Pooled CRISPR screening of high-content cellular phenotypes using ghost cytometry.

Recent advancements in image-based pooled CRISPR screening have facilitated the mapping of diverse genotype-phenotype associations within mammalian cells. However, the rapid enrichment of cells based on morphological information continues to pose a challenge, constraining the capacity for large-scale gene perturbation screening across diverse high-content cellular phenotypes. In this study, we demonstrate the applicability of multimodal ghost cytometry-based cell sorting, including both fluorescent and label-free high-content phenotypes, for rapid pooled CRISPR screening within vast cell populations. Using the high-content cell sorter operating in fluorescence mode, we successfully executed kinase-specific CRISPR screening targeting genes influencing the nuclear translocation of RelA. Furthermore, using the multiparametric, label-free mode, we performed large-scale screening to identify genes involved in macrophage polarization. Notably, the label-free platform can enrich target phenotypes without requiring invasive staining, preserving untouched cells for downstream assays and expanding the potential for screening cellular phenotypes even when suitable markers are absent.

Early restricted oxygen therapy after resuscitation from cardiac arrest (ER-OXYTRAC): protocol for a stepped-wedge cluster randomised controlled trial.

INTRODUCTION: Cardiac arrest is a critical condition, and patients often experience postcardiac arrest syndrome (PCAS) even after the return of spontaneous circulation (ROSC). Administering a restricted amount of oxygen in the early phase after ROSC has been suggested as a potential therapy for PCAS; however, the optimal target for arterial partial pressure of oxygen or peripheral oxygen saturation (SpO2) to safely and effectively reduce oxygen remains unclear. Therefore, we aimed to validate the efficacy of restricted oxygen treatment with 94%-95% of the target SpO2 during the initial 12 hours after ROSC for patients with PCAS. METHODS AND ANALYSIS: ER-OXYTRAC (early restricted oxygen therapy after resuscitation from cardiac arrest) is a nationwide, multicentre, pragmatic, single-blind, stepped-wedge cluster randomised controlled trial targeting cases of non-traumatic cardiac arrest. This study includes adult patients with out-of-hospital or in-hospital cardiac arrest who achieved ROSC in 39 tertiary centres across Japan, with a target sample size of 1000. Patients whose circulation has returned before hospital arrival and those with cardiac arrest due to intracranial disease or intoxication are excluded. Study participants are assigned to either the restricted oxygen (titration of a fraction of inspired oxygen with 94%-95% of the target SpO2) or the control (98%-100% of the target SpO2) group based on cluster randomisation per institution. The trial intervention continues until 12 hours after ROSC. Other treatments for PCAS, including oxygen administration later than 12 hours, can be determined by the treating physicians. The primary outcome is favourable neurological function, defined as cerebral performance category 1-2 at 90 days after ROSC, to be compared using an intention-to-treat analysis. ETHICS AND DISSEMINATION: This study has been approved by the Institutional Review Board at Keio University School of Medicine (approval number: 20211106). Written informed consent will be obtained from all participants or their legal representatives. Results will be disseminated via publications and presentations. TRIAL REGISTRATION NUMBER: UMIN Clinical Trials Registry (UMIN000046914).

Association of trabecular bone score and bone mineral apparent density with the severity of bone fragility in children and adolescents with osteogenesis imperfecta: A cross-sectional study.

