hTERT-RPE1 cells are genetically stable near diploid cells widely used to model cell division, DNA repair, or ciliogenesis in a non-transformed context. However, poor transfectability and limited ...homology-directed repair capacity hamper their amenability to gene editing. Here, we describe a protocol for rapid and efficient generation of diverse homozygous knockins. In contrast to other approaches, this strategy bypasses the need for molecular cloning. Our approach can also be applied to a variety of cell types including cancer and induced pluripotent stem cells (iPSCs).
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•Rapid and efficient generation of knockins in hTERT-RPE1 cells•Step-by-step guide for RNP-mediated editing bypassing the need for molecular cloning•Enhanced knockin efficiency by transient inhibition of NHEJ•Suitable for epitope tagging, point mutations, or fluorescent tagging
hTERT-RPE1 cells are genetically stable near diploid cells widely used to model cell division, DNA repair, or ciliogenesis in a non-transformed context. However, poor transfectability and limited homology-directed repair capacity hamper their amenability to gene editing. Here, we describe a protocol for rapid and efficient generation of diverse homozygous knockins. In contrast to other approaches, this strategy bypasses the need for molecular cloning. Our approach can also be applied to a variety of cell types including cancer and induced pluripotent stem cells (iPSCs).
Objective: The disinhibition scale of the Eating Inventory predicts weight loss outcome; however, it may include multiple factors. The purpose of this study was to examine the factor structure of the ...disinhibition scale and determine how its factors independently relate to long‐term weight loss outcomes.
Research Methods and Procedures: Exploratory factor analysis of the disinhibition scale was conducted on 286 participants in a behavioral weight loss trial (TRIM), and confirmatory factor analysis was conducted on 3345 members of the National Weight Control Registry (NWCR), a registry of successful weight loss maintainers. Multivariate regressions were used to examine the relationships between the disinhibition scale factors and weight over time in both samples.
Results: Using baseline data from TRIM, two factors were extracted from the disinhibition scale: 1) an “internal” factor that described eating in response to internal cues, such as feelings and thoughts; and 2) an “external” factor that described eating in response to external cues, such as social events. This factor structure was confirmed using confirmatory factor analysis in the NWCR. In TRIM, internal disinhibition significantly predicted weight loss at 6 months (p = 0.03) and marginally significantly predicted weight loss at 18 months (p = 0.06), with higher levels of internal disinhibition at baseline predicting less weight loss; external disinhibition did not predict weight loss at any time‐point. In NWCR, internal disinhibition significantly predicted one‐year weight change (p = 0.001), while external disinhibition did not.
Discussion: These results suggest that it is the disinhibition of eating in response to internal cues that is associated with poorer long‐term weight loss outcomes.
We present a two-point model to investigate the underlying source mechanisms for broadband shock-associated noise (BBSAN) in shock-containing supersonic jets. In the model presented, the generation ...of BBSAN is assumed to arise from the nonlinear interaction between downstream-propagating coherent structures with the quasi-periodic shock cells in the jet plume. The turbulent perturbations are represented as axially extended wavepackets and the shock cells are modelled as a set of stationary waveguide modes. Unlike previous BBSAN models, the physical parameters describing the hydrodynamic components are not scaled using the acoustic field. Instead, the source characteristics of both the turbulent and shock components are extracted from the hydrodynamic region of large-eddy simulation and particle image velocimetry datasets. Apart from using extracted data, a reduced-order description of the wavepacket structure is obtained using parabolised stability equations. The validity of the model is tested by comparing far-field sound pressure level predictions to azimuthally decomposed experimental acoustic data from a cold Mach 1.5 underexpanded jet. At polar angles and frequencies where BBSAN dominates, encouraging comparisons of the radiated noise spectra for the first three azimuthal modes, in both frequency and amplitude (${\pm }2\ \textrm {dB}\,\textrm {St}^{-1}$ at peak frequency), reinforce the suitability of using reduced-order wavepacket sources for predicting BBSAN peaks. On the other hand, wavepacket jitter is found to have a critical role in recovering sound amplitude at interpeak frequencies. The paper presents a quantitative demonstration that the wavepacket–shock interaction, carefully reconstructed by extracting components from data or linearised models, contains the correct essential flow physics that accounts for most features of the far-field BBSAN spectra.
The PIDDosome is often used as the alias for a multi-protein complex that includes the p53-induced death domain protein 1 (PIDD1), the bipartite linker protein CRADD (also known as RAIDD) and the ...pro-form of an endopeptidase belonging to the caspase family, i.e. caspase-2. Yet, PIDD1 variants can also interact with a number of other proteins that include RIPK1 (also known as RIP1) and IKBKG (also known as NEMO), PCNA and RFC5, as well as nucleolar components such as NPM1 or NCL. This promiscuity in protein binding is facilitated mainly by autoprocessing of the full-length protein into various fragments that contain different structural domains. As a result, multiple responses can be mediated by protein complexes that contain a PIDD1 domain. This suggests that PIDD1 acts as an integrator for multiple types of stress that need instant attention. Examples are various types of DNA lesion but also the presence of extra centrosomes that can foster aneuploidy and, ultimately, promote DNA damage. Here, we review the role of PIDD1 in response to DNA damage and also highlight novel functions of PIDD1, such as in centrosome surveillance and scheduled polyploidisation as part of a cellular differentiation program during organogenesis.
