Identification of an antiviral component from the venom of the scorpion Liocheles australasiae using transcriptomic and mass spectrometric analyses

Scorpion venom contains a variety of biologically active peptides. Among them, neurotoxins are major components in the venom, but it also contains peptides that show antimicrobial activity. Previously, we identified three insecticidal peptides from the venom of the Liocheles australasiae scorpion, but activities and structures of other venom components remained unknown. In this study, we performed a transcriptome analysis of the venom gland of the scorpion L. australasiae to gain a comprehensive understanding of its venom components. The result shows that potassium channel toxin-like peptides were the most diverse, whereas only a limited number of sodium channel toxin-like peptides were observed. In addition to these neurotoxin-like peptides, many non-disulfide-bridged peptides were identified, suggesting that these components have some critical roles in the L. australasiae venom. In this study, we also isolated a component with antiviral activity against hepatitis C virus using a bioassay-guided fractionation approach. By integrating mass spectrometric and transcriptomic data, we successfully identified LaPLA₂-1 as an anti-HCV component. LaPLA₂-1 is a phospholipase A₂ having a heterodimeric structure that is N-glycosylated at the N-terminal region. Since the antiviral activity of LaPLA₂-1 was inhibited by a PLA₂ inhibitor, the enzymatic activity of LaPLA₂-1 is likely to be involved in its antiviral activity

A Novel CLEIA for FGF23

Introduction: Measurement of fibroblast growth factor 23 (FGF23) has been reported to be clinically useful for the differential diagnosis of chronic hypophosphatemia. However, assays for research use only are available in Japan. Thus, the objective of this study was to examine the clinical utility of a novel and automated chemiluminescent enzyme immunoassay for the measurement of FGF23. Materials and Methods: Participants were recruited from July 2015 to January 2017 at six facilities in Japan. Thirty-eight patients with X-linked hypophosphatemic rickets (XLH; 15 males, 23 females, age 0–66 years), five patients with tumour-induced osteomalacia (TIO; 3 males, 2 females, age 60–73 years), and twenty-two patients with hypophosphatemia (11 males, 11 females, age 1–75 years) caused due to other factors participated in this study. Results: With the clinical cut-off value of FGF23 at 30.0 pg/mL indicated in the Diagnostic Guideline of Rickets/Osteomalacia in Japan, the sensitivity and specificity of FGF23-related hypophosphatemic rickets/osteomalacia without vitamin D deficiency (disease group-1) were 100% and 81.8%, respectively, which distinguished it from non-FGF23-related hypophosphatemia (disease group-2). Furthermore, the diagnostic sensitivity of FGF23-related hypophosphatemia with vitamin D deficiency remained at 100%. Among the four patients with FGF23 levels ≥ 30.0 pg/mL in disease group-2, two patients with relatively higher FGF23 values were suspected to have genuine FGF23-related hypophosphatemia, due to the ectopic production of FGF23 in pulmonary and prostate small cell carcinomas. Conclusion: The novel FGF23 assay tested in this study is useful for the differential diagnosis of hypophosphatemic rickets/osteomalacia in a clinical setting

Pyridoxal 5′-phosphate and related metabolites in hypophosphatasia: Effects of enzyme replacement therapy

Objective To investigate the utility of serum pyridoxal 5′-phosphate (PLP), pyridoxal (PL), and 4-pyridoxic acid (PA) as a diagnostic marker of hypophosphatasia (HPP) and an indicator of the effect of, and patient compliance with, enzyme replacement therapy (ERT), we measured PLP, PL, and PA concentrations in serum samples from HPP patients with and without ERT. Methods Blood samples were collected from HPP patients and serum was frozen as soon as possible (mostly within one hour). PLP, PL, and PA concentrations were analyzed using high-performance liquid chromatography with fluorescence detection after pre-column derivatization by semicarbazide. We investigated which metabolites are associated with clinical phenotypes and how these metabolites change with ERT. Results Serum samples from 20 HPP patients were analyzed. The PLP-to-PL ratio and PLP concentration were elevated in all HPP patients. They correlated negatively with serum alkaline phosphatase (ALP) activity and showed higher values in more severe phenotypes (perinatal severe and infantile HPP) compared with other phenotypes. PL concentration was reduced only in perinatal severe HPP. ERT reduced the PLP-to-PL ratio to mildly reduced or low-normal levels and the PLP concentration was reduced to normal or mildly elevated levels. Urine phosphoethanolamine (PEA) concentration did not return to normal levels with ERT in most patients. Conclusions The serum PLP-to-PL ratio is a better indicator of the effect of ERT for HPP than serum PLP and urine PEA concentrations, and a PLP-to-PL ratio of <4.0 is a good indicator of the effect of, and patient compliance with, ERT

