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Background/objective: Physical inactivity is one of the major cardiovascular disease risk factors; however, not much is known regarding lifetime bone-specific physical activity and arterial stiffness. The aim of this cross-sectional study was to determine whether total bone-specific physical activity score (tBPAQ) was related to arterial stiffness and body composition in healthy young adults., Methods: Healthy young women (n = 56, 20.3 ± 1.3 years) and men (n = 52, 21.0 ± 1.2 years) between 18 and 25 years were recruited for this study. The tBPAQ was used to obtain a comprehensive account of lifetime bone-loading physical activity. We measured the carotid to femoral pulse wave velocity (cfPWV) to evaluate arterial stiffness using the novel oscillometric device (SphygmoCor XCEL). Dual energy X-ray absorptiometry was used to measure bone free lean body mass (BFLBM, kg) and % total body fat., Results: Partial correlations analyses showed a significant inverse relationship between tBPAQ and cfPWV (r = - 0.371, p = 0.007) in young women; however, this relationship was not found in young men (p > 0.05). There were significant negative correlations between tBPAQ and % total body fat in both young women (r = - 0.265, p = 0.048) and men (r = - 0.327, p = 0.018). No significant relationships were found between tBPAQ and BFLBM (p > 0.05)., Conclusion: Our study provides preliminary evidence of sex-specificity of negative relationships of tBPAQ with i) cfPWV (women only) and ii) % total body fat (men and women). Whether bone-loading physical activity can lead to better cardiometabolic outcomes needs to be examined., Competing Interests: The authors have no conflicts of interest relevant to this article., (© 2021 The Society of Chinese Scholars on Exercise Physiology and Fitness. Published by Elsevier (Singapore) Pte Ltd.)

This study uses an unobtrusive eye tracking approach to examine understudied psychological mechanisms - message attention and credibility - when people are exposed to misinformation and correction on social media. We contrast humor versus non-humor correction strategies that point out rhetorical flaws in misinformation regarding the HPV vaccine, which was selected for its relevance and impact on public health. We randomly assigned participants to one of two experimental conditions: humor correction versus non-humor correction. Our analyses revealed that the humor correction increased attention to the image portion of the correction tweet, and this attention indirectly lowered HPV misperceptions by reducing the credibility of the misinformation tweet. The study also found that the non-humor correction outperformed the humor correction in reducing misperceptions via its higher credibility ratings. Practical implications for correcting misinformation on social media are discussed.

A close link has been established between self-harm and suicide risk in adolescents, and increasing attention is given to social media as possibly involved in this relationship. It is important to identify indicators of suicidality (i.e., suicide ideation or attempt) including aspects related to contagion in online and offline social networks and explore the role of social media in the relationship between social circumstances and suicidality in young adolescents with self-harm. This study explored characteristics of Korean adolescents with a recent history of self-harm and identified how behavioral and social features explain lifetime suicidality with emphasis on the impact of social media. Data came from a nationwide online survey among sixth- to ninth-graders with self-harm during the past 12 months ( n = 906). We used χ 2 tests of independence to explore potential concomitants of lifetime suicidality and employed a multivariate logistic regression model to examine the relationship between the explanatory variables and suicidality. Sensitivity analyses were performed with lifetime suicide attempt in place of lifetime suicidality. 33.9% ( n = 306) and 71.2% ( n = 642) reported to have started self-harm by the time they were fourth- and six-graders, respectively; 44.3% ( n = 400) reported that they have friends who self-harm. Having endorsed moderate/severe forms and multiple forms of self-harm (OR 5.36, p < 0.001; OR 3.13, p < 0.001), having engaged in self-harm for two years or more (OR 2.42, p = 0.001), having friends who self-harm (OR 1.92, p = 0.013), and having been bullied at school were associated with an increased odds of lifetime suicidality (OR 2.08, p = 0.004). Notably, having posted content about one's self-harm on social media during the past 12 months was associated with an increased odds of lifetime suicidality (OR 3.15, p < 0.001), whereas having seen related content in the same period was not. Sensitivity analyses yielded similar results with lifetime suicide attempt, supporting our findings from the logistic regression. The current study suggests that self-harm may be prevalent from early adolescence in South Korea with assortative gathering. The relationship of vulnerable adolescents' social circumstances to suicide risk may be compounded by the role of social media. As the role of social media can be linked to both risk (i.e., contagion) and benefit (i.e., social connection and support), pre-existing vulnerabilities alongside SH and what online communication centers on should be a focus of clinical attention., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Seong, Noh, Lee, Lee, Kim, Seo, Yoo, Hwang, Choi, Han, Hong and Kim.)

