Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9

CRISPR/Cas9 technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells. Similarly, certain endogenous genes have been tagged with fluorescent proteins. Often, the detection of tagged proteins requires high expression and sophisticated tools such as confocal microscopy and flow cytometry. Therefore, a simple, sensitive and robust transcriptional reporter system driven by endogenous promoter for studies into transcriptional regulation is desirable. We report a CRISPR/Cas9-based methodology for rapidly integrating a firefly luciferase gene in somatic cells under the control of endogenous promoter, using the TGFβ-responsive gene PAI-1. Our strategy employed a polycistronic cassette containing a non-fused GFP protein to ensure the detection of transgene delivery and rapid isolation of positive clones. We demonstrate that firefly luciferase cDNA can be efficiently delivered downstream of the promoter of the TGFβ-responsive gene PAI-1. Using chemical and genetic regulators of TGFβ signalling, we show that it mimics the transcriptional regulation of endogenous PAI-1 expression. Our unique approach has the potential to expedite studies on transcription of any gene in the context of its native chromatin landscape in somatic cells, allowing for robust high-throughput chemical and genetic screens

Two experiments for the price of one? -- The role of the second oscillation maximum in long baseline neutrino experiments

We investigate the quantitative impact that data from the second oscillation maximum has on the performance of wide band beam neutrino oscillation experiments. We present results for the physics sensitivities to standard three flavor oscillation, as well as results for the sensitivity to non-standard interactions. The quantitative study is performed using an experimental setup similar to the Fermilab to DUSEL Long Baseline Neutrino Experiment (LBNE). We find that, with the single exception of sensitivity to the mass hierarchy, the second maximum plays only a marginal role due to the experimental difficulties to obtain a statistically significant and sufficiently background-free event sample at low energies. This conclusion is valid for both water Cherenkov and liquid argon detectors. Moreover, we confirm that non-standard neutrino interactions are very hard to distinguish experimentally from standard three-flavor effects and can lead to a considerable loss of sensitivity to \theta_{13}, the mass hierarchy and CP violation.Comment: RevTex 4.1, 23 pages, 10 figures; v2: Typos corrected, very minor clarifications; matches published version; v3: Fixed a typo in the first equation in sec. III

Imaging of acute appendicitis in children: EU versus US ... or US versus CT? A European perspective

There is substantial evidence that imaging may reduce the negative appendectomy rate, also in children. However, controversy exists about the preferred method: US or CT, and the choice appears to be determined by the side of the Atlantic Ocean. This review brings forth several arguments in favour of U

Institutional logics and interorganizational learning in technological arenas: Evidence from standard-setting organizations in the mobile handset industry

© 2015, INFORMS. Conceptualizing standard-setting organizations (SSOs) as technological arenas within which firms from different countries interact and learn, we offer insights into the interplay between firms' institutional logics and their interorganizational learning outcomes. We suggest that firms' interorganizational learning is embedded in their macrolevel country contexts, characterized by more corporatist versus less corporatist (pluralist) institutional logics. Whereas corporatism spurs coordinated approaches, pluralism engenders competitive interactions that affect the extent to which firms span organizational and technological boundaries and learn from each other. We test our theory using longitudinal analysis of 181 dyads involving 26 firms participating in 17 SSOs in the global mobile handset industry. We find that interorganizational learning, as measured by patent citations, involving corporatist firm dyads significantly increases when the dominant logic within the arena is also corporatist. By making cooperative schemas more accessible, a dominant corporatist logic also enhances interorganizational learning across technologically distant dyads. When a pluralist logic dominates the arena, corporatist dyads learn less because firms in the dyad activate a contradictory logic that decouples them from their natural processes for interorganizational learning. These findings highlight the implications of institutional logics for interorganizational learning outcomes and provide insights into how firms attend to institutional contradictions in arenas that provide opportunities for interorganizational learning

Computerised cognitive assessment in patients with traumatic brain injury: an observational study of feasibility and sensitivity relative to established clinical scales