Osteogenesis imperfecta (OI) is a hereditary skeletal disease characterized by bone fragility. Areal bone mineral density (BMD), evaluated by dual-energy X-ray absorptiometry (DXA), is used to assess bone brittleness. The height-adjusted BMD Z-score (BMDHAZ) is calculated in children and adolescents with OI to reduce the confounding factor of short stature. However, even with the BMDHAZ, severity evaluation in children and adolescents with OI is challenging because certain abnormalities in bone quality cannot be accurately assessed by BMD analysis. The trabecular bone scores (TBS) and bone mineral apparent density (BMAD), which represent the structural integrity of bone and bone-size-associated BMD, respectively, are associated with fracture risk. Recently, age- and sex-specific reference ranges have been reported, enabling the calculation of Z-scores for children. To evaluate which density measurements show the highest correlation with fracture risk, we analyzed the associations between the Z-scores of TBS, BMAD, and BMDHAZ, fracture rate, and genetic variants. We retrospectively reviewed 42 participants with OI aged 5 to 20 years who underwent DXA. COL1A1/2 pathogenic variants were detected in 41 of the 42 participants. In participants with nonsense and frameshift variants (n = 17) resulting in haploinsufficiency and mild phenotype, the TBS Z-score was negatively correlated with fracture rate (FR) (r = -0.50, p = 0.042). In participants with glycine substitution (n = 9) causing the severe phenotype, the BMAD Z-scores were negatively correlated with FR (r = -0.74, p = 0.022). No correlation between the BMDHAZ and FR was observed in both groups. These findings suggest that the TBS and BMAD are useful in assessing children and adolescents with OI with specific genetic variants.

Association between neonatal phototherapy and sleep: The Japan Environment and Children's Study.

This observational cohort study aimed to evaluate the association between the duration of neonatal phototherapy and sleep-and-wakefulness states at 1 month, 1.5 years, and 3 years of age. We analysed data from 77,876 infants using the Japan Environment and Children's Study, a nationwide birth cohort study. The participants were divided into three groups: no phototherapy, short phototherapy (1-24 h), and long phototherapy (>24 h). Multiple regression analysis was performed to assess the effect of phototherapy duration on infant sleep at each age after adjusting for potential risk factors. A longer duration of phototherapy was associated with a shorter sleep time over 24 h at 1 month of age (ß, -0.62; SE, -0.77 to -0.47) when compared with a shorter duration of, or no, phototherapy, following the adjustment of confounding factors. Contrastingly, the short duration group, when compared with the no phototherapy group, was associated with later sleep onset (ß, 0.04; SE, 0.00-0.08) and later sleep offset (ß, 0.05; SE, 0.01-0.09) at 1.5 years of age. We concluded that the duration of phototherapy may be transiently associated with sleep duration in infants, as emphasised by the shortening of the total sleep time per 24 h at 1 month of age.

Phototherapy and risk of developmental delay: the Japan Environment and Children's Study.

This observational cohort study aimed to examine the association between the duration of phototherapy for neonatal jaundice and the risk of developmental delay at 3 years of age using nationwide birth cohort data. Data from 76,897 infants were analyzed. We divided participants into four groups: no phototherapy, short phototherapy (1-24 h), long phototherapy (25-48 h), and very long phototherapy (> 48 h). The Japanese version of the Ages and Stages Questionnaire-3 was used to evaluate the risk of developmental delay at 3 years of age. Logistic regression analysis was performed to assess the impact of phototherapy duration on the prevalence of developmental delay. After adjustment for potential risk factors, a dose-response relationship was identified between the duration of phototherapy and Ages and Stages Questionnaire-3, and the differences were significant in four domains; odds ratio for communication delay was associated with short, long, and very long phototherapy = 1.10 (95% confidence interval 0.97-1.26), 1.32 (1.04-2.66), and 1.48 (1.11-1.98), respectively; for gross motor delay = 1.01 (0.89-1.15), 1.28 (1.03-2.58), and 1.26 (0.96-1.67); for problem solving delay = 1.13 (1.03-1.25), 1.19 (0.99-1.43), and 1.41 (1.11-1.79); and for personal social delay = 1.15 (0.99-1.32), 1.10 (0.84-1.44), and 1.84 (1.38-2.45). CONCLUSION: Longer duration of phototherapy is a predictive factor for developmental delay, making it important to avoid extended periods of phototherapy. However, whether it increases the prevalence of developmental delay remains unclear. WHAT IS KNOWN: • Phototherapy is a common treatment for neonatal jaundice, associated with both short-term and long-term complications. • However, an association between phototherapy and the prevalence of developmental delay has not been revealed in a large cohort study. WHAT IS NEW: • We identified that a long duration of phototherapy was a predictive factor for developmental delay at 3 years of age. • However, whether a long duration of phototherapy increases the prevalence of developmental delay remains unclear.

