Induction of fetal meiotic oocytes from embryonic stem cells in cynomolgus monkeys.

Human in vitro oogenesis provides a framework for clarifying the mechanism of human oogenesis. To create its benchmark, it is vital to promote in vitro oogenesis using a model physiologically close to humans. Here, we establish a foundation for in vitro oogenesis in cynomolgus (cy) monkeys (Macaca fascicularis): cy female embryonic stem cells harboring one active and one inactive X chromosome (Xa and Xi, respectively) differentiate robustly into primordial germ cell-like cells, which in xenogeneic reconstituted ovaries develop efficiently into oogonia and, remarkably, further into meiotic oocytes at the zygotene stage. This differentiation entails comprehensive epigenetic reprogramming, including Xi reprogramming, yet Xa and Xi remain epigenetically asymmetric with, as partly observed in vivo, incomplete Xi reactivation. In humans and monkeys, the Xi epigenome in pluripotent stem cells functions as an Xi-reprogramming determinant. We further show that developmental pathway over-activations with suboptimal up-regulation of relevant meiotic genes impede in vitro meiotic progression. Cy in vitro oogenesis exhibits critical homology with the human system, including with respect to bottlenecks, providing a salient model for advancing human in vitro oogenesis.

Ex vivo reconstitution of fetal oocyte development in humans and cynomolgus monkeys.

In vitro oogenesis is key to elucidating the mechanism of human female germ-cell development and its anomalies. Accordingly, pluripotent stem cells have been induced into primordial germ cell-like cells and into oogonia with epigenetic reprogramming, yet further reconstitutions remain a challenge. Here, we demonstrate ex vivo reconstitution of fetal oocyte development in both humans and cynomolgus monkeys (Macaca fascicularis). With an optimized culture of fetal ovary reaggregates over three months, human and monkey oogonia enter and complete the first meiotic prophase to differentiate into diplotene oocytes that form primordial follicles, the source for oogenesis in adults. The cytological and transcriptomic progressions of fetal oocyte development in vitro closely recapitulate those in vivo. A comparison of single-cell transcriptomes among humans, monkeys, and mice unravels primate-specific and conserved programs driving fetal oocyte development, the former including a distinct transcriptomic transformation upon oogonia-to-oocyte transition and the latter including two active X chromosomes with little X-chromosome upregulation. Our study provides a critical step forward for realizing human in vitro oogenesis and uncovers salient characteristics of fetal oocyte development in primates.

Circadian protection against bacterial skin infection by epidermal CXCL14-mediated innate immunity.

The epidermis is the outermost layer of the skin and the body's primary barrier to external pathogens; however, the early epidermal immune response remains to be mechanistically understood. We show that the chemokine CXCL14, produced by epidermal keratinocytes, exhibits robust circadian fluctuations and initiates innate immunity. Clearance of the skin pathogen Staphylococcus aureus in nocturnal mice was associated with CXCL14 expression, which was high during subjective daytime and low at night. In contrast, in marmosets, a diurnal primate, circadian CXCL14 expression was reversed. Rhythmically expressed CXCL14 binds to S. aureus DNA and induces inflammatory cytokine production by activating Toll-like receptor (TLR)9-dependent innate pathways in dendritic cells and macrophages underneath the epidermis. CXCL14 also promoted phagocytosis by macrophages in a TLR9-independent manner. These data indicate that circadian production of the epidermal chemokine CXCL14 rhythmically suppresses skin bacterial proliferation in mammals by activating the innate immune system.

Long-term expansion with germline potential of human primordial germ cell-like cells in vitro.

Human germ cells perpetuate human genetic and epigenetic information. However, the underlying mechanism remains elusive, due to a lack of appropriate experimental systems. Here, we show that human primordial germ cell-like cells (hPGCLCs) derived from human-induced pluripotent stem cells (hiPSCs) can be propagated to at least ~106 -fold over a period of 4 months under a defined condition in vitro. During expansion, hPGCLCs maintain an early hPGC-like transcriptome and preserve their genome-wide DNA methylation profiles, most likely due to retention of maintenance DNA methyltransferase activity. These characteristics contrast starkly with those of mouse PGCLCs, which, under an analogous condition, show a limited propagation (up to ~50-fold) and persist only around 1 week, yet undergo cell-autonomous genome-wide DNA demethylation. Importantly, upon aggregation culture with mouse embryonic ovarian somatic cells in xenogeneic-reconstituted ovaries, expanded hPGCLCs initiate genome-wide DNA demethylation and differentiate into oogonia/gonocyte-like cells, demonstrating their germline potential. By creating a paradigm for hPGCLC expansion, our study uncovers critical divergences in expansion potential and the mechanism for epigenetic reprogramming between the human and mouse germ cell lineage.

