Generation of juvenile rainbow trout derived from cryopreserved whole ovaries by intraperitoneal transplantation of ovarian germ cells.

Cryopreservation of fish sperm offers the practical applications in the selective breeding and biodiversity conservation. However, because of the lack of cryopreservation methods for fish eggs and embryos, maternally inherited cytoplasmic compartments cannot be successfully preserved. We previously developed an alternative method to derive functional eggs and sperm from cryopreserved whole testis by transplanting testicular cells into female and male recipients. However, if target fish had ovaries, the previous method employing male-derived germ cells would be ineffective. Here, we aimed to generate functional gametes from cryopreserved whole ovaries by transplanting ovarian germ cells into peritoneal cavity of sterile hatchlings. Cryopreservation conditions for rainbow trout ovaries (1.0 M DMSO, 0.1 M trehalose, and 10% egg yolk) were optimized by testing several different cryoprotective agents. Ovarian germ cells from thawed ovaries were intraperitoneally transplanted into allogeneic triploid hatchlings. Transplanted germ cells migrated toward and were incorporated into recipient gonads, where they underwent gametogenesis. Transplantation efficiency of ovarian germ cells remained stable after cryopreservation period up to 1185 days. Although all triploid recipients that did not undergo transplantation were functionally sterile, 5 of 25 female recipients and 7 of 25 male recipients reached sexual maturity at 2.5 years post-transplantation. Inseminating the resultant eggs and sperm generated viable offspring displaying the donor characteristics of orange body color, green fluorescence, and chromosome numbers. This method is thus a breakthrough tool for the conservation of endangered fish species that are crucial to cryopreserve the genetic resources of female fish.

Germ Cell-Specific Excision of loxP-Flanked Transgenes in Rainbow Trout Oncorhynchus mykiss.

Cre/loxP-mediated DNA excision in germ cell lineages could contribute substantially to the study of germ cell biology in salmonids, which are emerging as a model species in this field. However, a cell type-specific Cre/loxPsystem has not been successfully developed for any salmonid species. Therefore, we examined the feasibility of Cre/loxP-mediated, germ cell-specific gene excision and transgene activation in rainbow trout. Double-transgenic (wTg) progeny were obtained by mating a transgenic male carryingcrewith a transgenic female carrying thehsc-LRLGgene;crewas driven by rainbow troutvasaregulatory regions and thehsc-LRLGgene was made up of the rainbow troutheat-shock-cognate71promoter, theDsRedgene flanked by twoloxPsites, and theEgfpgene. PCR analysis, fluorescence imaging, and histological analysis revealed that excision of theloxP-flanked sequence and activation ofEgfpoccurred only in germ cells of wTg fish. However, progeny tests revealed that the excision efficiency ofloxP-flanked sequence in germ cells was low (≤3.27%). In contrast, the other wTg fish derived from two differentcre-transgenic males frequently excised theloxP-flanked sequence in germ cells (≤89.25%). Thus, we showed for the first time successful germ cell-specific transgene manipulation via the Cre/loxPsystem in rainbow trout. We anticipate that this technology will be suitable for studies of cell function through cell targeting, cell-linage tracing, and generating cell type-specific conditional gene knockouts and separately for developing sterile rainbow trout in aquaculture.

Isolation of Shiga Toxin-Producing Escherichia coli from the Surfaces of Beef Carcasses in Slaughterhouses in Japan.

Shiga toxin-producing E. coli (STEC) is an important foodborne pathogen worldwide. It is necessary to control and prevent STEC contamination on beef carcasses in slaughterhouses because STEC infection is associated with beef consumption. However, the frequencies of STEC contamination of beef carcasses in various slaughterhouses in Japan are not well known. Herein, we investigated the contamination of beef carcasses with STEC in slaughterhouses to assess the potential risks of STEC. In total, 524 gauze samples were collected from the surfaces of beef carcasses at 12 domestic slaughterhouses from November 2020 to February 2023. The samples were measured for aerobic plate counts and tested for pathogenic genes (stx and eae) and major O-serogroups (O26, O45, O103, O111, O121, O145, and O157) by real-time PCR screening. Subsequently, immunomagnetic separation (IMS) was performed on samples positive for stx, eae, and at least one of the seven O-serogroups of STEC. Isolation process without IMS was performed on samples positive for stx, including those subjected to IMS. STEC O157:H7 and stx-positive E. coli other than serotype O157:H7 were isolated from 0.6% and 4.6% of beef carcass surfaces, respectively. Although the STEC O157:H7 isolation rate was low and stx-positive E. coli other than serotype O157:H7 belonged to minor O-serogroups, the results mean a risk of foodborne illness. Furthermore, a moderate correlation was observed between aerobic plate counts and detection rates of stx-positive samples by real-time PCR screening. The STEC O157:H7 isolated facilities showed higher values on aerobic plate counts and detection rates of stx-positive samples than the mean values of total samples. Therefore, these results suggest that it is important to evaluate hygiene treatments against beef carcasses for the reduction of STEC contamination risk, particularly in facilities with high aerobic plate counts.

