Detection of Active SARS-CoV-2 3CL Protease in Infected Cells Using Activity-Based Probes with a 2,6-Dichlorobenzoyloxymethyl Ketone Reactive Warhead.

The 3CL protease (3CLpro) is a viral cysteine protease of SARS-CoV-2 and is responsible for the main processing of the viral polyproteins involved in viral replication and proliferation. Despite the importance of 3CLpro as a drug target, the intracellular dynamics of active 3CLpro, including its expression and subcellular localization in SARS-CoV-2-infected cells, are poorly understood. Herein, we report an activity-based probe (ABP) with a clickable alkyne and an irreversible warhead for the SARS-CoV-2 3CL protease. We designed and synthesized two ABPs that contain a chloromethyl ketone (probe 2) or 2,6-dichlorobenzoyloxymethyl ketone (probe 3) reactive group at the P1' site. Labeling of recombinant 3CLpro by the ABPs in the purified and proteome systems revealed that probe 3 displayed ligand-directed and selective labeling against 3CLpro. Labeling of transiently expressed active 3CLpro in COS-7 cells also validated the good target selectivity of probe 3 for 3CLpro. We finally demonstrated that endogenously expressed 3CLpro in SARS-CoV-2-infected cells can be detected by fluorescence microscopy imaging using probe 3, suggesting that active 3CLpro at 5 h postinfection is localized in the juxtanuclear region. To the best of our knowledge, this is the first report investigating the subcellular localization of active 3CLpro by using ABPs. We believe that probe 3 will be a useful chemical tool for acquiring important biological knowledge of active 3CLpro in SARS-CoV-2-infected cells.

Di-n-butyl phthalate promotes the neural differentiation of mouse embryonic stem cells through neurogenic differentiation 1.

An understanding of the risk of gene deletion and mutation posed by endocrine-disrupting chemicals (EDCs) is necessary for the identification of etiological reagents for many human diseases. Therefore, the characterization of the genetic traits caused by developmental exposure to EDCs is an important research subject. A new regenerative approach using embryonic stem cells (ESCs) holds promise for the development of stem-cell-based therapies and the identification of novel therapeutic agents against human diseases. Here, we focused on the characterization of the genetic traits and alterations in pluripotency/stemness triggered by phthalate ester derivatives. Regarding their in vitro effects, we reported the abilities of ESCs regarding proliferation, cell-cycle control, and neural ectoderm differentiation. The expression of their stemness-related genes and their genetic changes toward neural differentiation were examined, which led to the observation that the tumor suppressor gene product p53/retinoblastoma protein 1 and its related cascades play critical functions in cell-cycle progression, cell death, and neural differentiation. In addition, the expression of neurogenic differentiation 1 was affected by exposure to di-n-butyl phthalate in the context of cell differentiation into neural lineages. The nervous system is one of the most sensitive tissues to exposure to phthalate ester derivatives. The present screening system provides a good tool for studying the mechanisms underlying the effects of EDCs on the developmental regulation of humans and rodents, especially on the neuronal development of ESCs.

Uniformly positive or negative correlation of cerebral gray matter regions with driving safety behaviors of healthy older drivers.

This study investigated the relationship between cerebral gray matter (GM) regions and driving safety behaviors (DSBs) of 98 older drivers without dementia (mean age, 77.72 ± 3.677 years). Their DSBs were evaluated on actual vehicles running on a closed-circuit course. The DSB was scored in six categories: DSB1, visual search behavior; DSB2, speeding; DSB3, signaling of the indicator; DSB4, vehicle stability; DSB5, positioning; and DSB6, steering. The scores were calculated by a single driving instructor; larger scores indicated safer driving performances. Regional GM volumes were measured with voxel-based morphometry by magnetic resonance imaging (MRI). Out of 56 GM regions, 18 were correlated with DSB categories except for DSB4. When a single GM region was correlated with multiple DSB categories, a positive or negative response was uniformly determined for the respective region despite clear differences in the DSB categories. This result suggests the possible existence of two contradictory mechanisms in the brain for DSB. The left postcentral gyrus may largely function in regulating DSBs because it was negatively correlated with five of six DSB categories. Thus, MRI's measurement of regional GM volumes may help deepen the understanding of the diversity and complexity inherent in brain functions for DSBs.

