Intranasal Sendai virus-based SARS-CoV-2 vaccine using a mouse model.

The coronavirus disease 2019 (COVID-19) epidemic remains worldwide. The usefulness of the intranasal vaccine and boost immunization against severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) has recently received much attention. We developed an intranasal SARS-CoV-2 vaccine by loading the receptor binding domain of the S protein (S-RBD) of SARS-CoV-2 as an antigen into an F-deficient Sendai virus vector. After the S-RBD-Fd antigen with trimer formation ability was intranasally administered to mice, S-RBD-specific IgM, IgG, IgA, and neutralizing antibody titers were increased in serum or bronchoalveolar lavage fluid for 12 weeks. Furthermore, in mice that received a booster dose at week 8, a marked increase in neutralizing antibodies in the serum and bronchoalveolar lavage fluid was observed at the final evaluation at week 12, which neutralized the pseudotyped lentivirus expressing the SARS-CoV-2 spike protein, indicating the usefulness of the Sendai virus-based SARS-CoV-2 intranasal vaccine.

Aberrant CHCHD2-associated mitochondriopathy in Kii ALS/PDC astrocytes.

Amyotrophic Lateral Sclerosis/Parkinsonism-Dementia Complex (ALS/PDC), a rare and complex neurological disorder, is predominantly observed in the Western Pacific islands, including regions of Japan, Guam, and Papua. This enigmatic condition continues to capture medical attention due to affected patients displaying symptoms that parallel those seen in either classical amyotrophic lateral sclerosis (ALS) or Parkinson's disease (PD). Distinctly, postmortem examinations of the brains of affected individuals have shown the presence of α-synuclein aggregates and TDP-43, which are hallmarks of PD and classical ALS, respectively. These observations are further complicated by the detection of phosphorylated tau, accentuating the multifaceted proteinopathic nature of ALS/PDC. The etiological foundations of this disease remain undetermined, and genetic investigations have yet to provide conclusive answers. However, emerging evidence has implicated the contribution of astrocytes, pivotal cells for maintaining brain health, to neurodegenerative onset, and likely to play a significant role in the pathogenesis of ALS/PDC. Leveraging advanced induced pluripotent stem cell technology, our team cultivated multiple astrocyte lines to further investigate the Japanese variant of ALS/PDC (Kii ALS/PDC). CHCHD2 emerged as a significantly dysregulated gene when disease astrocytes were compared to healthy controls. Our analyses also revealed imbalances in the activation of specific pathways: those associated with astrocytic cilium dysfunction, known to be involved in neurodegeneration, and those related to major neurological disorders, including classical ALS and PD. Further in-depth examinations revealed abnormalities in the mitochondrial morphology and metabolic processes of the affected astrocytes. A particularly striking observation was the reduced expression of CHCHD2 in the spinal cord, motor cortex, and oculomotor nuclei of patients with Kii ALS/PDC. In summary, our findings suggest a potential reduction in the support Kii ALS/PDC astrocytes provide to neurons, emphasizing the need to explore the role of CHCHD2 in maintaining mitochondrial health and its implications for the disease.

Genetic factors affecting survival in Japanese patients with sporadic amyotrophic lateral sclerosis: a genome-wide association study and verification in iPSC-derived motor neurons from patients.

