Endometrial Exosomes/Microvesicles in the Uterine Microenvironment: A New Paradigm for Embryo-Endometrial Cross Talk at Implantation

Exosomes are nanoparticles (∼100 nm diameter) released from cells, which can transfer small RNAs and mRNA via the extracellular environment to cells at distant sites. We hypothesised that exosomes or the slightly larger microvesicles (100–300 nm) are released from the endometrial epithelium into the uterine cavity, and that these contain specific micro (mi)RNA that could be transferred to either the trophectodermal cells of the blastocyst or to endometrial epithelial cells, to promote implantation. The aim of this study was to specifically identify and characterise exosomes/microvesicles (mv) released from endometrial epithelial cells and to determine whether exosomes/mv are present in uterine fluid. Immunostaining demonstrated that the tetraspanins, CD9 and CD63 used as cell surface markers of exosomes are present on the apical surfaces of endometrial epithelial cells in tissue sections taken across the menstrual cycle: CD63 showed cyclical regulation. Exosome/mv pellets were prepared from culture medium of endometrial epithelial cell (ECC1 cells) and from uterine fluid and its associated mucus by sequential ultracentifugation. Exosomes/mv were positively identified in all preparations by FACS and immunofluorescence staining following exosome binding to beads. Size particle analysis confirmed the predominance of particles of 50–150 nm in each of these fluids. MiRNA analysis of the ECC1 cells and their exosomes/mv demonstrated sorting of miRNA into exosomes/mv: 13 of the 227 miRNA were specific to exosomes/mv, while a further 5 were not present in these. The most abundant miRNA in exosomes/mv were hsa-miR-200c, hsa-miR-17 and hsa-miR-106a. Bioinformatic analysis showed that the exosome/mv-specific miRNAs have potential targets in biological pathways highly relevant for embryo implantation. Thus exosomes/mv containing specific miRNA are present in the microenvironment in which embryo implantation occurs and may contribute to the endometrial-embryo cross talk essential for this process.     

Identification and characterization of small molecule inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase

Plasmodium falciparum causes the most deadly form of malaria and accounts for over one million deaths annually. The malaria parasite is unable to salvage pyrimidines and relies on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHOD), a mitochondrially localized flavoenzyme, catalyzes the rate-limiting step of this pathway and is therefore an attractive antimalarial chemotherapeutic target. Using a target-based high throughput screen, we have identified a series of potent, species-specific inhibitors of P. falciparum DHOD (pfDHOD) that are also efficacious against three cultured strains (3D7, HB3, and Dd2) of P. falciparum. The primary antimalarial mechanism of action of these compounds was confirmed to be inhibition of pfDHOD through a secondary assay with transgenic malaria parasites, and the structural basis for enzyme inhibition was explored through in silico structure-based docking and site-directed mutagenesis. Compound-mediated cytotoxicity was not observed with human dermal fibroblasts or renal epithelial cells. These data validate pfDHOD as an antimalarial drug target and provide chemical scaffolds with which to begin medicinal chemistry efforts.     

Activation of the E3 ligase function of the BRCA1/BARD1 complex by polyubiquitin chains

Loss of the tumour suppressor BRCA1 results in profound chromosomal instability. The fundamental defect underlying this catastrophic phenotype is not yet known. In vivo, BRCA1 forms a heterodimeric complex with BARD1. Both proteins contain an N-terminal zinc RING-finger domain which confers E3 ubiquitin ligase activity. We have isolated full-length human BRCA1/BARD1 complex and have shown that it has a dual E3 ubiquitin ligase activity. First, it mediates the monoubiquitylation of nucleosome core histones in vitro, including the variant histone H2AX that co-localizes with BRCA1 at sites of DNA damage. Secondly, BRCA1/BARD1 catalyses the formation of multiple polyubiquitin chains on itself. Remarkably, this auto-polyubiquitylation potentiates the E3 ubiquitin ligase activity of the BRCA1/BARD1 complex >20-fold. Even though BRCA1 has been reported to associate with a C-terminal ubiquitin hydrolase, BAP1, this enzyme does not appear to function in the deubiquitylation of the BRCA1/BARD1 complex.     