Proton Pump Inhibitors and Cyclin-Dependent Kinase 4/6 Inhibitors in Patients With Breast Cancer.

BACKGROUND: Proton pump inhibitors (PPIs) reduce the bioavailability of several anticancer drugs. The impact of PPIs co-administered with cyclin-dependent kinase 4 and 6 inhibitors is controversial. We aimed to clarify whether the concomitant use of PPIs impacts palbociclib and abemaciclib effectiveness in breast cancer treatment. PATIENTS AND METHODS: This multicenter, retrospective, observational study, conducted across 4 medical institutions in Japan, consecutively included patients with endocrine-resistant metastatic breast cancer, receiving palbociclib or abemaciclib between December 2017 and August 2022. Propensity score-matched analyses were performed. Treatment efficacy and safety with and without PPIs were compared. Progression-free survival and overall survival were estimated using the Kaplan-Meier method and compared using a log-rank test. A Cox proportional hazards model was used to estimate the hazard ratio. RESULTS: The study included 240 patients. After 1:1 matching, 112 patients were treated with and without PPIs. The median progression-free survival period was 1.2 years in the PPI group and 1.3 years in the non-PPI group (hazard ratio, 1.19; 95% CI, 0.70-2.02). The median overall survival period was 3.6 years in the PPI group, whereas it was not reached in the non-PPI group (hazard ratio, 1.23; 95% CI, 0.61-2.47). Consistent results were obtained for subgroups receiving palbociclib (n = 177) and abemaciclib (n = 63) without propensity score matching. Adverse event incidence and severity were similar in both groups. CONCLUSION: The effectiveness of cyclin-dependent kinase 4/6 inhibitors is unlikely to be affected by concomitant PPI use. Future prospective pharmacokinetic studies are warranted.

Linking LRP12 CGG repeat expansion to inherited peripheral neuropathy.

BACKGROUND: The causative genes for over 60% of inherited peripheral neuropathy (IPN) remain unidentified. This study endeavours to enhance the genetic diagnostic rate in IPN cases by conducting screenings focused on non-coding repeat expansions. METHODS: We gathered data from 2424 unrelated Japanese patients diagnosed with IPN, among whom 1555 cases with unidentified genetic causes, as determined through comprehensive prescreening analyses, were selected for the study. Screening for CGG non-coding repeat expansions in LRP12, GIPC1 and RILPL1 genes was conducted using PCR and long-read sequencing technologies. RESULTS: We identified CGG repeat expansions in LRP12 from 44 cases, establishing it as the fourth most common aetiology in Japanese IPN. Most cases (29/37) exhibited distal limb weakness, without ptosis, ophthalmoplegia, facial muscle weakness or bulbar palsy. Neurogenic changes were frequently observed in both needle electromyography (97%) and skeletal muscle tissue (100%). In nerve conduction studies, 28 cases primarily showed impairment in motor nerves without concurrent involvement of sensory nerves, consistent with the phenotype of hereditary motor neuropathy. In seven cases, both motor and sensory nerves were affected, resembling the Charcot-Marie-Tooth (CMT) phenotype. Importantly, the mean CGG repeat number detected in the present patients was significantly shorter than that of patients with LRP12-oculopharyngodistal myopathy (p<0.0001). Additionally, GIPC1 and RILPL1 repeat expansions were absent in our IPN cases. CONCLUSION: We initially elucidate LRP12 repeat expansions as a prevalent cause of CMT, highlighting the necessity for an adapted screening strategy in clinical practice, particularly when addressing patients with IPN.

Evaluation of Medication Adherence Among Prevalent Users in Hypertension, Dyslipidemia, and Diabetes Using Health Insurance Claims: A Population-Based Cohort Study in Japan.

