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The ability to grow on media containing certain D-amino acids as a sole nitrogen source is widely utilized to differentiate Cryptococcus gattii from C. neoformans. We used the C. neoformans H99 and C. gattii R265 strains to dissect the mechanisms of D-amino acids utilization. We identified three putative D-amino acid oxidase (DAO) genes in both strains and showed that each DAO gene plays different roles in D-amino acid utilization in each strain. Deletion of DAO2 retarded growth of R265 on eleven D-amino acids suggesting its prominent role on D-amino acid assimilation in R265. All three R265 DAO genes contributed to growth on D-Asn and D-Asp. DAO3 was required for growth and detoxification of D-Glu by both R265 and H99. Although growth of H99 on most D-amino acids was poor, deletion of DAO1 or DAO3 further exacerbated it on four D-amino acids. Overexpression of DAO2 or DAO3 enabled H99 to grow robustly on several D-amino acids suggesting that expression levels of the native DAO genes in H99 were insufficient for growth on D-amino acids. Replacing the H99 DAO2 gene with a single copy of the R265 DAO2 gene also enabled its utilization of several D-amino acids. Results of gene and promoter swaps of the DAO2 genes suggested that enzymatic activity of Dao2 in H99 might be lower compared to the R265 strain. A reduction in virulence was only observed when all DAO genes were deleted in R265 but not in H99 indicating a pathobiologically exclusive role of the DAO genes in R265. These results suggest that C. neoformans and C. gattii divergently evolved in D-amino acid utilization influenced by their major ecological niches.

Cryptococcal meningoencephalitis is the most common fungal disease in the central nervous system. The mechanisms by which Cryptococcus neoformans invades the brain are largely unknown. In this study, we found that C. neoformans-derived microvesicles (CnMVs) can enhance the traversal of the blood-brain barrier (BBB) by C. neoformans invitro. The immunofluorescence imaging demonstrates that CnMVs can fuse with human brain microvascular endothelial cells (HBMECs), the constituents of the BBB. This activity is presumably due to the ability of the CnMVs to activate HBMEC membrane rafts and induce cell fusogenic activity. CnMVs also enhanced C. neoformans infection of the brain, found in both infected brains and cerebrospinal fluid. In infected mouse brains, CnMVs are distributed inside and around C. neoformans-induced cystic lesions. GFAP (glial fibrillary acidic protein)-positive astrocytes were found surrounding the cystic lesions, overlapping with the 14-3-3-GFP (14-3-3-green fluorescence protein fusion) signals. Substantial changes could be observed in areas that have a high density of CnMV staining. This is the first demonstration that C. neoformans-derived microvesicles can facilitate cryptococcal traversal across the BBB and accumulate at lesion sites of C. neoformans-infected brains. Results of this study suggested that CnMVs play an important role in the pathogenesis of cryptococcal meningoencephalitis.

Heteroresistance in Cryptococcus neoformans is an intrinsic adaptive resistance to azoles and the heteroresistant phenotype is associated with disomic chromosomes. Two chromosome 1 (Chr1) genes, ERG11, the fluconazole target, and AFR1, a drug transporter, were reported as major factors in the emergence of Chr1 disomy. In the present study, we show Chr4 to be the second most frequently formed disomy at high concentrations of fluconazole (FLC) and characterize the importance of resident genes contributing to disomy formation. We deleted nine Chr4 genes presumed to have functions in ergosterol biosynthesis, membrane composition/integrity or drug transportation that could influence Chr4 disomy under FLC stress. Of these nine, disruption of three genes homologous to Sey1 (a GTPase), Glo3 and Gcs2 (the ADP-ribosylation factor GTPase activating proteins) significantly reduced the frequency of Chr4 disomy in heteroresistant clones. Furthermore, FLC resistant clones derived from sey1Δglo3Δ did not show disomy of either Chr4 or Chr1 but instead had increased the copy number of the genes proximal to ERG11 locus on Chr1. Since the three genes are critical for the integrity of endoplasmic reticulum (ER) in Saccharomyces cerevisiae, we used Sec61ß-GFP fusion as a marker to study the ER in the mutants. The cytoplasmic ER was found to be elongated in sey1Δ but without any discernable alteration in gcs2Δ and glo3Δ under fluorescence microscopy. The aberrant ER morphology of all three mutant strains, however, was discernable by transmission electron microscopy. A 3D reconstruction using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) revealed considerably reduced reticulation in the ER of glo3Δ and gcs2Δ strains. In sey1Δ, ER reticulation was barely detectable and cisternae were expanded extensively compared to the wild type strains. These data suggest that the genes required for maintenance of ER integrity are important for the formation of disomic chromosomes in C. neoformans under azole stress.

MRI biomarkers of tumor edema, vascular permeability, blood volume, and average vessel caliber are increasingly being employed to assess the efficacy of tumor therapies. However, the dependence of these biomarkers on a number of physiological factors can compromise their sensitivity and complicate the assessment of therapeutic efficacy. Here we examine the response of these MRI tumor biomarkers to cediranib, a potent vascular endothelial growth factor receptor (VEGFR) inhibitor, in an orthotopic mouse glioma model. A significant increase in the tumor volume and relative vessel caliber index (rVCI) and a slight decrease in the water apparent diffusion coefficient (ADC) were observed for both control and cediranib treated animals. This contrasts with a clinical study that observed a significant decrease in tumor rVCI, ADC and volume with cediranib therapy. While the lack of a difference between control and cediranib treated animals in these biomarker responses might suggest that cediranib has no therapeutic benefit, cediranib treated mice had a significantly increased survival. The increased survival benefit of cediranib treated animals is consistent with the significant decrease observed for cediranib treated animals in the relative cerebral blood volume (rCBV), relative microvascular blood volume (rMBV), transverse relaxation time (T2), blood vessel permeability (K(trans)), and extravascular-extracellular space (ν(e)). The differential response of pre-clinical and clinical tumors to cediranib therapy, along with the lack of a positive response for some biomarkers, indicates the importance of evaluating the whole spectrum of different tumor biomarkers to properly assess the therapeutic response and identify and interpret the therapy-induced changes in the tumor physiology.

A recent large outbreak of fungal infections by Exserohilum rostratum from contaminated compounding solutions has highlighted the need to rapidly screen available pharmaceuticals that could be useful in therapy. The present study utilized two newly-developed high throughput assays to screen approved drugs and pharmaceutically active compounds for identification of potential antifungal agents. Several known drugs were found that have potent effects against E. rostratum including the triazole antifungal posaconazole. Posaconazole is likely to be effective against infections involving septic joints and may provide an alternative for refractory central nervous system infections. The anti-E. rostratum activities of several other drugs including bithionol (an anti-parasitic drug), tacrolimus (an immunosuppressive agent) and floxuridine (an antimetabolite) were also identified from the drug repurposing screens. In addition, activities of other potential antifungal agents against E. rostratum were excluded, which may avoid unnecessary therapeutic trials and reveals the limited therapeutic alternatives for this outbreak. In summary, this study has demonstrated that drug repurposing screens can be quickly conducted within a useful time-frame. This would allow clinical implementation of identified alternative therapeutics and should be considered as part of the initial public health response to new outbreaks or rapidly-emerging microbial pathogens.

