Basic evaluation of the CRISPR/Cas system stability for application to paper-based analytical devices.

Despite the promising features of the CRISPR/Cas system for application to point-of-care nucleic acid tests, there are only a few reports on its integration into paper-based analytical devices (PADs) for the purpose of assay simplification. In most cases, paper platforms have only been used for the final signal readout in an assay otherwise performed in a test tube. Therefore, there is very limited information on the suitability of the CRISPR/Cas system for on-device reagent storage. To fill this gap, the current work primarily investigated the influence of various factors, including the type of paper, reagent drying method, effect of stabilizers, and storage condition on the storage stability of reagents necessary for CRISPR-based assays on paper substrates, by comparing the fluorescence signal emitted by the trans-cleavage of the dsDNA-activated Cas12a complex. The results obtained in the form of fluorescence signals emitted after trans-cleavage of a ssDNA probe through a dsDNA-activated Cas12a complex on paper substrates showed that CRISPR-related reagents spontaneously dried at room temperature on BSA blocked paper retained over 70% of their initial activity when stored at -20 °C for 28 days, independent of the type of paper substrates, which was improved by the addition of sucrose as a stabilizer. In addition, reagents dried on paper substrates under the optimized conditions exhibited stronger heat tolerance at temperatures above 65 °C compared to their corresponding solutions. This work is expected to contribute to the future development of fully integrated PADs relying on CRISPR/Cas systems for point-of-care applications requiring no additional reagent handling.

A case of leukemia cutis showing annular erythema during the course of Philadelphia chromosome-positive acute B-lymphoblastic leukemia.

We report a case of leukemia cutis showing annular erythema during the course of Philadelphia chromosome-positive acute B-lymphoblastic leukemia. The annular appearance may be developed by immunomodulatory effects of blinatumomab.

LAG-3 expression in microglia regulated by IFN-γ/STAT1 pathway and metalloproteases.

Microglia are resident innate immune cells in the central nervous system (CNS) and play important roles in the development of CNS homeostasis. Excessive activation and neurotoxicity of microglia are observed in several CNS disorders, but the mechanisms regulating their activation remain unclear. Immune checkpoint molecules are expressed on activated immune cells and regulate their activation in peripheral immunity. However, the expression mechanism of immune checkpoint molecules in activated microglia is still unknown. Here, we analyzed the expression of immune checkpoint molecules in activated microglia using the mouse microglial cell line BV2 and primary cultured microglia. The expression of lymphocyte activation gene-3 (LAG-3), a type of immune checkpoint molecule, was increased in microglia activated by IFN-γ. IFN-γ-induced LAG-3 expression in microglia was suppressed by transfection of siRNA targeting STAT1. LAG-3 has two forms, membrane and soluble, and both forms were upregulated in microglia activated by IFN-γ. The production of soluble LAG-3 was suppressed by treatment with inhibitors of metalloproteinases such as ADAM10 and ADAM17. IFN-γ administration into cisterna magna of mice increased LAG-3 expression in spinal microglia. Furthermore, LAG-3 knockdown in microglia promoted nitric oxide production by IFN-γ. Our results demonstrate that LAG-3 expression in microglia is induced by the IFN-γ-STAT1 pathway and soluble LAG-3 production is regulated via cleavage of membranous LAG-3 by metalloproteinases including ADAM10 and ADAM17.

Sequence divergence and retrotransposon insertion underlie interspecific epigenetic differences in primates.

Changes in the epigenome can affect the phenotype without the presence of changes in the genomic sequence. Given the high identity of the human and chimpanzee genome sequences, a substantial portion of their phenotypic divergence likely arises from epigenomic differences between the two species. In this study, the transcriptome and epigenome were determined for induced pluripotent stem cells (iPSCs) generated from human and chimpanzee individuals. The transcriptome and epigenomes for trimethylated histone H3 at lysine-4 (H3K4me3) and lysine-27 (H3K27me3) showed high levels of similarity between the two species. However, there were some differences in histone modifications. Although such regions, in general, did not show significant enrichment of interspecies nucleotide variations, gains in binding motifs for pluripotency-related transcription factors, especially POU5F1 and SOX2, were frequently found in species-specific H3K4me3 regions. We also revealed that species-specific insertions of retrotransposons, including the LTR5_Hs subfamily in human and a newly identified LTR5_Pt subfamily in chimpanzee, created species-specific H3K4me3 regions associated with increased expression of nearby genes. Human iPSCs have more species-specific H3K27me3 regions, resulting in more abundant bivalent domains. Only a limited number of these species-specific H3K4me3 and H3K27me3 regions overlap with species-biased enhancers in cranial neural crest cells, suggesting that differences in the epigenetic state of developmental enhancers appear late in development. Therefore, iPSCs serve as a suitable starting material for studying evolutionary changes in epigenome dynamics during development.

