Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals.

Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.

An orange calcium-modulated bioluminescent indicator for non-invasive activity imaging.

Fluorescent indicators are used widely to visualize calcium dynamics downstream of membrane depolarization or G-protein-coupled receptor activation, but are poorly suited for non-invasive imaging in mammals. Here, we report a bright calcium-modulated bioluminescent indicator named Orange CaMBI (Orange Calcium-modulated Bioluminescent Indicator). Orange CaMBI reports calcium dynamics in single cells and, in the context of a transgenic mouse, reveals calcium oscillations in whole organs in an entirely non-invasive manner.

The LAMMER kinase is involved in morphogenesis and response to cell wall- and DNA-damaging stresses in Candida albicans.

Dual specificity LAMMER kinase has been reported to be conserved across species ranging from yeasts to animals and has multiple functions. Candida albicans undergoes dimorphic switching between yeast cells and hyphal growth forms as its key virulence factors. Deletion of KNS1, which encodes for LAMMER kinase in C. albicans, led to pseudohyphal growth on YPD media and defects in filamentous growth both on spider and YPD solid media containing 10% serum. These cells exhibited expanded central wrinkled regions and specifically reduced peripheral filaments. Among the several stresses tested, the kns1Δ strains showed sensitivity to cell-wall and DNA-replicative stress. Under fluorescent microscopy, an increase in chitin decomposition was observed near the bud necks and septa in kns1Δ cells. When the expression levels of genes for cell wall integrity (CWI) and the DNA repair mechanism were tested, the kns1 double-deletion cells showed abnormal patterns compared to wild-type cells; The transcript levels of genes for glycosylphosphatidylinositol (GPI)-anchored proteins were increased upon calcofluor white (CFW) treatment. Under DNA replicative stress, the expression of MluI-cell cycle box binding factor (MBF)-targeted genes, which are expressed during the G1/S transition in the cell cycle, was not increased in the kns1 double-deletion cells. This strain showed increased adhesion to the surface of an agar plate and zebrafish embryo. These results demonstrate that Kns1 is involved in dimorphic transition, cell wall integrity, response to DNA replicative stress, and adherence to the host cell surface in C. albicans.

A multilevel investigation of cultural competence among South Korean clinical nurses.

BACKGROUND: There is lack of empirical evidence on whether organisational variables affect the cultural competence of nurses. AIM: This study aimed to investigate individual and organisational characteristics associated with South Korean clinical nurses' cultural competence. METHODS: A descriptive cross-sectional research design was used. A convenient sample of 401 clinical nurses from 21 hospitals in South Korea was recruited between November 2015 and February 2016. Multilevel modelling was used to estimate the effects of individual- and organisation-level predictors. Data were analysed using the nlme package in R. RESULTS: Multilevel modelling indicated that professionalism (ß = 0.02, p < 0.001) and foreign language fluency (ß = 0.25, p = 0.012) were significant individual-level predictors. Education on foreign patient care was marginally significant at p = 0.069. At the organisational level, nurse-to-patient ratio (ß = 0.37, p = 0.038) and organisational cultural competence (ß = 0.02, p < 0.001) were significant predictors. In addition, hospital ownership and work environment were marginally significant at p = 0.054 and p = 0.050, respectively. Furthermore, there was a significant cross-level interaction between professionalism and hospital ownership (ß = 0.03, p = 0.003). CONCLUSION: To provide culturally competent care, nurse leaders should recognise the importance of organisational-level factors, such as nurse staffing and organisational cultural competence, and create an environment that is inclusive of diverse patients, as well as promote professionalism among individual nurses.

Quantification of an Antibody-Conjugated Drug in Fat Plasma by an Affinity Capture LC-MS/MS Method for a Novel Prenyl Transferase-Mediated Site-Specific Antibody-Drug Conjugate.

The novel prenyl transferase-mediated, site-specific, antibody-drug conjugate LCB14-0110 is comprised of a proprietary beta-glucuronide linker and a payload (Monomethyl auristatin F, MMAF, an inhibitor for tubulin polymerization) attached to human epidermal growth factor receptor 2 (HER2)-targeting trastuzumab. A LC-MS/MS method was developed to quantify the antibody-conjugated drug (acDrug) for in vitro linker stability and preclinical pharmacokinetic studies. The method consisted of affinity capture, enzymatic cleavage of acDrug, and LC-MS/MS analysis in the positive ion mode. A quadratic regression (weighted 1/concentration2), with the equation y = ax2 + bx + c, was used to fit calibration curves over the concentration range of 19.17~958.67 ng/mL for acDrug. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control (QC) samples. The overall recovery was 42.61%. The dilution integrity was for a series of 5-fold dilutions with accuracy and precision values ranging within ±25%. The stability results indicated that acDrug was stable at all stability test conditions (short-term: 1 day, long-term: 10 months, Freeze/Thaw (F/T): 3 cycles). This qualified method was successfully applied to in vitro linker stability and pharmacokinetic case studies of acDrug in rats.

