Membrane-free culture and real-time barrier integrity assessment of perfused intestinal epithelium tubes

In vitro models that better reflect in vivo epithelial barrier (patho-)physiology are urgently required to predict adverse drug effects. Here we introduce extracellular matrix-supported intestinal tubules in perfused microfluidic devices, exhibiting tissue polarization and transporter expression. Forty leak-tight tubules are cultured in parallel on a single plate and their response to pharmacological stimuli is recorded over 125 h using automated imaging techniques. A study comprising 357 gut tubes is performed, of which 93% are leak tight before exposure. EC50-time curves could be extracted that provide insight into both concentration and exposure time response. Full compatibility with standard equipment and user-friendly operation make this Organ-on-a-Chip platform readily applicable in routine laboratories

Mendelian randomization study of B-type natriuretic peptide and type 2 diabetes: evidence of causal association from population studies

<p>Background: Genetic and epidemiological evidence suggests an inverse association between B-type natriuretic peptide (BNP) levels in blood and risk of type 2 diabetes (T2D), but the prospective association of BNP with T2D is uncertain, and it is unclear whether the association is confounded.</p> <p>Methods and Findings: We analysed the association between levels of the N-terminal fragment of pro-BNP (NT-pro-BNP) in blood and risk of incident T2D in a prospective case-cohort study and genotyped the variant rs198389 within the BNP locus in three T2D case-control studies. We combined our results with existing data in a meta-analysis of 11 case-control studies. Using a Mendelian randomization approach, we compared the observed association between rs198389 and T2D to that expected from the NT-pro-BNP level to T2D association and the NT-pro-BNP difference per C allele of rs198389. In participants of our case-cohort study who were free of T2D and cardiovascular disease at baseline, we observed a 21% (95% CI 3%-36%) decreased risk of incident T2D per one standard deviation (SD) higher log-transformed NT-pro-BNP levels in analysis adjusted for age, sex, body mass index, systolic blood pressure, smoking, family history of T2D, history of hypertension, and levels of triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol. The association between rs198389 and T2D observed in case-control studies (odds ratio = 0.94 per C allele, 95% CI 0.91-0.97) was similar to that expected (0.96, 0.93-0.98) based on the pooled estimate for the log-NT-pro-BNP level to T2D association derived from a meta-analysis of our study and published data (hazard ratio = 0.82 per SD, 0.74-0.90) and the difference in NT-pro-BNP levels (0.22 SD, 0.15-0.29) per C allele of rs198389. No significant associations were observed between the rs198389 genotype and potential confounders.</p> <p>Conclusions: Our results provide evidence for a potential causal role of the BNP system in the aetiology of T2D. Further studies are needed to investigate the mechanisms underlying this association and possibilities for preventive interventions.</p&gt

Proteomic analysis of the Plasmodium male gamete reveals the key role for glycolysis in flagellar motility.

BACKGROUND: Gametogenesis and fertilization play crucial roles in malaria transmission. While male gametes are thought to be amongst the simplest eukaryotic cells and are proven targets of transmission blocking immunity, little is known about their molecular organization. For example, the pathway of energy metabolism that power motility, a feature that facilitates gamete encounter and fertilization, is unknown. METHODS: Plasmodium berghei microgametes were purified and analysed by whole-cell proteomic analysis for the first time. Data are available via ProteomeXchange with identifier PXD001163. RESULTS: 615 proteins were recovered, they included all male gamete proteins described thus far. Amongst them were the 11 enzymes of the glycolytic pathway. The hexose transporter was localized to the gamete plasma membrane and it was shown that microgamete motility can be suppressed effectively by inhibitors of this transporter and of the glycolytic pathway. CONCLUSIONS: This study describes the first whole-cell proteomic analysis of the malaria male gamete. It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization

Vinorelbine/carboplatin vs gemcitabine/carboplatin in advanced NSCLC shows similar efficacy, but different impact of toxicity

This randomised phase III study in advanced non-small cell lung cancer (NSCLC) patients was conducted to compare vinorelbine/carboplatin (VC) and gemcitabine/carboplatin (GC) regarding efficacy, health-related quality of life (HRQOL) and toxicity. Chemonaive patients with NSCLC stage IIIB/IV and WHO performance status 0–2 were eligible. No upper age limit was defined. Patients received vinorelbine 25 mg m−2 or gemcitabine 1000 mg m−2 on days 1 and 8 and carboplatin AUC4 on day 1 and three courses with 3-week cycles. HRQOL questionnaires were completed at baseline, before chemotherapy and every 8 weeks until 49 weeks. During 14 months, 432 patients were included (VC, n=218; GC, n=214). Median survival was 7.3 vs 6.4 months, 1-year survival 28 vs 30% and 2-year survival 7 vs 7% in the VC and GC arm, respectively (P=0.89). HRQOL, represented by global QOL, nausea/vomiting, dyspnoea and pain, showed no significant differences. More grade 3–4 anaemia (P<0.01), thrombocytopenia (P<0.01) and transfusions of blood (P<0.01) or platelets (P<0.01) were observed in the GC arm. There was more grade 3–4 leucopoenia (P<0.01) in the VC arm, but the rate of neutropenic infections was the same (P=0.87). In conclusion, overall survival and HRQOL are similar, while grade 3–4 toxicity requiring interventions are less frequent when VC is compared to GC in advanced NSCLC

