Analysis of histone H3K4me3 modifications in bovine placenta derived from different calf-production methods.

In ruminants, the overgrowth of offspring produced by in vitro fertilization (IVF) is a common problem. Abnormal epigenetic modifications caused by environmental factors during the early embryonic period are suspected as an aetiology of overgrowth. In this study, we investigated the genome-wide histone H3K4me3 profiles of bovine placentae that play a pivotal role in foetal development and compared their characteristics between artificial insemination (AI)- and IVF-derived samples. Cotyledons were harvested from the placentae obtained at parturition of 5 AI- and 13 IVF-derived calves, and chromatin immunoprecipitation sequencing was performed for H3K4me3. We confirmed no significant maternal tissue contamination in the samples we used. The revealed H3K4me3 profiles reflected the general characteristics of the H3K4me3 modification, which is abundantly distributed in the promoter region of active genes. By extracting common modifications from multiple samples, the genes involved in placenta-specific biological processes could be enriched. Comparison with the H3K4me3 modifications of blastocyst samples was also effective for enriching the placenta-specific features. Principal component analysis suggested the presence of differential H3K4me3 modifications in AI- and IVF-derived samples. The genes contributing to the difference were related to the developmental biological processes. Imprinted genes such as BEGAIN, ZNF215 and DLX5 were among the extracted genes. Principal component and discriminant analyses using only male samples categorized the samples into three groups based on foetal weight and calf-production methods. To our knowledge, this is the first study to profile the genome-wide histone modifications of bovine foetal placentae and reveal their differential characteristics between different calf-production methods.

Three-dimensional morphology of bovine blastocysts hatched against lipopolysaccharide exposure in vitro.

Embryo transfer in cattle is globally becoming more ubiquitous, but the pregnancy rate is lower than that of artificial insemination. The uterus contains its own bacteria, and concentrations of lipopolysaccharides (LPS) from gram-negative bacteria are higher in uteri affected by endometritis than in healthy uteri and they suppress embryogenesis. The purpose of this study was to investigate the morphological characteristics of bovine embryos with a higher viability and implantability, by analyzing the morphology of bovine blastocysts that successfully hatched under challenge of LPS, using an optical coherence tomography (OCT) system. Developing embryos produced by in vitro fertilization that had reached the blastocyst stage on Day 7 were three-dimensionally scanned using an OCT system, then were continued to culture with or without LPS until Day 9, when the presence or absence of hatching was determined. The OCT-captured three-dimensional images were used to quantify 20 different metrics, including inner cell mass (ICM), trophectoderm, blastocoel, and total embryo volume; each of the parameters was compared between the hatched and unhatched embryos. Under the LPS challenge, hatched embryos had higher ICM thickness and volume, and lower trophectoderm thickness than unhatched embryos. Furthermore, hatched embryos under LPS challenge had higher ICM thickness and ICM volume than hatched embryos without LPS challenge. The present results suggest the possibility that ICM thickness and ICM volume calculated by OCT system could be indices for good quality bovine embryos.

Hatchability evaluation of bovine IVF embryos using OCT-based 3D image analysis.

Although embryo transfer is widely applied in cattle, many of the transferred embryos do not result in pregnancy. To determine a new parameter for bovine embryo evaluation, we investigated the relationships between in vitro hatchability and embryo morphological parameters using optical coherence tomography (OCT) that we established recently. Bovine embryos were obtained from Japanese Black cattle by in vitro fertilization (IVF). The quality of the blastocysts was examined under an inverted microscope and confirmed as Codes 1-3 according to the IETS standards for embryo evaluation. The OCT images of the embryos were captured on Day 7 after IVF, and the embryos were cultured until Day 9 to determine their hatchability. During OCT, the embryos were irradiated with near-infrared light for a few minutes to obtain three-dimensional images. In total, 22 parameters were assessed for each of the 42 embryos, of which 25 hatched (H embryos) and 17 did not (NH embryos). The thickness of the trophectoderm (TE) and TE+zona pellucida (ZP) was lesser, and the volumes of the TE, ZP, blastocoel, and whole embryo and blastocoel diameter were greater in the H embryos than in the NH embryos. PCA identified that the increase in the blastocoel-related value along with the decrease in the thickness-related value of the TE and/or ZP could be indicators for evaluating the hatchability of bovine IVF embryos. These results support the idea that OCT-captured structural data of blastocyst-stage embryos can be used as a potential model to predict the quality of bovine embryos.

A Case of a Chronic Expanding Hematoma Caused by an Epidermal Cyst.

