Creation of Knock-In Alleles of Insulin Receptor Tagged by Fluorescent Proteins mCherry or EYFP in Fruit Fly Drosophila melanogaster.

The insulin/insulin-like growth factor-like signaling (IIS) pathway is highly conserved across metazoans and regulates numerous physiological functions, including development, metabolism, fecundity, and lifespan. The insulin receptor (InR), a crucial membrane receptor in the IIS pathway, is known to be ubiquitously expressed in various tissues, albeit at generally low levels, and its subcellular localization remains incompletely characterized. In this study, we employed CRISPR-mediated mutagenesis in the fruit fly Drosophila to create knock-in alleles of InR tagged with fluorescent proteins (InR::mCherry or InR::EYFP). By inserting the coding sequence of the fluorescent proteins mCherry or EYFP near the end of the coding sequence of the endogenous InR gene, we could trace the natural InR protein through their fluorescence. As an example, we investigated epithelial cells of the male accessory gland (AG), an internal reproductive organ, and identified two distinct patterns of InR::mCherry localization. In young AG, InR::mCherry accumulated on the basal plasma membrane between cells, whereas in mature AG, it exhibited intracellular localization as multiple puncta, indicating endocytic recycling of InR during cell growth. In the AG senescence accelerated by the mutation of Diuretic hormone 31 (Dh31), the presence of InR::mCherry puncta was more pronounced compared to the wild type. These findings raise expectations for the utility of the newly created InR::mCherry/EYFP alleles for studying the precise expression levels and subcellular localization of InR. Furthermore, this fluorescently tagged allele approach can be extended to investigate other membrane receptors with low abundance, facilitating the direct examination of their true expression and localization.

Intrinsic contractile dysfunction due to impaired sarcoplasmic reticulum Ca2+ release in compensatory hypertrophied muscle fibers following synergist ablation.

Synergist ablation (SA) is an experimental procedure for the induction of hypertrophy. However, SA causes a decrease in specific force (i.e., force per cross-sectional area), likely due to excessive muscle use. Here, we investigated the mechanisms behind the SA-induced intrinsic contractile dysfunction, especially focusing on the excitation-contraction (EC) coupling. Male Wistar rats had unilateral surgical ablation of gastrocnemius and soleus muscles to induce compensatory hypertrophy in the plantaris muscles. Two weeks after SA, plantaris muscle was dissected from each animal and used for later analyses. SA significantly increased the mean fiber cross-sectional area (+18%). On the other hand, the ratio of depolarization-induced force to the maximum Ca2+-activated specific force, an indicator of sarcoplasmic reticulum (SR) Ca2+ release, was markedly reduced in mechanically skinned fibers from the SA group (-51%). These functional defects were accompanied by an extensive fragmentation of the SR Ca2+ release channel, the ryanodine receptor 1 (RyR1), and a decrease in the amount of other triad proteins (i.e., DHPR, STAC3, and junctophilin1). SA treatment also caused activation of calpain-1 and increased the amount of NADPH oxidase 2, endoplasmic reticulum (ER) stress proteins (i.e., Grp78, Grp94, PDI, and Ero1), and lipid peroxidation [i.e., 4-hydroxynonenal (4-HNE)] in SA-treated muscles. Our findings show that SA causes skeletal muscle weakness due to impaired EC coupling. This is likely to be induced by Ca2+-dependent degradation of triad proteins, which may result from Ca2+ leak from fragmented RyR1 triggered by increased oxidative stress.NEW & NOTEWORTHY Synergist ablation (SA) has widely been used to understand the mechanisms behind skeletal muscle hypertrophy. However, compensatory hypertrophied muscles display intrinsic contractile dysfunction, i.e., a hallmark of overuse. Here, we demonstrate that SA-induced compensatory hypertrophy is accompanied by muscle weakness due to impaired sarcoplasmic reticulum Ca2+ release. This dysfunction may be caused by the degradation of triad proteins due to the reciprocal amplification of reactive oxygen species and Ca2+ signaling at the junctional space microdomain.