Surrounding Gastric Mucosa Findings Facilitate Diagnosis of Gastric Neoplasm as Gastric Adenoma or Early Gastric Cancer

Background and Aim. It is difficult to master the skill of discriminating gastric adenoma from early gastric cancer by conventional endoscopy or magnifying endoscopy combined with narrow-band imaging, because the colors and morphologies of these neoplasms are occasionally similar. We focused on the surrounding gastric mucosa findings in order to determine how to discriminate between early gastric cancer and gastric adenoma by analyzing the characteristics of the gastric background mucosa. Methods. We retrospectively examined 146 patients who underwent endoscopic submucosal dissection for gastric neoplasm between October 2009 and January 2015. The boundary of atrophic gastritis was classified endoscopically according to the Kimura-Takemoto classification system. Of 146 lesions, 63 early gastric cancers and 21 gastric adenomas were ultimately evaluated and assessed. Results. Almost all gastric adenomas were accompanied by open-type gastritis, whereas 47 and 16 early gastric cancers were accompanied by open-type and closed-type gastritis, respectively (p = 0.037). Conclusions. The evaluation of the boundary of atrophic gastritis associated with gastric neoplasms appears to be useful for discrimination between early gastric cancer and gastric adenoma. When gastric neoplasm is present in the context of surrounding localized gastric atrophy, gastric cancer is probable but not certain

Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector

Background: Disease modeling with patient-derived induced pluripotent stem cells (iPSCs) is a powerful tool forelucidating the mechanisms underlying disease pathogenesis and developing safe and effective treatments. Patientperipheral blood (PB) cells are used for iPSC generation in many cases since they can be collected with minimuminvasiveness. To derive iPSCs that lack immunoreceptor gene rearrangements, hematopoietic stem and progenitorcells (HSPCs) are often targeted as the reprogramming source. However, the current protocols generally requireHSPC mobilization and/or ex vivo expansion owing to their sparsity at the steady state and low reprogrammingefficiencies, making the overall procedure costly, laborious, and time-consuming.Methods: We have established a highly efficient method for generating iPSCs from non-mobilized PB-derivedCD34+ HSPCs. The source PB mononuclear cells were obtained from 1 healthy donor and 15 patients and werekept frozen until the scheduled iPSC generation. CD34+ HSPC enrichment was done using immunomagnetic beads,with no ex vivo expansion culture. To reprogram the CD34+-rich cells to pluripotency, the Sendai virus vectorSeVdp-302L was used to transfer four transcription factors: KLF4, OCT4, SOX2, and c-MYC. In this iPSC generationseries, the reprogramming efficiencies, success rates of iPSC line establishment, and progression time wererecorded. After generating the iPSC frozen stocks, the cell recovery and their residual transgenes, karyotypes, T cellreceptor gene rearrangement, pluripotency markers, and differentiation capability were examined.Results：We succeeded in establishing 223 iPSC lines with high reprogramming efficiencies from 15 patients with 8 different disease types. Our method allowed the rapid appearance of primary colonies (~ 8 days), all of which were expandable under feeder-free conditions, enabling robust establishment steps with less workload. After thawing, the established iPSC lines were verified to be pluripotency marker-positive and of non-T cell origin. A majority of the iPSC lines were confirmed to be transgene-free, with normal karyotypes. Their trilineage differentiation capability was also verified in a defined in vitro assay.Conclusion：This robust and highly efficient method enables the rapid and cost-effective establishment of transgene-free iPSC lines from a small volume of PB, thus facilitating the biobanking of patient-derived iPSCs and their use for the modeling of various diseases