Pyruvate dehydrogenase complex deficiency is linked to regulatory loop disorder in the αV138M variant of human pyruvate dehydrogenase.

The pyruvate dehydrogenase multienzyme complex (PDHc) connects glycolysis to the tricarboxylic acid cycle by producing acetyl-CoA via the decarboxylation of pyruvate. Because of its pivotal role in glucose metabolism, this complex is closely regulated in mammals by reversible phosphorylation, the modulation of which is of interest in treating cancer, diabetes, and obesity. Mutations such as that leading to the αV138M variant in pyruvate dehydrogenase, the pyruvate-decarboxylating PDHc E1 component, can result in PDHc deficiency, an inborn error of metabolism that results in an array of symptoms such as lactic acidosis, progressive cognitive and neuromuscular deficits, and even death in infancy or childhood. Here we present an analysis of two X-ray crystal structures at 2.7-Å resolution, the first of the disease-associated human αV138M E1 variant and the second of human wildtype (WT) E1 with a bound adduct of its coenzyme thiamin diphosphate and the substrate analogue acetylphosphinate. The structures provide support for the role of regulatory loop disorder in E1 inactivation, and the αV138M variant structure also reveals that altered coenzyme binding can result in such disorder even in the absence of phosphorylation. Specifically, both E1 phosphorylation at αSer-264 and the αV138M substitution result in disordered loops that are not optimally oriented or available to efficiently bind the lipoyl domain of PDHc E2. Combined with an analysis of αV138M activity, these results underscore the general connection between regulatory loop disorder and loss of E1 catalytic efficiency.

Organisational cultural competence needed to care for foreign patients: A focus on nursing management.

AIM: This study aimed to explore the organisational level of the cultural competence needed to care for foreign patients from the Korean clinical registered nurses' perspective. BACKGROUND: Owing to the increasing foreign resident population in Korea, health care organisations face challenges arising from cultural differences. However, there is a lack of research on the impact of these changes on nursing care and management. METHOD: Participants were 16 clinical registered nurses from six general hospitals. Data were collected through individual in-depth and focus group interviews. Transcribed data were analysed by deductive content analysis. RESULTS: Data were classified into three categories: information, resources and incentives. Information included a systematic staff training programme and clear practice guidelines. Resources included convenient and credible interpretation services and adequate tools and materials, and incentives were referred to managerial support. CONCLUSION: Korean registered nurses perceived a lack of organisational support for caring for foreign patients. System-wide support and active unit-level intervention of nursing managers are essential for successful transformation towards a culturally competent organisation. IMPLICATION FOR NURSING MANAGEMENT: Registered nurses should play leadership roles to establish a dedicated nursing team or committee to create a supportive working environment and implement a monitoring system so that their organisations move towards greater cultural competence.

Causal Information Over Facial Expression: Modulation of Facial Expression Processing by Congruency and Causal Factor of the Linguistic Cues in 5-Year-Old Japanese Children.

It is unknown whether linguistic cues influence preschoolers' recognition of facial expression when the emotion of the face is incongruent with the linguistic cues and what type of linguistic cue is influential in the modulation of facial expression. In a priming task, we presented 5-year-old children three types of linguistic information conveying happy or sad emotion (simple label, label with rich causal explanation of emotion, and label with poor causal explanation of emotion) prior to the presentation of a happy or a sad face. We asked participants to recognize emotion of facial expression and examined whether they follow linguistic information than the facial expression when the emotions of linguistic information and face were incongruent. As a result, children who were presented the label with rich causal explanation judged emotion of face according to the linguistic information, whereas children who were presented the simple label or the label with poor causal explanation followed the emotions of facial expression. This result indicated that children's autonomic reliance on situational cues depends on the emotional causality and the concreteness of emotional state conveyed by the linguistic cues. This finding contributes to our understanding of the face inferiority effect on children's developing notion of emotions.

Dexamethasone downregulates SIRT1 and IL6 and upregulates EDN1 genes in stem cells derived from gingivae via the AGE/RAGE pathway.