Resistance exercise in combination with aerobic exercise reduces the incidence of serious events in patients with liver cirrhosis: a meta-analysis of randomized controlled trials.

BACKGROUND: Exercise, particularly resistance exercise, is beneficial for sarcopenia in patients with liver cirrhosis. However, the effects of exercise on events remain unclear. We aimed to examine the effects of exercise on serious events in patients with liver cirrhosis using a meta-analysis of randomized controlled trials (RCTs). METHODS: A literature search was conducted in 2022. Eleven RCTs were selected for the meta-analysis (exercise group, n = 232; control group, n = 193). Serious events were defined as death or serious complications according to the original articles. A meta-analysis was performed using a random-effects model. The primary outcome was the incidence of serious events. RESULTS: In the 11 RCTs, the incidence of serious events was 5.6% (13/232) and 12.3% (24/193) in the exercise and control groups, respectively. However, a meta-analysis demonstrated no significant difference in the incidence of serious events between the two groups (risk difference [RD] - 0.03, 95% confidence intervals (CI) - 0.07 to 0.02). In a stratification analysis based on a combination of aerobic and resistance exercise, five RCTs (n = 185) were enrolled. The incidence of serious events was 6.25% (7/112) and 24.7% (18/73) in the combination exercise and control groups, respectively. A meta-analysis demonstrated a significant reduction in the incidence of serious events in the combination exercise group compared with the control group (RD - 0.12; 95% CI - 0.21 to - 0.03). CONCLUSIONS: Resistance exercise in combination with aerobic exercise reduces serious events in patients with liver cirrhosis. A combination of aerobic and resistance exercise may be beneficial to improve the prognosis of patients with liver cirrhosis.

Network-based diagnostic probability estimation from resting-state functional magnetic resonance imaging.

Brain functional connectivity is a useful biomarker for diagnosing brain disorders. Connectivity is measured using resting-state functional magnetic resonance imaging (rs-fMRI). Previous studies have used a sequential application of the graphical model for network estimation and machine learning to construct predictive formulas for determining outcomes (e.g., disease or health) from the estimated network. However, the resulting network had limited utility for diagnosis because it was estimated independent of the outcome. In this study, we proposed a regression method with scores from rs-fMRI based on supervised sparse hierarchical components analysis (SSHCA). SSHCA has a hierarchical structure that consists of a network model (block scores at the individual level) and a scoring model (super scores at the population level). A regression model, such as the multiple logistic regression model with super scores as the predictor, was used to estimate diagnostic probabilities. An advantage of the proposed method was that the outcome-related (supervised) network connections and multiple scores corresponding to the sub-network estimation were helpful for interpreting the results. Our results in the simulation study and application to real data show that it is possible to predict diseases with high accuracy using the constructed model.

A prospective randomized study of multimodal analgesia combined with single injection transversus abdominis plane block versus epidural analgesia against postoperative pain after laparoscopic colon cancer surgery.

PURPOSE: Transversus abdominis plane (TAP) block is a safe, effective, and promising analgesic procedure, but TAP block only cannot overcome postoperative pain. We conducted a prospective randomized study to evaluate postoperative pain control using multimodal analgesia (MA) combined with a single injection TAP block compared with epidural analgesia (EA) after laparoscopic colon cancer surgery. METHODS: Sixty-seven patients scheduled for elective laparoscopic colon cancer surgery were enrolled in this study and randomized into EA and MA groups. The primary endpoint was the frequency of additional analgesic use until postoperative day (POD) 2. The VAS score, blood pressure, time to bowel movement, time to mobilization, postoperative complications, and length of hospital stay were also compared between the two groups. RESULTS: Sixty-four patients (EA group, n = 33; MA group, n = 31) were analyzed. The patient characteristics did not differ markedly between the two groups. The frequency of additional analgesic use was significantly lower in the MA group than in the EA group (P < 0.001), whereas the VAS score did not differ markedly between the two groups. The postoperative blood pressure on the day of surgery was significantly lower in the MA group than in the EA group (P = 0.016), whereas urinary retention was significantly higher in the EA group than in the MA group (P < 0.001). CONCLUSION: MA combined with a single injection TAP block after laparoscopic colon cancer surgery may be comparable to EA in terms of analgesia and superior to EA in terms of urinary retention.