Two members of the Cryptococcus neoformans-gattii species complex, the etiologic agents of cryptococcosis, can be differentiated by biological, biochemical, serological and molecular typing techniques. Based on their differences in carbon and nitrogen utilization patterns, cost effective and very specific diagnostic tests using D-proline and canvanine-glycine-bromthymol blue (CGB) media have been formulated and are widely used for identification of the two species. However, these methods have yet to be tested for strains with confirmed molecular types to assess the degree of specificity for each molecular type in the two species. We collected global isolates of every major molecular type available and tested their patterns of nitrogen utilization. We confirmed specificity of the CGB test to be 100% regardless of molecular type while the D-proline test yielded 8-38% false negative results in three of the four C. gattii molecular types, VGI-VGIII. The utilization pattern of a new set of amino acids: D-alanine, L-tryptophan and L-phenylalanine, showed species specificity comparable to that of D-proline. We discovered that the transcription factor Gat1 (Are1) regulates the utilization of nitrogen differently between C. neoformans and C. gattii strains. Unlike in C. neoformans, expression of the genes encoding glycine decarboxylase complex in C. gatti was only partially suppressed by nitrogen catabolite repression in the presence of ammonium. GAT1 in C. neoformans controlled the induction of three of the four genes encoding the glycine decarboxylase complex when glycine was used as the sole nitrogen source while in C. gattii its regulation of these genes was less stringent. Moreover, while virulence of C. neoformans strains in mice was not affected by Gat1, the transcription factor positively influenced the virulence of C. gattii strain.

BackgroundAspergillus fumigatus is the most common etiologic agent of invasive aspergillosis in immunocompromised patients. Several studies have addressed the mechanism involved in host defense but only few have investigated the pathogen's response to attack by the host cells. To our knowledge, this is the first study that investigates the genes differentially expressed in conidia vs hyphae of A. fumigatus in response to neutrophils from healthy donors as well as from those with chronic granulomatous disease (CGD) which are defective in the production of reactive oxygen species.Methodology/principal findingsTranscriptional profiles of conidia and hyphae exposed to neutrophils, either from normal donors or from CGD patients, were obtained by using the genome-wide microarray. Upon exposure to either normal or CGD neutrophils, 244 genes were up-regulated in conidia but not in hyphae. Several of these genes are involved in the degradation of fatty acids, peroxisome function and the glyoxylate cycle which suggests that conidia exposed to neutrophils reprogram their metabolism to adjust to the host environment. In addition, the mRNA levels of four genes encoding proteins putatively involved in iron/copper assimilation were found to be higher in conidia and hyphae exposed to normal neutrophils compared to those exposed to CGD neutrophils. Deletants in several of the differentially expressed genes showed phenotypes related to the proposed functions, i.e. deletants of genes involved in fatty acid catabolism showed defective growth on fatty acids and the deletants of iron/copper assimilation showed higher sensitivity to the oxidative agent menadione. None of these deletants, however, showed reduced resistance to neutrophil attack.ConclusionThis work reveals the complex response of the fungus to leukocytes, one of the major host factors involved in antifungal defense, and identifies fungal genes that may be involved in establishing or prolonging infections in humans.

Interest has focused on quantifying the size and scope of environmental markets, particularly those that offset ecosystem impacts or restore natural infrastructure to improve habitat or promote clean air and water. In this paper, we focus on the US wetland and stream compensatory mitigation market, asking: what types of firms make up the mitigation "industry"? What are the economic impacts-i.e., the "size"-of the mitigation industry? How has this industry changed over time? We present the results of a national survey of mitigation firms and construct an input-output model of the industry's economic impacts and employment. We also develop a comparative, 2014 model of the industry using data from a previous study of the broader, ecological restoration economy. Our findings suggest that the (2019, pre-COVID) mitigation industry collects annual revenues (direct economic impacts) in excess of $3.5 billion, which, along with additional indirect (supply chain) and induced (spillover) economic impacts, combine to over $9.6 billion in total output and support over 53,000 total jobs. We estimate 2014-2019 growth of ~35.2 percent in revenues, ~32.6 percent in total economic impacts, and a compound annual growth rate (CAGR) of 5.25%. This places the mitigation industry within the range of other, well-established industries within the technical services sector. We suggest establishing North American Industry Classification System (NAICS) codes specifically for ecological restoration and mitigation firms, an essential step in generating accurate and consistent employment estimates in the future, particularly at sub-national geographic scales., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 BenDor et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Interpersonal communication includes verbal and nonverbal communication. Verbal communication comprises one-way (e.g., a speech or lecture) and interactive verbal communication (e.g., daily conversations or meetings), which we frequently encounter. Nonverbal communication has considerable influence on interpersonal communication, and body motion synchrony is known to be an important factor for successful communication and social interaction. However, most research on body motion synchrony has been elucidated by either the setting of one-way verbal transmission or the verbal interaction setting, and it remains unclear whether verbal directionality and interactivity affect body motion synchrony. One-way and two-way (interactive) verbal communication is implicated in designed or undesigned leader-follower relationships, and also in the complexity and diversity of interpersonal interactions, where two-way verbal communication is more complex and diverse than in the one-way condition. In this study, we tested head motion synchrony between the one-way verbal communication condition (in which the roles of the speaker and listener are fixed) and the two-way verbal communication condition (where the speaker and listener can freely engage in a conversation). Therefore, although no statistically significant difference in synchrony activity (relative frequency) was found, a statistically significant difference was observed in synchrony direction (temporal lead-lag structure as mimicry) and intensity. Specifically, the synchrony direction in two-way verbal communication was close to zero, but this in one-way verbal communication was synchronized with the listener's movement predominantly delayed. Furthermore, synchrony intensity, in terms of the degree of variation in the phase difference distribution, was significantly higher in the one-way verbal communication than in the two-way condition, with bigger time-shifts being observed in the latter. This result suggests that verbal interaction does not affect the overall frequency of head motion synchrony but does affect the temporal lead-lag structure and coherence., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kwon, Kotani. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Congenital heart defects are the most common type of birth defects in humans and frequently involve heart valve dysfunction. The current treatment for unrepairable heart valves involves valve replacement with an implant, Ross pulmonary autotransplantation, or conventional orthotopic heart transplantation. Although these treatments are appropriate for older children and adults, they do not result in the same efficacy and durability in infants and young children for several reasons. Heart valve implants do not grow with the. Ross pulmonary autotransplants have a high mortality rate in neonates and are not feasible if the pulmonary valve is dysfunctional or absent. Furthermore, orthotopic heart transplants invariably fail from ventricular dysfunction over time. Therefore, the treatment of irreparable heart valves in infants and young children remains an unsolved problem. The objective of this single-arm, prospective study is to offer an alternative solution based on a new type of transplant, which we call "partial heart transplantation." Partial heart transplantation differs from conventional orthotopic heart transplantation because only the part of the heart containing the heart valve is transplanted. Similar to Ross pulmonary autotransplants and conventional orthotopic heart transplants, partial heart transplants contain live cells that should allow it to grow with the recipient child. Therefore, partial heart transplants will require immunosuppression. The risks from immunosuppression can be managed, as seen in conventional orthotopic heart transplant recipients. Stopping immunosuppression will simply turn the growing partial heart transplant into a non-growing homovital homograft. Once this homograft deteriorates, it can be replaced with a durable adult-sized mechanical implant. The protocol for our single-arm trial is described. The ClinicalTrials.gov trial registration number is NCT05372757., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Rajab et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Among the various types of data augmentation strategies, the mixup-based approach has been particularly studied. However, in existing mixup-based approaches, object loss and label mismatching can occur if random patches are utilized when constructing augmented images, and additionally, patches that do not contain objects might be included, which degrades performance. In this paper, we propose a novel augmentation method that mixes patches in a non-overlapping manner after they are extracted from the salient regions in an image. The suggested method can make effective use of object characteristics, because the constructed image consists only of visually important regions and is robust to noise. Since the patches do not occlude each other, the semantically meaningful information in the salient regions can be fully utilized. Additionally, our method is more robust to adversarial attack than the conventional augmentation method. In the experimental results, when Wide ResNet was trained on the public datasets, CIFAR-10, CIFAR-100 and STL-10, the top-1 accuracy was 97.26%, 83.99% and 82.40% respectively, which surpasses other augmentation methods., Competing Interests: The authors have declared that no competing interests exist.

