Development of a nanobody-based amperometric immunocapturing assay for sensitive and specific detection of Toxocara canis excretory-secretory antigen

Introduction Human Toxocariasis (HT) is a zoonosis that, despite of its wide distribution around the world, remains poorly diagnosed. The identification of specific IgG immunoglobulins against the Toxocara canis Excretory-Secretory antigen (TES), a mix of glycoproteins that the parasite releases during its migration to the target organs in infected patients, is currently the only laboratory tool to detect the disease. The main drawbacks of this test are the inability to distinguish past and active infections together with lack of specificity. These factors seriously hamper the diagnosis, follow-up and control of the disease. Aim To develop an amperometric immunocapturing diagnostic assay based on single domain immunoglobulins from camelids (nanobodies) for specific and sensitive detection of TES. Methods After immunization of an alpaca (Vicugna pacos) with TES, RNA from peripheral blood lymphocytes was used as template for cDNA amplification with oligo dT primers and library construction. Isolation and screening of TES-specific nanobodies were carried out by biopanning and the resulting nanobodies were expressed in Escherichia coli. Two-epitopes amperometric immunocapturing assay was designed using paramagnetic beads coated with streptavidin and bivalent nanobodies. Detection of the system was carried out with nanobodies chemically coupled to horseradish peroxidase. The reaction was measured by amperometry and the limit of detection (LOD) was compared to conventional sandwich ELISA. Results We obtained three nanobodies that specifically recognize TES with no-cross reactivity to antigens of Ascaris lumbricoides and A. suum. The LOD of the assay using PBST20 0.05% as diluent was 100 pg/ml, 10 times more sensitive than sandwich ELISA. Conclusion Sensitive and specific detection of TES for discrimination of active and past infections is one of the most difficult challenges of T. canis diagnosis. The main advantage of our system is the use of two different nanobodies that specifically recognize two different epitopes in TES with a highly sensitive and straightforward readout. Considering that the amounts of TES available for detection in clinical samples are in the range of picograms or a few nanograms maximum, the LOD found in our experiments suggests that the test is potentially useful for the detection of clinically relevant cases of HT

Rotaviruses and Rotavirus Vaccines

This special edition of Pathogens brings together a breadth of information on rotavirus and global rotavirus vaccines, to highlight rotavirus research from across the world that represents recent advances in our knowledge of vaccine effectiveness, rotavirus epidemiology, genotypic diversity, and genomic characterisation

筑波大学計算科学研究センター 平成26年度 年次報告書

1 平成26年度重点施策および改善目標の達成状況　……　22 自己評価と課題　……　103 各研究部門の報告　……　13I.素粒子物理研究部門　……　13Ⅱ.宇宙・原子核物理研究部門　……　40 Ⅱ-1.宇宙分野　……　40 Ⅱ-2.原子核物理分野　……　63Ⅲ.量子物性研究部門　……　78Ⅳ.生命科学研究部門　……　96 Ⅳ-1.生命機能情報分野　……　96 Ⅳ-2.分子進化分野　……　108Ⅴ.地球環境研究部門　……　123Ⅵ.高性能計算システム研究部門　……　133Ⅶ.計算情報学研究部　……　166 Ⅶ-1.データ基盤分野　……　166 Ⅶ-2.計算メディア分野　……　18

Antioxidant and DPPH-Scavenging Activities of Compounds and Ethanolic Extract of the Leaf and Twigs of Caesalpinia bonduc L. Roxb.

Antioxidant effects of ethanolic extract of Caesalpinia bonduc and its isolated bioactive compounds were evaluated in vitro. The compounds included two new cassanediterpenes, 1α,7α-diacetoxy-5α,6β-dihydroxyl-cass-14(15)-epoxy-16,12-olide (1)and 12α-ethoxyl-1α,14β-diacetoxy-2α,5α-dihydroxyl cass-13(15)-en-16,12-olide(2); and others, bonducellin (3), 7,4’-dihydroxy-3,11-dehydrohomoisoflavanone (4), daucosterol (5), luteolin (6), quercetin-3-methyl ether (7) and kaempferol-3-O-α-L-rhamnopyranosyl-(1Ç2)-β-D-xylopyranoside (8). The antioxidant properties of the extract and compounds were assessed by the measurement of the total phenolic content, ascorbic acid content, total antioxidant capacity and 1-1-diphenyl-2-picryl hydrazyl (DPPH) and hydrogen peroxide radicals scavenging activities.Compounds 3, 6, 7 and ethanolic extract had DPPH scavenging activities with IC50 values of 186, 75, 17 and 102 μg/ml respectively when compared to vitamin C with 15 μg/ml. On the other hand, no significant results were obtained for hydrogen peroxide radical. In addition, compound 7 has the highest phenolic content of 0.81±0.01 mg/ml of gallic acid equivalent while compound 8 showed the highest total antioxidant capacity with 254.31±3.54 and 199.82±2.78 μg/ml gallic and ascorbic acid equivalent respectively. Compound 4 and ethanolic extract showed a high ascorbic acid content of 2.26±0.01 and 6.78±0.03 mg/ml respectively.The results obtained showed the antioxidant activity of the ethanolic extract of C. bonduc and deduced that this activity was mediated by its isolated bioactive compounds

Public health interventions to promote oral health and well-being in patients with type 2 diabetes: a systematic review

Background and objective: Poor oral health and type 2 diabetes mellitus (T2DM) are chronic conditions affecting a wide proportion of the population. Both conditions share many risk factors and are linked by a chronic inflammation state. This review aimed at identifying public health interventions that could promote oral health and diabetes control in patients with poor oral health and T2DM. Methods: The systematic review was performed according to the PRISMA Statement and registered in the International Prospective Register of Systematic Reviews (PROSPERO registration: CRD42022310974). Seven electronic databases were searched (PubMed, Scopus, Embase, Web of Science, CINAHL, PsycInfo, Cochrane Library) from inception to 21 January 2022, and additional hand searching was performed across reviews’ references. A qualitative analysis was conducted, including all primary studies on diabetic patients, about interventions whose effectiveness and/or feasibility was measured for at least one outcome related to oral health or T2DM. Results: Of the 3153 records obtained after deduplication, 89 studies were considered eligible for inclusion. The most frequently evaluated outcomes were HbA1c and fasting glucose for T2DM, and parameters such as probing depth, bleeding on probing and clinical attachment loss for periodontitis. Most studies assessed the use of non-surgical periodontal treatment (especially scaling and root planing, sometimes corroborated by antibiotics): evidence confirmed effectiveness on periodontal parameters, but was more contrasting regarding T2DM outcomes. Three studies evaluated interventions involving group education for lifestyle modification, which showed to be effective on both outcomes. Also, community-based oral hygiene interventions and glycaemic control appeared to improve T2DM and periodontal outcomes. Conclusions: A variety of interventions are described in the literature. Of those included in this review, many indicated that there is a potential opportunity to promote good oral health alongside T2DM. An integrated approach involving health education, oral hygiene and glycaemic control may offer synergic improvement of both conditions