High-intensity interval training in the form of isometric contraction improves fatigue resistance in dystrophin-deficient muscle.

Duchenne muscular dystrophy is a genetic muscle-wasting disorder characterized by progressive muscle weakness and easy fatigability. Here we examined whether high-intensity interval training (HIIT) in the form of isometric contraction improves fatigue resistance in skeletal muscle from dystrophin-deficient mdx52 mice. Isometric HIIT was performed on plantar flexor muscles in vivo with supramaximal electrical stimulation every other day for 4 weeks (a total of 15 sessions). In the non-trained contralateral gastrocnemius muscle from mdx52 mice, the decreased fatigue resistance was associated with a reduction in the amount of peroxisome proliferator-activated receptor γ coactivator 1-α, citrate synthase activity, mitochondrial respiratory complex II, LC3B-II/I ratio, and mitophagy-related gene expression (i.e. Pink1, parkin, Bnip3 and Bcl2l13) as well as an increase in the phosphorylation levels of Src Tyr416 and Akt Ser473, the amount of p62, and the percentage of Evans Blue dye-positive area. Isometric HIIT restored all these alterations and markedly improved fatigue resistance in mdx52 muscles. Moreover, an acute bout of HIIT increased the phosphorylation levels of AMP-activated protein kinase (AMPK) Thr172, acetyl CoA carboxylase Ser79, unc-51-like autophagy activating kinase 1 (Ulk1) Ser555, and dynamin-related protein 1 (Drp1) Ser616 in mdx52 muscles. Thus, our data show that HIIT with isometric contractions significantly mitigates histological signs of pathology and improves fatigue resistance in dystrophin-deficient muscles. These beneficial effects can be explained by the restoration of mitochondrial function via AMPK-dependent induction of the mitophagy programme and de novo mitochondrial biogenesis. KEY POINTS: Skeletal muscle fatigue is often associated with Duchenne muscular dystrophy (DMD) and leads to an inability to perform daily tasks, profoundly decreasing quality of life. We examined the effect of high-intensity interval training (HIIT) in the form of isometric contraction on fatigue resistance in skeletal muscle from the mdx52 mouse model of DMD. Isometric HIIT counteracted the reduced fatigue resistance as well as dystrophic changes in skeletal muscle of mdx52 mice. This beneficial effect could be explained by the restoration of mitochondrial function via AMP-activated protein kinase-dependent mitochondrial biogenesis and the induction of the mitophagy programme in the dystrophic muscles.

Analyses of BER-referred noise-to-signal ratio in online measurements of high-baud-rate coherent receivers.

Digital coherent transmission features a very large transmission bandwidth and has played a main role in core optical transmission networks. With the progress of semiconductor technologies, practical coherent transceivers with rates over 100 Gbaud are becoming feasible. With such advances, the transceiver components must have lower power consumption and lower costs, and it becomes important to know how each component contributes to the overall transmission performance. Here, to decompose the effects of noise factors in high-baud-rate DP-16QAM transmissions, we used the theoretical relationship between the bit error rate (BER) and noise-to-signal ratio (NSR) and performed linear analyses. The NSR could be decomposed into individual noise contributions according to dependences on the inverse signal and local photocurrents. The obtained parameters were shown to be useful for predicting required optical signal-to-noise ratio (ROSNR) characteristics.

Generalizable brain network markers of major depressive disorder across multiple imaging sites.

Many studies have highlighted the difficulty inherent to the clinical application of fundamental neuroscience knowledge based on machine learning techniques. It is difficult to generalize machine learning brain markers to the data acquired from independent imaging sites, mainly due to large site differences in functional magnetic resonance imaging. We address the difficulty of finding a generalizable marker of major depressive disorder (MDD) that would distinguish patients from healthy controls based on resting-state functional connectivity patterns. For the discovery dataset with 713 participants from 4 imaging sites, we removed site differences using our recently developed harmonization method and developed a machine learning MDD classifier. The classifier achieved an approximately 70% generalization accuracy for an independent validation dataset with 521 participants from 5 different imaging sites. The successful generalization to a perfectly independent dataset acquired from multiple imaging sites is novel and ensures scientific reproducibility and clinical applicability.