Background: Online technology could potentially revolutionise how patients are cognitively assessed and monitored. However, it remains unclear whether assessments conducted remotely can match established pen-and-paper neuropsychological tests in terms of sensitivity and specificity. Methods: This observational study aimed to optimise an online cognitive assessment for use in traumatic brain injury (TBI) clinics. The tertiary referral clinic in which this tool has been clinically implemented typically sees patients a minimum of 6 months post-injury in the chronic phase. Between March and August 2019, we conducted a cross-group, cross-device and factor analyses at the St. Mary's Hospital TBI clinic and major trauma wards at Imperial College NHS trust and St. George's Hospital in London (UK), to identify a battery of tasks that assess aspects of cognition affected by TBI. Between September 2019 and February 2020, we evaluated the online battery against standard face-to-face neuropsychological tests at the Imperial College London research centre. Canonical Correlation Analysis (CCA) determined the shared variance between the online battery and standard neuropsychological tests. Finally, between October 2020 and December 2021, the tests were integrated into a framework that automatically generates a results report where patients’ performance is compared to a large normative dataset. We piloted this as a practical tool to be used under supervised and unsupervised conditions at the St. Mary's Hospital TBI clinic in London (UK). Findings: The online assessment discriminated processing-speed, visual-attention, working-memory, and executive-function deficits in TBI. CCA identified two significant modes indicating shared variance with standard neuropsychological tests (r = 0.86, p < 0.001 and r = 0.81, p = 0.02). Sensitivity to cognitive deficits after TBI was evident in the TBI clinic setting under supervised and unsupervised conditions (F (15,555) = 3.99; p < 0.001). Interpretation: Online cognitive assessment of TBI patients is feasible, sensitive, and efficient. When combined with normative sociodemographic models and autogenerated reports, it has the potential to transform cognitive assessment in the healthcare setting. Funding: This work was funded by a National Institute for Health Research (NIHR) Invention for Innovation (i4i) grant awarded to DJS and AH ( II-LB-0715-20006)

MiniBooNE and LSND data: non-standard neutrino interactions in a (3+1) scheme versus (3+2) oscillations

The recently observed event excess in MiniBooNE anti-neutrino data is in agreement with the LSND evidence for electron anti-neutrino appearance. We propose an explanation of these data in terms of a (3+1) scheme with a sterile neutrino including non-standard neutrino interactions (NSI) at neutrino production and detection. The interference between oscillations and NSI provides a source for CP violation which we use to reconcile different results from neutrino and anti-neutrino data. Our best fit results imply NSI at the level of a few percent relative to the standard weak interaction, in agreement with current bounds. We compare the quality of the NSI fit to the one obtained within the (3+1) and (3+2) pure oscillation frameworks. We also briefly comment on using NSI (in an effective two-flavour framework) to address a possible difference in neutrino and anti-neutrino results from the MINOS experiment.Comment: 28 pages, 9 figures, discussion improved, new appendix added, conclusions unchange

Predicting the Tolerated Sequences for Proteins and Protein Interfaces Using RosettaBackrub Flexible Backbone Design

Predicting the set of sequences that are tolerated by a protein or protein interface, while maintaining a desired function, is useful for characterizing protein interaction specificity and for computationally designing sequence libraries to engineer proteins with new functions. Here we provide a general method, a detailed set of protocols, and several benchmarks and analyses for estimating tolerated sequences using flexible backbone protein design implemented in the Rosetta molecular modeling software suite. The input to the method is at least one experimentally determined three-dimensional protein structure or high-quality model. The starting structure(s) are expanded or refined into a conformational ensemble using Monte Carlo simulations consisting of backrub backbone and side chain moves in Rosetta. The method then uses a combination of simulated annealing and genetic algorithm optimization methods to enrich for low-energy sequences for the individual members of the ensemble. To emphasize certain functional requirements (e.g. forming a binding interface), interactions between and within parts of the structure (e.g. domains) can be reweighted in the scoring function. Results from each backbone structure are merged together to create a single estimate for the tolerated sequence space. We provide an extensive description of the protocol and its parameters, all source code, example analysis scripts and three tests applying this method to finding sequences predicted to stabilize proteins or protein interfaces. The generality of this method makes many other applications possible, for example stabilizing interactions with small molecules, DNA, or RNA. Through the use of within-domain reweighting and/or multistate design, it may also be possible to use this method to find sequences that stabilize particular protein conformations or binding interactions over others

