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ObjectivePrior research has implied that promoting sustaining physical activity through nudges is challenging and boosting health literacy is important for the long-term establishment of behaviors. This study aimed to investigate the effects of commitment-based health application on step count and health literacy.MethodsA control experiment was conducted involving employees from companies located in Shizuoka Prefecture, Japan. Participants were divided into three groups: the commitment app group (utilizing a commitment-based application "Minchalle," where teams of around five members were randomly assigned to declare a target step count and report daily step count with pictures), the self-commitment group (individuals declaring a target step count and endeavoring on their own), and the control group (no intervention). Changes in step count and health literacy were examined over one month.ResultsA total of 109 employees from 7 companies participated. The changes in step count were an increase of 893 steps for the commitment app group, 243 steps for the self-commitment group, and 178 steps for the control group, with a significant increase in the commitment app group compared to the control group. Regarding health literacy measures, there was significant progress in four items out of five for the commitment app group compared to the control group, and significant progress in one item for the self-commitment group compared to the control group.ConclusionCommunication within the app teams, such as commitment, sharing photos of their goal achievements and provide encouraging comments to others, functioned as social nudges, suggesting the potential for an immediate increase in step count and long-term behavioral reinforcement through improved health literacy.

Mutations in the non-structural protein regions of hepatitis C virus (HCV) are a cause of a non-sustained virological response (SVR) to treatment with direct-acting antivirals (DAAs) for chronic hepatitis; however, there are non-SVR cases without these mutations. In this study, we examined immune cell profiles in peripheral blood before and after ombitasvir/paritaprevir/ritonavir treatment and screened for genes that could be used to predict the therapeutic effects of DAAs. Fluorescence-activated cell sorting analysis indicated that the median frequencies of programmed cell death-1-positive (PD-1+) effector regulatory T cells (eTregs), PD-1+CD8+ T cells, and PD-1+Helper T cells were decreased significantly in SVR cases, but without significant changes in non-SVR cases. The frequency of PD-1+ naïve Tregs was significantly higher in the SVR group than in the non-SVR group before and after treatment. Similar results were found in patients treated with other DAAs (e.g., daclatasvir plus asunaprevir) and supported an immune response after HCV therapy. RNA-sequencing analysis indicated a significant increase in the expression of genes associated with the immune response in the SVR group, while genes related to intracellular and extracellular signal transduction were highly expressed in the non-SVR group. Therefore, we searched for genes associated with PD-1+ eTregs and CD8+ T cells that were significantly different between the SVR and non-SVR groups and found that T-box transcription factor 21 was associated with the non-SVR state. These results indicate that PD-1-related signaling pathways are associated with a non-SVR mechanism after DAAs treatment separate from mutation-related drug resistance.

The insulin-like growth factor 1 receptor (IGF1R) has been associated with growth and metastasis in various cancers. However, its role in postoperative recurrence and prognosis in lung cancer lacks clear consensus. Therefore, this study aimed to investigate the potential relationship between IGF1R and postoperative recurrence as well as long-term survival in a large cohort. Additionally, we assessed the relationship between IGF1R and programmed death ligand 1 (PD-L1) expression. Our study encompassed 782 patients with non-small cell lung cancer (NSCLC). Immunostaining of surgical specimens was performed to evaluate IGF1R and PD-L1 expression. Among the patients, 279 (35.8%) showed positive IGF1R expression, with significantly worse relapse-free survival (RFS) and overall survival (OS). Notably, no significant differences in RFS and OS were observed between IGF1R-positive and -negative groups in stages 2 and 3. However, in the early stages (0–1), the positive group displayed significantly worse RFS and OS. In addition, PD-L1 expression was detected in 100 (12.8%) patients, with a significant predominance in the IGF1R-positive. IGF1R may serve as a prognostic indicator and a guide for perioperative treatment strategies in early-stage lung cancer. In conclusion, our findings underscore an association between IGF1R expression and poor survival and PD-L1 expression in NSCLC. [ABSTRACT FROM AUTHOR]

N,N-diethyl-meta-toluamide (DEET) is a widely used insect repellent, with minimal skin permeation and sustained repellent activity in the superficial layers of the skin. In this study, we prepared a 10% DEET formulation consisting of 40% ethanol with or without 2% poly(oxyethylene)/poly(oxypropylene) butyl ether (POE-POP), an amphiphilic random copolymer. Further, we demonstrated the effects of POE-POP on tensile stress (stickiness), hydrophobicity, skin retention, permeation, and repellent activity of DEET. Stickiness was measured in male ICR mice (7-week old), and skin retention and permeation were evaluated in male Wistar rats (7-week old). In addition, female Aedes albopictus were used to measure the repellent action of DEET. The addition of POE-POP did not affect stickiness, volatility, and degradability but decreased logP and increased viscosity of DEET. Next, we demonstrated the behavior of DEET formulations in the rat skin. POE-POP prolonged the retention of DEET in the superficial layers of the rat skin (skin surface and stratum corneum) and decreased the penetration of DEET into rat skin tissues (epithelium and dermis). The repellent effect of DEET was also enhanced by the addition of POE-POP. However, severe skin damage was not observed after repetitive treatment with DEET formulations containing POE-POP for one month (twice a day). In conclusion, we demonstrated that a 10% DEET formulation consisting of 40% ethanol and 2% POE-POP attenuated the skin penetration and prolonged the repellent action of DEET without causing stickiness and skin damage. We conclude that the combination of ethanol and POE-POP is useful as a safe and effective delivery system for the development of insect repellent formulations containing DEET.

Recently, the utilization of real-world medical data collected from clinical sites has been attracting attention. Especially as the number of variables in real-world medical data increases, causal discovery becomes more and more effective. On the other hand, it is necessary to develop new causal discovery algorithms suitable for small data sets for situations where sample sizes are insufficient to detect reasonable causal relationships, such as rare diseases and emerging infectious diseases. This study aims to develop a new causal discovery algorithm suitable for a small number of real-world medical data using quantum computing, one of the emerging information technologies attracting attention for its application in machine learning. In this study, a new algorithm that applies the quantum kernel to a linear non-Gaussian acyclic model, one of the causal discovery algorithms, is developed. Experiments on several artificial data sets showed that the new algorithm proposed in this study was more accurate than existing methods with the Gaussian kernel under various conditions in the low-data regime. When the new algorithm was applied to real-world medical data, a case was confirmed in which the causal structure could be correctly estimated even when the amount of data was small, which was not possible with existing methods. Furthermore, the possibility of implementing the new algorithm on real quantum hardware was discussed. This study suggests that the new proposed algorithm using quantum computing might be a good choice among the causal discovery algorithms in the low-data regime for novel medical knowledge discovery.

Clinical isolates of Clostridioides difficile sometimes exhibit multidrug resistance and cause diarrhea after antibiotic administration. Metronidazole and vancomycin are often used as therapeutic agents, but resistance to these antibiotics has been found clinically. Therefore, the development of alternative antimicrobial agents is needed. Nisin A, produced by Lactococcus lactis, has been demonstrated to be effective against C. difficile infection. In this study, we evaluated the susceptibility of 11 C. difficile clinical isolates to nisin A and found that they could be divided into 2 groups: high and low susceptibility. Since CprABC and DltDABC, which are responsible for nisin A efflux and cell surface charge, respectively, have been reported to be related to nisin A susceptibility, we investigated the expression of cprA and dltA among the 11 strains. cprA expression in all strains was induced by nisin A, but dltA expression was not. The expression levels of both genes did not correlate with nisin A susceptibility in these clinical isolates. To evaluate cell surface charge, we performed a cytochrome C binding assay and found no relationship between charge and nisin A susceptibility. Then, we determined the whole genome sequence of each clinical isolate and carried out phylogenetic analysis. The 11 isolates separated into two major clusters, which were consistent with the differences in nisin A susceptibility. Furthermore, we found common differences in several amino acids in the sequences of CprA, CprB, and CprC between the two clusters. Therefore, we speculated that the different amino acid sequences of CprABC might be related to nisin A susceptibility. In addition, C. difficile strains could be divided in the same two groups based on susceptibility to epidermin and mutacin III, which are structurally similar to nisin A. These results suggest that genotypic variations in C. difficile strains confer different susceptibilities to bacteriocins.