Evaluation of the one-step nucleic acid amplification assay for detecting lymph node metastasis in patients with cervical and endometrial cancer: A multicenter prospective study.

OBJECTIVE: This multicenter study aimed to evaluate the accuracy of the one-step nucleic acid amplification (OSNA) assay in diagnosing lymph node metastasis (LNM) in patients with cervical and endometrial cancers. METHODS: Surgically removed LNs from patients with cervical and endometrial cancer were sectioned at 2-mm intervals along the short axis direction and alternately examined using the OSNA assay and conventional histopathological examination. Ultrastaging (200-µm LN sections) was performed for metastatic LNs using hematoxylin and eosin staining and immunostaining with an anti-CK19 antibody in cases where the OSNA assay and histopathological examination (performed using 2-mm LN sections) results showed discordance. RESULTS: A total of 437 LNs from 133 patients were included; 61 patients (14%) showed metastasis by histopathological examination, with a concordance rate of 0.979 (95% confidence interval [CI]: 0.961-0.991) with the OSNA assay. The sensitivity and specificity of the OSNA assay were 0.918 (95% CI: 0.819-0.973) and 0.989 (95% CI: 0.973-0.997), respectively. Discordance between the two methods was observed in nine LNs (2.1%), and allocation bias of metastatic foci was identified as the major cause of discordance. CONCLUSIONS: The OSNA assay showed equally accurate detection of LN metastasis as the histopathological examination. We suggest that the OSNA assay may be a useful tool for the rapid intraoperative diagnosis of LN metastasis in patients with cervical and endometrial cancers.

Development of a novel PET ligand, [11C]GO289 targeting CK2 expressed in the brain.

Positron emission tomography (PET) is a powerful imaging tool that enables early in vivo detection of Alzheimer's disease (AD). For this purpose, various PET ligands have been developed to image ß-amyloid and tau protein aggregates characteristically found in the brain of AD patients. In this study, we initiated to develop another type of PET ligand that targets protein kinase CK2 (formerly termed as casein kinase II), because its expression level is known to be altered in postmortem AD brains. CK2 is a serine/threonine protein kinase, an important component of cellular signaling pathways that control cellular degeneration. In AD, the CK2 level in the brain is thought to be elevated by its involvement in both phosphorylation of proteins such as tau and neuroinflammation. Decreased CK2 activity and expression levels lead to ß-amyloid accumulation. In addition, since CK2 also contributes to the phosphorylation of tau protein, the expression level and activity of CK2 is expected to undergo significant changes during the progression of AD pathology. Furthermore, CK2 could act as a potential target for modulating the inflammatory response in AD. Therefore, PET imaging targeting CK2 expressed in the brain could be a useful another imaging biomarker for AD. We synthesized and radiolabeled a CK2 inhibitor, [11C]GO289, in high yields from its precursor and [11C]methyl iodide under basic conditions. On autoradiography, [11C]GO289 specifically bound to CK2 in both rat and human brain sections. On baseline PET imaging, this ligand entered and rapidly washed out of the rat brain with its peak activity rather being small (SUV < 1.0). However, on blocking, there was no detectable CK2 specific binding signal. Thus, [11C]GO289 may be useful in vitro but not so in vivo in its current formulation. The lack of detectable specific binding signal in the latter may be due to a relatively high component of nonspecific binding signal in the overall rather weak PET signal, or it may also be related to the known fact that ATP can competitively binds to subunits of CK2, reducing its availability for this ligand. In the future, it will be necessary for PET imaging of CK2 to try out different non-ATP competitive formulations of CK2 inhibitor that can also provide significantly higher in vivo brain penetration.