To understand, predict, and help correct each other's actions we need to maintain accurate, up-to-date knowledge of people, and communication is a critical means by which we gather and disseminate this information. Yet the conditions under which we communication social information remain unclear. Testing hypotheses generated from our theoretical framework, we examined when and why social information is disseminated about an absent third party: i.e., gossiped. Gossip scenarios presented to participants (e.g., "Person-X cheated on their exam") were based on three key factors: (1) target (ingroup, outgroup, or celebrity), (2) valence (positive or negative), and (3) content. We then asked them (a) whether they would spread the information, and (b) to rate it according to subjective valence, ordinariness, interest level, and emotion. For ratings, the scenarios participants chose to gossip were considered to have higher valence (whether positive or negative), to be rarer, more interesting, and more emotionally evocative; thus showing that the paradigm was meaningful to subjects. Indeed, for target, valence, and content, a repeated-measures ANOVA found significant effects for each factor independently, as well as their interactions. The results supported our hypotheses: e.g., for target, more gossiping about celebrities and ingroup members (over strangers); for valence, more about negative events overall, and yet for ingroup members, more positive gossiping; for content, more about moral topics, with yet all domains of social content communicated depending on the situation-context matters, influencing needs. The findings suggest that social knowledge sharing (i.e., gossip) involves sophisticated calculations that require our highest sociocognitive abilities, and provide specific hypotheses for future examination of neural mechanisms., Competing Interests: The authors have declared that no competing interests exist.

Background: The available data are not sufficient to understand the clinical impact of statin intensity in elderly patients who undergo percutaneous coronary intervention (PCI) due to acute myocardial infarction (AMI)., Methods: Using the COREA-AMI registry, we sought to compare the clinical impact of high- versus low-to-moderate-intensity statin in younger (<75 years old) and elderly (≥75 years old) patients. Of 10,719 patients, we included 8,096 patients treated with drug-eluting stents. All patients were classified into high-intensity versus low-to-moderate-intensity statin group according to statin type and dose at discharge. The primary end point was target-vessel failure (TVF), a composite of cardiovascular death, target-vessel MI, or target-lesion revascularization (TLR) from 1 month to 12 months after index PCI., Results: In younger patients, high-intensity statin showed the better clinical outcomes than low-to-moderate-intensity statin (TVF: 79 [5.4%] vs. 329 [6.8%], adjusted hazard ratio [aHR] 0.76; 95% confidence interval [CI] 0.59-0.99; P = 0.038). However, in elderly patients, the incidence rates of the adverse clinical outcomes were similar between two statin-intensity groups (TVF: 38 [11.4%] vs. 131 [10.6%], aHR 1.1; 95% CI 0.76-1.59; P = 0.63)., Conclusions: In this AMI cohort underwent PCI, high-intensity statin showed the better 1-year clinical outcomes than low-to-moderate-intensity statin in younger patients. Meanwhile, the incidence rates of adverse clinical events between high- and low-to-moderate-intensity statin were not statistically different in elderly patients. Further randomized study with large elderly population is warranted., Competing Interests: The authors have declared that no competing interests exist.

Hydraulic fracturing of unconventional reservoirs has seen a boom in the last century, as a means to fulfill the growing energy demand in the world. The fracturing fluid used in the process plays a substantial role in determining the results. Hence, several research and development efforts have been geared towards developing more sustainable, efficient, and improved fracturing fluids. Herein, we present a dynamic binary complex (DBC) solution, with potential to be useful in the hydraulic fracturing domain. It has a supramolecular structure formed by the self-assembly of low molecular weight viscosifiers (LMWVs) oleic acid and diethylenetriamine into an elongated entangled network under alkaline conditions. With less than 2 wt% constituents dispersed in aqueous solution, a viscous gel that exhibits high viscosities even under shear was formed. Key features include responsiveness to pH and salinity, and a zero-shear viscosity that could be tuned by a factor of ~280 by changing the pH. Furthermore, its viscous properties were more pronounced in the presence of salt. Sand settling tests revealed its potential to hold up sand particles for extended periods of time. In conclusion, this DBC solution system has potential to be utilized as a smart salt-responsive, pH-switchable hydraulic fracturing fluid., Competing Interests: The authors have declared that no competing interests exist.

Soft robots have been extensively researched due to their flexible, deformable, and adaptive characteristics. However, compared to rigid robots, soft robots have issues in modeling, calibration, and control in that the innate characteristics of the soft materials can cause complex behaviors due to non-linearity and hysteresis. To overcome these limitations, recent studies have applied various approaches based on machine learning. This paper presents existing machine learning techniques in the soft robotic fields and categorizes the implementation of machine learning approaches in different soft robotic applications, which include soft sensors, soft actuators, and applications such as soft wearable robots. An analysis of the trends of different machine learning approaches with respect to different types of soft robot applications is presented; in addition to the current limitations in the research field, followed by a summary of the existing machine learning methods for soft robots., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Despite global recommendations for governments to implement a comprehensive suite of policies to address obesity, policy adoption has been deficient globally. This paper utilised political science theory and systems thinking methods to examine the dynamics underlying decisions regarding obesity prevention policy adoption within the context of the Australian state government initiative, Healthy Together Victoria (HTV) (2011-2016). The aim was to understand key influences on policy processes, and to identify potential opportunities to increase the adoption of recommended policies., Methods: Data describing government processes in relation to the adoption of six policy interventions considered as part of HTV were collected using interviews (n = 57), document analyses (n = 568) and field note observations. The data were analysed using multiple political science theories. A systematic method was then used to develop a Causal Loop Diagram (CLD) for each policy intervention. A simplified meta-CLD was generated from synthesis of common elements across each of the six policy interventions., Results: The dynamics of policy change could be explained using a series of feedback loops. Five interconnected balancing loops served to reduce the propensity for policy change. These pertained to an organisational norm of risk aversion, and the complexity resulting from a whole-of-government policy approach and in-depth stakeholder consultation. However, seven virtuous reinforcing loops helped overcome policy resistance through policy actor capabilities that were improved over time as policy actors gained experience in advocating for change., Conclusion: Policy processes for obesity prevention are complex and resistant to change. In order to increase adoption of recommended policies, several capabilities of policy actors, including policy skills, political astuteness, negotiation skills and consensus building, should be fostered and strengthened. Strategies to facilitate effective and broad-based consultation, both across and external to government, need to be implemented in ways that do not result in substantial delays in the policy process., Competing Interests: The authors have declared that no competing interests exist.