Objective: Prior research has implied that promoting sustaining physical activity through nudges is challenging and boosting health literacy is important for the long-term establishment of behaviors. This study aimed to investigate the effects of commitment-based health application on step count and health literacy. Methods: A control experiment was conducted involving employees from companies located in Shizuoka Prefecture, Japan. Participants were divided into three groups: the commitment app group (utilizing a commitment-based application "Minchalle," where teams of around five members were randomly assigned to declare a target step count and report daily step count with pictures), the self-commitment group (individuals declaring a target step count and endeavoring on their own), and the control group (no intervention). Changes in step count and health literacy were examined over one month. Results: A total of 109 employees from 7 companies participated. The changes in step count were an increase of 893 steps for the commitment app group, 243 steps for the self-commitment group, and 178 steps for the control group, with a significant increase in the commitment app group compared to the control group. Regarding health literacy measures, there was significant progress in four items out of five for the commitment app group compared to the control group, and significant progress in one item for the self-commitment group compared to the control group. Conclusion: Communication within the app teams, such as commitment, sharing photos of their goal achievements and provide encouraging comments to others, functioned as social nudges, suggesting the potential for an immediate increase in step count and long-term behavioral reinforcement through improved health literacy. [ABSTRACT FROM AUTHOR]

Unisexual hybrids that reproduce either clonally or hemiclonally are considered to be evolutionarily short-lived as they lack the ability to reduce deleterious mutations and increase genetic diversity. In the greenling (Teleostei: Hexagrammidae, genus Hexagrammos), unisexual hybrids that produce haploid eggs containing only the H. octogrammus (maternal species) genome generate hemiclonal offspring by fertilization with haploid sperm of H. agrammus (paternal species). When hemiclonal hybrids are backcrossed to a male of the maternal species, the offspring (BC-Hoc) are phenotypically similar to the maternal species and produce recombinant gametes through conventional meiosis. BC-Hoc (recombinant generation) individuals referred to as carriers harbor the genetic factor for hybridogenesis, thereby facilitating the production of new hemiclonal lineages through hybridization. Previous studies based on field research have suggested that the carriers produced by two-way backcrossing (mating pattern in which hemiclonal hybrids are backcrossed with both parental species) may overcome the evolutionary dead end imposed by the lack of recombination. The present study verified this hypothesis by regenerating a newly hemiclonal lineage through artificial hybridization. To clarify the genetic mode of hybrids produced by crosses between BC-Hoc and Hag, mature eggs were obtained from 16 individuals and fertilized with either Hag or Hoc sperm. Hybridogenesis was confirmed in one of the 16 individuals. Based on the low occurrence rate, these findings suggest that hemiclonal lineages can be regenerated, and that the hemiclonal factors are likely distributed across multiple genes on different chromosomes. The findings provide important evidence for the retention of a robust system for increasing genetic variability and maintaining evolutionary succession in unisexual hybrids that reproduce hemiclonally. [ABSTRACT FROM AUTHOR]

We present here a novel genetic algorithm-based random forest (GARF) modeling technique that enables a reduction in the complexity of large gene disease signatures to highly accurate, greatly simplified gene panels. When applied to 803 glioblastoma multiforme samples, this method allowed the 840-gene Verhaak et al. gene panel (the standard in the field) to be reduced to a 48-gene classifier, while retaining 90.91% classification accuracy, and outperforming the best available alternative methods. Additionally, using this approach we produced a 32-gene panel which allows for better consistency between RNA-seq and microarray-based classifications, improving cross-platform classification retention from 69.67% to 86.07%. A webpage producing these classifications is available at http://simplegbm.semel.ucla.edu.

The pedicle screw (PS) is widely used for spinal fixation surgery. However, PS malpositioning can cause critical complications; thus, the accuracy of ascertaining PS trajectory is paramount. This study aimed to demonstrate the accuracy and safety of a simple and cost-effective PS placement technique using a human internal reference frame for angle estimation. Ex vivo lumbar porcine spine samples were fixed to a wooden board with rostrocaudal and mediolateral rotational angles adjusted by two angle vises. PS entry points (EPs) were identified using clear anatomical vertebral landmarks. PS placement was performed on one side using the perpendicular probing and screwing technique (PPST), wherein the attitude angle of the sample was adjusted such that the longitudinal axis of the target pedicle was perpendicular to the ground. The pedicle probe and PS driver were manually maintained perpendicular to the ground during probing and PS placement. PS placement on the contralateral side was performed freehand as a control. Offsets between the preoperatively planned and implanted PS rotational angles measured using computed tomography for PPST and freehand method were analyzed. Pedicle wall penetration was also evaluated. The mean ± standard error of the medial rotational offsets was 5.83° ± 0.57° in the freehand group versus 2.89° ± 0.31° in the PPST group (p

ObjectiveWe evaluated the clinical characteristics of metabolic dysfunction-associated fatty liver disease (MAFLD) to evaluate the usefulness of the MAFLD diagnostic criteria in a resident health survey.MethodsIn 1056 participants of a health survey, we compared obesity, diabetes, metabolic dysregulation, FibroScan-aspartate aminotransferase (FAST) score, dietary habits, and gut microbiota between healthy individuals and participants with MAFLD and Nonalcoholic fatty liver disease (NAFLD).ResultsThe proportion of participants with MAFLD in the fatty liver was higher than that with NAFLD (88.1% vs. 75.5%, respectively). Of 36 participants with a FAST score > 0.35, 29 (80.6%) participants had MAFLD and 23 (63.9%) participants had NAFLD. Of 29 patients with liver fibrosis, 26 (89.7%) participants had obesity and metabolic dysregulation. In the evaluation of diet, the total energy, protein, dietary fiber, and salt intake were significantly higher in participants with MAFLD than those in participants without fatty liver. In the microbiota analysis, the results of the linear discriminant analysis effect size analysis revealed nine bacterial genera that were significantly different in participants with MAFLD in comparison with participants without fatty liver. Of these genera, the relative abundance of Blautia was especially low in participants with MAFLD.ConclusionIn a resident health survey, participants with MAFLD had a higher proportion of fatty liver than those with NAFLD. MAFLD criteria could help in improved screening of participants with liver fibrosis. Therefore, the MAFLD criteria could be a useful diagnostic tool for aggressively identifying participants with a high risk of fatty liver. Additionally, Blautia might be involved in the development of MAFLD.

Regular monitoring is an important component of the successful management of pelagic animals of interest to commercial fisheries. Here we provide a biomass estimate for Antarctic krill (Euphausia superba) in the eastern sector of the Commission for the Conservation of Antarctic Marine Living Resources (CCAMLR) Division 58.4.2 (55°E to 80°E; area = 775,732 km2) using data collected during an acoustic-trawl survey carried out in February and March 2021. Using acoustic data collected in day-time and trawl data, areal biomass density was estimated as 8.3 gm-2 giving a total areal krill biomass of 6.48 million tonnes, with a 28.9% coefficient of variation (CV). The inaccessibility of the East Antarctic makes fisheries-independent surveys of Antarctic krill expensive and time consuming, so we also assessed the efficacy of extrapolating smaller surveys to a wider area. During the large-scale survey a smaller scale survey (centre coordinates -66.28°S 63.35°E, area = 4,902 km2) was conducted. We examine how representative krill densities from the small-scale (Mawson box) survey were over a latitudinal range by comparing krill densities from the large-scale survey split into latitudinal bands. We found the small scale survey provided a good representation of the statistical distribution of krill densities within its latitudinal band (KS-test, D = 0.048, p-value = 0.98), as well as mean density (t-test p-value = 0.44), but not outside of the band. We recommend further in situ testing of this approach.

