New high-throughput screening method for Chinese hamster ovary cell lines expressing low reduced monoclonal antibody levels: application of a system controlling the gas phase over cell lysates in miniature bioreactors and facilitating multiple sample setup.

Interchain disulfide bonds in monoclonal antibodies may be reduced during large-scale mAb production using Chinese hamster ovary (CHO) cells. This reaction lowers the mAb product yield and purity; however, it may be prevented by screening cell lines that are unsusceptible to reduction and using them in mAb production. Antibody reduction susceptibility may be cell line-dependent. To the best of our knowledge, however, an efficient method of screening reduction-unsusceptible CHO cell lines has not been previously reported. Here, we report a novel screening method that can simultaneously detect and identify mAb reduction susceptibility in lysates containing ≤ 48 CHO cell lines. This evaluation system was equally effective and generated similar results at all culture scales, including 250 mL, 3 L, and 1000 L. Furthermore, we discovered that reduction-susceptible cell lines contained higher total intracellular nicotinamide adenine dinucleotide phosphate (NADPH) and NADP+ concentrations than reduction-unsusceptible cell lines, regardless of whether they expressed immunoglobulin (Ig)G4 or IgG1. NADPH or NADP+ supplementation in the lysate of reduction-unsusceptible cells resulted in mAb reduction. Application of the innovative CHO cell line screening approach could mitigate or prevent reductions in large-scale mAb generation from CHO cells.

Characteristics of pediatric ocular trauma in a pediatric emergency department in Japan.

INTRODUCTION: Pediatric ocular trauma is a common complaint in pediatric emergency departments (ED) and is a major cause of acquired monocular blindness. However, data on its epidemiology and management in the ED are lacking. The objective of this study was to describe the characteristics and management of pediatric ocular trauma patients who visited a Japanese pediatric emergency department (ED). METHODS: The present, retrospective, observational study was conducted in a pediatric ED in Japan between March 2010 and March 2021. Children younger than 16 years who visited our pediatric ED and received the diagnosis of ocular trauma were included. ED visits for follow-up examinations for the same complaint were excluded. The patients' sex, age, arrival time, mechanism of injury, signs and symptoms, examinations, diagnosis, history of urgent ophthalmological consultation, outcomes, and ophthalmological complications were extracted from electronic medical records. RESULTS: In total, 469 patients were included; of these, 318 (68%) were male, and the median age was 7.3 years. The incident leading to trauma occurred most frequently at home (26%) and most often involved being struck in the eye (34%). In 20% of the cases, the eye was struck by some body part. Tests performed in the ED included visual acuity testing (44%), fluorescein staining (27%), and computed tomography (19%). Thirty-seven (8%) patients underwent a procedure in the ED. Most patients had a closed globe injury (CGI), with only two (0.4%) having an open globe injury (OGI). Eighty-five (18%) patients required an urgent ophthalmological referral, and 12 (3%) required emergency surgery. Ophthalmological complications occurred in only seven patients (2%). CONCLUSION: Most cases of pediatric ocular trauma seen in the pediatric ED were CGI, with only a few cases leading to emergency surgery or ophthalmological complications. Pediatric ocular trauma can be safely managed by pediatric emergency physicians.

Metabolomic alterations in the blood plasma of older adults with mild cognitive impairment and Alzheimer's disease (from the Nakayama Study).

Alzheimer's disease (AD) is a progressive disease, and the number of AD patients is increasing every year as the population ages. One of the pathophysiological mechanisms of AD is thought to be the effect of metabolomic abnormalities. There have been several studies of metabolomic abnormalities of AD, and new biomarkers are being investigated. Metabolomic studies have been attracting attention, and the aim of this study was to identify metabolomic biomarkers associated with AD and mild cognitive impairment (MCI). Of the 927 participants in the Nakayama Study conducted in Iyo City, Ehime Prefecture, 106 were selected for this study as Control (n = 40), MCI (n = 26), and AD (n = 40) groups, matched by age and sex. Metabolomic comparisons were made across the three groups. Then, correlations between metabolites and clinical symptoms were examined. The blood mRNA levels of the ornithine metabolic enzymes were also measured. Of the plasma metabolites, significant differences were found in ornithine, uracil, and lysine. Ornithine was significantly decreased in the AD group compared to the Control and MCI groups (Control vs. AD: 97.2 vs. 77.4; P = 0.01, MCI vs. AD: 92.5 vs. 77.4; P = 0.02). Uracil and lysine were also significantly decreased in the AD group compared to the Control group (uracil, Control vs. AD: 272 vs. 235; P = 0.04, lysine, Control vs. AD: 208 vs. 176; P = 0.03). In the total sample, the MMSE score was significantly correlated with lysine, ornithine, thymine, and uracil. The Barthel index score was significantly correlated with lysine. The instrumental activities of daily living (IADL) score were significantly correlated with lysine, betaine, creatine, and thymine. In the ornithine metabolism pathway, the spermine synthase mRNA level was significantly decreased in AD. Ornithine was decreased, and mRNA expressions related to its metabolism were changed in the AD group compared to the Control and MCI groups, suggesting an association between abnormal ornithine metabolism and AD. Increased betaine and decreased methionine may also have the potential to serve as markers of higher IADL in elderly persons. Plasma metabolites may be useful for predicting the progression of AD.