BACKGROUND: Several genetic factors are associated with the pathogenesis of sporadic amyotrophic lateral sclerosis (ALS) and its phenotypes, such as disease progression. Here, in this study, we aimed to identify the genes that affect the survival of patients with sporadic ALS. METHODS: We enrolled 1076 Japanese patients with sporadic ALS with imputed genotype data of 7 908 526 variants. We used Cox proportional hazards regression analysis with an additive model adjusted for sex, age at onset and the first two principal components calculated from genotyped data to conduct a genome-wide association study. We further analysed messenger RNA (mRNA) and phenotype expression in motor neurons derived from induced pluripotent stem cells (iPSC-MNs) of patients with ALS. RESULTS: Three novel loci were significantly associated with the survival of patients with sporadic ALS-FGF1 at 5q31.3 (rs11738209, HR=2.36 (95% CI, 1.77 to 3.15), p=4.85×10-9), THSD7A at 7p21.3 (rs2354952, 1.38 (95% CI, 1.24 to 1.55), p=1.61×10-8) and LRP1 at 12q13.3 (rs60565245, 2.18 (95% CI, 1.66 to 2.86), p=2.35×10-8). FGF1 and THSD7A variants were associated with decreased mRNA expression of each gene in iPSC-MNs and reduced in vitro survival of iPSC-MNs obtained from patients with ALS. The iPSC-MN in vitro survival was reduced when the expression of FGF1 and THSD7A was partially disrupted. The rs60565245 was not associated with LRP1 mRNA expression. CONCLUSIONS: We identified three loci associated with the survival of patients with sporadic ALS, decreased mRNA expression of FGF1 and THSD7A and the viability of iPSC-MNs from patients. The iPSC-MN model reflects the association between patient prognosis and genotype and can contribute to target screening and validation for therapeutic intervention.

Preimplantation genetic testing for aneuploidy: a comparison of live birth rates in patients with recurrent pregnancy loss due to embryonic aneuploidy or recurrent implantation failure.

STUDY QUESTION: Can preimplantation genetic testing for aneuploidy (PGT-A) improve the live birth rate and reduce the miscarriage rate in patients with recurrent pregnancy loss (RPL) caused by an abnormal embryonic karyotype and recurrent implantation failure (RIF)? SUMMARY ANSWER: PGT-A could not improve the live births per patient nor reduce the rate of miscarriage, in both groups. WHAT IS KNOWN ALREADY: PGT-A use has steadily increased worldwide. However, only a few limited studies have shown that it improves the live birth rate in selected populations in that the prognosis has been good. Such studies have excluded patients with RPL and RIF. In addition, several studies have failed to demonstrate any benefit at all. PGT-A was reported to be without advantage in patients with unexplained RPL whose embryonic karyotype had not been analysed. The efficacy of PGT-A should be examined by focusing on patients whose previous products of conception (POC) have been aneuploid, because the frequencies of abnormal and normal embryonic karyotypes have been reported as 40-50% and 5-25% in patients with RPL, respectively. STUDY DESIGN, SIZE, DURATION: A multi-centre, prospective pilot study was conducted from January 2017 to June 2018. A total of 171 patients were recruited for the study: an RPL group, including 41 and 38 patients treated respectively with and without PGT-A, and an RIF group, including 42 and 50 patients treated respectively with and without PGT-A. At least 10 women in each age group (35-36, 37-38, 39-40 or 41-42 years) were selected for PGT-A groups. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients and controls had received IVF-ET for infertility. Patients in the RPL group had had two or more miscarriages, and at least one case of aneuploidy had been ascertained through prior POC testing. No pregnancies had occurred in the RIF group, even after at least three embryo transfers. Trophectoderm biopsy and array comparative genomic hybridisation (aCGH) were used for PGT-A. The live birth rate of PGT-A and non-PGT-A patients was compared after the development of blastocysts from up to two oocyte retrievals and a single blastocyst transfer. The miscarriage rate and the frequency of euploidy, trisomy and monosomy in the blastocysts were noted. MAIN RESULT AND THE ROLE OF CHANCE: There were no significant differences in the live birth rates per patient given or not given PGT-A: 26.8 versus 21.1% in the RPL group and 35.7 versus 26.0% in the RIF group, respectively. There were also no differences in the miscarriage rates per clinical pregnancies given or not given PGT-A: 14.3 versus 20.0% in the RPL group and 11.8 versus 0% in the RIF group, respectively. However, PGT-A improved the live birth rate per embryo transfer procedure in both the RPL (52.4 vs 21.6%, adjusted OR 3.89; 95% CI 1.16-13.1) and RIF groups (62.5 vs 31.7%, adjusted OR 3.75; 95% CI 1.28-10.95). Additionally, PGT-A was shown to reduce biochemical pregnancy loss per biochemical pregnancy: 12.5 and 45.0%, adjusted OR 0.14; 95% CI 0.02-0.85 in the RPL group and 10.5 and 40.9%, adjusted OR 0.17; 95% CI 0.03-0.92 in the RIF group. There was no difference in the distribution of genetic abnormalities between RPL and RIF patients, although double trisomy tended to be more frequent in RPL patients. LIMITATIONS, REASONS FOR CAUTION: The sample size was too small to find any significant advantage for improving the live birth rate and reducing the clinical miscarriage rate per patient. Further study is necessary. WIDER IMPLICATION OF THE FINDINGS: A large portion of pregnancy losses in the RPL group might be due to aneuploidy, since PGT-A reduced the overall incidence of pregnancy loss in these patients. Although PGT-A did not improve the live birth rate per patient, it did have the advantage of reducing the number of embryo transfers required to achieve a similar number live births compared with those not undergoing PGT-A. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Japan Society of Obstetrics and Gynecology and grants from the Japanese Ministry of Education, Science, and Technology. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

