Microphysiological Conditions Do Not Affect MDR1-Mediated Transport of Rhodamine 123 above an Artificial Proximal Tubule.

Despite disadvantages, such as high cost and their poor predictive value, animal experiments are still the state of the art for pharmaceutical substance testing. One reason for this problem is the inability of standard cell culture methods to emulate the physiological environment necessary to recapitulate in vivo processes. Microphysiological systems offer the opportunity to close this gap. In this study, we utilize a previously employed microphysiological system to examine the impact of pressure and flow on the transportation of substances mediated by multidrug resistance protein 1 (MDR1) across an artificial cell-based tubular barrier. By using a miniaturized fluorescence measurement device, we could continuously track the MDR1-mediated transport of rhodamine 123 above the artificial barrier over 48 h. We proved that applying pressure and flow affects both active and passive transport of rhodamine 123. Using experimental results and curve fittings, the kinetics of MDR1-mediated transport as well as passive transport were investigated; thus, a kinetic model that explains this transport above an artificial tubular barrier was identified. This kinetic model demonstrates that the simple Michaelis-Menten model is not an appropriate model to explain the MDR1-mediated transport; instead, Hill kinetics, with Hill slope of n = 2, is a better fit. The kinetic values, Km, Vmax, and apparent permeability (Papp), obtained in this study are comparable with other in vivo and in vitro studies. Finally, the presented proximal tubule-on-a-chip can be used for pharmaceutical substance testing and to investigate pharmacokinetics of the renal transporter MDR1.

Marker-assisted mapping enables forward genetic analysis in Aedes aegypti, an arboviral vector with vast recombination deserts.

Aedes aegypti is a major vector of arboviruses that cause dengue, chikungunya, yellow fever, and Zika. Although recent success in reverse genetics has facilitated rapid progress in basic and applied research, integration of forward genetics with modern technologies remains challenging in this important species, as up to 47% of its chromosome is refractory to genetic mapping due to extremely low rate of recombination. Here, we report the development of a marker-assisted mapping strategy to readily screen for and genotype only the rare but informative recombinants, drastically increasing both the resolution and signal-to-noise ratio. Using marker-assisted mapping, we mapped a transgene that was inserted in a >100-Mb recombination desert and a sex-linked spontaneous red-eye (re) mutation just outside the region. We subsequently determined, by CRISPR/Cas9-mediated knockout, that cardinal is the causal gene of re, which is the first forward genetic identification of a causal gene in Ae. aegypti. The identification of the causal gene of the sex-linked re mutation provides the molecular foundation for using gene editing to develop versatile and stable genetic sexing methods. To facilitate genome-wide forward genetics in Ae. aegypti, we generated and compiled a number of lines with markers throughout the genome. Thus, by overcoming the challenges presented by the vast recombination deserts and the scarcity of markers, we have shown that effective forward genetic analysis is increasingly feasible in this important arboviral vector species.

(Cyclopentadienone)iron-Catalyzed Transfer Dehydrogenation of Symmetrical and Unsymmetrical Diols to Lactones.

Air-stable iron carbonyl compounds bearing cyclopentadienone ligands with varying substitution were explored as catalysts in dehydrogenative diol lactonization reactions using acetone as both the solvent and hydrogen acceptor. Two catalysts with trimethylsilyl groups in the 2- and 5-positions, [2,5-(SiMe3)2-3,4-(CH2)4(η4-C4C═O)]Fe(CO)3 (1) and [2,5-(SiMe3)2-3,4-(CH2)3(η4-C4C═O)]Fe(CO)3 (2), were found to be the most active, with 2 being the most selective in the lactonization of diols containing both primary and secondary alcohols. Lactones containing five-, six-, and seven-membered rings were successfully synthesized, and no over-oxidations to carboxylic acids were detected. The lactonization of unsymmetrical diols containing two primary alcohols occurred with catalyst 1, but selectivity was low based on alcohol electronics and modest based on alcohol sterics. Evidence for a transfer dehydrogenation mechanism was found, and insight into the origin of selectivity in the lactonization of 1°/2° diols was obtained. Additionally, spectroscopic evidence for a trimethylamine-ligated iron species formed in solution during the reaction was discovered.

Epiphytic bacterial community composition on two common submerged macrophytes in brackish water and freshwater.

