Infantile survival and neurodevelopment at three years of age on delivery by the intended delivery mode in extremely preterm infants.

OBJECTIVE: To elucidate the impact of the intended delivery mode on long-term outcomes among extremely preterm infants. MATERIALS AND METHODS: Women who delivered singletons between 23 0/7 and 25 6/7 weeks of gestation from January 2010 to March 2014 and their infants were included in this study. The cases of fetal growth restriction and those with a chromosomal or major structural abnormality were excluded. The cases of fetal death that was diagnosed before labor onset and cases of non-reassuring fetal status, placental abruptions or umbilical cord prolapse that was diagnosed at labor onset were also excluded. The primary outcome was the incidence of composite adverse events, including death, cerebral palsy, or neurodevelopmental delay, at the age of three years. The composite adverse events, including death, grade III or IV intraventricular hemorrhage, cystic periventricular leukomalacia, necrotizing enterocolitis, focal intestinal perforation, and sepsis of neonatal period, were assessed as short-term outcomes. The association between the intended delivery mode and primary outcome, short-term outcome, and each component was analyzed using a multivariate logistic regression model. RESULTS: Eighty cases were included in the analyses. Primary outcomes could be assessed in 72 cases. Infantile composite adverse events before discharge were observed in 19 cases (24%). The prevalence of primary outcomes was 40% (29 cases). The intended delivery mode was not associated with primary and short-term outcomes and each component complication. CONCLUSION: An advantage of intended cesarean delivery in terms of prognosis at three years of age in extremely preterm infants was not observed.

Severe intrauterine adhesions after life-threatening event caused by Epstein-Barr virus-associated atraumatic spleen laceration: Case report.

This is the first known case report of severe intrauterine adhesion (IUA) following a life-threatening event caused by an Epstein-Barr virus-associated atraumatic spleen laceration. A 22-year-old nulligravid female suffered from infectious mononucleosis for approximately 1 month. Sudden severe hypovolemic shock with massive hemoperitoneum appeared and hemostasis was completely achieved by a splenectomy for an atraumatic spleen laceration, although that was followed by multiorgan failure and abdominal compartment syndrome. Complete recovery without any neurological sequelae was achieved by intensive treatment. A postoperative pathological evaluation revealed Epstein-Barr virus-associated splenomegaly. The patient was referred to our department because of secondary amenorrhea for approximately 5 months since the last menstruation, which occurred just prior to the event. Laboratory blood test results demonstrated normal thyroid and ovarian functions. Hysterofiberscopy revealed complete obstruction at the end of the cervical canal, indicating secondary uterine amenorrhea caused by severe IUA. Hysteroscopic adhesiolysis with a rigid hysteroscope reached the opening of the uterine cavity and menstruation was restored.

Vasa praevia with meandering fetal vessels despite placental and umbilical cord insertion on the same side of the uterine wall: A case report.

Vasa praevia with meandering fetal vessels is extremely rare, and it is difficult to diagnose this prenatally. When cesarean section is performed, a change in the site of uterine incision may be required for a safe delivery.

Abdominal pregnancy implanted on surface of pedunculated subserosal uterine leiomyoma: A case report.

Abdominal pregnancy is a rare form of ectopic pregnancy. Various sites of implantation in abdominal pregnancy have been reported. Uterine serosa is an extremely rare implantation site, with only a few cases reported to date. No case of abdominal pregnancy implanted on the surface of a subserosal uterine leiomyoma has been reported. We herein report the case of a 40-year-old primigravida woman who was diagnosed with abdominal pregnancy implanted on the surface of a pedunculated subserosal uterine leiomyoma. The uterine leiomyoma with gestational tissue was resected laparoscopically and the postoperative course was uneventful. It is necessary to remember the possibility of unexpected implantation sites and that laparoscopic surgery may be more difficult in such cases than that for fallopian tube pregnancy.

Analysis of cross-reactivity between flaviviruses with sera of patients with Japanese encephalitis showed the importance of neutralization tests for the diagnosis of Japanese encephalitis.

