Is overall and timing-specific physical activity associated with depression in older adults?

Objective: Regarding the circadian rhythm regulating an individual's response to external stimulation, it remains unclear whether older adults engaged in physical activity at different times of day may be differently related to depression symptoms. Thus, this study aimed to investigate the association between overall and timing-specific physical activity and depressive symptoms in older Taiwanese adults. Methods: This cross-sectional study was conducted at a medical center in Taipei City, Taiwan, between September 2020 and December 2021. The participants were community-dwelling older adults aged ≥65 who could walk independently and were not at high risk of cognitive dysfunction. Physical activity was measured using a triaxial accelerometer (GT3X+, ActiGraph) and categorized into timing-specific periods (morning: 06:01-12:00; afternoon: 12:01-18:00; evening: 18:01-24:00) as well as overall physical activity, which included both light physical activity (LPA) and moderate-to-vigorous physical activity (MVPA). A 15-item Geriatric Depression Scale was utilized to assess and measure depressive symptoms. Multivariate linear regression models were estimated for data analysis. Results: A total of 180 older adults (55.0% female; 80.5 ± 7.1 years old; 18.3% at risk of depression) were included. On average, the participants spent 237.3 (± 85.7) minutes in LPA per day and 12.8 (± 17.0) minutes in MVPA per day. The results showed that overall MVPA engagement was associated with lower depressive-symptom scores [B = -1.357, 95% CI (-2.561, -0.153)] in older adults. However, no significant associations were observed between overall LPA, timing-specific MVPA and LPA, and depression in older adults. Conclusion: To prevent depression in older adults, it is advisable to accumulate a higher amount of MVPA throughout the entire day rather than engage in LPA. Further studies employing a prospective design are necessary to validate and strengthen our findings.

Mental Health, Loneliness, and Social Support Among Undergraduate Students: A Multinational Study in Asia.

In this study, we aimed to investigate the prevalence of poor mental health and its association with loneliness and social support among 3531 undergraduate students in nine Asian countries. Mental health was assessed using the Self-Reporting Questionnaire, which was developed by the World Health Organization. Across the entire sample, we detected that nearly half of the students reported poor mental health according to the Self-Reporting Questionnaire and nearly one out of seven students felt lonely. While feeling lonely increased the odds of experiencing poor mental health (odds ratio [OR]), moderate (OR: 0.35) and strong social support (OR: 0.18) decreases the odds of experiencing poor mental health. The high prevalence of poor mental health calls for further in-depth investigations and implementation of mental health support interventions.

Comparing the effects of olfactory-based sensory stimulation and board game training on cognition, emotion, and blood biomarkers among individuals with dementia: A pilot randomized controlled trial.

Olfactory dysfunction can indicate early cognitive decline and is associated with dementia symptoms. We developed an olfactory-based sensory stimulation program and investigated its effects on cognition and emotion, and board game training were used as a comparison. In this parallel design pilot study, 30 participants with mild to moderate dementia were equal randomly assigned to the control (CONT), olfactory stimulation with cognitive training (OS), and board game (BG) groups. Two participants were withdrawn from CONT and OS groups, respectively. The intervention was a 12-week program with one 30-min session twice a week. We employed a blood-based biomarker technique and several cognitive and psychological tests to measure basal and after-intervention values. No significant differences were observed between the groups after intervention, as measured using the Mini-Mental State Examination, Loewenstein Occupational Therapy Cognitive Assessment (LOTCA), Top International Biotech Smell Identification Test, and Geriatric Depression Scale (GDS). The results showed that the OS group had a lower plasma Tau level than the other groups following intervention, whereas the CONT group had a significantly increased plasma amyloid ß1-42 level. OS participants had a lower concentration ratio of plasma Tau and amyloid Aß1-42 and showed more stable or improved cognition, olfactory function, and mood state. Both the OS and BG groups had a higher percentage of participants with stable or improved cognition and emotion. Taken together, these results suggest that olfactory-based sensory stimulation can be a beneficial intervention for patients with dementia. Clinical trial registration: [Clinicaltrials.gov], identifier [NCT05168098].