Easy preparation of a liposome-mediated protein delivery system by freeze-thawing a liposome-protein complex.

Homeostasis can be achieved by adding a protein supplement; however, an appropriate vector is required to deliver the protein into the cell because of the low stability of proteins in the blood and low cell membrane permeability. Here we report an easy one-step method of encapsulating proteins into liposomes for delivery. We used negatively charged superoxide dismutase (SOD) and a polycation liposome as protein and liposome models, respectively. Liposome-encapsulated SOD was prepared by freeze-thawing the SOD-liposome complex (lipoplexes). The amount of immobilized SOD within the lipoplex significantly increased on freeze-thawing. Surprisingly, subjecting the single-layered lipoplexes to freeze-thawing produced multilayered liposomes with SOD localized between the lipid layers. The amount of SOD delivered intracellularly significantly increased by freeze-thawing compared with that delivered by lipoplexes without freeze-thawing. SOD, liposomes, and endosomes were separately localized in the cells. The freeze-thawed lipoplex-encapsulated SOD samples were intravenously injected in mice. The SOD biodistribution was dramatically changed compared with the injection of free SOD or lipoplex. SOD was detached from the lipoplex in the bloodstream after the injection of non-freeze-thawed lipoplex, whereas the encapsulation of SOD in the liposomes upon freeze-thawing enabled the stable circulation of SOD with the liposomes in the bloodstream. This work paves the way for the application of the freeze-thawing technology for the easy one-step encapsulation of proteins into liposomes for protein delivery.

Predictive Modeling of Mental Illness Onset Using Wearable Devices and Medical Examination Data: Machine Learning Approach.

The prevention and treatment of mental illness is a serious social issue. Prediction and intervention, however, have been difficult because of lack of objective biomarkers for mental illness. The objective of this study was to use biometric data acquired from wearable devices as well as medical examination data to build a predictive model that can contribute to the prevention of the onset of mental illness. This was an observational study of 4,612 subjects from the health database of society-managed health insurance in Japan provided by JMDC Inc. The inputs to the predictive model were 3-months of continuous wearable data and medical examinations within and near that period; the output was the presence or absence of mental illness over the following month, as defined by insurance claims data. The features relating to the wearable data were sleep, activity, and resting heart rate, measured by a consumer-grade wearable device (specifically, Fitbit). The predictive model was built using the XGBoost algorithm and presented an area-under-the-receiver-operating-characteristic curve of 0.712 (SD = 0.02, a repeated stratified group 10-fold cross validation). The top-ranking feature importance measure was wearable data, and its importance was higher than the blood-test values from medical examinations. Detailed verification of the model showed that predictions were made based on disrupted sleep rhythms, mild physical activity duration, alcohol use, and medical examination data on disrupted eating habits as risk factors. In summary, the predictive model showed useful accuracy for grouping the risk of mental illness onset, suggesting the potential of predictive detection, and preventive intervention using wearable devices. Sleep abnormalities in particular were detected as wearable data 3 months prior to mental illness onset, and the possibility of early intervention targeting the stabilization of sleep as an effective measure for mental illness onset was shown.

Enhancement of target toxin neutralization effect in vivo by PEGylation of multifunctionalized lipid nanoparticles.

Protein-protein (e.g., antibody-antigen) interactions comprise multiple weak interactions. We have previously reported that lipid nanoparticles (LNPs) bind to and neutralize target toxic peptides after multifunctionalization of the LNP surface (MF-LNPs) with amino acid derivatives that induce weak interactions; however, the MF-LNPs aggregated after target capture and showed short blood circulation times. Here we optimized polyethylene glycol (PEG)-modified MF-LNPs (PEG-MF-LNPs) to inhibit the aggregation and increase the blood circulation time. Melittin was used as a target toxin, and MF-LNPs were prepared with negatively charged, hydrophobic, and neutral amino-acid-derivative-conjugated functional lipids. In this study, MF-LNPs modified with only PEG5k (PEG5k-MF-LNPs) and with both PEG5k and PEG2k (PEGmix-MF-LNPs) were prepared, where PEG5k and PEG2k represent PEG with a molecular weight of 5000 and 2000, respectively. PEGylation of the MF-LNPs did not decrease the melittin neutralization ability of nonPEGylated MF-LNPs, as tested by hemolysis assay. The PEGmix-MF-LNPs showed better blood circulation characteristics than the PEG5k-MF-LNPs. Although the nonPEGylated MF-LNPs immediately aggregated when mixed with melittin, the PEGmix-MF-LNPs did not aggregate. The PEGmix-MF-LNPs dramatically increased the survival rate of melittin-treated mice, whereas the nonPEGylated MF-LNPs increased slightly. These results provide a fundamental strategy to improve the in vivo toxin neutralization ability of MF-LNPs.

