Droplet-based microfluidic platform for detecting agonistic peptides that are self-secreted by yeast expressing a G-protein-coupled receptor.

BACKGROUND: Single-cell droplet microfluidics is an important platform for high-throughput analyses and screening because it provides an independent and compartmentalized microenvironment for reaction or cultivation by coencapsulating individual cells with various molecules in monodisperse microdroplets. In combination with microbial biosensors, this technology becomes a potent tool for the screening of mutant strains. In this study, we demonstrated that a genetically engineered yeast strain that can fluorescently sense agonist ligands via the heterologous expression of a human G-protein-coupled receptor (GPCR) and concurrently secrete candidate peptides is highly compatible with single-cell droplet microfluidic technology for the high-throughput screening of new agonistically active peptides. RESULTS: The water-in-oil microdroplets were generated using a flow-focusing microfluidic chip to encapsulate engineered yeast cells coexpressing a human GPCR [i.e., angiotensin II receptor type 1 (AGTR1)] and a secretory agonistic peptide [i.e., angiotensin II (Ang II)]. The single yeast cells cultured in the droplets were then observed under a microscope and analyzed using image processing incorporating machine learning techniques. The AGTR1-mediated signal transduction elicited by the self-secreted Ang II peptide was successfully detected via the expression of a fluorescent reporter in single-cell yeast droplet cultures. The system could also distinguish Ang II analog peptides with different agonistic activities. Notably, we further demonstrated that the microenvironment of the single-cell droplet culture enabled the detection of rarely existing positive (Ang II-secreting) yeast cells in the model mixed cell library, whereas the conventional batch-culture environment using a shake flask failed to do so. Thus, our approach provided compartmentalized microculture environments, which can prevent the diffusion, dilution, and cross-contamination of peptides secreted from individual single yeast cells for the easy identification of GPCR agonists. CONCLUSIONS: We established a droplet-based microfluidic platform that integrated an engineered yeast biosensor strain that concurrently expressed GPCR and self-secreted the agonistic peptides. This offers individually isolated microenvironments that allow the culture of single yeast cells secreting these peptides and gaging their signaling activities, for the high-throughput screening of agonistic peptides. Our platform base on yeast GPCR biosensors and droplet microfluidics will be widely applicable to metabolic engineering, environmental engineering, and drug discovery.

Improvement Effect of Upper Mediastinal Lymphadenectomy During Minimally Invasive Esophagectomy on the Prognosis in Squamous Cell Carcinoma: Efficacy Index and Propensity Score Matching Analyses.

BACKGROUND: The effect of lymphadenectomy around the recurrent laryngeal nerve (RLN) in open esophagectomy has been demonstrated with the efficacy index (EI). However, it remains unclear whether this effect exists for minimally invasive esophagectomy (MIE) in the prone position. The purpose of this study was to clarify whether the upper mediastinal lymphadenectomy contributed to improved prognosis in patients with esophageal squamous cell carcinoma. STUDY DESIGN: This study included 339 patients with esophageal squamous cell carcinoma treated with MIE in the prone position at Kobe University or Hyogo Cancer Center, Japan, from 2010 to 2015. EIs for each station, correlations between metastatic lymph nodes around the left RLN and RLN palsy, and survival of patients with and without upper mediastinal lymphadenectomy were investigated. RESULTS: Among 297 patients treated with upper mediastinal lymphadenectomy, Clavien- Dindo grade ≥ II left RLN palsy occurred in 59 patients (20%). Overall, EIs for the right RLN (7.4) and left RLN (6.6) were higher than EIs for other stations. For patients with upper-third or middle-third tumors, the trend was stronger. Left RLN palsy was more likely in patients with metastatic lymph nodes around the left RLN than in those without (44% vs 15%, p < 0.0001). After propensity score-matching, 42 patients were included in each group with and without upper mediastinal lymphadenectomy. In survival analyses, the 5-year overall survival rates were 55% vs 35% and cause-specific survival rates were 61% vs 43% for the patients with and without upper mediastinal lymphadenectomy respectively. Significant differences were confirmed in survival curves (overall survival: p = 0.03; cause-specific survival: p = 0.04, respectively). CONCLUSIONS: Upper mediastinal lymphadenectomy contributes to improved prognosis with high EIs in MIE in the prone position.