Multicenter retrospective study of stereotactic body radiotherapy for patients with previously untreated initial small hepatocellular carcinoma.

AIM: We aimed to verify the therapeutic efficacy and safety of stereotactic body radiotherapy (SBRT) for previously untreated initial small hepatocellular carcinoma (HCC) in a multicenter, retrospective study. METHODS: Patients who underwent SBRT for HCC at the Japanese Society of Clinical Oncology (JCOG) member hospitals in Japan between July 2013 and December 2017 and met the following eligibility criteria were included: (1) initial HCC; (2) ≤3 nodules, ≤5 cm in diameter; (3) Child-Pugh score of A or B; and (4) unsuitability for or refusal of standard treatment. We analyzed the overall survival, recurrence-free survival, and cumulative incidence of local recurrence rate, and adverse events directly related to SBRT. RESULTS: Seventy-three patients with 79 lesions from 14 hospitals were analyzed. The median age was 77 years (range: 50-89 years), and the median tumor size was 23 mm (range: 6-50 mm). The median radiation dose was 40 Gy (range: 35-60 Gy) in five fractions (range: 4-8). The median follow-up period was 45 months (range: 0-103 months). The 3-year overall survival, recurrence-free survival, and cumulative incidence of local recurrence rates were 69.9% (95% CI: 58.7%-81%), 57.9% (95% CI: 45.2%-70.5%), and 20.0% (95% CI: 11.2%-30.5%), respectively. Four cases (5.5%) of adverse events of grade 3 or higher were reported: three cases of grade 3 and one case of grade 4 (duodenal ulcer). No grade 5 toxicities were observed. CONCLUSION: SBRT is a promising treatment modality, particularly for small HCCs, as they are not suitable for standard treatment.

Treatment satisfaction with molidustat in CKD-related anemia in non-dialysis patients: a post-hoc analysis of two clinical trials.

BACKGROUND: Erythropoiesis-stimulating agents (ESAs) are the standard treatment for patients with renal anemia to increase hemoglobin (Hb) levels and reduce the need for blood transfusions. However, treatments targeting high Hb levels require high doses of ESAs administered intravenously, which is associated with an elevated risk of adverse cardiovascular events. Furthermore, there have been some problems such as hemoglobin variability and low achievement of target hemoglobin due to the shorter half-lives of ESAs. Consequently, erythropoietin-promoting medications, such as hypoxia-inducible factor-prolyl hydroxylase (HIF-PH) inhibitors, have been developed. This study aimed to evaluate changes in the Treatment Satisfaction Questionnaire for Medicine version II (TSQM-II) domain scores relative to baseline in each trial, to assess patient satisfaction with molidustat versus darbepoetin alfa. METHODS: This post-hoc analysis of two clinical trials compared treatment satisfaction with an HIF-PH inhibitor, molidustat, versus a standard ESA, darbepoetin alfa, as part of therapy in patients with non-dialysis chronic kidney disease (CKD) and renal anemia. RESULTS: Exploratory outcome data using the TSQM-II showed that both arms in both trials had enhanced treatment satisfaction over the course of the study period, as well as improvements in most TSQM-II domains at week 24 of treatment. Molidustat was associated with convenience domain scores at multiple time points depending on the trial. More patients were highly satisfied with the convenience of molidustat than that of darbepoetin alfa. Patients treated with molidustat had increased global satisfaction domain scores compared with those treated with darbepoetin alfa; however, the differences in global satisfaction domain scores were not significant. CONCLUSION: These patient-reported satisfaction outcomes support the use of molidustat as a patient-centered treatment option for CKD-related anemia. REGISTRATION OF CLINICAL TRIALS: ClinicalTrials.gov Identifier: NCT03350321 (November 22, 2017). CLINICALTRIALS: gov Identifier: NCT03350347 (November 22, 2017).

Are sleep disturbances a cause or consequence of autism spectrum disorder?

