Method to Assemble Genomic DNA Fragments or Genes on Human Artificial Chromosome with Regulated Kinetochore Using a Multi-Integrase System

The production of cells capable of carrying multiple transgenes to Mb-size genomic loci has multiple applications in biomedicine and biotechnology. In order to achieve this goal, three key steps are required: (i) cloning of large genomic segments; (ii) insertion of multiple DNA blocks at a precise location and (iii) the capability to eliminate the assembled region from cells. In this study, we designed the iterative integration system (IIS) that utilizes recombinases Cre, ΦC31 and ΦBT1, and combined it with a human artificial chromosome (HAC) possessing a regulated kinetochore (alphoid^tetO-HAC). We have demonstrated that the IIS-alphoid^tetO-HAC system is a valuable genetic tool by reassembling a functional gene from multiple segments on the HAC. IIS-alphoid^tetO-HAC has several notable advantages over other artificial chromosome-based systems. This includes the potential to assemble an unlimited number of genomic DNA segments; a DNA assembly process that leaves only a small insertion (<60 bp) scar between adjacent DNA, allowing genes reassembled from segments to be spliced correctly; a marker exchange system that also changes cell color, and counter-selection markers at each DNA insertion step, simplifying selection of correct clones; and presence of an error proofing mechanism to remove cells with misincorporated DNA segments, which improves the integrity of assembly. In addition, the IIS-alphoid^tetO-HAC carrying a locus of interest is removable, offering the unique possibility to revert the cell line to its pretransformed state and compare the phenotypes of human cells with and without a functional copy of a gene(s). Thus, IIS-alphoid^tetO-HAC allows investigation of complex biomedical pathways, gene(s) regulation, and has the potential to engineer synthetic chromosomes with a predetermined set of genes

Risk of nonalcoholic fatty liver disease and associations with gastrointestinal cancers

Metabolic syndrome may contribute to the rising incidence of multiple gastrointestinal (GI) cancers in recent birth cohorts. However, other than hepatocellular carcinoma, the association between nonalcoholic fatty liver disease (NAFLD) and risk of non-liver GI cancers is unexplored. We prospectively examined the associations of NAFLD risk with GI cancers among 319,290 participants in the UK Biobank (2006-2019). Baseline risk for NAFLD was estimated using the Dallas Steatosis Index, a validated prediction tool. Multivariable Cox models were used to estimate relative risks (RRs) and 95% confidence intervals (CIs) according to NAFLD risk categories: low (\u3c20%), intermediate (20%-49%), and high (≥50%). We also examined the associations by age of cancer diagnosis (earlier onset [\u3c60] vs. ≥60). A total of 273 incident liver cancer and 4789 non-liver GI cancer cases were diagnosed. Compared with individuals at low risk for NAFLD, those at high risk had 2.41-fold risk of liver cancer (RR = 2.41, 95% CI: 1.73-3.35) and 23% increased risk of non-liver GI cancers (RR = 1.23, 95% CI: 1.14-1.32) (all

Free carrier effects in gallium nitride epilayers: the valence band dispersion

The dispersion of the A-valence-band in GaN has been deduced from the observation of high-index magneto-excitonic states in polarised interband magneto-reflectivity and is found to be strongly non-parabolic with a mass in the range 1.2-1.8 m_{e}. It matches the theory of Kim et al. [Phys. Rev. B 56, 7363 (1997)] extremely well, which also gives a strong k-dependent A-valence-band mass. A strong phonon coupling leads to quenching of the observed transitions at an LO-phonon energy above the band gap and a strong non-parabolicity. The valence band was deduced from subtracting from the reduced dispersion the electron contribution with a model that includes a full treatment of the electron-phonon interaction.Comment: Revtex, 4 pages, 5 figure

Site-specific recombination in Schizosaccharomyces pombe and systematic assembly of a 400kb transgene array in mammalian cells using the integrase of Streptomyces phage ϕBT1

We have established the integrase of the Streptomyces phage ϕBT1 as a tool for eukaryotic genome manipulation. We show that the ϕBT1 integrase promotes efficient reciprocal and conservative site-specific recombination in vertebrate cells and in Schizosaccharomyces pombe, thus establishing the utility of this protein for genome manipulation in a wide range of eukaryotes. We show that the ϕBT1 integrase can be used in conjunction with Cre recombinase to promote the iterative integration of transgenic DNA. We describe five cycles of iterative integration of a candidate mouse centromeric sequence 80 kb in length into a human mini-chromosome within a human-Chinese hamster hybrid cell line. These results establish the generality of the iterative site-specific integration technique

Modelling chemistry in the nocturnal boundary layer above tropical rainforest and a generalised effective nocturnal ozone deposition velocity for sub-ppbv NOx conditions