Epithelial-to-mesenchymal transition (EMT) plays a critical role in cancer metastasis, and is regulated by growth factors such as transforming growth factor β (TGF-β) and fibroblast growth factors (FGF) secreted from the stromal and tumor cells. However, the role of growth factors in EMT has not been fully established. Several integrins are upregulated by TGF-β1 during EMT. Integrins are involved in growth factor signaling through integrin-growth factor receptor crosstalk. We previously reported that FGF1 directly binds to integrin αvβ3 and the interaction was required for FGF1 functions such as cell proliferation and migration. We studied the role of αvβ3 induced by TGF-β on TGF-β-induced EMT. Here, we describe that FGF1 augmented EMT induced by TGF-β1 in MCF10A and MCF12A mammary epithelial cells. TGF-β1 markedly amplified integrin αvβ3 and FGFR1 (but not FGFR2). We studied if the enhancing effect of FGF1 on TGF-β1-induced EMT requires enhanced levels of both integrin αvβ3 expression and FGFR1. Knockdown of β3 suppressed the enhancement by FGF1 of TGF-β1-induced EMT in MCF10A cells. Antagonists to FGFR suppressed the enhancing effect of FGF1 on EMT. Integrin-binding defective FGF1 mutant did not augment TGF-β1-induced EMT in MCF10A cells. These findings suggest that enhanced integrin αvβ3 expression in addition to enhanced FGFR1 expression is critical for FGF1 to augment TGF-β1-induced EMT in mammary epithelial cells.

Background Home delivery (HD) without skilled birth attendants (SBAs) are considered crucial risk factors increasing maternal and child mortality rates in Loa PDR. While a few studies in the literature discuss the choice of delivery in remote areas of minority ethnic groups; our work aims to identify factors that indicated their delivery place, at home or in the health facilities. Methods A community-based qualitative study was conducted between February and March 2020. Three types of interviews were implemented, In-depth interviews with 16 women of eight rural villages who delivered in the last 12 months in Xepon District, Savannakhet Province, Lao PDR. Also, three focus group discussions (FGDs) with nine HCPs and key-informant interviews of ten VHVs were managed. Factors affecting the choice of the delivery place were categorized according to the social-ecological model. Results Our sample included five Tri women and two Mangkong women in the HD group, while the FD group included three Tri women, two Mangkong women, one Phoutai woman, two Laolung women and one Vietnamese. Our investigation inside the targeted minority showed that both positive perceptions of home delivery (HD) and low-risk perception minorities were the main reasons for the choice of HD, on the individual level. On the other hand, fear of complication, the experience of stillbirth, and prolonged labour pain during HD were reasons for facility-based delivery (FD). Notably, the women in our minority reported no link between their preference and their language, while the HCPs dated the low knowledge to the language barrier. On the interpersonal level, the FD women had better communication with their families, and better preparation for delivery compared to the HD group. The FD family prepared cash and transportation using their social network. At the community level, the trend of the delivery place had shifted from HD to FD. Improved accessibility and increased knowledge through community health education were the factors of the trend. At the societal (national policy) level, the free delivery policy and limitation of HCPs’ assisted childbirth only in health facilities were the factors of increasing FD, while the absence of other incentives like transportation and food allowance was the factor of remaining of HD. Conclusions Based on the main findings of this study, we urge the enhancement of family communication on birth preparedness and birthplace. Furthermore, our findings support the need to educate mothers, especially those of younger ages, about their best options regarding the place of delivery. We propose implementing secondary services of HD to minimize the emergency risks of HD. We encourage local authorities to be aware of the medical needs of the community especially those of pregnant females and their right for a free delivery policy.

BackgroundIt has been reported that genetic factors are associated with risk factors and onset of lifestyle-related diseases, but this finding is still the subject of much debate.ObjectiveThe aim of the present study was to investigate the correlation of genetic factors, including salivary telomere length and three single nucleotide polymorphisms (SNPs) that may influence lifestyle-related diseases, with lifestyle-related diseases themselves.MethodsIn one year at a single facility, relative telomere length and SNPs were determined by using monochrome multiplex quantitative polymerase chain reaction and TaqMan SNP Genotyping Assays, respectively, and were compared with lifestyle-related diseases in 120 Japanese individuals near our university.ResultsIn men and all participants, age was inversely correlated with relative telomere length with respective p values of 0.049 and 0.034. In men, the frequency of hypertension was significantly higher in the short relative telomere length group than in the long group with unadjusted p value of 0.039, and the difference in the frequency of hypertension between the two groups was of borderline statistical significance after adjustment for age (p = 0.057). Furthermore, in men and all participants, the sum of the number of affected lifestyle-related diseases, including hypertension, was significantly higher in the short relative telomere length group than in the long group, with p values of 0.004 and 0.029, respectively. For ADIPOQ rs1501299, men's ankle brachial index was higher in the T/T genotype than in the G/G and G/T genotypes, with p values of 0.001 and 0.000, respectively. For SIRT1 rs7895833, men's body mass index and waist circumference and all participants' brachial-ankle pulse wave velocity were higher in the A/G genotype than in the G/G genotype, with respective p values of 0.048, 0.032 and 0.035. For FOXO3A rs2802292, women's body temperature and all participants' saturation of peripheral oxygen were lower in the G/T genotype than in the T/T genotype, with respective p values of 0.039 and 0.032. However, relative telomere length was not associated with physiological or anthropometric measurements except for height in men (p = 0.016). ADIPOQ rs1501299 in men, but not the other two SNPs, was significantly associated with the sum of the number of affected lifestyle-related diseases (p = 0.013), by genotype. For each SNPs, there was no significant difference in the frequency of hypertension or relative telomere length by genotype.ConclusionRelative telomere length and the three types of SNPs determined using saliva have been shown to be differentially associated with onset of and measured risk factors for lifestyle-related diseases consisting mainly of cardiovascular diseases and cancer.

Foot-and-mouth disease virus (FMDV) serotype O, topotype CATHAY is a known porcinophilic virus that has caused devastating damage to the pig industry. However, the minimum infectious dose via a natural infection route in pigs, the infection dynamics in cattle, and risk of viral transmission from infected cattle to pigs have not been quantitatively analyzed. The FMDV strain O/HKN/1/2015 was serially diluted and inoculated into pigs via an intraoral route to determine the infectious dose. We found that a 104.0 tissue culture infectious dose (TCID50) of the virus was insufficient, but 105.5 TCID50 was sufficient to infect pigs via the oral route. While cows inoculated with the strain showed increased temperature in their feet, typical clinical signs including vesicular development were not observed. The cows showed short-term and low levels of viremia and virus excretion only before the detection of virus neutralizing antibodies. FMDV genes were not detected in esophageal-pharyngeal fluid from cows after 14 days post inoculation. No genetic insertions that could be associated with host adaptation were observed in viruses isolated from infected cows. These findings indicate that cows infected with FMDV of O/CATHAY have a low risk of viral transmission or persistence. Information on the dynamics of virus infection is essential for ensuring the rapid and accurate diagnosis of this disease, and its surveillance.

Human hepatitis B virus (HBV) infection remains a serious health problem worldwide. However, the mechanism for the maintenance of HBV in a latent state within host cells remains unclear. Here, using single-cell RNA sequencing analysis, we identified four genes linked to the maintenance of HBV in a liver cell line expressing HBV RNA at a low frequency. These genes included DOCK11 and DENND2A, which encode small GTPase regulators. In primary human hepatocytes infected with HBV, knockdown of these two genes decreased the amount of both HBV DNA and covalently closed circular DNA to below the limit of detection. Our findings reveal a role for DOCK11 and DENND2A in the maintenance of HBV.