PURPOSE: Hypertension (HT), dyslipidemia (DL), and diabetes mellitus (DM) are major risk factors for cardiovascular diseases. Despite the wide availability of medications to reduce this risk, poor adherence to medications remains an issue. The aim of this study is to evaluate medication adherence of prevalent users in these disease medications (HT, DL, DM) using claims data. Factors associated with non-adherence were also examined. METHODS: Of 7538 participants of the Tsuruoka Metabolomics Cohort Study, 3693 (HT: 2702, DL: 2112, DM: 661) were identified as prevalent users of these disease medications. Information on lifestyle was collected through a questionnaire. Adherence was assessed by a proportion of days covered (PDC) and participants with PDC ≥0.8 were defined as adherent. Predictors of non-adherence were determined by performing multivariable logistic regression. RESULTS: Medication adherence differed by treatment status. Among those without comorbidities, those with HT-only showed the highest adherence (90.2%), followed by those with DM-only (81.2%) and those with DL-only (80.8%). Factors associated with non-adherence in each medication group were skipping breakfast and poor understanding of medications among those with HT medications, females, having comorbidities, having a history of heart disease, and drinking habit among those with DL medications, and good sleep quality and skipping breakfast among those with DM medications. CONCLUSION: While participants showed high medication adherence, differences were observed across medication groups. The identified predictors of non-adherence could help target those in need of adherence support.

Interprofessional competency in clinical students: validating the Clinical student version of the Japanese Self-assessment Scale of Interprofessional Competency (C-JASSIC).

In this study, we developed and validated the Clinical Student Version of the Japanese Interprofessional Competency Self-Assessment Scale (C-JASSIC) for healthcare students in their clinical practice phase. Data obtained from 331 students (medical, 98; nursing, 99; pharmacy, 134) during orientation for interprofessional education (pre-IPE) and from 319 students (medical, 94; nursing, 93; pharmacy, 132) within a week following IPE (post-IPE) were analyzed. Exploratory pre-IPE and Confirmatory post-IPE factor analyses revealed a consistent 6-factor structure aligning with the Japanese Interprofessional Competency domains. The scale exhibited strong internal consistency, with Cronbach's α values exceeding 0.8 for all factors both pre- and post-IPE. Scores for overall competency and individual domains increased post-IPE in 234 matched cases. A notable significant pre vs post difference concerned "Understanding of Others," indicating enhanced interprofessional comprehension after clinical practice. There was a weak but significant positive correlation between IPE satisfaction and difference in pre- and post-IPE scores. However, no significant differences were observed among medical, nursing, and pharmacy students. Despite its strengths, such as its competency-based design and cultural relevance to Japan, a limitation of the study may be potential self-reporting bias. Nonetheless, C-JASSIC represents a valuable tool for seamless competency evaluations from student to professional stages, with implications for broader Asian contexts.

[Validity assessment of self-reported medication use in a pharmacoepidemiologic study by comparison with prescription record review].

Objectives　Although self-reported questionnaires are widely used to collect information on medication use in epidemiological studies, their validity for studies involving older adults has not been sufficiently assessed. This study evaluated the validity of self-reported medication use using questionnaires in comparison with drug notebooks.Methods　The study enrolled 370 older community dwellers who participated in an aging sub-study survey of the Tsuruoka Metabolomics Cohort Study between April 2019 and March 2021. Medication use was assessed by comparing self-reported questionnaire data with drug notebook records. We analyzed medications used for hypertension, dyslipidemia, myocardial infarction, angina, diabetes, rheumatism, osteoporosis/metabolic bone disease, constipation, anxiety/depression, dementia, asthma, allergy, thrombosis, and thyroid disease. Moreover, gastrointestinal (GI) medications, steroids, and antipyretic analgesics were assessed, and data on injectable medications for osteoporosis/metabolic bone disease was collected. Using drug notebook records, we identified regular medication users by assessing whether they had received oral medication prescriptions covering over 28 days and took the medication within the 90 days preceding the day of their survey. To define medication categories, we used Anatomical Therapeutic Chemical (ATC) classification codes. Sensitivity, specificity, and kappa statistics were calculated for each medication using drug notebooks as standards. Those who did not bring their drug notebooks on the day of the survey were defined as non-medication users.Results　The mean age (standard deviation) of the 370 participants (146 men and 224 women) was 73.3 (4.0) years. The sensitivity and specificity for each medication were as follows: hypertension (0.97, 0.97), dyslipidemia (0.93, 0.98), myocardial infarction (0.24, 0.99), diabetes (0.94, 1.00), rheumatism (1.00, 1.00), osteoporosis/metabolic bone disease (0.82, 0.99), constipation (0.71, 0.98), GI conditions (0.63, 0.97), anxiety/depression (0.36, 1.00), dementia (0.67, 1.00), asthma (0.67, 0.98), allergy (0.57, 0.99), thrombosis (0.88, 0.98), steroids (0.80, 0.99), thyroid disease (1.00, 1.00) and antipyretic analgesics (0.75, 0.96).Conclusions　Although sensitivity and specificity differed by medication categories, the results of our population-based cohort study suggested that self-reported questionnaires on medication use among older adults are valid, especially for medications with high sensitivity (≥ 0.8).