Combining next-generation sequencing with microarray for transcriptome analysis in rainbow trout gonads.

Microarray technology is a powerful tool for studying genome-wide gene expression. As the genome of many fish has not yet been determined, however, cDNA microarrays can only be designed from limited expressed sequence tag data. In this study, we designed a microarray based on the sequencing data (337,466 reads) obtained by next-generation sequencing of RNA extracted from rainbow trout (Oncorhynchus mykiss) embryonic genital ridge, testis, and ovary. These data (307,264 reads) were assembled into 28,668 contigs; 3,298 reads could not be assembled and 26,904 reads were unique sequences that did not cluster with other reads. Based on this information, 55,928 microarray probes were designed for a microarray, which was validated by hybridization experiments with RNA extracted from type A spermatogonia (A-SG) and testicular somatic cells. Expression of known spermatogonial markers was confirmed to be higher in A-SG than in testicular somatic cells whereas supporting-cell markers were expressed at higher levels in testicular somatic cells. This microarray analysis revealed that 8,068 transcripts showed at least fourfold higher signal in A-SG than testicular somatic cells. Fourteen of 17 randomly selected transcripts were expressed at significantly higher-levels in A-SG than somatic cells, by quantitative RT-PCR. In addition, three transcripts analyzed with in situ hybridization showed A-SG-specific signals in immature trout testis, with one of them exhibiting a heterogeneous expression pattern in A-SG. The rainbow trout gonad microarray developed in this study therefore appears to be a useful tool to understand gametogenesis in rainbow trout.

Production of Germ Cell-Less Rainbow Trout by dead end Gene Knockout and their Use as Recipients for Germ Cell Transplantation.

In germ cell transplantation experiments, the use of sterile recipients that do not produce their own gametes is an important prerequisite. Triploidization and dnd gene knockdown (KD) methods have been widely used to produce sterile fish. However, triploidization does not produce complete sterility in some fish species, and gene KD is labor and time intensive since it requires microinjection into individual fertilized eggs. To overcome these problems, in this study, we generated homozygous mutants of the dead end (dnd) gene in rainbow trout (Oncorhynchus mykiss) using the clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, analyzed their reproductive capacity, and evaluated their suitability as recipients for germ cell transplantation. By crossing F1 heterozygous mutants produced from founders subjected to genome editing, an F2 generation consisting of approximately 1/4 homozygous knockout mutants (dnd KO) was obtained. The dnd KO hatchlings retained the same number of primordial germ cells (PGCs) as the wild-type (WT) individuals, after which the number gradually decreased. At 1 year of age, germ cells were completely absent in all analyzed individuals. To evaluate the dnd KO individuals as recipients for germ cell transplantation, germ cells prepared from donor individuals were transplanted into the abdominal cavity of dnd KO hatchlings. These cells migrated to the recipient gonads, where they initiated gametogenesis. The mature recipient individuals produced only donor-derived sperm and eggs in equivalent numbers to WT rainbow trout. These results indicate that dnd KO rainbow trout are suitable recipient candidates possessing a high capacity to nurse donor-derived germ cells.

The utility of transarterial embolization and computed tomography for life-threatening spontaneous retroperitoneal hemorrhage.

PURPOSE: To assess the safety and efficacy of transarterial embolization (TAE) and to evaluate the utility of contrast-enhanced computed tomography (CE-CT) for life-threatening spontaneous retroperitoneal hemorrhage (SRH). METHODS: Nineteen patients underwent TAE following CE-CT for life-threatening SRH. CE-CT and angiographic findings, technical successes, and clinical successes were evaluated. The diagnostic performance of CE-CT for the detection of active bleeding arteries was also assessed by two independent readers. RESULTS: Active extravasation of contrast material was accurately observed in 78.9‒84.2% of the patients on CE-CT. Angiograms revealed active extravasation in 37 arteries of 15 patients (78.9%), and 4 patients showed no sign of active bleeding. Sensitivity, positive predictive value, and accuracy rate of CE-CT for the detection of active bleeding vessels was 59.5%, 62.9‒71.0% and 55.6‒60.0% respectively. The successful embolization of 48 intended arteries was achieved in all the patients, including empirical TAE in four patients. Hemodynamic stabilization was achieved in 17 patients (89.5%) with a significant decrease in transfusion (p < 0.001). CONCLUSION: TAE is a technically safe and clinically effective treatment method for life-threatening SRH. CE-CT has moderate capability for accurate identification of active bleeding arteries. TAE including arteries that potentially distribute anatomic territory of the hematoma is essential.