BackgroundClinical sequencing using a panel of genes has recently been applied worldwide for patients with refractory solid tumors, but the significance of clinical sequencing using gene panel testing remains uncertain. Here we sought to clarify the feasibility and utility of clinical sequencing in the treatment of refractory tumors at our hospital.MethodsA total of 39 patients with advanced solid tumors treated at our hospital between 2018 and 2020 were enrolled in the clinical sequencing. Among them, we identified 36 patients whose tissue samples were of suitable quality for clinical sequencing, and we analyzed the genomic profiles of these tumors.ResultsPathogenic alterations were detected in 28 (78%) of the 36 patients. The most common mutation was TP53 (55%), followed by KRAS (22%), and the highest frequency of gene amplification was ERBB2 (17%). Nine of the 36 patients were identified as candidates for novel molecular-targeted therapy based on their actionable gene alterations, but only one case ended up receiving novel targeted therapy following the genetic tests.ConclusionsOur current results suggested that clinical sequencing might be useful for the detection of pathogenic alterations and the management of additional cancer treatment. However, molecular target based on actionable genomic alteration does not always bridge to subsequent therapy due to clinical deterioration, refusal for unapproved drug, and complexity of clinical trial access. Both improved optimal timing of clinical sequencing and a consensus about its off-label use might help patients receive greater benefit from clinical sequencing.

Fibroblast growth factor-1 (FGF1) and FGF2 play a critical role in angiogenesis, a formation of new blood vessels from existing blood vessels. Integrins are critically involved in FGF signaling through crosstalk. We previously reported that FGF1 directly binds to integrin αvβ3 and induces FGF receptor-1 (FGFR1)-FGF1-integrin αvβ3 ternary complex. We previously generated an integrin binding defective FGF1 mutant (Arg-50 to Glu, R50E). R50E is defective in inducing ternary complex formation, cell proliferation, and cell migration, and suppresses FGF signaling induced by WT FGF1 (a dominant-negative effect) in vitro. These findings suggest that FGFR and αvβ3 crosstalk through direct integrin binding to FGF, and that R50E acts as an antagonist to FGFR. We studied if R50E suppresses tumorigenesis and angiogenesis. Here we describe that R50E suppressed tumor growth in vivo while WT FGF1 enhanced it using cancer cells that stably express WT FGF1 or R50E. Since R50E did not affect proliferation of cancer cells in vitro, we hypothesized that R50E suppressed tumorigenesis indirectly through suppressing angiogenesis. We thus studied the effect of R50E on angiogenesis in several angiogenesis models. We found that excess R50E suppressed FGF1-induced migration and tube formation of endothelial cells, FGF1-induced angiogenesis in matrigel plug assays, and the outgrowth of cells in aorta ring assays. Excess R50E suppressed FGF1-induced angiogenesis in chick embryo chorioallantoic membrane (CAM) assays. Interestingly, excess R50E suppressed FGF2-induced angiogenesis in CAM assays as well, suggesting that R50E may uniquely suppress signaling from other members of the FGF family. Taken together, our results suggest that R50E suppresses angiogenesis induced by FGF1 or FGF2, and thereby indirectly suppresses tumorigenesis, in addition to its possible direct effect on tumor cell proliferation in vivo. We propose that R50E has potential as an anti-cancer and anti-angiogenesis therapeutic agent ("FGF1 decoy").

Pregnant women with diabetes mellitus (DM) are at high risk for hypertensive disorder of pregnancy (HDP). Women with poor control DM sometimes have heavy-for-dates infants. However, women with HDP sometimes have light-for-dates infants. We aim to clarify the relationship between glycemic control and fetal growth in women with DM and/or subsequent HDP. Of 7893 women gave singleton birth at or after 22 gestational weeks, we enrolled 154 women with type 1 DM (T1DM) or type 2 DM (T2DM) whose infants did not have fetal abnormalities. Among women with T1DM or T2DM, characteristics of the three groups (with HDP, without HDP, and with chronic hypertension [CH]) were compared. No women with T1DM had CH, but 19 (17.4%) of 109 with T2DM did. HDP incidence was similar between women with T1DM (22.2%) and T2DM without CH (16.7%). Among women with T1DM, the incidences of fetal growth restriction (FGR) with and without HDP were similar. However, among women with T2DM without CH, this incidence was significantly higher among those with HDP (33.3%) than among those without HDP (5.3%), was significantly more common with HbA1c levels at first trimester ≥ 7.2% (33.3%) than with those < 7.2% (5.6%), and significantly more numerous without pre-pregnancy therapies for DM (23.3%) than with them (3.3%). Among women with T2DM and HDP, those with FGR had smaller placenta SDs and higher insulin dosages at delivery than those without light-for-dates. In multivariate analysis, the presence of diabetic nephropathy was a predictor of T1DM and HDP (P = 0.0105), whereas HbA1c levels ≥ 7.2% before pregnancy was a predictor of T2DM and HDP (P = 0.0009). Insulin dosage ≥ 50U/day at delivery (P = 0.0297) and the presence of HDP (P = 0.0116) independently predicted T2DM, HDP, and FGR development. Insufficient pre-pregnancy treatment of DM increased the risk of HDP.

Phagocytes in patients with chronic granulomatous disease (CGD) do not generate reactive oxidative species (ROS), whereas nitric oxide (NO) production is increased in response to the calcium ionophore A23187 in CGD phagocytes compared with healthy phagocytes. Recently, patients with X-linked CGD (X-CGD) have been reported to show higher flow-mediated dilation, suggesting that endothelial cell function is affected by NO production from phagocytes. We studied NOS3 and EDN1 mRNA and protein expression in human umbilical vein endothelial cells (HUVECs) in a co-culture system with neutrophils from X-CGD patients. HUVECs were co-cultured for 30 minutes with human neutrophils from X-CGD or healthy participants in response to A23187 without cell-to-cell contact. The expression of NOS3 and EDN1 mRNA in HUVECs was quantified by real-time polymerase chain reaction. Moreover, we demonstrated the protein expression of eNOS, ET-1, and NFκB p65, including phosphorylation at Ser1177 of eNOS and Ser536 of NFκB p65. Neutrophils from X-CGD patients showed significantly higher NO and lower H2O2 production in response to A23187 than healthy neutrophils in vitro. Compared with healthy neutrophils, X-CGD neutrophils under A23187 stimulation exhibited significantly increased NO and decreased H2O2, and promoted downregulated NOS3 and EDN1 expression in HUVECs. The total expression and phosphorylation at Ser1177 of eNOS and ET-1 expression were significantly decreased in HUVECs co-cultures with stimulated X-CGD neutrophils. Also, phosphorylation at Ser536 of NFκB p65 were significantly decreased. In conclusions, eNOS and ET-1 significantly down-regulated in co-culture with stimulated X-CGD neutrophils through their excessive NO and the lack of ROS production. These findings suggest that ROS generated from neutrophils may mediate arterial tone affecting eNOS and ET-1 expression via their NO and ROS production.

MicroRNAs (miRNAs) inhibit protein function by silencing the translation of target mRNAs. However, in primary central nervous system lymphoma (PCNSL), the expression and functions of miRNAs are inadequately known. Here, we examined the expression of 847 miRNAs in 40 PCNSL patients with a microarray and investigated for the miRNA predictors associated with cancer immunity-related genes such as T helper cell type 1/2 (Th-1/Th-2) and regulatory T cell (T-reg) status, and stimulatory and inhibitory checkpoint genes, for prognosis prediction in PCNSL. The aim of this study is to find promising prognosis markers based on the miRNA expression in PCNSL. We detected 334 miRNAs related to 66 cancer immunity-related genes in the microarray profiling. Variable importance measured by the random survival forest analysis and Cox proportional hazards regression model elucidated that 11 miRNAs successfully constitute the survival formulae dividing the Kaplan-Meier curve of the respective PCNSL subgroups. On the other hand, univariate analysis shortlisted 23 miRNAs for overall survival times, with four miRNAs clearly dividing the survival curves-miR-101/548b/554/1202. These miRNAs regulated Th-1/Th-2 status, T-reg cell status, and immune checkpoints. The miRNAs were also associated with gene ontology terms as Ras/MAP-kinase, ubiquitin ligase, PRC2 and acetylation, CDK, and phosphorylation, and several diseases including acquired immunodeficiency syndrome, glioma, and those related to blood and hippocampus with statistical significance. In conclusion, the results demonstrated that the four miRNAs comprising miR-101/548b/554/1202 associated with cancer immunity can be a useful prognostic marker in PCNSL and would help us understand target pathways for PCNSL treatments.

