protein and mRNA levels by VEGF was recovered by Fc-saxatilin. Our findings suggest that Fc-saxatilin attenuates VEGF-induced endothelial permeability via the regulation of downstream signaling, and this may contribute to its protective effect against vascular leakage in the ischemic brain.

Pachypodol, a Methoxyflavonoid Isolated from Pogostemon cablin Bentham Exerts Antioxidant and Cytoprotective Effects in HepG2 Cells: Possible Role of ERK-Dependent Nrf2 Activation.

Oxidative stress has been implicated in the pathogenesis of many diseases including chronic liver diseases. Nrf2 is a master transcriptional factor regulating the induction of cellular antioxidant defense systems. Here, the Nrf2-activating effect of the crude methanol extract of dried leaves of Pogostemon cablin Bentham was demonstrated by measuring the antioxidant response element (ARE)-driven luciferase activity and pachypodol, 4',5-dihydroxy-3,3',7-trimethoxyflavone, was isolated by bioactivity-guided fractionation and further separation using chromatographic techniques. To our knowledge, this is the first study to evaluate the antioxidant and cytoprotective effects of pachypodol in HepG2 cells as well as the underlying molecular mechanisms. Indeed, pachypodol protected HepG2 cells from cell death caused by tert-butylhydroperoxide-induced oxidative stress and also attenuated ROS production. The ability of pachypodol to activate Nrf2/ARE pathway was further confirmed by observing Nrf2 expression in nuclear fraction, mRNA levels of Nrf2 target antioxidants, and cellular glutathione content in HepG2 cells. Extracellular signal-regulated kinase (ERK) is one of the important kinases involved in Nrf2 activation. Pachypodol increased ERK phosphorylation and ERK inhibition by PD98059 totally abrogated the increase in ARE luciferase activity, nuclear Nrf2 accumulation and mRNA levels of antioxidant enzymes by pachypodol. In conclusion, pachypodol isolated from P. cablin can protect hepatocytes from oxidative injury, possibly mediated by enhancing endogenous antioxidant defense system through ERK-dependent Nrf2 activation.

Histological features of intracranial thrombi in stroke patients with cancer.

The histological features of thrombus in stroke patients with cancer are not well known. Using immunohistochemical staining of thrombi retrieved during mechanical thrombectomy in stroke patients, thrombus compositions were compared between 16 patients with active cancer, 16 patients with inactive cancer, and 16 patients without any history of cancer. The active cancer group showed higher platelet and lower erythrocyte fractions than the inactive cancer or the control group. Four patients with vegetation showed very high platelet and low erythrocyte fractions. Patients with cryptogenic etiology in the active cancer group showed a similar pattern to those with vegetation. These findings may aid the determination of treatment strategies in cancer-associated stroke. ANN NEUROL 2019.

TNF-α-Induced YAP/TAZ Activity Mediates Leukocyte-Endothelial Adhesion by Regulating VCAM1 Expression in Endothelial Cells.

YAP/TAZ, a transcriptional co-activator of Hippo pathway, has emerged as a central player in vessel homeostasis such as sprouting angiogenesis and vascular barrier stabilization, during development. However, the role of YAP/TAZ in pathological angiogenesis remains unclear. Here, we demonstrated that YAP/TAZ is a critical mediator in leukocyte-endothelial adhesion induced by the vascular inflammatory cytokine TNF-α. YAP/TAZ was dephosphorylated, translocated from the cytosol to the nucleus, and activated by TNF-α in endothelial cells. A specific inhibitor of Rho GTPases suppressed the TNF-α-induced dephosphorylation of YAP. Knockdown of YAP/TAZ using siRNA significantly reduced the expression of the leukocyte adhesion molecule VCAM1 induced by TNF-α. The adhesion of monocytes to endothelial cells was also markedly reduced by YAP/TAZ silencing. However, knockdown of YAP/TAZ did not affect TNF-α-induced NF-κB signaling. Overall, these results suggest that YAP/TAZ plays critical roles in regulating TNF-α-induced endothelial cell adhesive properties without affecting the NF-κB pathway, and implicate YAP/TAZ as a potential therapeutic target for treating inflammatory vascular diseases.

