Evaluation of the Analytical Performance of the Fully Automated Gene Analyzer µTASWako g1 for the Detection of SARS-CoV-2.

BACKGROUND: The worldwide spread of coronavirus disease 2019 (COVID-19) has led to an urgent need for nucleic acid amplification test (NAAT) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Because NAAT has many manual processes, results may vary depending on the operator. Therefore, it has been required to develop a fully automated testing device and reagent that detects genetic material from SARS-CoV-2. The µTASWako g1 system (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), a genetic analyzer, provides results in 75 minutes by performing a fully automated PCR process. METHODS: We evaluated the analytical and clinical performance of the µTASWako g1 system for the detection of SARS-CoV-2 RNA. RESULTS: The µTASWako g1 system had the limit of detection at 2,000 copies/mL using a known concentration of RNA. In clinical samples, the µTASWako g1 system had a sensitivity of 88.0% and 100% specificity compared to conventional RT-PCR. The µTAS Wako g1 system could detect three variants of concern carrying spike mutations including N501Y, E484K, and L452R. CONCLUSIONS: As the assay on the µTASWako g1 system is highly accurate for the detection of SARS-CoV-2 regardless of the experience of operator, it can be widely applicable in clinical laboratories.

Inhibition of low-density lipoprotein uptake by Helicobacter pylori virulence factor CagA.

Helicobacter pylori (H. pylori) infection mainly causes gastroduodenal diseases, including chronic gastritis, peptic ulcer disease and gastric cancer. In recent years, several studies have demonstrated that infection with H. pylori, especially strains harboring the virulence factor CagA (cytotoxin-associated gene A), contribute to the development of non-gastric systemic diseases, including hypercholesterolemia and atherosclerotic cardiovascular diseases. However, mechanisms underlying this association has not been defined. In this study, we carried out a large-scale genetic screen using Drosophila and identified a novel CagA target low-density lipoprotein receptor (LDLR), which aids in the clearance of circulating LDL. We showed that CagA physically interacted with LDLR via its carboxy-terminal region and inhibited LDLR-mediated LDL uptake into cells. Since deficiency of LDLR-mediated LDL uptake has been known to increase plasma LDL and accelerate atherosclerosis, our findings may provide a novel mechanism for the association between infection with CagA-positive H. pylori and hypercholesterolemia leading to atherosclerotic cardiovascular diseases.

Characterization of MORN2 stability and regulatory function in LC3-associated phagocytosis in macrophages.

Microtubule-associated protein A1/B1-light chain 3 (LC3)-associated phagocytosis (LAP) is a type of non-canonical autophagy that regulates phagosome maturation in macrophages. However, the role and regulatory mechanism of LAP remain largely unknown. Recently, the membrane occupation and recognition nexus repeat-containing-2 (MORN2) was identified as a key component of LAP for the efficient formation of LC3-recruiting phagosomes. To characterize MORN2 and elucidate its function in LAP, we established a MORN2-overexpressing macrophage line. At a steady state, MORN2 was partially cleaved by the ubiquitin-proteasome system. MORN2 overexpression promoted not only LC3-II production but also LAP phagosome (LAPosome) acidification during Escherichia coli uptake. Furthermore, the formation of LAPosomes containing the yeast cell wall component zymosan was enhanced in MORN2-overexpressing cells and depended on reactive oxygen species (ROS). Finally, MORN2-mediated LAP was regulated by plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) such as SNAP-23 and syntaxin 11. Taken together, these findings demonstrate that MORN2, whose expression is downregulated via proteasomal digestion, is a limiting factor for LAP, and that membrane trafficking by SNARE proteins is involved in MORN2-mediated LAP.

OCT3/4-binding sequence-dependent maintenance of the unmethylated state of CTCF-binding sequences with DNA demethylation and suppression of de novo DNA methylation in the H19 imprinted control region.