The Duration of Neonatal Phototherapy and Allergic Disorders: The Japan Environment and Children's Study.

INTRODUCTION: Currently, the association between the duration of neonatal phototherapy and the risk of allergic disorders has not been reported. This observational cohort study aimed to examine the association between allergic disorders, including food allergies, that are present before 3 years of age and the duration of phototherapy using the nationwide birth cohort data. METHODS: The Japan Environment and Children's Study was a nationwide birth cohort study. Data of 77,064 infants aged 1 year, 1.5 years, 2 years, and 3 years were analyzed. We divided the participants into three groups: no phototherapy, short phototherapy (1-24 h), and long phototherapy (>24 h) and evaluated the cumulative incidence of allergic disorders before 3 years of age, including asthma, atopic dermatitis, and food allergies. Logistic regression analysis was performed to assess the impact of phototherapy duration on the cumulative incidence of allergic disorders. RESULTS: After adjustment for potential risk factors, long phototherapy was found to be positively associated with food allergies at age 2 years (OR: 1.16; 95% CI: 1.01-1.33) and all allergic disorders at age 3 years (OR: 1.12; 95% CI: 1.01-1.24), including food allergies (OR 1.18; 95% CI: 1.04-1.35). CONCLUSION: A long duration of neonatal phototherapy was positively associated with the risk of allergic disorders, especially food allergies.

Primary malignant pericardial mesothelioma with difficult antemortem diagnosis: A case report.

BACKGROUND: Primary malignant pericardial mesothelioma (PMPM) is an extremely rare malignant tumor, and it is difficult to diagnose definitively before death. We present a case in which PMPM was diagnosed at autopsy. We consider this case to be highly suggestive and report it here. CASE SUMMARY: A 78-year-old male presented with transient loss of consciousness and falls. The transient loss of consciousness was considered to result from complications of diastolic dysfunction due to pericardial disease, fever with dehydration, and paroxysmal atrial fibrillation. Ultrasound cardiography (UCG) and computed tomography showed cardiac enlargement and high-density pericardial effusion. We considered pericardial disease to be the main pathogenesis of this case. Cardiac magnetic resonance imaging and gadolinium contrast-enhanced T1-weighted images showed thick staining inside and outside the pericardium. Pericardial biopsy was considered to establish a definitive diagnosis, but the patient and his family refused further treatment and examinations, and the patient was followed conservatively. We noticed a thickening of the pericardium and massive changes in the pericardium on UCG over time. We performed an autopsy 60 h after the patient died of pneumonia. Giemsa staining of the autopsy tissue showed an epithelial-like arrangement in the pericardial tumor, and immunostaining showed positive and negative factors for the diagnosis of PMPM. Based on these findings, the final diagnosis of PMPM was made. CONCLUSION: PMPM has a poor prognosis, and early diagnosis and treatment are important. The temporal echocardiographic findings may provide a clue for the diagnosis of PMPM.

A behavioral paradigm for measuring perceptual distances in mice.

Perceptual similarities between a specific stimulus and other stimuli of the same modality provide valuable information about the structure and geometry of sensory spaces. While typically assessed in human behavioral experiments, perceptual similarities-or distances-are rarely measured in other species. However, understanding the neural computations responsible for sensory representations requires the monitoring and often manipulation of neural activity, which is more readily achieved in non-human experimental models. Here, we develop a behavioral paradigm that enables the quantification of perceptual similarity between sensory stimuli using mouse olfaction as a model system.