Key Message: OsPUB41 plays a negative role in drought stress response through the mediation of OsUBC25 and interacts with OsCLC6, suggesting a putative substrate. The notable expansion of Plant U-Box E3 ligases (PUB), compared with those in mammals, implies that PUB proteins have evolved to perform plant-specific functions. OsPUB41, a potential ortholog of CMPG1, was recently reported to regulate the cell wall degrading enzyme (CWDE)-induced innate immune response in rice. Here, we characterized the OsPUB41 gene, which encodes a dual-localized cytosolic and nuclear U-box E3 ligase in rice. OsPUB41 expression was specifically induced by dehydration among various abiotic stresses and abscisic acid (ABA) treatments. Furthermore, we revealed that the core U-box motif of OsPUB41 possesses the E3 ligase activity that can be activated by OsUBC25 in rice. The Ubi:RNAi-OsPUB41 knock-down and ospub41 suppression mutant plants exhibited enhanced tolerance to drought stress compared with the wild-type rice plants in terms of transpirational water loss, long-term dehydration response, and chlorophyll content. Moreover, the knock-down or suppression of the OsPUB41 gene did not cause adverse effect on rice yield-related traits. Yeast two-hybrid and an in vitro pull-down analyses revealed that OsCLC6, a chloride channel, is a putative substrate of OsPUB41. Overall, these results suggest that OsPUB41 acts as a negative regulator of dehydration conditions and interacts with OsCLC6, implying that it is a substrate of OsPUB41., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

This study was designed to investigate the roles information and communications technology (ICT) played during the current COVID-19 pandemic. Specifically, we focused on the relationships between ICT use and perceived importance of social connectedness and future anxiety, while considering relevant personality and psychosocial factors. A U.S. sample of 394 adults answered questions about ICT use, pandemic-related reactions and actions, demographics, and psychosocial factors via an online survey. Using logistic regression, findings indicated that personality (extraversion and conscientiousness) and psychosocial (need to belong and perceived attachment to phone) factors, types of ICT as news source, and gender were associated with perceived importance of social connectedness. Neuroticism, time spent on ICT for social purposes, and perceived threat of COVID-19 were associated with future anxiety. In addition, using Mann-Whitney U test, people who rated higher on importance of social connectedness had higher ICT use, both in terms of types and time spent on ICT. Overall, results are consistent with the idea that technology is a coping tool during the pandemic and balanced use can lead to feelings of social connectedness and less future anxiety. Therefore, it is important for authorities to align their messaging and outreach with people's psychosocial, personality, and health considerations through ICT channels while empowering ICT users to be responsible for their interactions with the technology.

The rapid increase of self-injurious behavior among Korean adolescents, and its reckless spread on social media, has driven the necessity to study this behavior. The present study investigates the characteristics of self-injurious behavior among adolescents in local communities, and the psychological factors affecting such behavior. Questionnaires were administered to 516 sixth graders in elementary school and first to third graders in middle school of both genders, nationwide. They measured the prevalence and characteristics of self-injurious behavior and the relevant psychological factors, such as levels of depression, anxiety, and self-esteem. Furthermore, group differences were assessed for self-injury experience and the characteristics relevant to self-injurious behavior. In addition, this study performs logistic regression to explore the risk factors predicting self-injurious behavior. In all, 166 participants (32.2%) reported self-injury, with a higher rate of self-injury in female students than in male students. Although the study finds high rates of mild forms of self-injury, such as "biting", "pulling hair," and "hitting self", it also finds relatively high reports of more risky methods, such as "cutting or carving". The logistic regression shows a significant effect of the negative self-image sub-factor of depression (CDI) and oversensitivity and physical and sleep problems sub-factors of anxiety (RCMAS) on self-injurious behavior. The rates of self-injury were higher in female participants than in male ones, and adolescents in local communities reported higher rates of mild forms of self-injury than the moderate/severe forms. The results of this study suggest that early screenings and interventions should be conducted through evaluation of self-image and emotional stability of early adolescents to hinder the risk of self-harm.

Strigolactones (SLs) are plant hormones that suppress shoot branching through perception by their receptor protein DWARF 14 (D14). The artificial regulation of SL signaling has been considered a potent agricultural technique because plant architecture is strongly related to crop yield. In this communication, we describe the development of a small-molecule D14 inhibitor that functions at sub-micromolar levels. This potent inhibitor may be a lead compound for a first-in-class plant growth regulator.

Objective: This investigation explores how using different e-health interventions facilitates positive psychosocial changes and how these changes reduce cancer concerns and improve quality of life in breast cancer patients over time., Methods: A total of 326 breast cancer patients were randomly assigned to one of three e-health interventions: (a) Internet only, (b) the Comprehensive Health Enhancement Support System information and support services (CHESS-IS), or (c) CHESS with mentor. Proximal health outcomes such as information overload, emotional functioning, and social support were measured alongside distal outcomes like cancer concerns and quality of life. Participants completed surveys at four time points: pretest as a baseline, 6 weeks, 3 months, and 6 months., Results: Both interventions were effective in improving patient health beyond Internet only but they differed in type of change mechanism and clinical benefit. The CHESS-IS enhanced proximal outcomes at 3 months through improved information competence. The CHESS with mentor intervention reduced breast cancer concerns at 6 months, mediated mainly by emotional-social competence and emotional functioning., Conclusions: Using e-health interventions like CHESS can help patients improve cancer information management skills and emotional functioning, contributing to better short-term health outcomes. Adding a human mentor can enhance the benefits of CHESS use, extending the experience among breast cancer patients. Theoretical, practical, and clinical implications of the study results are discussed., (© 2020 John Wiley & Sons Ltd.)

