On-site environmental DNA detection of species using ultrarapid mobile PCR.

Molecular methods, including those for environmental DNA (eDNA), provide essential information for biological and conservation sciences. Molecular measurements are often performed in the laboratory, which limits their scope, especially for rapid on-site analysis. eDNA methods for species detection provide essential information for the management and conservation of species and communities in various environments. We have developed an innovative novel method for on-site eDNA measurements using an ultrarapid mobile PCR platform. We tested the ability of our method to detect the distribution of silver carp, Hypophthalmichthys molitrix, in rivers and lakes. Our method reduced the measurement time to 30 min and provided high detectability of aquatic organisms compared to national observation surveys that use multiple fishing nets and laboratory extraction/detection using a benchtop qPCR platform. Our on-site eDNA method can be immediately applied to various taxa and environments.

Projection range of eDNA analysis in marshes: a suggestion from the Siberian salamander (Salamandrella keyserlingii) inhabiting the Kushiro marsh, Japan.

BACKGROUND: Freshwater ecosystems are rapidly declining. The Siberian salamander (Salamandrella keyserlingii) which inhabits the Kushiro marsh in Hokkaido, Japan has lost some habitat due to human activity. There are many challenges associated with conventional monitoring methods, including cost, the need for specialist personnel, environmental impact, and ability to detect the presence of this species; thus, we investigated the feasibility of using environmental DNA (eDNA) analysis to detect its presence and identify its breeding grounds. METHODS: We performed tank experiments to confirm eDNA emission from egg sacs, larvae, and adult Siberian salamanders in the water. We also performed water sampling and visual observation of egg sacs in the Kushiro marsh during the end of the breeding season and the larval season. RESULTS: The tank experiments found eDNA emission from all growth stages. It also implied concentrated emissions just after spawning and after hatching, and limited emissions during the incubation phase in egg sacs. We also detected eDNA in the field, likely reflecting the distribution of egg sacs or larvae. Combining this data with visual observations, it was determined that the eDNA results from the field were best explained by the number of egg sacs within 7-10 m of the sampling point. CONCLUSIONS: The results of this investigation show that the breeding sites and habitats of marshland species can successfully be monitored using eDNA analysis. They also suggest that the eDNA results from the marshes may reflect the biomass that is in close range to the sampling point. These results support the increased use of eDNA analysis in marshes and provide knowledge that could improve the interpretation of future results.

[A Case of Unresectable Advanced Gastric Cancer Treated by Seven Courses of Chemotherapy with Docetaxel, Cisplatin, and S-1Followed by Conversion Surgery Achieved Five Year Relapse Free Survival].

The patient is a 51-year-old man.Upper gastrointestinal endoscopy revealed gigantic type 3 gastric cancer.Enhanced abdominal CT demonstrated a gigantic mass of 15×7 cm in the stomach, and a large number of peritoneal dissemination with moderate amount of ascites.The patient was diagnosed with cT4aN3bM1(peritoneal dissemination), Stage IV gastric cancer(JPN ver8)and was treated by chemotherapy with docetaxel(40mg/m / 2 day 1)plus cisplatin(60mg/m2 day 1)plus S-1(80mg/m2 day 1-14).After 7 courses of chemotherapy, peritoneal dissemination was disappeared.The patient received total gastrectomy and D2 lymphadenectomy as a conversion surgery.The pathological findings revealed a T0N0M0, stage 0; the tumor was determined to be Grade 3 owing to the chemotherapeutic effect.Without postoperative adjuvant therapy, the patient is alive without recurrence at the 5 years follow-up after operation.

[A Case of Laparoscopic Repair of Internal Hernia after Laparoscope-Assisted Distal Gastrectomy with Antecolic Roux-en-Y Reconstruction].

A 72-year-old woman had undergone laparoscope-assisted distal gastrectomy with D1 plus lymph node dissection and antecolic Roux-en-Y reconstruction for early gastric cancer. She visited our department outpatient clinic with left upper abdominal pain 1 year and 9 months after the surgery. CT revealed a spiral sign of the superior mesenteric arteriovenous branch. An internal hernia was suspected on hospitalization. Although abdominal symptoms were relieved by conservative treatment, the hernia persisted. Laparoscopic surgery was performed and revealed that almost entire small intestine had been affected due to Petersen's defect. Since no ischemic changes were observed, the defect was repaired laparoscopically with suture closure. There has been no recurrence of internal hernia after the laparoscopic surgery. Internal hernia after distal gastrectomy is relatively rare. However, the risk of internal hernia is high due to the gap between the elevated jejunum and transverse colon mesentery in Roux-en-Y reconstruction and can lead to intestinal necrosis. Since an internal hernia can occur in patients who have undergone gastric resection with Roux-en-Y reconstruction, suture closure of Petersen's defect should be performed to prevent this occurrence.