Blood CDKN2A Gene Expression in Aging and Neurodegenerative Diseases.

BACKGROUND: Cyclin-dependent kinase inhibitor 2A (CDKN2A) is an important gene in cellular senescence and aging. OBJECTIVE: This study assessed the utility of blood CDKN2A mRNA expression levels and methylation status as a potential biomarker for aging and the pathogenesis of Alzheimer's disease (AD). METHODS: The correlation between CDKN2A mRNA expression levels and age was examined in 45 healthy subjects, after which mRNA expression levels were compared among 46 AD patients, 20 mild cognitive impairment due to AD patients, 21 Parkinson's disease patients, 21 dementia with Lewy bodies patients, and 55 older healthy controls. The methylation rates of the second exon of the CDKN2A gene, known to influence its expression levels, was also examined. RESULTS: A significant correlation between CDKN2A mRNA expression levels and age was found (Spearman's rank correlation coefficient: r = 0.407, p = 0.005). CDKN2A mRNA expression levels in blood were significantly decreased in AD patients, although those of healthy controls were significantly increased with age. Further, only in AD patients were CDKN2A mRNA expression levels significantly and positively correlated with methylation rates. CONCLUSION: Although further research with a larger sample size is needed to elucidate the relationships between CDKN2A gene expression in blood and the development of other neurodegenerative diseases, CDKN2A mRNA expression in blood may be a biomarker for differentiating AD from normal aging and other neurodegenerative diseases.

Detection of a Urethral Foreign Body in a Pediatric Patient: Another Useful Application of Point-of-Care Ultrasound.

BACKGROUND: Urethral foreign bodies (UFBs) are rare in pediatric emergency care, but require immediate diagnosis and intervention when they occur. Although radiography, computed tomography, and cystography are available for diagnosing UFBs, these modalities are undesirable because they involve radiation exposure. Point-of-care ultrasound (POCUS) is used as an alternative imaging modality for UFB detection in adult patients, but reports of its use in pediatric emergency departments are still scarce. We report a pediatric case of a UFB detected by POCUS. CASE REPORT: A 10-year-old boy with a history of a learning disorder presented to our pediatric emergency department with a paper clip in his penis, which he had intentionally inserted during play. He denied any symptoms, such as abdominal pain, vomiting, and hematuria. Physical examination failed to reveal the tip of the FB, but showed a palpable mass in the penile urethra accompanied by mild tenderness in the area. POCUS demonstrated a hyperechoic structure with reverberation artifact extending to the bulbar urethra. Endoscopic removal was planned, but the tip of the FB emerged from the external urethral meatus with postural change. Manual removal was successfully performed, after which the hyperechoic structure in the urethra was no longer visible on ultrasonography. The patient was discharged on the same day of the procedure. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: POCUS is a noninvasive procedure that can be useful for detecting UFBs in children.

Pediatric Case of Successful Point-of-Care Ultrasound-Guided Nasogastric Tube Placement.

