Search

Background: Gelatin hydrogel nonwoven fabrics (Genocel) are three-dimensional gelatin scaffolds that provide cells with space for proliferation, migration, and differentiation. They are expected to be an effective wound healing modality to treat intractable wounds, such as diabetic foot ulcers, because they enhance early neovascularization when used as a skin substitute. In this study, we explored the healing process of Genocel applied to skin defects in diabetic mice and compared it with that of a conventional skin substitute, Pelnac. Methods: Genocel and Pelnac sheets were used to treat skin defects on the backs of diabetic mice. On days 7 and 14, the remaining wound area was evaluated and specimens were harvested for HE, Azan, anti-CD31, CD68, and CD163 staining to assess neoepithelialization, granulation tissue formation, capillary formation, and macrophage infiltration. Results: Wounds treated with Genocel showed a wound healing process comparable to that of wounds treated with Pelnac. No significant differences were observed in the remaining wound area, neoepithelial length, granulation formation, number of pan-macrophages, or M2 ratio on days 7 and 14. The only significant difference was the number of induced M2 macrophages, which was higher in Pelnac group than in the Genocel group on day 7 (p

Abstract DNA analysis of large herbivore feces samples collected from seagrass beds at two distant sites (Irabu Island in Miyako Islands and Kushi in Okinawa Island) in the Ryukyu Islands proved that some of these feces were from dugongs, which had been treated in recent studies as extinct in this region since the last stranding of a deceased individual in 2019. In addition, local knowledge of sightings of animals thought to be dugongs and confirmed cases of dugong feeding trails since 2010 were compiled to estimate its recent distribution. This is the first scientific report on the presence of this mammal in the Ryukyu Islands within the last four years, and particularly in the Miyako Islands within the last half-century. As the Ryukyu Islands are known to be the northern limit of the dugong’s fragmented distribution in East Asia, conservation efforts are therefore needed.

Summary:. Midfacial reconstruction for extensive defects of the hard nasal structures and bilateral maxillae is challenging. Postoperative radiotherapy causes skin contracture, making secondary reconstruction extremely difficult. A 57-year-old man underwent resection of the nasal bone, nasal cartilage, and hard palate for cancer of the nasal cavity. Postoperative radiotherapy (70 Gy) resulted in bilateral osteoradionecrosis. Severe depression deformity of the midface causes a disorder in closing the mouth, resulting in difficulty in conversation and oral intake. We performed simultaneous reconstruction of the bilateral maxillary and nasal hard structures using double free flaps (fibular osteocutaneous and anterolateral thigh flaps). A 16-cm right fibular osteocutaneous flap was elevated, and an 8-cm proximal bone was resected to obtain the length of the peroneal vessels. The distal 8 cm was cut into three pieces while maintaining the blood flow. The removed nonvascularized fibula was processed into two pieces of cortex: nasal bridge and columella. All areas of the skin island were de-epithelialized to bilaterally fill the maxillary sinuses. Next, the ipsilateral anterolateral thigh flap was elevated with the central 6-cm part for closure of the palate and the proximal area to fill the nasal cavity. The distal area consisted of a fascial flap to cover the reconstructed nasal structure. The chimeric double flap allowed for oral intake, conversation, and nasomaxillary prominence. Computed tomography performed 8 months postoperatively showed maintained bony structures. We used the extra fibula as a nonvascularized cortex piece to prevent infection and exposure, which enabled simultaneous reconstruction of the bilateral maxillae and hard nasal structure.

Summary:. Reconstruction of extended total maxillectomy is challenging. This study aimed to isolate the skull base from the nasal cavity to avoid intracranial infection, cerebrospinal fluid fistula, and palate closure to maintain feeding and conversation. However, facial appearance and symmetry are important for quality of life. We report primary multiple buttress reconstruction using a removed nonvascularized fibula that reduced the risk of infection and exposure. A 74-year-old woman experienced a local recurrence of right maxillary sinus cancer after subtotal maxillectomy and postoperative radiotherapy (60 Gy). We performed extended total maxillectomy, including the right eyeball, orbit, temporal bone, palate, and zygomatic arch. Primary reconstruction was performed using fibular and anterolateral thigh free flaps. The proximal fibula bone was resected to obtain the length of the peroneal vessels, and the distal 9 cm of the fibula was made into two pieces while keeping the peroneal vessels attached. The nonvascularized 5-cm fibula was split sagittally with an L-shaped section to maintain the strength of the fragments. An anterolateral thigh flap was elevated from the ipsilateral thigh attached to the partial vastus lateralis muscle, which was divided into proximal (to the cheek skin and prosthetic eye bed) and distal (to the nasal cavity and palate) skin islands. Two nonvascularized bone fragments were fixed at the lateral and infraorbital rims. The dead space around the built-up pillar made of transferred bone was filled with vastus lateralis muscle to prevent infection and depression. This approach allowed for one-stage multiple buttress reconstruction for extended total maxillectomy.

Introductions: Silk elastin, a recombinant protein with repeats of elastin and silk fibroin, possesses a self-gelling ability and is a potential wound dressing material. The aim of this study is to elucidate the mechanism of the wound healing-promoting effect of silk elastin by comparing its in vivo behavior in a mouse wound model with that of a collagen sponge. Methods: Skin defects (8 mm in diameter) were created on the backs of C57BL/6J and BKS.Cg- + Lepr/+Lepr db male mice. Silk elastin sponges of 2.5 or 5.0 mm thickness, as well as collagen sponges, were placed on the wounds and secured with a polyurethane film. In the control group, only the polyurethane film was applied. The remaining wound area was grossly evaluated, and tissue samples were collected after 7, 14, and 21 days for histological evaluation, including neoepithelialization, wound contraction, granulation tissue formation, newly formed capillaries, and macrophages. Genetic analysis was conducted using real-time polymerase chain reaction. Results: In the study with C57BL/6J, there were no significant differences between the silk elastin and collagen sponge groups. Similarly, in the study using BKS.Cg- + Lepr/+Lepr db, no significant differences were found in the remaining wound area and granulation tissue formation between the silk elastin and collagen sponge groups. However, on day 14, the 5.0-mm-thick silk elastin sponge group showed increased macrophages, longer neoepithelialization, and more frequent angiogenesis compared to other groups. Gene expression of inducible nitric oxide synthase and arginase-1 was also higher in the 5.0 mm thick silk elastin sponge group. Conclusions: Silk elastin sponges demonstrated superior neoepithelialization and angiogenesis compared to collagen sponges. The results suggest that silk elastin and collagen sponges promote wound healing through different mechanisms, with silk elastin possibly enhancing wound healing by facilitating increased macrophage migration. Further studies are needed, but silk elastin shows great potential as a versatile wound dressing material.

Introduction: The regeneration of adipose tissue in patients after breast cancer surgery would be desirable without the use of growth factors or cells to avoid potential recurrence and metastasis. We reported that prolate spheroidal-shaped poly-L-lactic acid (PLLA) mesh implants of approximately 18-mm polar diameter and 7.5-mm greatest equatorial diameter containing collagen sponge (CS) would be replaced by regenerated adipose tissue after implantation, thereby suggesting an innovative method for breast reconstruction. Our study aimed to evaluate the adipose tissue regeneration ability of implant aggregates in a porcine model. Methods: We prepared implant aggregates consisting of thirty PLLA mesh implants containing CS packed in a woven poly (glycolic acid) bag. The implant aggregates were inserted under the mammary glands in the porcine abdomen for a year. Single and double groups were classified by inserting either one or two implant aggregates on each side of the abdomen, respectively. Results: In both groups, the volume of the implant aggregates decreased over time, and the formation of adipose tissue peaked between 6 and 9 months. Histologically, the formation of adipose tissue was confirmed in the area that was in contact with native adipose tissue. Conclusions: Our implant aggregates could induce the autologous adipose tissue after long term implantation in vivo, without the use of any growth factor or cell treatment, presenting a potential novel method of breast reconstruction.