Exosomes are a group of small membranous vesicles that are shed into the extracellular environment by tumoral or non-tumoral cells and contribute to cellular communication by delivering micro RNAs (miRNAs). In this study, we aimed to evaluate the role of exosomal miRNAs from colorectal cancer cell lines in tumorigenesis, by affecting cancer-associated fibroblasts (CAFs), which are vital constituents of the tumor microenvironment. To analyze the effect of exosomal miRNA on the tumor microenvironment, migration of the monocytic cell line THP-1 was evaluated via Transwell migration assay using CAFs isolated from colon cancer patients. The migration assay was performed with CAFs ± CCL7-blocking antibody and CAFs that were treated with exosomes isolated from colon cancer cell lines. To identify the associated exosomal miRNAs, miRNA sequencing and quantitative reverse transcription polymerase chain reaction were performed. The migration assay revealed that THP-1 migration was decreased in CCL7-blocking antibody-expressing and exosome-treated CAFs. Colon cancer cell lines contained miRNA let-7d in secreted exosomes targeting the chemokine CCL7. Exosomes from colorectal cancer cell lines affected CCL7 secretion from CAFs, possibly via the miRNA let-7d, and interfered with the migration of CCR2+ monocytic THP-1 cells in vitro., Competing Interests: The authors have declared that no competing interests exist.

Levels of protein translation by ribosomes are governed both by features of the translation machinery as well as sequence properties of the mRNAs themselves. We focus here on a striking three-nucleotide periodicity, characterized by overrepresentation of GCN codons and underrepresentation of G at the second position of codons, that is observed in Open Reading Frames (ORFs) of mRNAs. Our examination of mRNA sequences in Saccharomyces cerevisiae revealed that this periodicity is particularly pronounced in the initial codons-the ramp region-of ORFs of genes with high protein expression. It is also found in mRNA sequences immediately following non-standard AUG start sites, located upstream or downstream of the standard annotated start sites of genes. To explore the possible influences of the ramp GCN periodicity on translation efficiency, we tested edited ramps with accentuated or depressed periodicity in two test genes, SKN7 and HMT1. Greater conformance to (GCN)n was found to significantly depress translation, whereas disrupting conformance had neutral or positive effects on translation. Our recent Molecular Dynamics analysis of a subsystem of translocating ribosomes in yeast revealed an interaction surface that H-bonds to the +1 codon that is about to enter the ribosome decoding center A site. The surface, comprised of 16S/18S rRNA C1054 and A1196 (E. coli numbering) and R146 of ribosomal protein Rps3, preferentially interacts with GCN codons, and we hypothesize that modulation of this mRNA-ribosome interaction may underlie GCN-mediated regulation of protein translation. Integration of our expression studies with large-scale reporter studies of ramp sequence variants suggests a model in which the C1054-A1196-R146 (CAR) interaction surface can act as both an accelerator and braking system for ribosome translation., Competing Interests: The authors have declared that no competing interests exist.

We have previously demonstrated that endothelial targeting of gold nanoparticles followed by external beam irradiation can cause specific tumor vascular disruption in mouse models of cancer. The induced vascular damage may lead to changes in tumor physiology, including tumor hypoxia, thereby compromising future therapeutic interventions. In this study, we investigate the dynamic changes in tumor hypoxia mediated by targeted gold nanoparticles and clinical radiation therapy (RT). By using noninvasive whole-body fluorescence imaging, tumor hypoxia was measured at baseline, on day 2 and day 13, post-tumor vascular disruption. A 2.5-fold increase (P<0.05) in tumor hypoxia was measured two days after combined therapy, resolving by day 13. In addition, the combination of vascular-targeted gold nanoparticles and radiation therapy resulted in a significant (P<0.05) suppression of tumor growth. This is the first study to demonstrate the tumor hypoxic physiological response and recovery after delivery of vascular-targeted gold nanoparticles followed by clinical radiation therapy in a human non-small cell lung cancer athymic Foxn1nu mouse model., Competing Interests: The authors have declared that no competing interests exist.

It has been demonstrated that the performance of typical unimodal brain-computer interfaces (BCIs) can be noticeably improved by combining two different BCI modalities. This so-called "hybrid BCI" technology has been studied for decades; however, hybrid BCIs that particularly combine electroencephalography (EEG) and functional near-infrared spectroscopy (fNIRS) (hereafter referred to as hBCIs) have not been widely used in practical settings. One of the main reasons why hBCI systems are so unpopular is that their hardware is generally too bulky and complex. Therefore, to make hBCIs more appealing, it is necessary to implement a lightweight and compact hBCI system with minimal performance degradation. In this study, we investigated the feasibility of implementing a compact hBCI system with significantly less EEG channels and fNIRS source-detector (SD) pairs, but that can achieve a classification accuracy high enough to be used in practical BCI applications. EEG and fNIRS data were acquired while participants performed three different mental tasks consisting of mental arithmetic, right-hand motor imagery, and an idle state. Our analysis results showed that the three mental states could be classified with a fairly high classification accuracy of 77.6 ± 12.1% using an hBCI system with only two EEG channels and two fNIRS SD pairs., Competing Interests: The authors have declared that no competing interests exist.

Although the beneficial effects of probiotics in the prevention or treatment of metabolic disorders have been extensively researched, the precise mechanisms by which probiotics improve metabolic homeostasis are still not clear. Given that probiotics usually exert a comprehensive effect on multiple metabolic disorders, defining a concurrent mechanism underlying the multiple effects is critical to understand the function of probiotics. In this study, we identified the SIRT1-dependent or independent PGC-1α pathways in multiple organs that mediate the protective effects of a strain of Lactobacillus plantarum against high-fat diet-induced adiposity, glucose intolerance, and dyslipidemia. L. plantarum treatment significantly enhanced the expression of SIRT1, PPARα, and PGC-1α in the liver and adipose tissues under HFD-fed condition. L. plantarum treated mice also exhibited significantly increased expressions of genes involved in bile acid synthesis and reverse cholesterol transport in the liver, browning and thermogenesis of adipose tissue, and fatty acid oxidation in the liver and adipose tissue. Additionally, L. plantarum treatment significantly upregulated the expressions of adiponectin in adipose tissue, irisin in skeletal muscle and subcutaneous adipose tissue (SAT), and FGF21 in SAT. These beneficial changes were associated with a significantly improved HFD-induced alteration of gut microbiota. Our findings suggest that the PGC-1α-mediated pathway could be regarded as a potential target in the development of probiotics-based therapies for the prevention and treatment of metabolic disorders., Competing Interests: Hyunchae Joung, Byoung-Kook Kim, and In Suk Choi, who are employees of Chong Kun Dang Bio Research Institute, declare conflict of interest as they have collaborated in gut microbiota analysis and this study was funded by Chong Kun Dang Bio Research Institute. The other authors declare no conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Our decisions have a temporally distributed order, and different choice orders (e.g., choosing preferred items first or last) can lead to vastly different experiences. We previously found two dominant strategies (favorite-first and favorite-last) in a preference-based serial choice setting (the 'sushi problem'). However, it remains unclear why these two opposite behavioral patterns arise: i.e., the mechanisms underlying them. Here we developed a novel serial-choice task, using pictures based on attractiveness, to test for a possible shared mechanism with delay discounting, the 'peak-end' bias (i.e., preference for experienced sequences that end well), or working-memory capacity. We also collected psychological and clinical metric data on personality, depression, anxiety, and emotion regulation. We again found the two dominant selection strategies. However, the results of the delay, peak-end bias, and memory capacity tasks were not related to serial choice, while two key psychological metrics were: emotion regulation and conscientiousness (with agreeableness also marginally related). Favorite-first strategists actually regulated emotions better, suggesting better tolerance of negative outcomes. Whereas participants with more varied strategies across trials were more conscientious (and perhaps agreeable), suggesting that they were less willing to settle for a single, simpler strategy. Our findings clarify mechanisms underlying serial choice and show that it may reflect a unique ability to organize choices into sequences of events., Competing Interests: The authors have declared that no competing interests exist.