DNA demethylation and suppression of de novo DNA methylation are activities that maintain an unmethylated state. However, the strength of these two activities at the same locus has not been estimated separately. Furthermore, the association between these two activities and the unmethylated state remains unclear. Octamer-binding transcription factor-binding sequences (OBSs) and CCCTC-binding factor-binding sequences (CBSs) within the mouse H19-imprinted control region (ICR) are involved in the induction of DNA demethylation and maintenance of the unmethylated state in mouse undifferentiated embryonic cell lines. To reveal the association between the two cis-elements and the two unmethylated state maintenance activities in maintaining the unmethylated state of the ICR, we evaluated the altered DNA methylation levels at sites that were initially methylated or unmethylated using a stable transfection-based assay, and estimated the strength of the two unmethylated state maintenance activities separately via a Poisson process model that described the DNA methylation state regulatory process. Although DNA demethylation depending on OBSs affected almost the entire ICR, DNA demethylation depending on CBSs occurred near CBSs, resulting in redundant demethylation of CBS regions. Detailed analysis of the CBS4 region suggested that OBSs were required to induce unmethylated state maintenance activities, and that CBSs-dependent activities contributed, but diminished, during incubation when protection of the CBS4 region by OBSs-dependent activities was absent. Analysis via the Poisson process model indicated that the unmethylated state at the CBS4 region was maintained by OBSs-dependent suppression of de novo DNA methylation rather than DNA demethylation. We propose that the hierarchical regulation of redundant protection of the CBS region via cooperation between the two unmethylated state maintenance activities is a potential function of the ICR that effectively maintains allele-specific methylation status in the same DNA sequence.

Oct motif variants in Beckwith-Wiedemann syndrome patients disrupt maintenance of the hypomethylated state of the H19/IGF2 imprinting control region.

The methylation status of imprinting control center 1 (IC1) regulates the monoallelic transcription of H19 and Igf2 in mammalian cells. Several single nucleotide variants in Oct motifs within IC1 occur in patients with Beckwith-Wiedemann syndrome (BWS) who have hypermethylated maternal IC1. However, the importance of Oct motifs in the regulation of IC1 methylation status remains unclear. Here, we demonstrate that three variants found in BWS (BWS variants) suppress intensive induction of DNA demethylation, whereas consensus disruption of motifs unrelated to BWS only slightly affects the induction of demethylation. BWS variants reduce DNA demethylation levels and trigger the accumulation of DNA methylation downstream of the IC1 transgenes. Thus, the risk of IC1 hypermethylation is associated with inhibitory levels of Oct motif-dependent hypomethylation maintenance activities.

Motion analysis of operating a balance exercise assist robot system during forward and backward movements.

[Purpose] Stand-and-ride personal mobility devices controlled by movements of the user's center of gravity are used for balance training. We aimed to describe the physical activity required to operate this type of mobility device. [Participants and Methods] Eleven healthy males performed the following tasks: 1) moving their center of gravity forward or backward while standing on the floor (control task) and, 2) moving the mobility device forward or backward by moving their center of gravity (experimental task). [Results] We observed that the displacement of the center of gravity and the center of pressure, as well as angular displacements of the hips and knee joints, and maximum muscle activities of the biceps femoris, the medial head of the gastrocnemius and peroneus longus muscles were lesser during the experimental than during the control task. The distance moved by the device was significantly greater than the displacement of the user's center of gravity during the experimental task. [Conclusion] We observed that moving the device forward or backward required lesser physical activity than that required to shift the user's center of gravity forward or backward while standing on the floor. Additionally, we observed that even a small displacement of the user's center of gravity produced a large displacement of the device. We concluded that during balance training, the greater and more easily perceived movement of the mobility device would provide helpful feedback to the user.

Kinematic, kinetic, and electromyographic characteristics during quiet standing on a balance exercise assist robot.

[Purpose] The balance exercise assist robot is a training device based on a personal transport assistance robot ridden in the standing position. The personal transport assistance robot uses an inverted pendulum control system and moves in response to movements of the user's center of gravity. The purpose of this study was to describe the characteristics of postural control during the action of stopping the personal transport assistance robot. [Participants and Methods] Eleven healthy male participants were required to maintain a standing position for 30 s; each task was performed 10 times. The measurement conditions were as follows: (1) on the floor; (2) on the robot, touching the handlebars; and (3) on the robot, not touching the handlebars. [Results] During the robotic tasks, the total locus lengths of the center of gravity and total joint momentums of the hip, knee, and ankle joints were larger, and the amount of displacement of the center of pressure was smaller than that during the floor task. Posture control on the robot was performed actively by mechanical interaction of the ankle, knee, and hip joints within a small base of support. [Conclusion] The balance exercise assist robot can be useful for postural control exercises because maintaining a standing position on the personal transport assistance robot required active postural control.

