We have isolated a microsporidium from a laboratory stock of the European grape vine moth, Lobesia botrana Den. et Schiff. (Lepidoptera, Tortricidae). Screening of this stock showed an infection rate of more than 90%, whereas field collected larvae from three different locations in Rhineland-Palatinate (Germany) did not demonstrate any signs of infection. Light and electron microscopic investigations of infected insects showed that gross pathology, morphology, and ultrastructure of the microsporidium are similar to those described earlier for Pleistophora legeri. Comparative phylogenetic analysis of the small subunit rDNA using maximum likelihood, maximum parsimony, and neighbour joining distance methods showed that our isolate was closely related to Cystosporogenes operophterae. Based on our morphological and molecular investigations we propose to rename this species Cystosporogenes legeri nov. comb.

A large number of novel disease genes have been identified by homozygosity mapping and the positional candidate approach. In this study we used single nucleotide polymorphism (SNP) array-based, whole genome homozygosity mapping as the first step to a molecular diagnosis in the highly heterogeneous muscle disease, limb girdle muscular dystrophy (LGMD). In a consanguineous family, both affected siblings showed homozygous blocks on chromosome 15 corresponding to the LGMD2A locus. Direct sequencing of CAPN3, encoding calpain-3, identified a homozygous deletion c.483delG (p.Ile162SerfsX17). In a sporadic LGMD patient complete absence of caveolin-3 on Western blot was observed. However, a mutation in CAV3 could not be detected. Homozygosity mapping revealed a large homozygous block at the LGMD2I locus, and direct sequencing of FKRP encoding fukutin-related-protein detected the common homozygous c.826 C>A (p.Leu276Ile) mutation. Subsequent re-examination of this patient's muscle biopsy showed aberrant α-dystroglycan glycosylation. In summary, we show that whole-genome homozygosity mapping using low cost SNP arrays provides a fast and non-invasive method to identify disease-causing mutations in sporadic patients or sibs from consanguineous families in LGMD2. Furthermore, this is the first study describing that in addition to PTRF, encoding polymerase I and transcript release factor, FKRP mutations may cause secondary caveolin-3 deficiency.

Strict planetary protection practices are implemented during spacecraft assembly to prevent inadvertent transfer of earth microorganisms to other planetary bodies. Therefore, spacecraft are assembled in cleanrooms, which undergo strict cleaning and decontamination procedures to reduce total microbial bioburden. We wanted to evaluate if these practices selectively favor survival and growth of hardy microorganisms, such as pathogens. Three geographically distinct cleanrooms were sampled during the assembly of three NASA spacecraft: The Lockheed Martin Aeronautics' Multiple Testing Facility during DAWN, the Kennedy Space Center's Payload Hazardous Servicing Facility (KSC-PHSF) during Phoenix, and the Jet Propulsion Laboratory's Spacecraft Assembly Facility during Mars Science Laboratory. Sample sets were collected from the KSC-PHSF cleanroom at three time points: before arrival of the Phoenix spacecraft, during the assembly and testing of the Phoenix spacecraft, and after removal of the spacecraft from the KSC-PHSF facility. All samples were subjected to metagenomic shotgun sequencing on an Illumina HiSeq 2500 platform. Strict decontamination procedures had a greater impact on microbial communities than sampling location Samples collected during spacecraft assembly were dominated by Acinetobacter spp. We found pathogens and potential virulence factors, which determine pathogenicity in all the samples tested during this study. Though the relative abundance of pathogens was lowest during the Phoenix assembly, potential virulence factors were higher during assembly compared to before and after assembly, indicating a survival advantage. Decreased phylogenetic and pathogenic diversity indicates that decontamination and preventative measures were effective against the majority of microorganisms and well implemented, however, pathogen abundance still increased over time. Four potential pathogens, Acinetobacter baumannii, Acinetobacter lwoffii, Escherichia coli and Legionella pneumophila, and their corresponding virulence factors were present in all cleanroom samples. This is the first functional metagenomics study describing presence of pathogens and their corresponding virulence factors in cleanroom environments. The results of this study should be considered for microbial monitoring of enclosed environments such as schools, homes, hospitals and more isolated habitation such the International Space Station and future manned missions to Mars.