Correction: Filtered beauty in Oslo and Tokyo: A spatial frequency analysis of facial attractiveness.

[This corrects the article DOI: 10.1371/journal.pone.0227513.].

Filtered beauty in Oslo and Tokyo: A spatial frequency analysis of facial attractiveness.

Images of European female and male faces were digitally processed to generate spatial frequency (SF) filtered images containing only a narrow band of visual information within the Fourier spectrum. The original unfiltered images and four SF filtered images (low, medium-low, medium-high and high) were then paired in trials that kept constant SF band and face gender and participants made a forced-choice decision about the more attractive among the two faces. In this way, we aimed at identifying those specific SF bands where forced-choice preferences corresponded best to forced-choice judgements made when viewing the natural, broadband, facial images. We found that aesthetic preferences dissociated across SFs and face gender, but similarly for participants from Asia (Japan) and Europe (Norway). Specifically, preferences when viewing SF filtered images were best related to the preference with the broadband face images when viewing the highest filtering band for the female faces (about 48-77 cycles per face). In contrast, for the male faces, the medium-low SF band (about 11-19 cpf) related best to choices made with the natural facial images. Eye tracking provided converging evidence for the above, gender-related, SF dissociations. We suggest greater aesthetic relevance of the mobile and communicative parts for the female face and, conversely, of the rigid, structural, parts for the male face for facial aesthetics.

Grinding and chemical mechanical polishing process for micropore x-ray optics fabricated with deep reactive ion etching.

Silicon micropore optics using deep reactive ion etching of silicon wafers has been being developed for future x-ray astronomy missions. Sidewalls of the micropores through a thin wafer with a typical thickness of hundreds of micrometers and a pore width of ∼20 µm are used for x-ray mirrors. However, burr structures observed after etching with a typical height of a few micrometers at the micropore edges are known to significantly reduce x-ray reflectivity. A new grinding and chemical mechanical polishing process is introduced to remove the burr structures. Both sides of the silicon wafer were ground and precisely polished after etching. X-ray reflectivity measurements confirmed an increase of reflectivity by 2-15 times at incident angles of 0.8-0.2 deg. The surface microroughness worsened from 2.0±0.2 nm rms to 7.8-0.8+0.6 nm rms; however, an additional annealing recovered the smooth surface and the estimated surface microroughness was <1.4 nm rms. This new process enables not only removing the burr structures but also choosing a flat part of the sidewalls for better angular resolution.

The "face race lightness illusion": An effect of the eyes and pupils?

In an internet-based, forced-choice, test of the 'face race lightness illusion', the majority of respondents, regardless of their ethnicity, reported perceiving the African face as darker in skin tone than the European face, despite the mean luminance, contrast and numbers of pixels of the images were identical. In the laboratory, using eye tracking, it was found that eye fixations were distributed differently on the African face and European face, so that gaze dwelled relatively longer onto the locally brighter regions of the African face and, in turn, mean pupil diameters were smaller than for the European face. There was no relationship between pupils' size and implicit social attitude (IAT) scores. In another experiment, the faces were presented either tachistoscopically (140 ms) or longer (2500 ms) so that, when gaze was prevented from looking directly at the faces in the former condition, the tendency to report the African face as "dark" disappeared, but it was present when gaze was free to move for just a few seconds. We conclude that the presence of the illusion depends on oculomotor behavior and we also propose a novel account based on a predictive strategy of sensory acquisition. Specifically, by differentially directing gaze towards to facial regions that are locally different in luminance, the resulting changes in retinal illuminance yield respectively darker or brighter percepts while attending to each face, hence minimizing the mismatch between visual input and the learned perceptual prototypes of ethnic categories.

Effects of social anxiety on metaphorical associations between emotional valence and clothing brightness.