MMP2 and MMP9 are associated with the pathogenesis of recurrent pregnancy loss through protein expression rather than genetic polymorphism.

Matrix metalloproteinases (MMPs) degrade extracellular matrix proteins and are important for placenta formation during early pregnancy. Recurrent pregnancy loss (RPL) is associated with abnormalities in endometrial extracellular matrix remodeling. This study aimed to elucidate the roles of MMP2 and MMP9 in RPL pathogenesis. In total, 295 women with a history of RPL and 101 controls were included in this genetic study. Genotype analysis was performed using polymerase chain reaction (PCR) restriction fragment length polymorphisms. For proteolytic analysis, decidua and villi were collected from 10 RPL-miscarried women with normal fetal chromosomes (NC) and 19 women with fetal chromosome aberrations (AC). The expression of MMP2 and MMP9 in the decidua and villi was measured by IHC and ELISA. All samples were collected after obtaining informed consent. There were no statistically significant differences in MMP2-735 C/T and MMP9-1562 C/T frequencies between women with RPL and the controls. There was no significant difference in MMP2 expression levels in the villi; however, MMP9 expression was significantly higher in normal fetal chromosomes. In the decidua, the expression of MMP2 in the NC group was significantly lower, and MMP9 in the NC group was significantly higher than in the AC group. Although no differences in MMP2-735 C/T and MMP9-1562 C/T gene polymorphisms were observed in the present study, it is suggested that differences at the protein level are involved in the pathogenesis of RPL since MMP expression is not only regulated by genes but also by local inflammation and various inductive signals.

Common and rare genetic variants predisposing females to unexplained recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is a major reproductive health issue with multifactorial causes, affecting 2.6% of all pregnancies worldwide. Nearly half of the RPL cases lack clinically identifiable causes (e.g., antiphospholipid syndrome, uterine anomalies, and parental chromosomal abnormalities), referred to as unexplained RPL (uRPL). Here, we perform a genome-wide association study focusing on uRPL in 1,728 cases and 24,315 female controls of Japanese ancestry. We detect significant associations in the major histocompatibility complex (MHC) region at 6p21 (lead variant=rs9263738; P = 1.4 × 10-10; odds ratio [OR] = 1.51 [95% CI: 1.33-1.72]; risk allele frequency = 0.871). The MHC associations are fine-mapped to the classical HLA alleles, HLA-C*12:02, HLA-B*52:01, and HLA-DRB1*15:02 (P = 1.1 × 10-10, 1.5 × 10-10, and 1.2 × 10-9, respectively), which constitute a population-specific common long-range haplotype with a protective effect (P = 2.8 × 10-10; OR = 0.65 [95% CI: 0.57-0.75]; haplotype frequency=0.108). Genome-wide copy-number variation (CNV) calling demonstrates rare predicted loss-of-function (pLoF) variants of the cadherin-11 gene (CDH11) conferring the risk of uRPL (P = 1.3 × 10-4; OR = 3.29 [95% CI: 1.78-5.76]). Our study highlights the importance of reproductive immunology and rare variants in the uRPL etiology.