Centrosome amplification results into genetic instability and predisposes cells to neoplastic transformation. Supernumerary centrosomes trigger p53 stabilization dependent on the PIDDosome (a ...multiprotein complex composed by PIDD1, RAIDD and Caspase‐2), whose activation results in cleavage of p53’s key inhibitor, MDM2. Here, we demonstrate that PIDD1 is recruited to mature centrosomes by the centriolar distal appendage protein ANKRD26. PIDDosome‐dependent Caspase‐2 activation requires not only PIDD1 centrosomal localization, but also its autoproteolysis. Following cytokinesis failure, supernumerary centrosomes form clusters, which appear to be necessary for PIDDosome activation. In addition, in the context of DNA damage, activation of the complex results from a p53‐dependent elevation of PIDD1 levels independently of centrosome amplification. We propose that PIDDosome activation can in both cases be promoted by an ANKRD26‐dependent local increase in PIDD1 concentration close to the centrosome. Collectively, these findings provide a paradigm for how centrosomes can contribute to cell fate determination by igniting a signalling cascade.
SYNOPSIS
The PIDDosome is a Caspase‐2‐activating platform assembling in response to two stimuli: genotoxic stress and supernumerary centrosomes. Here, both triggers are shown to rely on centrosomal localization of PIDD1, highlighting the centrosome’s capability to influence cell fate.
Centriolar distal appendage protein ANKRD26 is the centrosomal PIDD1 receptor.
The centrosome recruits the PIDD1 precursor, which undergoes subsequent autoproteolysis.
Clustering of PIDD1‐positive extra‐centrosomes is required for PIDDosome activation.
P53‐dependent PIDD1 transactivation leads to centrosome‐dependent PIDDosome activation even in the absence of extra‐centrosomes.
ANKRD26‐dependent PIDD1 recruitment is involved in p53 activation both upon genotoxic stress and in the presence of supernumerary centrosomes.
A stage-matched expert system intervention was evaluated on 4144 smokers in a two-arm randomized control trial with four follow-ups over 24 months. Smokers were recruited by random digit-dial calls, ...and 80.0% of the eligible smokers were enrolled. Individualized and interactive expert system computer reports were sent at 0, 3, and 6 months. The reports provided feedback on 15 variables relevant for progressing through the stages. The primary outcomes were point prevalence and prolonged abstinence rates. At 24 months, the expert system resulted in 25.6% point prevalence and 12% prolonged abstinence, which were 30% and 56% greater than the control condition. Abstinence rates at each 6-month follow-up were significantly greater in the Expert System (ES) condition than in the comparison condition with the absolute difference increasing at each follow-up. A proactive home-based stage-matched expert system smoking cessation program can produce both high participation rates and relatively high abstinence rates.
Review article Calcium hydroxide has been used in endodontology for many years. The aim of this paper is to review the various formulations of calcium hydroxide that have been described, with ...specific reference to the vehicle used to carry the compound. The requirements for a vehicle are described, and ex vivo and in vivo studiesreviewed. Vehicles can be classified into aqueous, viscous and oily, the clinical properties of calcium hydroxide changing depending on the vehicle. The review also describes the use of various active components that have been added to calcium hydroxide, including antimicrobial and anti‐inflammatory agents. This review will help clinicians to make informed judgements about which formulations of calcium hydroxide should be used for specific endodontic procedures.
The purpose of this pilot study is to assess the reliability and construct validity of a measure of relationship-focused antecedents for smoking (RAS). The scale includes both positively-valenced ...items (e.g.. “I feel like smoking when I am relaxing with my partner”) and negatively-valenced items (e.g., “I feel like smoking when my partner criticizes me”). Participants included 123 individuals who smoke cigarettes with co-habitating smoking (n=63) or non-smoking (n=60) romantic partners. Participants completed the RAS and a series of measures associated with smoking outcomes. Principal component analysis with varimax rotation resulted in a 2-component solution. The RAS showed excellent internal consistency for the total scale (α=.96) and for the positive (α=.88) and negative (α=.97) subscales. Higher positive subscale scores were associated with lower motivation to quit while higher negative scores were associated with lower relationship satisfaction and dyadic efficacy to quit. Higher scores on both subscales were related to higher social motives, dependence motives, and social outcome expectances. Participants with smoking partners reported higher positive subscale scores and lower negative subscale scores. The RAS may be helpful in the design of smoking cessation interventions for couples.
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