BACKGROUND AND OBJECTIVES: Individuals with social anxiety have various types of deficiencies in emotional processing. Diversity of deficiencies may imply that socially anxious individuals have malfunctions in fundamental parts of emotional processing. Therefore, we hypothesized that social anxiety contributes to deficiencies in building on the metaphorical relationship between emotional experience and brightness. METHODS: We conducted a judgment task of valences of faces with manipulated clothing brightness (bright or dark). RESULTS: A congruency effect between the emotional valence and clothing brightness was observed in participants with low social anxiety. However, this pattern was not found in participants with high social anxiety. The results suggested that a deficiency in metaphorical associations leads to maladaptive emotional processing in individuals with social anxiety. LIMITATIONS: Our findings cannot be directly generalized to clinical populations. Such populations should be tested in the future studies. CONCLUSIONS: We may expand Lakoff and Johnson's (1999) conceptual metaphor theory by showing the relationships between social anxiety and malfunction in metaphorical processing. Malfunctions in metaphorical processing could lead to various types of psychological disorders which have deficiencies in emotional processing.

Transgenic Monkey Model of the Polyglutamine Diseases Recapitulating Progressive Neurological Symptoms.

Age-associated neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and the polyglutamine (polyQ) diseases, are becoming prevalent as a consequence of elongation of the human lifespan. Although various rodent models have been developed to study and overcome these diseases, they have limitations in their translational research utility owing to differences from humans in brain structure and function and in drug metabolism. Here, we generated a transgenic marmoset model of the polyQ diseases, showing progressive neurological symptoms including motor impairment. Seven transgenic marmosets were produced by lentiviral introduction of the human ataxin 3 gene with 120 CAG repeats encoding an expanded polyQ stretch. Although all offspring showed no neurological symptoms at birth, three marmosets with higher transgene expression developed neurological symptoms of varying degrees at 3-4 months after birth, followed by gradual decreases in body weight gain, spontaneous activity, and grip strength, indicating time-dependent disease progression. Pathological examinations revealed neurodegeneration and intranuclear polyQ protein inclusions accompanied by gliosis, which recapitulate the neuropathological features of polyQ disease patients. Consistent with neuronal loss in the cerebellum, brain MRI analyses in one living symptomatic marmoset detected enlargement of the fourth ventricle, which suggests cerebellar atrophy. Notably, successful germline transgene transmission was confirmed in the second-generation offspring derived from the symptomatic transgenic marmoset gamete. Because the accumulation of abnormal proteins is a shared pathomechanism among various neurodegenerative diseases, we suggest that this new marmoset model will contribute toward elucidating the pathomechanisms of and developing clinically applicable therapies for neurodegenerative diseases.

Tissue oxygen saturation levels from fetus to neonate.

AIM: Oxygen saturation during the term of delivery to the first cry, when fetal circulation dynamically changes, has not yet been examined. The aim of this study was therefore to determine whether the continuous measurement of regional tissue oxygen saturation (rSO2 ) from crowning until 5 min after delivery is possible using fetal tissue oximetry with a sensor attached to the examiner's finger. METHODS: Oxygen saturation levels in fetal cranial tissue between the second stage of delivery to crowning and up to 5 min after delivery were measured using fetal tissue oximetry with a sensor attached to the examiner's finger. Thirty-five deliveries were examined, and oxygen saturation was measured in seven infants from delivery of the head until 5 min after birth. Umbilical cord blood gas was measured in all cases. This clinical test was performed under the permission of the Ethics Committee of Hamamatsu University School of Medicine. RESULTS: Average tissue oxygen saturation in the second stage of delivery and at 5 min after delivery were 50.3 ± 16.3% and 56.8 ± 8.46%, respectively. In cases of continuous measurement, average rSO2 for crowning, immediately after delivery, and the first cry was 32.7 ± 9.5%, 30.0 ± 6.6%, and 31.6 ± 5.5%, respectively. CONCLUSION: We herein successfully measured oxygen saturation levels in fetal cranial tissue during crowning, delivery of the head, the first cry, and 5 min after delivery using fetal tissue oximetry with a sensor attached to the examiner's finger.

Myometrial relaxation of mice via expression of two pore domain acid sensitive K(+) (TASK-2) channels.

