It is now recognized that extensive expression heterogeneities among cells precede the emergence of lineages in the early mammalian embryo. To establish a map of pluripotent epiblast (EPI) versus ...primitive endoderm (PrE) lineage segregation within the inner cell mass (ICM) of the mouse blastocyst, we characterized the gene expression profiles of individual ICM cells. Clustering analysis of the transcriptomes of 66 cells demonstrated that initially they are non-distinguishable. Early in the segregation, lineage-specific marker expression exhibited no apparent correlation, and a hierarchical relationship was established only in the late blastocyst. Fgf4 exhibited a bimodal expression at the earliest stage analysed, and in its absence, the differentiation of PrE and EPI was halted, indicating that Fgf4 drives, and is required for, ICM lineage segregation. These data lead us to propose a model where stochastic cell-to-cell expression heterogeneity followed by signal reinforcement underlies ICM lineage segregation by antagonistically separating equivalent cells.
DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b cooperatively regulate cytosine methylation in CpG dinucleotides in mammalian genomes, providing an epigenetic basis for gene silencing and maintenance ...of genome integrity. Proper CpG methylation is required for the normal growth of various somatic cell types, indicating its essential role in the basic cellular function of mammalian cells. Previous studies using Dnmt1−/– or Dnmt3a−/–Dnmt3b−/– ES cells, however, have shown that undifferentiated embryonic stem (ES) cells can tolerate hypomethylation for their proliferation. In an attempt to investigate the effects of the complete loss of CpG DNA methyltransferase function, we established mouse ES cells lacking all three of these enzymes by gene targeting. Despite the absence of CpG methylation, as demonstrated by genome‐wide methylation analysis, these triple knockout (TKO) ES cells grew robustly and maintained their undifferentiated characteristics. TKO ES cells retained pericentromeric heterochromatin domains marked with methylation at Lys9 of histone H3 and heterochromatin protein‐1, and maintained their normal chromosome numbers. Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA methyltransferases.
DNA methylation is a major epigenetic mechanism that has been suggested to control developmental gene regulation during embryogenesis, but its regulatory mechanisms remain unclear. In this report, we ...show that CpG islands associated with the X-linked homeobox gene cluster Rhox, which is highly expressed in the extraembryonic trophectoderm, are differentially methylated in a stage- and lineage-specific manner during the post-implantation development of mice. Inactivation of both Dnmt3a and Dnmt3b, DNA methyltransferases essential for the initiation of de novo DNA methylation, abolished the establishment of DNA methylation and the silencing of Rhox cluster genes in the embryo proper. The Dnmt3-dependent CpG-island methylation at the Rhox locus extended for a large genomic region ( approximately 1 Mb) containing the Rhox cluster and surrounding genes. Complementation experiments using embryonic stem (ES) cells deficient in the DNA methyltransferases suggested that the CpG-island methylation by Dnmt3a and Dnmt3b was restricted within this large genomic region, and did not affect the neighboring genes outside it, implicating the existence of region-specific boundaries. Our results suggest that DNA methylation plays important roles in both long-range gene silencing and lineage-specific silencing in embryogenesis.
The transcription factors Nanog and Gata6 are critical to specify the epiblast versus primitive endoderm (PrE) lineages. However, little is known about the mechanisms that regulate the protein ...stability and activity of these factors in the developing embryo. Here we uncover an early developmental function for the Polycomb group member Bmi1 in supporting PrE lineage formation through Gata6 protein stabilization. We show that Bmi1 is enriched in the extraembryonic (endoderm XEN and trophectodermal stem TS) compartment and repressed by Nanog in pluripotent embryonic stem (ES) cells. In vivo, Bmi1 overlaps with the nascent Gata6 and Nanog protein from the eight-cell stage onward before it preferentially cosegregates with Gata6 in PrE progenitors. Mechanistically, we demonstrate that Bmi1 interacts with Gata6 in a Ring finger-dependent manner to confer protection against Gata6 ubiquitination and proteasomal degradation. A direct role for Bmi1 in cell fate allocation is established by loss-of-function experiments in chimeric embryoid bodies. We thus propose a novel regulatory pathway by which Bmi1 action on Gata6 stability could alter the balance between Gata6 and Nanog protein levels to introduce a bias toward a PrE identity in a cell-autonomous manner.
Epstein-Barr virus (EBV) reactivation in COVID-19 patients has been reported, but studies on its clinical significance are lacking. We herein report the occurrence of infectious mononucleosis (IM) ...due to EBV reactivation in a 60-year-old man with rheumatoid arthritis being treated with methotrexate and tocilizumab. The patient presented with a fever and tested positive for COVID-19. Laboratory findings revealed an increased atypical lymphocyte count, decreased platelet count, and elevated liver enzyme levels. Flow cytometry showed predominant expansion of reactive T cells. EBV reactivation was confirmed using real-time polymerase chain reaction. The patient was treated with remdesivir, and clinical improvement was observed after 10 days of treatment. Follow-up showed a gradual decrease in the EBV-DNA load with no recurrence of atypical lymphocytes. These findings suggest that COVID-19 in immunocompromised patients may lead to unexpected EBV reactivation and IM, even for patients outside the age at which IM is likely to occur.