BACKGROUND:We aimed to determine the optimal approach with endoscopic biliary drainage (EBD) and corticosteroid (CS) for the treatment of IgG4-related sclerosing cholangitis (ISC). METHODS:To evaluate the safety of EBD for treatment of biliary stricture caused by ISC, we assessed the risk of stent dislodgement and sought to determine the most appropriate time for stent removal. We also assessed the safety of treatment with CS alone for patients with obstructive jaundice, and the rate of and risk factors for biliary tract complications. RESULTS:Sixty-nine patients with ISC treated with CS were enrolled. Twenty-eight patients (40.6%) were treated with EBD for biliary stricture before CS initiation. Intentional stent removal was performed in thirteen (46.4%) after confirming CS-induced improvement. Eleven of thirteen patients (84.6%) underwent stent removal within 1 month after CS initiation and all their stent removals were safely carried out without early (within two weeks) recurrence of obstructive jaundice. Ten of twenty-eight patients (35.7%) experienced spontaneous stent dislodgement after CS initiation, and seven (70%) of them developed stent dislodgement two weeks to two months after CS initiation. Among forty-one patients treated with CS alone without EBD, 10 patients had obstructive jaundice at the time of CS initiation and all of them achieved clinical improvement without biliary tract infection. During the follow-up, three patients (4.3%), all of whom had undergone EBD, developed bile-duct stones, while none of those treated with CS alone developed bile-duct stones (p = 0.032). Long-term biliary stenting was a risk factor for bile-duct stones. CONCLUSIONS:Biliary stent removal should be carried out within 2 weeks after CS initiation if biliary stricture improves to prevent stent dislodgement. Obstructive jaundice can be treated safely with CS alone in patients without infection. Clinicians should be aware of the possibility of bile-duct stones in patients treated with EBD.

AIM:Metabolic reprograming is crucial in the proliferation of hepatocellular carcinoma (HCC). Canagliflozin (CANA), a sodium-glucose cotransporter 2 (SGLT2) inhibitor, affects various metabolisms. We investigated the effects of CANA on proliferation and metabolic reprograming of HCC cell lines using multi-omics analysis of metabolomics and absolute quantification proteomics (iMPAQT). METHODS:The cells were counted 72 hours after treatment with CANA (10 μM; n = 5) or dimethyl sulfoxide (control [CON]; n = 5) in Hep3B and Huh7 cells. In Hep3B cells, metabolomics and iMPAQT were used to evaluate the levels of metabolites and metabolic enzymes in the CANA and CON groups (each n = 5) 48 hours after treatment. RESULTS:Seventy-two hours after treatment, the number of cells in the CANA group was significantly decreased compared to that in the CON group in Hep3B and Huh7 cells. On multi-omics analysis, there was a significant difference in the levels of 85 metabolites and 68 metabolic enzymes between the CANA and CON groups. For instance, CANA significantly downregulated ATP synthase F1 subunit alpha, a mitochondrial electron transport system protein (CON 297.28±20.63 vs. CANA 251.83±22.83 fmol/10 μg protein; P = 0.0183). CANA also significantly upregulated 3-hydroxybutyrate, a beta-oxidation metabolite (CON 530±14 vs. CANA 854±68 arbitrary units; P

BACKGROUND:CD4-positive T cells are the main target of human T-cell leukemia virus type 1 (HTLV-1). Interferon-γ release assays rely on the fact that T-lymphocytes release this cytokine when exposed to tuberculosis-specific antigens and are useful in testing for latent tuberculosis infection before initiating biologic therapy, such as anti-tumor necrosis factor agents. However, the reliability of interferon-γ release assays in detecting tuberculosis infection among HTLV-1-positive patients with rheumatoid arthritis (RA) remains unclear. The present study aimed to evaluate the use of the T-SPOT.TB assay in HTLV-1-positive RA patients. METHODS:Overall, 29 HTLV-1-positive RA patients and 87 age- and sex-matched HTLV-1-negative RA patients (controls) were included from the HTLV-1 RA Miyazaki Cohort Study. Results of the T-SPOT.TB assay for latent tuberculosis infection screening were collected from medical records of patients. RESULTS:Approximately 55% of the HTLV-1-positive RA patients showed invalid T-SPOT.TB assay results (odds ratio: 108, 95% confidence interval: 13.1-890, p < 0.0001) owing to a spot count of >10 in the negative controls. HTLV-1 proviral load values were significantly higher in patients with invalid results compared with those without invalid results (p = 0.003). CONCLUSION:HTLV-1 infection affects T-SPOT.TB assay results in RA patients. Assay results in HTLV-1 endemic regions should be interpreted with caution when screening for latent tuberculosis infection before initiation of biologic therapy.

Meiosis and oocyte maturation are tightly regulated processes. The meiosis arrest female 1 (MARF1) gene is essential for meiotic progression in animals; however, its detailed function remains unclear. In this study, we examined the molecular mechanism of dMarf1, a Drosophila homolog of MARF1 encoding an OST and RNA Recognition Motif (RRM) -containing protein for meiotic progression and oocyte maturation. Although oogenesis progressed in females carrying a dMarf1 loss-of-function allele, the dMarf1 mutant oocytes were found to contain arrested meiotic spindles or disrupted microtubule structures, indicating that the transition from meiosis I to II was compromised in these oocytes. The expression of the full-length dMarf1 transgene, but none of the variants lacking the OST and RRM motifs or the 47 conserved C-terminal residues among insect groups, rescued the meiotic defect in dMarf1 mutant oocytes. Our results indicate that these conserved residues are important for dMarf1 function. Immunoprecipitation of Myc-dMarf1 revealed that several mRNAs are bound to dMarf1. Of those, the protein expression of nanos (nos), but not its mRNA, was affected in the absence of dMarf1. In the control, the expression of Nos protein became downregulated during the late stages of oogenesis, while it remained high in dMarf1 mutant oocytes. We propose that dMarf1 translationally represses nos by binding to its mRNA. Furthermore, the downregulation of Nos induces cycB expression, which in turn activates the CycB/Cdk1 complex at the onset of oocyte maturation.

Liquid biopsy has become widely applied in clinical medicine along with the progress in innovative technologies, such as next generation sequencing, but the origin of circulating tumor DNA (ctDNA) has not yet been precisely established. We reported bimodal peaks of long fragment circulating free DNA (cfDNA) of 5 kb and short fragment cfDNA of 170 bp in patients with advanced lung cancer, and both contained ctDNA. In this paper, we demonstrate that the total amount of cfDNA is higher when patients with lung cancer have extrathoracic metastases, and the amount of long fragment cfDNA is significantly higher in those patients. To investigate the origin of long fragment cfDNA, conditioned media isolated from lung cancer cell lines was fractionated. Long fragment cfDNA was found concomitant with extracellular vesicles (EVs), but short fragment cfDNA was not observed in any fractions. However, in peripheral blood from a metastatic animal model both fragments were detected even with those same lung cancer cell lines. In human plasma samples, long fragment cfDNA was observed in the same fraction as that from conditioned media, and short fragment cfDNA existed in the supernatant after centrifugation at 100,000g. Concentration of ctDNA in the supernatant was two times higher than that in plasma isolated by the conventional procedure. Long fragment cfDNA associated with tumor progression might therefore be released into peripheral blood, and it is possible that the long fragment cfDNA escapes degradation by co-existing with EVs. Examination of the biological characteristics of long fragment cfDNA is a logical subject of further investigation.