Proteomic insights into extracellular vesicles in ALS for therapeutic potential of Ropinirole and biomarker discovery.

BACKGROUND: Extracellular vesicles (EVs) hold the potential for elucidating the pathogenesis of amyotrophic lateral sclerosis (ALS) and serve as biomarkers. Notably, the comparative and longitudinal alterations in the protein profiles of EVs in serum (sEVs) and cerebrospinal fluid (CSF; cEVs) of sporadic ALS (SALS) patients remain uncharted. Ropinirole hydrochloride (ROPI; dopamine D2 receptor [D2R] agonist), a new anti-ALS drug candidate identified through induced pluripotent stem cell (iPSC)-based drug discovery, has been suggested to inhibit ALS disease progression in the Ropinirole Hydrochloride Remedy for Amyotrophic Lateral Sclerosis (ROPALS) trial, but its mechanism of action is not well understood. Therefore, we tried to reveal longitudinal changes with disease progression and the effects of ROPI on protein profiles of EVs. METHODS: We collected serum and CSF at fixed intervals from ten controls and from 20 SALS patients participating in the ROPALS trial. Comprehensive proteomic analysis of EVs, extracted from these samples, was conducted using liquid chromatography/mass spectrometer (LC/MS). Furthermore, we generated iPSC-derived astrocytes (iPasts) and performed RNA sequencing on astrocytes with or without ROPI treatment. RESULTS: The findings revealed notable disparities yet high congruity in sEVs and cEVs protein profiles concerning disease status, time and ROPI administration. In SALS, both sEVs and cEVs presented elevated levels of inflammation-related proteins but reduced levels associated with unfolded protein response (UPR). These results mirrored the longitudinal changes after disease onset and correlated with the revised ALS Functional Rating Scale (ALSFRS-R) at sampling time, suggesting a link to the onset and progression of SALS. ROPI appeared to counteract these changes, attenuating inflammation-related protein levels and boosting those tied to UPR in SALS, proposing an anti-ALS impact on EV protein profiles. Reverse translational research using iPasts indicated that these changes may partly reflect the DRD2-dependent neuroinflammatory inhibitory effects of ROPI. We have also identified biomarkers that predict diagnosis and disease progression by machine learning-driven biomarker search. CONCLUSIONS: Despite the limited sample size, this study pioneers in reporting time-series proteomic alterations in serum and CSF EVs from SALS patients, offering comprehensive insights into SALS pathogenesis, ROPI-induced changes, and potential prognostic and diagnostic biomarkers.

Impaired decidualization and relative increase of PROK1 expression in the decidua of patients with unexplained recurrent pregnancy loss showing insulin resistance.

A recent meta-analysis revealed that patients with unexplained recurrent pregnancy loss (RPL) show higher insulin resistance compared to healthy controls. However, the etiology of RPL remains unknown. Prokineticin (PROK1), a pleiotropic uterine endometrial protein, is important for implantation and decidualization and is regulated by hypoxia and insulin. In this study, we investigated the decidualization status and the role of PROK1 in the decidua of patients with unexplained RPL showing insulin resistance. Thirty-two patients with unexplained RPL were included in this study. Following the diagnosis of a miscarriage, the decidua and villi of the patient were surgically collected. Fasting blood glucose and insulin levels were measured, and HOMA-ß was calculated. Using IHC and ELISA, the expression of IGFBP-1, PRL and PROK1 in the decidua and IGF-2 in the villi were analyzed in patients with euploid miscarriage with a high HOMA-ß index (n = 8) and compared to controls (euploid miscarriage with normal HOMA-ß: n = 12, aneuploid miscarriage with normal HOMA-ß: n = 12). The co-localization of PROK1 and IGFBP-1 was observed in the decidua by IHC. In the decidua of RPL patients with high HOMA-ß, the expression levels of IGFBP-1 and PRL were significantly lower, whereas the PROK1/IGFBP-1 ratio was significantly higher compared to that of the controls. IGF-2 expression in villi was significantly lower in RPL patients with high HOMA-ß. Impaired decidualization and excessive PROK1 production may have pathological implications in patients with unexplained RPL with insulin resistance, especially under the state of hyper insulin production.