Functional analyses of a novel missense and other mutations of the vitamin D receptor in association with alopecia

WOS: 000405172600069PubMed ID: 28698609Hereditary 1,25-dihydroxyvitamin D-resistant rickets (HVDRR) is a rare disorder, caused by bialellic mutations of the vitamin D receptor (VDR) gene, sometimes associated with alopecia. The aim of this study is to elucidate the mechanism of functional disruption of a novel mutation, detected in a patient with HVDRR, comparing to other mutations with or without alopecia. The patient was a 2-year-old girl with alopecia, who was clinically diagnosed as HVDRR. Genetic analysis revealed a novel homozygous mutation, S360P, located in ligand binding domain (LBD). The mutation was predicted as not disease causing by Polyphen2 and SIFT. But the transcriptional activity of S360P was disrupted as well as other reported mutations, Q152X (located in the hinge lesion), and R274L, H305Q (located in LBD). Following assays revealed no ligand binding affinity, no interaction with cofactors or RXR and no functioning of nuclear localization signals. Our results provide an additional evidence for the previous findings suggesting that DNA binding by the VDR/RXR heterodimer is essential for the function of the VDR in hair development. In conclusion, we identified a novel missense mutation of VDR causing HVDRR with alopecia. Functional analyses revealed that the single amino acid substitution could disrupt the function of the protein.Ministry of Education, Culture, Sports, Science, and Technology JapanMinistry of Education, Culture, Sports, Science and Technology, Japan (MEXT) [24791042, 23591489]; JSPS KAKENHIMinistry of Education, Culture, Sports, Science and Technology, Japan (MEXT)Japan Society for the Promotion of ScienceGrants-in-Aid for Scientific Research (KAKENHI) [JP16J04675]We are grateful to the patient and her parents for all their help and participation in this study. We thank Ms. Reiko Onai for technical support. This study was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology Japan (to TI: 24791042 and SK: 23591489), JSPS KAKENHI (to MT: JP16J04675)

Hereditary 1,25-dihydroxyvitamin D-resistant rickets (HVDRR) caused by a VDR mutation: A novel mechanism of dominant inheritance

Hereditary 1,25-dihydroxyvitamin D-resistant rickets (HVDRR) is caused by mutations in the VDR gene, and its inheritance is autosomal recessive. In this report, we aimed to confirm whether HVDRR is occasionally inherited as a dominant trait. An 18-month-old Japanese boy was evaluated for short stature and bowlegs. His father had been treated for rickets during childhood, and his paternal grandfather had bowlegs. We diagnosed him with HVDRR based on laboratory data and radiographic evidence of rickets. Sequence analyses of VDR were performed, and the functional consequences of the detected mutations were analyzed for transcriptional activity, ligand binding, and interaction with the retinoid X receptor, cofactors, and the vitamin D response element (VDRE). A novel mutation (Q400LfsX7) and a reported variant (R370H) were identified in the patient. Heterozygous Q400LfsX7 was detected in his father, and heterozygous R370H was detected in his healthy mother. Functional studies revealed that the transcriptional activity of Q400LfsX7-VDR was markedly disturbed. The mutant had a dominant-negative effect on wild-type-VDR, and the ligand binding affinity of Q400LfsX7-VDR was completely impaired. Interestingly, Q400LfsX7-VDR had a strong interaction with corepressor NCoR and could interact with VDRE without the ligand. R370H-VDR was functionally similar to wild-type-VDR. In conclusion, we found a dominant-negative mutant of VDR causing dominantly inherited HVDRR through a constitutive corepressor interaction, a mechanism similar to that in dominantly inherited thyroid hormone receptor mutations. Our report together with a reported pedigree suggested a distinct inheritance of HVDRR and enriched our understanding of VDR abnormalities

Endothelin regulates neural crest deployment and fate to form great vessels through Dlx5/Dlx6-independent mechanisms

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