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Detection of allergen-specific immunoglobulin E (IgE) antibodies (Abs) in serum would allow for screening of the causative allergen in patients with type-I allergy. In this study, we developed a new assay method to detect allergen-specific IgE Abs, which involved crosslinking the plural FcεRIα molecules with an allergen and detection using an amplified luminescence proximity homogeneous assay (AlphaCL). First, the allergen concentration, bead concentrations, and incubation time were optimized for the detection of anti-2,4-dinitrophenyl (DNP) IgE Abs in buffer. Under optimal conditions, AlphaCL was able to detect DNP-specific IgE Abs in simulated human serum at levels comparable to those in serum from type-I allergic patients. When AlphaCL was used to detect anti-DNP IgE Abs, no signal counts were obtained with the monovalent allergen 2,4-dinitrophenylated poly-γ-glutamic acid, whereas high signal counts were obtained with the multivalent allergen DNP-BSA. This confirmed that AlphaCL could specifically detect allergen-specific IgE Abs with the ability to crosslink a multivalent allergen. In summary, we have established a new assay model using AlphaCL to detect allergen-specific IgE Abs with FcεRIα crosslinking ability in human serum. This simple and practical assay model may be applied as a new diagnostic tool for patients with type-I allergy., (© 2023. The Author(s).)

COVID-19 antibody testing has been developed to investigate humoral immune response in SARS-CoV-2 infection. To assess the serological dynamics and neutralizing potency following SARS-CoV-2 infection, we investigated the neutralizing (NT) antibody, anti-spike, and anti-nucleocapsid antibodies responses using a total of 168 samples obtained from 68 SARS-CoV-2 infected patients. Antibodies were measured using an authentic virus neutralization assay, the high-throughput laboratory measurements of the Abbott Alinity quantitative anti-spike receptor-binding domain IgG (S-IgG), semiquantitative anti-spike IgM (S-IgM), and anti-nucleocapsid IgG (N-IgG) assays. The quantitative measurement of S-IgG antibodies was well correlated with the neutralizing activity detected by the neutralization assay (r = 0.8943, p < 0.0001). However, the kinetics of the SARS-CoV-2 NT antibody in severe cases were slower than that of anti-S and anti-N specific antibodies. These findings indicate a limitation of using the S-IgG antibody titer, detected by the chemiluminescent immunoassay, as a direct quantitative marker of neutralizing activity capacity. Antibody testing should be carefully interpreted when utilized as a marker for serological responses to facilitate diagnostic, therapeutic, and prophylactic interventions., (© 2022. The Author(s).)

Despite the poor prognosis of systemic sclerosis (SSc) due to the co-occurrence of left ventricular diastolic dysfunction (LVDD), presence of occult LVDD has not been sufficiently investigated. This retrospective study aimed to reveal the prevalence and determinants of occult LVDD in patients with SSc by exercise stress test. Forty-five SSc patients (age, 63 ± 13 years; men/women, 6/39) with normal pulmonary artery pressure and pulmonary artery wedge pressure (PAWP) at rest underwent a symptom-limited exercise test with right heart catheterization using a supine cycle ergometer; haemodynamic parameters at rest, leg raise and during exercise were evaluated. Occult LVDD defined PAWP ≥ 25 mmHg during exercise was seen in 13 patients (29%). Higher PAWP, lower pulmonary vascular resistance and diastolic pulmonary pressure gradient, larger left atrium at rest, and higher PAWP during leg raise (15 ± 4 vs 10 ± 4 mmHg in non-LVDD group, p < 0.001) were observed in the occult LVDD group. The area under the ROC curve for PAWP after leg raise was largest at 0.83 (95% CI: 0.70-0.95, p = 0.001). About one-third (29%) of SSc patients with normal haemodynamics at rest showed occult LVDD. A higher PAWP after leg raise could be useful for detecting occult LVDD., (© 2022. The Author(s).)

A variety of eye-related symptoms due to the overuse of digital devices is collectively referred to as computer vision syndrome (CVS). In this study, a web-based survey about mind and body functions, including eye strain, was conducted on 1998 Japanese volunteers. To investigate the biological mechanisms behind CVS, a multi-trait genome-wide association study (GWAS), a multivariate analysis on individual-level multivariate data, was performed based on the structural equation modeling methodology assuming a causal pathway for a genetic variant to influence each symptom via a single common latent variable. Twelve loci containing lead variants with a suggestive level of significance were identified. Two loci showed relatively strong signals and were associated with TRABD2B relative to the Wnt signaling pathway and SDK1 having neuronal adhesion and immune functions, respectively. By utilizing publicly available eQTL data, colocalization between GWAS and eQTL signals for four loci was detected, and a locus on 2p25.3 showed a strong colocalization (PPH 4  > 0.9) on retinal MYT1L, known to play an important role in neuronal differentiation. This study suggested that the use of multivariate questionnaire data and multi-trait GWAS can lead to biologically reasonable findings and enhance our genetic understanding of complex relationships among symptoms related to CVS.