BACKGROUND: Plants and their heterotrophic bacterial biofilm communities possibly strongly interact, especially in aquatic systems. We aimed to ascertain whether different macrophytes or their habitats determine bacterial community composition. We compared the composition of epiphytic bacteria on two common aquatic macrophytes, the macroalga Chara aspera Willd. and the angiosperm Myriophyllum spicatum L., in two habitats, freshwater (Lake Constance) and brackish water (Schaproder Bodden), using fluorescence in situ hybridization. The bacterial community composition was analysed based on habitat, plant species, and plant part. RESULTS: The bacterial abundance was higher on plants from brackish water [5.3 x 10(7) cells (g dry mass)-1] than on plants from freshwater [1.3 x 10(7) cells (g dry mass)-1], with older shoots having a higher abundance. The organic content of freshwater plants was lower than that of brackish water plants (35 vs. 58%), and lower in C. aspera than in M. spicatum (41 vs. 52%). The content of nutrients, chlorophyll, total phenolic compounds, and anthocyanin differed in the plants and habitats. Especially the content of total phenolic compounds and anthocyanin was higher in M. spicatum, and in general higher in the freshwater than in the brackish water habitat. Members of the Cytophaga-Flavobacteria-Bacteroidetes group were abundant in all samples (5-35% of the total cell counts) and were especially dominant in M. spicatum samples. Alphaproteobacteria were the second major group (3-17% of the total cell counts). Betaproteobacteria, gammaproteobacteria, and actinomycetes were present in all samples (5 or 10% of the total cell counts). Planctomycetes were almost absent on M. spicatum in freshwater, but present on C. aspera in freshwater and on both plants in brackish water. CONCLUSION: Bacterial biofilm communities on the surface of aquatic plants might be influenced by the host plant and environmental factors. Distinct plant species, plant part and habitat specific differences in total cell counts and two bacterial groups (CFB, planctomycetes) support the combined impact of substrate (plant) and habitat on epiphytic bacterial community composition. The presence of polyphenols might explain the distinct bacterial community on freshwater M. spicatum compared to that of M. spicatum in brackish water and of C. aspera in both habitats.

Quorum sensing by N-acylhomoserine lactones is not required for Aeromonas hydrophila during growth with organic particles in lake water microcosms.

It was investigated whether quorum sensing (QS) mediated by N-acylhomoserine lactones (AHLs) was important for heterotrophic bacteria from the littoral zone of the oligotrophic Lake Constance for growth with organic particles. More than 900 colonies from lake water microcosms with artificial organic aggregates consisting of autoclaved unicellular algae embedded in agarose beads were screened for AHL-production. AHL-producing bacteria of the genus Aeromonas enriched in the microcosms but AHLs could not be detected in any microcosm. To test for a potential function of AHL-mediated QS, growth experiments with the wild type and an AHL-deficient mutant of Aeromonas hydrophila in lake water microcosms were performed. Growth of both strains did not differ in single cultures and showed no mutual influence in co-cultures. In co-cultures with a competitor bacterium belonging to the Cytophaga-Flavobacterium group, growth of both A. hydrophila strains was reduced while growth of the competitor bacterium was not affected. Exogenous AHL-addition did not influence growth of the Aeromonas strains in any microcosm experiment. These results showed that AHL-mediated QS was not required for A. hydrophila during colonization and degradation of organic particles in lake water microcosms, suggesting that cell-cell signalling of heterotrophic bacteria in oligotrophic waters relies on novel signal molecules.

Phylogeography of the vairone (Leuciscus souffia, Risso 1826) in Central Europe.

The vairone Leuciscus souffia is a cyprinid fish that inhabits river systems in and around the Alps. The complete mitochondrial DNA control region (945 bp) was sequenced in 295 vairone from 22 populations in Central Europe. A total of 51 haplotypes were identified with a maximum pairwise distance between haplotypes of 5.6%. Phylogenetic analyses revealed two major clades in L. souffia, an 'Italian' clade, and an 'Alpine' clade. Two hybrid zones exist, in the Mediterranean Alps and in the Soca basin. The position of the sister species of L. souffia, L. turskyi, to the 'Alpine' and the 'Italian' clade could not be resolved unambiguously. However, a linearized tree analysis indicated that L. turskyi represents a third lineage, that originated at the same time as the 'Alpine' and the 'Italian' clades of L. souffia. In the 'Alpine' clade two groups were resolved, a subclade with haplotypes from the Rhône and Var basins and a cluster with haplotypes from the Danube and Rhine systems. Our data suggest a long history of the vairone in Central Europe, predating Pleistocene glacial cycles. Two main refugia during glaciations must have existed, one in Italy and another one most probably in the Danube system. However, age estimates based on molecular clock calibrations suggest the survival of 'Alpine' haplotypes in several drainages during the last glaciation cycles. The Rhine system was only recently colonized.