A kinematic and kinetic analysis of the hip and knee joints in patients with posterior tibialis tendon dysfunction; comparison with healthy age-matched controls.

BACKGROUND: The biomechanical abnormalities in patients with posterior tibial tendon dysfunction (PTTD) have been described, but few studies have investigated biomechanical chains of adjacent joints. Therefore, we examined the gait pattern of the lower extremity in subjects with PTTD, focusing on the hip and knee joints. METHODS: We compared 19 PTTD patients (average age: 67.1) with 30 age-matched control subjects (average age: 65.1). Gait analysis was performed with a nine-camera motion-capture system and four force plates, using the Vicon Plug-In-Gait and Vicon Nexus software. Temporal-spatial parameters were compared between PTTD and control subjects, and motion and ground reaction force data were compared between the affected limb, the contralateral limb, and the right limb in control subjects. RESULTS: Subjects with PTTD had increased stance phase ratio and decreased stride length, cadence, and gait speed. The limbs of subjects with PTTD showed increased knee internal rotation at lording response, which was biased to abduction in the knee joint during the gait cycle, and irregular hip flexion and knee extension moment in the terminal stance, even under control of gait speed. SIGNIFICANCE: We believe that the subjects with PTTD have an increased risk of knee osteoarthritis in both the affected and contralateral limbs.

Gait training using a stationary, one-leg gait exercise assist robot for chronic stroke hemiplegia: a case report.

[Purpose] The Gait Exercise Assist Robot (GEAR) is a stationary, one-leg robot for gait training. The purpose of this case study was to evaluate the efficacy of rehabilitation using GEAR training for chronic stroke hemiplegia. [Participant and Methods] The participant was a 66-year-old male stroke survivor with left hemiparesis due to a right putaminal hemorrhage. He could walk slowly under supervision, although his gait had a constant forward trunk lean, with flexed knee, and a lack of hip extension movement on the affected side. Gait training using GEAR and physical therapy were performed for 14 days. Under both training conditions, the physical therapist made the participant conscious of extension movement of the hip joint in the affected-side stance phase. The robotic assistance was adjusted to maximize voluntary movement while observing gait. Physical function and gait ability parameters were evaluated before and after training. [Results] After training, extension motion of the hip joint increased in the affected-side stance phase, and body weight was transferred smoothly onto the affected-side limb, leading to an improvement in gait speed. [Conclusion] Gait training using GEAR and physical therapy may improve gait pattern and speed in patients with chronic stroke hemiplegia.

[Contribution of socioeconomic status to smoking behavior of parents of 4th grade elementary school students in Japan].

OBJECTIVES: The present study was performed to elucidate the effects of individual and social factors on smoking behavior of parents of fourth grade elementary school students in Japan. METHODS: A self-administered questionnaire was sent to a total of 4,179 households of fourth grade elementary school students. A total of 3,522 responses including actual numbers of children, smoking behavior of parents, and marital status were available for the analysis. RESULTS: Current smoking rate in mothers was 21.2%. In mothers, "smoking of spouse" "single mother", "under the age of 34", "not taking child-care leave;", "mother's parents not alive", "mothers from Chiba", "nursery use", "not use parenting circles", "unvaccinated measles or inoculation unknown" and "life dissatisfaction" were statistically associated with smoking behavior. The current smoking rate in fathers was 51.4%. Four factors of "smoking of spouse", "under the age of 34", "non-skilled labor, sales work" and "employees of private companies of less than 1,000 employees" were statistically associated with smoking behavior. CONCLUSION: The present study demonstrated a close link between smoking behavior and individual socioeconomic status in Japanese parents. Especially, smoking of spouse and being a single female parent were important factors for smoking.

A case of IgG4-related sclerosing disease complicated by sclerosing cholangitis, retroperitoneal fibrosis and orbital pseudotumour.

We present a case of IgG4-related sclerosing disease complicated by sclerosing cholangitis (SC), idiopathic retroperitoneal fibrosis (IRF) and orbital pseudotumour (OPT). Clinical, radiographic and pathological findings later suggested that the patient had SC complicated by IRF. The patient's SC and IRF were well controlled for the first 10 years of the follow-up period; OPT developed in the tenth year. During investigation of the OPT, serum IgG4 level was found to be significantly elevated. The patient was then diagnosed with IgG4-related sclerosing disease complicated by SC, IRF and OPT. This is a rare manifestation of IgG4-related sclerosing disease, which was diagnosed incidentally during OPT work-up. We suggest that this is a variation of the so-called IgG4-related sclerosing disease or hyper-IgG4 disease.