Bariatric surgery is currently one of the most effective treatments for obesity and leads to significant weight reduction, improved cardiovascular risk factors and overall survival in treated patients. To date, most studies focused on short-term effects of bariatric surgery on the metabolic profile and found high variation in the individual responses to surgery. The aim of this study was to identify relevant metabolic changes not only shortly after bariatric surgery (Roux-en-Y gastric bypass) but also up to one year after the intervention by using untargeted metabolomics. 132 serum samples taken from 44 patients before surgery, after hospital discharge (1-3 weeks after surgery) and at a 1-year follow-up during a prospective study (NCT01271062) performed at two study centers (Austria and Switzerland). The samples included 24 patients with type 2 diabetes at baseline, thereof 9 with diabetes remission after one year. The samples were analyzed by using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS, HILIC-QExactive). Raw data was processed with XCMS and drift-corrected through quantile regression based on quality controls. 177 relevant metabolic features were selected through Random Forests and univariate testing and 36 metabolites were identified. Identified metabolites included trimethylamine-N-oxide, alanine, phenylalanine and indoxyl-sulfate which are known markers for cardiovascular risk. In addition we found a significant decrease in alanine after one year in the group of patients with diabetes remission relative to non-remission. Our analysis highlights the importance of assessing multiple points in time in subjects undergoing bariatric surgery to enable the identification of biomarkers for treatment response, cardiovascular benefit and diabetes remission. Key-findings include different trend pattern over time for various metabolites and demonstrated that short term changes should not necessarily be used to identify important long term effects of bariatric surgery.

Experimental and bioinformatic studies of transcription initiation by RNA polymerase II (RNAP2) have revealed a mechanism of RNAP2 transcription initiation less uniform across gene promoters than initially thought. However, the general transcription factor TFIIB is presumed to be universally required for RNAP2 transcription initiation. Based on bioinformatic analysis of data and effects of TFIIB knockdown in primary and transformed cell lines on cellular functionality and global gene expression, we report that TFIIB is dispensable for transcription of many human promoters, but is essential for herpes simplex virus-1 (HSV-1) gene transcription and replication. We report a novel cell cycle TFIIB regulation and localization of the acetylated TFIIB variant on the transcriptionally silent mitotic chromatids. Taken together, these results establish a new paradigm for TFIIB functionality in human gene expression, which when downregulated has potent anti-viral effects.

It is unknown if cholecalciferol is able to modify defects in regulatory T cells (Tregs) in type 1 diabetes (T1D). In this randomized, double-blind, placebo controlled trial 30 young patients with new-onset T1D were assigned to cholecalciferol (70IU/kgbodyweight/day) or placebo for 12months. Tregs were determined by FACS-analysis and functional tests were assessed with ex vivo suppression co-cultures at months 0, 3, 6 and 12. Suppressive capacity of Tregs increased (p<0.001) with cholecalciferol from baseline (-1.59±25.6%) to 3 (30.5±39.4%), 6 (44.6±23.8%) and 12months (37.2±25.0%) and change of suppression capacity from baseline to 12months was significantly higher (p<0.05) with cholecalciferol (22.2±47.2%) than placebo (-16.6±21.1%). Serum calcium and parathormone stayed within normal range. This is the first study, which showed that cholecalciferol improved suppressor function of Tregs in patients with T1D and vitamin D could serve as one possible agent in the development of immunomodulatory combination therapies for T1D.

The pharmacokinetic properties of liraglutide, a glucagon-like peptide-1 receptor agonist approved for the treatment of type 2 diabetes mellitus (T2D), have been established in healthy individuals and subjects with T2D. Liraglutide has been under investigation as adjunct treatment to insulin in type 1 diabetes mellitus (T1D). This single-center, double-blind, placebo-controlled, crossover, clinical pharmacology trial is the first to analyze the pharmacokinetic properties of liraglutide as add-on to insulin in T1D.

Inhalation treatment with nanoparticle containing aerosols appears a promising new therapeutic option but new formulations have to be assessed for efficacy and toxicity. We evaluated the utility of a VITROCELL®6 PT-CF+PARI LC SPRINT® Baby Nebulizer (PARI BOY) system compared with a conventional MicroSprayer. A549 cells were cultured in the air-liquid interface, exposed to nanoparticle aerosols and characterized by measurement of transepithelial electrical resistance and staining for tight junction proteins. Deposition and distribution rates of polystyrene particles and of carbon nanotubes on the cells were assessed. In addition, cytotoxicity of aerosols containing polystyrene particles was compared with cytotoxicity of polystyrene particles in suspension tested in submersed cultures. Exposure by itself in both exposure systems did not damage the cells. Deposition rates of aerosolized polystyrene particles were about 700 times and that of carbon nanotubes about 4 times higher in the MicroSprayer than in the VITROCELL®6 PT-CF system. Cytotoxicity of amine-functionalized polystyrene nanoparticles was significantly higher when applied as an aerosol on cell cultured in air-liquid interface culture compared with nanoparticle suspensions tested in submersed culture. The higher cytotoxicity of aerosolized nanoparticles underscores the importance of relevant exposure systems.

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I-IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab.

To evaluate the short-term cost-effectiveness of insulin degludec (degludec) vs insulin glargine 100 units/mL (glargine U100) from a Canadian public healthcare payer perspective in patients with type 2 diabetes (T2D) who are at high risk of cardiovascular events and hypoglycaemia.