Population Pharmacokinetics, Pharmacogenomics, and Adverse Events of Osimertinib and its Two Active Metabolites, AZ5104 and AZ7550, in Japanese Patients with Advanced Non-small Cell Lung Cancer: a Prospective Observational Study.

BACKGROUND: Potential novel strategies for adverse event (AE) management of osimertinib therapy, including therapeutic drug monitoring and the use of biomarkers, have not yet been fully investigated. This study aimed to evaluate (1) the relationship between exposure to osimertinib, especially its active metabolites (AZ5104 and AZ7550), and AEs, and (2) the relationship between germline polymorphisms and AEs. METHODS: We conducted a prospective, longitudinal observational study of 53 patients with advanced non-small cell lung cancer receiving osimertinib therapy from February 2019 to April 2022. A population pharmacokinetic model was developed to estimate the area under the serum concentration-time curve from 0 to 24 h (AUC0-24) of osimertinib and its metabolites. Germline polymorphisms were analyzed using TaqMan® SNP genotyping and CycleavePCR® assays. RESULTS: There was a significant association between the AUC0-24 of AZ7550 and grade ≥ 2 paronychia (p = 0.043) or anorexia (p = 0.011) and between that of osimertinib or AZ5104 and grade ≥ 2 diarrhea (p = 0.026 and p = 0.049, respectively). Furthermore, the AUC0-24 of AZ5104 was significantly associated with any grade ≥ 2 AEs (p = 0.046). EGFR rs2293348 and rs4947492 were associated with severe AEs (p = 0.019 and p = 0.050, respectively), and ABCG2 rs2231137 and ABCB1 rs1128503 were associated with grade ≥ 2 AEs (p = 0.008 and p = 0.038, respectively). CONCLUSION: Higher exposures to osimertinib, AZ5104, and AZ7550 and polymorphisms in EGFR, ABCG2, and ABCB1 were related to higher severity of AEs; therefore, monitoring these may be beneficial for osimertinib AE management.

Clinical phenotypic diversity of NOTCH2NLC-related disease in the largest case series of inherited peripheral neuropathy in Japan.

BACKGROUND: NOTCH2NLC GGC repeat expansions have been associated with various neurogenerative disorders, including neuronal intranuclear inclusion disease and inherited peripheral neuropathies (IPNs). However, only a few NOTCH2NLC-related disease studies in IPN have been reported, and the clinical and genetic spectra remain unclear. Thus, this study aimed to describe the clinical and genetic manifestations of NOTCH2NLC-related IPNs. METHOD: Among 2692 Japanese patients clinically diagnosed with IPN/Charcot-Marie-Tooth disease (CMT), we analysed NOTCH2NLC repeat expansion in 1783 unrelated patients without a genetic diagnosis. Screening and repeat size determination of NOTCH2NLC repeat expansion were performed using repeat-primed PCR and fluorescence amplicon length analysis-PCR. RESULTS: NOTCH2NLC repeat expansions were identified in 26 cases of IPN/CMT from 22 unrelated families. The mean median motor nerve conduction velocity was 41 m/s (range, 30.8-59.4), and 18 cases (69%) were classified as intermediate CMT. The mean age of onset was 32.7 (range, 7-61) years. In addition to motor sensory neuropathy symptoms, dysautonomia and involuntary movements were common (44% and 29%). Furthermore, the correlation between the age of onset or clinical symptoms and the repeat size remains unclear. CONCLUSIONS: These findings of this study help us understand the clinical heterogeneity of NOTCH2NLC-related disease, such as non-length-dependent motor dominant phenotype and prominent autonomic involvement. This study also emphasise the importance of genetic screening, regardless of the age of onset and type of CMT, particularly in patients of Asian origin, presenting with intermediate conduction velocities and dysautonomia.

