Cell Death Induced by the Combination of Ephedra sinica Extract and Radiation in HNSCC is Positively Related to BAX and p-MLKL Expression.

BACKGROUND: Numerous studies have proven the efficacy and safety of natural products, and are widely used as attractive cancer treatments. The investigation of effective natural products for improving cancer treatment is a promising strategy. Combination treatment with radiosensitizers and radiotherapy (RT) is considered necessary for therapeutic improvement in head and neck squamous cell carcinoma(HNSCC). OBJECTIVE: This study aims to investigate whether Ephedra sinica (ES) extract could induce selective cell death in cancer cells and serve as a radiosensitizer for HNSCC. METHODS: HNSCC cells were pretreated with ES extract before radiation, and the radiosensitizing activity was assessed using a colony formation assay. Radiation-induced cell death was evaluated using an annexinV-FITC assay. Western blotting was performed to confirm cell death-related gene expression, including apoptosis and necrosis markers. RESULTS: ES extract significantly inhibited HNSCC cell viability (FaDu and SNU1076), while having minimal effect on normal HaCaT cells. When HNSCC cells were irradiated with 2, 4, or 8 Gy and cultured with ES extract (25 µg/mL), they exhibited increased radiation sensitivity compared to non-treated cells. The combination of ES extract and radiation resulted in increased cell death compared to non-treated, ES-treated, or irradiated cells. The apoptosis marker BAX and necrosis marker p-MLKL expression levels were also elevated following the combination treatment. CONCLUSION: ES extract demonstrated significant cytotoxic potential in HNSCC cells without affecting normal cells. It enhanced the radiosensitivity of HNSCC cells by upregulating BAX and p-MLKL expression, leading to increased cell death. These results suggest ES extract exhibits a potential radiosensitizing capacity in HNSCC.

Identification of Biomarkers for the Diagnosis of Sepsis-Associated Acute Kidney Injury and Prediction of Renal Recovery in the Intensive Care Unit.

PURPOSE: Acute kidney injury (AKI) following sepsis is associated with higher mortality; however, reliable biomarkers for AKI development and recovery remain to be elucidated. MATERIALS AND METHODS: Patients with sepsis admitted to the medical intensive care unit (ICU) of Severance Hospital between June 2018 and May 2019 were prospectively analyzed. Patients were divided into those with and without AKI within 48 hours. Patients with septic AKI were subdivided into AKI-recovery and non-recovery groups based on whether their kidney injury recovered within 7 days. RESULTS: A total of 84 patients were enrolled. The baseline creatinine (2.9 mg/dL vs. 0.8 mg/dL vs. 1.2 mg/dL, p<0.001), Charlson Comorbidity Index (4.5 vs. 2.0 vs. 3.0, p=0.002), Sequential Organ Failure Assessment (10.0 vs. 6.5 vs. 8.0, p<0.001), and Acute Physiology and Chronic Health Evaluation II scores (32.0 vs. 21.5 vs. 30.5, p=0.004) were higher in the non-recovery AKI group compared to the non-AKI and AKI-recovery groups. The Kaplan-Meier curves revealed that non-recovery from AKI was associated with lower survival (p<0.001). High-lactate (p≤0.05) and kynurenine levels (p≤0.05) were associated with non-recovery of renal function following AKI. The areas under the curve for predicting non-recovery from AKI were 0.693 and 0.721 for lactate and kynurenine, respectively. The survival rate was lower in the high-kynurenine (p=0.040) and high-lactate (p=0.010) groups. CONCLUSION: The mortality of patients who recovered from AKI was comparable to that of patients without AKI. Lactate and kynurenine could be useful biomarkers for the diagnosis and recovery of AKI following sepsis.

Association between measured or calculated small dense low-density lipoprotein cholesterol and oxidized low-density lipoprotein in subjects with or without type 2 diabetes mellitus.