Regulation of anaphylactic responses by phosphatidylinositol phosphate kinase type I {alpha}.

The membrane phospholipid phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P(2)] is a critical signal transducer in eukaryotic cells. However, the physiological roles of the type I phosphatidylinositol phosphate kinases (PIPKIs) that synthesize PI(4,5)P(2) are largely unknown. Here, we show that the alpha isozyme of PIPKI (PIPKIalpha) negatively regulates mast cell functions and anaphylactic responses. In vitro, PIPKIalpha-deficient mast cells exhibited increased degranulation and cytokine production after Fcepsilon receptor-I cross-linking. In vivo, PIPKIalpha(-/-) mice displayed enhanced passive cutaneous and systemic anaphylaxis. Filamentous actin was diminished in PIPKIalpha(-/-) mast cells, and enhanced degranulation observed in the absence of PIPKIalpha was also seen in wild-type mast cells treated with latrunculin, a pharmacological inhibitor of actin polymerization. Moreover, the association of FcepsilonRI with lipid rafts and FcepsilonRI-mediated activation of signaling proteins was augmented in PIPKIalpha(-/-) mast cells. Thus, PIPKIalpha is a negative regulator of FcepsilonRI-mediated cellular responses and anaphylaxis, which functions by controlling the actin cytoskeleton and dynamics of FcepsilonRI signaling. Our results indicate that the different PIPKI isoforms might be functionally specialized.

CD69-null mice protected from arthritis induced with anti-type II collagen antibodies.

CD69, known as an early activation marker antigen on T and B cells, is also expressed on platelets and activated neutrophils, suggesting certain roles in inflammatory diseases. In order to address the role of CD69 in the pathogenesis of arthritis, we established CD69-null mice. CD69-null mice displayed a markedly attenuated arthritic inflammatory response when injected with anti-type II collagen antibodies. Cell transfer experiments with neutrophils, but not T cells or spleen cells, from wild-type mice into CD69-null mice restored the induction of arthritis. These results indicate a critical role for CD69 in neutrophil function in arthritis induction during the effector phase. Thus, CD69 would be a possible therapeutic target for arthritis in human patients.

Role of IgE in Th2 cell-mediated allergic airway inflammation.

Recent studies with gene knockout mice have demonstrated that T helper 2 (Th2) cell-derived cytokines, including IL-4, IL-5, and IL-13, play important roles in causing allergic airway inflammation. In addition to Th2 cytokines, IgE-dependent activation of mast cells has been suggested to play a role in allergic airway inflammation. In this review, we will discuss the role of IgE in Th2 cell-mediated allergic airway inflammation. We used IgE transgenic mice, which enabled us to investigate the role of IgE without the influence of activated T cells and other immunoglobulins. Whereas IgE cross-linking by antigens did not induce eosinophil recruitment into the airways or airway hyperreactivity, IgE cross-linking induced CD4+ T cell recruitment into the airways. In addition, when antigen-specific Th2 cells were transferred to IgE transgenic mice, IgE cross-linking significantly enhanced antigen-induced eosinophil recruitment into the airways. These findings suggest that IgE-dependent mast cell activation plays an important role in allergic airway inflammation by recruiting Th2 cells into the site of allergic inflammation.

Chronic inflammation of the skin can be induced in IgE transgenic mice by means of a single challenge of multivalent antigen.