Detection of an endangered aquatic heteropteran using environmental DNA in a wetland ecosystem.

The use of environmental DNA (eDNA) has recently been employed to evaluate the distribution of various aquatic macroorganisms. Although this technique has been applied to a broad range of taxa, from vertebrates to invertebrates, its application is limited for aquatic insects such as aquatic heteropterans. Nepa hoffmanni (Heteroptera: Nepidae) is a small (approx. 23 mm) aquatic heteropteran that inhabits wetlands, can be difficult to capture and is endangered in Japan. The molecular tool eDNA was used to evaluate the species distribution of N. hoffmanni in comparison to that determined using hand-capturing methods in two regions of Japan. The eDNA of N. hoffmanni was detected at nearly all sites (10 eDNA-detected sites out of 14 sites), including sites where N. hoffmanni was not captured by hand (five eDNA-detected sites out of six captured sites). Thus, this species-specific eDNA technique can be applied to detect small, sparsely distributed heteropterans in wetland ecosystems. In conclusion, eDNA could be a valuable technique for the detection of aquatic insects inhabiting wetland habitats, and could make a significant contribution to providing distribution data necessary to species conservation.

[A Case of HER2-Positive Siewert Type I Adenocarcinoma of the Esophagogastric Junction Treated via Neoadjuvant Chemotherapy Followed by Radical Resection].

A 63-year-old man with dysphagia was referred to our hospital. He was found to have a type 2 tumor extending from the lower thoracic esophagus to the esophagogastric junction via upper gastrointestinal endoscopy. A biopsy revealed adenocarcinoma with overexpression of the human epidermal growth factor type 2(HER2). The tumor was type I according to Siewert's classification, as the epicenter of the tumor was 27mm to the oral side from the esophago-gastric junction. The clinical diagnosis was T3N1M1, stage IV according to the Japanese Classification of Gastric Carcinoma, and T3N2M0, stage III per the Japanese Classification of Esophageal Cancer. He was treated with neoadjuvant chemotherapy consisting of 6 courses of capecitabine(1,000mg/m / / 2: days 1-14)plus cisplatin(80mg/m2: day 1)and trastuzumab(8mg/kg: day 1 of the first course, 6mg/kg: day 1 after the second course). Computed tomography(CT)and upper gastrointestinal endoscopy showed shrinkage of the primary esophagogastric cancer and lymph node metastases. The patient had a partial response and underwent radical esophagectomy. The pathological findings revealed a T3N2M0, stage III tumor; the tumor was determined to be Grade 1b owing to the chemotherapeutic effect. At a follow-up examination 1 year and 7 months after the start of chemotherapy, the patient is alive without recurrence.

[A Case of Synchronous Esophageal and Gastric Cancer Successfully Treated Using Multimodal Therapy].

We report a case of advanced esophageal and gastric cancer that was successfully treated via multimodal therapy. A 65- year-old man with hoarseness was referred to our hospital. He was diagnosed with clinical T4aN2M0, Stage IV esophageal squamous cell carcinoma and clinical T3N1M0, Stage II B gastric adenocarcinoma. He was treated with 3 courses of chemotherapy, administered over 4weeks, with S-1(80mg/m / / 2: day 1-14), cisplatin(60mg/m2: day 1), and docetaxel(40mg/m2: day 1). Computed tomography(CT)revealed shrinkage of the primary esophageal tumor, gastric tumor, and lymph node metastases. Next, we selected definitive radiation chemotherapy(CRT), because lymph node metastases remained around the bilateral recurrent laryngeal nerves. After CRT with a total 60 Gy plus administration of 5-fluorouracil and cisplatin, CT showed that the primary esophageal tumor and lymph node metastases had disappeared. Then, distal gastrectomy was performed for the remaining gastric cancer, as part of the multimodal therapy. After gastrectomy, no systemic chemotherapy was performed. At a follow-up examination 5 years and 6 months after the start of chemotherapy, the patient is alive without recurrence.

[A Case of Long-Term Survival after Gastrectomy and Metachronous Brain Metastasis].

A30 -year-old woman underwent total gastrectomy with D2 lymph node dissection after being diagnosed with clinical T3, N2, M0, Stage III B gastric cancer. The postoperative pathological findings revealed a T3(SE), N2, M0, Stage III B tumor. Headache, dizziness, and vomiting occurred during chemotherapy for peritoneal recurrence, using weekly paclitaxel on days 1, 8, and 15. Head CT showed a solitary tumor with a diameter of 28mm in the cerebellum, as well as cerebellar swelling and hydrocephalus. She underwent an emergency craniotomy and tumor enucleation. Pathological examination revealed a metastatic brain tumor from the gastric cancer. She received 12 courses of CPT-11 plus cisplatin until discontinuation because of an adverse event. The patient is alive 6 years after the diagnosis of the cerebellar metastasis without recurrence.