Type II Endoleak After Endovascular Aortic Aneurysm Repair Using the Endurant Stent Graft System for Abdominal Aortic Aneurysm with Occluded Inferior Mesenteric Artery.

PURPOSE: To evaluate the incidence of type II endoleak (EL-II) and aneurysm enlargement after endovascular aneurysm repair (EVAR) using the Endurant stent graft in patients with abdominal aortic aneurysm (AAA) with occluded inferior mesenteric artery (IMA). MATERIALS AND METHODS: Between 2012 and 2017, 103 patients who underwent EVAR using the Endurant stent graft for AAA with occluded IMA (50 patients with prophylactic embolized IMA and 53 with spontaneous occluded IMA) were retrospectively reviewed. The incidence of EL-II and aneurysm enlargement was evaluated. Predictive factors for persistent EL-II were evaluated based on patient characteristics, preprocedural anatomical characteristics, intraprocedural details, and postprocedural complications. RESULTS: Incidence rates of early EL-II and persistent EL-II were 6.8% (7/103 patients) and 4.9% (5/103 patients), respectively. Aneurysm enlargement was found in 10 patients (9.7%), including all 5 patients with persistent EL-II, 3 with de novo EL-II, and 2 with no EL-II. The rates of freedom from aneurysm enlargement at 1, 2, and 3 years were 98.7%, 97.0%, and 93.1% for the group without persistent EL-II, and 80.0%, 60.0%, and 20.0% for the group with persistent EL-II (p < 0.001), respectively. The maximum aneurysm diameter (odds ratio (OR), 1.16; 95% confidence interval (CI), 1.01-1.34; p = 0.0362) and the number of patent lumbar arteries (OR, 2.72; 95% CI, 1.07-6.90; p = 0.0357) were predictive of persistent EL-II. CONCLUSIONS: The incidence of EL-II after EVAR using the Endurant stent graft for AAA with occluded IMA was low, but most early EL-II persisted and resulted in aneurysm enlargement. Level of Evidence Level 4, Case Series.

The influence of horizontal cephalic rotation on the deviation of mandibular position.

PURPOSE: When performing an occlusal procedure, it is recommended that the patient should be sitting straight with the head in a natural position. An inappropriate mandibular position caused by an incorrect occlusal record registration or occlusal adjustment can result in damaged teeth and cause functional disorders in muscles and temporomandibular joints. The purpose of this study was to clarify the influence of horizontal cephalic rotation on mandibular position by investigating the three-dimensional positions of condylar and incisal points. MATERIALS AND METHODS: A three-dimensional jaw movement measurement device with six degrees of freedom (the WinJaw System) was used to measure condylar and incisal points. The subjects were asked to sit straight with the head in a natural position. The subjects were then instructed to rotate their head horizontally 0°, 10°, 20°, 30°, 40°, 50°and 60° in the right or left direction. RESULTS: The results indicated that horizontal cephalic rotation made the condyle on the rotating side shift forward, downward, and toward the inside, and the condyle on the counter rotating side shift backward, upward, and toward the outside. Significant differences in deviations were found for angles of rotation higher than 20°. The incisal point shifted in the forward and counterrotating directions, and significant differences were found for angles of rotation higher than 20°. CONCLUSION: The mandibular position was altered by horizontal cephalic rotations of more than 20°. It is essential to consider the possibility of deviation of the mandibular position during occlusal procedures.

Classification of uterine artery angiographic images: a predictive factor of failure in uterine artery embolization for postpartum hemorrhage.