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by core symptoms such as atypical social communication, stereotyped behaviors, and restricted interests. One of the comorbid symptoms of individuals with ASD is sleep disturbance. There are two major hypotheses regarding the neural mechanism underlying ASD, i.e., the excitation/inhibition (E/I) imbalance and the altered neuroplasticity hypotheses. However, the pathology of ASD remains unclear due to inconsistent research results. This paper argues that sleep is a confounding factor, thus, must be considered when examining the pathology of ASD because sleep plays an important role in modulating the E/I balance and neuroplasticity in the human brain. Investigation of the E/I balance and neuroplasticity during sleep might enhance our understanding of the neural mechanisms of ASD. It may also lead to the development of neurobiologically informed interventions to supplement existing psychosocial therapies.

Changes in preterm and extremely preterm birth rates in Japan after the introduction of obstetrical practice guidelines in 2008.

AIM: Obstetrical guidelines were established in Japan in 2008, and obstetrical diagnoses and treatments were subsequently standardized nationally. We examined changes in the preterm birth rate (PTBR) and extremely preterm birth rate (EPTBR) following the introduction of such guidelines. METHODS: Information on 50 706 432 live births in Japan between 1979 and 2021, including Japanese reproductive medicine, the childbearing age of pregnant women, and the employment status of reproductive-age women between 2007 and 2020, were obtained from the Japanese government and academic societies. Regression analysis was used to compare chronological changes nationally and those of eight Japanese regions. Regional and national average PTBRs and EPTBRs from 2007 to 2020 were compared by using a repeated measures analysis of variance. RESULTS: From 1979 to 2007, PTBRs and EPTBRs in Japan increased significantly. However, from 2008, the national PTBR and EPTBR decreased until 2020 (p < 0.001) and 2019 (p = 0.02), respectively. From 2007 to 2020, overall PTBR and EPTBR were 5.68% and 0.255%, respectively. A significant difference in the PTBR and EPTBR existed between the eight Japanese regions. During this period, the number of pregnancies using assisted reproductive technology increased from 19 595 to 60 381, pregnant women became older, the employment rate of those of reproductive age increased, and nonregular employment was 54%, which was 2.5 times higher than for men. CONCLUSIONS: In Japan, after obstetrical guidelines were enacted in 2008, PTRBs decreased significantly even under the pressure of increasing preterm births. Countermeasures may be necessary for regions showing high PTBRs.

Limitations of minimally invasive posterior cervical foraminotomy-a decompression method of posteriorly shifting the nerve root-in cases of large anterior osteophytes in cervical radiculopathy: A retrospective multicenter cohort study.

BACKGROUND: Posterior cervical foraminotomy against anterior osteophyte is an indirect decompression procedure but less invasive compared to anterior cervical discectomy and fusion. Residual compression to the nerve root may lead to poor surgical outcomes. Although clinical results of posterior cervical foraminotomy for osteophytes are not considered better than those of disk herniation, osteophyte size and the association of the decompression area with poor surgical outcomes remain unclear. This study aimed to identify the limitations of minimally invasive posterior cervical foraminotomy for cervical radiculopathy and discuss the methods to improve surgical outcomes. METHODS: We analyzed 55 consecutive patients with degenerative cervical radiculopathy who underwent minimally invasive posterior cervical foraminotomy. Minimum postoperative follow-up duration was 1 year. We divided the patients into nonimproved and improved groups. The cutoff value between preoperative and postoperative Neck Disability Index scores was 30% improvement. Preoperative imaging data comprised disk height, local kyphosis, spinal cord compression, anterior osteophytes in the foramen, and anterior osteophytes of >50% of the intervertebral foramen diameter. Postoperative imaging data comprised craniocaudal length and lateral width of decompressed lamina, preserved superior facet width, and area of decompressed lamina. RESULTS: Fifty-five patients were divided into two groups: nonimproved (n = 19) and improved (n = 36). The presence of osteophytes itself was not significant; however, the presence of osteophytes of >50% of the foramen diameter increased in the nonimproved group (P = 0.004). Mean lateral width and mean area of decompressed lamina after surgery significantly increased in the improved group (P = 0.001, P = 0.03). CONCLUSION: The presence of anterior osteophytes >50% of the diameter of the foramen led to poor improvement of clinical outcomes in minimally invasive posterior cervical foraminotomy. However, the larger the lateral width and area of the decompressed lamina, the better the surgical outcome.