The inherent conformational preferences of the neutral β-peptide foldamer series, Ac-(ACHC) n -NHBn, n = 2-4, are studied in the gas phase using conformation-specific IR-UV double resonance methods. The cyclically constrained chiral β-amino acid cis -2-aminocyclohexane carboxylic acid (ACHC) is designed to bring both right- and left-handed helices into close energetic proximity. Comparison of the infrared spectra in the NH stretch and amide I/II regions with the predictions of DFT calculations lead to the unambiguous assignment of four out of the six observed conformations of the molecules in this series, while corroborating computational and spectral evidence, affords tentative assignments of the remaining two conformers for which IR data were not recorded. The observed structures fall into one of two conformational families: a right-handed 12/10-mixed helix or its "cap-disrupted" left-handed helical analogue, which coexist with significant populations. Site-specific and stereospecific methylation on the cyclohexane backbone at the dipeptide ( n = 2) level is also tested as a means to sterically lock in a predetermined cyclohexane chair conformation. These substitutions are proven to be a means of selectively driving formation of one helical screw sense or the other. Calculated relative energies and free energies of all possible structures for the molecules provide strong supporting evidence that the rigid nature of the ACHC residue confers unusual stability to the 12/10-mixed helix conformation, regardless of local environment, temperature, or C-terminal capping unit. The simultaneous presence of both handed helices offers unique opportunities for future studies of their interconversion.

Purpose: Generation of antigen-specific T cells from patients with cancer employs large numbers of peripheral blood cells and/or tumor-infiltrating cells to generate antigen-presenting and effector cells commonly requiring multiple rounds of restimulation ex vivo . We used a novel paramagnetic, nanoparticle-based artificial antigen-presenting cell (nano-aAPC) that combines anti-CD28 costimulatory and human MHC class I molecules that are loaded with antigenic peptides to rapidly expand tumor antigen-specific T cells from patients with melanoma., Experimental Design: Nano-aAPC-expressing HLA-A*0201 molecules and costimulatory anti-CD28 antibody and HLA-A*0201 molecules loaded with MART-1 or gp100 class I-restricted peptides were used to stimulate CD8 T cells purified from the peripheral blood of treatment-naïve or PD-1 antibody-treated patients with stage IV melanoma. Expanded cells were restimulated with fresh peptide-pulsed nano-aAPC at day 7. Phenotype analysis and functional assays including cytokine release, cytolysis, and measurement of avidity were conducted., Results: MART-1-specific CD8 T cells rapidly expanded up to 1,000-fold by day 14 after exposure to peptide-pulsed nano-aAPC. Expanded T cells had a predominantly stem cell memory CD45RA + /CD62L + /CD95 + phenotype; expressed ICOS, PD-1, Tim3, and LAG3; and lacked CD28. Cells from patients with melanoma were polyfunctional; highly avid; expressed IL2, IFNγ, and TNFα; and exhibited cytolytic activity against tumor cell lines. They expanded 2- to 3-fold after exposure to PD-1 antibody in vivo , and expressed a highly diverse T-cell receptor V beta repertoire., Conclusions: Peptide-pulsed nano-aAPC rapidly expanded polyfunctional antigen-specific CD8 T cells with high avidity, potent lytic function, and a stem cell memory phenotype from patients with melanoma., (©2020 American Association for Cancer Research.)

Background: Accumulated evidence suggests that individuals with social anxiety disorder (SAD) are at particular risk of developing alcohol use disorder (AUD). Yet, little is known about the mechanisms under this high comorbidity. This study aimed to elucidate the process of the development of alcohol related problems among individuals with elevated social anxiety. We investigated the moderation effect of difficulties in emotion regulation on the relationship between symptoms of social anxiety, coping and conformity motives and alcohol related problems., Methods: In a sample of university students (N = 647) in South Korea, we examined whether cognitive (fear of negative evaluation), behavioral (social avoidance), and physiological symptoms (concerns over physiological symptoms) of social anxiety affect alcohol related problems with the mediation of coping and conformity motives. Furthermore, difficulties in emotion regulation were hypothesized to moderate each mediational path., Results: Results showed that the fear of negative evaluation and concerns over physiological symptoms were associated with alcohol related problems with the mediation of conformity and coping motives, respectively. As hypothesized, each path was moderated by difficulties in emotion regulation., Conclusions: Findings suggest that coping and conformity motives to cope with cognitive and physiological symptoms of social anxiety were related to alcohol related problems. In addition, individuals with high levels of difficulties in emotion regulation were prone to exhibit more alcohol related problems.

Objective: Limited data exist on non-suicidal self-injury (NSSI) and suicide attempts among psychiatric patients in Korea. In this study, we investigated the clinical characteristics of patients who engaged in NSSI and/or suicide attempts., Methods: We performed a retrospective medical chart review of patients with NSSI and/or suicide attempts at the psychiatric department of a university medical center in Seoul between 2017 and 2019. According to their history, patients were allocated to one of three groups: NSSI only, suicide attempts only and NSSI and suicide attempts group. Groups were compared based on sociodemographic characteristics and psychological assessments., Results: Overall, 80 patients with NSSI and/or suicide attempts were evaluated. Patients with NSSI and suicide attempts were more likely to be female than the other two groups. Patients with NSSI and suicide attempts were more likely to suffer from Cluster B personality disorder than the other groups. And patients with NSSI and suicide attempts scored significantly higher on novelty-seeking in TCI and RC8, RC9 in MMPI-2., Conclusion: Patients with NSSI and/or suicide attempts were more likely to be female, younger, and showed higher levels of psychological disturbances. These findings highlight the importance of early detection and intervention for patients with NSSI.

