Clinicians should have a high index of suspicion and be aware of the clinical and radiologic characteristics of TOM.

Summary Pig embryonic stem cells (pESCs) have been considered an important candidate for preclinical research on human therapies. However, the lack of understanding of pig pluripotent networks has hampered establishment of authentic pESCs. Here, we report that FGF2, ACTVIN, and WNT signaling are essential to sustain pig pluripotency in vitro . Newly derived pESCs were stably maintained over an extended period, and capable of forming teratomas that contained three germ layers. Transcriptome analysis showed that pESCs were developmentally similar to late epiblasts of preimplantation embryos and in terms of biological functions resembled human rather than mouse pluripotent stem cells. However, the pESCs had distinct features such as coexpression of SSEA1 and SSEA4, two active X chromosomes, and a unique transcriptional pattern. Our findings will facilitate both the development of large animal models for human stem cell therapy and the generation of pluripotent stem cells from other domestic animals for agricultural use.

X-chromosome inactivation (XCI) is an epigenetic mechanism that occurs in the eutherian embryo development to equalize the dosage of X-linked genes between males and females. This event is regulated by various factors, and the genes located in the X-chromosome inactivation center (XIC), which is known to be an evolutionary conserved region, are associated with XCI; however, a number of studies regarding this epigenetic event and genomic region are primarily performed in mouse models despite its species-specific features. Thus, in this study, the porcine XIC was identified, and we analyzed the expression of XIC-linked genes in porcine preimplantation embryos. Comparative sequence analysis revealed that the porcine XIC is synteny with that of human and the non-coding RNAs were less conserved compared with the protein coding genes in the XIC. Among the XIC-linked genes, the expression levels of CHIC1 and RLIM were decreased from morula to blastocyst development and their dosage was compensated between the male and female blastocysts. Additionally, the CpG sites of CHIC1 were approximately 50% methylated in parthenote blastocysts. Contrary to these genes, XIST and LOC102165544, an uncharacterized non-coding gene, showed dramatically increased expression levels after the morula stage and preferential female expression in blastocysts. Imprinted XIST expression was not observed, and their CpG sites were hypo-methylated in parthenogenic blastocysts. These results demonstrate that the porcine XIC consists of an evolutionary conserved structure with fewer sequences conserved non-coding RNAs. In addition, a few XIC-linked genes would likely achieve dosage compensation, but XCI would not be completed in porcine blastocysts.

Cellular reprogramming of committed cells into a pluripotent state can be induced by ectopic expression of genes such as OCT4, SOX2, KLF4, and MYC. Reprogrammed cells can be maintained by activating endogenous pluripotent networks without transgene expression. Although various research groups have attempted to generate pig induced pluripotent stem cells (iPSCs), authentic iPSCs have not be obtained, instead showing dependence on transgene expression. In this study, iPSCs were derived from porcine fetal fibroblasts via drug-inducible vectors carrying human transcription factors (OCT4, SOX2, KLF4, and MYC). Therefore, this study investigated characteristics of iPSCs and reprogramming mechanisms in pig. The iPSCs were stably maintained over an extended period with potential in vitro differentiation into three germ layers. In addition, the pluripotent state of iPSCs was regulated by modulating culture conditions. They showed naive- or primed-like pluripotent states in LIF or bFGF supplemented culture conditions, respectively. However, iPSCs could not be maintained without ectopic expression of transgenes. The cultured iPSCs expressed endogenous transcription factors such as OCT4 and SOX2, but not NANOG (a known gateway to complete reprogramming). Endogenous genes related to mesenchymal-to-epithelial transition (DPPA2, CDH1, EPCAM, and OCLN) were not sufficiently reactivated, as measured by qPCR. DNA methylation analysis for promoters of OCT4, NANOG, and XIST showed that epigenetic reprogramming did not occur in female iPSCs. Based on our results, expression of exogenous genes could not sufficiently activate the essential endogenous genes and remodel the epigenetic milieu to achieve faithful pluripotency in pig. Accordingly, investigating iPSCs could help us improve and develop reprogramming methods by understanding reprogramming mechanisms in pig.