Insulin dosing based on carbohydrate counting is the gold standard for improving glycaemic control in type 1 diabetes (T1D). This post hoc analysis aimed to explore the efficacy and safety of fast-acting insulin aspart (faster aspart) according to bolus dose adjustment method in people with T1D.

Reprimo (RPRM), a member of the RPRM gene family, is a tumor-suppressor gene involved in the regulation of the p53-mediated cell cycle arrest at G2/M. RPRM has been associated with malignant tumor progression and proposed as a potential biomarker for early cancer detection. However, the expression and role of RPRM, as well as its family, are poorly understood and their physiology is as yet unstudied. In this scenario, a model system like the zebrafish could serve to dissect the role of the RPRM family members in vivo. Phylogenetic analysis reveals that RPRM and RPRML have been differentially retained by most species throughout vertebrate evolution, yet RPRM3 has been retained only in a small group of distantly related species, including zebrafish. Herein, we characterized the spatiotemporal expression of RPRM (present in zebrafish as an infraclass duplication rprma/rprmb), RPRML and RPRM3 in the zebrafish. By whole-mount in situ hybridization (WISH) and fluorescent in situ hybridization (FISH), we demonstrate that rprm (rprma/rprmb) and rprml show a similar spatiotemporal expression profile during zebrafish development. At early developmental stages rprmb is expressed in somites. After one day post-fertilization, rprm (rprma/rprmb) and rprml are expressed in the notochord, brain, blood vessels and digestive tube. On the other hand, rprm3 shows the most unique expression profile, being expressed only in the central nervous system (CNS). We assessed the expression patterns of RPRM gene transcripts in adult zebrafish and human RPRM protein product in tissue samples by RT-qPCR and immunohistochemistry (IHC) staining, respectively. Strikingly, tissue-specific expression patterns of the RPRM transcripts and protein are conserved between zebrafish and humans. We propose the zebrafish as a powerful tool to elucidate the both physiological and pathological roles of the RPRM gene family.

Dominant congenital spinal muscular atrophy (DCSMA) is a disorder of developing anterior horn cells and shows lower-limb predominance and clinical overlap with hereditary spastic paraplegia (HSP), a lower-limb-predominant disorder of corticospinal motor neurons. We have identified four mutations in bicaudal D homolog 2 (Drosophila) (BICD2) in six kindreds affected by DCSMA, DCSMA with upper motor neuron features, or HSP. BICD2 encodes BICD2, a key adaptor protein that interacts with the dynein-dynactin motor complex, which facilitates trafficking of cellular cargos that are critical to motor neuron development and maintenance. We demonstrate that mutations resulting in amino acid substitutions in two binding regions of BICD2 increase its binding affinity for the cytoplasmic dynein-dynactin complex, which might result in the perturbation of BICD2-dynein-dynactin-mediated trafficking, and impair neurite outgrowth. These findings provide insight into the mechanism underlying both the static and the slowly progressive clinical features and the motor neuron pathology that characterize BICD2-associated diseases, and underscore the importance of the dynein-dynactin transport pathway in the development and survival of both lower and upper motor neurons.

The common cutworm (Agrotis segetum) and the black cutworm (Agrotis ipsilon) are serious soil pests of many vegetable and field crops all over the world. We have demonstrated the cross-infectivity of two baculoviruses, A. segetum nucleopolyhedrovirus (AgseNPV) and A. ipsilon nucleopolyhedrovirus (AgipNPV) for these two insect pests. The susceptibility of A. segetum to AgipNPV was confirmed by DNA restriction endonuclease analyses of DNA isolated from virus harvested from infected A. segetum larvae. For an initial comparison of both viruses, partial polyhedrin sequences were amplified by PCR, cloned, and sequenced. Both viruses shared a very similar polyhedrin gene sequence resulting in only three amino acid substitutions. Phylogenetic analyses clearly demonstrated that both viruses belong to NPV group II and are most closely related to a clade consisting of Spodoptera exigua NPV, Spodoptera frugiperda NPV, and Spodoptera littoralis NPV. Since AgipNPV shows high virulence for both cutworm species, it appears to be a suitable candidate as a single biological control agent of A. segetum and A. ipsilon.