Intravitreal injection of anti-VEGF drugs has become standard therapy for patients with exudative age-related macular degeneration (AMD). However, some patients do not exhibit sufficient response to ...the drugs for suppression of choroidal neovascularization activity. We investigated the efficacy of switchback from ranibizumab to aflibercept in patients with AMD who could not achieve further benefit beyond initial therapy of aflibercept injection.
Eleven eyes of eleven patients were included in this study. Two patients were nonresponders, and nine exhibited tachyphylaxis to aflibercept. All patients received three monthly injections of ranibizumab as an initial phase of switching and received aflibercept as a switchback drug. We investigated changes in injection interval, visual acuity, and central retinal thickness.
In four patients (36.4%), injection interval was extended. The interval was 6.73 weeks before switch and 9.27 weeks after switchback (
=0.96). LogMAR visual acuity was 0.22 before switch and 0.24 after switchback (
=0.62). Central retinal thickness was 306.8 µm before switch and 256.1 after switchback (
=0.13). In all patients who were nonresponders to aflibercept, injection interval could not be extended.
A switchback from ranibizumab to aflibercept may be beneficial in some patients with AMD who exhibit tachyphylaxis to aflibercept.
Patients with exudative age-related macular degeneration (AMD) who did not respond to ranibizumab at the induction phase were assessed and referred to as initial non-responders.
We retrospectively ...reviewed the medical records of 215 patients (218 eyes) with exudative AMD. For the initial treatments, patients received three intravitreal injections of ranibizumab (IVR) every 4 weeks. Minimum follow-up period was 12 months. We defined patients with no improvement of best corrected logMAR visual acuity (BCVA), and with no decrease of central retinal thickness (CRT) at the end of the initial treatment, as initial non-responders. Patients who had previous treatment history prior to this investigation were included, but patients who had photodynamic therapy (PDT) with IVR were excluded.
Twenty-two eyes (10.1%) were identified as initial non-responders. The mean BCVA of initial non-responders before IVR and after induction phase were 0.39 and 0.36, respectively. There was no significant difference between these values, however the mean BCVA decreased significantly to 0.55 at 12 months after the beginning of the induction phase (P = 0.021). The mean greatest linear dimension (GLD) of the lesion before IVR of initial non-responders was 4,121 μm. We found 16 eyes with typical AMD, and six eyes with polypoidal choroidal vasculopathy. One eye had predominantly classic choroidal neovascularization (CNV), and others had occult CNV of typical AMD. As additional treatments, twelve eyes received PDT, and in three of the eyes exudation remained after PDT.
Initial non-responders were more prevalent in patients with occult CNV than in patients with other CNV types. Some of the initial non-responders did not respond to PDT. This study suggested possible involvement of other factors, in addition to vascular endothelial growth factor, in the occurrence of CNV in initial non-responder patients.
To evaluate the effect of photodynamic therapy (PDT) using a modified procedure on exudative age-related macular degeneration having been conventionally difficult to treat.
The medical records of ...eight consecutive patients (eight eyes) with age-related macular degeneration treated with modified PDT were reviewed retrospectively. Modified PDT was used for the lesions that could not be covered by conventional use of PDT, either because the lesion was too large or too close to the optic disc. A moving PDT laser spot at constant speed, for 83 seconds, was used to cover the entire lesion, and was named "Ironing PDT." This retrospective study was performed with informed patient consent. It was approved by the Institutional Review Board of Kansai Medical University.
No exudation could be found 36 months after treatment in five eyes (62.5%). There was no significant difference between the best-corrected visual acuity before PDT (0.95 logMAR) and after PDT (1.09 logMAR). The logMAR best-corrected visual acuity was improved in one eye, maintained in five eyes, and deteriorated in two eyes.
Ironing PDT decreased subfoveal fluid and preserved visual acuity in some patients with age-related macular degeneration difficult to treat with conventional therapy.
We studied the transport mechanism of pirarubicin (THP) in mononuclear cells (MNCs) obtained from healthy human donors. The THP uptake was time-, temperature-, concentration- and energy (in ...part)-dependent. The uptake of daunorubicin (DNR) and doxorubicin (ADR) was also concentration-dependent, and the transport of ADR consisted of saturable and nonsaturable components. In cis-inhibition experiments, ADR inhibited both THP and DNR uptake noncompetitively, while DNR showed competitive inhibition of the uptake of THP. The THP uptake rate appeared to be increased by preloading DNR, indicating a trans-stimulatory effect, but not with ADR. These results suggest that THP and DNR were taken up into MNCs via a common carrier-mediated transport system, but that the carrier of ADR might differ from that of THP and DNR. Furthermore, apparent differences in the affinity for the carrier, transport efficacy and substrate specificity of the transporter between MNCs and the human leukemia cell lines (HL60 and K562) were indicated.
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