Differential Response of Oral Mucosal and Gingival Cells to Corynebacterium durum, Streptococcus sanguinis, and Porphyromonas gingivalis Multispecies Biofilms.

Polymicrobial interactions with oral mucosal surfaces determine the health status of the host. While a homeostatic balance provides protection from oral disease, a dysbiotic polymicrobial community promotes tissue destruction and chronic oral diseases. How polymicrobial communities transition from a homeostatic to a dysbiotic state is an understudied process. Thus, we were interested to investigate this ecological transition by focusing on biofilm communities containing high abundance commensal species and low abundance pathobionts to characterize the host-microbiome interactions occurring during oral health. To this end, a multispecies biofilm model was examined using the commensal species Corynebacterium durum and Streptococcus sanguinis and the pathobiont Porphyromonas gingivalis. We compared how both single and multispecies biofilms interact with different oral mucosal and gingival cell types, including the well-studied oral keratinocyte cell lines OKF4/TERT-1and hTERT TIGKs as well as human primary periodontal ligament cells. While single species biofilms of C. durum, S. sanguinis, and P. gingivalis are all characterized by unique cytokine responses for each species, multispecies biofilms elicited a response resembling S. sanguinis single species biofilms. One notable exception is the influence of P. gingivalis upon TNF-α and Gro-α production in hTERT TIGKs cells, which was not affected by the presence of other species. This study is also the first to examine the host response to C. durum. Interestingly, C. durum yielded no notable inflammatory responses from any of the tested host cells, suggesting it functions as a true commensal species. Conversely, S. sanguinis was able to induce expression and secretion of the proinflammatory cytokines IL-6 and IL-8, demonstrating a much greater inflammatory potential, despite being health associated. Our study also demonstrates the variability of host cell responses between different cell lines, highlighting the importance of developing relevant in vitro models to study oral microbiome-host interactions.

Investigation of Normative Value of Commercialized Taiwan Smell Identification Test.

BACKGROUND: The Taiwan Smell Identification Test (TWSIT) was developed and successfully administered in Taiwanese population since 2015. However, for sanitation reason, the original liquid-jar form of this test is not appropriate. The commercialized TWSIT was then re-designed as "scratch-and-sniff" version: the TIBSIT (Top International Biotech, Taipei, Taiwan). This project aims to investigate the normative value of TIBSIT in different age groups and genders. METHODS: Volunteers aged 20 to 80 with no subjective smell loss were recruited in a rhinology clinic. The participants were given full instructions on using the TIBSIT. RESULTS: Two hundred and nine healthy subjects (female: male =107: 102) were enrolled into this study. The median TIBSIT score in female and male subjects is 47 and 46, respectively. Subjects were further divided into three age groups: (A) 20∼34 years (n = 77), (B) 35∼54 years (n = 77), and (C) 55∼80 years (n = 55). The TIBSIT scores at the tenth percentile value at each age group of male patients are 43, 42 and 37; while the scores at the tenth percentile value at each age group of female patients are 42, 42 and 41. TIBSIT score was significantly and inversely correlated with age (Spearman rho = - 0.20, p = 0.004.). CONCLUSION: We established the normative values of the new TIBSIT in different age groups in Taiwan. Future nation-wide screening is needed for more sophisticated norms establishment.

When Confucius meets Erikson: An innovative life review program on Chinese seniors with probable dementia.

Before Erikson proposed the life stages, Confucius concluded an autobiographic review of his entire life in about 500 BC. Confucius' life stages may be closer to the life experiences of Chinese older adults. Therefore, the purpose of this study was to examine the effect of the life review program on cognitive ability, life satisfaction, meaning of life, and ego integrity. The experiment was based on a single-group time-series design with a four-time test across 21 weeks. The evoking materials were horticultural activities held in seven weekly sessions. The innovative life review program was in accordance with a combination of Confucius' and Erikson's life stages. Nine participants completed all sessions. There was a significant positive effect of time on participants' cognitive ability and meaning of life after the program. A combination of Confucius' and Erikson's life stages can be more advantageous for Chinese older adults than only Erikson's life stage.