BACKGROUND: It is now widely accepted that IgE mediates immediate-type allergic response. However, the pathologic role of IgE is controversial in the chronic allergic inflammation observed in atopic diseases, such as asthma and atopic dermatitis. OBJECTIVE: We investigated the role of IgE in cutaneous allergic reactions by using 2 newly developed lines of antigen-specific IgE transgenic mice. METHODS: IgE transgenic mice were administered subcutaneously with corresponding antigens, and the subsequent ear swelling was measured. RESULTS: A single subcutaneous administration of TNP-conjugated ovalbumin (OVA) into the ears of nonimmunized mice carrying the TNP-specific IgE transgene elicited immediate-phase and late-phase ear swelling as expected, which peaked at 20 minutes and 8 hours later, respectively. Interestingly, however, 2 to 3 days after the antigen challenge, more intense ear swelling appeared. Its magnitude and duration were dependent on the valency of TNP in OVA, as well as the dose of TNP-OVA, and it lasted over 1 month when 100 microg of OVA conjugated with 11 molecules of TNP was given. Interestingly, administration of OVA to OVA-specific IgE transgenic mice elicited immediate-phase and late-phase ear swelling but not third-phase ear swelling. Massive infiltration of inflammatory cells was observed in the third-phase ear swelling of TNP-specific IgE transgenic mice. Cyclosporine A almost completely inhibited the third-phase ear swelling and cellular infiltration, whereas an antihistamine, cyproheptadine, did not show any significant effect on the third-phase reaction. CONCLUSION: These results indicate that IgE can trigger not only immediate-type hypersensitivity but also chronic allergic inflammation. Our findings highlight a novel immunopathologic role of IgE in chronic atopic disorders.

Long term maintenance of IgE-mediated memory in mast cells in the absence of detectable serum IgE.

Mast cells and basophils involved in allergic responses do not have clonotypic Ag receptors. However, they can acquire Ag specificity through binding of Ag-specific IgE to FcepsilonRI expressed on their surface. Previous studies demonstrated that IgE binding induced the stabilization and accumulation of FcepsilonRI on the cell surface and resulted in up-regulation of FcepsilonRI. In this study we have further analyzed the maintenance of IgE-mediated memory in mast cells and basophils in vivo by comparing kinetics of serum IgE levels, FcepsilonRI expression, and ability to induce systemic anaphylaxis. A single i.v. injection of trinitrophenyl-specific IgE induced 8-fold up-regulation of FcepsilonRI expression on peritoneal mast cells in B cell-deficient (micro m(-/-)) mice. Serum IgE levels became undetectable by day 6, but the treatment of mice with anti-IgE mAb induced a significant drop in body temperature on days 14, 28, and 42. The administration of trinitrophenyl -BSA, but not BSA, in place of anti-IgE mAb gave similar results, indicating the Ag specificity of the allergic response. This long term maintenance of Ag-specific reactivity in the allergic response was also observed in normal mice passively sensitized with IgE even though the duration was shorter than that in B cell-deficient mice. The appearance of IgE with a different specificity did not interfere with the maintenance of IgE-mediated memory of mast cells and basophils. These results suggest that IgE-mediated stabilization and up-regulation of FcepsilonRI enables mast cells and basophils not only to acquire Ag specificity, but also to maintain memory in vivo for lengthy periods of time.

CD4(+)CD25(+) T-cell development is regulated by at least 2 distinct mechanisms.

It has recently been shown that CD4(+)CD25(+) T cells are immunoregulatory T cells that prevent CD4(+) T-cell-mediated organ-specific autoimmune diseases. In this study, the regulatory mechanism of CD4(+)CD25(+) T-cell development were investigated using T-cell receptor (TCR) transgenic mice. It was found that CD4(+)CD25(+) T cells preferentially expressed the endogenous TCRalpha chain in DO10(+) TCR transgenic mice compared with CD4(+)CD25(-) T cells. Moreover, it was found that CD4(+)CD25(+) thymocytes were severely decreased in DO10(+) TCR-alpha(-/-) mice in positively selecting and negatively selecting backgrounds, whereas CD4(+)CD25(-) thymocytes efficiently developed by transgenic TCR in DO10(+) TCR-alpha(-/-) mice in positively selecting backgrounds, indicating that the appropriate affinity of TCR to major histocompatibility complex (MHC) for the development of CD4(+)CD25(+) thymocytes is different from that of CD4(+)CD25(-) thymocytes and that a certain TCR-MHC affinity is required for the development of CD4(+)CD25(+) thymocytes. Finally, it was found that, in contrast to thymus, CD4(+)CD25(+) T cells were readily detected in spleen of DO10(+) TCR-alpha(-/-) mice in positively selecting backgrounds and that splenic CD4(+)CD25(+) T cells, but not CD4(+)CD25(+) thymocytes, were significantly decreased in B-cell-deficient mice, suggesting that B cells may control the peripheral pool of CD4(+)CD25(+) T cells. Together, these results indicate that the development of CD4(+)CD25(+) T cells in thymus and the homeostasis of CD4(+)CD25(+) T cells in periphery are regulated by distinct mechanisms.