Purpose: We have previously reported that lamellar dissection of the cornea transects stromal nerves, and that regenerating neurites form a dense net along the surgical plane. In these experiments, we have disrupted the stromal nerve trunks in situ, without incising the cornea, to determine the regeneration events in the absence of a surgical plane., Methods: Thy1-YFP mice were anesthetized and in vivo images of the corneal nerves were obtained with a wide-field stereofluorescent microscope. A far infrared XYRCOS Laser attached to 20X objective of an upright microscope was used to perform in situ transection of the stromal nerves. 3 types of laser transections were performed (n = 5/group): (i) point transection (a single cut); (ii) segmental transection (two cuts enclosing a segment of nerve trunk); and (iii) annular transection (cuts on all nerve trunks crossing the perimeter of a 0.8 mm diameter circular area centered on the corneal apex). Mice were imaged sequentially for 4 weeks thereafter to assess nerve degeneration (disappearance or weakening of original fluorescence intensity) or regeneration (appearance of new fluorescent fronds). Beta-3-tubulin immunostaining was performed on corneal whole-mounts to demonstrate nerve disruption., Results: The pattern of stromal nerves in corneas of the same mouse and in corneas of littermates was dissimilar. Two distinct patterns were observed, often within the same cornea: (i) interconnected trunks that spanned limbus to limbus; or (ii) dichotomously branching trunks that terminate at the corneal apex. Point transections did not cause degeneration of proximal or distal segment in interconnected trunks, but resulted in degeneration of distal segment of branching trunks. In segmental transections, the nerve segment enclosed within the two laser cuts degenerated. Lack of beta-3 tubulin staining at transection site confirmed nerve transection. In interconnected trunks, at 4 weeks, a hyperfluorescent plaque filled the gap created by the transection. In annular transections, some nerve trunks degenerated, while others regained or retained fluorescence., Conclusions: Interconnected stromal nerves in murine corneas do not degenerate after in situ point transection and show evidence of healing at the site of disruption. Presence or absence of a surgical plane influences corneal nerve regeneration after transection., Competing Interests: The authors have declared that no competing interests exist.

Ulmus macrocarpa Hance as an oriental medicinal plant has shown enormous potential for the treatment of several metabolic disorders in Korea. Hyperlipidemia, which is characterized by the excess accumulation of lipid contents in the bloodstream, may lead to several cardiovascular diseases. Therefore, in this study, anti-hyperlipidemic potential of U. macrocarpa water extract (UME) was examined in vitro and in vivo using HepG2 cells and experimental rats, respectively. The hyperlipidemia in experimental rats was induced by the high-cholesterol diet (HCD) followed by oral administration of various concentrations (25, 50 and 100 mg/kg) of UME for 6 weeks. As a result, the UME significantly improved the biochemical parameters such as increased the level of triglyceride, total cholesterol, and low-density lipoprotein cholesterol as well as reduced the high-density lipoprotein cholesterol in the HCD-fed rats. In addition, UME also prevented lipid accumulation through regulating AMPK activity and lipid metabolism proteins (ACC, SREBP1 and HMGCR) in the HCD-fed rats as compared to the controls. Moreover, similar pattern of gene expression levels was confirmed in oleic acid (OA)-treated HepG2 cells. Taken together, our results indicate that UME prevents hyperlipidemia via activating the AMPK pathway and regulates lipid metabolism. Thus, based on the above findings, it is estimated that UME could be a potential therapeutic agent for preventing the hyperlipidemia., Competing Interests: The authors have read the journal's policy and have the following conflicts: authors THK and JJL are affiliated with Naturetech Co. Ltd.. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products to declare.

Shark meat is consumed as a food source worldwide, especially in Asian countries. However, since sharks are apex predators in the ocean food chain, they are prone to bioaccumulation of heavy metals. More than 100 million sharks are caught annually for human consumption, and the safety of shark meat cannot be overemphasized. Here, we examined heavy metal concentration in the muscle tissue of 6 shark species including 3 migratory species (Carcharhinus brachyurus, Carcharhinus obscurus, and Isurus oxyrinchus) and 3 local species (Triakis scyllium, Mustelus manazo, and Cephaloscyllium umbratile) from fish markets in Jeju Island, Republic of Korea. The concentrations of 11 heavy metals (Cr, Fe, Cu, Zn, As, Se, Cd, Sn, Sb, Pb, and Hg) and MeHg were analyzed. The result showed that the average concentrations of all metals, except for that of As, were below the regulatory maximum limits of many organizations, including the Codex standard. Hg and MeHg were significantly correlated with body length, body weight, and age, and the concentration of Hg was expected to exceed the limit in C. brachyurus with a body length or weight of over 130 cm or 25 kg, respectively. Our results indicate that shark meat can expose consumers to a high level of As and that copper sharks bigger than the predicted size should be avoided for excessive Hg. Considering these findings, a detailed guideline on consumption of meat of different shark species should be suggested based on further investigation., Competing Interests: The authors have declared that no competing interests exist.

Objective: This study aimed to evaluate the risk for hepatotoxicity with nimesulide, a non-steroidal anti-inflammatory drug (NSAID) available in Republic of Korea but withdrawn from the market in several countries., Methods: A systematic review and meta-analysis were conducted of studies retrieved from PubMed, EMBASE, Cochrane, the Research Information Sharing Service and ClinicalTrials.gov up to September 2017. All studies reporting nimesulide-associated hepatotoxicity in patients as compared with the unexposed or the exposed to other NSAIDs were included. Studies using spontaneous reporting databases were included to estimate reporting odds ratio (ROR) of hepatotoxicity associated with nimesulide exposure. The association between nimesulide use and hepatotoxicity was estimated using relative risk (RR) and ROR with 95% confidence interval (CI)., Results: A total of 25 observational studies were eligible for review. In a meta-analysis of five observational studies, nimesulide was significantly associated with hepatotoxicity [RR 2.21, 95% CI 1.72-2.83]. From studies using spontaneous reporting databases (n = 6), rates of reported hepatotoxicity were significantly higher in patients using nimesulide, compared with those treated with other NSAIDs [pooled ROR 3.99, 95% CI 2.86-5.57]. Of a total of 33 patients from case studies and series, the majority (n = 28, 84.8%) were female, and the mean age (± standard deviation) was 56.8 (± 15.6) years. Almost half of the patients on nimesulide (45.5%) either required liver transplantation or died due to fulminant hepatic failure, of whom a third developed hepatotoxicity within less than 15 days of nimesulide administration., Conclusions: Our study findings support previous reports of an increased risk for hepatotoxicity with nimesulide use and add to existing literature by providing risk estimates for nimesulide-associated hepatotoxicity. As the limited number of studies with primarily observational study designs were included in the analysis, more studies are needed to further describe the effects of dose and length of treatment on the risk for hepatotoxicity., Competing Interests: The authors have declared that no competing interests exist.