Minimal Clinically Important Difference of the Loewenstein Occupational Therapy Cognitive Assessment-Geriatric (LOTCA-G) in People With Dementia.

IMPORTANCE: The Loewenstein Occupational Therapy Cognitive Assessment-Geriatric (LOTCA-G) can provide multifaceted and comprehensive measures of cognitive function with reduced literacy demands; however, its minimal clinically important difference (MCID) has not been determined for interpreting the change scores. OBJECTIVE: To establish the LOTCA-G MCID for people with dementia. DESIGN: Psychometric field study. SETTING: Data were collected in communities. PARTICIPANTS: Forty-nine participants with mild to moderate dementia who were enrolled in previous research investigating the effects of cognitive stimulation-related interventions with equivalent training hours. OUTCOMES AND MEASURES: The Mini-Mental State Examination and LOTCA-G were used to assess cognitive function before and after the interventions. The anchor-based and distribution-based approaches were used to estimate the LOTCA-G MCID. RESULTS: Using the anchor-based method, we defined 16 participants as the improvement group, with a mean change score of 5.75 points (the first MCID estimate). The second MCID estimate was 6.23 points, which we calculated from the 49 participants by using the distribution-based method with the medium effect size. CONCLUSIONS AND RELEVANCE: Current results suggest that a LOTCA-G change score greater than 5.75 points should be considered as meaningful change for people with dementia. It is important to consider the LOTCA-G MCID as one of the indexes for determining the success of interventions in people with dementia. WHAT THIS ARTICLE ADDS: This study is the first to report the LOTCA-G MCID for dementia. Establishing an MCID for standardized cognitive assessment tools for the aging population can be used to support evidence-based practice in rehabilitation clinics.

Population based norms for the box and blocks test in healthy right-handed Taiwanese adults.

BACKGROUND: This study establishes norms for the Box and Block Test (BBT) in healthy Taiwanese adults between 15 and 75 years of age. METHODS: 621 right-handed healthy adults (296 males and 325 females) completed the study. All participants performed the BBT following the standard protocol. An age by gender by testing hand analysis of variance (ANOVA) was performed to determine differences for the variables of interest. RESULTS: On average, females performed better on the BBT than males by approximately 2 points (p < 0.001). Across all participants, dominant hand performance was 2.8 points higher than non-dominant hand performance (p < 0.001). Significant changes of BBT scores across life span were observed at the ages of 30, 45 and 60 years old. Average scores across all age groups are at least one standard deviation below the previously established American norms for each corresponding age group. CONCLUSION: When using the BBT test with adult Taiwanese clients, clinical practitioners should strongly consider using right-handed normative data from Taiwanese individuals as the norms for this population differ from the previously established norms from American adult participants.

The Benefits of Continuous Leisure Participation in Relocation Adjustment Among Residents of Long-Term Care Facilities.

BACKGROUND: The rising population of older adults is transforming Taiwan society. Although many long-term care facilities now operate for older adults, the transition from the home environment to long-term facilities may cause multiple issues, including relocation stress syndrome, for new residents. Autonomy is a critical element of the human experience. Leisure, as an expression of autonomy, has been shown to enhance self-image and promote feelings of competence and mastery. PURPOSE: The aim of this study was to assess the relationship between participation in leisure activities and adjustment to residential care using the continuity theory. METHODS: One hundred sixty-three qualified individuals from 11 long-term care institutions were recruited and completed the questionnaire. The sampling criteria for the study were age (55+ years), appropriate cognitive skills, and residency (less than 5 years). Interviews conducted by trained interviewers were used to collect data. Measured outcomes included leisure participation; physical, psychological, and social adjustments; and background information. Cluster analysis, descriptive analysis, multivariate analysis of variance, and least significant difference test were used in analysis. RESULTS: Type of admission (voluntary/mandated) and type of leisure participation significantly affected the level of relocation adjustment success. Four leisure behavior categories were identified, including reduced participation, expanded participation, active participation, and continuing participation. The participants in the active and continuing participation categories had significantly higher psychological adjustment than their peers in the reduced participation category. Moreover, the continuing participation category had a significantly better level of social adjustment than their expanded participation category peers. Continuous and active participation categories had a beneficial outcome in relocation adjustment. CONCLUSIONS: The findings support that, in general, residents of long-term care facilities who participate continuously in leisure activities adjust better psychologically and socially to their new life in these facilities than individuals in the other participation categories. As continuity of participation in leisure activities is important for successful aging, institutions should facilitate the continuing leisure behavior of new residents.