The association between chronic deciduitis and recurrent pregnancy loss.

Chronic deciduitis (CD) is defined as the presence of lymphocytes or plasma cells in decidual tissue. CD suggests the presence of chronic endometritis (CE) which is associated with recurrent pregnancy loss (RPL). In this study, we examined the role CD plays in RPL patients with aneuploid and euploid miscarriage. The frequency of CD in 49 RPL patients (22 euploid and 27 aneuploid miscarriages) and 17 control women was assessed and the subsequent live birth rate (LBR) in the presence and absence of CD were compared. When only one CD138-positive endometrial stromal plasma cell (ESPC) was found per high-power field (HPF), we diagnosed small-positive CD (Grade 1). When a cluster of two or more CD138-positive ESPCs was found per HPF, we diagnosed it as CD Grade 2. The prevalence of Grade 1 was 18.2% (4/22) in patients with euploid miscarriage, 37.0% (10/27) in patients with aneuploid miscarriage and 23.5% (4/17) in control women. The prevalence of Grade 2 was 45.5% (10/22) in patients with euploid miscarriage, 55.6% (15/27) in patients with aneuploid miscarriage and 23.5% (4/17) in control women. There was a significant difference in the prevalence of CD (p = 0.015). The LBR of patients with CD was similar to that of patients without CD. CD was associated with RPL, especially in patients with aneuploid miscarriage. However, since there was no difference in the LBR of patients with or without CD in the next pregnancy, it was unclear whether CD was a contributing cause of RPL.

A human induced pluripotent stem cell model from a patient with hereditary cerebral small vessel disease carrying a heterozygous R302Q mutation in HTRA1.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is an inherited cerebral small vessel disease (CSVD) caused by biallelic mutations in the high-temperature requirement serine peptidase A1 (HTRA1) gene. Even heterozygous mutations in HTRA1 are recently revealed to cause cardinal clinical features of CSVD. Here, we report the first establishment of a human induced pluripotent stem cell (hiPSC) line from a patient with heterozygous HTRA1-related CSVD. Peripheral blood mononuclear cells (PBMCs) were reprogrammed by the transfection of episomal vectors encoding human OCT3/4 (POU5F1), SOX2, KLF4, L-MYC, LIN28, and a murine dominant-negative mutant of p53 (mp53DD). The established iPSCs had normal morphology as human pluripotent stem cells and normal karyotype (46XX). Moreover, we found that the HTRA1 missense mutation (c.905G>A, p.R302Q) was heterozygous. These iPSCs expressed pluripotency-related markers and had the potential to differentiate into all three germ layers in vitro. HTRA1 and the supposed disease-associated gene NOG were differentially expressed in the patient iPSCs at mRNA levels compared to those of control lines. The iPSC line would facilitate in vitro research for understanding the cellular pathomechanisms caused by the HTRA1 mutation including its dominant-negative effect.

Cervicovaginal microbiome in patients with recurrent pregnancy loss.