Japanese encephalitis (JE) is one of the most important viral encephalitis in Asia. JE is caused by the Japanese encephalitis virus (JEV), which belongs to the genus Flavivirus, family Flaviviridae. The diagnosis of JE is usually based on serological assays, and it has been reported that cross-reactivity between flaviviruses has complicated the interpretations of results from serological assays. Therefore, analysis of the cross-reactivity is an important subject for serological diagnosis of JE and other diseases caused by flaviviruses. In the present study, the cross-reactivity of the sera of patients with JE to other flaviviruses was analyzed using enzyme-linked immunosorbent assay (ELISA) and neutralization tests. Sixteen serum samples were collected from patients with JE and were tested for: i) IgM antibody against West Nile virus (WNV), dengue virus (DENV), zika virus (ZIKV), and tick-borne encephalitis virus (TBEV) using IgM-ELISA, ii) IgG antibody against DENV and TBEV using IgG-ELISA, and iii) neutralization tests with DENV 1-4, ZIKV, TBEV, and WNV. Out of the 16 samples tested using ELISA, 11 and 14 samples were positive for IgM and IgG, respectively, against at least one of the other flaviviruses. In neutralization tests, neutralizing potency against DENV, ZIKV, or TBEV was not detected in any samples. Although 13 samples showed neutralizing potency against WNV, their neutralizing antibody titers were equal to or less than one-eighth of those against JEV. These results show that neutralization tests are more specific than ELISA, indicating the importance of the neutralization tests in the diagnosis of JE.

Neutralization Potency of Sera from Vietnamese Patients with Japanese Encephalitis (JE) against Genotypes I and V JE Viruses.

Japanese encephalitis virus (JEV) is classified into 5 genotypes (GI, GII, GIII, GIV, and GV), and the GI and GIII strains are the most widely distributed in JE endemic areas. In recent years, GV JEV has been detected in China and Korea, suggesting that GV JEV may invade other JE endemic areas, including Vietnam, and that more attention should be paid to the JEV strains circulating in these areas. In this study, we investigated the neutralization ability of the sera collected from 22 Vietnamese patients with JE who lived in northern Vietnam against the GI and GV JEV strains. In most cases, the ratios of the titer against GV to that against GI (GV:GI) were equal to or less than 1:4. However, the titer against GV JEV was equivalent (1:1) to that against GI JEV in only a few cases, and no serum had a ratio higher than 1:1. Thus, our results did not show convincing evidence that GV JEV was emerging in northern Vietnam in 2014.

Comparison of Neutralizing Antibody Titers against Japanese Encephalitis Virus Genotype V Strain with Those against Genotype I and III Strains in the Sera of Japanese Encephalitis Patients in Japan in 2016.

Japanese encephalitis (JE) is an acute viral disease caused by the Japanese encephalitis virus (JEV). JEV strains are classified into 5 genotypes (I-V). JEV genotype V strains have never been detected in Japan to date, but they were recently detected in South Korea. In the present analysis, we tried to determine if a JEV genotype V strain caused any JE case in Japan in 2016. Serum and cerebrospinal fluid samples were collected from 10 JE patients reported in Japan in 2016. JEV RNA was not detected in any of the samples. Although JEV is a single-serotype virus, it can be expected that the neutralizing antibody titers against JEV genotype V strains are higher than those against genotype I and III strains in the serum of patients with JE in Japan whose causative JEV was the genotype V strain. The neutralizing antibody titers against the JEV genotype V strain were not higher than those against the genotype I or III strain in any serum samples. Therefore, the evidence that the JEV genotype V strain caused any JE case in Japan in 2016 was absent.

Whole Genome Sequencing-Based Molecular Epidemiologic Analysis of Autochthonous Dengue Virus Type 1 Strains Circulating in Japan in 2014.