Poly-L-lactic acid (PLLA) mesh implants containing collagen sponge (CS) were replaced with autologous adipose tissue regeneration in vivo. Herein, we investigated the optimal external frames and internal fillings using poly (lactic-co-ε-caprolactone) (P (LA/CL)), PLLA, and low-molecular-weight PLLA (LMW-PLLA) as the external frame and polyglycolic acid (PGA) nanosheets and CS as the internal filling. We prepared six implants: P (LA/CL) with PGA nano, PLLA with PGA nano, PLLA with CS, PLLA with 1/2 CS, PLLA with 1/4 CS, and LMW-PLLA with CS, and evaluated adipogenesis at 6 and 12 months using a rat inguinal model. The internal spaces in the P (LA/CL) and LMW-PLLA implants collapsed at 6 months, whereas those in the other four implants collapsed at 12 months. Adipose tissue regeneration was not significantly different between the PLLA-implanted groups at 6 and 12 months and was greater than that in the P (LA/CL) with PGA nano and LMW-PLLA with CS groups. The PGA nanosheet inside PLLA was comparable to the CS inside PLLA in the regeneration of adipose tissue and macrophage infiltration. In summary, PLLA is a promising external frame material in which the internal space can be replaced with adipose tissue. Thus, PGA nanosheets are an alternative internal filling material for adipose tissue regeneration.

Introduction: An oronasal fistula is a challenging post-operative complication of palatoplasty due to impaired velopharyngeal function or its high recurrence rate. Muscle repositioning, a key procedure in palatoplasty, causes dead space at the junction between the hard and soft palates. Consequently, thin oral and nasal mucosae are prone to break down and form fistulas. In this study, we used basic fibroblast growth factor-impregnated collagen gelatin sponge (bFGF-CGS) in primary palatoplasty to reduce fistula formation. Methods: This retrospective study assessed the complications and efficacy of bFGF-CGS to reduce fistula formation. Patients who underwent primary palatoplasty with bFGF-CGS were included. The same number of patients who underwent primary palatoplasty without bFGF-CGS was included as a control group. The outcomes included post-operative oronasal fistula formation, delayed healing, bleeding, and infection. Results: Both groups included 44 patients. Except for age at palatoplasty, there were no statistically significant demographic differences between the two groups; however, the rates of fistula formation in the study and control group were 2.3% and 13.6%, respectively. There were no infections among the patients. Conclusions: The grafting of bFGF-CGS in primary palatoplasty was safe and probably effective in reducing post-operative oronasal fistula formation.

Background: A novel treatment has been developed to reconstruct large skin defects caused by the excision of giant congenital melanocytic nevi. It involves the reimplantation of high-hydrostatic pressurized nevus tissue as a cell-inactivated autologous scaffold for dermal regeneration, followed by the implantation of cultured epithelial autografts on the regenerated dermis. Because this treatment has shown promise in a first-in-human clinical trial which used a prototype pressure machine, a novel pressure device was specifically designed for clinical use. Methods: In a prospective investigator-initiated clinical trial involving three patients, we evaluated the safety and efficacy of the skin regeneration treatment using a pressure device. All three patients underwent surgical excision of the nevus tissue, primary reimplantation of the inactivated nevus tissue, and secondary implantation of cultured epithelial autografts. Results: Engraftment of inactivated nevus tissue and cultured epithelial autografts was successful in all three cases, with over 90% epithelialization at 8 weeks post-surgery. No serious adverse events or device malfunction were observed during the trial. Conclusion: The novel pressure device safely and effectively enabled dermal regeneration using the nevus tissue as an autologous scaffold. This innovative approach offers several advantages, including reduced invasiveness due to minimal sacrifice of normal skin for skin grafting and high curative potential resulting from full-thickness removal of the nevus tissue.

Summary:. We must take special care when treating postoperative fluid accumulation around breast implants (BIs) to exclude any serious complications, including BI-associated anaplastic large cell lymphoma. However, most late-onset fluid accumulation is caused by other conditions, such as traumatic hematoma and residual postoperative seroma. Surgeons must choose whether to conservatively observe or remove such BIs, while also determining whether to perform partial capsulectomy or total capsulectomy to solve the problem of fluid accumulation. We treated a 72-year-old woman who noticed swelling in her right breast 4 years after undergoing bilateral BI reconstruction. Before she was referred to our hospital, the fluid had been drained by needle aspiration five times, but the swelling returned to a similar size within a month. No malignant findings were observed by needle-aspirated cytology or flow cytometry. The patient requested the simultaneous removal of the left BI. Therefore, we performed both BI removal with total capsulectomy on the right side and partial capsulectomy of the superficial layer on the left side. A pathological examination of the capsule on the right side indicated a chronic expanding hematoma and synovial metaplasia characterized by papillary projections rich in CD68-positive cells, thus indicating reactive synovial cells. In contrast, the left superficial capsule was much thinner and showed less synovial metaplasia. Our findings indicate the advantages of total capsulectomy to solve the problem of repeated serous fluid accumulation around BIs, according to histological changes in the capsule.

The use of artificial light at night (ALAN) has been increasing globally and has been reported to affect a wide range of organisms. However, the effects of ALAN on wild fish communities remain unknown. In this study, we investigated the effects of ALAN on the species distribution and composition of a fish community in a canal. We hypothesized that the fish species composition in areas subjected to ALAN would differ from that in the control area. To test this hypothesis, we conducted a three-week manipulative field experiment in which real-world ALAN conditions were simulated by illuminating the water surface at night using LED lights for two weeks. During the experiment, water samples were collected from ALAN and control conditions four times a week, from which environmental DNA (eDNA) were extracted. Additionally, the number of arthropods in the ALAN and control environments was recorded daily to investigate whether ALAN impacts distribution patterns of fish prey, which may have indirect effect on fish through changed prey-predator relationships. Collected water samples were analyzed using eDNA metabarcoding with MiFish primers and real-time PCR targeting six fish species to obtain qualitative and quantitative data on fish species composition. We compared the fish species composition data between the ALAN and control environments. Our results suggest that ALAN did not significantly influence the overall fish species composition and that the sampling location had a more significant impact. Our findings also point to the possibility that the effect of ALAN on habitat selection may vary depending on the diet of the individual fish. Overall, the effect of ALAN on fish was less significant than expected. By combining eDNA methods with manipulative field experiment, this study shows the applicability of eDNA methods in investigating the effect of pollutants and offers a promising area for future investigation.