Measurements of atmospheric composition have been made over a remote rainforest landscape. A box model has previously been demonstrated to model the observed daytime chemistry well. However the box model is unable to explain the nocturnal measurements of relatively high [NO] and [O3], but relatively low observed [NO2]. It is shown that a one-dimensional (1-D) column model with simple O3 -NOx chemistry and a simple representation of vertical transport is able to explain the observed nocturnal concentrations and predict the likely vertical profiles of these species in the nocturnal boundary layer (NBL). Concentrations of tracers carried over from the end of the night can affect the atmospheric chemistry of the following day. To ascertain the anomaly introduced by using the box model to represent the NBL, vertically-averaged NBL concentrations at the end of the night are compared between the 1-D model and the box model. It is found that, under low to medium [NOx] conditions (NOx <1 ppbv), a simple parametrisation can be used to modify the box model deposition velocity of ozone, in order to achieve good agreement between the box and 1-D models for these end-of-night concentrations of NOx and O3. This parametrisation would could also be used in global climate-chemistry models with limited vertical resolution near the surface. Box-model results for the following day differ significantly if this effective nocturnal deposition velocity for ozone is implemented; for instance, there is a 9% increase in the following day’s peak ozone concentration. However under medium to high [NOx] conditions (NOx > 1 ppbv), the effect on the chemistry due to the vertical distribution of the species means no box model can adequately represent chemistry in the NBL without modifying reaction rate constants

CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity.

Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases

Empirical Power and Sample Size Calculations for Cluster-Randomized and Cluster-Randomized Crossover Studies

In recent years, the number of studies using a cluster-randomized design has grown dramatically. In addition, the cluster-randomized crossover design has been touted as a methodological advance that can increase efficiency of cluster-randomized studies in certain situations. While the cluster-randomized crossover trial has become a popular tool, standards of design, analysis, reporting and implementation have not been established for this emergent design. We address one particular aspect of cluster-randomized and cluster-randomized crossover trial design: estimating statistical power. We present a general framework for estimating power via simulation in cluster-randomized studies with or without one or more crossover periods. We have implemented this framework in the clusterPower software package for R, freely available online from the Comprehensive R Archive Network. Our simulation framework is easy to implement and users may customize the methods used for data analysis. We give four examples of using the software in practice. The clusterPower package could play an important role in the design of future cluster-randomized and cluster-randomized crossover studies. This work is the first to establish a universal method for calculating power for both cluster-randomized and cluster-randomized clinical trials. More research is needed to develop standardized and recommended methodology for cluster-randomized crossover studies

Reactive fragments targeting carboxylate residues employing direct to biology, high-throughput chemistry

The screening of covalent or ‘reactive’ fragment libraries against proteins is becoming an integral approach in hit identification, enabling the development of targeted covalent inhibitors and tools. To date, reactive fragment screening has been limited to targeting cysteine residues, thus restricting applicability across the proteome. Carboxylate residues present a unique opportunity to expand the accessible residues due to high proteome occurrence (∼12%). Herein, we present the development of a carboxylate-targeting reactive fragment screening platform utilising 2-aryl-5-carboxytetrazole (ACT) as the photoreactive functionality. The utility of ACT photoreactive fragments (ACT-PhABits) was evaluated by screening a 546-membered library with a small panel of purified proteins. Hits identified for BCL6 and KRASG12D were characterised by LC-MS/MS studies, revealing the selectivity of the ACT group. Finally, a photosensitised approach to ACT activation was developed, obviating the need for high energy UV-B light

Sleep-wake sensitive mechanisms of adenosine release in the basal forebrain of rodents : an in vitro study

Adenosine acting in the basal forebrain is a key mediator of sleep homeostasis. Extracellular adenosine concentrations increase during wakefulness, especially during prolonged wakefulness and lead to increased sleep pressure and subsequent rebound sleep. The release of endogenous adenosine during the sleep-wake cycle has mainly been studied in vivo with microdialysis techniques. The biochemical changes that accompany sleep-wake status may be preserved in vitro. We have therefore used adenosine-sensitive biosensors in slices of the basal forebrain (BFB) to study both depolarization-evoked adenosine release and the steady state adenosine tone in rats, mice and hamsters. Adenosine release was evoked by high K+, AMPA, NMDA and mGlu receptor agonists, but not by other transmitters associated with wakefulness such as orexin, histamine or neurotensin. Evoked and basal adenosine release in the BFB in vitro exhibited three key features: the magnitude of each varied systematically with the diurnal time at which the animal was sacrificed; sleep deprivation prior to sacrifice greatly increased both evoked adenosine release and the basal tone; and the enhancement of evoked adenosine release and basal tone resulting from sleep deprivation was reversed by the inducible nitric oxide synthase (iNOS) inhibitor, 1400 W. These data indicate that characteristics of adenosine release recorded in the BFB in vitro reflect those that have been linked in vivo to the homeostatic control of sleep. Our results provide methodologically independent support for a key role for induction of iNOS as a trigger for enhanced adenosine release following sleep deprivation and suggest that this induction may constitute a biochemical memory of this state