A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing

1. Download high-res image (150KB)
2. Download full-size image

     

Immunolocalization of integrin-like proteins in Arabidopsis and Chara

Integrins are a large family of integral plasma membrane proteins that link the extracellular matrix to the cytoskeleton in animal cells. As a first step in determining if integrin-like proteins are involved in gravitropic signal transduction pathways, we have used a polyclonal antibody against the chicken β₁ integrin subunit in western blot analyses and immunofluorescence microscopy to gain information on the size and location of these proteins in plants. Several different polypeptides are recognized by the anti-integrin antibody in roots and shoots of Arabidopsis and in the intemodal cells and rhizoids of Chara . These cross-reactive polypeptides are associated with cellular membranes, a feature which is consistent with the known location of integrins in animal systems. In immunofluorescence studies of Arabidopsis roots, a strong signal was obtained from labeling integrin-like proteins in root cap cells, and there was little or no immunolabel in other regions of the root tip. While the antibody stained throughout Chara rhizoids, the highest density of immunolabel was at the tip. Thus, in both Arabidopsis roots and Chara rhizoids, the sites of gravity perception/transduction appear to be enriched in integrin-like molecules.     

Average niche breadths of species in lake macrophyte communities respond to ecological gradients variably in four regions on two continents

Different species’ niche breadths in relation to ecological gradients are infrequently examined within the same study and, moreover, species niche breadths have rarely been averaged to account for variation in entire ecological communities. We investigated how average environmental niche breadths (climate, water quality and climate–water quality niches) in aquatic macrophyte communities are related to ecological gradients (latitude, longitude, altitude, species richness and lake area) among four distinct regions (Finland, Sweden and US states of Minnesota and Wisconsin) on two continents. We found that correlations between the three different measures of average niche breadths and ecological gradients varied considerably among the study regions, with average climate and average water quality niche breadth models often showing opposite trends. However, consistent patterns were also found, such as widening of average climate niche breadths and narrowing of average water quality niche breadths of aquatic macrophytes along increasing latitudinal and altitudinal gradients. This result suggests that macrophyte species are generalists in relation to temperature variations at higher latitudes and altitudes, whereas species in southern, lowland lakes are more specialised. In contrast, aquatic macrophytes growing in more southern nutrient-rich lakes were generalists in relation to water quality, while specialist species are adapted to low-productivity conditions and are found in highland lakes. Our results emphasise that species niche breadths should not be studied using only coarse-scale data of species distributions and corresponding environmental conditions, but that investigations on different kinds of niche breadths (e.g., climate vs. local niches) also require finer resolution data at broad spatial extents.     

Chemical Composition,Antibacterial, Antibiofilm and Synergistic Properties of Essential Oils from Eucalyptus globulus Labill. and Seven Mediterranean Aromatic Plants

Essential oils (EOs) from Eucalyptus globulus Labill . ssp. globulus and from Mediterranean autochthonous aromatic plants – Thymus mastichina L., Mentha pulegium L., Rosmarinus officinalis L., Calamintha nepeta (L.) Savi ssp. nepeta, Cistus ladanifer L., Foeniculum vulgare L., Dittrichia viscosa (L.) Greuter ssp. viscosa – were extracted by hydrodistillation and characterized by GC‐FID and NMR spectroscopy. EOs were evaluated for antimicrobial properties against several bacterial strains, using diverse methods, namely, the agar disc‐diffusion method, the microdilution method, the crystal violet assay and the Live/Dead staining for assessment of biofilm formation. Potential synergy was assessed by a checkerboard method. EOs of R. officinalis and C. ladanifer showed a predominance in monoterpene hydrocarbons (> 60%); EOs of C. nepeta, M. pulegium, T. mastichina, E. globulus and F. vulgare were rich in oxygenated monoterpenes (62 – 96%) whereas EO of D. viscosa was mainly composed of oxygenated sesquiterpenes (54%). All EOs showed antimicrobial activity; M. pulegium and E. globulus generally had the strongest antimicrobial activity. EO of C. nepeta was the most promising in hampering the biofilm formation. The combinations D. viscosa/C. nepeta and E. globulus/T. mastichina were synergistic against Staphylococcus aureus. These results support the notion that EOs from the aromatic plants herein reported should be further explored as potential pharmaceuticals and/or food preservatives.     

