RNA-binding protein LIN28A upregulates transcription factor HIF1α by posttranscriptional regulation via direct binding to UGAU motifs.

Hypoxia-inducible factor 1α (HIF1α) is a transcription factor that regulates angiogenesis under hypoxic conditions. To investigate the posttranscriptional regulatory mechanism of HIF1α, we performed a cell-based screening to reveal potential cis-elements and the regulatory RNA-binding proteins that act as trans-factors. We found that LIN28A promoted HIF1α protein expression independently of the downregulation of microRNA let-7, which is also directly mediated by LIN28A. Transcriptome analysis and evaluation of RNA stability using RNA-seq and SLAM-seq analyses, respectively, revealed that LIN28A upregulates HIF1A expression via mRNA stabilization. To investigate the physical association of LIN28A with HIF1A mRNA, we performed enhanced crosslinking immunoprecipitation in 293FT cells and integrally analyzed the transcriptome. We observed that LIN28A associates with HIF1A mRNA via its cis-element motif "UGAU". The "UGAU" motifs are recognized by the cold shock domain of LIN28A, and the introduction of a loss-of-function mutation to the cold shock domain diminished the upregulatory activities performed by LIN28A. Finally, the microvessel density assay showed that the expression of LIN28A promoted angiogenesis in vivo. In conclusion, our study elucidated the role of LIN28A in enhancing the HIF1α axis at the posttranscription layer.

Evaluation of fibrinogen concentration by clot firmness using a dielectric blood coagulation test system.

PURPOSE: To determine if fibrinogen concentration can be evaluated by dielectric permittivity changes in dielectric blood coagulation testing (DBCM) during cardiovascular surgery with cardiopulmonary bypass (CPB). METHODS: We performed a single-center prospective observational study at a university hospital. One hundred patients undergoing cardiovascular surgery with CPB were enrolled. Whole-blood samples were obtained after weaning from CPB, and dielectric clot strength (DCS) was measured by intrinsic pathway testing with or without heparinase in DBCM. The FIBTEM test was performed during rotational thromboelastometry using the same samples, and maximum clot firmness (MCF) was evaluated. Spearman's correlation analysis was performed, and receiver operating characteristics (ROC) curve analyses were used to evaluate the performance of hypofibrinogenemia detection. RESULTS: DCS showed a strong positive correlation with plasma fibrinogen concentration (Rs = 0.76, P < 0.0001). The area under the ROC curve for evaluating plasma fibrinogen concentration < 200 mg/dL was 0.91 (95% confidence interval (CI) 0.85-0.97) for DCS, compared with 0.88 (95% CI 0.81-0.94) for FIBTEM MCF. The optimal cutoff value of DCS was 17.0 (sensitivity 94%, specificity 80%). CONCLUSIONS: DCS variables showed a significantly strong correlation with plasma fibrinogen concentration, and the diagnostic performance for hypofibrinogenemia was comparable to that for FIBTEM MCF. This novel methodology has the potential to provide a point-of-care test with sufficient accuracy for the detection of perioperative hypofibrinogenemia during cardiovascular surgery with CPB.

TEG6s Platelet Mapping assay for the estimation of plasma fibrinogen concentration during cardiovascular surgery: a single-center prospective observational study.

PURPOSE: The Activator F (ActF) test on the TEG6s Platelet Mapping assay system is a means of quantifying blood viscoelasticity caused by fibrin network formation, triggered by reptilase and factor XIII, while platelets are inhibited. This unique methodology enables the measurement of blood viscoelasticity, even in highly heparinized blood. Here, we investigated whether fibrinogen concentration could be estimated using the ActF test in blood samples obtained during cardiopulmonary bypass (CPB) and after CPB in patients undergoing cardiovascular surgery. METHODS: We performed a single-center prospective observational study at a university hospital. Forty patients aged ≥ 18 years who underwent elective cardiovascular surgery with CPB were enrolled. Blood samples were drawn after the induction of anesthesia, after declamping of the aorta during CPB, and after the reversal of heparinization using protamine (after CPB). Coagulation profiles were evaluated using the Platelet Mapping assay and standard laboratory tests. RESULTS: There were strong correlations between the maximal amplitude of clot strength (MA) in the ActF test and fibrinogen concentration in samples drawn during CPB (R = 0.84, 95% confidence interval [CI] 0.72-0.91; P < 0.001) and after CPB (R = 0.83, 95% CI 0.70-0.91; P < 0.001). The areas under the receiver-operating characteristic curve for the ActF MA for fibrinogen concentrations < 150 mg/dL were 0.86 (95% CI 0.73-1.0) during CPB and 0.98 (95% CI 0.94-1.0) after CPB. CONCLUSION: TEG6s Platelet Mapping ActF MA values strongly correlated with plasma fibrinogen concentration in highly heparinized blood during CPB and yielded highly accurate measurements of low fibrinogen concentrations.

