Progress and Challenges for Live-cell Imaging of Genomic Loci Using CRISPR-based Platforms

Chromatin conformation,localization,and dynamics are crucial regulators of cellular behaviors. Although fluorescence in situ hybridization-based techniques have been widely utilized for investigating chromatin architectures in healthy and diseased states,the requirement for cell fix-ation precludes the comprehensive dynamic analysis necessary to fully understand chromatin activ-ities. This has spurred the development and application of a variety of imaging methodologies for visualizing single chromosomal loci in the native cellular context. In this review,we describe currently-available approaches for imaging single genomic loci in cells,with special focus on clus-tered regularly interspaced short palindromic repeats (CRISPR)-based imaging approaches. In addition,we discuss some of the challenges that limit the application of CRISPR-based genomic imaging approaches,and potential solutions to address these challenges. We anticipate that,with continued refinement of CRISPR-based imaging techniques,significant understanding can be gained to help decipher chromatin activities and their relevance to cellular physiology and pathogenesis.     

Two-step method to isolate target recombinant protein from co-purified bacterial contaminant SlyD after immobilised metal affinity chromatography

As part of a study to purify the internal domain of HER2 (ICD) from recombinant expression, through metal immobilised affinity chromatography (IMAC), we encountered a contaminant, SlyD, a 29 kDa native E. coli protein. SlyD is a recurrent contaminant, with a histidine rich domain enabling binding to IMAC columns and thus co-elution with the target protein. Research has been carried out on this protein and its purification, however, no work mentions how to treat it as a true contaminant or describe procedures to isolate it from target proteins. In this report, we described a two-step chromatographic method for the purification of ICD, including IMAC as a capture step and size exclusion chromatography (SEC) as a polishing step. IMAC allowed us to purify ICD from bacterial crude with SlyD co-eluting. SEC then allowed us to resolve ICD from SlyD and achieve a purity greater than 95% for ICD. However, this method has been developed to accommodate any protein whose molecular weight is different enough from SlyD to be separated by SEC.     

Application of conformationally restricted peptidomimetics to modeling the bound conformation of peptide antagonists with the IL-1 receptor

A collection of complementary peptide caricatures that closely mimic low-energy (presumably highly populated) conformations of amino acids of interest would constitute a valuable tool set to study the interactions of small peptide ligands with their biological targets. Our general strategy for the design, synthesis and application of peptidomimetics is presented. An illustration of how structural information from mimetics combined with cutting edge biophysical data can be used to derive a model for the bound conformation of an 11-mer peptide antagonist with the IL-1 receptor is given.     

Improved <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and direct plant regeneration in four cultivars of finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.)

We have developed an improved Agrobacterium-mediated transformation and rapid regeneration system for four cultivars (‘CO(Ra)-14’, ‘PR-202’, ‘Try-1’ and ‘Paiyur-2’) of finger millet using optimized transformation and direct plant regeneration conditions. The shoot apical meristems (SAMs) were used as explants in this study. Agrobacterium strain EHA105 carrying binary vector pCAMBIA1301 was used to optimize the transformation conditions. Concentration of hygromycin, the optical density of the culture, infection time, age of the explants, co-cultivation period, the concentrations of acetosyringone and antibiotics were optimized to improve the transformation frequency. The highest frequency of mean transient gus expression (85.1%) was achieved in cultivar ‘CO(Ra)-14’. The entire transformation procedure, from initiating SAMs to planting putative transgenic plantlets in the greenhouse, was completed within 45 days with the highest stable transformation frequency of 11.8% for ‘CO(Ra)-14’. PCR, gus staining and Southern blot analyses were performed in T₀ and T₁ generations to confirm the gene integration. Six events from T₀ had a single copy of the transgene and showed a normal Mendelian pattern of segregation. To our knowledge, this is the first report on the high frequency transformation of finger millet by Agrobacterium and subsequent recovery of transgenic plants via direct plant regeneration without a callus phase, in short duration (45 days). The proposed protocol could be supportive in breaking through the bottleneck in transformation and regeneration of finger millet cultivars.     

Manganese (hyper)accumulation within Australian Denhamia (Celastraceae): an assessment of the trait and manganese accumulation under controlled conditions

Plant and Soil - The genus Denhamia(Celastraceae) includes fifteen Australian species, many of which have a propensity for manganese (Mn) (hyper)accumulation. Among the key aims of this study were...     

Which features of UK farmland are important in retaining territories of the rapidly declining Turtle Dove Streptopelia turtur?

Capsule Turtle Doves continue to show a strong population decline; territories were more likely to be retained in areas with more nesting habitat, and more suitable foraging habitat.

Aim To determine which features of farmland in England are important for retaining Turtle Dove territories

Methods Fifty-eight grid squares with recent records of territorial Turtle Doves were resurveyed, and squares retaining Turtle Dove territories compared with those from which Turtle Doves had been lost.

Results Turtle Dove territories were detected in 48% of squares resurveyed. When correcting for the 70% detection rate of the survey methodology, territories were present in 66% of squares surveyed suggesting a 34% decline over a 2-year period. Established scrub and hedgerows?>?4 m tall positively influenced Turtle Dove presence and abundance, as did standing water. Bare ground and fallow had positive effects on Turtle Dove abundance whereas grazed land negatively impacted abundance.

Conclusion The positive effects of area of established scrub and volume of large hedgerows are likely to represent a declining density of birds selecting the best quality nest sites. We suggest instead that foraging habitat may be limiting distribution.