The Genus Gluconobacter Oxydans: Comprehensive Overview of Biochemistry and Biotechnological Applications

ABSTRACT

The genus Gluconobacter comprises some of the most frequently used microorganisms when it comes to biotechnological applications. Not only has it been involved in “historical” production processes, such as vinegar production, but in the last decades many bioconversion routes for special and rare sugars involving Gluconobacter have been developed. Among the most recent are the biotransformations involved in the production of L-ribose and miglitol, both very promising pharmaceutical lead molecules. Most of these processes make use of Gluconobacter's membrane-bound polyol dehydrogenases. However, recently other enzymes have also caught the eye of industrial biotechnology. Among them are dextran dextrinase, capable of transglucosylating substrate molecules, and intracellular NAD-dependent polyol dehydrogenases, of interest for co-enzyme regeneration. As such, Gluconobacter is an important industrial microbial strain, but it also finds use in other fields of biotechnology, such as biosensor-technology. This review aims to give an overview of the myriad of applications for Gluconobacter, with a special focus on some recent developments.     

In-Frame Deletions Allow Functional Characterization of Complex Cellulose Degradation Phenotypes in Cellvibrio japonicus

The depolymerization of the recalcitrant polysaccharides found in lignocellulose has become an area of intense interest due to the role of this process in global carbon cycling, human gut microbiome nutritional contributions, and bioenergy production. However, underdeveloped genetic tools have hampered study of bacterial lignocellulose degradation, especially outside model organisms. In this report, we describe an in-frame deletion strategy for the Gram-negative lignocellulose-degrading bacterium Cellvibrio japonicus. This method leverages optimized growth conditions for conjugation and sacB counterselection for the generation of markerless in-frame deletions. This method produces mutants in as few as 8 days and allows for the ability to make multiple gene deletions per strain. It is also possible to remove large sections of the genome, as shown in this report with the deletion of the nine-gene (9.4-kb) gsp operon in C. japonicus. We applied this system to study the complex phenotypes of cellulose degradation in C. japonicus. Our data indicated that a Δcel5B Δcel6A double mutant is crippled for cellulose utilization, more so than by either single mutation alone. Additionally, we deleted individual genes in the two-gene cbp2ED operon and showed that both genes contribute to cellulose degradation in C. japonicus. Overall, these described techniques substantially enhance the utility of C. japonicus as a model system to study lignocellulose degradation.     

Carry‐over effects provide linkages across the annual cycle of a Neotropical migratory bird,the Louisiana Waterthrush Parkesia motacilla

Population limitation models of migratory birds have sought to include impacts from events across the full annual cycle. Previous work has shown that events occurring in winter result in some individuals transitioning to the breeding grounds earlier or in better physical condition than others, thereby affecting reproductive success (carry‐over effects). However, evidence for carry‐over effects from breeding to wintering grounds has been shown less often. We used feather corticosterone (CORT_f) levels of the migratory Louisiana Waterthrush Parkesia motacilla as a measure of the physiological state of birds at the time of moult on the breeding territory to investigate whether carry‐over effects provide linkages across the annual cycle of this stream‐obligate bird. We show that birds arriving on wintering grounds with lower CORT_f scores, indicating reduced energetic challenges or stressors at the time of moult, occupied higher quality territories, and that these birds then achieved a better body condition during the overwinter period. Body condition, in turn, was important in determining whether adult birds returned the following winter, with birds in better condition returning at higher rates. Together these data suggest a carry‐over effect from the breeding grounds to the wintering grounds that is further extended with respect to annual return rates. Very few other studies have linked conditions during the previous breeding season with latent effects during the subsequent overwintering period or with annual survival. This study shows that the effects of variation in energetic challenges or stressors can potentially carry over from the natal stream and accumulate over more than one life‐history period before being manifested in reduced survival. This is of particular relevance to models of population limitation in migratory birds.