Low-Energy Probes of a Warped Extra Dimension

We investigate a natural realization of a light Abelian hidden sector in an extended Randall-Sundrum (RS) model. In addition to the usual RS bulk we consider a second warped space containing a bulk U(1)_x gauge theory with a characteristic IR scale of order a GeV. This Abelian hidden sector can couple to the standard model via gauge kinetic mixing on a common UV brane. We show that if such a coupling induces significant mixing between the lightest U(1)_x gauge mode and the standard model photon and Z, it can also induce significant mixing with the heavier U(1)_x Kaluza-Klein (KK) modes. As a result it might be possible to probe several KK modes in upcoming fixed-target experiments and meson factories, thereby offering a new way to investigate the structure of an extra spacetime dimension.Comment: 26 pages, 1 figure, added references, corrected minor typos, same as journal versio

Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders

Abstract CRISPR/Cas9 holds immense potential to treat a range of genetic disorders. Allele-specific gene disruption induced by non-homologous end-joining (NHEJ) DNA repair offers a potential treatment option for autosomal dominant disease. Here, we successfully delivered a plasmid encoding S. pyogenes Cas9 and sgRNA to the corneal epithelium by intrastromal injection and acheived long-term knockdown of a corneal epithelial reporter gene, demonstrating gene disruption via NHEJ in vivo. In addition, we used TGFBI corneal dystrophies as a model of autosomal dominant disease to assess the use of CRISPR/Cas9 in two allele-specific systems, comparing cleavage using a SNP-derived PAM to a guide specific approach. In vitro, cleavage via a SNP-derived PAM was found to confer stringent allele-specific cleavage, while a guide-specific approach lacked the ability to distinguish between the wild-type and mutant alleles. The failings of the guide-specific approach highlights the necessity for meticulous guide design and assessment, as various degrees of allele-specificity are achieved depending on the guide sequence employed. A major concern for the use of CRISPR/Cas9 is its tendency to cleave DNA non-specifically at “off-target” sites. Confirmation that S. pyogenes Cas9 lacks the specificity to discriminate between alleles differing by a single base-pair regardless of the position in the guide is demonstrated

Force of tuberculosis infection among adolescents in a high HIV and TB prevalence community: a cross-sectional observation study

BACKGROUND: Understanding of the transmission dynamics of tuberculosis (TB) in high TB and HIV prevalent settings is required in order to develop effective intervention strategies for TB control. However, there are little data assessing incidence of TB infection in adolescents in these settings. METHODS: We performed a tuberculin skin test (TST) and HIV survey among secondary school learners in a high HIV and TB prevalence community. TST responses to purified protein derivative RT23 were read after 3 days. HIV-infection was assessed using Orasure(R) collection device and ELISA testing. The results of the HIV-uninfected participants were combined with those from previous surveys among primary school learners in the same community, and force of TB infection was calculated by age. RESULTS: The age of 820 secondary school participants ranged from 13 to 22 years. 159 participants had participated in the primary school surveys. At a 10 mm cut-off, prevalence of TB infection among HIV-uninfected and first time participants, was 54% (n = 334/620). HIV prevalence was 5% (n = 40/816). HIV infection was not significantly associated with TST positivity (p = 0.07). In the combined survey dataset, TB prevalence was 45% (n = 645/1451), and was associated with increasing age and male gender. Force of infection increased with age, from 3% to 7.3% in adolescents [greater than or equal to]20 years of age. CONCLUSIONS: We show a high force of infection among adolescents, positively associated with increasing age. We postulate this is due to increased social contact with infectious TB cases. Control of the TB epidemic in this setting will require reducing the force of infection