Abstract Recent developments in environmental DNA (eDNA) analysis allow more rapid and extensive biomonitoring than traditional capture‐based surveys do. However, detection of eDNA not derived from living organisms may lead to false‐positive inferences of species presence. Such limitations may be overcome by utilizing RNA molecules present in the environment (environmental RNA [eRNA]) because of their physiochemical instability. Nevertheless, the biomonitoring performance of eRNA analysis remains unclarified because of the substantial lack of knowledge regarding basic eRNA properties, such as its persistence and degradation mechanisms. Here, we performed a factorial aquarium experiment to assess the effects of water temperature (10, 20, and 30°C) and pH (4, 7, and 10) conditions on the degradation of zebrafish (Danio rerio) eDNA and eRNA, targeting the mitochondrial cytochrome b (CytB) and nuclear beta‐2‐microglobulin (b2m) genes. A linear mixed‐model analysis showed that the degradation of eRNA was significantly faster than that of eDNA. Higher water temperatures promoted both eDNA and eRNA degradation, and alkaline conditions substantially promoted eRNA but not eDNA degradation. This might be explained by the physicochemical characteristics of DNA and RNA molecules, the membranous structure surrounding them, and their susceptibility to environmental microbial activity. Moreover, the relative concentrations of zebrafish eRNA to eDNA decreased over time, inferring that the ratio of eRNA to eDNA concentrations can be used for estimating the elapsed time since the genomic material was released and the freshness of the target eDNA signal in the field. Nevertheless, given that the confidence intervals of the eDNA and eRNA decay rates tended to overlap for each treatment level, this study indicates that fish eRNA is not always degraded rapidly and is, in fact, more abundant in water than previously expected. This result favors the application of eRNA analysis to indicate living biotic assemblages.

Introduction: From previous research, an emerging material composed of gelatin hydrogel nonwoven fabric (Genocel) has shown potential as a skin substitute, by improving neovascularization promotion in the early phase of wound healing. However, Genocel was inferior in terms of granulation formation compared to Pelnac. To solve this problem, we modified the manufacturing process of Genocel to reduce its water content, extend the degradation time (Genocel-L), and evaluate its healing process as a skin substitute. Methods: Genocel with a low water content (Genocel-L) was prepared and the difference in water content compared to that of the conventional Genocel was confirmed. Degradation tests were performed using collagenase and compared among Genocel-L, Genocel, and Pelnac sheets. In the in vivo study, sheets of Genocel-L or Pelnac were applied to skin defects created on the backs of C57BL/6JJcl mice. On days 7, 14, and 21, the remaining wound area was evaluated and specimens were harvested for Hematoxylin and Eosin, Azan, anti-CD31, CD68, and CD163 staining to assess neoepithelialization, granulation tissue, capillary formation, and macrophage infiltration. Results: Genocel-L had a lower water content than the conventional Genocel and a slower degradation than Genocel and Pelnac. In the in vivo experiment, no significant differences were observed between Genocel-L and Pelnac in relation to the wound area, neoepithelium length, granulation formation, and the number of newly formed capillaries. The area of newly formed capillaries in the Pelnac group was significantly larger than that in the Genocel-L group on day 21 (p

Objectives: Selective orbicularis neuromyectomy for postparetic periocular synkinesis was first reported in 2015. This study aims to statistically evaluate the accumulated outcomes and stability of this treatment. Methods: Twenty-five patients with postparetic periocular synkinesis were treated with this procedure between March 2010 and February 2020. Every patient was evaluated using the Sunnybrook scale, and their palpebral fissure width was measured using preoperative and postoperative videos. Patients who underwent >1-year follow-up were included in the study. Chemodenervation or physical therapy was not performed during the follow-up periods. Results: Of the 25 patients, 20 were included in this study. The follow-up period was 13-96 months (median 32 months). The mean of the patients' synkinesis scores fell by 4.6 points. Palpebral fissure width while puckering and at rest was significantly different between the preoperative and postoperative measurements (p < 0.01). Lower lid drooping gradually deteriorated in one patient 7 years after the treatment and was corrected secondarily. Conclusions: This study demonstrates that selective orbicularis neuromyectomy is a simple and sustainable treatment for periocular synkinesis. However, further longer follow-up of elderly patients and objective analysis are warranted.

Surgical resection of malignant bone tumors leads to significant defects in the normal surrounding tissues that should be reconstructed to avoid amputation. Our research aimed to inactivate osteosarcoma (OS)-affected bone to obtain autologous bone grafts for bone defect reconstruction using a novel therapy called high hydrostatic pressurization (HHP) therapy. The key points are complete tumor death and preservation of the non-denatured native extracellular matrix (ECM) and bone tissue by HHP. Previously, we found that HHP at 200 MPa for 10 min can completely inactivate cells in normal skin and skin tumors, including malignant melanoma and squamous cell carcinoma while maintaining their original biochemical properties and biological components. Based on our previous research, this study used HHP at 200 MPa for 10 min to eradicate OS. We prepared an OS cell line (LM8), pressurized it at 200 MPa for 10 min, and confirmed its inactivation through morphological observation, WST-8 assay, and live/dead assay. We then injected OS cells with or without HHP into the bone marrow of the murine tibia, after which we implanted tumor tissues with or without HHP into the anterior surface of the tibia. After HHP, OS cells did not proliferate and were assessed using a live/dead assay. The pressurized cells and tumors did not grow after implantation. The pressurized bone was well prepared as tumor-free autologous bone tissues, resulting in the complete eradication of OS. This straightforward and short-pressing treatment was proven to process the tumor-affected bone to make a transplantable and tumor-free autologous bone substitute.

Introduction: Autologous cultured epidermis (CE) is an effective approach for overcoming the deficiency of donor sites to treat extensive burns. However, the production of autologous CE takes 3–4 weeks, which prevents its use during the life-threatening period of severe burns. In contrast, allogeneic CE can be prepared in advance and used as a wound dressing, releasing several growth factors stimulating the activity of recipient cells at the application site. Dried CE is prepared by drying CEs under controlled temperature and humidity conditions until all the water is completely removed and no viable cells are present. Dried CE accelerates wound healing in a murine skin defect model and is potentially a new therapeutic strategy. However, the dried CE safety and efficacy have not yet been studied in large animal models. Therefore, we studied the safety and efficacy of human-dried CE in wound healing using a miniature swine model. Methods: Human CE was manufactured using Green's method from donor keratinocytes. Three types of CEs (Fresh, Cryopreserved, and Dried) were prepared, and the ability of each CE to promote keratinocyte proliferation was confirmed in vitro. Extracts of the three CEs were added to keratinocytes seeded in 12-well plates, and cell proliferation was evaluated using the WST-8 assay for 7 days. Next, we prepared a partial-thickness skin defect on the back of a miniature swine and applied three types of human CE to evaluate wound healing promotion. On days 4 and 7, the specimens were harvested for hematoxylin-eosin, AZAN, and anti-CD31 staining to assess epithelialization, granulation tissue, and capillary formation. Results: The conditioned medium containing dried CE extract significantly enhanced keratinocyte proliferation compared to the control group (P

Summary:. Indocyanine green (ICG) fluorescence imaging is useful for assessing flap viability; however, it is associated with a risk of anaphylactic shock, even in patients with no history of drug allergies. SnapshotNIR is a noncontact, camera-type handheld tissue oximeter that can measure the tissue oxygen saturation of the body surface. The device emits red and near infrared light wavelengths and then optimizes the measurement of the differential reflectance from oxygenated and deoxygenated hemoglobin, and StO2 is calculated. A 20 × 15 cm surgical field can be evaluated in less than 3 seconds by holding the camera at a distance of 30 cm. We applied this device at zone II in a deep inferior epigastric perforator (DIEP) flap, and compared the findings with the border of flap perfusion detected by ICG imaging. Left breast reconstruction using a free DIEP flap was performed for a 60-year-old woman. The DIEP flap was vascularized by a perforator vessel coursing to the right abdominis muscle. First, Diagnogreen (5 mg; Daiichi Sankyo Co., Tokyo, Japan) was intravenously injected, and the ICG fluorescence perfusion border detected by PDE-neo (Hamamatsu Photonics, Hamamatsu City, Shizuoka, Japan) was determined. The ICG border was defined by two reconstructive surgeons after fluorescence had spread out for 2 minutes. Next, zones Ⅱ and Ⅳ of the DIEP flap, contralateral to the perforator, were evaluated using photographs obtained by SnapshotNIR. There were significant StO2 value differences between the ICG-negative area and ICG-positive area. This device can be widely applied in the noninvasive evaluation of flap viability.