Pancreatic ductal adenocarcinoma is one of the most aggressive types of cancer. Certain proteins in the tumor microenvironment have attracted considerable attention owing to their association with tumor invasion and metastasis. Here, we used proteomics to identify proteins associated with lymph-node metastasis, which is one of the prognostic factors. We selected lymph node metastasis-positive and -negative patients (n = 5 each) who underwent pancreatectomy between 2005 and 2015 and subjected to comprehensive proteomic profiling of tumor stroma. A total of 490 proteins were detected by mass spectrometry. Software analysis revealed that nine of these proteins were differentially expressed between the two patient groups. We focused on hemopexin and ferritin light chain based on immunohistochemistry results. We assessed the clinicopathological data of 163 patients and found that hemopexin expression was associated with UICC N2 (p = 0.0399), lymph node ratio (p = 0.0252), venous invasion (p = 0.0096), and lymphatic invasion (p = 0.0232). Notably, in vitro assays showed that hemopexin promotes invasion of the pancreatic cancer cells. Our findings suggest that hemopexin is a lymph node metastasis-associated protein that could potentially serve as a useful therapeutic target or biomarker of pancreatic ductal adenocarcinoma.

Several cohort studies have shown that periodontal disease is associated with an increased risk for stroke. However, it remains unclear whether serum antibody titers for a specific periodontal pathogen are associated with outcome after ischemic stroke, and which kinds of pathogens are associated with ischemic stroke. We examined the relationship between serum IgG titers to periodontal pathogens and outcome in ischemic stroke patients. A total of 445 patients with acute ischemic stroke (194 female [44.0%], mean age 71.9±12.3 years) were registered in this study. Serum IgG titers to 9 periodontal pathogens (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Prevotella nigrescens, Fusobacterium nucleatum, Treponema denticola, Tannerella forsythensis, Campylobacter rectus, Eikenella corrodens) were evaluated using the enzyme-linked immunosorbent assay (ELISA) method. An unfavorable outcome was defined as a 3 or higher on the modified Rankin Scale. The proportion of patients with unfavorable outcome was 25.4% (113 patients). Based on multivariate logistic regression analysis, numbers of IgG antibodies positive for periodontal pathogens (odds ratio 1.20, 95% CI 1.02-1.41, p = 0.03) were independent predictors of unfavorable outcome in ischemic stroke patients.

Oral health conditions and cerebral small vessel disease, such as white matter lesions or cerebral microbleeds (CMBs), are associated with the incidence of stroke. The purpose of this study was to examine the associations between oral health conditions (serum IgG titers of periodontal pathogens) with the presence or severity of CMBs in acute stroke patients. From January 2013 to April 2016, acute stroke patients were registered in two hospitals. Serum samples were evaluated for antibody titers against 9 periodontal pathogens using the ELISA method. The cut-off points for reactivity (the positive decision point) to each antigen were defined as more than a mean ELISA unit + 1 standard deviation (after logarithmic transformation) in all subjects. CMBs were evaluated on T2*-weighted MRI. In all, 639 patients were evaluated (ischemic, n = 533 and hemorrhagic, n = 106; 73.1 ± 12.9 years old). Among these patients, 627 were available for CMB evaluation. Among the 9 evaluated periodontal pathogens, only Campylobacter rectus (C. rectus) was associated with the presence of CMBs. the prevalence of positive serum antibody titers against C. rectus was higher among patients with CMBs than among those without CMBs (14.6% vs. 8.7%, P = 0.025). In addition, positive serum antibody titers against C. rectus remained one of the factors associated with the presence of CMBs in multivariate logistic analysis (odds ratio 2.03, 95% confidence interval 1.19-3.47, P = 0.010). A positive serum antibody titer against C. rectus was associated with the presence of CMBs in acute stroke patients.

To assess the influence of periodontal disease on cerebral hemorrhage and its clinical course, we examined the association of the serum IgG titer of periodontal pathogens with hemorrhage growth and 3-month outcome. We consecutively enrolled 115 patients with acute cerebral hemorrhage (44 females, aged 71.3 ± 13.1 years) and used ELISA to evaluate the serum IgG titers of 9 periodontal pathogens: Porphyromonas gingivalis, Aggregatibacter (A.) actinomycetemcomitans, Prevotella intermedia, Prevotella nigrescens, Fusobacterium (F.) nucleatum, Treponema denticola, Tannerella forsythensis, Campylobacter rectus, and Eikenella corrodens. Significant hematoma growth was defined as an increase in the volume of >33% or an absolute increase in the volume of >12.5 mL. A poor outcome was defined as a 3 or higher on the modified Rankin Scale. We observed hemorrhage growth in 13 patients (11.3%). Multivariate analysis revealed that increased IgG titers of A. actinomycetemcomitans independently predicted the elevated hemorrhage growth (odds ratio 5.26, 95% confidence interval 1.52-18.25, p = 0.01). Notably, augmented IgG titers of F. nucleatum but not A. actinomycetemcomitans led to a poorer 3-month outcome (odds ratio 7.86, 95% confidence interval 1.08-57.08, p = 0.04). Thus, we demonstrate that elevated serum IgG titers of A. actinomycetemcomitans are an independent factor for predicting cerebral hemorrhage growth and that high serum IgG titers of F. nucleatum may predict a poor outcome in patients with this disease. Together, these novel data reveal how systemic periodontal pathogens may affect stroke patients, and, should, therefore, be taken into consideration in the management and treatment of these individuals.

Ascertaining the accuracy of the pedicle screw (PS) trajectories is important as PS malpositioning can cause critical complications. We aimed to determine the angle range over which estimation is unreliable; build a low-cost PS placement support system that uses an inertial measurement unit (IMU) to enable the monitoring of surgical tools and PS trajectories, and determine the situations where IMU support would be most beneficial. In PS insertion experiments, we used cadaver samples that included lumbar porcine spines. Computed tomography images obtained before and after PS insertion were viewed. Offsets between the planned and implanted PS trajectories in the freehand and IMU-assisted groups were analyzed. The PS cortical bone breaches were classified according to the Gertzbein and Robbins criteria (GRC). Added head-down tilted sample experiments were repeated wherein we expected a decreased rostro-caudal rotational accuracy of the PS according to the angle estimation ability results. Evaluation of the PS trajectory accuracy revealed no significant advantage of IMU-assisted rostro-caudal rotational accuracy versus freehand accuracy. According to the GRC, IMU assistance significantly increased the rate of clinically acceptable PS positions (RoCA) than the freehand technique. In the head-down tilted sample experiments, IMU assist provided increased accuracies with both rostro-caudal and medial rotational techniques when compared with the freehand technique. In the freehand group, RoCA was significantly decreased in samples with rostral tilting relative to that in the samples without. However, In the IMU-assisted group, no significant difference in RoCA between the samples with and without head-down tilting was observed. Even when the planned PS medial and/or rostro-caudal rotational angle was relatively large and difficult to reproduce manually, IMU-support helped maintain the PS trajectory accuracy and positioning safety. IMU assist in PS placement was more beneficial, especially for larger rostro-caudal and/or medial rotational pedicle angles.

Matrix metalloproteinases (MMPs) contribute to many physiological and pathological phenomena via the proteolysis of extracellular matrix components. Specific blocking of the active site of each MMP sheds light on its particular role. However, it remains difficult to acquire an active-site inhibitor with high specificity for only the target MMP due to the highly conserved structure around the active site of MMPs. Recently, we reported that potent and specific inhibitors of serine proteases were obtained from our proprietary engineered serine protease inhibitor Kazal type 2 (SPINK2) library. In this research, using this library, we succeeded in obtaining potent and specific MMP-9 inhibitors. The obtained inhibitors bound to the active site of MMP-9 and inhibited MMP-9 with low nanomolar Ki values. The inhibitors did not cross-react with other MMPs that we tested. Further analysis using MMP-9 mutants demonstrated that the inhibitors recognize not only the residues around the conserved active site of MMP-9 but also different and unique residues in exosites that are distant from each other. This unique recognition manner, which can be achieved by the large interface provided by engineered SPINK2, may contribute to the generation of specific active-site inhibitors of MMPs.