Angiomyxolipoma (vascular myxolipoma) is a recently described rare variant of lipoma, four cases of which have been reported to date. Microscopically, the lesion consists of adipose tissue without lipoblasts, extensive myxoid areas, and numerous blood vessels. The main differential diagnosis of this lesion is myxoid liposarcoma, and other adipocytic lesions such as myxolipoma, myxoid spindle cell lipoma should be included. We report two cases of angiomyxolipoma located in the subcutaneous tissue of the forearm and the wrist.

Pneumatic otoscopy is most helpful for optimally assessing the presence of a middle ear effusion (MEE). To evaluate the diagnostic usefulness and to obtain objective parameters of video pneumatic otoscope (VPO) in ears with MEE, we measured the minimal and maximal pressures in the external auditory canal and recorded the movement of the tympanic membrane (TM) during VPO in 28 ears with MEE and 13 healthy ears. The movements of the TM at static stage, positive and negative pressure stages of VPO were analyzed in terms of the position of the umbo and the angle and length of the malleus. The percent volume of air space out of the total tympanomastoid air cell system (the air index) was obtained from the temporal bone CT, and was used as a reference standard. As a result, minimal pressure and the movement of the umbo from negative pressure stage in ears with MEE were significantly different from the normal ears. The minimal pressure, maximal pressure and the movement of umbo from negative pressure to static stage were significantly correlated with the air index. These results may suggest useful parameters for quantitative analysis of VPO for the visual diagnosis of MEE.

Objectives Gene regulation in early embryos has been widely studied for a long time because lineage segregation gives rise to the formation of a pluripotent cell population, known as the inner cell mass (ICM), during pre‐implantation embryo development. The extraordinarily longer pre‐implantation embryo development in pigs leads to the distinct features of the pluripotency network compared with mice and humans. For these reasons, a comparative study using pre‐implantation pig embryos would provide new insights into the mammalian pluripotency network and help to understand differences in the roles and networks of genes in pre‐implantation embryos between species. Materials and methods To analyse the functions of SOX2 in lineage segregation and cell proliferation, loss‐ and gain‐of‐function studies were conducted in pig embryos using an overexpression vector and the CRISPR/Cas9 system. Then, we analysed the morphological features and examined the effect on the expression of downstream genes through immunocytochemistry and quantitative real‐time PCR. Results Our results showed that among the core pluripotent factors, only SOX2 was specifically expressed in the ICM. In SOX2‐disrupted blastocysts, the expression of the ICM‐related genes, but not OCT4, was suppressed, and the total cell number was also decreased. Likewise, according to real‐time PCR analysis, pluripotency‐related genes, excluding OCT4, and proliferation‐related genes were decreased in SOX2‐targeted blastocysts. In SOX2‐overexpressing embryos, the total blastocyst cell number was greatly increased but the ICM/TE ratio decreased. Conclusions Taken together, our results demonstrated that SOX2 is essential for ICM formation and cell proliferation in porcine early‐stage embryogenesis.

OCT4 plays pivotal roles in maintaining pluripotency during early mammalian embryonic development and in embryonic stem cells. It is essential to establish a reporter system based on the OCT4 promoter region to study pluripotency. However, there is still a lack of information about the porcine OCT4 upstream reporter system. To improve our understanding of the porcine OCT4 regulatory region, we identified conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. The similarity of nucleotide sequences in the 5′ upstream region was low among mammalian species. However, the OCT4 promoter and four regulatory regions, including distal and proximal enhancer elements, had high similarity. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay results indicated that the porcine OCT4 distal enhancer and proximal enhancer were highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively. A comparison analysis of naïve and primed state marker gene expression in a dual-reporter assay showed that the expression levels of naïve and primed markers differed in fluorescence signal between high-expressing cells and low-expressing cells. Similar to OCT4 upstream-based reporter systems derived from other species, the porcine OCT4 upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work provides basic information about the porcine OCT4 upstream region and various porcine OCT4 fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and the establishment of embryonic stem cells in pigs.