Introduction: Genocel is an emerging material, used in cell culture, with high mechanical strength and good cytocompatibility. Based on these characteristics, Genocel is considered a promising skin substitute for wound healing. In this study, we explored the possibility of using Genocel as a skin substitute for murine skin defects and compared it with a conventional skin substitute. Methods: Sheets of Genocel and Pelnac were applied to skin defects created on the backs of mice. On days 7, 14, and 21, the remaining wound area was evaluated and specimens were harvested for HE, Azan, anti-CD31, CD68, and CD163 staining to assess neoepithelialization, granulation tissue, capillary formation, and macrophage infiltration. Results: No significant differences in the wound area or neoepithelium length were observed between groups. The number of newly formed capillaries in the Genocel group was significantly higher than that in the Pelnac group on day 7 (p

For traditional artificial dermises, a waiting period of approximately three weeks is required after the first implantation before they are adequately vascularized. The objective of this retrospective case series was to investigate whether full-thickness skin defects, requiring surgical reconstruction, could be successfully treated by implantation of a basic fibroblast growth factor (bFGF)-impregnated artificial dermis and secondary skin grafting with a shorter waiting period. Between January 2019 and January 2021, 19 skin defects in 14 patients (7 male and 7 female) were treated with two-stage skin grafting using bFGF-impregnated collagen-gelatin sponge (CGS). All of them were included in this case series, and the waiting period for skin grafting, success rate of skin grafting, infection during the waiting period, and scar quality 6–12 months postoperatively were retrospectively investigated. As a result, all skin grafting surgeries were successfully performed with a waiting period of 13.3 ± 4.3 days. Infection during the waiting period was observed in three lesions (15.8%); however, all infections were controllable. Postoperative scar quality was acceptable (Vancouver Scar Scale score range, 1–8). In conclusion, compared to traditional artificial dermises, bFGF-impregnated CGSs have the potential to shorten the waiting period without decreasing the success rate of skin grafting. Further studies are required to confirm this finding.

Establishing realistic targets for fish community composition is needed to assess the effectiveness of river restoration projects. We used environmental DNA (eDNA) metabarcoding with MiFish primers to obtain estimates of fish community composition across 17 sites upstream, downstream and within a restoration mitigation project area (Kaihotsu–Kasumi) located in the Shigenobu River system, Ehime Prefecture, Japan. We evaluate the benefits of using eDNA to quickly, sensitively, and extensively gather data to establish existing fish community composition in the restoration area, as well as potential future short-term, medium-term, and long-term targets of species assemblages that could realistically emerge following dispersal into the project area from upstream and downstream populations. We compare results from eDNA metabarcoding with species lists obtained from contemporaneous capture surveys and historical information. Nonmetric multidimensional scaling plots of community composition obtained from eDNA surveys showed that the Kaihotsu–Kasumi restoration area and surrounding river reaches were divided into three clusters: upper reaches, middle and lower reaches, and estuarine reaches. The Kaihotsu–Kasumi restoration area sites were included in the group containing the middle and lower reaches of the inflow and outflow rivers that were near the restoration area. We detected a total of twenty-six species in this group, twenty-one native species and five non-native species. Therefore, these native species were considered suitable as short-term target species with high potential for dispersal into Kaihotsu–Kasumi restoration area. By comparison, only 14 species would have been selected as target species based on capture surveys and historical literature. One factor increasing the resolution of our eDNA surveys was our ability to identify the presence of intraspecific lineages of Misgurnus anguillicaudatus (Clades A and B), which were missed by the capture surveys. These results indicate that the eDNA metabarcoding method can provide more comprehensive and realistic short-term target species estimates than capture surveys, as well as provide higher resolution monitoring through intraspecific lineage detection.

Summary:. Local flaps from the upper lip and cheeks have been the first choice for two-thirds to total resection of the lower lip. However, these local flap techniques involve many clinical problems, including small a mouth, drooling, scarring, and hypesthesia. The improvement of free anterolateral thigh (ALT) flap transfer can solve these problems with expansion of the application of free flaps for lower lip reconstruction. The patient in this case was a 56-year-old man with squamous cell carcinoma of the lower lip (cT3N1M0). Subtotal lower lip resection preserving both corners of the mouth with bilateral neck dissection was performed. Simultaneously, a sensory ALT flap was elevated with an 8 × 6 cm skin island and a lateral femoral cutaneous nerve. The lateral and medial sides of the fascia lata were processed into 1-cm-wide strings, which were tunneled through the orbicularis oris muscle of the upper lip and sutured to the orbicularis oris muscle at the mucosal side of the philtrum. The lateral femoral cutaneous nerve and right mental nerve were sutured. At 3 months, a second surgery was performed to replace the ALT flap on the white labial side with a clavicle full-thickness skin graft. This surgery achieved four important factors: opening and closing of the mouth, sensory function of the lower lip, cosmetic appearance, and minimization of donor-site damage. We believe the worldwide improvement of microsurgery techniques enables lower lip reconstruction using the sensory ALT flap to be selected as the first choice for two-thirds to total lower lip defects.

Summary:. Infantile hemangioma (IH) is a common pediatric vascular tumor and is easily diagnosed in most cases based on the clinical course and appearance, but deep IHs are difficult to diagnose based on external appearance alone. Clinical and imaging findings are therefore important clues to the diagnosis of soft tissue tumors; however, a definitive diagnosis is decided based on the pathological examination of biopsy or resection specimens. A 1-year-old girl with a subcutaneous mass on her glabella was referred to our hospital. At 3 months of age, her mother noticed a tumor that swelled when she cried. It gradually enlarged, and ultrasonography and magnetic resonance imaging were performed at 12 months of age. Doppler ultrasonography showed a hypo-vascular mass. Magnetic resonance imaging revealed a subcutaneous mass with low-intensity on T1-weighted image and slightly high-intensity on T2-weighted image, with tiny flow voids. Computed tomography showed no frontal bone defect. The soft tissue tumor could not be diagnosed based on these imaging findings; thus, we decided to perform total resection under general anesthesia. A histopathological examination showed a highly cellular tumor with capillaries with opened small vascular channels and glucose transporter 1 positivity. Thus, it was diagnosed as deep IH transitioning from the proliferative phase to the involuting phase. Deep IHs are difficult to diagnose because characteristic imaging findings disappear during the involuting phase. We emphasize the importance of performing Doppler ultrasonography in the early phase (eg, at 6 months of age) for soft tissue tumors of infancy.

