The Membrane Activity of the Amphibian Temporin B Peptide Analog TB_KKG6K Sheds Light on the Mechanism That Kills Candida albicans

Fungal infections with the opportunistic human pathogen C. albicans are associated with high mortality rates in immunocompromised patients. This is partly due to the yeast's ability to rapidly develop resistance toward currently available antifungals. Temporin B (TB) is a 13-amino-acid-long, cationic peptide secreted by the granular glands of the European frog Rana temporaria. We recently showed that the modified TB peptide analog TB_KKG6K rapidly killed planktonic and sessile Candida albicans at low micromolar concentrations and was neither hemolytic nor cytotoxic to mammalian cells in vitro. The present study aimed to shed light into its mechanism of action, with a focus on its fungal cell membrane activity. We utilized different fluorescent dyes to prove that it rapidly induces membrane depolarization and permeabilization. Studies on model membrane systems revealed that the TB analog undergoes hydrophobic and electrostatic membrane interactions, showing a preference for anionic lipids, and identified phosphatidylinositol and cardiolipin as possible peptide targets. Fluorescence microscopy using fluorescein isothiocyanate-labeled TB_KKG6K in the presence of the lipophilic dye FM4-64 indicated that the peptide compromises membrane integrity and rapidly enters C. albicans cells in an energy-independent manner. Peptide-treated cells analyzed by cryo-based electron microscopy exhibited no signs of cell lysis; however, subcellular structures had disintegrated, suggesting that intracellular activity may form part of the killing mechanism of the peptide. Taken together, this study proved that TB_KKG6K compromises C. albicans membrane function, which explains the previously observed rapid, fungicidal mode of action and supports its great potential as a future anti-Candida therapeutic. IMPORTANCE Fungal infections with the opportunistic human pathogen C. albicans are associated with high mortality rates in immunocompromised patients. This is partly due to the yeast's ability to rapidly develop resistance toward currently available antifungals. Small, cationic, membrane-active peptides are promising compounds to fight against resistance development, as many of them effectuate rapid fungal cell death. This fast killing is believed to hamper the development of resistance, as the fungi do not have sufficient time to adapt to the antifungal compound. We previously reported that the synthetic variant of the amphibian TB peptide, TB_KKG6K, rapidly kills C. albicans. In the current study, the mechanism of action of the TB analog was investigated. We show that this TB analog is membrane-active and impairs cell membrane function, highlighting its potential to be developed as an attractive alternative anti-C. albicans therapeutic that may hinder the development of resistance

Kernel Lot Distribution Assessment (KeLDA): a Comparative Study of Protein and DNA-Based Detection Methods for GMO Testing

Monitoring of market products for detection of genetically modified organisms (GMO) is needed to comply with legislation in force in many regions of the world, to enforce traceability and to allow official control along the production and the distribution chains. This objective can be more easily achieved if reliable, time and cost-effective analytical methods are available. A GMO can be detected using either DNA-based or protein-based methods; both present advantages and disadvantages. The objective of this work was to assess the performance of a protein-based (lateral flow strips—LFT) and of a DNA-based (polymerase chain reaction—PCR) detection method for GMO analysis. One thousand five hundred samples of soybean, deriving from the sampling of 15 independent bulk lots in large shipments, were analysed to assess and compare the performance of the analytical methods and evaluate their suitability for GMO testing. Several indicators were used to compare the performance of the methods, including the percentage correlation between the PCR and LFT results. The GMO content of the samples ranged from 0 up to 100 %, allowing a full assessment of both analytical approaches with respect to all possible GMO content scenarios. The study revealed a very similar performance of the two methodologies, with low false-negative and false-positive results, and a very satisfactory capacity of both methods in detecting low amounts of target. While determining the fitness for purpose of both analytical approaches, this study also underlines the importance of alternative method characteristics, like costs and time

The primary structure of a monoclonal λ-type immunoglobulin L-chain of subgroup II (Bence-Jones protein NEI): Evolutionary origin of antibody variability

ObjectiveThe objective of this study is to assess the gestational age at detection and prevalence of anencephaly in the North of The Netherlands over a 5-year period. MethodsA case list of all cases of anencephaly from two fetal medicine units was compiled. Cases were included if the estimated due date was between 1 August 2008 and 31 July 2013. ResultsOverall prevalence of anencephaly was 5.4 per 10.000 pregnancies (n=110). The majority of cases (69%) was detected before 18 weeks' gestation. Factors determining successful early diagnosis were competence level of the sonographers, with a significantly higher detection rate when scans were performed by a sonographer licensed by the Fetal Medicine Foundation (FMF) for nuchal translucency measurement (p=0.001), and gestational age at or beyond 11weeks of gestation (p=0.024). ConclusionImproving detection of anencephaly in the first trimester requires ultrasound screening at or after 11weeks of gestation, performed by experienced sonographers trained in recognizing fetal anomalies. Sonographers should be instructed that the goal of the first trimester scan is not only to measure nuchal translucency thickness but also to exclude major anomalies. (c) 2015 John Wiley & Sons, Ltd

The German National Registry of Primary Immunodeficiencies (2012-2017)

Introduction: The German PID-NET registry was founded in 2009, serving as the first national registry of patients with primary immunodeficiencies (PID) in Germany. It is part of the European Society for Immunodeficiencies (ESID) registry. The primary purpose of the registry is to gather data on the epidemiology, diagnostic delay, diagnosis, and treatment of PIDs. Methods: Clinical and laboratory data was collected from 2,453 patients from 36 German PID centres in an online registry. Data was analysed with the software Stata® and Excel. Results: The minimum prevalence of PID in Germany is 2.72 per 100,000 inhabitants. Among patients aged 1–25, there was a clear predominance of males. The median age of living patients ranged between 7 and 40 years, depending on the respective PID. Predominantly antibody disorders were the most prevalent group with 57% of all 2,453 PID patients (including 728 CVID patients). A gene defect was identified in 36% of patients. Familial cases were observed in 21% of patients. The age of onset for presenting symptoms ranged from birth to late adulthood (range 0–88 years). Presenting symptoms comprised infections (74%) and immune dysregulation (22%). Ninety-three patients were diagnosed without prior clinical symptoms. Regarding the general and clinical diagnostic delay, no PID had undergone a slight decrease within the last decade. However, both, SCID and hyper IgE- syndrome showed a substantial improvement in shortening the time between onset of symptoms and genetic diagnosis. Regarding treatment, 49% of all patients received immunoglobulin G (IgG) substitution (70%—subcutaneous; 29%—intravenous; 1%—unknown). Three-hundred patients underwent at least one hematopoietic stem cell transplantation (HSCT). Five patients had gene therapy. Conclusion: The German PID-NET registry is a precious tool for physicians, researchers, the pharmaceutical industry, politicians, and ultimately the patients, for whom the outcomes will eventually lead to a more timely diagnosis and better treatment