Efficient Allele-Specific Targeting of LRRK2 R1441 Mutations Mediated by RNAi

Since RNA interference (RNAi) has the potential to discriminate between single nucleotide changes, there is growing interest in the use of RNAi as a promising therapeutical approach to target dominant disease-associated alleles. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been linked to dominantly inherited Parkinson's disease (PD). We focused on three LRRK2 mutations (R1441G/C and the more prevalent G2109S) hoping to identify shRNAs that would both recognize and efficiently silence the mutated alleles preferentially over the wild-type alleles. Using a luciferase-based reporter system, we identified shRNAs that were able to specifically target the R1441G and R1441C alleles with 80% silencing efficiency. The same shRNAs were able to silence specifically mRNAs encoding either partial or full-length mutant LRRK2 fusion proteins, while having a minimal effect on endogenous wild-type LRRK2 expression when transfected in 293FT cells. Shifting of the mutant recognition site (MRS) from position 11 to other sites (4 and 16, within the 19-mer window of our shRNA design) reduced specificity and overall silencing efficiency. Developing an allele-specific RNAi of G2019S was problematic. Placement of the MRS at position 10 resulted in efficient silencing of reporters (75–80%), but failed to discriminate between mutant and wild-type alleles. Shifting of the MRS to positions 4, 5, 15, 16 increased the specificity of the shRNAs, but reduced the overall silencing efficiency. Consistent with previous reports, these data confirm that MRS placement influences both allele-specificity and silencing strength of shRNAs, while further modification to hairpin design or MRS position may lead to the development of effective G2019S shRNAs. In summary, the effective shRNA against LRRK2 R1441 alleles described herein suggests that RNAi-based therapy of inherited Parkinson's disease is a viable approach towards developing effective therapeutic interventions for this serious neurodegenerative disease

Rethinking Proteasome Evolution: Two Novel Bacterial Proteasomes

The proteasome is a multisubunit structure that degrades proteins. Protein degradation is an essential component of regulation because proteins can become misfolded, damaged, or unnecessary. Proteasomes and their homologues vary greatly in complexity: from HslV (heat shock locus v), which is encoded by 1 gene in bacteria, to the eukaryotic 20S proteasome, which is encoded by more than 14 genes. Despite this variation in complexity, all the proteasomes are composed of homologous subunits. We searched 238 complete bacterial genomes for structures related to the proteasome and found evidence of two novel groups of bacterial proteasomes. The first, which we name Anbu, is sparsely distributed among cyanobacteria and proteobacteria. We hypothesize that Anbu must be very ancient because of its distribution within the cyanobacteria, and that it has been lost in many more recent species. We also present evidence for a fourth type of bacterial proteasome found in a few β-proteobacteria, which we call β-proteobacteria proteasome homologue (BPH). Sequence and structural analyses show that Anbu and BPH are both distinct from known bacterial proteasomes but have homologous structures. Anbu is encoded by one gene, so we postulate a duplication of Anbu created the 20S proteasome. Anbu’s function appears to be related to transglutaminase activity, not the general stress response associated with HslV. We have found different combinations of Anbu, BPH, and HslV within these bacterial genomes, which raises questions about specialized protein degradation systems

Genomic aberrations associated with outcome in anaplastic oligodendroglial tumors treated within the EORTC phase III trial 26951

Despite similar morphological aspects, anaplastic oligodendroglial tumors (AOTs) form a heterogeneous clinical subgroup of gliomas. The chromosome arms 1p/19q codeletion has been shown to be a relevant biomarker in AOTs and to be perfectly exclusive from EGFR amplification in gliomas. To identify new genomic regions associated with prognosis, 60 AOTs from the EORTC trial 26951 were analyzed retrospectively using BAC-array-based comparative genomic hybridization. The data were processed using a binary tree method. Thirty-three BACs with prognostic value were identified distinguishing four genomic subgroups of AOTs with different prognosis (p < 0.0001). Type I tumors (25%) were characterized by: (1) an EGFR amplification, (2) a poor prognosis, (3) a higher rate of necrosis, and (4) an older age of patients. Type II tumors (21.7%) had: (1) loss of prognostic BACs located on 1p tightly associated with 19q deletion, (2) a longer survival, (3) an oligodendroglioma phenotype, and (4) a frontal location in brain. Type III AOTs (11.7%) exhibited: (1) a deletion of prognostic BACs located on 21q, and (2) a short survival. Finally, type IV tumors (41.7%) had different genomic patterns and prognosis than type I, II and III AOTs. Multivariate analysis showed that genomic type provides additional prognostic data to clinical, imaging and pathological features. Similar results were obtained in the cohort of 45 centrally reviewed–validated cases of AOTs. Whole genome analysis appears useful to screen the numerous genomic abnormalities observed in AOTs and to propose new biomarkers particularly in the non-1p/19q codeleted AOTs

An Insect Herbivore Microbiome with High Plant Biomass-Degrading Capacity

Herbivores can gain indirect access to recalcitrant carbon present in plant cell walls through symbiotic associations with lignocellulolytic microbes. A paradigmatic example is the leaf-cutter ant (Tribe: Attini), which uses fresh leaves to cultivate a fungus for food in specialized gardens. Using a combination of sugar composition analyses, metagenomics, and whole-genome sequencing, we reveal that the fungus garden microbiome of leaf-cutter ants is composed of a diverse community of bacteria with high plant biomass-degrading capacity. Comparison of this microbiome's predicted carbohydrate-degrading enzyme profile with other metagenomes shows closest similarity to the bovine rumen, indicating evolutionary convergence of plant biomass degrading potential between two important herbivorous animals. Genomic and physiological characterization of two dominant bacteria in the fungus garden microbiome provides evidence of their capacity to degrade cellulose. Given the recent interest in cellulosic biofuels, understanding how large-scale and rapid plant biomass degradation occurs in a highly evolved insect herbivore is of particular relevance for bioenergy