The purpose of this cross-sectional study was to determine the differences in areal bone mineral density (aBMD) based on alcohol consumption behaviors, bone-loading history as assessed by a bone-specific physical activity questionnaire (BPAQ), and the body mass index (BMI). College-aged female students ( N = 112) were recruited from the universities in Seoul and Gyeonggi province, South Korea. The aBMD of the lumbar spine and non-dominant side of the proximal femur (total hip, TH; femoral neck, FN; femoral trochanter, FT) were measured using dual energy X-ray absorptiometry (DXA). Alcohol consumption was determined by the frequency and amount of alcohol intake during the past 12 months using a self-reported questionnaire. The X-scan plus II was used to measure height (cm), body mass (kg), fat-free mass (FFM, kg), and % body fat. Drinking two or more times alcohol per week was associated with greater aBMD of the TH ( p = 0.04-0.002) and FN ( p = 0.043) compared to a lower frequency of alcohol consumption and 2-4 times per month, respectively. Based on the drinking amount per occasion, there were no significant group differences ( p > 0.05) in aBMD at any of the sites. The highest group of total BPAQ had greater aBMD of the TH, FN, and FT versus the lowest ( p = 0.023-0.009) and mid of total BPAQ groups ( p = 0.004-0.009). Additionally, the highest group had greater aBMD of the lumbar spine compared to the mid group ( p = 0.001). No significant group differences in aBMD at any of the sites were noted based on the BMI ( p > 0.05). Young college-aged women with greater bone-loading physical activity showed greater aBMD at the TH, FN, FT, and lumbar spine, while a moderate alcohol intake was associated with greater aBMD of the TH and FN. These findings have clinical implications for young women who may not participate in high-impact physical activity and are binge drinkers.

Protein aggregation involves the assembly of partially misfolded proteins into oligomeric and higher-order structures that have been associated with several neurodegenerative diseases. However, numerous questions relating to protein aggregation remain unanswered due to the lack of available tools for visualization of these species in living cells. We recently developed a fluorogenic method named aggregation tag (AggTag), and presented the AggTag probe P1, based on a Halo-tag ligand, to report on the aggregation of a protein of interest (POI) in live cells. However, the Halo-tag-based AggTag method only detects the aggregation of one specific POI at a time. In this study, we have expanded the AggTag method by using SNAP-tag technology to enable fluorogenic and biorthogonal detection of the aggregation of two different POIs simultaneously in live cells. A new AggTag probe-P2, based on a SNAP-tag ligand bearing a green solvatochromic fluorophore-was synthesized for this purpose. Using confocal imaging and chemical crosslinking experiments, we confirmed that P2 can also report both on soluble oligomers and on insoluble aggregates of a POI fused with SNAP-tag in live cells. Ultimately, we showed that the orthogonal fluorescence of P1 and P2 allows for simultaneous visualization of two different pathogenic protein aggregates in the same cell., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This study examined the interplay of depression and different types of e-health interventions on breast cancer patients' perceived healthcare competence, emotional processing, and social well-being over time. The three e-health interventions--Internet Only as a control condition, CHESS (Comprehensive Health Enhancement Support System) Only, and CHESS with a Human Mentor, a cancer information specialist--provided varying degrees of interactivity and presence. A total of 328 women with breast cancer participated in one of the three interventions for a 6-month period. Women were further split into two groups based on reported levels of depression. For perceived healthcare competence and social well-being, results revealed significant interaction effects for intervention type by depression over time, such that breast cancer patients with higher levels of depression benefited most from the CHESS with Mentor intervention over the 6-month study period. For emotional processing, depressed cancer patients benefited more from the CHESS with Mentor than the other two interventions, regardless of time. These findings have (a) theoretical implications on how mental health factors can intersect with interactivity and presence to influence psychosocial outcomes, (b) conceptual implications for the role of human interaction within e-health systems, and (c) practical implications for the development of e-health interventions for cancer patients with depression.

Base editing directly converts a target base pair into a different base pair in the genome of living cells without introducing double-stranded DNA breaks. While cytosine base editors (CBE) and adenine base editors (ABE) are used to install and correct point mutations in a wide range of organisms, the extent and distribution of off-target edits in mammalian embryos have not been studied in detail. We analyze on-target and proximal off-target editing at 13 loci by a variety of CBEs and ABE in more than 430 alleles generated from mouse zygotic injections using newly generated and published sequencing data. ABE predominantly generates anticipated A•T-to-G•C edits. Among CBEs, SaBE3 and BE4, result in the highest frequencies of anticipated C•G-to-T•A products relative to editing byproducts. Together, these findings highlight the remarkable fidelity of ABE in mouse embryos and identify preferred CBE variants when fidelity in vivo is critical.