OBJECTIVES: To evaluate the effects of dexamethasone on the aging of mesenchymal stem cells from human gingiva using next-generation sequencing. RESULTS: Four mRNAs were upregulated and 12 were downregulated when the results of dexamethasone at 24 h were compared with the control at 24 h. Expressions of SIRT1 and IL6 were decreased in dexamethasone at 24 h but expression of EDN1 was increased. CONCLUSIONS: Application of dexamethasone reduced the expression of SIRT1 and IL6 but enhanced the expression of EDN1 of stem cells.

sAPP modulates iron efflux from brain microvascular endothelial cells by stabilizing the ferrous iron exporter ferroportin.

A sequence within the E2 domain of soluble amyloid precursor protein (sAPP) stimulates iron efflux. This activity has been attributed to a ferroxidase activity suggested for this motif. We demonstrate that the stimulation of efflux supported by this peptide and by sAPPα is due to their stabilization of the ferrous iron exporter, ferroportin (Fpn), in the plasma membrane of human brain microvascular endothelial cells (hBMVEC). The peptide does not bind ferric iron explaining why it does not and thermodynamically cannot promote ferrous iron autoxidation. This peptide specifically pulls Fpn down from the plasma membrane of hBMVEC; based on these results, FTP, for ferroportin-targeting peptide, correctly identifies the function of this peptide. The data suggest that in stabilizing Fpn via the targeting due to the FTP sequence, sAPP will increase the flux of iron into the cerebral interstitium. This inference correlates with the observation of significant iron deposition in the amyloid plaques characteristic of Alzheimer's disease.

Elucidation of the interaction loci of the human pyruvate dehydrogenase complex E2·E3BP core with pyruvate dehydrogenase kinase 1 and kinase 2 by H/D exchange mass spectrometry and nuclear magnetic resonance.

The human pyruvate dehydrogenase complex (PDC) comprises three principal catalytic components for its mission: E1, E2, and E3. The core of the complex is a strong subcomplex between E2 and an E3-binding protein (E3BP). The PDC is subject to regulation at E1 by serine phosphorylation by four kinases (PDK1-4), an inactivation reversed by the action of two phosphatases (PDP1 and -2). We report H/D exchange mass spectrometric (HDX-MS) and nuclear magnetic resonance (NMR) studies in the first attempt to define the interaction loci between PDK1 and PDK2 with the intact E2·E3BP core and their C-terminally truncated proteins. While the three lipoyl domains (L1 and L2 on E2 and L3 on E3BP) lend themselves to NMR studies and determination of interaction maps with PDK1 and PDK2 at the individual residue level, HDX-MS allowed studies of interaction loci on both partners in the complexes, PDKs, and other regions of the E2·E3BP core, as well, at the peptide level. HDX-MS suggested that the intact E2·E3BP core enhances the binding specificity of L2 for PDK2 over PDK1, while NMR studies detected lipoyl domain residues unique to interaction with PDK1 and PDK2. The E2·E3BP core induced more changes on PDKs than any C-terminally truncated protein, with clear evidence of greater plasticity of PDK1 than of PDK2. The effect of L1L2S paralleled HDX-MS results obtained with the intact E2·E3BP core; hence, L1L2S is an excellent candidate with which to define interaction loci with these two PDKs. Surprisingly, L3S' induced moderate interaction with both PDKs according to both methods.

Color temperature's impact on task performance and brainwaves of school-age children.

[Purpose] This study investigated color temperature's impact on task performance. It presents a scientific analysis of brainwave and task performance time changes, and the results of a self-report type survey. [Subjects] Twenty-four elementary school fifth-grade boys and girls with no visual problems participated in the experiment. [Methods] Physiological reaction times of task performance were measured in a laboratory that could fix and maintain color temperature. Brainwave changes and the task performance times were measured, and a self-report questionnaire was conducted in order to measure of emotional reactions. [Results] Regarding the brainwave changes associated with color temperature, alpha waves were emitted in the O2 area when puzzle tasks were illuminated by orange light and low and high beta waves were emitted in the F3 area under white light. Five items (Brilliant, Soft, Lively, Relaxed, Open) were reported predominantly in responses to orange light in the self-report questionnaire. [Conclusion] The results of this study show that relaxation and stability are not assured when the color temperature is low, and that concentration and cognitive activity are not necessarily easier when the color temperature is high. The color temperature change when performing tasks promoted emotional factors more than brainwave, a biological change.