PURPOSE: To justify a classification system for angiographic images of uterine artery embolization (UAE) for postpartum hemorrhage (PPH) and identify new risk factors associated with failed embolization. MATERIALS AND METHODS: A retrospective analysis of 63 consecutive patients who underwent UAE for severe PPH was performed. Uterine artery angiography (UA) before embolization was classified into two types: type 1 was defined as complete staining and type 2 was defined as partial staining of the uterine arteries. The clinical outcome, UA classification, and other possible factors previously reported were evaluated. Univariate and multivariate analyses were performed to determine the factors related to clinical outcomes. RESULTS: Sixty-three patients were enrolled (type 1, 22; type 2, 41). The clinical success rates of the primary UAE session were 90.9% (20/22) for type 1 and 61.0% (25/41) for type 2 (p = 0.018). Univariate and multivariate analyses demonstrated that the only UA classification was significantly associated with primary UAE failure (p = 0.033). CONCLUSIONS: The UA classification is an independent predictive factor of the clinical success rate of the primary UAE session for PPH; thus, it is an intuitive and optimal predictor for interventional radiologists to decide whether additional therapy is necessary.

Successful Pulmonary Artery Embolization for the Management of Hemoptysis in a Patient with Eisenmenger Syndrome Caused by Patent Ductus Arteriosus.

The patient was a 19-year-old woman who was diagnosed with patent ductus arteriosus complicating Eisenmenger syndrome at a previous medical institution. She was referred to our hospital and arranged for lung transplantation. She developed hemoptysis after the introduction of i.v. epoprostenol, which was administered as a bridging treatment while the patient awaited lung transplantation. She continued to suffer from recurrent hemoptysis, even after switching from i.v. epoprostenol to i.v. treprostinil. Angiography of the systemic and pulmonary arteries revealed the vessel responsible for the recurrent hemoptysis and pulmonary artery embolization was successfully performed. It is essential to identify the culprit vessel and physicians must not hesitate in performing embolization when patients develop lethal hemoptysis.

Intra-arterially infused carbon dioxide-saturated solution for sensitizing the anticancer effect of cisplatin in a rabbit VX2 liver tumor model.

The present study aimed to evaluate the efficacy of an intra-arterially infused carbon dioxide (CO2)-saturated solution in sensitizing the anticancer effect of cisplatin in a rabbit VX2 liver tumor model. Forty VX2 liver tumor-bearing Japanese white rabbits were randomly divided into four groups and infused via the proper hepatic artery with a saline solution (control group), CO2-saturated solution (CO2 group), cisplatin solution (cisplatin group), or CO2-saturated solution and cisplatin solution (combined group). The tumor volume (TV) and the relative tumor volume (RTV), RTV = (TV on day 3 or 7)/(TV on day 0) x 100, were calculated using contrast-enhanced computed tomography. Hypoxia-inducible factor-1α (HIF­1α) and carbonic anhydrase IX (CA IX) staining were used to evaluate cellular hypoxia. Cleaved caspase-3 and cleaved caspase-9 were analyzed to assess tumor apoptosis. The mean RTV on days 3 and 7 were 202.6±23.7 and 429.2±94.8%, respectively, in the control group; 172.2±38.1 and 376.5±61.1% in the CO2 group; 156.1±15.1 and 269.6±45.2% in the cisplatin group; and 118.3±28.1 and 210.3±55.1% in the combined group. RTV was significantly lower in the CO2 group than in the control group (day 3; P<0.05), and in the combined group than in the cisplatin group (days 3 and 7; P<0.05). HIF-1α and CA IX suppression, and increased cleaved caspase-3 and cleaved caspase-9 expression, were detected in the CO2 and combined groups, compared with the other two groups. An intra-arterially infused CO2-saturated solution inhibits liver VX2 tumor growth and sensitizes the anticancer effect of cisplatin.

Production of viable trout offspring derived from frozen whole fish.

Long-term preservation of fish fertility is essential for the conservation of endangered fishes. However, cryopreservation techniques for fish oocytes and embryos have not yet been developed. In the present study, functional eggs and sperm were derived from whole rainbow trout that had been frozen in a freezer and stored without the aid of exogenous cryoprotectants. Type A spermatogonia retrieved from frozen-thawed whole trout remained viable after freezing duration up to 1,113 days. Long-term-frozen trout spermatogonia that were intraperitoneally transplanted into triploid salmon hatchlings migrated toward the recipient gonads, where they were incorporated, and proliferated rapidly. Although all triploid recipients that did not undergo transplantation were functionally sterile, 2 of 12 female recipients and 4 of 13 male recipients reached sexual maturity. Eggs and sperm obtained from the salmon recipients were capable of producing donor-derived trout offspring. This methodology is thus a convenient emergency tool for the preservation of endangered fishes.