Abstract Clarifying the effect of the sampling protocol on the detection of environmental DNA (eDNA) is essential for appropriately designing biodiversity research. However, technical issues influencing eDNA detection in the open ocean, which consists of water masses with varying environmental conditions, have not been thoroughly investigated. This study evaluated the sampling effort for the metabarcoding‐based detection of fish eDNA using replicate sampling with filters of different pore sizes (0.22 and 0.45 μm) in the subtropical and subarctic northwestern Pacific Ocean and Arctic Chukchi Sea. The asymptotic analysis predicted that the accumulation curves for detected taxa did not saturate in most cases, indicating that our sampling effort (7 or 8 replicates, corresponding to 10.5–40 L of filtration in total) was insufficient to fully assess the species diversity in the open ocean and that tens of replicates or a substantial filtration volume were required. The Jaccard dissimilarities between filtration replicates were comparable with those between the filter types at any site. In subtropical and subarctic sites, turnover dominated the dissimilarity, suggesting that the filter pore size had a negligible effect. In contrast, nestedness dominated the dissimilarity in the Chukchi Sea, implying that the 0.22 μm filter could collect a broader range of eDNA than the 0.45 μm filter. Therefore, the effect of filter selection on the collection of fish eDNA likely varies depending on the region. These findings highlight the highly stochastic nature of fish eDNA collection in the open ocean and the difficulty of standardizing the sampling protocol across various water masses.

Abstract The eDNA Society was founded in Japan in 2018 to develop eDNA science as a discipline that contributes to the human well‐being and the sustainable use of ecosystems. Since then, the society has expanded attracting a wide variety of people from international universities, research institutes, consultant companies, and local/national governments. Here we report edna2021, the fourth annual meeting of The eDNA Society, which was held mainly online on November 20–21, 2021. We had c.a. 500 participants in total, from at least 15 countries. In the meeting, we discussed not only the technical aspects of environmental DNA but also its potential for various applications including its combinations with environmental RNA, sediment core samples, population genetic analyses, etc.

Abstract Cryopreserved allogeneic cultured epidermis (CE) is used for treating second-degree burn wounds and diabetic foot ulcers; however, the need for cryopreservation limits its use. We have previously reported that CE accelerates wound healing irrespective of its viability and hypothesized that dehydrated CEs lacking living cells may act as an effective wound dressing. We prepared dried CE and investigated its morphological and physical properties and wound-healing effects and compared them with those of cryopreserved CE. Hematoxylin–eosin staining, immunostaining for basement membrane, and electron microscopy revealed that the morphologies of dried CE and cryopreserved CE were comparable and that the membrane structure was not damaged. The breaking strength, modulus of elasticity, and water permeability of dried CE were comparable with those of the cryopreserved CE. Furthermore, the levels of various active cytokines and chemokines in dried CE were comparable with those in cryopreserved CE. Dried CE applied to skin defect in diabetic mice significantly reduced the wound area and increased the new epithelium length 4 and 7 days after implantation, similar to that observed for cryopreserved CE. Consequently, dried CE had similar morphological and physical properties and wound-healing effects compared with those of cryopreserved CE and can be a physiological and versatile wound-dressing.

Machine learning is known as an effective technique to classify big data, and coding methods for input into neural networks are critical. In this study, we have applied the machine learning to environmental deoxyribonucleic acid (DNA) metabarcoding. We propose three coding methods of the nucleic acid sequences, and one of them is two-bit coding. In this coding method, one base is represented by two bits, and four types of bases are associated with two-bit binary numbers. A three-layer perceptron is used as a neural network, and backpropagation is used as a learning rule. The accuracy for the two-bit coding is slightly higher than those for the other coding methods, and it can be decided that the two-bit coding is the best coding method mainly because of its advantage, namely, the amount of information required to indicate the nucleic acid sequences is relatively small. We conclude that the environmental DNA metabarcoding is an interesting example as a novel application of machine learning.

Summary:. The medical imaging projection system (MIPS) is a real-time surgical navigation device using indocyanine green (ICG) emission signals and active projection mapping. The difference between the object and the projected image is within 1 mm, and the time lag is within 0.1 seconds. We herein report the application of the MIPS to vascularized lymph-node transplantation (VLNT) surgery for lower extremity lymphedema to detect inguinal lymph nodes and perform color-coded navigation surgery for lymph-node resection. A left superficial inguinal lymph node was planned to be used as a donor for VLNT to the right lower leg in a 73-year-old woman with lower extremity lymphedema. Under general anesthesia, multiple intradermal injections of 0.1 ml of ICG were administered around the left inguinal donor site. The MIPS showed a clear linear projection image from a lateral injected point connecting to a lateral superficial inguinal lymph node. The left superficial circumflex iliac artery and vein were dissected for vascularized VLNT. Intraoperative real-time MIPS navigation continuously guided the transection plane colored by ICG fluorescence signals without shifting the visual focus from the surgical field. This is the first report of the intraoperative use of ICG projection mapping for VLNT donor-site surgery. The MIPS was able to visualize functional lymph nodes to facilitate minimally invasive donor-site surgery.

Mandibular reconstruction involving the central segment after malignant tumor resection requires the achievement of both functional and aesthetic quality. Three-dimensional reproduction based on the concept of a double arc composed of a marginal arc and an occlusal arc is important. Most reports of fibula flaps applied three-segmented closed wedge fibula osteotomy (bilateral and central segments); however, the aesthetic outcome sometimes became too stout for female patients because of the large central segment. We performed four-segmented fibula osteotomy for a 78-year-old woman using a semiopen wedge technique characterized by a half-open wedge and half-closed wedge. This procedure obtained a slim mandibular contour and made double-barrel reconstruction easier to apply. We used titanium plates that were prebent according to the shape of a three-dimensional model constructed from CT data. Small bone cortex fragments made from a surplus fibula segment were inserted in a half-opened area. The build-up preparation for central mandibular reconstruction was all performed at the lower leg area before cutting the peroneal pedicle. This prefabricated double-barrel fibula free flap was transferred to the mandibular defect with arterial and venous anastomosis to the right superior thyroid artery, right external jugular vein, and right common facial vein. Although the patient was nearly 80 years of age, the bone segments, including free cortex tips, were fused with smooth remodeling. Semiopen wedge osteotomy can be a key to reproducing an aesthetically slim feminine chin with a functional height of mandibular bone maintained for stability of the dental prosthesis.

Background:. A micrograft (MG) suspension produced by the Rigenera protocol has been used to stimulate tissue regeneration. Recently, a combination therapy of an artificial dermis and skin MG has been used to promote angiogenesis and granulation tissue formation in the artificial dermis. There are no reports comparing the differences in MG impregnation efficiency between different artificial dermis products. Therefore, we compared the impregnation of skin MG in Pelnac Gplus and Integra. Methods:. Skin MG was prepared from the skin of C57BL/6JJcl mice using Rigeneracons and administered onto Pelnac Gplus and Integra sheets. The amount of MG suspension impregnated in Pelnac Gplus and Integra was evaluated. Pelnac Gplus and Integra sheets combined with MG were applied to murine defects, and wound area, neoepithelium length, granulation tissue formation, and newly formed capillaries were compared with the control groups on days 7 and 14. Results:. The weight percentage of the MG absorbed by Pelnac Gplus and Integra was 88.8% ± 3.5% and 28.2% ± 7.0%, respectively (P < 0.05). In the in vivo experiment, the area of newly formed granulation tissue and both the number and area of newly formed capillaries in the PelnacG + MG group were significantly larger than those in the control group at 14 days after implantation (P < 0.05). Conclusions:. Skin MG was successfully impregnated into Pelnac Gplus by simple administration but not into Integra. Administration of skin MG into the Pelnac Gplus promoted granulation formation and angiogenesis. Pelnac Gplus was more suitable than Integra in the combination therapy.