Effect of an exercise program for posture correction on musculoskeletal pain.

[Purpose] The present study investigated the effect of an exercise program for posture correction on musculoskeletal pain. [Subjects] Between September 2, 2013 and November 3, 2013, an exercise program was performed in 88 students from S University in K city (male students, n = 34; female students, n = 54). [Methods] The exercise program for posture correction was performed for 20 minutes per session, 3 times a week for 8 weeks. Pain levels were measured using a pain scale, and pain levels before and after the exercise program were compared. [Results] Overall, pain levels of the participants were lower after the exercise program than before the program, and significant differences in pain levels were noted in the shoulders, middle back, and lower back. [Conclusion] In conclusion, shoulder pain, mid back pain, and low back pain were relieved with the exercise program for posture correction. Therefore, the findings of this study can be used to improve the work efficiency of students as well as people engaged in sedentary work.

A study on the relationships between age, work experience, cognition, and work ability in older employees working in heavy industry.

[Purpose] The purpose of this study was to examine the correlation of age, work experience, cognition, and work ability in older employees working in heavy industry. [Subjects and Methods] The study was conducted using 100 subjects who were over 55 years old and worked in heavy industry. To obtain data, we first had the subjects complete the MoCA-K test and Work Ability Index (WAI). The data were then analyzed by frequency and correlation using statistical software (SPSS 21.0). [Results] Through this study, we discovered a significant positive correlation between WAI and MoCA-K, age, and work experience. [Conclusion] This study revealed that work ability in older employees increases not with the number of years worked but with the enhancement of cognitive ability. Special management that focuses on cognition is therefore required for senior employees working in the field of heavy industry.

Insight to the interaction of the dihydrolipoamide acetyltransferase (E2) core with the peripheral components in the Escherichia coli pyruvate dehydrogenase complex via multifaceted structural approaches.

Multifaceted structural approaches were undertaken to investigate interaction of the E2 component with E3 and E1 components from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), as a representative of the PDHc from Gram-negative bacteria. The crystal structure of E3 at 2.5 Å resolution reveals similarity to other E3 structures and was an important starting point for understanding interaction surfaces between E3 and E2. Biochemical studies revealed that R129E-E2 and R150E-E2 substitutions in the peripheral subunit-binding domain (PSBD) of E2 greatly diminished PDHc activity, affected interactions with E3 and E1 components, and affected reductive acetylation of E2. Because crystal structures are unavailable for any complete E2-containing complexes, peptide-specific hydrogen/deuterium exchange mass spectrometry was used to identify loci of interactions between 3-lipoyl E2 and E3. Two peptides from the PSBD, including Arg-129, and three peptides from E3 displayed statistically significant reductions in deuterium uptake resulting from interaction between E3 and E2. Of the peptides identified on E3, two were from the catalytic site, and the third was from the interface domain, which for all known E3 structures is believed to interact with the PSBD. NMR clearly demonstrates that there is no change in the lipoyl domain structure on complexation with E3. This is the first instance where the entire wild-type E2 component was employed to understand interactions with E3. A model for PSBD-E3 binding was independently constructed and found to be consistent with the importance of Arg-129, as well as revealing other electrostatic interactions likely stabilizing this complex.

Study of the Physical Condition of Middle-aged Office and Field Workers Aged 50 and Older.

[Purpose] We investigated the physical condition of middle-aged workers in Korea by job category (office job vs. production job). [Subjects] In total, 3,650 middle-aged workers (production job, n = 2,615; office job, n = 1,035) ≥50 years of age from eight geographical areas were evaluated from 2007 to 2008. [Methods] The subjects' subjective physical condition, exercise status, smoking status, and drinking status were investigated, and precise physical measurements, cardiovascular endurance, muscular strength, muscular endurance, flexibility, reflexes, and reactions were measured. [Results] Subjectively, the physical condition and exercise status of those with office jobs were superior to those of production job workers. Office workers exhibited a higher rate of drinking than production workers, and also had better cardiovascular endurance, muscular strength, muscular endurance, reflexes, and reactions. However, production workers were more flexible than office workers. [Conclusion] Middle-aged production job workers in Korea have poorer overall physical condition than middle-aged office job workers. The physical condition of middle-aged and elderly workers must be managed through various health management programs to secure a productive aging society.

Fission yeast LAMMER kinase Lkh1 regulates the cell cycle by phosphorylating the CDK-inhibitor Rum1.