In vivo Pharmacokinetics/Pharmacodynamics Profiles for Appropriate Doses of Cefditoren pivoxil against S. pneumoniae in Murine Lung-Infection Model.

PURPOSE: Cefditoren, the active form of cefditoren pivoxil, is an oral cephalosporin antimicrobial drug. Although cefditoren exhibits high antimicrobial activity against Streptococcus pneumoniae, its pharmacokinetics/pharmacodynamics (PK/PD) characteristics remain unknown. This study aimed to determine its PK/PD parameter with target values for cefditoren against S. pneumoniae in S. pneumoniae lung-infected mice and to simulate MIC range of S. pneumoniae that can be expected to be treated at approved cefditoren doses in human using population pharmacokinetic (PPK) data from patients. METHODS: Susceptibility testing and time-kill assays against S. pneumoniae ATCC® 49619 were performed for in vitro PD evaluation. Based on the results of a PK study in healthy mice and PD studies in S. pneumoniae lung-infected mice, optimal PK/PD parameters were determined using the correlation curve between the PK/PD parameters and lung bacterial count changes. The target value was calculated to achieve a 2 log10 reduction in the lung bacterial counts. RESULTS: In vitro PD evaluation showed that cefditoren had a potent antimicrobial effect against S. pneumoniae in a time-dependent manner at concentrations above the MIC. In PK/PD analyses, both fAUC24/MIC and fCmax/MIC were well correlated with bactericidal efficacy, achieving 2 log10-kill with fAUC24/MIC ≥ 63 and fCmax/MIC ≥ 16. CONCLUSIONS: Cefditoren pivoxil has good therapeutic efficacy against acute pneumonia caused by S. pneumoniae with a MIC ≤ 0.031-0.063 mg/L at approved doses in adults and children.

HTLV-1-associated myelopathy/tropical spastic paraplegia with sporadic late-onset nemaline myopathy: a case report.

BACKGROUND: Sporadic late onset nemaline myopathy (SLONM) is a muscle disorder characterized by the presence of nemaline rods in muscle fibers. SLONM has no known genetic cause but has been associated with monoclonal gammopathy of undetermined significance and with human immunodeficiency virus (HIV) infection. Human T-cell leukemia virus-1 (HTLV-1) is a known causative agent of adult T-cell leukemia/lymphoma and HTLV-1 associated myelopathy/tropical spastic paraplegia (HAM/TSP), a chronic inflammatory neurological disease. HTLV-1 has been reported to be implicated in inflammatory myopathies, as well as in HIV infection.; however, there have been no reports of an association between HTLV-1 infection and SLONM to date. CASE PRESENTATION: A 70-year-old Japanese woman presented with gait disturbance, lumbar kyphosis, and respiratory dysfunction. The diagnosis of HAM/TSP with SLONM was made based on characteristic clinical symptoms of HAM/TSP, such as spasticity in the lower extremities, and cerebrospinal fluid test results; and of SLONM, such as generalized head drooping, respiratory failure, and muscle biopsy results. Steroid treatment was initiated and improvement in her stooped posture was observed after 3 days of treatment. CONCLUSION: This is the first case report of SLONM combined with HTLV-1 infection. Further studies are needed to elucidate the relationship between retroviruses and muscle diseases.

Development of a novel drug information provision system for Kampo medicine using natural language processing technology.

BACKGROUND: Kampo medicine is widely used in Japan; however, most physicians and pharmacists have insufficient knowledge and experience in it. Although a chatbot-style system using machine learning and natural language processing has been used in some clinical settings and proven useful, the system developed specifically for the Japanese language using this method has not been validated by research. The purpose of this study is to develop a novel drug information provision system for Kampo medicines using a natural language classifier® (NLC®) based on IBM Watson. METHODS: The target Kampo formulas were 33 formulas listed in the 17th revision of the Japanese Pharmacopoeia. The information included in the system comes from the package inserts of Kampo medicines, Manuals for Management of Individual Serious Adverse Drug Reactions, and data on off-label usage. The system developed in this study classifies questions about the drug information of Kampo formulas input by natural language into preset questions and outputs preset answers for the questions. The system uses morphological analysis, synonym conversion by thesaurus, and NLC®. We fine-tuned the information registered into NLC® and increased the thesaurus. To validate the system, 900 validation questions were provided by six pharmacists who were classified into high or low levels of knowledge and experience of Kampo medicines and three pharmacy students. RESULTS: The precision, recall, and F-measure of the system performance were 0.986, 0.915, and 0.949, respectively. The results were stable even with differences in the amount of expertise of the question authors. CONCLUSIONS: We developed a system using natural language classification that can give appropriate answers to most of the validation questions.