Population pharmacokinetics of cefazolin before, during and after cardiopulmonary bypass in adult patients undergoing cardiac surgery.

PURPOSE: The aims of the present study were to establish a population pharmacokinetic (PPK) model of cefazolin for adult patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and to assess the probability of target attainment (PTA) for the prophylaxis of surgical site infection (SSI) using cefazolin. METHODS: Adult patients who underwent cardiac surgery with CPB were enrolled in the prospective study. Blood samples for plasma cefazolin assay were collected, and total and unbound drug concentrations were measured and analysed using the nonlinear mixed-effects modelling (NONMEM) software considering saturable plasma protein binding. Using the PPK model, plasma unbound cefazolin concentration-time courses with current prophylaxis protocols were simulated, and the PTA for common SSI pathogens was estimated. RESULTS: A total of 199 blood samples were obtained from 27 patients. A one-compartment model with first-order elimination plus an on/off CPB compartment best described the data. The population mean for systemic drug clearance (CL) was reduced and that for the volume of distribution (V) was increased during CPB compared with the pre-CPB values. CPB-induced hypoalbuminemia was associated with reduced maximum protein binding (Bmax). The simulation studies suggested that the current dosing protocols are insufficient for attaining PTA > 0.9 throughout surgery against pathogens with minimum inhibitory concentrations (MICs) >8 mg/L. A new dosing protocol that achieves a PTA > 0.9 for pathogens with a MIC of 16 mg/L was proposed. CONCLUSION: PPK modelling with simulation may be valuable for devising a cefazolin prophylaxis protocol for patients undergoing cardiac surgery with CPB.

Incidence and predictive factors of hypoglycemia after pheochromocytoma resection.

OBJECTIVES: To determine the incidence and preoperative risk factors of post-excisional hypoglycemia in patients undergoing pheochromocytoma resection. METHODS: Patients who underwent surgical resection of pheochromocytoma at a single institution were retrospectively enrolled in the present study. The primary end-point was the development of post-excisional hypoglycemia; that is, a serum glucose level <70 mg/dL. The serum levels of immunoreactive insulin and glucose levels during the preoperative oral glucose-tolerance test and surgery were analyzed to elucidate the mechanism of hypoglycemia. RESULTS: A total of 49 patients underwent surgical resection of pheochromocytoma, of which 21 patients (43%) developed post-excisional hypoglycemia. The incidence of hypoglycemia was not statistically different between patients with adrenal tumors and those with extra-adrenal tumors (18/41 [44%] vs 3/8 [38%], respectively, P = 0.73). There was no difference in the immunoreactive insulin/glucose ratio during the preoperative oral glucose-tolerance test between patients with and those without post-excisional hypoglycemia. The intraoperative immunoreactive insulin/glucose ratio was significantly higher in patients with hypoglycemia than in those without hypoglycemia. A higher 24-h urinary epinephrine level, but not norepinephrine level, was a predictive factor for post-excisional hypoglycemia. CONCLUSIONS: Post-excisional hypoglycemia is a frequent complication of pheochromocytoma resection, irrespective of the tumor location, and might be common in patients with epinephrine-predominant tumors. All patients undergoing resection of adrenal and extra-adrenal pheochromocytoma require intensive monitoring of serum glucose levels during and after surgery.