Comparison between cryoballoon double stop and single stop in patients with paroxysmal atrial fibrillation.

BACKGROUND: Currently, cryoballoon (CB) thawing after single stop is generally performed. Previous research had reported that long thawing time using a single stop affects pulmonary veins tissue injury. However, it is uncertain whether CB thawing after single stop affects clinical outcomes. OBJECTIVE: This study aimed to clarify clinical significance of CB thawing in patients with paroxysmal atrial fibrillation. METHODS: Two hundred ten patients with paroxysmal atrial fibrillation who underwent CB from January 2018 to October 2019 were analyzed. We compared the clinical outcomes of patients whose CB applications were completely stopped with only the double stop technique (DS group, n = 99) and patients with single stop (SS group, n = 111). In DS group, we performed double stop technique for all CB application regardless of phrenic nerve injury or the temperature of esophagus. RESULTS: The atrial arrhythmia free-survival rate at 2 years after CB was significantly lower for the DS group than the SS group (76.8% vs 87.4%; p = 0.045). Complications occurred in 2 patients from the DS group and no complications were observed in patients from the SS group (p = 0.13). Mean procedural time was shorter in the DS group than in the SS group (53.1 vs 58.1 min; p = 0.046) CONCLUSION: DS group had higher recurrence rate than SS group. There was no significant difference regarding safety between both the groups. We found that the thawing process after single stop is very important for CB application.

Toward the development of a training program that promotes a "social model of disability" for physical therapists: a discussion on experiential learning surrounding the use of "acceptance of disability" by physical therapists.

[Purpose] This study aimed to extract knowledge for the development of a training program for creating a social model of disability for physical therapists, focusing on the experiential learning of those physical therapists who did not use acceptance of disability according to their subjective judgment. [Participants and Methods] The study included 11 physical therapists who were interviewed about their use of acceptance of disability and the circumstances leading to its non-use. [Results] The study identified the past and current use of acceptance of disability, as well as cases and reasons for its discontinuation, along with changes in clinical content. [Conclusion] The study extracted knowledge for the development of training programs in line with the components of the experiential learning model.

Dissociation of SH3 and cysteine-rich domain 3 and junctophilin 1 from dihydropyridine receptor in dystrophin-deficient muscles.

The disruption of excitation-contraction (EC) coupling and subsequent reduction in Ca2+ release from the sarcoplasmic reticulum (SR) have been shown to account for muscle weakness seen in patients with Duchenne muscular dystrophy (DMD). Here, we examined the mechanisms underlying EC uncoupling in skeletal muscles from mdx52 and DMD-null/NSG mice, animal models for DMD, focusing on the SH3 and cysteine-rich domain 3 (STAC3) and junctophilin 1 (JP1), which link the dihydropyridine receptor (DHPR) in the transverse tubule and the ryanodine receptor 1 in the SR. The isometric plantarflexion torque normalized to muscle weight of whole plantar flexor muscles was depressed in mdx52 and DMD-null/NSG mice compared with their control mice. This was accompanied by increased autolysis of calpain-1, decreased levels of STAC3 and JP1 content, and dissociation of STAC3 and JP1 from DHPR-α1s in gastrocnemius muscles. Moreover, in vitro mechanistic experiments demonstrated that STAC3 and JP1 underwent Ca2+-dependent proteolysis that was less pronounced in dystrophin-deficient muscles where calpastatin, the endogenous calpain inhibitor, was upregulated. Eccentric contractions further enhanced autolysis of calpain-1 and proteolysis of STAC3 and JP1 that were associated with severe torque depression in gastrocnemius muscles from DMD-null/NSG mice. These data suggest that Ca2+-dependent proteolysis of STAC3 and JP1 may be an essential factor causing muscle weakness due to EC coupling failure in dystrophin-deficient muscles.NEW & NOTEWORTHY The mechanisms underlying the disruption of excitation-contraction (EC) coupling in dystrophin-deficient muscles are not well understood. Here, using animal models for Duchenne muscular dystrophies (DMD), we show a Ca2+-dependent protease (calpain-1)-mediated proteolysis of SH3 and cysteine-rich domain 3 (STAC3) and junctophilin 1 (JP1), essential EC coupling proteins, in dystrophin-deficient muscle, and highlighting the dissociation of STAC3 and JP1 from dihydropyridine receptor as a causative factor in EC uncoupling of dystrophic muscles.

