Habitat selection by Calomys musculinus (Muridae, Sigmodontinae) in crop areas of the pampean region, Argentina

Calomys musculinus (Muridae, Sigmodontinae) is a small rodent species found in most central and northern Argentina. It is the reservoir of the Junin virus, ethiological agent of the Argentine Hemorrhagic Fever (FHA). In the present work we studied habitat selection by C. musculinus at different spatial scales in rural habitats where the landscape is mainly formed by cropfields, surrounded by weedy margins (borders). We found that C. musculinus selects borders over cropfields, but there were not differences between types of fields or types of borders. The structure of the spatial heterogeneity, which is mainly grouped between macrohabitats due to agrarian labors, did not allow to detect habitat selection, within cropfields and borders, for habitat patches larger than individual trap sites. Distribution between fields and borders was related to the green plant cover in early autumn, probably because of specific requirements of reproductive individuals. Within these habitats, we found differences in captures according to the presence of some plant species, which varied according to the season and the habitat

Differential clearance rates of microbial phylotypes by four appendicularian species

Appendicularians are abundant planktonic filter feeders that play a significant role in the pelagic food web due to their high clearance rates. Their diet and feeding rates have typically been measured as bulk chlorophyll or cell removal, with some attention given to prey size but no differentiation between the microbial phylotypes. Using a combination ofin situand laboratory incubations with flow cytometry and next-generation sequencing, we found species-specific differences in clearance rates and diet compositions of 4 common species:Oikopleura albicans,O. fusiformis,O. longicauda, andO. dioica. WhileO. albicansmost efficiently removed nano-eukaryotic algae, the other smaller species preferentially removed micron-sized pico-eukaryotic algae. Pico- and nano-eukaryotic cells constituted the major food source of the studied appendicularians despite their occurrence in oligotrophic water dominated by prokaryotic cells. Across species, pico- and nano-planktonic microalgae biomass comprised 45 to 75% of the appendicularian diets. Although non-photosynthetic bacteria were removed at lower rates than all other prey groups, their total contribution to the appendicularian diet was not trivial, representing 5 to 19% of the planktonic carbon in the appendicularian diet; pico-cyanobacteria contributed an additional 9 to 18%. Removal rates and efficiencies of pico-eukaryotes were higher than those of prokaryotes of similar size. Strikingly different clearance rates were observed for different prokaryotic phylotypes, indicating that factors other than size are involved in determining the capturability of the cells. Collectively, our findings provide additional evidence for differential retention of microbial prey among mucous-mesh grazers and its substantial effect on the upper-ocean microbial community.</jats:p

Hyperglycaemia induces metabolic dysfunction and glycogen accumulation in pancreatic β-cells

Insulin secretion from pancreatic β-cells is impaired in all forms of diabetes. The resultant hyperglycaemia has deleterious effects on many tissues, including β-cells. Here we use a mouse model of human neonatal diabetes to show that chronic hyperglycemia impairs glucose metabolism and alters expression of metabolic genes in pancreatic islets. This results in marked glycogen accumulation, and increased apoptosis in β-cells. Sulphonylurea therapy rapidly normalizes blood glucose levels, dissipates glycogen stores, increases autophagy, and restores β-cell metabolism. Insulin therapy has the same effect but with slower kinetics. Similar changes are observed in mice expressing an activating glucokinase mutation, in in vitro models of hyperglycaemia, and in islets from type-2 diabetes patients. Altered β-cell metabolism may underlie both the progressive impairment of insulin secretion and reduced β-cell mass in diabetes

Controlled assembly of SNAP-PNA-fluorophore systems on DNA templates to produce fluorescence resonance energy transfer

The SNAP protein is a widely used self-labeling tag that can be used for tracking protein localization and trafficking in living systems. A model system providing controlled alignment of SNAP-tag units can provide a new way to study clustering of fusion proteins. In this work, fluorescent SNAP-PNA conjugates were controllably assembled on DNA frameworks forming dimers, trimers, and tetramers. Modification of peptide nucleic acid (PNA) with the O6-benzyl guanine (BG) group allowed the generation of site-selective covalent links between PNA and the SNAP protein. The modified BG-PNAs were labeled with fluorescent Atto dyes and subsequently chemo-selectively conjugated to SNAP protein. Efficient assembly into dimer and oligomer forms was verified via size exclusion chromatography (SEC), electrophoresis (SDS-PAGE), and fluorescence spectroscopy. DNA directed assembly of homo- and hetero-dimers of SNAP-PNA constructs induced homo- and hetero-FRET, respectively. Longer DNA scaffolds controllably aligned similar fluorescent SNAP-PNA constructs into higher oligomers exhibiting homo-FRET. The combined SEC and homo-FRET studies indicated the 1:1 and saturated assemblies of SNAP-PNA-fluorophore:DNA formed preferentially in this system. This suggested a kinetic/stoichiometric model of assembly rather than binomially distributed products. These BG-PNA-fluorophore building blocks allow facile introduction of fluorophores and/or assembly directing moieties onto any protein containing SNAP. Template directed assembly of PNA modified SNAP proteins may be used to investigate clustering behavior both with and without fluorescent labels which may find use in the study of assembly processes in cells

Functional Neuromuscular Junctions Formed by Embryonic Stem Cell-Derived Motor Neurons

A key objective of stem cell biology is to create physiologically relevant cells suitable for modeling disease pathologies in vitro. Much progress towards this goal has been made in the area of motor neuron (MN) disease through the development of methods to direct spinal MN formation from both embryonic and induced pluripotent stem cells. Previous studies have characterized these neurons with respect to their molecular and intrinsic functional properties. However, the synaptic activity of stem cell-derived MNs remains less well defined. In this study, we report the development of low-density co-culture conditions that encourage the formation of active neuromuscular synapses between stem cell-derived MNs and muscle cells in vitro. Fluorescence microscopy reveals the expression of numerous synaptic proteins at these contacts, while dual patch clamp recording detects both spontaneous and multi-quantal evoked synaptic responses similar to those observed in vivo. Together, these findings demonstrate that stem cell-derived MNs innervate muscle cells in a functionally relevant manner. This dual recording approach further offers a sensitive and quantitative assay platform to probe disorders of synaptic dysfunction associated with MN disease

Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells

Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.National Institutes of Health (U.S.) (Grant RO1-GM34277)National Institutes of Health (U.S.) (Grant R01-CA133404)National Cancer Institute (U.S.) (Grant PO1-CA42063)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)National Institutes of Health (U.S.) (Director's Pioneer Award 1DP1-MH100706)Damon Runyon Cancer Research FoundationKinship Foundation. Searle Scholars ProgramSimons Foundatio