Cytotoxicity of propofol in human induced pluripotent stem cell-derived cardiomyocytes.

PURPOSE: Propofol infusion syndrome (PRIS) is a lethal condition caused by propofol overdose. Previous studies suggest that pathophysiological mechanisms underlying PRIS involve mitochondrial dysfunction; however, these mechanisms have not been fully elucidated. This study aimed to establish an experimental model of propofol-induced cytotoxicity using cultured human induced pluripotent stem cell (iPSC)-derived cardiomyocytes to determine the mechanisms behind propofol-induced mitochondrial dysfunction, and to evaluate the protective effects of coenzyme Q10 (CoQ10). METHODS: Human iPSC-derived cardiomyocytes were exposed to propofol (0, 2, 10, or 50 µg/ml) with or without 5 µM CoQ10. Mitochondrial function was assessed by measuring intracellular ATP, lactate concentrations in culture media, NAD+/NADH ratio, and the mitochondrial membrane potential. Propofol-induced cytotoxicity was evaluated by analysis of cell viability. Expression levels of genes associated with mitochondrial energy metabolism were determined by PCR. Intracellular morphological changes were analyzed by confocal microscopy. RESULTS: Treatment with 50 µg/ml propofol for 48 h reduced cell viability. High concentrations of propofol (≥ 10 µg/ml) induced mitochondrial dysfunction accompanied by downregulation of gene expression of PGC-1alpha and its downstream targets (NDUFS8 and SDHB, which are involved in the respiratory chain reaction; and CPT1B, which regulates beta-oxidation). Cardiomyocytes co-treated with 5 µM CoQ10 exhibited resistance to propofol-induced toxicity through recovery of gene expression. CONCLUSIONS: Propofol-induced cytotoxicity in human iPSC-derived cardiomyocytes may be associated with mitochondrial dysfunction via downregulation of PGC-1alpha-regulated genes associated with mitochondrial energy metabolism. Co-treatment with CoQ10 protected cardiomyocytes from propofol-induced cytotoxicity.

Polymyxin B hemoperfusion prevents acute kidney injury in sepsis model.

BACKGROUND: Direct hemoperfusion with a polymyxin B-immobilized column (PMX-DHP) adsorbs endotoxin and has been used for the treatment of septic shock. Yet, the mechanisms by which PMX-DHP acts on acute kidney injury are only partially understood. MATERIALS AND METHODS: Rats were anesthetized, tracheostomized, and placed on mechanical ventilation. The animals were randomized to three groups: a cecal ligation and puncture (CLP) + dummy-DHP group (n = 10), a CLP + PMX-DHP group (n = 10), and a sham group (n = 4). Four hours after CLP, a dummy-DHP or PMX-DHP was performed for 1 h. The heart rate, mean arterial pressure, arterial blood gases, and plasma concentrations of creatinine, lactate, potassium, interleukin (IL)-6, and IL-10 were measured at 0 h and 8 h. Eight hours after CLP, the kidney was harvested, and histopathologic examination was performed. The expressions of cleaved poly (ADP-ribose) polymerase (PARP) and nuclear factor (NF)-κB p65 were examined by immunohistochemistry. A terminal deoxynucleotide transferase dUTP nick-end labeling assay was performed to detect apoptotic nuclei in kidney sections. RESULTS: PMX-DHP maintained hemodynamics and the acid-base balance and significantly (P < 0.05) decreased the plasma concentrations of lactate, creatinine, potassium, IL-6, and IL-10 compared with dummy-DHP. PMX-DHP significantly (P < 0.001) attenuated the expressions of cleaved PARP and NF-κB p65 in renal tubular cells and renal tubular cell apoptosis compared with dummy-DHP. CONCLUSIONS: These findings suggest that PMX-DHP may protect against acute kidney injury not only by inhibiting the NF-κB signaling pathway but also by preventing renal tubular cell apoptosis.

[How to establish and organize research activities].

