Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System.

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, including biopharmaceuticals. Glycan structures exhibit significant heterogeneity, varying with production cell types, culture conditions, and purification methods. Consequently, monitoring and evaluating the glycan structures of recombinant proteins is vital, particularly in biopharmaceutical production. The lectin microarray, a technique complementary to mass spectrometry, boasts high sensitivity and ease of use. However, it typically requires more than a day to yield results. To adapt it to non-glycoscience research or drug product process development, an automated, high-throughput alternative is needed. Therefore, the world's first fully automated lectin-based glycan profiling system was developed, utilizing the "bead array in a single tip (BIST)" technology concept. This system allows for the preparation and storage of lectin-immobilized beads in units of 1,000, with customizable parallel insertion orders for various purposes. This article presents a practical protocol for research involving "glyco-qualified" recombinant proteins. After testing their reactivity against 12 polyacrylamide-glycan conjugates, 15 lectins were selected to increase the system's versatility. In addition, the sample labeling process was optimized by switching from Cy3 to biotin, reducing the overall processing time by 30 min. For immediate data qualification, lectin-binding signals are displayed as a dotcode on the top monitor. The system's reliability was confirmed through day-to-day reproducibility tests, repeatability tests, and long-term storage tests, with a coefficient of variation of <10%. This user-friendly and rapid glyco-analyzer has potential applications in the quality monitoring of endogenous glycoproteins for biomarker evaluation and validation. This method facilitates analysis for those new to glycoscience, thereby broadening its practical utility.

The Clinical Course and Treatment of a Case of Refractory Systemic Juvenile Myasthenia Gravis Successfully Treated with Thymectomy.

Juvenile myasthenia gravis (JMG) exhibits a more favorable response to glucocorticoids and has a better prognosis than adult myasthenia gravis. However, no established treatment exists for refractory JMG. Although thymectomy has been performed in several patients with refractory systemic JMG, there are few detailed clinical descriptions of patients who underwent thymectomy. Here, we present the case of a 10-year-old boy with refractory systemic JMG who was successfully treated with thymectomy. The patient developed symptoms, including dysphagia, malaise, diurnal ptosis, and weakness in the trunk muscles, and he was diagnosed with generalized JMG. Despite undergoing various treatments, including steroids, tacrolimus, steroid pulse therapy, intravenous immunoglobulin, azathioprine (AZT), and rituximab, his symptoms did not improve. Therefore, he underwent a thoracoscopic thymectomy 24 months after disease onset. Thymectomy led to remission, as demonstrated by a significant reduction in the quantitative myasthenia gravis score and anti-acetylcholine receptor antibody levels, which persisted for 43 months after surgery. Our case demonstrates the effectiveness of thymectomy in systemic JMG patients with positive anti-acetylcholine receptor antibodies, despite therapeutic failure with AZT and rituximab, within 2 years of disease onset.

Auto-Lectin Dotcoding by Two Octopuses: Rapid Analysis of Fluorescence-Labeled Glycoproteins by an 8-channel Fully-Automatic Bead Array Scanner with a Rolling-Circle Detector.

Protein glycosylation is a crucial factor that must be evaluated in biological pharmaceuticals. The glycoform profile of a protein can vary depending on the conditions of the cultivation, purification process, and the selection of a host cell. Lectin microarrays are reliable bioanalytical methods used in the early phases of bioprocesses for the detection of glycosylation. The concept of a fully automated glycan detection with a bead array has been previously reported; however, no simple system has been constructed on fluorescence-based detection using a microarray. Here, we present a fully automated detection system equipped with a novel fluorescence detector for a 13-lectin bead array with a single tip. The lattice-like arrangement of a set of fibers proximate to the tip of the light emitting diode and photomultiplier tube detector minimized the noise caused by the reflection of incident light on the plastic capillary tip and bead. A unique rolling-circle fiber unit with quadruple lattices stacked in two layers realizes the 8-parallel automeasurement with a drastic reduction in scanning time and machine size. The 8-glycan profiles obtained automatically within 25 min were identical with those obtained with the conventional lectin microarray after overnight incubation. The signals obtained were represented as lectin dotcodes. Therefore, autolectin dotcoding assisted by the twin 8 legs named as "detection and irradiation octopuses" may be a rapid glyco-evaluation system during the production and development of biopharmaceuticals.

Successful administration of eltrombopag in preparation for peritoneal dialysis catheter placement in a girl with MYH9-related disease.