Recently, modulation of gut microbiota by probiotics treatment has been emerged as a promising strategy for treatment of metabolic disorders. Apart from lactic acid bacteria, Bacillus species (Bacillus spp.) have also been paid attention as potential probiotics, but nevertheless, the molecular mechanisms for their protective effect against metabolic dysfunction remain to be elucidated. In this study, we demonstrate that a probiotic mixture composed of 5 different Bacillus spp. protects mice from high-fat diet (HFD)-induced obesity, insulin resistance and non-alcoholic fatty liver disease (NAFLD). Probiotic Bacillus treatment substantially attenuated body weight gain and enhanced glucose tolerance by sensitizing insulin action in skeletal muscle and epididymal adipose tissue (EAT) of HFD-fed mice. Bacillus-treated HFD-fed mice also exhibited significantly suppressed chronic inflammation in the liver, EAT and skeletal muscle, which was observed to be associated with reduced HFD-induced intestinal permeability and enhanced adiponectin production. Additionally, Bacillus treatment significantly reversed HFD-induced hepatic steatosis. In Bacillus-treated mice, hepatic expression of lipid oxidative genes was significantly increased, and lipid accumulation in subcutaneous and mesenteric adipose tissues were significantly decreased, commensurate with down-regulated expression of genes involved in lipid uptake and lipogenesis. Although, in Bacillus-treated mice, significant alterations in gut microbiota composition was not observed, the enhanced expression of tight junction-associated proteins showed a possibility of improving gut barrier function by Bacillus treatment. Our findings provide possible explanations how Bacillus probiotics protect diet-induced obese mice against metabolic disorders, identifying the treatment of probiotic Bacillus as a potential therapeutic approach., Competing Interests: Yosep Ji and Wilhelm Holzapfel, who are board members of Holzapfel Effective Microbes (HEM), declare conflict of interest as they have collaborated in gut microbiota analysis and this study was partially funded by HEM. The other authors declare no conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Direct laryngoscopy using a Macintosh laryngoscope is the most widely used approach; however, this skill is not easy for novices and trainees. We evaluated the performance of novices using a laryngoscope with a three-dimensional (3D)-printed ergonomic grip on an airway manikin. Forty second-year medical students were enrolled. Endotracheal intubation was attempted using a conventional Macintosh laryngoscope with or without a 3D-printed ergonomic support grip. Primary outcomes were intubation time and overall success rate. Secondary outcomes were number of unsuccessful attempts, first-attempt success rate, airway Cormack-Lehane (CL) grade, and difficulty score. In the easy airway scenario, intubation time, and the overall success rate were similar between two group. CL grade and ease-of-use scores were significantly better for those using the ergonomic support grip (P < 0.05). In the difficult airway scenario, intubation time (49.7±37.5 vs. 35.5±29.2, P = 0.013), the first-attempt success rate (67.5% vs. 90%, P = 0.029), number of attempts (1.4±0.6 vs. 1.1±0.4, P = 0.006), CL grade (2 [2, 2] vs. 2 [1, 1], P = 0.012), and ease-of-use scores (3.5 [2, 4] vs. 4 [3, 5], P = 0.008) were significantly better for those using the ergonomic support grip. Linear mixed model analysis showed that the ergonomic support grip had a favorable effect on CL grade (P<0.001), ease-of-use scores (P<0.001), intubation time (P = 0.015), and number of intubation attempts (P = 0.029). Our custom 3D-printed ergonomic laryngoscope support grip improved several indicators related to the successful endotracheal intubation in the easy and difficult scenario simulated on an airway manikin. This grip may be useful for intubation training and practice., Competing Interests: The authors have declared that no competing interests exist.

We investigate the influence of the visual appearance of a negotiator on persuasiveness within the context of negotiations. Psychological experiments were conducted to quantitatively analyze the relationship between visual appearance and the use of language. Male and female participants were shown three female and male photographs, respectively. They were asked to report how they felt about each photograph using a seven-point semantic differential (SD) scale for six affective factors (positive impression, extraversion, intelligence, conscientiousness, emotional stability, and agreeableness). Participants then answered how they felt about each negotiation scenario (they were presented with pictures and a situation combined with negotiation sentences) using a seven-point SD scale for seven affective factors (positive impression, extraversion, intelligence, conscientiousness, emotional stability, agreeableness, and degree of persuasion). Two experiments were conducted using different participant groups depending on the negotiation situations. Photographs with good or bad appearances were found to show high or low degrees of persuasion, respectively. A multiple regression equation was obtained, indicating the importance of the three language factors (euphemistic, honorific, and sympathy expressions) to impressions made during negotiation. The result shows that there are optimal negotiation sentences based on various negotiation factors, such as visual appearance and use of language. For example, persons with good appearance might worsen their impression during negotiations by using certain language, although their initial impression was positive, and persons with bad appearance could effectively improve their impressions in negotiations through their use of language, although the final impressions of their negotiation counterpart might still be more negative than those for persons with good appearance. In contrast, the impressions made by persons of normal appearance were not easily affected by their use of language. The results of the present study have significant implications for future studies of effective negotiation strategies considering visual appearance as well as gender.

Hyperphosphatemia is associated with mortality in patients with chronic kidney disease, and is common in critically ill patients with acute kidney injury (AKI); however, its clinical implication in these patients is unknown. We conducted an observational study in 1144 patients (mean age, 63.2 years; male, 705 [61.6%]) with AKI who received continuous renal replacement therapy (CRRT) between January 2009 and September 2016. Phosphate levels were measured before (0 h) and 24 h after CRRT initiation. We assessed disease severity using various clinical parameters. Phosphate at 0 h positively correlated with the Acute Physiology and Chronic Health Evaluation II (APACHE II; P < 0.001) and Sequential Organ Failure Assessment (SOFA; P < 0.001) scores, and inversely with mean arterial pressure (MAP; P = 0.02) and urine output (UO; P = 0.01). In a fully adjusted linear regression analysis for age, sex, Charlson comorbidity index (CCI), MAP, and estimated glomerular filtration rate (eGFR), higher 0 h phosphate level was significantly associated with high APACHE II (P < 0.001) and SOFA (P = 0.04) scores, suggesting that phosphate represents disease severity. A multivariable Cox model also showed that hyperphosphatemia was significantly associated with increased 28-day (HR 1.05, 95% CI 1.02-1.08, P = 0.001) and 90-day (HR 1.05, 95% CI 1.02-1.08, P = 0.001) mortality. Furthermore, patients with increased phosphate level during 24 h were at higher risk of death than those with stable or decreased phosphate levels. Finally, c-statistics significantly increased when phosphate was added to a model that included age, sex, CCI, body mass index, eGFR, MAP, hemoglobin, serum albumin, C-reactive protein, and APACHE II score. This study shows that phosphate is a potential biomarker that can reflect disease severity and predict mortality in critically ill patients receiving CRRT.