Patients with previous diabetic foot ulcer are prone to re-ulceration and (re)amputation, to various comorbidities, have significantly impaired quality of life and increased mortality. We aimed to evaluate the risk of foot related complications and mortality in a high-risk population of patients with healed diabetic foot syndrome over a decade. 91 patients with recently healed diabetic foot ulcer were invited for follow-up at 1, 6 and 11 years after inclusion. Patient characteristics at inclusion were: 40 women, 65 ± 11 years, diabetes type 1 (n = 6) or 2 (n = 85), BMI 28.5 ± 4.4 kg/m2, and HbA1c 68 ± 17 mmol/mol. Comorbidities included neuropathy (n = 91), peripheral artery disease (PAD), history of minor (n = 25) or major (n = 5, 5.5%) amputation, nephropathy (n = 40) and retinopathy (n = 53). Ulceration recurred in 71 (65%) patients, time to first recurrence was 1.8 ± 2.4 years (mean ± SD). 21 patients had to undergo (re)amputation (minor n = 19, major n = 2), time to amputation was 3.6 ± 1.9 years. Over time, 3 further major amputations were required in patients with an initial minor amputation. Thirty-three (36%) of the initially included patients completed the follow-up period of 11.0 ± 0.6 years. 58 patients (64%) died during the observational period, time to death was 5 ± 3 years in this group. We found overall high mortality of 64% throughout the follow-up period of 11 years in high-risk patients with healed diabetic foot syndrome. Presence of PAD, prior amputation and nephropathy as well as poor glycemic control were significantly predictive for death.

Caloric restriction and intermittent fasting are known to prolong life- and healthspan in model organisms, while their effects on humans are less well studied. In a randomized controlled trial study (ClinicalTrials.gov identifier: NCT02673515), we show that 4 weeks of strict alternate day fasting (ADF) improved markers of general health in healthy, middle-aged humans while causing a 37% calorie reduction on average. No adverse effects occurred even after >6 months. ADF improved cardiovascular markers, reduced fat mass (particularly the trunk fat), improving the fat-to-lean ratio, and increased β-hydroxybutyrate, even on non-fasting days. On fasting days, the pro-aging amino-acid methionine, among others, was periodically depleted, while polyunsaturated fatty acids were elevated. We found reduced levels sICAM-1 (an age-associated inflammatory marker), low-density lipoprotein, and the metabolic regulator triiodothyronine after long-term ADF. These results shed light on the physiological impact of ADF and supports its safety. ADF could eventually become a clinically relevant intervention.

Continuous subcutaneous insulin infusion (CSII) catheters are considered the weak link of insulin pump therapy. Wear-time considerably varies between patients and the choice of catheter material is based on personal preferences rather than scientific facts. Therefore, we systematically assessed and quantified the inflammatory tissue response to steel versus Teflon CSII catheters over a maximum wear-time of 7 days in swine. Tissue surrounding catheters was analysed using histopathology and quantitative real-time PCR. The area of inflammation increased significantly over time independent of material which was confirmed by an increase in CD68 expression and an increase in mononuclear and neutrophil cell infiltrate around the catheters. We observed substantially higher fibrin deposition (p < 0.05) around steel on day 4 of wear-time. IL-6 gene expression increased within 24 hours after insertion, returned to normal levels around Teflon (p < 0.05) but remained high around steel (p < 0.05). IL-10 and TGF-β levels did not resolve over time, indicating impaired wound healing. In conclusion, there was a major temporal effect in the acute inflammatory response to CSII catheters but we found little difference between materials. This study setup presents a robust tool for the systematic analysis of the tissue response to CSII catheters.

To investigate the mechanisms behind the lower postprandial glucose (PPG) concentrations achieved with fast-acting insulin aspart (faster aspart) than with insulin aspart (IAsp).

The Trial Comparing Cardiovascular Safety of Insulin Degludec vs Insulin Glargine in Patients with Type 2 Diabetes at High Risk of Cardiovascular Events (DEVOTE) was a double-blind, randomised, event-driven, treat-to-target prospective trial comparing the cardiovascular safety of insulin degludec with that of insulin glargine U100 (100 units/ml) in patients with type 2 diabetes at high risk of cardiovascular events. This paper reports a secondary analysis investigating associations of day-to-day fasting glycaemic variability (pre-breakfast self-measured blood glucose [SMBG]) with severe hypoglycaemia and cardiovascular outcomes.

The formation of appropriately patterned blood vessel networks requires endothelial cell migration and proliferation. Signaling through the Vascular Endothelial Growth Factor A (VEGFA) pathway is instrumental in coordinating these processes. mRNA splicing generates short (diffusible) and long (extracellular matrix bound) Vegfa isoforms. The differences between these isoforms in controlling cellular functions are not understood. In zebrafish, vegfaa generates short and long isoforms, while vegfab only generates long isoforms. We found that mutations in vegfaa had an impact on endothelial cell (EC) migration and proliferation. Surprisingly, mutations in vegfab more strongly affected EC proliferation in distinct blood vessels, such as intersegmental blood vessels in the zebrafish trunk and central arteries in the head. Analysis of downstream signaling pathways revealed no change in MAPK (ERK) activation, while inhibiting PI3 kinase signaling phenocopied vegfab mutant phenotypes in affected blood vessels. Together, these results suggest that extracellular matrix bound Vegfa might act through PI3K signaling to control EC proliferation in a distinct set of blood vessels during angiogenesis.