BACKGROUND: Small dense low-density lipoprotein (sdLDL) possesses atherogenic potential and is predicted to be susceptible to atherogenic modifications, which further increases its atherogenicity. However, studies on the association between measured or estimated sdLDL cholesterol (sdLDL-C) levels and atherogenic modification in diverse population groups are lacking. METHODS: Surplus serum samples were collected from male subjects with type 2 diabetes mellitus (DM) under treatment (n = 300) and without DM (non-DM; n = 150). sdLDL and oxidized LDL (oxLDL) levels were measured using the Lipoprint LDL subfractions kit (Quantimetrix Corporation) and the Mercodia oxidized LDL competitive enzyme-linked immunosorbent assay kit (Mercodia), respectively. The estimated sdLDL-Cs were calculated from two relevant equations. The effects of sdLDL-C on oxLDL were assessed using multiple linear regression (MLR) models. RESULTS: The mean (±SD) of measured sdLDL-C and oxLDL concentrations were 11.8 ± 10.0 mg/dl and 53.4 ± 14.2 U/L in the non-DM group and 0.20 ± 0.81 mg/dl and 46.0 ± 15.3 U/L in the DM group, respectively. The effects of measured sdLDL-Cs were significant (p = 0.031), whereas those of estimated sdLDL-Cs were not (p = 0.060, p = 0.116) in the non-DM group in the MLR models. The effects of sdLDL-Cs in the DM group were not significant. CONCLUSION: In the general population, high level of sdLDL-C appeared to be associated with high level of oxLDL. The equation for estimating sdLDL-C developed from a general population should be applied with caution to a special population, such as patients with DM on treatment.

Flow dynamics of surgical and transcatheter aortic valves: Past to present.

Objective: To perform an in vitro characterization of surgical aortic valves (SAVs) and transcatheter aortic valves (TAVs) to highlight the development of the flow dynamics depending on the type of valve implanted and assess the basic differences in the light of flow turbulence and its effect on blood damage likelihood and hemodynamic parameters that shed light on valve performance. Methods: A Starr-Edwards ball and cage valve of internal diameter 22 mm, a 23-mm Medtronic Hancock II SAV, a 23-mm St Jude Trifecta SAV, a 23-mm St Jude SJM (mechanical valve) SAV, a 26-mm Medtronic Evolut TAV, and a 26-mm Edwards SAPIEN 3 TAV were assessed in a pulse duplicator under physiological conditions. Particle image velocimetry was performed for each valve. Pressure gradient and effective orifice area (EOA) along with velocity flow field, Reynolds shear stress (RSS), and viscous shear stress (VSS) were calculated. Results: The SJM mechanical valve exhibited the greatest EOA (1.96 ± 0.02 cm2), showing superiority of efficiency compared with the same-size Trifecta (1.87 ± 0.07 cm2) and Hancock II (1.05 ± 0.01 cm2) (P < .0001). The TAVs show close EOAs (2.10 ± 0.06 cm2 with Evolut and 2.06 ± 0.03 cm2 with SAPIEN 3; P < .0001). The flow characteristics and behavior downstream of the valves differed depending on the valve type, design, and size. The greater the RSS and VSS the more turbulent the downstream flow. Hancock II displays the greatest range of RSS and VSS magnitudes compared with the same-size Trifecta and SJM. The Evolut displays the greatest range of RSS and VSS compared with the SAPIEN 3. Conclusions: The results of this study shed light on numerous advancements in the design of aortic valve replacement prosthesis and the subsequent hemodynamic variations. Future surgical and transcatheter valve designs should aim at not only concentrating on hemodynamic parameters but also at optimizing downstream flow properties.

Immune-Enhancing Effect of Submerged Culture of Ceriporia lacerata Mycelia on Cyclophosphamide-Induced Immunosuppressed Mice and the Underlying Mechanisms in Macrophages.