There have been few studies concerning an association between unexplained recurrent pregnancy loss (RPL) and the microbiome. A recent study including 67 patients demonstrated that an increase in Ureaplasma species in the endometrium raised the risk of miscarriage with an euploid karyotype. While endometrial sampling is invasive, cervicovaginal sampling is not. We compared vaginal and cervical microbiomes with a 16 S ribosomal RNA sequence between 88 patients with unexplained RPL and 17 healthy women with no history of miscarriage. We prospectively assessed risk factors for maternal colonization at a subsequent miscarriage without an aneuploid karyotype in patients. Cervicovaginal bacteria were dominated by Lactobacillus iners, Gardnerella vaginalis, Atopobium vaginae and Bifidobacterium breve in Japanese population. The proportions of Delftia and unknown bacteria in the cervix were significantly higher in patients with RPL than in controls. Streptococcus, Microbacterium, Delftia, Anaerobacillus and Chloroplast in the cervix were significantly higher in patients with a history of chorioamnionitis compared to the controls. The abundance of Cutibacterium and Anaerobacillus in the cervix was significantly higher in patients who had subsequently miscarried compared to those who gave birth. The miscarriage rate in patients with higher proportions of both Cutibacterium and Anaerobacillus (66.7%, 2/3) was significantly greater than that of patients who lacked these bacteria (9.2%, 6/65, adjusted odds ratio 16.90, 95% confidence interval 1.27-225.47, p = 0.032). The presence of certain bacteria could be a predictor of subsequent miscarriage without an aneuploid karyotype. The cervicovaginal microbiome might be useful for investigating a possible cause of RPL.

Phase 1/2a clinical trial in ALS with ropinirole, a drug candidate identified by iPSC drug discovery.

iPSC-based drug discovery led to a phase 1/2a trial of ropinirole in ALS. 20 participants with sporadic ALS received ropinirole or placebo for 24 weeks in the double-blind period to evaluate safety, tolerability, and therapeutic effects. Adverse events were similar in both groups. During the double-blind period, muscle strength and daily activity were maintained, but a decline in the ALSFRS-R, which assesses the functional status of ALS patients, was not different from that in the placebo group. However, in the open-label extension period, the ropinirole group showed significant suppression of ALSFRS-R decline and an additional 27.9 weeks of disease-progression-free survival. iPSC-derived motor neurons from participants showed dopamine D2 receptor expression and a potential involvement of the SREBP2-cholesterol pathway in therapeutic effects. Lipid peroxide represents a clinical surrogate marker to assess disease progression and drug efficacy. Limitations include small sample sizes and high attrition rates in the open-label extension period, requiring further validation.

Abnormal ciliogenesis in decidual stromal cells in recurrent miscarriage.

Primary cilia regulate cellular signaling and are involved in both sensing and transducing extracellular stimuli. A recent study of patients with recurrent miscarriage (RM) identified mutations affecting DYNC2H1, which were involved in ciliary biogenesis. However, there has been no study concerning primary cilia in the decidua. We compared the number and the length of primary cilia in the decidua of 15 patients with unexplained RM with those of 7 pregnant controls who underwent an artificial termination of pregnancy. Immunohistochemistry was performed using antibodies against primary cilia, extravillous trophoblasts (EVTs), macrophages, uterine Natural Killer (uNK) cells, decidual stromal cells, and the activation of TGF-ß and CREB signaling in the decidua of early pregnancy was studied. The density of decidual stromal cells, but not EVTs, macrophages or uNK cells, was found to be significantly higher in the decidua of patients compared to controls. The percentage of ciliated decidual stromal cells was significantly decreased in patients. There was no difference in the primary ciliary length. Regarding TGF-ß signaling, p-Smad2 in these cells was diminished significantly in patients, and most of the TGF-ß-activated decidual stromal cells of both patients and controls had primary cilia. No difference in the activation of CREB was found. Abnormal primary cilia on decidual stromal cells may be one of the explanatory factors for unknown RM. The inactivation of TGF-ß signaling may lead to abnormal ciliogenesis in the decidua.

APOE Alleles With Tau and Aß Pathology in Patients With Amyotrophic Lateral Sclerosis and Parkinsonism-Dementia Complex in the Kii Peninsula.