The aim of this study was to determine if bone-specific physical activity questionnaire (BPAQ) scores were positively related to bone health in healthy young and middle-aged premenopausal women. The total BPAQ was a stronger predictor of bone strength and bone mineral density of hip in young women as compared to middle-aged premenopausal women., Purpose: The purpose of this study was to determine whether the BPAQ scores were predictive indices of volumetric BMD (vBMD), bone strength, and bone geometry in young and middle-aged premenopausal women., Methods: Healthy young (n = 60) and middle-aged premenopausal women (n = 54) between the ages of 18 and 50 years were recruited for this study. Areal bone mineral density (aBMD) of lumbar spine and dual proximal femur (FN; femoral neck) was measured using DXA. We assessed vBMD of the tibia 4%, 38%, and 66% by peripheral quantitative computed tomography (pQCT). The BPAQ was used to obtain a comprehensive account of lifetime physical activity related to bone health., Results: Pearson's correlation tests showed positive correlations between total BPAQ and aBMD of the right FN (r = 0.313, p = 0.015) and the left FN (r = 0.307, p = 0.017) in young women while not found in middle-aged premenopausal women (p > 0.05). A positive relationship was only observed between total BPAQ and tibia 38% vBMD in middle-aged premenopausal women (r = 0.283, p = 0.038). All bone geometry variables were associated with total BPAQ (r = 0.280-0.422, p = 0.03-0.001) in young women. The Strength-Strain Index of tibia 38% (r = 0.350, p = 0.006) and 66% (r = 0.406, p = 0.001) was associated with total BPAQ in young women. In both young and middle-aged premenopausal women, when age, bone-free lean body mass (BFLBM), and total BPAQ were included in a stepwise multiple linear regression analysis, BFLBM was a significant predictor of all aBMD variables, accounting for 7-25.7% (p = 0.043-0.001)., Conclusions: The total BPAQ score-derived physical activity was more predictive of positive bone characteristics in young women than in middle-aged premenopausal women.

Background: Inappropriate use of antibiotics increases resistance and reduces their effectiveness. Despite evidence-based guidelines, antibiotics are still commonly used to treat infections likely caused by respiratory viruses. In this study, we examined the temporal relationships between antibiotic usage and respiratory infections in the Republic of Korea., Methods: The number of monthly antibiotic prescriptions and the incidence of acute respiratory tract infections between 2010 and 2015 at all primary care clinics were obtained from the Korean Health Insurance Review and Assessment Service. The monthly detection rates of respiratory viruses, including adenovirus, respiratory syncytial virus, influenza virus, human coronavirus, and human rhinovirus, were collected from Korea Centers for Disease Control and Prevention. Cross-correlation analysis was conducted to quantify the temporal relationship between antibiotic use and respiratory virus activities as well as respiratory infections in primary clinics., Results: The monthly use of different classes of antibiotic, including penicillins, other beta-lactam antibacterials, macrolides and quinolones, was significantly correlated with influenza virus activity. These correlations peaked at the 0-month lag with cross-correlation coefficients of 0.45 ( p  < 0.01), 0.46 ( p  < 0.01), 0.40 ( p  < 0.01), and 0.35 (< 0.01), respectively. Furthermore, a significant correlation was found between acute bronchitis and antibiotics, including penicillin (0.73, p  < 0.01), macrolides (0.74, p  < 0.01), and quinolones (0.45, p  < 0.01), at the 0-month lag., Conclusions: Our findings suggest that there is a significant temporal relationship between influenza virus activity and antibiotic use in primary clinics. This relationship indicates that interventions aimed at reducing influenza cases in addition to effort to discourage the prescription of antibiotics by physicians may help to decrease unnecessary antibiotic consumption., Competing Interests: This study was exempted from ethical approval by the Institutional Review Board designated by Korean Ministry of Health and Welfare (P01-201711-21-017).The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Here, we report that CRISPR guide RNAs (gRNAs) with a 5'-triphosphate group (5'-ppp gRNAs) produced via in vitro transcription trigger RNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5'-ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to ∼80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting 5'-hydroxyl gRNAs in complex with Cas9 or Cpf1 avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4 + T cells. These results are in line with previous findings that chemically synthesized sgRNAs with a 5'-hydroxyl group are much more efficient than in vitro-transcribed (IVT) sgRNAs in human and other mammalian cells. The phosphatase treatment of IVT sgRNAs is a cost-effective method for making highly active sgRNAs, avoiding innate immune responses in human cells., (© 2018 Kim et al.; Published by Cold Spring Harbor Laboratory Press.)

Competing Interests: Conflict of Interests The authors declare that there is no conflict of interests.

We report that engineered Cas9 variants with improved specificity-eCas9-1.1 and Cas9-HF1-are often poorly active in human cells, when complexed with single guide RNAs (sgRNAs) with a mismatch at the 5' terminus, relative to target DNA sequences. Because the nucleotide at the 5' end of sgRNAs, expressed under the control of the commonly-used U6 promoter, is fixed to a guanine, these attenuated Cas9 variants are not useful at many target sites. By using sgRNAs with matched 5' nucleotides, produced by linking them to a self-cleaving ribozyme, the editing activity of Cas9 variants can be rescued without sacrificing high specificity.