Objective: The study aimed to evaluate the quantitative characteristics of the macular vessel density (VD) and foveal avascular zone (FAZ) in glaucoma using optical coherence tomography angiography (OCT-A)., Design: Cross-sectional, age- and sex-matched case-control study., Methods: Fifty-two eyes of 52 patients with primary open angle glaucoma and 52 eyes from 52 healthy participants were recruited retrospectively. OCT-A was performed on a 3 x 3-mm macular region centered on the fovea. OCT-A scans were manually graded to define the FAZ. Parafoveal VD in superficial and deep retina were analyzed in the circular- and quadrant-segmented zone. The FAZ parameters included size, perimeter, and circularity index. The regression analysis among VD and FAZ-related parameters and ocular parameters was performed, and the diagnostic ability was calculated with refractive error adjusted., Results: For both groups, the mean age and the sex ratio was not different between groups. With refractive error adjusted, the average macular VD was lower in glaucoma than in the control group for superficial (P = 0.013), deep (P<0.001), and the whole retina (P = 0.002). There were increased FAZ perimeter and decreased FAZ circularity index in glaucoma when compared with controls (P<0.001). In the multivariate regression models, FAZ circularity index were significantly associated with decreased peripapillary RNFL thickness (P = 0.007) and macular GCIPL thickness (P = 0.009) measured by OCT. The refractive-error adjusted area under receiver operating characteristics was highest for FAZ circularity index (0.905; 95% CI, 0.844-0.966), followed by temporal deep retinal VD (0.870; 95% CI, 0.803-0.937) and FAZ perimeter (0.858; 95% CI, 0.784-0.932)., Conclusions: Decreased macular VD, increased FAZ perimeter, and decreased FAZ circularity index were observed in eyes with glaucoma using OCT-A. With refractive error adjusted, these parameters showed considerable diagnostic value for glaucoma. FAZ circularity index may be a novel biomarker representing disruption of the parafoveal capillary network in glaucoma, as supported by its association with structural parameters.

We propose that when individuals believe in fixed traits of personality (entity theorists), they are likely to expect a world of "uniformity." As such, they easily infer a population statistic from a small sample of data with confidence. In contrast, individuals who believe in malleable traits of personality (incremental theorists) are likely to presume a world of "diversity," such that they "hesitate" to infer a population statistic from a similarly sized sample. In four laboratory experiments, we found that compared to incremental theorists, entity theorists estimated a population mean from a sample with a greater level of confidence (Studies 1a and 1b), expected more homogeneity among the entities within a population (Study 2), and perceived an extreme value to be more indicative of an outlier (Study 3). These results suggest that individuals are likely to use their implicit self-theory orientations (entity theory versus incremental theory) to see a population in general as a constitution either of homogeneous or heterogeneous entities., Competing Interests: The authors have declared that no competing interests exist.

Objective: To characterize the 24-h habitual-position intraocular pressure (IOP) patterns of optic disc phenotypes (ODPs) in untreated normal-tension glaucoma (NTG) and the relationships between nocturnal IOP elevation and various clinical factors., Design: Prospective, cross-sectional, observational study., Methods: Eighty-two NTG patients with focal ischemic (FI) ODP and 82 age- and disease severity-matched NTG patients with myopic glaucomatous (MG) ODP were recruited prospectively over 3 years. The IOP was recorded 11 times over a 24-hour (h) period by a single ophthalmologist using a hand-held tonometer (TonoPen®XL). A cosinor model was used to describe the 24-h IOP rhythm. Associations between nocturnal IOP elevation and both ocular and demographic variables were evaluated using the generalized estimating equation (GEE)., Results: Mean habitual-position IOP was significantly higher during nighttime than daytime in the FI group (16.44 vs. 14.23 mmHg, P < 0.001), but not in the MG group (15.91 vs. 15.70 mmHg, P = 0.82). The FI group also exhibited a significantly higher peak IOP during sleeping hours (P = 0.01) and lower trough IOP during the 24-h period than the MG group (P < 0.01). The MG group showed a significantly higher peak IOP during waking hours than the FI group (P < 0.01). Therefore, 24-h IOP fluctuation range was significantly higher in the FI group than the MG group (P = 0.013). In the FI group, peak habitual-position IOP and the highest frequency of IOP peaks occurred during sleeping hours (12 AM-6 AM). By contrast, IOP peaks in the MG group occurred during morning hours (8 AM-12 PM). The FI group showed an overall nocturnal acrophase in habitual-position IOP, with 45 patients (54.9%) having a nocturnal acrophase; 10 (12.2%), a diurnal acrophase; and 27 (32.9%), no evident acrophase. By contrast, the MG group showed no evident peak in habitual-position IOP, with 9 patients (10.9%) having a nocturnal acrophase; 43 (52.4%), a diurnal acrophase; and 30 (36.6%), no evident acrophase. In multivariate modeling using the GEE, ODP (P < 0.001) and spherical equivalent (SE, P = 0.001) were independently associated with nocturnal IOP elevation., Conclusions: Based on 24-h habitual-position IOP data, FI is associated with significant nocturnal IOP elevation, while no such nocturnal IOP elevation is observed in MG ODP. In untreated NTG, there are also significant differences in the 24-h IOP pattern between FI and MG ODPs., Competing Interests: The authors have declared that no competing interests exist.

ADAM10 (A Disintegrin and Metalloprotease domain-containing protein 10) is a cell surface protein with a unique structure possessing both potential adhesion and protease domains. However, the role of ADAM10 in preimplantation stage embryos is not clear. In this study, we examined the expression patterns and functional roles of ADAM10 in porcine parthenotes during preimplantation development. The transcription level of ADAM10 dramatically increased from the morula stage onward. Immunostaining revealed that ADAM10 was present in both the nucleus and cytoplasm in early cleavage stage embryos, and localized to the apical region of the outer cells in morula and blastocyst embryos. Knockdown (KD) of ADAM10 using double strand RNA did not alter preimplantation embryo development until morula stage, but resulted in significantly reduced development to blastocyst stage. Moreover, the KD blastocyst showed a decrease in gene expression of adherens and tight junction (AJ/TJ), and an increase in trophectoderm TJ permeability by disrupting TJ assembly. Treatment with an ADAM10 specific chemical inhibitor, GI254023X, at the morula stage also inhibited blastocyst development and led to disruption of TJ assembly. An in situ proximity ligation assay demonstrated direct interaction of ADAM10 with coxsackie virus and adenovirus receptor (CXADR), supporting the involvement of ADAM10 in TJ assembly. In conclusion, our findings strongly suggest that ADADM10 is important for blastocyst formation rather than compaction, particularly for TJ assembly and stabilization in preimplantation porcine parthenogenetic development.

Oocyte maturation and embryonic development are sensitive to DNA damage. Compared with somatic cells or oocytes, little is known about the response to DNA damage in early preimplantation embryos. In this study, we examined DNA damage checkpoints and DNA repair mechanisms in parthenogenetic preimplantation porcine embryos. We found that most of the etoposide-treated embryos showed delay in cleavage and ceased development before the blastocyst stage. In DNA-damaged embryos, the earliest positive TUNEL signals were detected on Day 5 of in vitro culture. Caffeine, which is an ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related protein) kinase inhibitor, and KU55933, which is an ATM kinase inhibitor, were equally effective in rescuing the etoposide-induced cell-cycle blocks. This indicates that ATM plays a central role in the regulation of the checkpoint mechanisms. Treating the embryos with histone deacetylase inhibitors (HDACi) increased embryonic development and reduced etoposide-induced double-strand breaks (DSBs). The mRNA expression of genes involved in non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways for DSB repair was reduced upon HDACi treatment in 5-day-old embryos. Furthermore, HDACi treatment increased the expression levels of pluripotency-related genes (OCT4, SOX2 and NANOG) and decreased the expression levels of apoptosis-related genes (CASP3 and BAX). These results indicate that early embryonic cleavage and development are disturbed by etoposide-induced DNA damage. ATMi (caffeine or KU55933) treatment bypasses the checkpoint while HDACi treatment improves the efficiency of DSB repair to increase the cleavage and blastocyst rate in porcine early preimplantation embryos.