Cases of autochthonous infections of dengue virus type 1 (DENV-1) were detected in Japan after a 70-year period devoid of dengue outbreaks. We previously showed that E gene sequences are identical in 11 of the 12 DENV-1 strains autochthonous to Japan. However, the E sequence represents only 14% of the DENV-1 genome. In the present study, we have sequenced the entire genome of 6 autochthonous DENV-1 strains that were isolated from patients during the 2014 outbreak. Sequencing of 5 Yoyogi group strains with identical E sequences and 1 Shizuoka strain with a different E sequence revealed that the first Yoyogi group strain differed from the Shizuoka strain by 18 amino acid residues. Furthermore, 2 Yoyogi group strains had different genomic sequences while the other 3 had identical genomes. Phylogenetic analyses indicated that the Hyogo strain, a Yoyogi group strain, was the first to diverge from the other 4 Yoyogi group strains. The E gene sequence of the Yoyogi group strains exhibits the highest homology to those of the strains isolated in Malaysia and Singapore between 2013 and 2014. The patient infected with the Hyogo strain visited Malaysia before the onset of dengue fever, suggesting that this was a case of dengue infection imported from Malaysia.

Two different dengue virus strains in the Japanese epidemics of 2014.

In late August 2014, dengue cases were reported in Japan, and a total of 162 cases were confirmed. In the present study, the envelope (E) gene sequences of 12 specimens from the dengue patients were determined. A dengue virus serotype 1 (DENV1) strain (D1/Hu/Shizuoka/NIID181/2014), which was clearly different from the first reported strain (D1/Hu/Saitama/NIID100/2014), was identified, although the other 11 specimens showed the same nucleotide sequences as D1/Hu/Saitama/NIID100/2014. The E gene sequences of two different strains were compared with those of nine DENV1 strains of imported cases in Japan in 2014. Phylogenetic analysis based on the E gene sequences showed that the D1/Hu/Saitama/NIID100/2014 strain was closely related to a strain isolated from an imported case from Singapore. Although no strain closely related to D1/Hu/Shizuoka/NIID181/2014 was found in these imported strains, the strain was closely related to isolates in Thailand and Taiwan in 2009. These data indicate that the dengue cases in Japan were caused by two different dengue virus strains that entered Japan through different means.

Zika fever imported from Thailand to Japan, and diagnosed by PCR in the urines.

In July 2014, a Japanese traveller returning from Thailand was investigated for fever, headache, rash and conjunctivitis. Zika virus RNA was detected in his urine sample by real-time reverse transcription polymerase chain reaction. Serological tests showed cross reactivity of IgM against the dengue virus. Zika fever could be misdiagnosed or missed and should be considered in febrile patients with a rash, especially those returning from Thailand.

Detecting Dengue Virus Nonstructural Protein 1 (NS1) in Urine Samples Using ELISA for the Diagnosis of Dengue Virus Infection.

Dengue virus (DENV) infection is a serious global health threat. For the surveillance and control of dengue, there is a need for robust diagnostic tools that are relatively easy to use and reliable in various clinical settings. We investigated the applicability of NS1 antigen detection in urine samples for the diagnosis of DENV. About 118 urine samples, obtained from 96 dengue patients at various phases of disease, were used for this study. NS1 antigen was detected by ELISA in the urine samples obtained from patients after 2-17 days of disease onset. Positive detection rates of NS1 antigen ranged between 13-43%. Based on real-time RT-PCR, positive detection rates of viral genome in the urine samples ranged between 20-33% on days 0 to ≥15. On days 11 to ≥15 after the disease onset, NS1 antigen was detected at similar rates in serum and urine samples. Additionally, NS1 antigen was detected in 2 urine samples, but not in the serum samples, on days 7 and 16 after the onset of the disease. The results confirm the applicability of NS1 antigen detection in urine samples using ELISA to diagnose acute DENV infection and suggests that the assay is potentially useful when only limited amounts of serum samples are available and in limited resource settings.

The majority of early primordial germ cells acquire pluripotency by AKT activation.