MYH9-related disease is an autosomal dominant disorder characterized by macrothrombocytopenia, nephropathy, inclusion bodies in leukocytes, sensorineural hearing loss, and cataract. Severe cases require kidney replacement therapy in the patient's second decade of life; thrombocytopenia constitutes a major risk factor for hemorrhagic complications during dialysis initiation or kidney transplantation. Prophylactic platelet transfusion prior to surgery is commonly administered to affected patients in these cases. However, transfusion in such patients has limitations other than the general risk of allergic reactions and blood-borne infections; it may also trigger alloimmunization, leading to platelet transfusion resistance or the development of anti-donor antibodies in potential kidney transplant recipients. Here, we describe prophylactic administration of eltrombopag, an oral thrombopoietin receptor agonist, prior to laparoscopic peritoneal dialysis catheter placement in a 15-year-old girl with MYH9-related disease. Her platelet count was approximately 30 × 103/µL at baseline; it increased to 61 × 103/µL on the day before surgery, thereby avoiding the need for platelet transfusions. There were no major bleeding or adverse events associated with eltrombopag administration. Thus, eltrombopag may be a safe and effective alternative to prophylactic platelet transfusions in patients with MYH9-related disease.

Decreased liver damage in rat models of short bowel syndrome through DPP4 inhibition.

PURPOSE: Total parenteral nutrition causes liver damage in patients with short bowel syndrome (SBS), in whom intestinal failure-associated liver disease (IFALD) is the strongest risk factor for mortality. We previously demonstrated the efficacy of dipeptidyl peptidase-4 inhibitors (DPP4-Is) for nutritional absorption and intestinal barrier function enhancement. Herein, we investigated the efficacy of DPP4-Is in preventing liver damage in SBS rat models. METHODS: Rats were allocated to one of five groups: normal saline (NS) + sham, DPP4-I + sham, NS + SBS, DPP4-I + SBS, and GLP-2 + SBS. DPP4-I or NS was administered orally once daily. Serum aspartate aminotransferase, alanine aminotransferase (ALT), alkaline phosphatase, and total bile acid levels were measured to assess liver function. Moreover, we evaluated liver damage using the SAF (steatosis activity fibrosis) score, which is also used to assess nonalcoholic steatohepatitis. RESULTS: ALT levels and SAF scores were significantly lower in the DPP4-I + SBS group than in the NS + SBS group. Jejunal and ileal villus heights were significantly higher in the DPP4-I + SBS group than in the GLP-2 + SBS group. CONCLUSIONS: The downregulation of ALT levels and SAF scores triggered by DPP4-I use may be correlated with DPP4-I-induced adiposis inhibition in SBS and NASH models. Therefore, DPP4-I may be used to reduce IFALD in patients with SBS.

Development of a Fully Automated Desktop Analyzer and Ultrahigh Sensitivity Digital Immunoassay for SARS-CoV-2 Nucleocapsid Antigen Detection.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has had a significant impact on public health and the global economy. Several diagnostic tools are available for the detection of infectious diseases, with reverse transcription-polymerase chain reaction (RT-PCR) testing specifically recommended for viral RNA detection. However, this diagnostic method is costly, complex, and time-consuming. Although it does not have sufficient sensitivity, antigen detection by an immunoassay is an inexpensive and simpler alternative to RT-PCR. Here, we developed an ultrahigh sensitivity digital immunoassay (d-IA) for detecting SARS-CoV-2 nucleocapsid (N) protein as antigens using a fully automated desktop analyzer based on a digital enzyme-linked immunosorbent assay. METHODS: We developed a fully automated d-IA desktop analyzer and measured the viral N protein as an antigen in nasopharyngeal (NP) swabs from patients with coronavirus disease. We studied nasopharyngeal swabs of 159 and 88 patients who were RT-PCR-negative and RT-PCR-positive, respectively. RESULTS: The limit of detection of SARS-CoV-2 d-IA was 0.0043 pg/mL of N protein. The cutoff value was 0.029 pg/mL, with a negative RT-PCR distribution. The sensitivity of RT-PCR-positive specimens was estimated to be 94.3% (83/88). The assay time was 28 min. CONCLUSIONS: Our d-IA system, which includes a novel fully automated desktop analyzer, enabled detection of the SARS-CoV-2 N-protein with a comparable sensitivity to RT-PCR within 30 min. Thus, d-IA shows potential for SARS-CoV-2 detection across multiple diagnostic centers including small clinics, hospitals, airport quarantines, and clinical laboratories.