Simultaneous Multicolor Single-Molecule Tracking with Single-Laser Excitation via Spectral Imaging.

Single-molecule tracking (SMT) offers rich information on the dynamics of underlying biological processes, but multicolor SMT has been challenging due to spectral cross talk and a need for multiple laser excitations. Here, we describe a single-molecule spectral imaging approach for live-cell tracking of multiple fluorescent species at once using a single-laser excitation. Fluorescence signals from all the molecules in the field of view are collected using a single objective and split between positional and spectral channels. Images of the same molecule in the two channels are then combined to determine both the location and the identity of the molecule. The single-objective configuration of our approach allows for flexible sample geometry and the use of a live-cell incubation chamber required for live-cell SMT. Despite a lower photon yield, we achieve excellent spatial (20-40 nm) and spectral (10-15 nm) resolutions comparable to those obtained with dual-objective, spectrally resolved Stochastic Optical Reconstruction Microscopy. Furthermore, motions of the fluorescent molecules did not cause loss of spectral resolution owing to the dual-channel spectral calibration. We demonstrate SMT in three (and potentially more) colors using spectrally proximal fluorophores and single-laser excitation, and show that trajectories of each species can be reliably extracted with minimal cross talk.

Quality of life in older adults: Benefits from the productive engagement in physical activity.

BACKGROUND/OBJECTIVE: Productive engagement can be a strategy to reduce stress and chaos. Physical activity, a basic type of productive activity, could benefit older adults. Therefore, the purpose of this study was to explore how productive engagement in physical activity may influence older adults in maintaining their health-related quality of life when they live in a long-term care facility. METHODS: We used purposive sampling to recruit 163 participants from 14 long-term care facilities in Taiwan. Data were collected through individual interviews with a structured questionnaire. Descriptive statistics and independent t-test were used. RESULTS: The result demonstrated that the preferred type of physical activities for the older adults was similar after the relocation. Older adults with increasing productive engagement in physical activity reported better scores of Mental Component Summary, social and emotional role functioning than those with decreasing productive engagement in physical activity. CONCLUSION: Older adults can have a positive perceived health-related quality of life by consistently or increasingly engaging in productive physical activity, especially when encountering a life event.

A Systematic Review of Reminiscence Therapy for Older Adults in Taiwan.

BACKGROUND: Population aging is an increasing phenomenon in many countries around the world. It has been estimated that more than one quarter of all older adults experience a mental or neurological disorder or disease such as dementia, depression, anxiety, and substance abuse. Reminiscence is a popular treatment for enhancing well-being in older adults. Previous studies have shown that reminiscence therapy improves the cognitive functions, behaviors, and other psychosocial outcomes in older adult patients. The themes and materials that are used in reminiscence treatments for the older adults in Taiwan differ from those used in other countries because of Taiwan's unique historical background and culture. PURPOSE: The main purpose of the current study was to review systematically the application and outcomes of reminiscence therapy in Taiwan. METHOD: The terms "reminiscence," "dementia," "Alzheimer's disease," and "Taiwan" were used in an Integrated Resources Search that was conducted at National Taiwan Normal University. Sixteen articles were systematically reviewed using a synthesis of two matrices of experimental designs and intervention designs. RESULT: Reminiscence group sizes ranged between 7 and 12 participants, session frequencies were all once a week for periods lasting between 4 and 16 weeks, and session durations ranged from 30 minutes to 2 hours. The memories of the participants were stimulated using materials dating from their youth such as Taiwanese folk songs, toys, photos, radio programs, newspapers, and food and drinks. In terms of research methodology, most of the studies that were reviewed used quasi-experimental designs without random assignment or control group due to sample size. A minority of the reviewed studies applied the randomized controlled treatment method. Only a few of the studies evaluated indicators in pretest, posttest, and follow-up periods. The results of these studies identified several benefits of reminiscence therapy in Taiwanese older adults, including improvements in cognitive function, anxiety, depressive symptoms, self-esteem, life satisfaction, and personal interaction. CONCLUSIONS: Reminiscence is a good noninvasive treatment for the prevention and treatment of mental diseases in Taiwanese older adults. Furthermore, reminiscence promotes mental health in older adults. Finally, this study provides recommendations for further study and clinical application of reminiscence therapy in Taiwan.