Anti-spike protein antibody responses to BNT162b2 mRNA vaccine: A single-center survey in a COVID-19 non-epidemic area in Japan.

Background: There are a few reports on antibody responses after a two-dose BNT162b2 vaccination in non-epidemic areas. We evaluated this phenomenon. Methods: A total of 344 healthcare workers were vaccinated, and the serum anti-receptor-binding domain (RBD) antibody concentrations before and after two weeks following the two-dose BNT162b2 vaccination were measured using electro chemiluminescence immunoassay system. Results: Before vaccination, the antibody titers of all participants were less than 0.6 U/mL. After two doses of the BNT162b2 vaccine injection in 342 participants (2 excluded), a high seroconversion rate (99.7%) was observed. The average (±standard deviation) serum anti-RBD antibody titers were 2324 ± 1739 U/mL. Antibody levels in females and males were 2443 ± 1833 U/mL and 1908 ± 1287 U/mL, respectively (p = 0.037). Conclusion: In a non-epidemic area, two BNT162b2 doses induced a satisfactory antibody response, and the antibody concentrations in females were higher than in males.

Predictors of Opioid Resistance: An Investigation in Cancer Pain.

Appropriate use of opioid analgesics according to the World Health Organization pain relief ladder has provided pain relief to many patients with cancer pain. However, a proportion of patients fail to achieve sufficient pain relief and develop opioid resistance. Individual risk factors may relate to opioid resistance. Therefore, we conducted a historical cohort study to identify risk factors for opioid resistance and to construct an index to predict it. We investigated salient factors at the time of opioid initiation in the medical records of 233 patients. The outcome was the achievement of stable pain at 14 days after opioid introduction. We identified factors contributing to opioid resistance by multivariate analysis (p < 0.05). We created a resistance score from the regression equation of the identified factors to predict opioid resistance. Forty-nine (21.0%) patients were opioid resistant without achieving the outcome. Age, neuropathic pain, and alkaline phosphatase were extracted as significant factors for opioid resistance (p < 0.05). A resistance score was created from these factors and classified into binary values, the sensitivity was 80.6% and the negative predictive value was 91.6%. The findings suggest that the resistance score could be a sensitive predictor of opioid resistance before opioid initiation.

[Pediatric Multiple Endocrine Neoplasia Type 2B with Pheochromocytoma Diagnosed after Perforation of a Colonic Diverticulum : A Case Report].

A 12-year-old girl was found to have decending colon diverticulum perforation and retroperitoneal abscess on computed tomography (CT) carried out to determine the cause of fever and stomachache. CT-guided drainage tube placement was performed. She was suspected of having MEN2B from her specific facial appearance, Marfan-like body shape and lingual mucosa neuroma. Cervical ultrasonography and serum tumor marker revealed medullary thyroid carcinoma and metastasis to cervical lymph node. Genetic examination revealed a mutation of RET gene codon 918. Therefore, she was diagnosed as having MEN2B. Laboratory data showed elevated urinary catecholamines. Metaiodobenzylguanidine (MIBG) adrenal scintigraphy showed bilateral adrenal uptake and a definitive diagnosis of bilateral adrenal pheochromocytomas was made. Discharge from the drainage tube persisted and it was difficult to continue conservative treatment. Therefore, laparoscopic bilateral adrenalectomy and transverse colon colostomy were performed. Subsequently, total thyroidectomy and cervical lymph node dissection were performed. At five years of follow up, bilateral lung metastases were observed, but the serum calcitonin level was normal and the patient is under observation.

Primary aldosteronism due to bilateral micronodular hyperplasia and concomitant subclinical Cushing's syndrome: A case report.