Summary:. We applied double-flap reconstruction with a vascularized fibula osteo flap and a Y-shaped anterolateral thigh (ALT) flap, which was separated into a fascial flap and a de-epithelialized fat flap to replace the temporomandibular joint capsule and temporal soft tissue volume, respectively. This technique achieved excellent functional and cosmetic results with acceptable operation time and donor site scarring. A 16-year-old girl had a rapidly growing mandibular osteosarcoma. Right mandibulectomy from the right first premolar tooth to right condylar head (including temporalis muscle, masseter muscle, buccal mucosa, and part of the right maxilla) was performed. A left fibula osteo flap and left ALT flap were prepared in a prefabricated manner using a three-dimensional model and a prebent titanium plate. The area of the ALT flap was 23 × 6 cm. A proximal 8 × 4 cm skin flap was positioned to replace the left cheek mucosa, and the distal part was divided into two layers: a de-epithelialized fat flap and a fascial flap. The de-epithelialized fat flap was used to prevent the depression deformation at the temporal area, and the fascial flap was used to cover around the alternate condylar head made by the fibula instead of the resected temporomandibular joint capsule. The peroneal artery and veins were microscopically anastomosed to the distal end of the artery and veins of the descending branch of the ALT flap in a flow-through manner. At 12 months after surgery, the occlusal function and aesthetic balance were excellent.

Abstract Reliable abundance estimation is a primary challenge in environmental DNA (eDNA) analysis, which has been addressed by considering the effects of eDNA transport and degradation. However, these eDNA spatial dynamics depend on the cellular and molecular structure of eDNA, with its persistence state (particle size and DNA fragment length) being essential for improved abundance estimation. To address the issue, we used datasets obtained from two types of aquarium experiments (targeting zebrafish [Danio rerio] and Japanese jack mackerel [Trachurus japonicus]) and compared the relationships between eDNA concentration and species abundance among different eDNA size fractions and target marker lengths. We reared the fish in experimental tanks with different individual numbers or biomass densities, filtered rearing water using different pore size filters, and quantified eDNA concentrations targeting different fragment lengths or genetic regions. Consequently, both experiments showed that the accuracy and sensitivity in abundance estimation were improved (i.e., R2 values and slopes of linear regressions increased) when targeting eDNA at the 3‐ to 10 μm size fraction. On the contrary, targeting eDNA at the >10 μm size fraction yielded a lower R2 value. This result indicates that the relationship between eDNA concentration and species abundance can be worsened when extremely larger size fractions are targeted. Conversely, the target marker length negatively affected the R2 value. This study proposes that the relationship between eDNA concentration and species abundance relies on the complex interactions between the particle size, persistence, and spatial heterogeneity of eDNA in water.

Abstract As an alternative/supplement to conventional biodiversity survey methods, environmental DNA (eDNA) analysis has developed rapidly during the past decade, and is widely used for the biomonitoring of wildlife. However, the interpretation of eDNA results has been limited to the presence/absence or biomass/abundance of the target species because of uncertainty regarding the dynamics of eDNA in natural environments. This limitation may be ameliorated by targeting environmental RNA (eRNA). RNA is more prone to degrade than DNA and the pattern of messenger RNA (mRNA) expression changes depending on physiological conditions, meaning that the presence or concentration of mRNA could reflect the organism's presence with higher temporal resolution and provide information beyond simple presence/absence. Technical developments in the detection of eRNA focusing on mRNA with these distinct features could permit the advanced usage of genetic materials in water. In advancing this technique, we initiated this study asking that if we can detect elevated levels of eRNA whose genes are specific to a tissue source (e.g., gills or skin), then could not we infer the tissue origin of the genetic material detected. To this end, we developed gene‐specific primer sets for the target genes with biased expression in the gills, skin, and intestine, and conducted reverse transcription–polymerase chain reaction on zebrafish breeding tank water samples, obtaining positive results for all assays. The result of our experiment confirmed that the specific target tissues can be the source of genetic materials detected in water and with that we offer a proof of concept for eRNA analysis targeting specific mRNAs of aquatic vertebrates. In this commentary, we provide information on the experimental steps used for mRNA typing of eRNA from zebrafish as well as the limitations and challenges of this technique and the prospect of mRNA typing of eRNA in the future.

Abstract Environmental DNA (eDNA)‐based assessments of macro‐organisms have now become an essential approach for biomonitoring. eDNA survey methods have a number of advantages over conventional survey methods. However, the value of the data that will accumulate would be greatly enhanced by standardizing the analysis methods, which would allow us to compare data from multiple monitoring sites at different points in time. The eDNA Society (http://ednasociety.org/en/about), whose founding members consist of Japanese researchers conducting eDNA studies on macro‐organisms, was established in 2018, with the aim of expanding eDNA technology and science. Here, we introduce our key publication, “Environmental DNA Sampling and Experiment Manual” (http://ednasociety.org/en/manual), which was published under the initiative of the eDNA Society. Detailed methods for the surveys and experiments are described in the manual, including the selection of sampling sites, sampling methods, filtration methods, DNA extraction, species‐specific detection by real‐time polymerase chain reaction, and fish eDNA metabarcoding. The manual assists users in conducting standardized surveys and quality experiments, and provides a basis for collecting comparable data. Given that the efficacy of methods can be context dependent and variable, and that procedures may sometimes conflict with standardization, it is difficult to ensure that all processes are equally effective. However, even in such cases, it is important to maintain sufficiently high data quality by setting the minimum standards to be followed. Implementation of such standardized methodologies will enable the systematic and frequent collection of flawless, comparable eDNA data from around the world; this will provide important fundamental information for biodiversity conservation, as well as the sustainable use of fisheries resources.

Abstract Aqueous environmental DNA (eDNA) analysis has been applied to the monitoring of various ecosystems and taxa, and the characteristics of aqueous eDNA have been previously studied. In contrast, although sedimentary eDNA has been used to restore past information, the characteristics of sedimentary eDNA are not well understood. In this study, we compared the properties of sedimentary and aqueous eDNA of macro‐organisms. First, to clarify the preservation ability of sediments, we compared the difference in decay rates between aqueous and sedimentary eDNA using samples collected from a biotope (an artificial pond prepared with concrete). Next, to clarify the biological information retained in sedimentary eDNA both qualitatively and quantitatively, we compared eDNA concentrations between sediment and water samples collected simultaneously from a lake, and the fish species detected by eDNA metabarcoding were also compared. The results demonstrated the following: (a) the decay rate (decreased eDNA copy number divided by the initial eDNA copy number per unit time) of sedimentary eDNA (0.00033 ± 0.000049 [mean ± SE]/hr) was lower than that of aqueous eDNA (0.01863 ± 0.0011/hr); (b) sedimentary eDNA concentration of the mitochondrial marker of three fish species was higher than aqueous eDNA concentration for the same sample weight (12.5–1,456.9 times); and (c) the species composition obtained by metabarcoding was not significantly different between sediment and water; however, considering the lower decay rate of sedimentary eDNA, using both sample types may provide more comprehensive information of species distribution. Thus, sedimentary eDNA analysis will expand future biomonitoring and ecological studies by providing a difference in timescale.