The pathophysiology and treatment of acute decompensated heart failure (HF) in the presence of chronic kidney disease (CKD) remain ill defined. Here we compared the prognostic factors for 1-year mortality in patients with acute HF with and without CKD. We retrospectively studied 392 consecutive patients with acute decompensated HF. CKD as a comorbidity in these patients was defined by an estimated glomerular filtration rate of <60 mL/min/1.73 m 2 . Potential risk factors for 1-year mortality were selected by univariate analyses; then multivariate Cox regression analysis with forward selection (likelihood ratio) was performed to identify significant factors. Across the study cohort, 65% of patients had CKD, and the 1-year mortality rate was 9.2%. In the HF with CKD group, older age, lower systolic blood pressure at admission, discharge medications without beta-blockers, and discharge medications without diuretics were independent risk factors for 1-year mortality. In contrast, coexisting chronic obstructive pulmonary disease and higher C-reactive protein levels were independent risk factors for 1-year mortality in the HF without CKD group. Kaplan-Meier survival curves showed that discharge medications with no beta-blockers or diuretics correlated with significantly lower survival rates in patients with CKD (P < 0.001 in both groups, log-rank test), but not in patients without CKD (P = 0.822 and P = 0.374, respectively, log-rank test). Thus, there were significant differences in the prognostic factors for 1-year mortality between acute HF patients with and without CKD including beta-blocker and diuretic treatments. These findings suggest that patients with HF might benefit from individualized therapies.

In photosynthetic organisms, photoprotection to avoid overexcitation of photosystems is a prerequisite for survival. Green algae have evolved light-inducible photoprotective mechanisms mediated by genes such as light-harvesting complex stress-related (LHCSR). Studies on the light-dependent regulation of LHCSR expression in the green alga Chlamydomonas reinhardtii have revealed that photoreceptors for blue light (phototropin) and ultraviolet light perception (UVR8) play key roles in initiating photoprotective signal transduction. Although initial light perception via phototropin or UVR8 is known to result in increased LHCSR3 and LHCSR1 gene expression, respectively, the mechanisms of signal transduction from the input (light perception) to the output (gene expression) remain unclear. In this study, to further elucidate the signal transduction pathway of the photoprotective response of green algae, we established a systematic screening protocol for UV-inducible LHCSR1 gene expression mutants using a bioluminescence reporter assay. Following random mutagenesis screening, we succeeded in isolating mutants deficient in LHCSR1 gene and protein expression after UV illumination. Further characterization revealed that the obtained mutants could be separated into 3 different phenotype groups, the "UV-specific", "LHCSR1-promoter/transcript-specific" and "general photoprotective" mutant groups, which provided further insight into photoprotective signal transduction in C. reinhardtii.

Pancreatic cancer has the lowest 5 year survival rate among all cancers. Several extracellular factors are involved in the development and metastasis of pancreatic cancer to distant organs. Exosomes are lipid-bilayer, membrane-enclosed nanoparticles that are recognised as important mediators of cell-to-cell communications. However, the role of exosomes released from pancreatic cancer cells in tumour micro-environment remains unknown. Here, we show that exosomes released from pancreatic cancer PK-45H cells activate various gene expressions in human umbilical vein endothelial cells (HUVECs) by in vitro analyses. In addition, these exosomes released from PK-45H cells promote phosphorylation of Akt and ERK1/2 signalling pathway molecules and tube formation via dynamin-dependent endocytosis in HUVECs. Our findings suggested that exosomes released from pancreatic cancer cells may act as a novel angiogenesis promoter.

Transfer of nitrogen fixation ability to plants, especially crops, is a promising approach to mitigate dependence on chemical nitrogen fertilizer and alleviate environmental pollution caused by nitrogen fertilizer run-off. However, the need to transfer a large number of nitrogen fixation (nif) genes and the extreme vulnerability of nitrogenase to oxygen constitute major obstacles for transfer of nitrogen-fixing ability to plants. Here we demonstrate functional expression of a cyanobacterial nitrogenase in the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803). A 20.8-kb chromosomal fragment containing 25 nif and nif-related genes of the diazotrophic cyanobacterium Leptolyngbya boryana was integrated into a neutral genome site of Synechocystis 6803 by five-step homologous recombination together with the cnfR gene encoding the transcriptional activator of the nif genes to isolate CN1. In addition, two other transformants CN2 and CN3 carrying additional one and four genes, respectively, were isolated from CN1. Low but significant nitrogenase activity was detected in all transformants. This is the first example of nitrogenase activity detected in non-diazotrophic photosynthetic organisms. These strains provide valuable platforms to investigate unknown factors that enable nitrogen-fixing growth of non-diazotrophic photosynthetic organisms, including plants.