A prospective cohort study of periostin as a serum biomarker in patients with idiopathic pulmonary fibrosis treated with nintedanib.

This study investigated the utility of periostin, a matricellular protein, as a prognostic biomarker in patients with idiopathic pulmonary fibrosis (IPF) who received nintedanib. Monomeric and total periostin levels were measured by enzyme-linked immunosorbent assay in 87 eligible patients who participated in a multicenter prospective study. Forty-three antifibrotic drug-naive patients with IPF described in previous studies were set as historical controls. Monomeric and total periostin levels were not significantly associated with the change in forced vital capacity (FVC) or diffusing capacity of the lungs for carbon monoxide (DLCO) during any follow-up period. Higher monomeric and total periostin levels were independent risk factors for overall survival in the Cox proportional hazard model. In the analysis of nintedanib effectiveness, higher binarized monomeric periostin levels were associated with more favorable suppressive effects on decreased vital capacity (VC) and DLCO in the treatment group compared with historical controls. Higher binarized levels of total periostin were associated with more favorable suppressive effects on decreased DLCO but not VC. In conclusion, higher periostin levels were independently associated with survival and better therapeutic effectiveness in patients with IPF treated with nintedanib. Periostin assessments may contribute to determining therapeutic strategies for patients with IPF.

Noninvasive Neurally Adjusted Ventilatory Assist in Infants With Bronchiolitis: Respiratory Outcomes in a Single-Center, Retrospective Cohort, 2016-2018.

OBJECTIVES: To describe our experience of using noninvasive neurally adjusted ventilatory assist (NIV-NAVA) in infants with bronchiolitis, its association with the evolution of respiratory effort, and PICU outcomes. DESIGN: Retrospective analysis of a prospectively curated, high-frequency electronic database. SETTING: A PICU in a university-affiliated maternal-child health center in Canada. PATIENTS: Patients younger than 2 years old who were admitted with a diagnosis of acute bronchiolitis and treated with NIV-NAVA from October 2016 to June 2018. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Patient characteristics, as well as respiratory and physiologic parameters, including electrical diaphragmatic activity (Edi), were extracted from the electronic database. Respiratory effort was estimated using the modified Wood Clinical Asthma Score (mWCAS) and the inspiratory Edi. A comparison in the respiratory effort data was made between the 2 hours before and 2 hours after starting NIV-NAVA. In the two seasons, 64 of 205 bronchiolitis patients were supported with NIV-NAVA. These 64 patients had a median (interquartile range [IQR]) age of 52 days (32-92 d), and there were 36 of 64 males. Treatment with NIV-NAVA was used after failure of first-tier noninvasive respiratory support; 25 of 64 patients (39%) had at least one medical comorbidity. NIV-NAVA initiation was associated with a moderate decrease in mWCAS from 3.0 (IQR, 2.5-3.5) to 2.5 (IQR, 2.0-3.0; p < 0.001). NIV-NAVA initiation was also associated with a statistically significant decrease in Edi (p < 0.01). However, this decrease was only clinically relevant in infants with a 2-hour baseline Edi greater than 20 µV; here, the before and after Edi was 44 µV (IQR, 33-54 µV) compared with 27 µV (IQR, 21-36 µV), respectively (p < 0.001). Overall, six of 64 patients (9%) required endotracheal intubation. CONCLUSIONS: In this single-center retrospective cohort, in infants with bronchiolitis who were considered to have failed first-tier noninvasive respiratory support, the use of NIV-NAVA was associated with a rapid decrease in respiratory effort and a 9% intubation rate.

Medical equipment that improve safety and outcomes of inter-facility transportation of critically ill patients: A systematic review.