Quantitating morphological changes in biological samples during scanning electron microscopy sample preparation with correlative super-resolution microscopy.

Sample preparation is critical to biological electron microscopy (EM), and there have been continuous efforts on optimizing the procedures to best preserve structures of interest in the sample. However, a quantitative characterization of the morphological changes associated with each step in EM sample preparation is currently lacking. Using correlative EM and superresolution microscopy (SRM), we have examined the effects of different drying methods as well as osmium tetroxide (OsO4) post-fixation on cell morphology during scanning electron microscopy (SEM) sample preparation. Here, SRM images of the sample acquired under hydrated conditions were used as a baseline for evaluating morphological changes as the sample went through SEM sample processing. We found that both chemical drying and critical point drying lead to a mild cellular boundary retraction of ~60 nm. Post-fixation by OsO4 causes at least 40 nm additional boundary retraction. We also found that coating coverslips with adhesion molecules such as fibronectin prior to cell plating helps reduce cell distortion from OsO4 post-fixation. These quantitative measurements offer useful information for identifying causes of cell distortions in SEM sample preparation and improving current procedures.

Development of novel alternative biodiesel fuels for reducing PM emissions and PM-related genotoxicity.

This paper intend to investigate the effects of biodiesel fuel blends comprising of waste-cooking oil and butanol-diesel (B10W10-B10W40) under steady-state conditions. Both particulate organic carbon (OC) and PM including PM2.5 and PM10 significantly decreased with the increasing percentage of biodiesel fuel blends. The fuel blend of B10W40 also demonstrated the most effective function in reducing the emissions of PM10 and PM2.5 in the volume by 59.4% and 57.7%, respectively. Moreover, the emissions of nitrogen oxides decreased with the blending of B10W10-B10W40 (13.9-28.5%), while the brake specific fuel consumption was substantially increased (5.69-13.4%). The overall biological toxicity of PM10 generated from the fuel tested in this study was determined according to Single Cell Gel Electrophoresis assay in human alveolar basal epithelial A549 cells and micronucleus assay in CHO-K1 cells. In addition, the volume of more than 20% waste-cooking oil (B10W20 and B10W40) significantly reduced diesel-induced genotoxicity in lung cells and micronucleus formation in CHO-K1 cells. Collectively, these results indicated that biodiesel fuel blends with the butanol could be a potential alternative fuels for diesel engines because of its substantial property with a significant reduction of the PM-related genotoxicity and the emissions of PM, particulate OC, and NOX.

Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway.

Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRas(G12D), a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRas(G12D) is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM).

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 °C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Effect of Labeling Density and Time Post Labeling on Quality of Antibody-Based Super Resolution Microscopy Images.

Super resolution microscopy (SRM) has overcome the historic spatial resolution limit of light microscopy, enabling fluorescence visualization of intracellular structures and multi-protein complexes at the nanometer scale. Using single-molecule localization microscopy, the precise location of a stochastically activated population of photoswitchable fluorophores is determined during the collection of many images to form a single image with resolution of ~10-20 nm, an order of magnitude improvement over conventional microscopy. One of the key factors in achieving such resolution with single-molecule SRM is the ability to accurately locate each fluorophore while it emits photons. Image quality is also related to appropriate labeling density of the entity of interest within the sample. While ease of detection improves as entities are labeled with more fluorophores and have increased fluorescence signal, there is potential to reduce localization precision, and hence resolution, with an increased number of fluorophores that are on at the same time in the same relative vicinity. In the current work, fixed microtubules were antibody labeled using secondary antibodies prepared with a range of Alexa Fluor 647 conjugation ratios to compare image quality of microtubules to the fluorophore labeling density. It was found that image quality changed with both the fluorophore labeling density and time between completion of labeling and performance of imaging study, with certain fluorophore to protein ratios giving optimal imaging results.