BACKGROUND: Nasogastric tube (NGT) placement is commonly performed in pediatric emergency care and is classically confirmed by any one of several methods, among which auscultation or aspiration and radiography comprise the currently recognized as the reference standard. Point-of-care ultrasound (POCUS) is used to confirm NGT insertion, especially in adults or prehospital patients, but reports of its use in the pediatric emergency department (ED) are still scarce. We report a case of successful POCUS-guided NGT placement in a pediatric ED. CASE REPORT: A 3-year-old male undergoing remission therapy for acute lymphocytic leukemia presented to our ED with fever and decreased appetite. Tumor lysis syndrome was diagnosed, and endotracheal intubation was required because of the need for emergency hemodialysis for hypercalcemia. Because of difficulty in guiding the tube through the nose, ultrasound-guided placement was attempted. In the transverse view over the neck below the level of the cricoid cartilage, the 10-Fr NGT was visualized under ultrasound guidance as it passed through the esophagus. Subsequently, the entry of the NGT tip into the gastric cardia was confirmed on the subxiphoid longitudinal view. A chest radiograph confirmed the presence of the NGT in the stomach. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Although the utility of POCUS for NGT placement was reported in adult patients, reports of its use in pediatric cases are still few. POCUS is a real-time, noninvasive, time-saving procedure that can be a useful alternative to radiography for confirming correct NGT placement.

Early Diagnosis of a Pediatric Pancreatic Tumor by Abdominal Physical Examination: A Case Report.

BACKGROUND: Detecting a neoplasm in pediatric patients with acute abdominal pain is difficult, especially when there is no palpable mass. Ihara's maneuver, which allows the physician to apply manual pressure directly on the pancreatic body, is a useful palpation method of diagnosing acute pancreatitis in children. We report a case of solid pseudopapillary neoplasm of the pancreas (SPNP) detected by Ihara's maneuver. CASE REPORT: An otherwise healthy, 15-year-old male visited our emergency department with acute abdominal pain and nausea. His vital signs were appropriate for his age. A physical examination denied peritoneal signs, but Ihara's maneuver induced strong tenderness. His serum amylase and lipase were normal. A contrast-enhanced computed tomography scan revealed a well-defined, 2.2-cm, nonenhanced mass in the pancreatic tail. Laparoscopic distal pancreatectomy was performed, and the diagnosis of SPNP was confirmed. The patient was well postoperatively without any metastasis. SPNP is a rare neoplasm with low malignant potential. Although it typically occurs in young females, it has also been reported in children. The early diagnosis of SPNP is usually challenging because most patients do not have specific symptoms or laboratory findings. In the present case, the SPNP was difficult to detect by palpation because of its size, but Ihara's maneuver induced strong tenderness of the pancreas and led to a diagnosis. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Ihara's maneuver has the potential to enable early diagnosis not only of pancreatitis but also of pancreatic tumors, such as SPNP.

Another Useful Application of Point-of-Care Ultrasound: Detection of Esophageal Foreign Bodies in Pediatric Patients.

Foreign body ingestion is a common pediatric complaint. Esophageal foreign body (EFB) requires early diagnosis and emergency removal of the object. We report 3 cases of EFB detected by point-of-care ultrasound demonstrating characteristic sonographic findings. Point-of-care ultrasound can be a potentially useful modality for identifying EFB.

INPP5D mRNA Expression and Cognitive Decline in Japanese Alzheimer's Disease Subjects.

Microglial dysfunction and inflammation have recently been shown to be related to the development of Alzheimer's disease (AD). Inositol polyphosphate-5-phosphatase (INPP5D) functions broadly as a negative regulator of immune signaling, and its locus was associated with development of AD in a large-scale genome-wide association study. Thus, we examined INPP5D mRNA expression and methylation rates of the CpG sites in the upstream region of INPP5D exon 1 in peripheral leukocytes in 50 AD and age- and sex-matched control subjects. INPP5D mRNA expression in AD subjects was significantly higher than that in control subjects (1.16±0.39 versus 1.0±0.23, p = 0.049) and was correlated with the Mini-Mental State Examination score (p = 0.002, r = 0.426) and the total score of the Alzheimer's Disease Assessment Scale (p < 0.001, r = -0.697). Methylation rates in the upstream region of INPP5D exon 1 were not significantly different between AD and control subjects (average rate: 3.5±3.0 versus 2.8±1.3, p = 0.551). Our results suggested that INPP5D mRNA expression was elevated in the early stage and decreased with cognitive decline in AD. INPP5D mRNA expression in leukocytes may be a useful biomarker for the early stage of AD.

Membrane fusion between baculovirus budded virus-enveloped particles and giant liposomes generated using a droplet-transfer method for the incorporation of recombinant membrane proteins.