BACKGROUND: Although many critically ill patients require inter-facility transport for definitive or specialized therapy, the medical equipment required to enhance transport safety remains unclear. This review was performed to summarize the evidence regarding devices used to improve the safety and survival in patients requiring such transport. METHODS: We searched MEDLINE, the Cochrane Central Register of Controlled Trials, and Igaku Chuo Zasshi for randomized controlled trials and observational studies comparing outcomes according to the presence or absence of devices (or new vs conventional devices) during transfer of critically ill patients. RESULTS: Four studies focusing on continuous blood pressure monitoring, extracorporeal membrane oxygenation, pelvic circumferential compression devices, and cuffed tracheal tubes, respectively, met the inclusion criteria. A meta-analysis was not performed because the 4 studies focused on different devices. Near-continuous blood pressure monitoring increased interventions such as intravenous fluid administration during transport, shortened the intensive care unit and hospital lengths of stay, and reduced the incidence of multiple-organ failure compared with use of oscillometric devices. Despite the small sample size and varying severity of illness among the groups, transport of patients with severe respiratory failure under extracorporeal membrane oxygenation resulted in fewer hypoxemic events during transport than transport on conventional ventilators. During transport of patients with pelvic fractures, pelvic belts may help to reduce mortality and the transfusion volume. Cuffed (vs uncuffed) tracheal tubes may reduce post-transport tube replacement events in pediatric patients. CONCLUSION: Studies on devices needed for inter-facility transport of critically ill patients are scarce, but some devices may be beneficial.

Histological validation of atrial structural remodelling in patients with atrial fibrillation.

BACKGROUND AND AIMS: This study aimed to histologically validate atrial structural remodelling associated with atrial fibrillation. METHODS AND RESULTS: Patients undergoing atrial fibrillation ablation and endomyocardial atrial biopsy were included (n = 230; 67 ± 12 years old; 69 women). Electroanatomic mapping was performed during right atrial pacing. Voltage at the biopsy site (Vbiopsy), global left atrial voltage (VGLA), and the proportion of points with fractionated electrograms defined as ≥5 deflections in each electrogram (%Fractionated EGM) were evaluated. SCZtotal was calculated as the total width of slow conduction zones, defined as regions with a conduction velocity of <30 cm/s. Histological factors potentially associated with electroanatomic characteristics were evaluated using multiple linear regression analyses. Ultrastructural features and immune cell infiltration were evaluated by electron microscopy and immunohistochemical staining in 33 and 60 patients, respectively. Fibrosis, intercellular space, myofibrillar loss, and myocardial nuclear density were significantly associated with Vbiopsy (P = .014, P < .001, P < .001, and P = .002, respectively) and VGLA (P = .010, P < .001, P = .001, and P < .001, respectively). The intercellular space was associated with the %Fractionated EGM (P = .001). Fibrosis, intercellular space, and myofibrillar loss were associated with SCZtotal (P = .028, P < .001, and P = .015, respectively). Electron microscopy confirmed plasma components and immature collagen fibrils in the increased intercellular space and myofilament lysis in cardiomyocytes, depending on myofibrillar loss. Among the histological factors, the severity of myofibrillar loss was associated with an increase in macrophage infiltration. CONCLUSION: Histological correlates of atrial structural remodelling were fibrosis, increased intercellular space, myofibrillar loss, and decreased nuclear density. Each histological component was defined using electron microscopy and immunohistochemistry studies.

Systematic Surveillance of an Emerging Picornavirus among Cattle and Sheep in China.

Emerging viruses are a constant threat to human and animal health. Boosepivirus is a novel picornavirus considered a gastrointestinal pathogen and has broken out in recent years. In 2020, we identified a strain of boosepivirus NX20-1 from Chinese calf feces and performed genetic characterization and evolutionary analysis. NX20-1 was closely related to the Japanese strain Bo-12-38/2009/JPN and belonged to Boosepivirus B. We found that 64 of 603 samples (10.6%) from 20 different provinces across the country were positive for boosepivirus by reverse transcription (RT)-PCR. Further, coinfection with other diarrheal pathogens was also present in 35 of these positive samples. Importantly, we found the prevalence of boosepivirus in sheep as well, indicating that Boosepivirus can infect different domestic animals. Our data suggest that boosepivirus is a potential diarrheal pathogen, but the pathogenicity and the mechanism of pathogenesis need further study. IMPORTANCE We identified a novel picornavirus, boosepivirus, for the first time in China. Genetic evolutionary analysis revealed that NX20-1 strain was closely related to the Japanese strain Bo-12-38/2009/JPN and belonged to Boosepivirus B. In addition, we found that the virus was prevalent in China with an overall positivity rate of 10.6% (64 of 603 samples), and there was significant coinfection with other pathogens. Importantly, we found the prevalence of boosepivirus in sheep as well, suggesting that boosepivirus has a risk of spillover and can be transmitted across species.