At a signalized intersection, there exists an area where drivers become indecisive as to either stop their car or proceed through when the traffic signal turns yellow. This point, called a dilemma zone, has remained a safety concern for drivers due to the great possibility of a rear-end or right-angle crash occurring. In order to reduce the risk of car crashes at the dilemma zone, Institute of Transportation Engineers (ITE) recommended a dilemma zone model. The model, however, fails to provide precise calculations on the decision of drivers because it disregards the supplemental roadway information, such as whether a red light camera is present. Hence, the goal of this study was to incorporate such roadway environmental factors into a more realistic driver decision-making model for the dilemma zone. A driving simulator was used to determine the influence of roadway conditions on decision-making of real drivers. Following data collection, each driver's decision outcomes were implemented in an Agent-Based Simulation (ABS) so as to analyze behaviors under realistic road environments. The experimental results revealed that the proposed dilemma zone model was able to accurately predict the decisions of drivers. Specifically, the model confirmed the findings from the driving simulator study that the changes in the roadway environment reduced the number of red light violations at an intersection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Although Yoga has the potential to be an alternative physical activity to enhance bone health, there is a lack of high quality evidence for this type of intervention. The purpose of this randomized controlled trial was to examine the effects of a progressive 8-month Ashtanga-based Yoga program on bone turnover markers (BTM), areal bone mineral density (aBMD) and volumetric bone characteristics in premenopausal women. Thirty-four premenopausal women (35-50 years) were randomly assigned either to a Yoga group (YE, n = 16) or a control group (CON, n = 18). Participants in YE group performed 60 minutes of an Ashtanga-based Yoga series 2 times/week with one day between sessions for 8 months, and the session intensity was progressively increased by adding the number of sun salutations (SS). Participants in CON were encouraged to maintain their normal daily lifestyles monitored by the bone specific physical activity questionnaire (BPAQ) at 2 month intervals for 8 months. Body composition was measured by dual energy x-ray absorptiometry (DXA). Bone formation (bone alkaline phosphatase, Bone ALP) and bone resorption (Tartrate-Resistant Acid Phosphatase-5b, TRAP5b) markers were assessed at baseline and after 8 months. aBMD of total body, lumbar spine and dual proximal femur and tibia bone characteristics were measured using DXA and peripheral Quantitative Computed Tomography (pQCT), respectively. We found that the serum Bone ALP concentrations were maintained in YE, but significantly (p = 0.005) decreased in CON after the 8 month intervention, and there were significant (p = 0.002) group differences in Bone ALP percent changes (YE 9.1 ± 4.0% vs. CON -7.1 ± 2.3%). No changes in TRAP5b were found in either group. The 8-month Yoga program did not increase aBMD or tibia bone strength variables. Body composition results showed no changes in weight, fat mass, or % fat, but small significant increases in bone free lean body mass occurred in both groups. The findings of this study suggest that regular long-term Ashtanga Yoga had a small positive effect on bone formation but did not alter aBMD or tibia bone characteristics in premenopausal women. Key pointsRegular long-term Ashtanga-based Yoga program had a small positive effect on bone formation, but no effects were found on bone resorption.None of the bone density or geometry variables were changed by the 8-month Ashtanga-based Yoga intervention.Future Yoga interventions should focus on longer duration and greater frequency to elicit improvements in bone mineral density.

Objectives: Although diffuse splenic (18)F-fluorodeoxyglucose (F-18 FDG) uptake exceeding hepatic activity, is considered abnormal, its clinical significance is rarely discussed in the literature. The aim of this study was to determine the contributing factors causing diffusely increased splenic FDG uptake in patients with cholangiocarcinoma., Methods: From January 2010 to March 2013, 140 patients (84 men, 56 women) were enrolled in this study. All patients had been diagnosed with cholangiocarcinoma and underwent F-18 FDG positron emission tomography/computed tomography (PET/CT) for the pretreatment staging work up. Clinical records were reviewed retrospectively. Various hematological parameters, C-reactive protein (CRP) level, CEA, CA19-9, pancreatic enzymes and liver function tests were conducted within 2 days after the F-18 FDG PET/CT study., Results: Diffuse splenic uptake was observed in 23 patients (16.4%). Of those, 19 patients (82.6%) underwent endoscopic retrograde cholangiopancreastography (ERCP) 7 days before F-18 FDG PET/CT. The CRP level (p < 0.001) and white blood cell count (p = 0.023) were significantly higher in the group of patients with diffuse splenic FDG uptake. The hemoglobin (p < 0.001) and the hematocrit (p < 0.001) were significantly lower in patients with diffuse splenic FDG uptake. Pancreatic enzymes, liver function test results, and tumor markers were not significantly different between the patients who did or did not have diffusely increased splenic FDG uptake. The significant factors for diffuse splenic F-18 FDG uptake exceeding hepatic F-18 FDG uptake on multivariate analysis included: performing ERCP before F-18 FDG PET-CT (odds ratio [OR], 77.510; 95% CI, 7.624-132.105), and the presence of leukocytosis (OR, 12.436; 95% CI, 2.438-63.445) or anemia (OR, 1.211; 95% CI, 1.051-1.871)., Conclusion: In conclusion, our study demonstrated that concurrent inflammation could be associated with diffusely increased splenic FDG uptake. We suggest that performing ERCP before F-18 FDG PET/CT could cause acute inflammation which may induce splenic FDG activity.