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis and is an attractive therapeutic target for combating obesity and related diseases. Human BAT activity has been evaluated by 18F-fluorodeoxyglucose-positron emission tomography/computed tomography (18FDG-PET/CT) under acute cold exposure, but the method has some serious limitations, including radiation exposure. Infrared thermography (IRT) may be a simple and less-invasive alternative to evaluate BAT activity. In the present study, to establish an optimal condition for IRT, using a thermal imaging camera, skin temperature was measured in the supraclavicular region close to BAT depots (Tscv) and the control chest region (Tc) in 24 young healthy volunteers. Their BAT activity was assessed as the maximal standardized uptake value (SUVmax) by 18FDG-PET/CT. Under a warm condition at 24-27°C, no significant correlation was found between the IRT parameters (Tscv, Tc,, and the difference between Tscv and Tc,, Δtemp) and SUVmax, but 30-120 min after cold exposure at 19°C, Tscv and Δtemp were significantly correlated with SUVmax (r = 0.40-0.48 and r = 0.68-0.76). Δtemp after cold exposure was not affected by mean body temperature, body fatness, and skin blood flow. A lower correlation (r = 0.43) of Δtemp with SUVmax was also obtained when the participant's hands were immersed in water at 18°C for 5 min. Receiver operating characteristic analysis revealed that Δtemp after 30-60 min cold exposure can be used as an index for BAT evaluation with 74% sensitivity, 92% specificity, and 79% diagnostic accuracy. Thus, IRT may be useful as a simple and less-invasive method for evaluating BAT, particularly for large-scale screening and longitudinal repeat studies.

BACKGROUND:Diffuse axonal injury (DAI) is difficult to identify in the early phase of traumatic brain injury (TBI) using common diagnostic methods. Tau protein is localized specifically in nerve axons. We hypothesized that serum level of tau can be a useful biomarker to diagnose DAI in the early phase of TBI. METHODS & RESULTS:We measured serum tau levels in 40 TBI patients who were suspected of DAI within 6 hours after TBI to evaluate the accuracy of the tau level as a diagnostic marker for DAI. Diagnosis of DAI was confirmed according to magnetic resonance imaging (MRI) findings. The serum tau level in the DAI group (n = 13) was significantly higher than that in the non-DAI group (n = 27) (DAI vs. non-DAI, 25.3 [0 to 99.1] pg/mL vs. 0 [0 to 44.4] pg/mL, P = 0.03)). A receiver-operating characteristic curve to evaluate the diagnostic ability of serum tau level within 6 hours for DAI showed an area under the curve of 0.690 with 74.1% for sensitivity and 69.2% for specificity. Serum tau level was not significantly higher in unfavorable outcome group (Glasgow Outcome scale [GOS] score = 1-3 at hospital discharge) compared with favorable outcome group (GOS score = 4-5) (P = 0.19). CONCLUSIONS:Tau protein may be a useful biomarker for diagnosis of DAI in the early phase of TBI.

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 fusion protein, encoded by the Philadelphia chromosome, have drastically improved the outcomes for patients with chronic myeloid leukemia (CML). Although several real-time quantitative polymerase chain reaction (RQ-PCR) kits for the detection of BCR-ABL1 transcripts are commercially available, their accuracy and efficiency in laboratory practice require reevaluation. We have developed a new in-house RQ-PCR method to detect minimal residual disease (MRD) in CML cases. MRD was analyzed in 102 patients with CML from the DOMEST study, a clinical trial to study the rationale for imatinib mesylate discontinuation in Japan. The BCR-ABL1/ABL1 ratio was evaluated using the international standard (IS) ratio, where IS < 0.1% was defined as a major molecular response. At enrollment, BCR-ABL1 transcripts were undetectable in all samples using a widely-applied RQ-PCR method performed in the commercial laboratory, BML (BML Inc., Tokyo, Japan); however, the in-house method detected the BCR-ABL1 transcripts in five samples (5%) (mean IS ratio: 0.0062 ± 0.0010%). After discontinuation of imatinib, BCR-ABL1 transcripts were detected using the in-house RQ-PCR in 21 patients (21%) that were not positive using the BML method. Nineteen samples were also tested using a commercially available RQ-PCR assay kit with a detection limit of IS ratio, 0.0032 (ODK-1201, Otsuka Pharmaceutical Co., Tokyo, Japan). This method detected low levels of BCR-ABL1 transcripts in 14 samples (74%), but scored negative for five samples (26%) that were positive using the in-house method. From the perspective of the in-house RQ-PCR method, number of patients confirmed loss of MMR was 4. These data suggest that our new in-house RQ-PCR method is effective for monitoring MRD in CML.

We proposed a method for segmentation of brain tissues-gray matter, white matter, and cerebrospinal fluid-using multi-contrast images, including a T1 map and a uniform T1-weighted image, from a magnetization-prepared 2 rapid acquisition gradient echoes (MP2RAGE) sequence at 7 Tesla. The proposed method was evaluated with respect to the processing time and the similarity of the segmented masks of brain tissues with those obtained using FSL, FreeSurfer, and SPM12. The processing time of the proposed method (28 ± 0 s) was significantly shorter than those of FSL and SPM12 (444 ± 4 s and 159 ± 2 s for FSL and SPM12, respectively). In the similarity assessment, the tissue mask of the brain obtained by the proposed method showed higher consistency with those obtained using FSL than with those obtained using SPM12. The proposed method misclassified the subcortical structures and large vessels since it is based on the intensities of multi-contrast images obtained using MP2RAGE, which uses a similar segmentation approach as FSL but is not based on a template image or a parcellated brain atlas, which are used for FreeSurfer and SPM12, respectively. However, the proposed method showed good segmentation in the cerebellum and white matter in the medial part of the brain in comparison with the other methods. Thus, because the proposed method using different contrast images of MP2RAGE sequence showed the shortest processing time and similar segmentation ability as the other methods, it may be useful for both neuroimaging research and clinical diagnosis.

With rapid aging of the world's population, the demand for research, for a better understanding of aging and aging-related disorders, is increasing. Ideally, such research should be conducted on human subjects. However, due to ethical considerations, animals such as rodents and monkeys are used as alternatives. Among these alternative models, non-human primates are preferred because of their similarities with humans. The small South American common marmoset (Callithrix jacchus) may offer several advantages over other non-human primates in terms of its smaller size, shorter life-span, and dental anatomy identical to humans. The purpose of this study was to determine the viability of using the marmoset as a human oral disease model. We collected saliva samples from eight marmosets and eight human subjects. Prokaryotic DNA was extracted from the saliva samples, and 16S bacterial rRNA gene sequencing was performed on each of the samples. Our results indicated that the types of oral microbiomes detected among human and marmoset samples were nearly indistinguishable. In contrast, the oral microbiomes of our human and marmoset subjects were distinctly different from those reported for rats and dogs, which are currently popular research animals. The oral microbiomes of marmosets showed greater diversity than those of humans. However, the oral microbiota of marmosets exhibited less variation than those of humans, which may be attributed to the fact that the marmoset subjects were kept in a controlled environment with identical lifestyles. The characteristics of its oral microbiota, combined with other technical advantages, suggest that the marmoset may provide the best animal model thus far for the study of oral health. This study characterized the oral microbes of the marmoset, thereby providing information to support future application of the marmoset as a model for age-related oral disease.

We propose the fabrication of surface relief holograms via selective SiO2 deposition on soda-lime silicate glass substrates. Initially, the original hologram was recorded on an azobenzene photosensitive polymer film coated on the soda-lime silicate glass by irradiation with a conventional continuous wave Ar+ laser with a wavelength of 514.5 nm. The hologram was transferred to the soda-lime silicate glass surface via a corona discharge treatment as an index modulation hologram, which was created by partial substitution of protons for sodium ions during the corona discharge treatment in air. After the corona discharge treatment, the polymer film was removed from the substrate. The diffraction efficiency of the index hologram on the soda-lime silicate glass was estimated to be 5.8 × 10-2% at a wavelength of 532 nm. Finally, the glass substrate was subjected to corona discharge treatment in air with vaporized cyclic siloxane. A surface relief hologram with the diffraction efficiency of 2.3% was successfully fabricated on the soda-lime silicate glass.