A recurrent de novo ZSWIM6 variant in a Japanese patient with severe neurodevelopmental delay and frequent vomiting.

A recurrent ZSWIM6 variant, NM_020928.2:c.2737C>T [p.Arg913*], was identified in a Japanese male patient with severe neurodevelopmental delay, epilepsy, distinctive facial features, microcephaly, growth deficiency, abnormal behavior, and frequent vomiting but without frontonasal or limb malformations. In this patient, distinctive facial features gradually became apparent with age, and severe vomiting caused by gastroesophageal reflux continued even after percutaneous endoscopic gastrostomy.

Nationwide survey of pediatric gastrointestinal endoscopy in Japan.

BACKGROUND AND AIM: The implementation rates of pediatric gastrointestinal endoscopy are increasing with advancements in the devices used and pediatricians' skills. As part of the Japan Pediatric Endoscopy Study Group, we aimed to investigate the rates of pediatric gastrointestinal endoscopy use and the associated adverse events through a nationwide survey. METHODS: A questionnaire was sent to 630 institutions in Japan. The numbers of pediatric gastrointestinal endoscopy cases and adverse events occurring during endoscopy, from April 2011 to March 2016, were investigated. RESULTS: Responses were obtained from 445 facilities. The total number of pediatric gastrointestinal endoscopies was 37 447 and that of endoscopic examinations was 32 219 (86.0%), with esophagogastroduodenoscopy accounting for 18 484 cases; ileal colonoscopy, 11 936; endoscopic retrograde cholangiopancreatography, 389; wireless capsule endoscopy, 897; and balloon-assisted enteroscopy, 513. The number of endoscopic treatments was 5228, followed by balloon dilatation (1703), foreign body removal (989), and polypectomy (822); 201 adverse events (0.54%) occurred, 79 of which presented during endoscopic examination (0.25%). Eight serious perforations were noted in 0.0054% and 0.025% of those undergoing esophagogastroduodenoscopy and colonoscopy, respectively. Overall, 122 adverse events (2.33%) occurred in association with endoscopic treatment. One case of cardiopulmonary arrest occurred because of accidental extubation. However, no deaths occurred. CONCLUSION: Endoscopic examinations had a slightly higher adverse event rate, because of an increase in endoscopic retrograde cholangiopancreatography and small intestine enteroscopy, than that reported in previous studies, but the adverse event rate of endoscopic treatment did not increase.

GlycoBIST: A System for Automatic Glycan Profiling of a Target Protein Using Milli-Bead Array in a Tip.

Lectin is a biomolecule that recognizes a specific part of glycans and, thus, has been used widely as a probe for glycoprotein analysis. Owing to the wide repertoire in nature combined with the recent two decades of advances in microarray technology, the multiplexed use of lectins has been widely used for glycan profiling of endogenous proteins. Because protein glycosylation is recognized as being biologically important and is expected to be a reliable disease marker, lectin microarray analysis with highly sensitive detection has been used to discover disease-relevant glycosylation alterations. However, the conventional system is limited to research purposes; thus, its implementation in clinical settings is warranted. Here, we provide an automatic glycan profiling method using GlycoBIST. A unique array format is used for 10-plexed lectin-glycoprotein interaction analysis on 1-mm-sized beads, which are arranged vertically in a capillary-shaped plastic tip. Using a one-boxed autopipetting machine, the whole process (including interaction, washing, and detection) is performed automatically and serially, resulting in reproducible measurements. In this article, a typical method for glycan profiling of a purified glycoprotein and the fabrication of GlycoBIST tips is explained. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Fabrication of a GlycoBIST tip Basic Protocol 2: Automatic profiling of a target glycoprotein using GlycoBIST.

Lectin Bead Array in a Single Tip Facilitates Fully Automatic Glycoprotein Profiling.

A quantitative description of glyco-alteration/differences in diseases can lead to the development of a diagnostic agent for use in vitro to monitor the degree of change in target glycoproteins. Analytical systems have been developed along with the progress of omics-oriented technologies. For clinical implementation, their full automation is required with an apparatus that is simple to operate. Here, we report an automatic analysis system for quantitative characterization of glyco-alteration/differences that depends on the unique strategy of "bead arrays in a single tip." The alternative lectin array can obtain a minimum characterization of the glycan profile for nanogram quantities of an endogenous glycoprotein. A simple autopipetting robot produces the precise chemiluminescence detection of glycan-lectin interactions with a wide dynamic range that is superior to fluorescence-based lectin arrays. The tip-based array format enables automatic glycan profiling from sample pretreatment to detection with low variation and linear detection, which may facilitate the use of this lectin array in clinical practice.