Chronic expanding hematoma (CEH) is a rare type of hematoma that expands slowly and continuously without compromising coagulation. Its etiology is often unknown. However, we experienced a rare case of CEH, in which an epidermal cyst was thought to be the cause. A 57-year-old man had developed a painless soft-tissue tumor in his left buttock 45 years earlier, which slowly grew to 11 cm in diameter. Preoperative magnetic resonance imaging suggested a large cyst containing several masses. Surgery revealed a hematoma and keratin debris surrounded by a white fibrous cyst wall and a fibrous nodule measuring 4 cm in diameter. On histopathological examination of the white fibrous wall, an epidermal component was observed contralateral to the superficial punctum, but the epidermal component was absent from most of the wall, including the fibrous nodule. Based on a pathological examination, the CEH was suggested to have been caused by partial rupturing and inflammation of an epidermal cyst. To the best of our knowledge, there are no reports of epidermal cysts causing CEH. In addition, the large fibrous nodule protruding from the CEH cyst wall was considered to be rare. This was considered to be a rare CEH that may have originated from an epidermal cyst.

Costal Cartilage Graft to Prevent Drooping after Free Flap Reconstruction of the Lower Lip.

For large lower lip defects, a thin flap combined with a tendon is the standard reconstructive option. However, this method can result in flap ptosis, which occurred in two of our patients. To correct the ptosis, we transplanted costal cartilage into the reconstructed lower lips, which produced good or moderate results. We report our experience based on long-term follow-up. In case 1, reconstruction was performed with a latissimus dorsi myocutaneous flap. Within 10 years of the first cartilage transplant, two additional surgeries were required due to cartilage/screw breakage. These problems may have been triggered by the bulkiness of the flap and/or the angle at which the cartilage was anchored in place. There have not been any further problems for 3 years. In case 2, reconstruction was performed with a free anterolateral thigh flap. The skin around the flap had poor extensibility, and the patient had marked Class II occlusion. We grafted cartilage without fixing it to the mandible. However, temporary interference with the maxillary dentition was observed. In conclusion, costal cartilage grafts are effective against flap ptosis after free flap reconstruction of the lower lip in patients without Class II occlusion. To achieve long-term stability, the optimal angle and positioning of the cartilage and the extensibility of the skin must be thoroughly investigated before surgery, and a thick piece of cartilage must be firmly fixed in place.

A case of cryptococcal necrotizing fasciitis and immune reconstitution inflammatory syndrome in a renal transplantation recipient.

INTRODUCTION AND IMPORTANCE: Immunocompromised patients are at high risk of unexpectedly serious infections caused by uncommon bacteria or fungi. We experienced a case of Cryptococcus neoformans-induced necrotizing fasciitis (NF) of the lower extremities. The progress so far has been reported by the urology department [1]. Moreover, after the NF had been treated, the patient developed immune reconstitution inflammatory syndrome (IRIS). We report from surgeon's view point. CASE PRESENTATION: A 51-year-old male renal transplant patient complained of pain in both lower extremities (LE). After the initial debridement, periodic acid-Schiff after diastase digestion (D-PAS) staining confirmed the diagnosis. No symptoms were seen in the lungs or cerebrospinal system. The patient was reluctant to undergo surgical treatment but several debridement improved patient's condition. After the LE wound healed, prednisolone was discontinued, then painful nodules appeared on both LE. Based on the negative culture results and the fact that the patient had been treated with flucytosine and fluconazole, we suspected that the nodules had been caused by IRIS. CLINICAL DISCUSSION: It was difficult to diagnose Cryptococcus-induced NF and paradoxical IRIS. Cooperation from other specialists was essential. CONCLUSION: We think this patient needed earlier and more definitive debridement. Fortunately, we were able to save the patient's life and maintain his LE function. In immunocompromised patients, cryptococcus can be a pathogen. In addition, IRIS can occur during treatment. Management of IRIS is the capital point of sepsis management, careful anti-inflammatory drug control by specialists is required.

Three-Dimensional Live Imaging of Bovine Preimplantation Embryos: A New Method for IVF Embryo Evaluation.

Conception rates for transferred bovine embryos are lower than those for artificial insemination. Embryo transfer (ET) is widely used in cattle but many of the transferred embryos fail to develop, thus, a more effective method for selecting bovine embryos suitable for ET is required. To evaluate the developmental potential of bovine preimplantation embryos (2-cell stage embryos and blastocysts), we have used the non-invasive method of optical coherence tomography (OCT) to obtain live images. The images were used to evaluate 22 parameters of blastocysts, such as the volume of the inner cell mass and the thicknesses of the trophectoderm (TE). Bovine embryos were obtained by in vitro fertilization (IVF) of the cumulus-oocyte complexes aspirated by ovum pick-up from Japanese Black cattle. The quality of the blastocysts was examined under an inverted microscope and all were confirmed to be Code1 according to the International Embryo Transfer Society standards for embryo evaluation. The OCT images of embryos were taken at the 2-cell and blastocyst stages prior to the transfer. In OCT, the embryos were irradiated with near-infrared light for a few minutes to capture three-dimensional images. Nuclei of the 2-cell stage embryos were clearly observed by OCT, and polynuclear cells at the 2-cell stage were also clearly found. With OCT, we were able to observe embryos at the blastocyst stage and evaluate their parameters. The conception rate following OCT (15/30; 50%) is typical for ETs and no newborn calves showed neonatal overgrowth or died, indicating that the OCT did not adversely affect the ET. A principal components analysis was unable to identify the parameters associated with successful pregnancy, while by using hierarchical clustering analysis, TE volume has been suggested to be one of the parameters for the evaluation of bovine embryo. The present results show that OCT imaging can be used to investigate time-dependent changes of IVF embryos. With further improvements, it should be useful for selecting high-quality embryos for transfer.