Abstract Environmental DNA (eDNA) analysis has seen rapid development in the last decade, as a novel biodiversity monitoring method. Previous studies have evaluated optimal strategies, at several experimental steps of eDNA metabarcoding, for the simultaneous detection of fish species. However, optimal sampling strategies, especially the season and the location of water sampling, have not been evaluated thoroughly. To identify optimal sampling seasons and locations, we performed sampling monthly or at two‐monthly intervals throughout the year in three dam reservoirs. Water samples were collected from 15 and nine locations in the Miharu and Okawa dam reservoirs in Fukushima Prefecture, respectively, and five locations in the Sugo dam reservoir in Hyogo Prefecture, Japan. One liter of water was filtered with glass‐fiber filters, and eDNA was extracted. By performing MiFish metabarcoding, we successfully detected a total of 21, 24, and 22 fish species in Miharu, Okawa, and Sugo reservoirs, respectively. From these results, the eDNA metabarcoding method had a similar level of performance compared to conventional long‐term data. Furthermore, it was found to be effective in evaluating entire fish communities. The number of species detected by eDNA survey peaked in May in Miharu and Okawa reservoirs, and in March and June in Sugo reservoir, which corresponds with the breeding seasons of many of fish species inhabiting the reservoirs. In addition, the number of detected species was significantly higher in shore, compared to offshore samples in the Miharu reservoir, and a similar tendency was found in the other two reservoirs. Based on these results, we can conclude that the efficiency of species detection by eDNA metabarcoding could be maximized by collecting water from shore locations during the breeding seasons of the inhabiting fish. These results will contribute in the determination of sampling seasons and locations for fish fauna survey via eDNA metabarcoding, in the future.

Abstract Recent advances in environmental DNA (eDNA) analysis using high‐throughput sequencing (HTS) provide a noninvasive way to evaluate the intraspecific genetic diversity of aquatic macroorganisms. However, erroneous sequences present in HTS data can result in false positive haplotypes; therefore, reliable strategies are necessary to eliminate such erroneous sequences when evaluating intraspecific genetic diversity using eDNA metabarcoding. In this study, we propose an approach combining denoising using amplicon sequence variant (ASV) method and the removal of haplotypes with low detection rates. A mixture of rearing water of Ayu (Plecoglossus altivelis altivelis) was used as an eDNA sample. In total, nine haplotypes of Ayu mitochondrial D‐loop region were contained in the sample and amplified by two‐step tailed PCR. The 15 PCR replicates indexed with different tags were prepared from the eDNA sample to compare the detection rates between true haplotypes and false positive haplotypes. All PCR replications were sequenced by HTS, and the total number of detected true haplotypes and false positive haplotypes was compared with and without denoising using the two types of ASV methods, Divisive Amplicon Denoising Algorithm 2 (DADA2) and UNOISE3. The use of both ASV methods considerably reduced the number of false positive haplotypes. Moreover, all true haplotypes were detected in all 15 PCR replicates, whereas false positive haplotypes had detection rates varying from 1/15 to 15/15. Thus, by removing haplotypes with lower detection rates than 15/15, the number of false positive haplotypes was further reduced. The approach proposed in this study successfully eliminated most of the false positive haplotypes in the HTS data obtained from eDNA samples, which allowed us to improve the detection accuracy for evaluating intraspecific genetic diversity using eDNA analysis.

Summary:. Surgical invasion is a risk factor of pyoderma gangrenosum (PG). A total of 25% of postoperative PG cases were reported to occur after breast surgeries, including bilateral breast reduction and breast reconstruction following cancer resection. Immunosuppressive therapy and less-invasive wound therapy are necessary; however, the complete healing of ulcers takes 5.1 months on average. We herein report a case of skin grafting under a surgical concept of less-invasive and short-term treatment. An 82-year-old woman complained of a high fever and severe pain at her breast wounds after bilateral breast cancer resection. Although we performed emergency debridement surgery to remove the necrotic tissue, suspecting surgical site infection and inflammation, her high fever persisted. She was diagnosed with PG because of the physical findings of characteristic painful, sterile ulcerations, bullae and pustules, and the pathological abundance of neutrophils in the absence of infection and vasculitis. Oral administration of prednisolone 30 mg/day improved the symptoms, and we applied negative-pressure wound therapy (NPWT) from day 16 following debridement surgery. After the gradual reduction of oral steroid intake to 12.5 mg/day, we performed skin grafting surgery. To limit the surgical invasion, we used the surplus skin around the ulcers. Split-thickness mesh skin grafts were fixed by NPWT to avoid the use of tie-over sutures. We achieved short-term treatment of PG with a less-invasive surgical strategy using skin around the ulcers and NPWT.

Background:. Postoperative free flap monitoring is essential for immediately detecting obstruction of anastomosed vessels with successive recovery surgery for salvaging flaps. We performed postoperative nursing monitoring using handheld Doppler sonography, but nurses reported feeling anxious with this approach and demanded a clear-cut evaluation method. Therefore, we implemented monitoring with the fingerstall-type tissue oximeter Toccare, a noninvasive device that enables easy flap checking by simply touching the flap with a probe. Method:. Handheld Doppler was used for nursing monitoring from April to October 2020, with anxiety associated with its use reported. We collected information via an anonymous questionnaire to determine the reason for the anxiety. Toccare was subsequently applied for postoperative free flap monitoring by nurses. The protocol involved measuring tissue oxygen saturation by touching the flap with a Toccare probe every 4 hours from 24 to 100 hours postoperatively. Seven months later, a second anonymous questionnaire was conducted, and results were compared. Result:. Free deep inferior epigastric artery perforator flaps and anterolateral thigh flaps (n = 5 each) were included. The average tissue oxygen saturation values in the deep inferior epigastric artery perforator and anterolateral thigh flaps were 52.0% and 52.4%, respectively. According to the second questionnaire about Toccare, 7% felt anxious, 62% felt slightly anxious, and 31% did not feel anxious. Toccare was preferred by 89% of nurses who had used both methods. Conclusions:. Flap monitoring using Toccare reduced nurses’ anxiety. A numerical evaluation method with easy handling and clear doctor call criteria is essential for low-anxiety nursing monitoring.

Summary:. Patients with severe group A Streptococcus (GAS) induced necrotizing soft tissue infection sometimes develop Streptococcal toxic shock syndrome, which is a life-threatening condition with an extremely high fatality rate. Obtaining survival is the most important goal; however, an early diagnosis for debridement surgery and quick granulation formation for skin grafting surgery can better preserve the extremity functions. The patient was a 47-year-old man with a history of atopic dermatitis who presented with GAS-induced necrotizing soft tissue infection in his left lower extremity. His vital signs indicated shock, and he was diagnosed with streptococcal toxic shock syndrome. Emergency surgery was performed with his body pressure maintained with noradrenaline. Intraoperatively, rapid antigen detection tests (RADTs) were negative in the medial thigh and positive in the lower leg, which helped in decision-making regarding the area of aggressive debridement surgery. The wound culture results matched the intraoperative rapid antigen detection test results. A collagen/gelatin sponge with the sustained release of basic fibroblast growth factor was used as an artificial dermis before skin grafting. Excellent granulation was obtained, and skin grafting surgery was performed on the 11th day after collagen/gelatin sponge placement. He was discharged home on the 42nd day with normal lower extremity functions. First, an intraoperative diagnosis using GAS-rapid antigen detection tests with an appropriate sampling method from small incisions avoided excessive surgical debridement. Second, collagen/gelatin sponge with the sustained release of basic fibroblast growth factor promoted quick granulation tissue formation for wound bed preparation. These efforts resulted in the successful less-invasive treatment of a patient with streptococcal toxic shock syndrome caused by GAS-induced necrotizing soft tissue infection.