Primordial germ cells (PGCs) are undifferentiated germ cells in embryos, the fate of which is to become gametes; however, mouse PGCs can easily be reprogrammed into pluripotent embryonic germ cells (EGCs) in culture in the presence of particular extracellular factors, such as combinations of Steel factor (KITL), LIF and bFGF (FGF2). Early PGCs form EGCs more readily than do later PGCs, and PGCs lose the ability to form EGCs by embryonic day (E) 15.5. Here, we examined the effects of activation of the serine/threonine kinase AKT in PGCs during EGC formation; notably, AKT activation, in combination with LIF and bFGF, enhanced EGC formation and caused ∼60% of E10.5 PGCs to become EGCs. The results indicate that the majority of PGCs at E10.5 could acquire pluripotency with an activated AKT signaling pathway. Importantly, AKT activation did not fully substitute for bFGF and LIF, and AKT activation without both LIF and bFGF did not result in EGC formation. These findings indicate that AKT signal enhances and/or collaborates with signaling pathways of bFGF and of LIF in PGCs for the acquisition of pluripotency.

Detection of dengue virus nonstructural protein 1 (NS1) by using ELISA as a useful laboratory diagnostic method for dengue virus infection of international travelers.

BACKGROUND: Dengue virus ( DENV) nonstructural protein 1 ( NS1) has been used as a novel diagnostic marker during the early phase of DENV infection. METHODS: Presence of NS1 antigen was examined using 336 serum samples obtained from 276 travelers returning to Japan from Asia, Central and South America, Pacific Islands, and Africa with dengue. Assay specificity was evaluated using 148 non-dengue samples. RESULTS: Positive rates among four DENV serotypes were 68%-89%. NS1 antigen positive rates were at similar levels between primary infection and secondary infection. NS1 antigen positive rates were 88%-96% on days 1-5, 75%-100% on days 6-10, and 36-60% on ≥ day 11. Positive rates using real-time polymerase chain reaction (RT-PCR) were over 70% on days 1-5, but decreased thereafter. CONCLUSIONS: The results indicate that NS1 antigen positive rates were higher than those of RT-PCR during longer period of early phase in DENV infection. Thus, NS1 antigen ELISA is a useful tool for confirming DENV infection in international travelers, when it is used in combination with anti-DENV IgM ELISA.

Max is a repressor of germ cell-related gene expression in mouse embryonic stem cells.

Embryonic stem cells and primordial germ cells (PGCs) express many pluripotency-associated genes, but embryonic stem cells do not normally undergo conversion into primordial germ cells. Thus, we predicted that there is a mechanism that represses primordial germ cell-related gene expression in embryonic stem cells. Here we identify genes involved in this putative mechanism, by using an embryonic stem cell line with a Vasa reporter in an RNA interference screen of transcription factor genes expressed in embryonic stem cells. We identify five genes that result in the expression of Vasa when silenced. Of these, Max is the most striking. Transcriptome analysis reveals that Max knockdown in embryonic stem cells results in selective, global derepression of germ cell-specific genes. Max interacts with histone H3K9 methyltransferases and associates with the germ cell-specific genes in embryonic stem cells. In addition, Max knockdown results in a decrease in histone H3K9 dimethylation at their promoter regions. We propose that Max is part of protein complex that acts as a repressor of germ cell-related genes in embryonic stem cells.

Cell cycle gene-specific control of transcription has a critical role in proliferation of primordial germ cells.

Transcription elongation is stimulated by positive transcription elongation factor b (P-TEFb), for which activity is repressed in the 7SK small nuclear ribonucleoprotein (7SK snRNP) complex. We show here a critical role of 7SK snRNP in growth control of primordial germ cells (PGCs). The expression of p15(INK4b), a cyclin-dependent kinase inhibitor (CDKI) gene, in PGCs is selectively activated by P-TEFb and its recruiting molecule, Brd4, when the amount of active P-TEFb is increased due to reduction of the 7SK snRNP, and PGCs consequently undergo growth arrest. These results indicate that CDKI gene-specific control of transcription by 7SK snRNP plays a pivotal role in the maintenance of PGC proliferation.

REST and its downstream molecule Mek5 regulate survival of primordial germ cells.