The white-rot fungi Ceriporia lacerata is used in bioremediation, such as lignocellulose degradation, in nature. Submerged cultures and extracts of C. lacerata mycelia (CLM) have been reported to contain various active ingredients, including ß-glucan and extracellular polysaccharides, and to exert anti-diabetogenic properties in mice and cell lines. However, the immunostimulatory effects have not yet been reported. This study aimed to identify the immunomodulatory effects, and underlying mechanisms thereof, of submerged cultures of CLM using RAW264.7 macrophages and cyclophosphamide (CTX)-induced immunosuppression in mice. Compared to CTX-induced immunosuppressed mice, the spleen and thymus indexes in mice orally administered CLM were significantly increased; body weight loss was alleviated; and natural killer (NK) cytotoxicity, lymphocyte proliferation, and cytokine (tumor necrosis factor [TNF]-α, interferon [IFN]-γ, and interleukin [IL]-2) production were elevated in the serum. In RAW264.7 macrophages, treatment with CLM induced phagocytic activity, increased the production of nitric oxide (NO), and promoted mRNA expression of the immunomodulatory cytokines TNF-α, IFN-γ, IL-1ß, IL-6, IL-10, and IL-12. In addition, CLM increased the inducible NO synthase (iNOS) concentration in macrophages, similar to lipopolysaccharide (LPS) stimulation. Mechanistic studies showed that CLM induced the activation of the NF-κB, PI3k/Akt, ERK1/2, and JNK1/2 pathways. Moreover, the phosphorylation of NF-κB and IκB induced by CLM in RAW264.7 cells was suppressed by specific MAPKs and PI3K inhibitors. Further experiments with a TLR4 inhibitor demonstrated that the production of TNF-α, IL-1ß, and IL-6 induced by CLM was decreased after TLR4 was blocked. Overall, CLM protected against CTX-induced adverse reactions by enhancing humoral and cellular immune functions, and has potential as an immunomodulatory agent.

Effect of Outlier Elimination on the 99th Percentile Upper Reference Limits of High-Sensitivity Cardiac Troponin I Assays Based on a Strictly Selected Healthy Reference Population.

BACKGROUND: We established high-sensitivity cardiac troponin I (hsTnI) 99th percentile upper reference limits (URLs) for the Centaur XPT High-Sensitivity Troponin I assay (Centaur hsTnI; Siemens, Erlangen, Germany) and Atellica IM High-Sensitivity Troponin I assay (Atellica hsTnI; Siemens) and assessed the effect of outlier elimination. METHODS: The reference population comprised 380 men and 387 women, satisfying the strict systematic reference population criteria. After reference population verification by the N-terminal pro-B-type natriuretic peptide (NT-proBNP) assay, 99th percentile URLs for Centaur hsTnI and Atellica hsTnI were calculated before and after outlier elimination. RESULTS: The 99th percentile URL for Centaur hsTnI was 60.4 (men, 74.7; women, 57.5) ng/L and that for Atellica hsTnI was 59.6 (men, 75.2; women, 55.1) ng/L. After the elimination of 61 (8.0%) outlier samples in Centaur hsTnI and 58 (7.6%) in Atellica hsTnI, the 99th percentile URLs were 13.5 ng/L (men, 15.3 ng/L; women, 11.9 ng/L) and 13.4 ng/L (men, 15.5 ng/L; women, 12.9 ng/L), respectively, significantly lower than those before outlier elimination. The CVs at the 99th percentile URLs were 5.2% and 3.5%, respectively. The measurable fractions among the reference population were 91.5% and 93.4%, respectively. Performance evaluation of Atellica B-type natriuretic peptide (BNP), Atellica NT-proBNP, Centaur hsTnI, and Atellica hsTnI showed outstanding results. CONCLUSIONS: The Korean hsTnI 99th percentile URLs calculated in this study were significantly lower after outlier elimination than before. Centaur hsTnI and Atellica hsTnI meet the "Guideline acceptable" and "Level 3 (second generation, high sensitivity)" requirements, satisfying international standards.

Development and validation of a novel sepsis biomarker based on amino acid profiling.

BACKGROUND & AIMS: Sepsis is a potentially fatal condition influenced by pathogens and host factors. Current sepsis biomarkers such as white blood cell count and C-reactive protein and procalcitonin levels show unsatisfactory performance in terms of diagnostic sensitivity and specificity in clinical practice. Thus, we developed and validated a new sepsis biomarker based on amino acid profiling. METHODS: We used two independent groups. The training and validation groups included 161 and 22 healthy controls, 123 and 50 patients with systemic inflammatory response syndrome, and 115 and 45 patients with sepsis, respectively. Using mass spectrometry, we measured and analyzed serum amino acid levels to select candidate amino acids that could differentiate sepsis from other conditions. Then, several possible multivariate indexes were developed by generating formulae with different combinations of candidate amino acids. The formula showing the best performance was selected and validated further. RESULTS: Kynurenine, tryptophan, phenylalanine, arginine, aspartic acid, glutamic acid, and glutamine were selected as candidate amino acids. Ten possible formulae were generated, and the formula with the highest diagnostic performance, which included kynurenine, tryptophan, phenylalanine, and arginine, was selected. In the validation group, the area under the receiving operating characteristic curve of the selected multivariate index (0.931) was similar to that of procalcitonin (0.945). Moreover, the generated multivariate index showed potential as a prognostic marker. CONCLUSIONS: Serum amino acid composition in patients with sepsis differs significantly from that in healthy individuals and patients with inflammation only. The newly developed multivariate index is expected to be implementable as a sepsis biomarker in clinical practice in the near future.