Giant proteoliposomes are generally useful as artificial cell membranes in biochemical and biophysical studies, and various procedures for their preparation have been reported. We present here a novel preparation technique that involves the combination of i) cell-sized lipid vesicles (giant unilamellar vesicles, GUVs) that are generated using the droplet-transfer method, where lipid monolayer-coated water-in-oil microemulsion droplets interact with oil/water interfaces to form enclosed bilayer vesicles, and ii) budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus) that express recombinant transmembrane proteins on their envelopes. GP64, a fusogenic glycoprotein on viral envelopes, is activated by weak acids and is thought to cause membrane fusion with liposomes. Using confocal laser scanning microscopy (CLSM), we observed that the single giant liposomes fused with octadecyl rhodamine B chloride (R18)-labeled wild-type BV envelopes with moderate leakage of entrapped soluble compounds (calcein), and the fusion profile depended on the pH of the exterior solution: membrane fusion occurred at pH ∼4-5. We further demonstrated that recombinant transmembrane proteins, a red fluorescent protein (RFP)-tagged GPCR (corticotropin-releasing hormone receptor 1, CRHR1) and envelope protein GP64 could be partly incorporated into membranes of the individual giant liposomes with a reduction of the pH value, though there were also some immobile fluorescent spots observed on their circumferences. This combination may be useful for preparing giant proteoliposomes containing the desired membranes and inner phases.

An Algorithmic Approach Using Ultrasonography in the Diagnosis of Pediatric Nasal Bone Fracture.

BACKGROUND: Ultrasonography (US) was recently reported as a reliable modality for diagnosing nasal bone fractures. However, whether US is reliable as a screening tool in the pediatric emergency department (ED) remains unknown. This prospective cohort study had a 2-fold aim: to assess the utility of US in the diagnosis of pediatric nasal bone fracture, and to evaluate the validity of our protocol for managing pediatric nasal bone fractures (Fuchu-Kids algorithm). PATIENTS AND METHODS: Among the patients who presented at the ED with facial trauma, those with a suspected nasal bone fracture were enrolled in the study. Patients were treated according to Fuchu-Kids algorithm, and the validity of the protocol using US imaging was evaluated. RESULTS: Among 81 patients who were enrolled during the 1-year study period, 63 patients were able to complete the process described in our protocol for further examination. The diagnostic power of the Fuchu-Kids algorithm had a sensitivity of 91.7%, a specificity of 92.3%, a positive predictive value (PPV) of 88%, and a negative predictive value (NPV) of 94.7%. However, when the performance of US was assessed as a single examination, its sensitivity, specificity, PPV, and NPV were 75%, 92.3%, 85.7%, and 85.7%, respectively. CONCLUSION: Using our algorithm, the majority of patients with nasal bone fracture were successfully diagnosed and screened out successfully. Repeated US imaging is effective when clinical symptoms persist even if the first US imaging was negative for nasal bone fracture. However, a detailed medical interview and clinical examination are mandatory, regardless of the use of US.

TREM2 mRNA Expression in Leukocytes Is Increased in Alzheimer's Disease and Schizophrenia.

UNLABELLED: TREM2 and TYROBP are causal genes for Nasu-Hakola disease (NHD), a rare autosomal recessive disease characterized by bone lesions and early-onset progressive dementia. TREM2 forms a receptor signaling complex with TYROBP, which triggers the activation of immune responses in macrophages and dendritic cells, and the functional polymorphism of TREM2 is reported to be associated with neurodegenerative disorders such as Alzheimer's disease (AD). The objective of this study was to reveal the involvement of TYROBP and TREM2 in the pathophysiology of AD and schizophrenia. METHODS: We investigated the mRNA expression level of the 2 genes in leukocytes of 26 patients with AD and 24 with schizophrenia in comparison with age-matched controls. Moreover, we performed gene association analysis between these 2 genes and schizophrenia. RESULTS: No differences were found in TYROBP mRNA expression in patients with AD and schizophrenia; however, TREM2 mRNA expression was increased in patients with AD and schizophrenia compared with controls (P < 0.001). There were no genetic associations of either gene with schizophrenia in Japanese patients. CONCLUSION: TREM2 expression in leukocytes is elevated not only in AD but also in schizophrenia. Inflammatory processes involving TREM2 may occur in schizophrenia, as observed in neurocognitive disorders such as AD. TREM2 expression in leukocytes may be a novel biomarker for neurological and psychiatric disorders.

Incorporation of adenylate cyclase into membranes of giant liposomes using membrane fusion with recombinant baculovirus-budded virus particles.

Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.