A developmental coordinate of pluripotency among mice, monkeys and humans.

The epiblast (EPI) is the origin of all somatic and germ cells in mammals, and of pluripotent stem cells in vitro. To explore the ontogeny of human and primate pluripotency, here we perform comprehensive single-cell RNA sequencing for pre- and post-implantation EPI development in cynomolgus monkeys (Macaca fascicularis). We show that after specification in the blastocysts, EPI from cynomolgus monkeys (cyEPI) undergoes major transcriptome changes on implantation. Thereafter, while generating gastrulating cells, cyEPI stably maintains its transcriptome over a week, retains a unique set of pluripotency genes and acquires properties for 'neuron differentiation'. Human and monkey pluripotent stem cells show the highest similarity to post-implantation late cyEPI, which, despite co-existing with gastrulating cells, bears characteristics of pre-gastrulating mouse EPI and epiblast-like cells in vitro. These findings not only reveal the divergence and coherence of EPI development, but also identify a developmental coordinate of the spectrum of pluripotency among key species, providing a basis for better regulation of human pluripotency in vitro.

Dynamic molecular conformational change leading to energy transfer in F8-5% BSP copolymer revealed by single-molecule spectroscopy.

Polyfluorene-based copolymers such as poly(9,9-dioctylfluorene)-alt-5% [bis-N,N'-(4-butylphenyl)-bis-N,N'-phenyl-1,4-phenylenediamine] (F8-5% BSP) are efficient blue-emitting polymers with various electronic phases: F8 blue-emitting glassy phase, F8 ordered more red-emitting ß-phase, and F8/BSP charge transfer (CT) state. Polymer light-emitting device performance and color purity can be significantly improved by forming ß-phase segments. However, the role of the ß-phase on energy transfer (ET) among glassy F8, ß-phase, and F8/BSP CT state is unclear. Herein, we identify dynamic molecular conformation-controlled ET from locally excited states to either the CT state or ß-phase in light-emitting copolymers. By conducting single-molecule spectroscopy for single F8-5% BSP chains, we find inefficient intra-chain ET from glassy segments to the CT state, while efficient ET from the glassy to the ß-phase. Spontaneous and reversible CT on-off emission is observed both in the presence and absence of the ß-phase. The density functional theory calculations reveal the origin of the on-chain CT state and indicate this CT emission on-off switching behavior could be related to molecule torsional motion between BSP and F8 units. The population of the CT state by ET can be increased via through-space interaction between the F8 block and the BSP unit on a self-folded chain. Temperature-dependent single-molecule spectroscopy confirms such interaction showing a gradual increase in intensity of the CT emission with the temperature. Based on these observations, we propose the dynamic molecular motion-induced conformation change as the origin of the glassy-to-CT ET, and thermal energy may provide the activation for such a change to enhance the ET from glassy or ß-phases to the CT state.

Simultaneous quantification of dasatinib, nilotinib, bosutinib, and ponatinib using high-performance liquid chromatography-Photodiode array detection.

BACKGROUND: Dasatinib, nilotinib, and bosutinib, second-generation tyrosine kinase inhibitors (TKIs), and ponatinib, a third-generation TKI, are approved pharmaceuticals used in the treatment of chronic myeloid leukemia (CML). Although liquid chromatography-tandem mass spectrometry assays for simultaneous quantification of the four TKIs in human serum have been reported in the literature, a high-performance liquid chromatography (HPLC) assay that simultaneously quantifies these compounds has not yet been developed. This study aims to establish and validate an efficient HPLC analytical method using a photodiode array (PDA) detector for the simultaneous quantification of the four TKIs. METHODS: Calibration standards were prepared by serial dilution of serum samples containing the four TKIs, followed by solid-phase extraction. The four TKIs were eluted in order within 10 min using a binary HPLC gradient system. RESULTS: The calibration ranges were 2-500 ng/ml for dasatinib, 100-5000 ng/ml for nilotinib, and 10-500 ng/ml for bosutinib and ponatinib. Intra-day and inter-day precision and accuracy values were found to be in accordance with the U.S. Food and Drug Administration guidelines. The recovery rates were 92.9%-96.0%, 80.7%-86.1%, 91.6%-99.0%, and 86.4%-92.6% for dasatinib, nilotinib, bosutinib, and ponatinib, respectively. CONCLUSION: To the best of our knowledge, this is the first report of an HPLC-PDA analytical method that allows efficient simultaneous quantification of the four TKIs in the serum of patients with CML. We believe that the method developed herein can improve the efficiency of therapeutic drug monitoring in patients with CML in clinical practice.