Interleukin (IL)-23 and IL-12 are closely related in structure, and these cytokines regulate both innate and adaptive immunity. However, the precise signaling networks that regulate the production of each in Toxoplasma gondii-infected THP-1 monocytic cells, particularly the PI3K/AKT and MAPK signaling pathways, remain unknown. In the present study, T. gondii infection upregulated the expression of IL-23 and IL-12 in THP-1 cells, and both cytokines increased with parasite dose. IL-23 secretion was strongly inhibited by TLR2 monoclonal antibody (mAb) treatment in a dose-dependent manner and by TLR2 siRNA transfection, whereas IL-12 secretion was strongly inhibited by TLR4 mAb treatment dose-dependently and by TLR4 siRNA transfection. IL-23 production was dose-dependently inhibited by the PI3K inhibitors LY294002 and wortmannin, whereas IL-12 production increased dose-dependently. THP-1 cells exposed to live T. gondii tachyzoites underwent rapid p38 MAPK, ERK1/2 and JNK activation. IL-23 production was significantly upregulated by the p38 MAPK inhibitor SB203580 dose-dependently, whereas pretreatment with 10 μM SB203580 significantly downregulated IL-12 production. ERK1/2 inhibition by PD98059 was significantly downregulated IL-23 production but upregulated IL-12 production. JNK inhibition by SP600125 upregulated IL-23 production, but IL-12 production was significantly downregulated dose-dependently. T. gondii infection resulted in AKT activation, and AKT phosphorylation was inhibited dose-dependently after pretreatment with PI3K inhibitors. In T. gondii-infected THP-1 cells, ERK1/2 activation was regulated by PI3K; however, the phosphorylation of p38 MAPK and JNK was negatively modulated by the PI3K signaling pathway. Collectively, these results indicate that IL-23 production in T. gondii-infected THP-1 cells was regulated mainly by TLR2 and then by PI3K and ERK1/2; however, IL-12 production was mainly regulated by TLR4 and then by p38 MAPK and JNK. Our findings provide new insight concerning the intracellular networks of the PI3K/AKT and MAPK signaling cascades for regulating T. gondii-induced IL-23 and IL-12 secretion in human monocytic cells.

Nonverbal communication is an important factor in human communication, and body movement synchronization in particular is an important part of nonverbal communication. Some researchers have analyzed body movement synchronization by focusing on changes in the amplitude of body movements. However, the definition of "body movement synchronization" is still unclear. From a theoretical viewpoint, phase difference is the most important factor in synchronization analysis. Therefore, there is a need to measure the synchronization of body movements using phase difference. The purpose of this study was to provide a quantitative definition of the phase difference distribution for detecting body movement synchronization in human communication. The phase difference distribution was characterized using four statistical measurements: density, mean phase difference, standard deviation (SD) and kurtosis. To confirm the effectiveness of our definition, we applied it to human communication in which the roles of speaker and listener were defined. Specifically, we examined the difference in the phase difference distribution between two different communication situations: face-to-face communication with visual interaction and remote communication with unidirectional visual perception. Participant pairs performed a task supposing lecture in the face-to-face communication condition and in the remote communication condition via television. Throughout the lecture task, we extracted a set of phase differences from the time-series data of the acceleration norm of head nodding motions between two participants. Statistical analyses of the phase difference distribution revealed the characteristics of head nodding synchronization. Although the mean phase differences in synchronized head nods did not differ significantly between the conditions, there were significant differences in the densities, the SDs and the kurtoses of the phase difference distributions of synchronized head nods. These results show the difference in nonverbal synchronization between different communication types. Our study indicates that the phase difference distribution is useful in detecting nonverbal synchronization in various human communication situations.

The CRISPR/Cas9 system has proven to be an efficient gene-editing tool for genome modification of cells and organisms. However, the applicability and efficiency of this system in pig embryos have not been studied in depth. Here, we aimed to remove porcine OCT4 function as a model case using the CRISPR/Cas9 system. Injection of Cas9 and single-guide RNA (sgRNA) against OCT4 decreased the percentages of OCT4-positive embryos to 37-50% of total embryos, while ~100% of control embryos exhibited clear OCT4 immunostaining. We assessed the mutation status near the guide sequence using polymerase chain reaction (PCR) and DNA sequencing, and a portion of blastocysts (20% in exon 2 and 50% in exon 5) had insertions/deletions near protospacer-adjacent motifs (PAMs). Different target sites had frequent deletions, but different concentrations of sgRNA made no impact. OCT4 mRNA levels dramatically decreased at the 8-cell stage, and they were barely detectable in blastocysts, while mRNA levels of other genes, including NANOG, and CDX2 were not affected. In addition, the combination of two sgRNAs led to large-scale deletion (about 1.8 kb) in the same chromosome. Next, we injected an enhanced green fluorescent protein (eGFP) vector targeting the OCT4 exon with Cas9 and sgRNA to create a knockin. We confirmed eGFP fluorescence in blastocysts in the inner cell mass, and also checked the mutation status using PCR and DNA sequencing. A significant portion of blastocysts had eGFP sequence insertions near PAM sites. The CRISPR/CAS9 system provides a good tool for gene functional studies by deleting target genes in the pig.

To elucidate the roles of metalloproteinases and the Bcl-2 family of proteins in Trichovaginalis. vaginalis-induced apoptosis in human cervical cancer cells (SiHa cells) and vaginal epithelial cells (MS74 cells), SiHa cells and MS74 cells were incubated with live T. vaginalis, T. vaginalis excretory and secretory products (ESP), and T. vaginalis lysates, either with or without the specific metalloproteinase inhibitor 1,10-phenanthroline (1,10-PT), and examined apoptotic events and Bcl-2 signaling. The live T. vaginalis and the T. vaginalis ESP induced the release of cytochrome c into the cytosol, the activation of caspase-3 and caspase-9, and the cleavage of PARP. Additionally, the live T. vaginalis, but not the T. vaginalis lysate, induced the cleavage of the proapoptotic Bim protein. The live T. vaginalis and the T. vaginalis ESP, but not the T. vaginalis lysate, induced the dose-dependent cleavage of the antiapoptotic Bcl-xL and Mcl-1 proteins and decreased the association levels of Bcl-xL/Bim and Mcl-1/Bim complexes. We performed gelatin zymography and casein-hydrolysis assays on the live T. vaginalis and the T. vaginalis ESP to identify the apoptosis-inducing factor. Both the live T. vaginalis and the ESP contained high levels of metalloproteinases, of which activities were significantly inhibited by 1,10-PT treatment. Furthermore, the 1,10-PT blocked the cleavage of Bcl-xL, Mcl-1, PARP, caspase-3, and caspase-9, as well as the release of cytochrome c into the cytosol, and it significantly increased the association levels of the Bcl-xL/Bim and Mcl-1/Bim protein complexes, returning them to normal levels. Our results demonstrate that T. vaginalis induces mitochondria-dependent apoptosis in SiHa cells through the dissociation of Bcl-xL/Bim and Mcl-1/Bim complexes and that the apoptosis is blocked by the metalloproteinase inhibitor 1,10-PT. These results expand our understanding of the role of metalloproteinases in T. vaginalis-induced apoptosis and the signaling pathway in trichomoniasis of the cervicovaginal epithelial cells.