Effect of the recreational life review program on patients with dementia in an outpatient clinic: a preliminary study.

The purpose of this study is to investigate the effect of the Life Review Program-Taiwan (LRP-TW) on outpatients with mild to moderate dementia. Ten individuals were selected through purposive sampling and tested using a single group pretest-posttest design. The Mini-Mental State Examination (MMSE), the Loewenstein Occupational Therapy Cognitive Assessment-Geriatric (LOTCA-G) and the Geriatric Depression Scale-Short Form (GDS-SF) were used as outcome measures. The total scores and three subtests of the LOTCA-G revealed significant differences after the intervention. Affective function remained stable through the experiment. The LRP-TW may offer a clinical intervention program for supporting cognitive and mental performance in individuals with dementia.

Matrix softness regulates plasticity of tumour-repopulating cells via H3K9 demethylation and Sox2 expression.

Tumour-repopulating cells (TRCs) are a self-renewing, tumorigenic subpopulation of cancer cells critical in cancer progression. However, the underlying mechanisms of how TRCs maintain their self-renewing capability remain elusive. Here we show that relatively undifferentiated melanoma TRCs exhibit plasticity in Cdc42-mediated mechanical stiffening, histone 3 lysine residue 9 (H3K9) methylation, Sox2 expression and self-renewal capability. In contrast to differentiated melanoma cells, TRCs have a low level of H3K9 methylation that is unresponsive to matrix stiffness or applied forces. Silencing H3K9 methyltransferase G9a or SUV39h1 elevates the self-renewal capability of differentiated melanoma cells in a Sox2-dependent manner. Mechanistically, H3K9 methylation at the Sox2 promoter region inhibits Sox2 expression that is essential in maintaining self-renewal and tumorigenicity of TRCs both in vitro and in vivo. Taken together, our data suggest that 3D soft-fibrin-matrix-mediated cell softening, H3K9 demethylation and Sox2 gene expression are essential in regulating TRC self-renewal.

Photoactivated localization microscopy with bimolecular fluorescence complementation (BiFC-PALM) for nanoscale imaging of protein-protein interactions in cells.

Bimolecular fluorescence complementation (BiFC) has been widely used to visualize protein-protein interactions (PPIs) in cells. Until now, however, the resolution of BiFC has been limited by the diffraction of light to ∼250 nm, much larger than the nanometer scale at which PPIs occur or are regulated. Cellular imaging at the nanometer scale has recently been realized with single molecule superresolution imaging techniques such as photoactivated localization microscopy (PALM). Here we have combined BiFC with PALM to visualize PPIs inside cells with nanometer spatial resolution and single molecule sensitivity. We demonstrated that PAmCherry1, a photoactivatable fluorescent protein commonly used for PALM, can be used as a BiFC probe when split between residues 159 and 160 into two fragments. PAmCherry1 BiFC exhibits high specificity and high efficiency even at 37°C in detecting PPIs with virtually no background from spontaneous reconstitution. Moreover, the reconstituted protein maintains the fast photoconversion, high contrast ratio, and single molecule brightness of the parent PAmCherry1, which enables selective PALM localization of PPIs with ∼18 nm spatial precision. With BiFC-PALM, we studied the interactions between the small GTPase Ras and its downstream effector Raf, and clearly observed nanoscale clustering and diffusion of individual KRas G12D/CRaf RBD (Ras-binding domain) complexes on the cell membrane. These observations provided novel insights into the regulation of Ras/Raf interaction at the molecular scale, which would be difficult with other techniques such as conventional BiFC, fluorescence co-localization or FRET.