BACKGROUND AND OBJECTIVES: To examine the association of the APOE ε4 and ε2 alleles with the pathologic features of patients with amyotrophic lateral sclerosis and parkinsonism-dementia complex cases in the Kii peninsula of Japan (Kii ALS/PDC). METHODS: We analyzed APOE variants in 18 autopsy patients with ALS/PDC, consisting of 9, 8, and 1 patient with PDC, ALS, and PDC followed by ALS, respectively. Moreover, we revealed the relationship between APOE variants and Aß and tau pathologies. RESULTS: The frequency of the ε4 allele was not different between patients with Kii ALS/PDC and control participants. APOE ε4 was associated with increased Aß pathology (p = 0.005 by the χ 2 test), but not with increased tau pathology (p = 0.984). The frequency of the ε2 allele was apparently higher than that of control participants (p = 0.254). The APOE ε2 allele was associated with increased tau pathology (p = 0.009) and not with reduced Aß pathology (p = 0.383) in patients with Kii ALS/PDC. DISCUSSION: Although there was no overrepresentation of the frequency of the ε4 or ε2 allele, our findings suggest that the ε2 allele is associated with increased tau pathology and not with reduced Aß pathology in patients with Kii ALS/PDC.

Characteristic DNA methylation profiles of chorionic villi in recurrent miscarriage.

Dysregulation of transcriptional programs that are tightly regulated by DNA methylation during placental and fetal development at different gestational stages, may cause recurrent miscarriage. Here, we examined genome-wide DNA methylation in chorionic villi and decidual tissues from patients suffering RM and from healthy women who had undergone artificial abortion (n = 5 each). We found that 13,426 and 5816 CpG sites were differentially methylated in chorionic villi and decidua, respectively. DNA methylation profiles of chorionic villi, but not decidua, in RM patients was clearly distinct from AA controls. Among the differentially methylated genes, the enhancer region of SPATS2L was significantly more highly methylated in RM patients (n = 19) than AA controls (n = 19; mean methylation level, 52.0%-vs.-28.9%, P < 0.001), resulting in reduced expression of SPATS2L protein in the former. Functionally, depletion of SPATS2L in extravillous trophoblast cells decreased their invasion and migration abilities. Our data indicate that particularly the chorionic villi in RM patients exhibit distinct DNA methylation profiles compared with normal pregnancies and that this changed DNA methylation status may impede the progression of embryo development via the altered expression of genes such as SPATS2L in the villi.

Abnormal accumulation of OFD1 in endometrial cancer with poor prognosis inhibits ciliogenesis.

The aim of the present study was to examine primary cilia in endometrial tissue during the menstrual cycle and to clarify their morphological changes with different grades of endometrial cancer. Images of fluorescence immunostaining taken by confocal microscopy were used to count the number of primary cilia in normal endometrium and endometrioid carcinoma Grade 1 and Grade 3 specimens. To examine the association between autophagy and ciliogenesis in endometrioid carcinoma, the expression of p62/Sequestosome-1, a selective substrate for autophagy, and oral-facial-digital syndrome 1 protein (OFD1), a protein associated with ciliogenesis, were examined using images of fluorescence immunostaining taken by confocal microscopy. The level of p62 expression was confirmed by western blotting. In proliferative and secretory endometrial stromal cells, the percentage of cells that were ciliated was 7.2 and 32.7% (95% confidence interval=21.61-39.79; P<0.01), and the length of the primary cilia was 1.24 µm and 2.34 µm (0.92-1.26; P<0.01), respectively. In stromal cells of endometrioid carcinoma Grade 1 and Grade 3, the percentage of ciliated cells was 13.5 and 2.9% (7.89-15.05; P<0.001), and the length of the primary cilia was 2.02 and 1.14 µm (0.76-0.99; P<0.001), respectively. In both normal menstrual cycle tissue and endometrial carcinomas, the percentage of primary cilia was lower and their length was shorter in tissues with higher proliferative potential. The expression of OFD1 was significantly higher in Grade 3 compared with Grade 1 as indicated by quantifying the intensity of the fluorescence images (133-12248; P=0.046). To the best of our knowledge, this is the first study concerning the distribution of primary cilia in normal endometrium and endometrial cancer tissues. Overall, fewer ciliated cells in the highly malignant endometrial cancer tissues may be associated not only to the proliferation of cancer cells, but also to the excessive accumulation of OFD1 due to dysfunctional autophagy.