Objective: The objective of this study was to investigate the predictive role of volumetric parameters such as metabolic tumor volume (MTV) and total lesion glycolysis (TLG) measured by F-18 FDG PET/CT for regional lymph node (rLN) metastasis in rectal cancer patients., Methods: Retrospectively, 74 rectal cancer patients were enrolled. All patients received surgical treatment and F-18 FDG PET/CT at diagnosis. The F-18 FDG PET/CT findings of primary cancer and rLN involvement were compared with the pathologic diagnosis within 5 weeks after operation. Univariate and multivariate analyses were used to analyze the associations among the pathologic LN status and age, sex, T stage, AJCC stage, SUVmax, lymphatic invasion, venous invasion, neural invasion, and volumetric parameters., Results: The LN (+) group showed statistically significant higher values of MTV2.5 (p < 0.0001), MTV3 (p < 0.0001), MTV3.5 (p = 0.0001), TLG2.5 (p = 0.0007), TLG3 (p = 0.0011), and TLG3.5 (p = 0.0024). In univariate analysis, T stage, AJCC stage, neural invasion, and volumetric parameters were factors significantly associated with pathologic LN involvement. However, in multivariate analysis, advanced T stage, high AJCC stage, MTV2.5, and TLG2.5 were associated with pathologic LN involvement in rectal cancer., Conclusion: This study showed that, not only T stage and AJCC stage, but also volumetric parameters such as MTV2.5 and TLG2.5 are useful factors for the prediction of pathologic LN status in rectal cancer patients.

RNA-guided endonucleases (RGENs), derived from the prokaryotic adaptive immune system known as CRISPR/Cas, enable targeted genome engineering in cells and organisms. RGENs are ribonucleoproteins that consist of guide RNA and Cas9, a protein component originated from Streptococcus pyogenes. These enzymes cleave chromosomal DNA, whose sequence is complementary, to guide RNA in a targeted manner, producing site-specific DNA double-strand breaks (DSBs), the repair of which gives rise to targeted genome modifications. Despite broad interest in RGEN-mediated genome editing, these nucleases are limited by off-target mutations and unwanted chromosomal translocations associated with off-target DNA cleavages. Here, we show that off-target effects of RGENs can be reduced below the detection limits of deep sequencing by choosing unique target sequences in the genome and modifying both guide RNA and Cas9. We found that both the composition and structure of guide RNA can affect RGEN activities in cells to reduce off-target effects. RGENs efficiently discriminated on-target sites from off-target sites that differ by two bases. Furthermore, exome sequencing analysis showed that no off-target mutations were induced by two RGENs in four clonal populations of mutant cells. In addition, paired Cas9 nickases, composed of D10A Cas9 and guide RNA, which generate two single-strand breaks (SSBs) or nicks on different DNA strands, were highly specific in human cells, avoiding off-target mutations without sacrificing genome-editing efficiency. Interestingly, paired nickases induced chromosomal deletions in a targeted manner without causing unwanted translocations. Our results highlight the importance of choosing unique target sequences and optimizing guide RNA and Cas9 to avoid or reduce RGEN-induced off-target mutations.

Purpose: Remnant thyroid ablation and 1-year stimulated thyroglobulin (sTg) measurement are recommended for those who have undergone total thyroidectomy for differentiated thyroid cancer. The serum Tg kinetics in such patients are still unclear. This study was designed to evaluate whether the periablative change in serum markers can predict biochemical remission in papillary thyroid cancer (PTC) patients., Methods: We reviewed the medical records of 185 patients who were given high-dose radioactive iodine ablation therapy from January 2006 to December 2008. Serum Tg, TSH, and anti-Tg antibody (TgAb) were measured on the day and the following 10th day of radioactive iodine administration. We defined preablative sTg as Tg-1, postablative Tg measured on the 10th day of ablation as Tg-2, and the 1-year sTg as Tg-3. ΔTg means Tg2-Tg1. The same definition was applied to TgAb., Results: A biochemical remission defined as Tg-3 < 2 ng/ml was achieved in 144 patients. Among the patients who achieved biochemical remission, PTC recurred in six during a median follow-up of 54 months. Tg-1 < 3.3 ng/ml (p < 0.0001) predicted biochemical remission. Neither the ΔTg nor ΔTgAb was useful for predicting biochemical remission. On the evaluation of recurrence after biochemical remission, Tg-1 > 5.32 (p < 0.0001) and Tg-3 > 2.9 (p = 0.01) were proven to be statistically significant cutoff values for predicting recurrence. The ΔTg and ΔTgAb were not able to predict recurrence., Conclusion: For the prediction of biochemical remission or recurrence after biochemical remission, preablative sTg was demonstrated to be a statistically significant serum marker. However, short-term changes in biochemical markers including Tg and TgAb around the day of ablation could not provide useful clinical information about biochemical remission or disease recurrence. In conclusion, 1-year sTg measurement cannot be omitted with short-term change.

Activation of naïve cluster of differentiation (CD)8(+) cytotoxic T lymphocytes (CTLs) is a tightly regulated process, and specific dendritic cell (DC) subsets are typically required to activate naive CTLs. Potential pathways for antigen presentation leading to CD8(+) T-cell priming include direct presentation, cross-presentation, and cross-dressing. To distinguish between these pathways, we designed single-chain trimer (SCT) peptide-MHC class I complexes that can be recognized as intact molecules but cannot deliver antigen to MHC through conventional antigen processing. We demonstrate that cross-dressing is a robust pathway of antigen presentation following vaccination, capable of efficiently activating both naïve and memory CD8(+) T cells and requires CD8α(+)/CD103(+) DCs. Significantly, immune responses induced exclusively by cross-dressing were as strong as those induced exclusively through cross-presentation. Thus, cross-dressing is an important pathway of antigen presentation, with important implications for the study of CD8(+) T-cell responses to viral infection, tumors, and vaccines.