In eukaryotes, LAMMER kinases are involved in various cellular events, including the cell cycle. However, no attempt has been made to investigate the mechanisms that underlie the involvement of LAMMER kinase. In this study, we performed a functional analysis of LAMMER kinase using the fission yeast, Schizosaccharomyces pombe. FACS analyses revealed that deletion of the gene that encodes the LAMMER kinase Lkh1 made mutant cells pass through the G1/S phase faster than their wild-type counterparts. Co-immunoprecipitation and an in vitro kinase assay also revealed that Lkh1 can interact with and phosphorylate Rum1 to activate this molecule as a cyclin-dependent kinase inhibitor, which blocks cell cycle progression from the G1 phase to the S phase. Peptide mass fingerprinting and kinase assay with Rum1(T110A) confirmed T110 as the Lkh1-dependent phosphorylation residue. In this report we present for the first time a positive acting mechanism that is responsible for the CKI activity of Rum1, in which the LAMMER kinase-mediated phosphorylation of Rum1 is involved.

A study on the necessity of medical facilities safety design adoption.

[Purpose] The purpose of this study was to investigate the requirements of the introduction of a safety design and certification system for medical facilities. [Subjects] A survey was carried out of one hundred nurses, physical therapists, occupational therapists, speech and language therapists from May to August in 2012. [Methods] The survey was conducted after giving subjects some information about safety design. [Results] The participants were aware of the need for establishing a safety design certification system. Total responses to services, facilities and space were analyzed in order to evaluate the priorities of safety, user characteristics, functionality, convenience and aesthetics. Regarding the application of a safety design certification system to services, items were prioritized in the order of children's items, household supplies and hospital supplies. For facilities, the priorities were, living space, social welfare and medical facilities; space, they were public and transportation-related places. The requirements for operating a safety design system were in order development of: highly skilled manpower, the legal system, educational promotion and qualifying facilities. [Conclusion] In conclusion, in order to implement safety design in medical facilities, a safety design certification system should be introduced first, and to do this a systematic and comprehensive study is needed.

Registered Nurses' perspective of systemic factors affecting nursing home care quality decline: A qualitative descriptive study.

AIM: A growing number of older people are living in nursing homes worldwide, but their safety and quality of care are not guaranteed. This study explores registered nurses' (RNs) perspectives on systemic factors affecting the quality of care and safety decline of nursing home. DESIGN: Qualitative descriptive study. METHODS: In this study, semi-structured interviews were conducted with 10 RNs working in six nursing homes, who were chosen through purposive sampling. Data were collected from 1 August-19 September 2019, and analysed using thematic analysis. RESULTS: The following five themes were derived: lack of sufficient number of RNs, poor work conditions, unclear job descriptions for RNs, no official position of nursing director and absence of transition care system. PATIENT OR PUBLIC CONTRIBUTION: All reports of RNs affecting resident safety and quality of care decline were related to systemic factors. Therefore, improving quality of care in nursing homes should be supported by changes in systemic factors, such as maintaining an appropriate number of RNs and improving their working conditions.

The LAMMER kinase homolog, Lkh1, regulates Tup transcriptional repressors through phosphorylation in Schizosaccharomyces pombe.

Disruption of the fission yeast LAMMER kinase, Lkh1, gene resulted in diverse phenotypes, including adhesive filamentous growth and oxidative stress sensitivity, but an exact cellular function had not been assigned to Lkh1. Through an in vitro pull-down approach, a transcriptional repressor, Tup12, was identified as an Lkh1 binding partner. Interactions between Lkh1 and Tup11 or Tup12 were confirmed by in vitro and in vivo binding assays. Tup proteins were phosphorylated by Lkh1 in a LAMMER motif-dependent manner. The LAMMER motif was also necessary for substrate recognition in vitro and cellular function in vivo. Transcriptional activity assays using promoters negatively regulated by Tup11 and Tup12 showed 6 or 2 times higher activity in the Delta lkh1 mutant than the wild type, respectively. Northern analysis revealed derepressed expression of the fbp1(+) mRNA in Delta lkh1 and in Delta tup11 Delta tup12 mutant cells under repressed conditions. Delta lkh1 and Delta tup11 Delta tup12 mutant cells showed flocculation, which was reversed by co-expression of Tup11 and -12 with Ssn6. Here, we presented a new aspect of the LAMMER kinase by demonstrating that the activities of global transcriptional repressors, Tup11 and Tup12, were positively regulated by Lkh1-mediated phosphorylation.