Genome screen for bone mineral density phenotypes in Fisher 344 and Lewis rat strains

In humans, peak bone mineral density (BMD) is the primary determinant of osteoporotic fracture risk among older individuals, with high peak BMD levels providing protection against osteoporosis in the almost certain event of bone loss later in life. A genome screen to identify quantitative trait loci (QTLs) contributing to areal BMD (aBMD) and volumetric BMD (vBMD) measurements at the lumbar spine and femoral neck was completed in 595 female F₂ rats produced from reciprocal crosses of inbred Fischer 344 and Lewis rats. Significant evidence of linkage was detected to rat Chromosomes 1, 2, 8, and 10, with LOD scores above 8.0. The region on rat Chromosome 8 is syntenic to human Chromosome 15, where linkage to spine and femur BMD has been previously reported and confirmed in a sample of premenopausal women.     

Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events

Recently, certain lots of heparin have been associated with an acute, rapid onset of serious side effects indicative of an allergic-type reaction. To identify potential causes for this sudden rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high-resolution analytical techniques. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked beta1-->3 to a beta-N-acetylgalactosamine. The disaccharide unit has an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. Given the nature of this contaminant, traditional screening tests cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be used to determine whether or not heparin lots contain the contaminant reported here.     

Synthesis, biological evaluation and molecular modelling of N-heterocyclic dipeptide aldehydes as selective calpain inhibitors

A series of N-heterocyclic dipeptide aldehydes 4-13 have been synthesised and evaluated as inhibitors of ovine calpain 1 (o-CAPN1) and ovine calpain 2 (o-CAPN2). 5-Formyl-pyrrole 9 (IC(50) values of 290 and 25nM against o-CAPN1 and o-CAPN2, respectively) was the most potent and selective o-CAPN2 inhibitor, displaying >11-fold selectivity. The amino acid sequences of o-CAPN1 and o-CAPN2 have been determined. Because of the lack of available structural information on the ovine calpains, in silico homology models of the active site cleft of o-CAPN1 and o-CAPN2 were developed based on human calpain 1 (h-CAPN1) X-ray crystal structure (PDB code 1ZCM). These models were used to rationalise the observed SAR for compounds 4-13 and the selectivity observed for 9. The o-CAPN2 selective inhibitor 9 (CAT0059) was assayed in an in vitro ovine lens culture system and shown to successfully protect the lens from calcium-induced opacification.     

Hyper-dispersed cache distributions reduce pilferage: a laboratory study

Many animals use hoarding as a long-term strategy to ensure a food supply at times of shortage. We suggest that long-term scatter hoarders, whose caches are vulnerable to potentially high pilferage, should hoard in ways to reduce cache loss. This could be achieved by manipulating the density and dispersal patterns of caches to reduce the foraging efficiency of pilferers. This study explores the effect of distribution patterns on cache loss in the laboratory. We recorded the discovery of food items in different dispersal patterns by two bird species: coal tits Periparus ater (a hoarder) and great tits Parus major (a non-hoarder). Hyper-dispersed distributions reduced foraging efficiency because both species used systematic local search patterns. This study shows that hyper-dispersed distributions would be advantageous to hoarding animals to reduce cache loss.     

Self-organizing actin waves as planar phagocytic cup structures

Actin waves that travel on the planar membrane of a substrate-attached cell underscore the capability of the actin system to assemble into dynamic structures by the recruitment of proteins from the cytoplasm. The waves have no fixed shape, can reverse their direction of propagation and can fuse or divide. Actin waves separate two phases of the plasma membrane that are distinguished by their lipid composition. The area circumscribed by a wave resembles in its phosphoinositide content the interior of a phagocytic cup, leading us to explore the possibility that actin waves are in-plane phagocytic structures generated without the localized stimulus of an attached particle. Consistent with this view, wave-forming cells were found to exhibit a high propensity for taking up particles. Cells fed rod-shaped particles produced elongated phagocytic cups that displayed a zonal pattern that reflected in detail the actin and lipid pattern of free-running actin waves. Neutrophils and macrophages are known to spread on surfaces decorated with immune complexes, a process that has been interpreted as “frustrated” phagocytosis. We suggest that actin waves enable a phagocyte to scan a surface for particles that might be engulfed.Key words: actin waves, Dictyostelium, membrane tension, pattern formation, phagocytosis, PI3-kinase, PI(3,4,5)P3, self-organization