A case of cervical esophageal duplication cyst in a newborn infant.

Esophageal duplication cyst is a rare congenital anomaly resulting from a foregut budding error during the fourth to sixth week of embryonic development. Cervical esophageal duplication cysts are very rare and may cause respiratory distress in infancy. A full-term newborn girl who was born by normal delivery was transferred to our hospital because of swelling of the right anterior neck since birth. Cervical ultrasonography showed a 40 × 24 × 33 mm simple cyst on the right neck. Tracheal intubation was required at 2 weeks of age because of worsening external compression of the trachea. Fine-needle aspiration cytology revealed the existence of ciliated epithelium. At 1 month of age, exploration was performed through a transverse neck incision. The cyst had a layer of muscle connected to the lateral wall of the esophagus. Histopathological diagnosis was a cervical esophageal duplication cyst. We describe the clinical features of infantile cervical esophageal duplication cysts based on our experience of this rare disease in a neonate, along with a review of 19 cases previously reported in literature.

Current Practice and Outcomes of Thoracoscopic Esophageal Atresia and Tracheoesophageal Fistula Repair: A Multi-institutional Analysis in Japan.

BACKGROUND: In order to better understand the current practice and outcomes of thoracoscopic repair of esophageal atresia (EA)/tracheoesophageal fistula (TEF), a multi-institutional analysis was conducted among seven Japanese institutes. MATERIALS AND METHODS: A survey was sent to the seven institutes regarding the surgical technique, postoperative management, and outcomes of thoracoscopic repair of EA/TEF. RESULTS: The operation was uniformly performed via an intrapleural approach in the 0-45° prone position. The TEF was occluded with suture ligature in four (57.1%) institutes and clips in the remaining three (42.9%) institutes. Anastomosis was performed using the extracorporeal knot-tying technique in four institutes and the intracorporeal technique in three institutes. Patients were routinely left intubated and paralyzed for 3-7 days postoperatively in four institutes. In total, 58 patients underwent thoracoscopic repair of EA/TEF. Fifty-two (89.7%) of the patients underwent successful thoracoscopic repair. Six (10.3%) operations were converted to open thoracotomy because of a long gap (n=4), right aortic arch (n=1), and intraoperative instability (n=1). The body weight at operation ranged from 1.2 to 4.6 kg, and the operative time ranged from 115 to 428 minutes. There were no major intraoperative complications. Eleven patients (19.0%) suffered from anastomotic leakage. Twenty-eight patients (48.3%) developed anastomotic stricture. One patient died during the postoperative period because of an unrelated disease. Recurrent TEF developed in three patients (5.2%). Thirteen patients (22.4%) later required fundoplication. CONCLUSIONS: The outcome of thoracoscopic repair of EA/TEF was comparable to that of the open procedure. As considerable variability was observed among the seven institutes with respect to the surgical technique and management, standardizing the surgical management may improve the outcome.

Skill qualifications in pediatric minimally invasive surgery.

PURPOSE: In 2006, The Japanese Society of Pediatric Endoscopic Surgeons devised a plan to develop a pediatric endoscopic surgical skill qualification (ESSQ) system. This system is controlled by The Japan Society for Endoscopic Surgery. The standard requirement for skills qualification is the ability of each applicant to complete common types of laparoscopic surgery. The main goal of the system is to decrease complications of laparoscopic surgery by evaluating the surgical skills of each applicant and subsequently certify surgeons with adequate skills to perform laparoscopic operations safely. METHODS: A committee of pediatric ESSQ created a checklist to assess the applicant's laparoscopic surgical skills. Skills are assessed in a double-blinded fashion by evaluating an unedited video recording of a fundoplication for pediatric gastroesophageal reflux disease. RESULTS: The initial pediatric ESSQ system was started in 2008. In 2008 and 2009, respectively, 9 out of 17 (53%) and 6 out of 12 (50%) applicants were certified as expert pediatric laparoscopic surgeons. CONCLUSIONS: Our ultimate goal is to provide safe and appropriate pediatric minimally invasive procedures and to avoid severe complications. To prove the predictive validity of this system, a survey of the outcomes of operations performed by certified pediatric surgeons is required.

Interleukin-18 promoter polymorphisms and the disease progression of Hepatitis B virus-related liver disease.