Bone morphogenetic protein and retinoic acid synergistically specify female germ-cell fate in mice.

The mechanism for sex determination in mammalian germ cells remains unclear. Here, we reconstitute the female sex determination in mouse germ cells in vitro under a defined condition without the use of gonadal somatic cells. We show that retinoic acid (RA) and its key effector, STRA8, are not sufficient to induce the female germ-cell fate. In contrast, bone morphogenetic protein (BMP) and RA synergistically induce primordial germ cells (PGCs)/PGC-like cells (PGCLCs) derived from embryonic stem cells (ESCs) into fetal primary oocytes. The induction is characterized by entry into the meiotic prophase, occurs synchronously and recapitulates cytological and transcriptome progression in vivo faithfully. Importantly, the female germ-cell induction necessitates a proper cellular competence-most typically, DNA demethylation of relevant genes-which is observed in appropriately propagated PGCs/PGCLCs, but not in PGCs/PGCLCs immediately after induction. This provides an explanation for the differential function of BMP signaling between PGC specification and female germ-cell induction. Our findings represent a framework for a comprehensive delineation of the sex-determination pathway in mammalian germ cells, including humans.

In vitro expansion of mouse primordial germ cell-like cells recapitulates an epigenetic blank slate.

The expansion of primordial germ cells (PGCs), the precursors for the oocytes and spermatozoa, is a key challenge in reproductive biology/medicine. Using a chemical screening exploiting PGC-like cells (PGCLCs) induced from mouse embryonic stem cells (ESCs), we here identify key signaling pathways critical for PGCLC proliferation. We show that the combinatorial application of Forskolin and Rolipram, which stimulate cAMP signaling via different mechanisms, expands PGCLCs up to ~50-fold in culture. The expanded PGCLCs maintain robust capacity for spermatogenesis, rescuing the fertility of infertile mice. Strikingly, during expansion, PGCLCs comprehensively erase their DNA methylome, including parental imprints, in a manner that precisely recapitulates genome-wide DNA demethylation in gonadal germ cells, while essentially maintaining their identity as sexually uncommitted PGCs, apparently through appropriate histone modifications. By establishing a paradigm for PGCLC expansion, our system reconstitutes the epigenetic "blank slate" of the germ line, an immediate precursory state for sexually dimorphic differentiation.

Cyclosporin A and FGF signaling support the proliferation/survival of mouse primordial germ cell-like cells in vitro†.

Primordial germ cells (PGCs) are the founding population of the germ cell lineage that undergo a multistep process to generate spermatozoa or oocytes. Establishing an appropriate culture system for PGCs is a key challenge in reproductive biology. By a chemical screening using mouse PGC-like cells (mPGCLCs), which were induced from mouse embryonic stem cells, we reported previously that forskolin and rolipram synergistically enhanced the proliferation/survival of mPGCLCs with an average expansion rate of ~20-fold. In the present study, we evaluated other chemicals or cytokines to see whether they would improve the current mPGCLC culture system. Among the chemicals and cytokines examined, in the presence of forskolin and rolipram, cyclosporin A (CsA) and fibroblast growth factors (FGFs: FGF2 and FGF10) effectively enhanced the expansion of mPGCLCs in vitro (~50-fold on average). During the expansion by CsA or FGFs, mPGCLCs comprehensively erased their DNA methylation to acquire a profile equivalent to that of gonadal germ cells in vivo, while maintaining their highly motile phenotype as well as their transcriptional properties as sexually uncommitted PGCs. Importantly, these mPGCLCs robustly contributed to spermatogenesis and produced fertile offspring. Furthermore, mouse PGCs (mPGCs) cultured with CsA ex vivo showed transcriptomes and DNA methylomes similar to those of cultured mPGCLCs. The improved culture system for mPGCLCs/mPGCs would be instructive for addressing key questions in PGC biology, including the mechanisms for germ cell migration, epigenetic reprogramming, and sex determination of the germline.