LDB2 regulates the expression of DLL4 through the formation of oligomeric complexes in endothelial cells.

Delta-like ligand 4 (DLL4) expression in endothelial cells is intimately associated with angiogenic sprouting and vascular remodeling, but the precise mechanism of transcriptional regulation of DLL4 remains incompletely understood. Here, we showed that LIM-domain binding protein 2 (LDB2) plays an important role in regulating basal DLL4 and VEGF-induced DLL4 expression. Knockdown of LDB2 using siRNA enhanced endothelial sprouting and tubular network formation in vitro. Injection of ldb2-morpholino resulted in defective development of intersegmental vessels in zebrafish. Reduction or overexpression of LDB2 in endothelial cells decreased or increased DLL4 expression. LDB2 regulated DLL4 promoter activity by binding to its promoter region and the same promoter region was occupied and regulated by the LMO2/TAL1/GATA2 complex. Interestingly, LDB2 also mediated VEGF-induced DLL4 expression in endothelial cells. The regulation of DLL4 by the LDB2 complex provides a novel mechanism of DLL4 transcriptional control that may be exploited to develop therapeutics for aberrant vascular remodeling. [BMB Reports 2018; 51(1): 21-26].

Role of Protein Kinases and Their Inhibitors in Radiation Response of Tumor Cells.

Phosphorylation, the addition of a phosphate group to a molecule, is an effective way of regulating the biological properties of that molecule. Protein phosphorylation is a post-translational modification of proteins and affects cellular signaling transduction. Protein kinases induce phosphorylation by catalyzing the transfer of phosphate groups to serine, threonine, and tyrosine residues on protein substrates. Consistent with their roles in cancer, protein kinases have emerged as one of the most clinically useful target molecules in pharmacological cancer therapy. Intrinsic or acquired resistance of cancers against anti-cancer therapeutics, such as ionizing radiation, is a major obstacle for the effective treatment of many cancers. In this review, we describe key aspects of various kinases acting on proteins. We also discuss the roles of protein kinases in the pathophysiology and treatment of cancer. Because protein kinases correlate with radiation resistance in various types of cancer, we focus on several kinases responsible for radiation resistance and/or sensitivity and their therapeutic implications. Finally, we suggest some ongoing radiation-sensitization strategies using genetic loss and/or kinase inhibitors that can counteract radiation resistance-related protein kinases.

Deficiencies of Circulating Mucosal-associated Invariant T Cells and Natural Killer T Cells in Patients with Multiple Trauma.

Mucosal-associated invariant T (MAIT) cells and natural killer T (NKT) cells are known to play important roles in autoimmunity, infectious diseases and cancers. However, little is known about the roles of these invariant T cells in multiple trauma. The purposes of this study were to examine MAIT and NKT cell levels in patients with multiple trauma and to investigate potential relationships between these cell levels and clinical parameters. The study cohort was composed of 14 patients with multiple trauma and 22 non-injured healthy controls (HCs). Circulating MAIT and NKT cell levels in the peripheral blood were measured by flow cytometry. The severity of injury was categorised according to the scoring systems, such as Acute Physiology and Chronic Health Evaluation (APACHE) II score, Simplified Acute Physiology Score (SAPS) II, and Injury Severity Score (ISS). Circulating MAIT and NKT cell numbers were significantly lower in multiple trauma patients than in HCs. Linear regression analysis showed that circulating MAIT cell numbers were significantly correlated with age, APACHE II, SAPS II, ISS category, hemoglobin, and platelet count. NKT cell numbers in the peripheral blood were found to be significantly correlated with APACHE II, SAPS II, and ISS category. This study shows numerical deficiencies of circulating MAIT cells and NKT cells in multiple trauma. In addition, these invariant T cell deficiencies were found to be associated with disease severity. These findings provide important information for predicting the prognosis of multiple trauma.