Performance Evaluation of Thyroid Function Tests on a Novel Chemiluminescent Microparticle Immunoassay System.

BACKGROUND: The Alinity i system (Abbott), a recently developed automated immunoassay analyzer, has a compact footprint and a throughput of 200 tests per hour. Here, we present the first performance evaluation of thyroid function test (TFT) on the Alinity i system. METHODS: We performed precision, linearity, comparison, functional sensitivity, carryover, and reference interval verification of 4 hormones (Thyroid Stimulation Hormone; TSH, total T3, free T4, and total T4) using the reagents provided by the manufacturer following the guidelines of the Clinical Laboratory Standards Institute. The performance of Alinity i was compared to that of the Architect i2000 immunoassay analyzer (Abbott, US). RESULTS: The within-laboratory coefficients of variation for all the hormones were 1.6-3.6%. Linearity was observed for all the hormones over the entire tested analytical range (R2 ≥ 0.99). The results for all the evaluated hormones using Alinity i system strongly correlated (r ≥ 0.978) with those from Architect i2000. Functional sensitivity was lower than the lower limit of the analytical measurement range. Sample carryover was less than 1.0%. CONCLUSIONS: TFTs on the Alinity i system showed acceptable performance in terms of precision, linearity, comparison, functional sensitivity, and carryover. Therefore, this system could be a useful laboratory tool for performing TFTs.

Safety and efficacy of sucroferric oxyhydroxide in pediatric patients with chronic kidney disease.

BACKGROUND: Pediatric patients with advanced chronic kidney disease (CKD) are often prescribed oral phosphate binders (PBs) for the management of hyperphosphatemia. However, available PBs have limitations, including unfavorable tolerability and safety. METHODS: This phase 3, multicenter, randomized, open-label study investigated safety and efficacy of sucroferric oxyhydroxide (SFOH) in pediatric and adolescent subjects with CKD and hyperphosphatemia. Subjects were randomized to SFOH or calcium acetate (CaAc) for a 10-week dose titration (stage 1), followed by a 24-week safety extension (stage 2). Primary efficacy endpoint was change in serum phosphorus from baseline to the end of stage 1 in the SFOH group. Safety endpoints included treatment-emergent adverse events (TEAEs). RESULTS: Eighty-five subjects (2-18 years) were randomized and treated (SFOH, n = 66; CaAc, n = 19). Serum phosphorus reduction from baseline to the end of stage 1 in the overall SFOH group (least squares [LS] mean ± standard error [SE]) was - 0.488 ± 0.186 mg/dL; p = 0.011 (post hoc analysis). Significant reductions in serum phosphorus were observed in subjects aged ≥ 12 to ≤ 18 years (LS mean ± SE - 0.460 ± 0.195 mg/dL; p = 0.024) and subjects with serum phosphorus above age-related normal ranges at baseline (LS mean ± SE - 0.942 ± 0.246 mg/dL; p = 0.005). Similar proportions of subjects reported ≥ 1 TEAE in the SFOH (75.8%) and CaAc (73.7%) groups. Withdrawal due to TEAEs was more common with CaAc (31.6%) than with SFOH (18.2%). CONCLUSIONS: SFOH effectively managed serum phosphorus in pediatric patients with a low pill burden and a safety profile consistent with that reported in adult patients.

HIV-Associated CKDs in Children and Adolescents.