Efficacy and safety of once-daily single-inhaler triple therapy for mild-to-moderate chronic obstructive pulmonary disease: a study protocol for a randomised and interventional study.

INTRODUCTION: Bronchodilators, including long-acting muscarinic antagonists (LAMA) and long-acting beta 2 agonists (LABA), are the main treatments for chronic obstructive pulmonary disease (COPD). The efficacy of triple therapy (inhaled corticosteroids/LAMA/LABA) has also been reported. However, the effect of triple therapy on patients with mild-to-moderate COPD has not yet been clarified. This study aims to investigate the safety and efficacy of triple therapy, compared with LAMA/LABA combination therapy, for lung function and health-related quality of life in patients with mild-to-moderate COPD and identify baseline characteristics and biomarkers to predict responders and non-responders to triple therapy. METHODS AND ANALYSIS: This is a multicentre, prospective, open-label, randomised, parallel-group study. Mild-to-moderate patients with COPD will be randomised to receive fluticasone furoate/umeclidinium/vilanterol or umeclidinium/vilanterol for 24 weeks. A total of 668 patients will be enrolled from March 2022 to September 2023 from 38 sites in Japan. The primary endpoint is the change in the trough forced expiration volume in 1 s after 12 weeks of treatment. Secondary endpoints are responder rates based on the COPD assessment test score and the St. George's Respiratory Questionnaire total score after 24 weeks of treatment. The safety endpoint is the occurrence of any adverse events. We will also investigate safety in terms of changes in microbial colonisation in sputum and antimycobacterium avium complex antibodies. ETHICS AND DISSEMINATION: The study protocol and informed consent documents were approved by the Saga University Clinical Research Review Board (approval number: CRB7180010). Written informed consent will be obtained from all patients. Recruitment of the patients began in March 2022. The results will be disseminated through scientific peer-reviewed publications and domestic and international medical conferences. TRIAL REGISTRATION NUMBERS: UMIN000046812 and jRCTs031190008.

Hemodynamic assessment in children after cardiac surgery: A pilot study on the value of infrared thermography.

Introduction: Low cardiac output syndrome in the postoperative period after cardiac surgery leads to an increase in tissue oxygen extraction, assessed by the oxygen extraction ratio. Measurement of the oxygen extraction ratio requires blood gases to be taken. However, the temperature of the skin and various parts of the body is a direct result of blood flow distribution and can be monitored using infrared thermography. Thus, we conducted a prospective clinical study to evaluate the correlation between the thermal gradient obtained by infrared thermography and the oxygen extraction ratio in children at risk for low cardiac output after cardiac surgery. Methods: Children aged 0 to 18 years, having undergone cardiac surgery with cardio-pulmonary bypass in a pediatric intensive care unit were included in the study. One to 4 thermal photos were taken per patient using the FLIR One Pro thermal imaging camera. The thermal gradient between the central temperature of the inner canthus of the eye and the peripheral temperature was compared to the concomitant oxygen extraction ratio calculated from blood gases. Results: 41 patients were included with a median age of 6 months (IQR 3-48) with median Risk Adjustment for Congenital Heart Surgery-1 score was 2 (IQR 2-3). Eighty nine thermal photos were analyzed. The median thermal gradient was 2.5 °C (IQR 1,01-4.04). The median oxygen extraction ratio was 35% (IQR 26-42). Nine patients had an oxygen extraction ratio ≥ 50%. A significant but weak correlation was found between the thermal gradient and the oxygen extraction ratio (Spearman's test p = 0.25, p = 0.016). Thermal gradient was not correlated with any other clinical or biologic markers of low cardiac output. Only young age was an independent factor associated with an increase in the thermal gradient. Conclusion: In this pilot study, which included mainly children without severe cardiac output decrease, a significant but weak correlation between thermal gradient by infrared thermography and oxygen extraction ratio after pediatric cardiac surgery was observed. Infrared thermography is a promising non-invasive technology that could be included in multimodal monitoring of postoperative cardiac surgery patients. However, a clinical trial including more severe children is needed.