BACKGROUND: Adrenal incidentaloma (AI) has been frequently encountered in the clinical setting. It has been shown that primary aldosteronism (PA) or subclinical Cushing's syndrome (SCS) are the representative causative diseases of AI. However, the coexistence of PA and SCS has been reportedly observed. Recently, we encountered a case of AI, in which PA and SCS coexisted, confirmed by histopathological examinations after a laparoscopic adrenalectomy. We believe that there were some clinical implications in the diagnosis of the present case. CASE SUMMARY: A 58-year-old man presented with lower right abdominal pain with a blood pressure of 170/100 mmHg. A subsequent computed tomography scan revealed right ureterolithiasis, which was the cause of right abdominal pain, and right AI measuring 22 mm × 25 mm. After the disappearance of right abdominal pain, subsequent endocrinological examinations were performed. Aldosterone-related evaluations, including adrenal venous sampling, revealed the presence of bilateral PA. In addition, several cortisol-related evaluations showed the presence of SCS on the right adrenal adenoma. A laparoscopic right adrenalectomy was then performed. The histopathological examination of the resected right adrenal revealed the presence of a cortisol-producing adenoma, while CYP11B2 immunoreactivity was absent in this adenoma. However, in the adjacent non-neoplastic adrenal, multiple CYP11B2-positive adrenocortical micronodules were detected, showing the presence of aldosterone-producing adrenocortical micronodules. CONCLUSION: Careful clinical and pathological examination should be performed when a patient harboring AI presents with concomitant SCS and PA.

Predictive Factors of Opioid-Induced Nausea in Cancer Patients.

Approximately 30% of patients experience nausea after initiation of opioid therapy, which can lead to poor quality of life. We aimed to identify risk factors for opioid-induced nausea at the initiation of opioid therapy by conducting a retrospective review of medical records of patients diagnosed by palliative care specialists with solid cancer and pain at the lesion site at Showa University Hospital between June 2005 and June 2011. The primary endpoint was the development of nausea grade ≥1 according to the Common Terminology Criteria for Adverse Events version 4.0 within 48 hours of initiation of opioid therapy. The median age of the 134 enrolled patients was 67.7 (range 28-95) years. Fifty-three percent were male and 44% had gastrointestinal cancer. Furthermore, 22.4% had opioid-induced nausea. Age (odds ratio (OR) 1.74; 95% confidence interval (CI), 1.13-2.69), edema (OR 5.83; 95% CI, 1.22-28.19), and gastrointestinal cancer (OR 2.61, 95% CI 1.07-6.36) were significantly associated with opioid-induced nausea. Prophylactic antiemetics were found to be ineffective.

Enhanced Pb and Zn stabilization in municipal solid waste incineration fly ash using waste fishbone hydroxyapatite.

The present research focused on evaluating the role of waste fishbone hydroxyapatite (FB-HAP) in stabilizing heavy metals, particularly Pb and Zn, in incineration fly ash (IFA). Bones were collected from various fish species and processed for batch experiments. A commercial apatite product (Apatite II™) was also obtained for a comparative analysis. The experiments were performed at fishbone/fly ash ratios of 0.0 (control group) and 1:10 (by weight), settling times of 6, 12, 24, and 672 h (28 days), and W/S ratios of 1.0 and 1.5 mL/g. The highest Pb removal efficiency reached 86.39% at 28 days settling periods, when the FB-HAP dose was only 10% at W/S 1.5 mL/g. FB-HAP was found noticeably more effective (approximately 1.5 to 2 times) than Apatite II™, particularly at shorter settling periods. Stabilization of Zn was efficient at longer settling period (28 days) using FB-HAP. The highest stabilization rate of Zn was 62.67% at W/S 1.0 mL/g. The results indicated that settling time and W/S ratio were the most important factors to enhance the stabilization of Pb and Zn in IFA. Utilization of waste fishbone is expected to be a low-cost and eco-friendly technology.

Coping styles associated with glucose control in individuals with type 2 diabetes mellitus.