This study aimed to elucidate spatial characteristics for magnetic resonance imaging (MRI) of cardiogenic cerebral embolism, to determine imaging biomarkers predicting patient outcome and cerebral herniation in cardioembolic stroke. This retrospective study assessed 90 patients with cardiogenic cerebral embolism. All images from MRI were normalized using a voxel-based symptom lesion mapping technique. Patients were categorized into two subgroups based on the outcome and presence of cerebral herniation. Each subgroup was assessed individually. The distribution map of all analyzed patients revealed accumulated ischemic lesions in bilateral middle cerebral artery areas. Ischemic lesions for the poor outcome group accumulated at the corona radiata on the right side and throughout the entire left hemisphere. Receiver operating characteristic (ROC) analysis suggested that a normalized ischemic volume of 62.8 mL allowed optimal differentiation between good and poor outcomes (sensitivity, 0.923; specificity, 0.923; area under the curve (AUC), 0.91) for left-side-dominant infarction. The distribution map for the cerebral herniation group revealed large ischemic areas in the left hemisphere. The analysis of differential involvement map with random permutation analysis showed that left anterior circulation infarcts were associated with midline shift. Receiver operating characteristic analysis revealed that a normalized infarction volume of 192.9 mL was highly predictive of cerebral herniation (sensitivity, 0.929; specificity, 0.750; AUC, 0.895). The medial frontal and occipital lobes, caudate head and basal ganglia were significantly involved in those patients who developed cerebral herniation. Ischemic volume contributed to outcomes and cerebral herniation. Ischemic lesions of the anterior and posterior cerebral arteries and basal ganglia in addition to middle cerebral artery area were identified as differences on MRI images between with and without cerebral herniation patients.

To obtain genetic information about the germplasm of tea (Camellia sinensis L.) in Japan, 167 accessions including 138 var. sinensis (96 Japanese var. sinensis and 42 exotic var. sinensis) and 29 Assam hybrids were analyzed using single nucleotide polymorphisms (SNPs) markers identified by double-digest restriction-site-associated DNA sequencing (ddRAD-seq) analysis. Approximately 10,000 SNPs were identified by ddRAD-seq and were mapped across the whole genome. The 167 tea accessions were classified into three genetic subgroups: (1) Japanese var. sinensis; (2) Japanese and exotic var. sinensis; (3) Assam hybrids and exotic var. sinensis. Leaf morphology varied widely within each genetic subgroups. The 96 Japanese var. sinensis were classified into four genetic subgroups as follows; two subgroups of Shizuoka (the largest tea production region) landraces, Uji (most ancient tea production region) landraces, and the pedigree of 'Yabukita', the leading green tea cultivar in Japan. These results indicated that the SNP markers obtained from ddRAD-seq are a useful tool to investigate the geographical background and breeding history of Japanese tea. This genetic information revealed the ancestral admixture situation of the 'Yabukita' pedigree, and showed that the genome structure of 'Yabukita' is clearly different from those of other Japanese accessions.

[This corrects the article DOI: 10.1371/journal.pone.0219436.].

RAS is the founding member of a superfamily of GTPases and regulates signaling pathways involved in cellular growth control. While recent studies have shown that the activation state of RAS can be controlled by lysine ubiquitylation and acetylation, the existence of lysine methylation of the RAS superfamily GTPases remains unexplored. In contrast to acetylation, methylation does not alter the side chain charge and it has been challenging to deduce its impact on protein structure by conventional amino acid substitutions. Herein, we investigate lysine methylation on RAS and RAS-related GTPases. We developed GoMADScan (Go language-based Modification Associated Database Scanner), a new user-friendly application that scans and extracts posttranslationally modified peptides from databases. The GoMADScan search on PhosphoSitePlus databases identified methylation of conserved lysine residues in the core GTPase domain of RAS superfamily GTPases, including residues corresponding to RAS Lys-5, Lys-16, and Lys-117. To follow up on these observations, we immunoprecipitated endogenous RAS from HEK293T cells, conducted mass spectrometric analysis and found that RAS residues, Lys-5 and Lys-147, undergo dimethylation and monomethylation, respectively. Since mutations of Lys-5 have been found in cancers and RASopathies, we set up molecular dynamics (MD) simulations to assess the putative impact of Lys-5 dimethylation on RAS structure. Results from our MD analyses predict that dimethylation of Lys-5 does not significantly alter RAS conformation, suggesting that Lys-5 methylation may alter existing protein interactions or create a docking site to foster new interactions. Taken together, our findings uncover the existence of lysine methylation as a novel posttranslational modification associated with RAS and the RAS superfamily GTPases, and putative impact of Lys-5 dimethylation on RAS structure.

BackgroundBlocking the PD-1 pathway induces immune-related adverse events (irAEs) which often involve the thyroid gland (thyroid irAEs). Clinical features of a thyroid irAE including its predictability and relationship to prognosis remain to be elucidated.MethodsTwo hundred consecutive patients treated with nivolumab at Kyoto University Hospital between September 1, 2014 and August 31, 2017 were included in a retrospective cohort study. We systematically determined and classified subclinical and overt thyroid irAEs based on data collected of serum free T4 and TSH levels. Baseline characteristics and detailed clinical data were analyzed, and analyses of overall survival (OS) excluded patients censored within 1 month from the first administration of nivolumab.ResultsSixty-seven patients (33.5%) developed thyroid irAEs and these were divided into a subclinical thyroid irAE group (n = 40, 20.0%) and an overt thyroid irAE group (n = 27, 13.5%). Patients with thyroid uptake of FDG-PET before treatment showed high incidences of overt thyroid irAE (adjusted odds ratio 14.48; 95% confidence interval [CI] 3.12-67.19), while the same relationship was not seen with subclinical thyroid irAE. Regarding the total cohort, the thyroid irAE (+) group had a significantly longer median OS than the thyroid irAE (-) group (16.1 versus 13.6 months, hazard ratio [HR] 0.61; 95% CI 0.39-0.93). In 112 non-excluded patients with lung cancer, the thyroid irAE (+) group similarly had a longer median OS than the thyroid irAE (-) group (not reached versus 14.2 months, HR 0.51; 95% CI 0.27-0.92). However, this observation was not seen in 41 non-excluded patients with malignant melanoma (12.0 versus 18.3 months, HR 1.54; 95% CI 0.67-3.43).ConclusionsBy thyroid uptake of FDG-PET, overt thyroid irAEs could be predicted before nivolumab therapy. Thyroid irAEs related to good prognosis in lung cancer but might be inconclusive in malignant melanoma.

MicroRNAs (miRNAs) are small RNA molecules that inhibit gene function by suppressing translation of target genes. However, in primary central nervous system lymphoma (PCNSL), the biological significance of miRNAs is largely unknown, although some miRNAs are known to be prognosis markers. Here, we analyzed 847 miRNAs expressed in 27 PCNSL specimens using microarray profiling and surveyed miRNA signature for prognostic prediction. Of these, 16 miRNAs were expressed in 27 PCNSL specimens at a frequency of 48%. Their variable importance measured by Random forest model revealed miR-192, miR-486, miR-28, miR-52, miR-181b, miR-194, miR-197, miR-93, miR-708, and let-7g as having positive effects; miR-29b-2*, miR-126, and miR-182 as having negative effects; and miR-18a*, miR-425, and miR-30d as neutral. After principal component analysis, the prediction formula for prognosis, consisting of the expression values of the above-mentioned miRNAs, clearly divided Kaplan-Meier survival curves by the calculated Z-score (HR = 6.4566, P = 0.0067). The 16 miRNAs were enriched by gene ontology terms including angiogenesis, cell migration and proliferation, and apoptosis, in addition to signaling pathways including TGF-β/SMAD, Notch, TNF, and MAPKinase. Their target genes included BCL2-related genes, HMGA2 oncogene, and LIN28B cancer stem cell marker. Furthermore, three miRNAs including miR-181b, miR-30d, and miR-93, selected from the 16 miRNAs, also showed comparable results for survival (HR = 8.9342, P = 0.0007), suggestive of a miRNA signature for prognostic prediction in PCNSL. These results indicate that this miRNA signature is useful for prognostic prediction in PCNSL and would help us understand target pathways for therapies in PCNSL.