Usefulness of blood biomarkers for predicting venous thromboembolism in Japanese patients with cancer.

Prospective studies on risk factors for the occurrence of venous thromboembolism (VTE) in Asian patients with cancer are limited. Therefore, the present study assessed risk factors for VTE, including multiple blood biomarkers and risk scores consisting of several risk factors, in Japanese patients receiving anticancer drug therapy. In this single-center, prospective, observational study, 200 patients with six types of cancer were enrolled and followed for 1 year to observe the occurrence of symptomatic or asymptomatic VTE. The present study evaluated risk factors, Khorana and Vienna cancer and thrombosis study (CATS) scores at enrollment, and longitudinal data on various blood biomarkers. A Vienna CATS score of ≥3 was significantly associated with VTE occurrence (HR, 2.8; 95% CI, 0.9-8.7; P=0.045). In multivariable analysis, there was a significant association between VTE and the presence of pancreatic cancer (HR, 3.2; 95% CI, 1.1-8.8; P=0.028) and high soluble fibrin (HR, 3.7; 95% CI, 1.1-7.8; P=0.036). Covariate analysis using the propensity score also showed a significant association with hemoglobin dichotomized at <100 g/l (HR, 3.9; 95% CI, 1.1-14.0; P=0.034). Longitudinal data indicated that VTE was associated with soluble fibrin baseline values and an increase in D-dimer levels over time. The present results suggested that blood biomarkers are beneficial for predicting the risk of VTE in Japanese patients with cancer. The present study also provided novel evidence for the importance of measuring soluble fibrin in patients with cancer.

[Endoscopic variceal ligation and percutaneous transhepatic obliteration difficulty in esophageal variceal bleeding: a case report].

A 48-year-old male patient with a history of alcoholic cirrhosis was admitted to our hospital due to hematemesis with a 7-day history of melena. Emergency esophagogastroduodenoscopy revealed esophageal variceal bleeding. We attempted hemostasis with endoscopic variceal ligation (EVL). The esophageal mucosa was not aspirated into the EVL device although the patient had no history of endoscopic injection sclerotherapy or EVL. Percutaneous transhepatic obliteration (PTO) was performed and esophageal variceal bleeding was successfully hemostasis. PTO is a viable option for refractory esophageal bleeding.

Microenvironmental elements singularity synergistically regulate the behavior and chemosensitivity of endometrioid carcinoma.

The importance of the microenvironment is widely recognized as it regulates not only malignant cell behavior but also drug sensitivity. The cancer cell microenvironment is composed of biological, physical and chemical elements, and simultaneous reproduction of these three elements are important conditions investigated in cancer research. In the present study, we focused on the epidemiological and anatomical specificities of endometrioid carcinoma, obesity (biological), fluid flow (physical) and anticancer agents (chemical) to target the specific microenvironmental elements of endometrioid carcinoma. To elucidate the individual effects of these elements on endometrioid carcinoma and to investigate the relationships between these factors, we developed an adipose tissue fragments (ATFs)-embedded cell disc under a rotational culture method to generate carcinoma-stroma interactions and to create fluid flow. ATFs and fluid flow individually or synergistically influenced proliferative cellular behavior and the morphological changes underlying endometrioid carcinoma. ATFs and fluid flow also governed the expression of extracellular signal-regulated kinase and p38 signaling synergistically or individually, depending on the endometrioid carcinoma cell type. Adipose tissue induced chemoresistance to cis-diamminedichloro-platinum (CDDP) in endometrioid cancer, but the resistance effect was abolished by fluid flow. Thus, a simple reconstructed model was established to investigate three elements of the microenvironment of endometrioid carcinoma in vitro. This culture model unequivocally demonstrated the individual and synergistic effects of the three elements on endometrioid carcinoma. This new culture model is a promising tool for elucidating the mechanisms underlying endometrioid carcinoma and for developing further treatment strategies.

Zinc binding of a Cys2His2-type zinc finger protein is enhanced by the interaction with DNA.