Myometrial relaxation of mouse via expression of two-pore domain acid sensitive (TASK) channels was studied. In our previous report, we suggested that two-pore domain acid-sensing K(+) channels (TASK-2) might be one of the candidates for the regulation of uterine circular smooth muscles in mice. In this study, we tried to show the mechanisms of relaxation via TASK-2 channels in marine myometrium. Isometric contraction measurements and patch clamp technique were used to verify TASK conductance in murine myometrium. Western blot and immunehistochemical study under confocal microscopy were used to investigate molecular identity of TASK channel. In this study, we showed that TEA and 4-AP insensitive non-inactivating outward K(+) current (NIOK) may be responsible for the quiescence of murine pregnant longitudinal myometrium. The characteristics of NIOK coincided with two-pore domain acid-sensing K(+) channels (TASK-2). NIOK in the presence of K(+) channel blockers was inhibited further by TASK inhibitors such as quinidine, bupivacaine, lidocaine, and extracellular acidosis. Furthermore, oxytocin and estrogen inhibited NIOK in pregnant myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed stronger inhibition of NIOK by quinidine and increased immunohistochemical expression of TASK-2. Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretch-activated channels in the longitudinal myometrium of mouse. Activation of TASK-2 channels seems to play an essential role for relaxing uterus during pregnancy and it might be one of the alternatives for preventing preterm delivery.

Examiner's finger-mounted fetal tissue oximetry: a preliminary report on 30 cases.

OBJECTIVE: To describe preliminary experience with a finger-mounted fetal tissue oximetry probe during the 2nd stage of labor. MATERIALS AND METHODS: A total of 30 term pregnant women without pregnancy complications were recruited. We measured fetal tissue oxygen saturation (FtO2) by using a finger-mounted fetal tissue oximetry during cervical examinations in the 2nd stage of labor. The data capturing rate of FtO2 and the interclass correlation coefficient were also examined. The mean FtO2 was compared to the neonatal condition assessed by the levels of umbilical cord blood. RESULTS: FtO2 was obtained in all cases, regardless of wetness, hair color, the part of the fetal head that was exposed, rotation of the fetus, color of amniotic fluid, and caput succedaneum. The mean FtO2 was 65.5%±8.58% in normal neonates [Apgar score >7 (1 min), n=25]. The mean FtO2 was significantly correlated with umbilical cord arterial pH (r=0.52, P=0.0030, n=30), but not with umbilical cord arterial partial pressure of oxygen. The interclass correlation coefficient was 0.94. CONCLUSIONS: Tissue oxygen saturation of the fetal head was obtained easily by the examiner's finger-mounted fetal tissue oximetry.

Effects of acupuncture stimulation on blood glucose concentration in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, an animal model for type-2 diabetes mellitus.

BACKGROUND: Effects of acupuncture stimulation on blood glucose concentration and body weight were investigated in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a model for type-2 diabetes. MATERIAL AND METHODS: Three groups of rats were used: OLETF, acupuncture-treated OLETF (AcOLETF), and Long-Evans Tokushima Otsuka (LETO) rats (as control for the OLETF rats). In AcOLETF rats, acupuncture stimulation was applied twice a week to 6 points (zhongwan, tianshu, qihai, ganshu, pishu, shenshu) and changes in blood glucose concentration and body weight were measured. RESULTS: Initially, at 6 weeks old, there was no significant difference in blood glucose levels between groups. Blood glucose levels increased with age in each group, reaching a maximum of about 430 mg/dl at 37 weeks in OLETF rats. In AcOLETF rats, blood glucose levels increased at a slower rate than in OLETF rats, reaching a maximum concentration of about 280 mg/dl at 37 weeks of age, significantly lower than that in OLETF rats. The concentration of blood glucose in LETO rats had stabilized at a maximum value of 120~140 mg/dl by 16 weeks, remaining at this level for up to 39 weeks. In each group, body weight increased with age and was not affected by acupuncture treatment. CONCLUSIONS: In OLETF rats, acupuncture treatment significantly reduced blood glucose levels, but not their body weight, suggesting that acupuncture therapy was effective in preventing the development of type-2 diabetes mellitus.

[A Japanese version of the FLANDERS handedness questionnaire].

Quantitative assessment of handedness is required in various clinical and research settings in psychology, neuroscience, and medicine. In the present study we tested the reliability and validity of a Japanese version of the FLANDERS handedness questionnaire, which was a new measure of skilled hand preference originally reported by Nicholls, Thomas, Loetscher, and Grimshaw (2013). Participants (N=431) completed three types of handedness questionnaires: the FLANDERS handedness questionnaire, Edinburgh Handedness Inventory, and H · N handedness test. Factor analysis revealed that the Japanese version of FLANDERS handedness questionnaire had a single-factor structure and high internal consistency. This questionnaire also posssed high test-retest reliability and criterion-referenced validity. These results indicate that the Japanese version of the FLANDERS handedness questionnaire is a valid and useful measure of skilled hand preference for Japanese participants.