Previous studies have indicated that Yoga exercise has a positive effect on reducing blood pressure and heart rate. However, no randomized controlled studies to date have investigated its effects on arterial compliance. The purpose of this study was to investigate the effects of an 8-month Yoga intervention on arterial compliance and muscle strength in normal premenopausal women 35-50 years of age. Thirty-four women were randomly assigned either to a Yoga exercise group (YE, n = 16) or a control group (CON, n = 18). Participants in YE group performed 60 minutes of an Ashtanga Yoga series 2 times/week with one day between sessions for 8 months. Each Yoga session consisted of 15 minutes of warm-up exercises, 35 minutes of Ashtanga Yoga postures and 10 minutes of cool-down with relaxation; and the session intensity was progressively increased during the 8 months. Participants in CON were encouraged to maintain their normal daily lifestyles monitored by the bone-specific physical activity questionnaire at 2 month intervals for 8 months. Arterial compliance (pulse contour analysis) and muscle strength (1 Repetition Maximum) were assessed at baseline and after the intervention. Arterial compliance of the large and small arteries was not affected by the 8 month Yoga training (p > 0.05). Also, there were no significant (p > 0.05) group, time, or group × time interaction effects for cardiovascular variables. YE group significantly (p < 0.01) improved leg press muscle strength compared to CON (11.4% vs. -6.5%). Eight months of Ashtanga Yoga training was beneficial for improving leg press strength, but not arterial compliance in premenopausal women. Key pointsThe 8 month Yoga training did not affect arterial compliance of the large and small arteries.None of the cardiovascular variables were changed by the Yoga intervention.Isotonic muscle strength was not altered by the Yoga intervention, with the exception of leg press.

Several nonclassical major histocompatibilty antigens (class Ib molecules) have emerged as key players in the early immune response to pathogens or stress. Class Ib molecules activate subsets of T cells that mount effector responses before the adaptive immune system, and thus are called innate T cells. MR1 is a novel class Ib molecule with properties highly suggestive of its regulation of mucosal immunity. The Mr1 gene is evolutionarily conserved, is non-Mhc linked, and controls the development of mucosal-associated invariant T (MAIT) cells. MAIT cells preferentially reside in the gut, and their development is dependent on commensal microbiota. Although these properties suggest that MAIT cells function as innate T cells in the mucosa, this has been difficult to test, due to the (i) paucity of MAIT cells that display MR1-specific activation in vitro and (ii) lack of knowledge of whether or not MR1 presents antigen. Here we show that both mouse and human MAIT cells display a high level of cross-reactivity on mammalian MR1 orthologs, but with differences consistent with limited ligand discrimination. Furthermore, acid eluates from recombinant or cellular MR1 proteins enhance MAIT cell activation in an MR1-specific and cross-species manner. Our findings demonstrate that the presentation pathway of MR1 to MAIT cells is highly evolutionarily conserved.

Like CD1d-restricted iNKT cells, mucosal-associated invariant T cells (MAITs) are "innate" T cells that express a canonical TCRalpha chain, have a memory phenotype, and rapidly secrete cytokines upon TCR ligation. Unlike iNKT cells, MAIT cells require the class Ib molecule MHC-related protein I (MR1), B cells, and gut flora for development and/or expansion, and they preferentially reside in the gut lamina propria. Evidence strongly suggests that MAIT cell activation is ligand-dependent, but the nature of MR1 ligand is unknown. In this study, we define a mechanism of endogenous antigen presentation by MR1 to MAIT cells. MAIT cell activation was dependent neither on a proteasome-processed ligand nor on the chaperoning by the MHC class I peptide loading complex. However, MAIT cell activation was enhanced by overexpression of MHC class II chaperones Ii and DM and was strikingly diminished by silencing endogenous Ii. Furthermore, inhibiting the acidification of the endocytic compartments reduced MR1 surface expression and ablated MAIT cell activation. The importance of the late endosome for MR1 antigen presentation was further corroborated by the localization of MR1 molecules in the multivesicular endosomes. These findings demonstrate that MR1 traffics through endocytic compartments, thereby allowing MAIT cells to sample both endocytosed and endogenous antigens.

To investigate the genotoxic effect of marine sediments on aquatic organism, sediment samples were collected from 13 sites along the coast of Gwangyang Bay (Korea). Concentrations of polycyclic aromatic hydrocarbons (PAHs) in sediments were determined and the relationship between exposure of flounder blood cells to sediment extracts and DNA single-strand breakage in the blood cells was examined using the comet assay. Levels of DNA damage were proportionally increased by exposure concentration and the highest sediment-associated DNA damage was observed at the station showing the highest PAHs contamination. DNA damage in blood cells exposed to five types of PAHs (benzo[a]pyrene, fluoranthene, anthracene, pyrene and phenanthrene) and in flounder (Paralichthys olivaceus) exposed to benzo[a]pyrene (BaP) for 0, 2 and 4 days were assessed by measuring comet tail length. The tail lengths of five PAHs-exposed groups at 50 and 100 ppb were significantly different from the non-exposed group, and the genotoxic effect of BaP correlated with both concentration and duration of exposure. Throughout the study, significant differences in DNA breakage were recorded between cells exposed to sediment extracts or PAHs and non-exposed control. This study demonstrated the comet assay as a successful tool in monitoring contamination of marine sediments and assessing genotoxicity of PAHs in marine organisms, either in vitro or in vivo.