Development of antigen-prediction algorithm for personalized neoantigen vaccine using human leukocyte antigen transgenic mouse.

Immunotherapy is currently recognized as the fourth modality in cancer therapy. CTL can detect cancer cells via complexes involving human leukocyte antigen (HLA) class I molecules and peptides derived from tumor antigens, resulting in antigen-specific cancer rejection. The peptides may be predicted in silico using machine learning-based algorithms. Neopeptides, derived from neoantigens encoded by somatic mutations in cancer cells, are putative immunotherapy targets, as they have high tumor specificity and immunogenicity. Here, we used our pipeline to select 278 neoepitopes with high predictive "SCORE" from the tumor tissues of 46 patients with hepatocellular carcinoma or metastasis of colorectal carcinoma. We validated peptide immunogenicity and specificity by in vivo vaccination with HLA-A2, A24, B35, and B07 transgenic mice using ELISpot assay, in vitro and in vivo killing assays. We statistically evaluated the power of our prediction algorithm and demonstrated the capacity of our pipeline to predict neopeptides (area under the curve = 0.687, P < 0.0001). We also analyzed the potential of long peptides containing the predicted neoepitopes to induce CTLs. Our study indicated that the short peptides predicted using our algorithm may be intrinsically present in tumor cells as cleavage products of long peptides. Thus, we empirically demonstrated that the accuracy and specificity of our prediction tools may be potentially improved in vivo using the HLA transgenic mouse model. Our data will help to design feedback algorithms to improve in silico prediction, potentially allowing researchers to predict peptides for personalized immunotherapy.

Larger improvements in fatigue resistance and mitochondrial function with high- than with low-intensity contractions during interval training of mouse skeletal muscle.

Interval training (IT) results in improved fatigue resistance in skeletal muscle mainly due to an increased aerobic capacity, which involves increased muscle mitochondrial content and/or improved mitochondrial function. We hypothesized that IT with high-intensity contractions is more effective in increasing mitochondrial function, and hence fatigue resistance, than low-intensity contractions. To study this hypothesis without interference from differences in muscle fiber recruitment obliged to occur during voluntary contractions, IT was performed with in situ supramaximal electrical stimulation where all muscle fibers are recruited. We compared the effect of IT with repeated low-intensity (20 Hz stimulation, IT20) and high-intensity (100 Hz stimulation, IT100) contractions on fatigue resistance and mitochondrial content and function in mouse plantar flexor muscles. Muscles were stimulated every other day for 4 weeks. The averaged peak torque during IT bouts was 4.2-fold higher with IT100 than with IT20. Both stimulation protocols markedly improved in situ fatigue resistance, although the improvement was larger with IT100. The citrate synthase activity, a biomarker of mitochondrial content, was similarly increased with IT20 and IT100. Conversely, increased expression of mitochondrial respiratory chain (MRC) complexes I, III, and IV was only observed with IT100 and this was accompanied by increases in MRC supercomplex formation and pyruvate-malate-driven state 3 respiration in isolated mitochondria. In conclusion, the IT-induced increase in fatigue resistance is larger with high-intensity than with low-intensity contractions and this is linked to improved mitochondrial function due to increased expression of MRC complexes and assembly of MRC supercomplexes.