In this study, we aimed to explore whether interleukin-18 (IL-18) gene-promoter polymorphisms are associated with the outcome of hepatitis B virus (HBV) infection. In all, 204 chronically HBV-infected patients were recruited in this study. Of the 204 HBV-infected patients, 43 were considered to be inactive HBV carriers based on the sustained normalization of serum alanine aminotransferase (ALT) together with seropositivity for the antibody to hepatitis B e-antigen (anti-HBe). A total of 161 patients were found to have chronic progressive liver disease, which included cirrhosis. In these HBV-infected patients, the frequencies of AA genotype of IL-18 gene-promoter polymorphisms at position -607 and C allele at position -137 were significantly higher in inactive HBV carriers compared with those in patients with chronic progressive liver disease. These polymorphisms of the IL-18 promoter regions (-607 and -137) could be associated with different outcomes of HBV infection.

Laparoscopy-assisted percutaneous endoscopic gastrostomy.

OBJECTIVE: Percutaneous endoscopic gastrostomy (PEG) placement is associated with considerable complications. We added laparoscopic monitoring to improve outcome. METHODS: Thirty-four patients who had laparoscopy-assisted PEG (LAP-PEG) were reviewed. A 5 mm supraumbilical trocar and two 5 mm working ports were required for LAP-PEG. A needle was placed percutaneously into the stomach under laparoscopic and gastroscopic control. A wire was placed through the needle, encircled with a snare, and the PEG completed. The anterior wall of the stomach was then anchored to the abdominal wall. RESULTS: Thirty-one subjects had cerebral palsy. Age at LAP-PEG ranged from 5 months to 25 years (mean, 8.1 years). Weight ranged from 4.7 kg to 25.9 kg (mean, 12.2 kg). In 23 patients, LAP-PEG was performed with laparoscopic Nissen fundoplication. In 11 patients, it was performed for reasons such as gastric volvulus and nutritional supplementation. Mean operating time was 67 minutes, and all procedures were performed safely without intra- or postoperative complications. CONCLUSION: LAP-PEG is our method of choice for gastrostomy because it allows the first and last parts of a conventional PEG procedure to be well controlled and safe instead of being blind.

Development of the Handy Bio-Strand and its application to genotyping of OPRM1 (A118G).

We previously developed a three-dimensional microarray system, the Bio-Strand, which exhibits advantages in automated DNA analysis in combination with our Magtration Technology. In the current study, we have developed a compact system for the Bio-Strand, the Handy Bio-Strand, which consists of several tools for the preparation of Bio-Strand Tip, hybridization, and detection. Using the Handy Bio-Strand, we performed single nucleotide polymorphism (SNP) genotyping of OPRM1 (A118G) by allele-specific oligonucleotide competitive hybridization (ASOCH). DNA fragments containing SNP sites were amplified from genomic DNA by PCR and then were fixed on a microporous nylon thread. Thus, prepared Bio-Strand Tip was hybridized with allele-specific Cy5 probes (<15mer), on which the SNP site was designed to be located in the center. By optimizing the amount of competitors, the selectivity of Cy5 probes increased without a drastic signal decrease. OPRM1 (A118G) genotypes of 23 human genomes prepared from whole blood samples were determined by ASOCH using the Handy Bio-Strand. The results were perfectly consistent with those determined by PCR direct sequencing. ASOCH using the Handy Bio-Strand would be a very simple and reliable method for SNP genotyping for small laboratories and hospitals.

Development of an automated SNP analysis method using a paramagnetic beads handling robot.

Biological and medical importance of the single nucleotide polymorphism (SNP) has led to development of a wide variety of methods for SNP typing. Aiming for establishing highly reliable and fully automated SNP typing, we have developed the adapter ligation method in combination with the paramagnetic beads handling technology, Magtration(R). The method utilizes sequence specific ligation between the fluorescently labeled adapter and the sample DNAs at the cohesive end produced by a type IIS restriction enzyme. Evaluation of the method using human genomic DNA showed clear discrimination of the three genotypes without ambiguity using the same reaction condition for any SNPs examined. The operations following PCR amplification were automatically performed by the Magtration(R)-based robot that we have previously developed. Multiplex typing of two SNPs in a single reaction by using four fluorescent dyes was successfully preformed at the almost same sensitivity and reliability as the single typing. These results demonstrate that the automated paramagnetic beads handling technology, Magtration(R), is highly adaptable to the automated SNP analysis and that our method best fits to an automated in-house SNP typing for laboratory and medical uses.