INTRODUCTION: Limited information is available describing the current prevalence of proteinuria and HIV-associated CKDs (HIV-CKDs) in children and adolescents living with HIV and receiving antiretroviral therapy in the United States. METHODS: To address this issue, we performed a retrospective study of children and adolescents living with HIV who received medical care at Children's National Hospital in Washington, DC, between January 2012 and July 2019. Demographic data, clinical parameters (mode of HIV transmission, viral loads, CD4 cell counts, serum creatinine, glomerular filtration rate [GFR], plasma lipid levels, proteinuria, blood pressure, renal biopsies), and medical treatments, all done as a standard of clinical care, were collected and analyzed. RESULTS: The majority of the 192 patients enrolled were of African descent (88%) and acquired HIV through vertical transmission (97%). The prevalence of all HIV-CKDs was 6%. Of these patients, 39% had intermittent or persistent proteinuria, and 7% percent had proteinuria with a mild decline in GFR (60-80 ml/min per 1.73 m2), and 6% had a mild decline in GFR without proteinuria. Documented hypertension was present in 6% of the patients, mainly in association with HIV-CKD. Patients with persistent proteinuria (3%) and biopsy-proven HIV-CKD had a slow but constant progression of their renal diseases. CONCLUSIONS: The prevalence of persistent proteinuria and HIV-CKD was lower than that reported in previous studies conducted in the United States. However, intermittent proteinuria, mild reductions in GFR, and progression of established HIV-CKD were common findings in this group of patients with predominantly vertically acquired HIV who were receiving antiretroviral therapy.

Performance evaluation of an amplicon-based next-generation sequencing panel for BRCA1 and BRCA2 variant detection.

BACKGROUND: As next-generation sequencing (NGS) technology matures, various amplicon-based NGS tests for BRCA1/2 genotyping have been introduced. This study was designed to evaluate an NGS test using a newly released amplicon-based panel, AmpliSeq for Illumina BRCA Panel (AmpliSeq panel), for detection of clinically significant BRCA variants, and to compare it to another amplicon-based NGS test confirmed by Sanger sequencing. METHODS: We reviewed BRCA test results done by NGS using the TruSeq Custom Amplicon kit from patients suspected of hereditary breast/ovarian cancer syndrome (HBOC) in 2018. Of those, 96 residual samples with 100 clinically significant variants were included in this study using predefined criteria: 100 variants were distributed throughout the BRCA1 and BRCA2 genes. All target variants were confirmed by Sanger sequencing. Duplicate NGS testing of these samples was performed using the AmpliSeq panel, and the concordance of results from the two amplicon-based NGS tests was assessed. RESULTS: Ninety-nine of 100 variants were detected in duplicate BRCA1/2 genotyping using the AmpliSeq panel (sensitivity, 99%; specificity, 100%). In the discordant case, one variant (BRCA1 c.3627dupA) was found only in repeat 1, but not in repeat 2. Automated nomenclature of all variants, except for two indel variants, was in consensus with Human Genome Variation Society nomenclature. CONCLUSION: Our findings confirm that the analytic performance of the AmpliSeq panel is satisfactory, with high sensitivity and specificity.

GeneReader NGS System Is a Useful Sequencing Platform for Clinical Testing of BRCA1 and BRCA2.

Although next-generation sequencing (NGS) is widely used for BRCA1/2 sequencing analysis, it involves a fragmented workflow along with complex bioinformatic analysis and interpretation. In this study, the performance characteristics and workflow of the GeneReader NGS System (QIAGEN), including BRCA1/2 sequencing, were evaluated. For BRCA1/2 genetic testing, we conducted library preparation, emulsion PCR, and sequencing. QCI Analyze software was used for read alignment, quality control, variant calling, and clinical report generation. GeneReader and Sanger sequencing utilized 63 patients with breast or ovarian cancer for comparison. Reproducibility, precision, variant calling, turnaround time, and hands-on time were evaluated. The read percentage in the on-target regions was 90.5%. More than 99.99% of target regions showed read depths ≥100x. Variants generated from GeneReader showed 100% accuracy compared to the Sanger sequencing results. Annotation with GeneReader showed >99.8% concordance with HGVS nomenclature. Single-nucleotide variations and indel variants showed 100% calling reproducibility; the precision for variant frequency showed a 0.3-3.6% coefficient of variation. Most processes involved hands-off time (3714 min, 88.6% of total run time). The GeneReader NGS System for BRCA1 and BRCA2 testing showed good analytical performance and a short hands-on time. Because of its integrated sample preparation for bioinformatic interpretation, this system is practical for clinical laboratories.

