Analysis of Fluorescent Proteins for Observing Single Gene Locus in a Live and Fixed Escherichia coli Cell.

Fluorescent proteins (FPs) are essential tools for advanced microscopy techniques such as super-resolution imaging, single-particle tracking, and quantitative single-molecule counting. Various FPs fused to DNA-binding proteins have been used to observe the subcellular location and movement of specific gene loci in living and fixed bacterial cells. However, quantitative assessments of the properties of FPs for gene locus measurements are still lacking. Here, we assessed various FPs to observe specific gene loci in live and fixed Escherichia coli cells using a fluorescent repressor-operator binding system (FROS), tet operator-Tet repressor proteins (TetR). Tsr-fused FPs were used to assess the intensity and photostability of various FPs (five red FPs: mCherry2, FusionRed, mRFP, mCrimson3, and dKatushka; and seven yellow FPs: SYFP2, Venus, mCitrine, YPet, mClover3, mTopaz, and EYFP) at the single-molecule level in living cells. These FPs were then used for gene locus measurements using FROS. Our results indicate that TetR-mCrimson3 (red) and TetR-EYFP (yellow) had better properties for visualizing gene loci than the other TetR-FPs. Furthermore, fixation procedures affected the clustering of diffusing TetR-FPs and altered the locations of the TetR-FP foci. Fixation with formaldehyde consistently disrupted proper DNA locus observations using TetR-FPs. Notably, the foci measured using TetR-mCrimson3 remained close to their original positions in live cells after glyoxal fixation. This in vivo study provides a cell-imaging guide for the use of FPs for gene-locus observation in E. coli and a scheme for evaluating the use of FPs for other cell-imaging purposes.

Zero-Mode Waveguide Nanowells for Single-Molecule Detection in Living Cells.

Single-molecule fluorescence imaging experiments generally require sub-nanomolar protein concentrations to isolate single protein molecules, which makes such experiments challenging in live cells due to high intracellular protein concentrations. Here, we show that single-molecule observations can be achieved in live cells through a drastic reduction in the observation volume using overmilled zero-mode waveguides (ZMWs- subwavelength-size holes in a metal film). Overmilling of the ZMW in a palladium film creates a nanowell of tunable size in the glass layer below the aperture, which cells can penetrate. We present a thorough theoretical and experimental characterization of the optical properties of these nanowells over a wide range of ZMW diameters and overmilling depths, showing an excellent signal confinement and a 5-fold fluorescence enhancement of fluorescent molecules inside nanowells. ZMW nanowells facilitate live-cell imaging as cells form stable protrusions into the nanowells. Importantly, the nanowells greatly reduce the cytoplasmic background fluorescence, enabling the detection of individual membrane-bound fluorophores in the presence of high cytoplasmic expression levels, which could not be achieved with TIRF microscopy. Zero-mode waveguide nanowells thus provide great potential to study individual proteins in living cells.

Third-wave cognitive behavioral therapies for caregivers of cancer patients: a scoping review.

BACKGROUND: Cancer caregivers extend comprehensive support covering all aspects of patients' daily lives. It has been reported that a significant proportion of cancer caregivers experience emotional distress. As one way to solve this problem, third-wave cognitive behavioral therapies (CBT), which involves integrating acceptance and mindfulness into cognitive‒behavioral therapy, has been applied to improve caregiver outcomes. METHODS: A scoping review was conducted based on the scoping review guidelines proposed by the Jonna Briggs Institute (JBI). The population was caregivers of cancer patients, the concept was third-wave CBT, and the context remained open. English and Korean publications published from 2001 to June 2022 were identified from PubMed, Embase, CINAHL, PsycINFO, Cochrane, Korea Med, and RISS. RESULTS: A total of 12 studies were included in this scoping review. Mindfulness-Based Stress Reduction (MBSR) and Acceptance and Commitment Therapy (ACT) was the most frequently applied intervention (n = 3, each). Among the components of third-wave CBT, 'mindfulness' was identified in all the studies reviewed (n = 12). Dyadic interventions comprised the majority (n = 9). Interventions using digital technologies such as mobile application/web page (n = 3), telephone (n = 3), and FaceTime (n = 2) have increased since 2017. Depression was the most frequently evaluated outcome (n = 8), followed by anxiety and mindfulness (n = 6, each). CONCLUSIONS: The current review explored available third-wave CBT intervention studies for cancer caregivers and targeted outcomes. Most of the interventions were dyadic interventions and utilized mindfulness. Delivery methods were continuously updated with digital technologies. Further RCTs with robust research designs and a synthesis of the results of the trials would provide evidence about how to effectively apply third-wave CBTs for cancer caregivers.

Simplified Brillouin optical time-domain sensor based on direct modulation of a laser diode.

We propose and experimentally demonstrate a novel kind of Brillouin optical time-domain sensor based on direct modulation of a laser diode (LD) which is free from the use of any microwave device. The Brillouin pump and the probe waves are alternately generated by the LD modulation, and an optical time-domain analysis adopted for distributed measurement. Maps of Brillouin frequency shift are obtained with a spatial resolution of 2 m and an accuracy of ±2 MHz in a 2 km optical fiber.

Transcription and translation contribute to gene locus relocation to the nucleoid periphery in E. coli.

Transcription by RNA polymerase (RNAP) is coupled with translation in bacteria. Here, we observe the dynamics of transcription and subcellular localization of a specific gene locus (encoding a non-membrane protein) in living E. coli cells at subdiffraction-limit resolution. The movement of the gene locus to the nucleoid periphery correlates with transcription, driven by either E. coli RNAP or T7 RNAP, and the effect is potentiated by translation.

Contribution of RNA polymerase concentration variation to protein expression noise.

Cell-to-cell variation in gene expression, or noise, is a general phenomenon observed within cell populations. Transcription is known to be the key stage of gene expression where noise is generated, however, how variation in RNA polymerase (RNAP) concentration contributes to gene expression noise is unclear. Here, we quantitatively investigate how variations in absolute amounts of RNAP molecules affect noise in the expression of two fluorescent protein reporters driven by identical promoters. We find that intrinsic noise is independent of variation in RNAP concentrations, whereas extrinsic noise, which is variation in gene expression due to varying cellular environments, scales linearly with variation in RNAP abundance. Specifically, the propagation of RNAP abundance variation to expressed protein noise is inversely proportional to the concentration of RNAP, which suggests that the change in noise that results from RNAP fluctuations is determined by the fraction of promoters that is not occupied by RNAP.