AIMS/OBJECTIVES: Glycemic control varies according to stress level and the efficacy of control measures, affecting the outcomes of diabetes. Although detailed coping styles have not been well studied in patients with type 2 diabetes mellitus, problem-focused coping strategies are believed to be related to better control of blood glucose. Associations between coping profiles/dimensions and blood glucose control were examined in individuals with type 2 diabetes. MATERIALS AND METHODS: The participants included 503 Japanese patients (mean age 63.9 ± 12.6 years) with type 2 diabetes. The average glycated hemoglobin A1c (HbA1c) levels were calculated from HbA1c levels measured more than four times within the 12 months before the assessment. Coping profiles were assessed using the Brief Scale for Coping Profile. Lifestyle factors were also included in the analyses. RESULTS: Factors other than age were not associated with HbA1c levels in patients who used insulin. Conversely, habitual alcohol consumption, single status, the adaptive emotion-focused coping dimension, and changing mood and changing one's point of view profiles were associated with HbA1c levels. CONCLUSIONS: These findings suggest that adaptive emotion-focused coping supports glycemic control in type 2 diabetes patients who do not use insulin. Additional studies using a longitudinal design are required to further examine the relationships between psychological factors and glycemic control.

Identification of children with chronic kidney disease through school urinary screening using urinary protein/creatinine ratio measurement: an observational study.

BACKGROUND: School urinary screening has been performed in Japan. METHODS: Ikeda City and Toyono Town introduced, in 2012 and 2013, urinary protein/creatinine (Cr) ratio measurement into the urine-screening protocols designed for students aged between 4 and 15 years. For each student whose urinary protein/Cr ratio was ≥ 0.15 g/gCr (positive case), an appointment was made with a specialist at Ikeda City Hospital. The results of these screening urinalyses conducted through 2018 are summarized. RESULTS: 14,606 junior high and elementary school students aged between 6 and 15 years were included. On average, they underwent 4.16 screening tests. 77 positive cases were detected, and seven students were diagnosed with high-risk chronic kidney disease (CKD). Of these, four underwent renal biopsy, and two, one, and one were diagnosed with IgA nephropathy, MPGN, and FSGS, respectively. In three students, detection of CKD would have been difficult without urinary screening. Incident rates of high-risk CKD and IgA nephropathy are estimated as 11.5 and 3.3 cases/100,000 students/year. 78.0% of positive cases without high-risk CKD showed no urinary abnormality after one year. 2301 kindergarten students aged between 4 and 6 years received an average of 1.74 screening urinalyses; none was positive or high-risk CKD. The estimated cost of detecting one high-risk CKD student whose detection would have been difficult without this screening was 3,156,711 Japanese yen. CONCLUSION: School urinary screening using the urinary protein/Cr ratio can efficiently refer to a specialist. It detects a few children with high-risk CKD early with spending high cost.

Personality Traits Do Not Have Influence on Glycemic Control in Outpatients with Type 2 Diabetes Mellitus.

OBJECTIVE: Glycemic control varies based on lifestyle factors and stress coping mechanisms, which are influenced by personality. The psychological factors associated with glycemic control have not yet been established in patients with type 2 diabetes mellitus (T2DM). The relationship between a 5-factor model of personality and glycemic control was evaluated in individuals with T2DM. METHODS: The subjects were 503 Japanese outpatients with T2DM. Glycated hemoglobin A1c (HbA1c) levels, depressive status, insomnia and personality traits were assessed. Lifestyle factors of the patients, such as habitual alcohol consumption and smoking, were also included in the analyses. RESULTS: Because the influence of insulin therapy on HbA1c is so strong, we stratified the patients according to insulin use. Simple regression analysis showed a significant correlation between HbA1c and neuroticism in patients who did not use insulin. After adjustment for confounders, multiple regression analyses revealed that none of the personality factors, including neuroticism, were found to be associated with HbA1c. CONCLUSION: These findings suggest that personality traits do not have a large impact on glycemic control. Further studies are required to confirm the relationships between psychological factors and glycemic control using a longitudinal study design.