Accuracy evaluation of automated electrochemiluminescence immunoassay for everolimus and sirolimus compared to liquid chromatography-tandem mass spectrometry.

BACKGROUND: We evaluated the analytical performance of a newly developed electrochemiluminescence immunoassay for everolimus and sirolimus compared to that of liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: According to Clinical and Laboratory Standards Institute guidelines, the analytical performance including precision, recovery, linearity, and carryover was evaluated. For correlation evaluation, the results of Elecsys® analysis of everolimus and sirolimus were compared with those of LC-MS/MS using 120 samples from patients treated with everolimus or sirolimus. RESULTS: The within-run and total imprecision values were as follows: 2.3%-4.5% and 4.5%-6.4% for the everolimus assay; 3.3%-4.8% and 4.7%-8.1% for the sirolimus assay, respectively. The measured concentration was linear over the range of 0.718-27.585 ng/mL for everolimus analysis and 0.789-26.880 ng/mL for sirolimus analysis (all R2  > 0.99). Recovery was 93.5%-105.5% for the everolimus assay and 99.2%-109.1% for the sirolimus assay (except lowest levels). Carryover was -1.09% for the everolimus assay and -0.12% for the sirolimus assay. The results of the two chemiluminescence immunoassays showed acceptable correlations with those of LC-MS/MS (R = 0.9585 and R = 0.9799, respectively). The two immunoassays showed slightly proportional biases compared to LC-MS/MS. CONCLUSION: Elecsys® Everolimus and Sirolimus assays showed acceptable analytical performance in precision, linearity, and correlation compared to LC-MS/MS These methods can be adopted in the clinical laboratory for rapid therapeutic drug monitoring of patients who require treatment with immunosuppressants.

Quality Improvement Initiative to Reduce Admissions for Nephrotic Syndrome Relapse in Pediatric Patients.

Introduction: Childhood nephrotic syndrome is frequently seen in pediatric nephrology practice and often requires patient hospitalization for management. Numerous complications of this disease can be managed in an outpatient setting if brought to the attention of the medical team in a timely manner. Outpatient management will reduce healthcare cost and improve patient safety. The goal of this quality improvement initiative was to reduce admissions for nephrotic syndrome relapse from 8 to <5 admissions at a single center in a 3-month period. Methods: Fish-bone analysis was used to determine barriers to early recognition of relapse and successful outpatient care. Patient education about the disease process was identified as the primary barrier. A standardized approach to patient education as well as educational materials were developed. Champions were identified within each stakeholder group to train and disseminate the new process. Admission counts were compared from 3 years prior to implementation to 2 years post-implementation. Clinic visits for nephrotic syndrome were tallied as a balancing measure. Patients were surveyed in the outpatient clinics about whether they had ever received the education as a process measure. Results: Admission counts were reduced and met goal for the first 3 quarters that were examined; however, the number of admissions went above target in the last quarter. Clinic visit numbers did not change over the study period. Process measure showed that 75-80% of families were provided with nephrotic syndrome education. Conclusion: A standardized approach to patient and family education about idiopathic nephrotic syndrome can reduce admissions for management of relapse. This will reduce healthcare expenditure as well as improve patient safety.

Hyponatremia: An Unusual Presentation in a Neonate With Chromosome 1q21.1 Deletion Syndrome.

Chromosome 1q21.1 deletion syndrome is associated with a wide variety of clinical features including mild to moderate mental retardation, microcephaly, cardiac abnormalities, and cataracts. We report an unusual case of a premature neonate with persistent hyponatremia, markedly elevated plasma arginine vasopressin level (32.7 pg/mL), and clinical findings consistent with the syndrome of inappropriate antidiuretic hormone secretion (SIADH). The patient, who also had microcephaly and dextrocardia, was subsequently diagnosed with chromosome 1q21.1 deletion syndrome. Further evaluation revealed hypothalamic abnormalities, features not previously described with this syndrome. To our knowledge, this is the first report of SIADH associated with congenital hypothalamic anomalies in a neonate with chromosome 1q21.1 deletion syndrome. We also report our experience using tolvaptan, a vasopressin receptor antagonist, in this patient to effectively maintain eunatremia.