There is, I think, something ethereal about i —the square root of minus one. I remember first hearing about it at school. It seemed an odd beast at that time—an intruder hovering on the edge of reality.

Usually familiarity dulls this sense of the bizarre, but in the case of i it was the reverse: over the years the sense of its surreal nature intensified. It seemed that it was impossible to write mathematics that described the real world in …

I and i

Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter]-[coding sequence]-[3' tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and internal ribosome entry sequence-driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large-scale projects testing the functions of libraries of regulatory or coding sequences.

https://anatomypubs.onlinelibrary.wiley.com/doi/epdf/10.1002/dvdy.21343

The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.

*Summary Gateway cloning technology facilitates high-throughput cloning of target sequences by making use of the bacteriophage lambda site-specific recombination system. Target sequences are first captured in a commercially available ‘entry vector’ and are then recombined into various ‘destination vectors’ for expression in different experimental organisms. Gateway technology has been embraced by a number of plant laboratories that have engineered destination vectors for promoter specificity analyses, protein localization studies, protein/protein interaction studies, constitutive or inducible protein expression studies, gene knockdown by RNA interference, or affinity purification experiments. We review the various types of Gateway destination vectors that are currently available to the plant research community and provide links and references to enable additional information to be obtained concerning these vectors. We also describe a set of ‘pEarleyGate’ plasmid vectors for Agrobacterium-mediated plant transformation that translationally fuse FLAG, HA, cMyc, AcV5 or tandem affinity purification epitope tags onto target proteins, with or without an adjacent fluorescent protein. The oligopeptide epitope tags allow the affinity purification, immunolocalization or immunoprecipitation of recombinant proteins expressed in vivo. We demonstrate the utility of pEarleyGate destination vectors for the expression of epitope-tagged proteins that can be affinity captured or localized by immunofluorescence microscopy. Antibodies detecting the FLAG, HA, cMyc and AcV5 tags show relatively little cross-reaction with endogenous proteins in a variety of monocotyledonous and dicotyledonous plants, suggesting broad utility for the tags and vectors.

/pdf/gateway-compatible-vectors-for-plant-functional-genomics-and-1jp90m60g7.pdf

Gateway-compatible vectors for plant functional genomics and proteomics.

2458 Despite dramatic improvements in treatment over the past 40 years, acute lymphoblastic leukemia (ALL) remains one of the most common causes of death from disease in childhood. Glucocorticoids are among the most effective agents used in the treatment of lymphoid malignancies, and patient response to treatment is an important determinant of long-term outcome in childhood ALL. In spite of its clinical significance, the molecular basis of glucocorticoid resistance is still poorly understood. The aim of this study was to develop an experimental model system to define clinically relevant mechanisms of glucocorticoid resistance in childhood ALL. An in vivo model of childhood ALL has been developed in our laboratory, using patient biopsies established as xenografts in immune-deficient nonobese diabetic severe-combined immunodeficient (NOD/SCID) mice. This model is highly representative of the human disease (Lock et al., Blood, 99: 4100-4108, 2002). The in vivo responses of these xenografts to the glucocorticoid dexamethasone (DEX) correlated significantly with patient outcome (p 1 μM) in xenografts from six patients, five of whom died of their disease. In contrast, four DEX-sensitive xenografts (IC50 values 2-fold in sensitive xenografts within 8 hours of treatment. In contrast, Bim induction was dramatically attenuated in DEX-resistant xenografts. These results have identified a clinically significant and novel mechanism of glucocorticoid resistance in childhood ALL, which occurs downstream of receptor-ligand interactions, but upstream of the signalling pathway resulting in Bim induction and apoptosis.

Novel mechanisms of glucocorticoid resistance in childhood acute lymphoblastic leukemia.

Three-dimensional vibration analysis of rotating pre-twisted variable thickness blades composed of spanwise graded functional materials

CRISPR/Cas9 and associated base editing systems are used to precisely modify target genes in diverse cell types and organisms. However, CRISPR/Cas-induced mutants often contain small insertions and deletions (indels) or single nucleotide variations (SNVs) at edited sites with different genotypes, and gene editing frequency (GEF) typically varies between editing sites and editing systems. Therefore, methods are needed to identify mutants and their genotypes, and evaluate GEF. Herein, we describe a novel SMART approach for simultaneous analysis of CRISPR/Cas-induced mutants, genotypes and GEF, using DNA-guided Thermus thermophilus Argonaute ( TtAgo) in vitro cleavage-mediated quantitative real-time PCR (qPCR) or digital PCR. SMART proved versatile for induced mutant identification, genotyping, and GEF evaluation using CRISPR/Cas9 gene-editing offspring lines harbouring the rice RNA-directed DNA methylation 3-like (RdDM3l) gene. SMART is higher specific, sensitive, and accurate than previously methods. SMART detected all types of mutations, including small indels, large indels and nucleotide substitutions, with good sensitivity regardless of mutation type. TtAgo has few strict requirements or restrictions for target sequences, such as restriction sites, GC content or protospacer-adjacent motifs (PAMs). SMART facilitates versatile CRISPR/Cas-induced mutant screening, genotyping, and GEF quantification, and it could be developed for clinical detection of rare mutations involving SNVs and indels. • SMART demonstrated a new application feature of TtAgo in mutant analysis • SMART is a novel approach to screen, genotype and evaluate GEF of mutants • SMART is higher specific, sensitive, and accurate than previously methods 

Dabing Zhang

Papers

Three-dimensional vibration analysis of rotating pre-twisted variable thickness blades composed of spanwise graded functional materials

In vitro Argonaute cleavage-mediated quantitative PCR facilitates versatile CRISPR/Cas-induced mutant analysis

Effect of post-treatment on the microstructure and properties of Ti-27Nb-5Zr-2Cr alloy prepared by laser powder deposition

Translational plant biology.

One Versatile Cas9 Integrated Single-Tube Pcr System for Rapid Analysis of Crispr/Cas Induced Mutants