Environmental DNA (eDNA) analysis has enabled more sensitive and efficient biological monitoring than traditional methods. However, since the target species is not directly observed, interpretation of results cannot preclude process Type I errors. Specifically, there may be a spatial or temporal gap between the target eDNA and the eDNA source in the sampled area. Moreover, eDNA surveillance lacks the ability to distinguish whether eDNA originated from a living or non-living source. This kind of Type I error is difficult to control for, in part, because the relationship between the state of eDNA (i.e., intracellular or extracellular) and the degradation rate is still unclear. Here, we applied PMA (Propidium monoazide) to eDNA analysis which enabled us to differentiate “intact cells” from “disrupted cells.” PMA is a dye that has a high affinity for double-stranded DNA and forms a covalent bond with double-stranded DNA and inhibits amplification of the bonded DNA molecules by PCR. Since PMA is impermeable to the cell membrane, DNA protected by an intact cell membrane can be selectively detected. In this study, we investigated the workability of PMA on vertebrate eDNA using zebrafish, Danio rerio. Aquarium water was incubated for 1 week to monitor the eDNA degradation process of both intracellular and extracellular eDNA. We developed ten species-specific quantitative PCR assays for D. rerio with different amplification lengths that enabled independent quantification of total eDNA (sum of intracellular and extracellular eDNA, commonly measured in other studies) and intracellular eDNA (DNA in intact cells) and allow for analyses of sequence length-dependent eDNA degradation in combination with PMA. We confirmed that PMA is effective at differentiating “intact” and “disrupted” fish cells. We found that total eDNA and intracellular eDNA have different degradation processes that are dependent on the length of target sequence. For future conservation efforts using eDNA analyses, it is necessary to increase the reliability of the analysis results. The research presented here provides new analysis tools that expand our understanding of the ecology of eDNA, so that more accurate and reliable conclusions can be determined.

Summary:. Replacing an infected prosthetic thoracic aorta graft carries a high re-infection risk. We previously reported two clinical cases successfully treated with a new muscular wrapping technique: latissimus dorsi (LD) muscle flap with a distally based serratus anterior (SA) extension; however, a cadaveric study to prove the regular existence of the distal attachment area was lacking. We tried to establish an appropriate way of elevating the combined muscle flap safely. All of the cadavers were preserved using the Thiel embalming technique to retain flexibility. We checked for the existence of the distal attachment area between the LD and SA. Combined muscle flaps were elevated proximally while identifying the thoracodorsal artery, including the LD and SA branches. After the SA branch was ligated and cut, the SA muscle was manually peeled from the LD muscle with only the distal tight attachment area remaining. Contrast-enhanced computed tomography was performed using a multislice computed tomography system. Six human cadavers (three men, three women: 91 years old, on average) were examined. All six LD and SA combined muscle flaps showed a distal tight attachment area at the level from the seventh rib to the ninth rib. The tip of the SA muscle easily reached the sternum. Contrast-enhanced computed tomography failed to reconfirm the distal vascular flow from the LD to the reverse SA muscle, which we had visualized in a clinical case. We demonstrated the anatomical reliability of the new Y-shaped muscular flaps, which are suitable for preventing re-infection of aortic graft replacement.

Summary:. Surgical excision of a giant congenital melanocytic nevus (GCMN) results in a full-thickness skin defect that is usually difficult to reconstruct even with tissue expansion or skin grafting. Here, we report the first case of GCMN treated with a combination of cultured epithelial autografts (CEAs) and mesh-skin grafts to reconstruct a large skin defect after surgical excision. A 14-month-old girl had a GCMN occupying 20% of the total body surface area of her neck and back. A 5-stage, full-thickness excision was performed between the age of 14 and 25 months. In each intervention, the wound after excision was covered with 1:6 mesh-skin grafts and CEAs, except for the neck, where patch skin grafts and CEAs were used. The skin grafts and CEAs were engrafted without shedding and epithelialization was completed within 3-4 weeks. Eighteen months after the last surgery, a mesh-like scar remained, with no recurrence or severe contracture observed. The cosmetic appearances of the donor sites (the scalp and lower abdomen) were acceptable. The application of CEAs with mesh-skin grafts has been established for the treatment of severe burns. This combined method also provides a possible option for the treatment of GCMNs.

Summary:. Edwardsiella tarda is an anaerobe associated with freshwater and marine life. Necrotizing soft tissue infection caused by E. tarda is rare, but its mortality rate is extremely high (61.1%). We experienced a survival case of Fournier’s gangrene with E. tarda. A key clinical feature for the diagnosis was gas production; however, there have been no previous reports of such a phenomenon in relation to E. tarda. A 64-year-old man was admitted with a 3-day history of a high fever. His medical history, which included paraplegia from the Th6 level down, made the focus of inflammation at the perineum difficult to notice. Whole-body CT revealed subcutaneous gas from the posterior scrotum around the entire circumference of the anus. During emergency surgery, an incision at the posterior scrotum revealed extensive fascia necrosis, and E. tarda and Streptococcus anginosus were identified. We speculated the gas production in this case to be the product of a mixed infection of aerobes and anaerobes, which acted synergistically to induce bacterial growth. Timely surgical debridement and antibiotic therapy led to the patient’s survival.

Summary. Curettage is common in the treatment of a giant congenital melanocytic nevus (GCMN) in infants and should generally be performed before 6 months of age. Post-curettage retarded epithelialization often interferes with the ability to perform multiple operations within a short interval, and thus, it is difficult to treat large lesions in the neonatal period. We herein report a case of a GCMN comprising 20% of the total body surface area, which required multi-stage curettage, in which a cultured epithelial autograft was used to promote epithelialization of the post-curettage wound. The patient was a 1-month-old boy with a GCMN in his head, neck, chest, back, buttock, left upper arm, and a few satellite lesions. A four-stage operation was performed between 3 and 6 months of age; the cultured epithelial autograft took well after each operation, and complete epithelialization was observed at postoperative days 20, 23, 27, and 12, respectively. Seven months after the last surgery, hypertrophic scar formation was only observed in a small area of the left upper arm without axillary contracture. The color of the treated area improved, except for slight partial re-pigmentation. A skin biopsy was obtained from the re-pigmented area. The results demonstrated that nevus cells remained in the basal layer of the epidermis, hair follicles, and deep layer of the remaining dermis, suggesting that the recurrent nevus cells in the regenerated epidermis migrated from hair follicles. We conclude that the combination of curettage and the application of a cultured epithelial autograft is a promising option for GCMN treatment.

The abundance of Japanese eel Anguilla japonica has rapidly decreased in recent decades. Following a re-evaluation of the possibility of extinction, the Japanese Ministry of the Environment and the International Union for Conservation of Nature listed the Japanese eel as an endangered species in 2013 and 2014, respectively. However, their abundance and precise distribution have never been clarified owing to their nocturnality and difficulty in their capture. In this study, the distribution of Japanese eels was investigated by monitoring for environmental DNA (eDNA), a non-invasive and efficient detection method. A total of 365 water samples were collected from 265 rivers located throughout Japan. High concentrations of eDNA of Japanese eels were detected in rivers on the Pacific side, but were low in the Sea of Japan side. In particular, very little eDNA amplification was confirmed from Hokkaido and the north of the Sea of Japan. The eDNA distribution in Japanese rivers coincides with the transport of the larvae in the ocean, as estimated by numerical simulations. Generalized linear mixed models were developed to explain the distribution of eDNA concentrations. The total nitrogen concentration emerged as an important factor in the best model. These results indicate that the distribution of Japanese eel is mostly determined by the maritime larval transport, and their survival and growth depend on the abundance of food in the river. The findings of the present study are useful for the management of populations and in the conservation of Japanese eels.