The goal of this article is to introduce how to establish and organize research activities. There are many questions to be answered to make our clinical levels higher and more sophisticated, and some of them may also trigger research activities. We have two options, clinical study and basic research, and combination of these two methods is an ideal way to find answers to these questions. Also, cutting edge methodologies often result in some breakthroughs in our research field. Collaboration with experts is helpful for application of these new methodologies, and it facilitates establishing network of researchers. Finally, based on results of many excellent studies (both basic and clinical), we design large scale randomized clinical trials, to verify whether these research products contribute to improvement in our clinical activity. Excellent results give some hints to a next series of studies, and in this context, reviewing and updating the achievements are also important. Organizing study groups facilitates these activities, and the Japanese Society of Anesthesiologists has recently started a project to activate research activity.

Elevated levels of angiopoietin-2 as a biomarker for respiratory failure after cardiac surgery.

OBJECTIVES: Angiopoietin-1 and angiopoietin-2 are important factors in regulating endothelial vascular permeability. This study evaluated perioperative changes in serum levels of angiopoietin-1 and -2 in patients undergoing cardiac surgery. DESIGN: Measurement of serum levels of angiopoietin-1 and angiopoietin-2 in samples collected during a previously conducted prospective, multicenter, observational study. SETTING: Three university hospitals. PARTICIPANTS: Eighty-four adult patients undergoing cardiac surgery. INTERVENTION: Serum levels of angiopoietins were measured at baseline, immediately after surgery, and the day after surgery (POD-1). MEASUREMENTS AND MAIN RESULTS: Serum levels of angiopoietin-2 were elevated by POD-1 (median 3.3 ng/mL, interquartile range [IQR] 2.5-4.6 ng/mL) compared with baseline (median 1.6 ng/mL, IQR 1.3-2.1 ng/mL, p < 0.0001), and angiopoietin-1 levels were decreased immediately after surgery (baseline median 23.2 ng/mL, IQR 10.2-32.8 ng/mL; postoperative median 8.0 ng/mL, IQR 1.5-13.2 ng/mL, p<0.0001). Angiopoietin-2 levels on POD-1 in patients undergoing off-pump coronary artery bypass grafting were significantly lower than those in patients undergoing aortic surgery (p = 0.0009) and valve surgery (p = 0.008). Angiopoietin-2 levels on POD-1 had a predictive performance of the area under the curve (AUC) of the receiver operating characteristic curve 0.74 for mechanical ventilation>3 days. Angiopoietin-1 levels and the angiopoietin-2/angiopoietin-1 ratio showed lower predictive performance (AUC values 0.58 and 0.68, respectively). CONCLUSIONS: Angiopoietin-2 serum levels were elevated after cardiac surgery. Elevated angiopoietin-2 had a good predictive performance for respiratory failure after cardiac surgery, perhaps reflecting the severity of lung dysfunction related to postoperative increases in vascular permeability.

[Potential influence of pre and intraoperative factors on postoperative recurrence and survival in patients undergoing radical resection of esophageal cancer].

BACKGROUND: Several papers report that preoperative and intraoperative factors influence postoperative recurrence of malignancy. The purpose of this study is to define which factors affect the recurrence and survival of patients after the surgical resection of the esophageal cancer. METHODS: Ninety five patients underwent complete elective resection of the esophageal cancer. All patients were without preoperative chemotherapy and radiotherapy. We extracted 12 parameters, and cox regression analyses were used to assess the relation of 12 factors and the outcomes of patients. The 12 factors included preoperative factors (age, sex, weight stage of cancer, ASA PS, serum creatinine and total bilirubin), intraoperative variables (duration of anesthesia, blood transfusion, fluid balance, hypotensive episodes) and surgical Apgar score. Hypotensive episodes were defined as the systolic pressure lower than 70 mmHg occurring from the introduction of anesthesia to the end of anesthesia. RESULTS: Hypotensive episodes and blood transfusion significantly affected 1 year cancer specific survival. Stage of cancer and blood transfusion affected 5 year cancer specific survival. CONCLUSIONS: We found that intraoperative hypotension affected 1-year cancer specific survival; however, the stage of cancer affected long-term survival instead of intraoperative factors. A low 5-year survival rate in esophageal cancer may have affected this result.