Three-dimensional live imaging of bovine embryos by optical coherence tomography.

While embryo transfer (ET) is widely practiced, many of the transferred embryos fail to develop in cattle. To establish a more effective method for selecting bovine embryos for ET, here we quantified morphological parameters of living embryos using three-dimensional (3D) images non-invasively captured by optical coherence tomography (OCT). Seven Japanese Black embryos produced by in vitro fertilization that had reached the expanded blastocyst stage after 7 days of culture were transferred after imaged by OCT. Twenty-two parameters, including thickness and volumes of the inner cell mass, trophectoderm, and zona pellucida, and volumes of blastocoel and whole embryo, were quantified from 3D images. Four of the seven recipients became pregnant. We suggest that these 22 parameters can be potentially employed to evaluate the quality of bovine embryos before ET.

Squamous cell carcinoma developed in a chronic radiation-induced chest wall ulcer that is difficult to undergo thorough preoperative histological examination.

INTRODUCTION: We report a case in which squamous cell carcinoma (SCC) developed in a large chronic radiation-induced thoracic ulcer after flap surgery in areas where preoperative histological examinations are difficult. PRESENTATION OF CASE: The patient was a 75-year-old female. She had undergone resection and radiotherapy for left breast cancer 15 years earlier. Six years ago, the ulcer expanded from the subclavian to xiphoid levels, exposing the lung and pericardium. A histopathological examination, which avoided the lung and pericardium, was performed. Inflammation was diagnosed. We reconstructed the chest wall with a pedicled rectus abdominis flap. Eighteen months later, three verrucous tissue-lined fistulas formed. A histological examination revealed well-differentiated SCC. Six months later, the patient died of massive bleeding from a fistula. DISCUSSION: It is unclear exactly when the SCC occurred. As three fistulas formed at the margins of the flap around the pericardium, we suspect that the cancer developed within or near the pericardial region. We need to reflect on the lack of a thorough biopsy. As no pericardial biopsy was performed, we should have asked a thoracic/cardiac surgeon to conduct a biopsy during the debridement operation. If the tumor had been localized to the pericardium, it could have been removed. CONCLUSION: It is necessary to consider the best method for performing the most thorough histological examination possible, even in areas where histological examinations are difficult, as all ulcers can contain tumors.

Function Preservation of the Upper Lip after Tumor Resection Using Residual Orbicularis Oris Muscle and Attached Levator Labii Superioris Alaeque Nasi.

We report a case, function preservation of the upper lip after tumor resection was possible using residual orbicularis oris muscle and attached levator labii superioris alaeque nasi. Patient was 67-year-old male with squamous cell carcinoma at the vermilion border. The tumor was resected with an 8-mm margin, leaving the oral mucosa as intact as possible. To reconstruct the red lip, we used the oral mucosa as a rotational transposition flap. The white lip was reconstructed with a cheek rotation flap. A levator labii superioris alaque nasi muscle flap, which was attached to the remaining orbicularis oris muscle, was used to increase marginal lip volume. The movement of the reconstructed lip was good. At 9 postoperative months, induration of the red lip was palpable, and we suspected that the blood supply to the levator labii superioris alaque nasi was borderline insufficient. Slight drooping of the reconstructed lip occurred. We dissected this was caused by dissection of mid facial muscles from orbicularis oris muscle to ease downward rotation of the cheek flap and obscure the original nasolabial fold. Although some drooping and induration of the lip occurred, the white and red lip were reconstructed in a single-stage procedure, which resulted in good movement and preserved the function of the orbicularis oris muscle.

Biological effects of chicken type III interferon on expression of interferon-stimulated genes in chickens: comparison with type I and type II interferons.

Interferons (IFNs) are key mediators that activate host defense mechanisms against viruses. The recently identified mammalian Type III IFN has biological effects similar to type I IFN. However, the biological effects of type III IFN have not yet been characterized in birds. We compared the effects of chicken type III IFN (IFN-λ) with type I (IFN-ß) and type II (IFN-γ) IFNs on IFN-stimulated genes (ISGs) using recombinant proteins expressed in Escherichia coli. Recombinant chicken IFN-λ inhibited influenza virus replication and induced the mRNA expression of the ISGs, Mx and OAS, in chicken embryonic fibroblasts (CEFs) in a dose-dependent manner. However, the effective dose of IFN-λ was higher than that of IFN-ß and IFN-γ. Furthermore, the effect of IFN-λ on induction of Mx and OAS was lesser than that of IFN-ß, but comparable to that of IFN-γ. These results indicate that chicken IFN-λ has the potential to induce ISGs and inhibit viral replication in chicken cells.