Coral reef ecosystems rely on stable symbiotic relationship between the dinoflagellate Symbiodinium spp. and host cnidarian animals. The collapse of such symbiosis could cause coral 'bleaching' and subsequent host death. Despite huge interest on Symbiodinium, lack of mutant strains and readily available genetic tools have hampered molecular research. A major issue was the tolerance to marker antibiotics. Here, we isolated Symbiodinium mutants requiring uracil for growth, and hence, useful in transformation screening. We cultured Symbiodinium spp. cells in the presence of 5-fluoroorotic acid (5FOA), which inhibits the growth of cells expressing URA3 encoding orotidine-5'-monophosphate decarboxylase, and isolated cells that require uracil for growth. Sequence analyses and genetic complementation tests using yeast demonstrated that one of the mutant cell lines had a point mutation in URA3, resulting in a splicing error at an unusual exon-intron junction, and consequently, loss of enzyme activity. This mutant could maintain a symbiotic relationship with the model sea anemone Exaiptasia pallida only in sea water containing uracil. Results show that the URA3 mutant will be a useful tool for screening Symbiodinium transformants, both ex and in hospite, as survival in the absence of uracil is possible only upon successful introduction of URA3.

Vici syndrome (VICIS) is a rare, autosomal recessive neurodevelopmental disorder with multisystem involvement characterized by agenesis of the corpus callosum, cataracts, cardiomyopathy, combined immunodeficiency, developmental delay, and hypopigmentation. Mutations in EPG5, a gene that encodes a key autophagy regulator, have been shown to cause VICIS, however, the precise pathomechanism underlying VICIS is yet to be clarified. Here, we describe detailed clinical (including brain MRI and muscle biopsy) and genetic features of nine Japanese patients with VICIS. Genetic dissection of these nine patients from seven families identified 14 causative mutations in EPG5. These included five nonsense, two frameshift, three splicing, one missense, and one multi-exon deletion mutations, and two initiation codon variants. Furthermore, cultured skin fibroblasts (SFs) from two affected patients demonstrated partial autophagic dysfunction. To investigate the function of EPG5, siRNA based EPG5 knock-down, and CRISPR/Cas9 mediated EPG5 knock-out HeLa cells were generated. EPG5-depleted cells exhibited a complete block of autophagic flux caused by defective autophagosome-lysosome fusion. Unexpectedly, endocytic degradation was normal in both VICIS SFs and EPG5 depleted cells, suggesting that EPG5 function is limited to the regulation of autophagosome-lysosome fusion.

Smith-Lemli-Opitz syndrome is an autosomal recessive disease caused by mutations in 7-dehydrocholesterol reductase ( DHCR7 ), which is rarely observed in Japan. We report a Japanese case with 46,XY disorder of sex development and Y-shaped 2-3 toe syndactyly. DHCR7 gene analysis revealed compound heterozygous mutations including the novel mutation H442R. Early diagnosis led to starting cholesterol treatment at an early age., Competing Interests: The authors declare no conflict of interest.

Prostacyclin synthase (PGIS) and microsomal prostaglandin E synthase-1 (mPGES-1) are prostaglandin (PG) terminal synthases that function downstream of inducible cyclooxygenase (COX)-2 in the PGI2 and PGE2 biosynthetic pathways, respectively. mPGES-1 has been shown to be involved in various COX-2-related diseases such as inflammatory diseases and cancers, but it is not yet known how PGIS is involved in these COX-2-related diseases. Here, to clarify the pathophysiological role of PGIS, we investigated the phenotypes of PGIS and mPGES-1 individual knockout (KO) or double KO (DKO) mice. The results indicate that a thioglycollate-induced exudation of leukocytes into the peritoneal cavity was suppressed by the genetic-deletion of PGIS. In the PGIS KO mice, lipopolysaccharide-primed pain nociception (as assessed by the acetic acid-induced writhing reaction) was also reduced. Both of these reactions were suppressed more effectively in the PGIS/mPGES-1 DKO mice than in the PGIS KO mice. On the other hand, unlike mPGES-1 deficiency (which suppressed azoxymethane-induced colon carcinogenesis), PGIS deficiency up-regulated both aberrant crypt foci formation at the early stage of carcinogenesis and polyp formation at the late stage. These results indicate that PGIS and mPGES-1 cooperatively exacerbate inflammatory reactions but have opposing effects on carcinogenesis, and that PGIS-derived PGI2 has anti-carcinogenic effects.