In mouse embryos, some primordial germ cells (PGCs) are eliminated by apoptosis, but the molecular pathways that lead to PGC survival versus apoptosis have not been fully characterized. Here, we found that REST (repressor element 1-silencing transcription factor), a transcription factor that binds a conserved regulatory element, NRSE/RE1, played a role in PGC survival. REST expression was higher in PGCs than in surrounding somatic cells. Moreover, in mouse embryos with a PGC-specific conditional REST mutation, the PGC population experienced more apoptosis and was significantly smaller than that in control embryos; these findings indicated that REST functioned in a cell-autonomous fashion that was critical for PGC survival. Several anti-apoptotic genes were among the previously identified REST-target gene candidates; moreover, some of these genes were downregulated in the REST-deficient PGCs. Mek5, which encodes a component in the a MAP kinase cascade, was one of these downregulated REST-target gene candidates, and a Mek5 mutation, like the REST mutation, caused an increase in PGC apoptosis; these finding suggested that REST promoted PGC survival via regulation of the Mek5 expression. Importantly, there were a normal number of PGCs in the REST mutants at birth, and both the male and female REST-mutant adults were fertile; these final observations revealed that the PGC population was very robust and could recover from a genetically induced reduction in cell number.

Expression and proliferation-promoting role of Diversin in the neuronally committed precursor cells migrating in the adult mouse brain.

The subventricular zone (SVZ) is the largest neurogenic region in the adult rodent brain. In the adult SVZ, unlike in the embryonic brain, neuronally committed precursor cells (neuroblasts) maintain their proliferative activity while migrating toward the olfactory bulb (OB), suggesting that they are inhibited from exiting the cell cycle. Little is known about the mechanisms underlying the unique ability of adult neuroblasts to proliferate during migration. Here, we studied the expression and function of Diversin, a component of the Wnt signaling pathways. In the neonatal and adult mouse brain, Diversin expression was observed in neuroblasts and mature neurons in the SVZ and hippocampus. Retrovirus-mediated overexpression of Diversin promoted the proliferation of neuroblasts and increased the number of neuroblasts that reached the OB. Conversely, the knockdown of Diversin decreased the proliferation of neuroblasts. Our results indicate that Diversin plays an important role in the proliferation of neuroblasts in the SVZ of the adult brain.

Identification of antisense RNA stem-loops that inhibit RNA-protein interactions using a bacterial reporter system.

Many well-characterized examples of antisense RNAs from prokaryotic systems involve hybridization of the looped regions of stem-loop RNAs, presumably due to the high thermodynamic stability of the resulting loop-loop and loop-linear interactions. In this study, the identification of RNA stem-loops that inhibit U1A protein binding to the hpII RNA through RNA-RNA interactions was attempted using a bacterial reporter system based on phage lambda N-mediated antitermination. As a result, loop sequences possessing 7-8 base complementarity to the 5' region of the boxA element important for functional antitermination complex formation, but not the U1 hpII loop, were identified. In vitro and in vivo mutational analysis strongly suggested that the selected loop sequences were binding to the boxA region, and that the structure of the antisense stem-loop was important for optimal inhibitory activity. Next, in an attempt to demonstrate the ability to inhibit the interaction between the U1A protein and the hpII RNA, the rational design of an RNA stem-loop that inhibits U1A-binding to a modified hpII was carried out. Moderate inhibitory activity was observed, showing that it is possible to design and select antisense RNA stem-loops that disrupt various types of RNA-protein interactions.

Cyclin-dependent kinase 5 is required for control of neuroblast migration in the postnatal subventricular zone.

At the lateral wall of the lateral ventricles in the adult rodent brain, neuroblasts form an extensive network of elongated cell aggregates called chains in the subventricular zone and migrate toward the olfactory bulb. The molecular mechanisms regulating this migration of neuroblasts are essentially unknown. Here, we report a novel role for cyclin-dependent kinase 5 (Cdk5), a neuronal protein kinase, in this process. Using in vitro and in vivo conditional knock-out experiments, we found that Cdk5 deletion impaired the chain formation, speed, directionality, and leading process extension of the neuroblasts in a cell-autonomous manner. These findings suggest that Cdk5 plays an important role in neuroblast migration in the postnatal subventricular zone.