Zinc finger proteins specifically recognize DNA sequences and, therefore, play a crucial role in living organisms. In this study the Zn(II)-, and DNA-binding of 1MEY#, an artificial zinc finger protein consisting of three finger units was characterized by multiple methods. Fluorimetric, circular dichroism and isothermal calorimetric titrations were applied to determine the accurate stability constant of a zinc finger protein. Assuming that all three zinc finger subunits behave identically, the obtained thermodynamic data for the Zn(II) binding were ΔHbinding site = - (23.5 - 28.0) kcal/mol (depending on the applied protonation state of the cysteines) and logß'pH 7.4 = 12.2 ± 0.1, being similar to those of the CP1 consensus zinc finger peptide. The specific DNA binding of the protein can be characterized by logß'pH 7.4 = 8.20 ± 0.08, which is comparable to the affinity of the natural zinc finger proteins (Sp1, WT1, TFIIIA) toward DNA. This value is ~ 1.9 logß' unit higher than those determined for semi- or nonspecific DNA binding. Competitive circular dichroism and electrophoretic mobility shift measurements revealed that the conditional stability constant characteristic for Zn(II) binding of 1MEY# protein increased by 3.4 orders of magnitude in the presence of its target DNA sequence.

Regional variation of pediatric interhospital critical care transport in Japan.

BACKGROUND: An in-depth understanding of the epidemiology of ill or injured children transported between hospitals is crucial in building regional medical transport services in public health. Although the epidemiological situation varies by nation and region, it has not been well documented in Japan. In this report we described the number of pediatric interhospital transportations and examined the regional variations and trends in the recent decade. METHODS: We performed repeated, cross-sectional analyses of children (<15 years) undergoing interhospital transportation in 2010, 2013, and 2016-19, using the national database of public ambulances of the Fire and Disaster Management Agency in Japan. We stratified the cases into critical care transport (CCT) or non-critical care transport (NonCCT) by the illness/injury severity of the transported children. We calculated the national population-adjusted number of CCTs and described prefectural variations in CCT numbers with analytical thinking. RESULTS: There were 23,506 CCTs and 138,347 NonCCTs. The national average of population-adjusted CCT numbers was 255 per 1,000,000 person-years. The statistics varied by prefectures, ranging from 25-536 per 1,000,000 person-years. The annual trends were also diverse across prefectures, increasing in nine, decreasing in six, and static in 31 prefectures. In analytical thinking of regional variations, potential contributing factors included available interhospital transport services and the threshold of direct admission and referral to tertiary-care hospitals, whereas regional variations were not well associated with geographical patterns or population size. CONCLUSIONS: Public ambulance services were substantially used for CCTs and NonCCTs in Japan. Regional variations should be taken into account for the future policymaking on pediatric interhospital transportation.

Dual targeting of aberrant DNA and histone methylation synergistically suppresses tumor cell growth in ATL.

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature CD4+ T cells caused by human T-cell lymphotropic virus type 1 (HTLV-1)-induced T-cell transformation. After infection with HTLV-1, it takes several decades for HTLV-1 carriers to develop ATL. The prognosis of ATL remains poor despite several new agents being approved in the last few years. Recently, it has been noted that epigenetic abnormalities, both DNA methylation and trimethylation at histone H3Lys27 (H3K27me3), contribute to ATL leukemogenesis. Here, we investigated the effect of combination treatment with DNA demethylating agents (azacitidine [AZA], decitabine (DAC), and OR-2100 (OR21), which is a silylated derivative of DAC) and inhibitors of enhancer of zeste homolog 2 (EZH2) (EPZ-6438 and DS-3201b), which catalyze trimethylation of H3K27, in ATL. The combination of DAC and OR21 but not AZA with EZH inhibitors exhibited synergistic anti-ATL effects in vitro and in vivo, concomitant with DNA demethylation and reduction of H3K27me3. The combination induced gene expression reprogramming. Dual-specificity phosphatase 5 (DUSP5), an extracellular signal-regulated kinase (ERK)-specific phosphatase, was identified as a key molecule that mediated the inhibitory effect of combination treatment by inactivating the ERK signaling pathway. DUSP5 was downregulated by DNA methylation and H3K27me3 accumulation in the promoter region in HTLV-1-infected cells from patients with ATL during ATL leukemogenesis. The present results demonstrate that dual targeting of aberrant DNA and histone methylation synergistically suppresses tumor cell growth by restoring DUSP5, and that dual targeting of aberrant DNA and histone methylation is a feasible therapeutic approach for ATL.