[Radical Chemoradiotherapy for Recurrent Esophageal Cancer after Curative Esophagectomy].

Recurrent esophageal cancer has a poor prognosis.However, we sometimes encounter cases with long-term survival after radical treatment for recurrent esophageal cancer.We perform radical chemoradiotherapy aggressively when recurrent esophageal cancer is present in a limited area and is sufficiently localized to be treated by radiation therapy.From June 2010 to December 2014, 150 patients underwent curative esophagectomy for esophageal cancer.Forty -one cases relapsed and we treated 13 of them with radical chemoradiotherapy.Complete response(CR), non-CR/non-PD, and progressive disease(PD) were observed in 5, 6, and 2 cases, respectively.The CR rate was 38.4%.The median survival time from recurrence was 500± 39.7 days, and the 1-year and 3-year survival rates were 84.6% and 28.7%, respectively. Four out of 5 CR cases were single site recurrences.The other case was multiple and regrowth of the cancer was identified 253 days after the CR.These results suggest that radical chemoradiotherapy for recurrent esophageal cancer after curative esophagectomy can achieve long time survival, especially in cases with single site lymph node recurrence.

[Three Cases of Adenocarcinoma of the Advanced Esophago-Gastric Junction Confirmed as Pathological Complete Response Following Neoadjuvant Chemotherapyand Gastrectomy].

Neoadjuvant chemotherapy(NAC)is thought to be effective to improve the outcomes in patients with adenocarcinoma of the esophago-gastric junction(AEG). We encountered 3 patients who were confirmed as having a pathological complete response following gastrectomy after NAC. The first patient had Siewert type II and clinical Stage III AEG, the second patient had Siewert type I and clinical Stage III AEG, while the third had Siewert type II and clinical Stage II AEG. Two patients received NAC with 2 courses of S-1 plus cisplatin, while the third patient received 2 courses of docetaxel, S-1, and cisplatin. All of the patients were treated with D2 gastrectomy after the NAC, and remain alive at more than 40 months after initial chemotherapy treatment without recurrence. We report the details of these 3 cases and review the literature.

[A Case of Recurrent Esophageal Cancer Treated with Salvage Lymphadenectomy after Definitive Chemoradiotherapy].

A 74-year-old man admitted with dysphagia was found to have advanced esophageal squamous cell carcinoma, clinical stage IV (T4N2M0). We initiated definitive chemoradiotherapy(dCRT)with combined 5-fluorouracil and cisplatin chemotherapy( FP therapy)and 50.4 Gy irradiation, followed by boost FP therapy, to which the patient showed confirmed complete response(CR). Local recurrence was detected in the scar of the primary lesion at 4 months after the boost FP therapy. Photodynamic therapy(PDT)for this lesion resulted in CR. Thirteen months later, right hilar and right pericardial lymph node metastases were found. The right hilar lymph node metastases were not visible on CT after triweekly docetaxel therapy, but the pericardial lesions remained. The patient underwent salvage lymphadenectomy without further chemotherapy, and at 5 months after surgery, he was alive and recurrence-free.

Investigation of the potential for triterpene synthesis in rice through genome mining and metabolic engineering.

The first committed step in sterol biosynthesis in plants involves the cyclization of 2,3-oxidosqualene by the oxidosqualene cyclase (OSC) enzyme cycloartenol synthase. 2,3-Oxidosqualene is also a precursor for triterpene synthesis. Antimicrobial triterpenes are common in dicots, but seldom found in monocots, with the notable exception of oat. Here, through genome mining and metabolic engineering, we investigate the potential for triterpene synthesis in rice. The first two steps in the oat triterpene pathway are catalysed by a divergent OSC (AsbAS1) and a cytochrome P450 (CYP51). The genes for these enzymes form part of a metabolic gene cluster. To investigate the origins of triterpene synthesis in monocots, we analysed systematically the OSC and CYP51 gene families in rice. We also engineered rice for elevated triterpene content. We discovered a total of 12 OSC and 12 CYP51 genes in rice and uncovered key events in the evolution of triterpene synthesis. We further showed that the expression of AsbAS1 in rice leads to the accumulation of the simple triterpene, ß-amyrin. These findings provide new insights into the evolution of triterpene synthesis in monocots and open up opportunities for metabolic engineering for disease resistance in rice and other cereals.