The investigation of calpain in human placenta with fetal growth restriction.

PROBLEM: The mechanism of fetal growth restriction (FGR) is not fully understood. In this study, we explored the contribution of the calpain-calpastatin system and the activated states of calpains in human FGR placenta. METHOD OF STUDY: The placentas were collected from patients of FGR (n = 17) and controls (n = 23) at elective cesarean sections in Nagoya City University Hospital and used for experiments upon informed consent. The existence and the expression of calpains and calpastatin in human placenta were compared between FGR and controls using immunohistochemistry, SDS-PAGE, and Western blotting. RESULTS: Staining of calpains (pre-, post-µ-calpain, pre-, post-m-calpain, and calpain-6) and calpastatin was observed in cytoplasm of trophoblast cells, both in FGR and control placenta. Pre-µ-calpain was located in the cytoplasm, and post-µ-calpain was located mainly in proximity to the cytoplasmic membrane. The expression of pre-µ-calpain was significantly higher (P < .001) and calpain-6 was significantly lower (P = .01) in FGR placentas. The inactive µ-calpain (80 kDa) was significantly elevated (P < .01), and active µ-calpain (76 kDa) was significantly decreased (P = .01) in FGR placentas. CONCLUSION: The results demonstrate that activation of µ-calpain is suppressed in FGR placentas and that calpain-6 in human placenta is involved in the pathology of FGR.

Generation of an ALS human iPSC line KEIOi001-A from peripheral blood of a Charcot disease-affected patient carrying TARDBP p.N345K heterozygous SNP mutation.

Amyotrophic Lateral Sclerosis is the most common motor neuron degenerative disease in adults, and TARDBP gene mutations have been reported to be involved in the pathogenesis. We present here how we generated the human induced pluripotent stem cell (hiPSC) line KEIOi001-A/SM4-4-5 from the peripheral blood of a 63-year-old male patient presenting the c.1035C > G heterozygous SNP mutation in the TARDBP gene locus. The established hiPSC line does not express the exogenous reprogramming factors oriP nor EBNA1 and shows no karyotypic abnormalities, while it expresses pluripotent stem cell markers, presents the SNP mutation and is capable of three-germ layers differentiation in vitro.

Brain Transcriptome Analysis Links Deficiencies of Stress-Responsive Proteins to the Pathomechanism of Kii ALS/PDC.

Amyotrophic lateral sclerosis and Parkinsonism-dementia complex (ALS/PDC) is a unique endemic neurodegenerative disease, with high-incidence foci in Kii Peninsula, Japan. To gather new insights into the pathological mechanisms underlying Kii ALS/PDC, we performed transcriptome analyses of patient brains. We prepared frozen brains from three individuals without neurodegenerative diseases, three patients with Alzheimer's disease, and 21 patients with Kii ALS/PDC, and then acquired microarray data from cerebral gray and white matter tissues. Microarray results revealed that expression levels of genes associated with heat shock proteins, DNA binding/damage, and senescence were significantly altered in patients with ALS/PDC compared with healthy individuals. The RNA expression pattern observed for ALS-type brains was similar to that of PDC-type brains. Additionally, pathway and network analyses indicated that the molecular mechanism underlying ALS/PDC may be associated with oxidative phosphorylation of mitochondria, ribosomes, and the synaptic vesicle cycle; in particular, upstream regulators of these mechanisms may be found in synapses and during synaptic trafficking. Furthermore, phenotypic differences between ALS-type and PDC-type were observed, based on HLA haplotypes. In conclusion, determining the relationship between stress-responsive proteins, synaptic dysfunction, and the pathogenesis of ALS/PDC in the Kii peninsula may provide new understanding of this mysterious disease.