Harmonization of resting-state functional MRI data across multiple imaging sites via the separation of site differences into sampling bias and measurement bias.

When collecting large amounts of neuroimaging data associated with psychiatric disorders, images must be acquired from multiple sites because of the limited capacity of a single site. However, site differences represent a barrier when acquiring multisite neuroimaging data. We utilized a traveling-subject dataset in conjunction with a multisite, multidisorder dataset to demonstrate that site differences are composed of biological sampling bias and engineering measurement bias. The effects on resting-state functional MRI connectivity based on pairwise correlations because of both bias types were greater than or equal to psychiatric disorder differences. Furthermore, our findings indicated that each site can sample only from a subpopulation of participants. This result suggests that it is essential to collect large amounts of neuroimaging data from as many sites as possible to appropriately estimate the distribution of the grand population. Finally, we developed a novel harmonization method that removed only the measurement bias by using a traveling-subject dataset and achieved the reduction of the measurement bias by 29% and improvement of the signal-to-noise ratios by 40%. Our results provide fundamental knowledge regarding site effects, which is important for future research using multisite, multidisorder resting-state functional MRI data.

[11C]NCGG401, a novel PET ligand for imaging of colony stimulating factor 1 receptors.

Colony-stimulating factor 1 receptors (CSF1R) are expressed exclusively on microglia in the central nervous system. The receptors regulate immune responses by controlling the survival and activity of microglia and are intricately involved in the pathophysiology of Alzheimer's disease. In this study, we developed [11C]NCGG401, a positron emission tomography (PET) ligand, targeting for CSF1R as an imaging biomarker for microglial pathophysiology in Alzheimer's disease. NCGG401 showed a high potency to inhibit human CSF1R kinase activity and a high binding affinity to human CSF1R. PET imaging with [11C]NCGG401 in healthy rats showed a good brain permeability. Furthermore, the specific binding component was determined by postmortem autoradiography in rat brain and human hippocampal sections. The knowledge of the characteristics of [11C]NCCC401, our initial CSF1R compound, we have obtained may be useful for further development and optimization of CSF1R radioligands for PET imaging of microglia.

The importance of specific citrullinated clusterin and vimentin found in a multi-coloured bead-based citrulline-peptide array system in rheumatoid arthritis.

OBJECTIVES: The importance of citrullination in rheumatoid arthritis (RA) has been reported, but the degree to which individual citrullinated proteins affect the onset and progression of RA is still unclear. We aimed to identify citrullinated proteins that may play an important role in the onset and progression of RA using an individualised anti-citrullinated protein antibody (ACPA) evaluation system with citrullinated peptides as probes. METHODS: Serum samples from 50 normal donors and 51 RA patients were evaluated using a custom MagPlexTM bead array with 13 types of citrullinated peptide. The presence/absence of ACPAs that react to each citrullinated peptide in each subject was determined using the Z-score, which was calculated based on the fluorescence intensity distribution of a sample from a normal donor. Whether the fluorescence intensity was inhibited when free citrullinated peptides were added to a system was also evaluated. RESULTS: Median fluorescence intensities obtained from beads coupled with the 13 types of citrullinated peptide were all significantly higher in RA patients versus normal donors. With a Z-score ≥2 as the cut-off value for the presence of ACPAs, ACPAs that recognised five types of citrullinated peptides derived from fibrinogen A, fillagrin, clusterin, and vimentin were widely detected in RA patients. In addition, inhibition experiments showed that citrullinated vimentin, clusterin, and enolase 1A peptides inhibited coupling of ACPAs to other citrullinated peptides. CONCLUSIONS: ACPAs to many citrullinated proteins exhibited cross-reactivity to citrullinated clusterin and vimentin, suggesting the importance of citrullinated clusterin and vimentin in the early stages of RA pathogenesis.