[Eight mm Port-Site Hernia Following Robot-Assisted Abdominoperineal Resection for Rectal Cancer-A Case Report].

A 70s woman with a history of asthma and dyslipidemia underwent a robot-assisted abdominoperineal resection for rectal cancer. The ports were placed as per the method of Shizuoka Cancer Center and no intraoperative complications were observed. The colostomy was constructed in the left lower abdomen by the retroperitoneal route. The 12-mm port part was closed in 2 layers, the fascia and dermis, and the 8-mm port part was closed only in the dermis. The postoperative course was good; however, the patient vomited 10 days after surgery. Abdominal computed tomography revealed an incarcerated small intestine in the 8-mm port of the left abdomen, and it was diagnosed as port-site hernia incarceration. Emergency laparotomy hernia repair was performed on the day. A part of the 8-mm port was incised to 30-mm and the fascia dilatation to 30-mm was observed. The color tone of the incarcerated small intestine was good. Only adhesion peeling was performed, the small intestine was returned, and the fascia was closed. The postoperative course was uneventful and the patient was discharged 17 days after the second surgery. At the 1 year postoperative follow-up, recurrence of hernia or rectal cancer was not observed.

Enhanced squalene production by modulation of pathways consuming squalene and its precursor.

Fermentative production of squalene in yeast as an alternative approach to extracting squalene from sharks or plants has attracted significant interest. However, squalene accumulation is limited due to its inevitable high-flux allocation toward ergosterol synthesis. In this study, we described expression control of squalene monooxygenase (Erg1p), the first-step enzyme of ergosterol synthesis from squalene, to significantly reduce squalene loss. We replaced the ERG1 promoter (PERG1) with three natural yeast promoters with different activities (PPCL2, PHCM1, and PTHI2). ERG1 controlled by PTHI2 showed 20 times higher squalene production compared with the wild-type strain, whereas the other two strains exhibited no significant difference. By combining the overexpression of rate-limiting enzyme and the deletion of non-essential competing pathway gene, the yeast Saccharomyces cerevisiae produced up to 379 mg/L of squalene.

Engineering of Synthetic Transcriptional Switches in Yeast.

Transcriptional switches can be utilized for many purposes in synthetic biology, including the assembly of complex genetic circuits to achieve sophisticated cellular systems and the construction of biosensors for real-time monitoring of intracellular metabolite concentrations. Although to date such switches have mainly been developed in prokaryotes, those for eukaryotes are increasingly being reported as both rational and random engineering technologies mature. In this review, we describe yeast transcriptional switches with different modes of action and how to alter their properties. We also discuss directed evolution technologies for the rapid and robust construction of yeast transcriptional switches.

Proposed modification of the eighth edition of the AJCC-ypTNM staging system of esophageal squamous cell cancer treated with neoadjuvant chemotherapy: Unification of the AJCC staging system and the Japanese classification.

BACKGROUND: The eighth edition of the American Joint Committee on Cancer (AJCC) tumor node metastasis (AJCC-TNM 8th) system adopted the newly separate post-neoadjuvant pathologic stage group (ypTNM). However, it is not compatible with the Japanese pathologic classification after neoadjuvant chemotherapy (JPN-CT-pTNM). The aim of this study is to clarify the subjects of the AJCC-ypTNM 8th and propose a unification of the AJCC and Japanese systems to create novel AJCC-CT-pTNM 8th. METHODS: Participants were 304 esophageal squamous cell carcinoma (ESCC) patients who underwent neoadjuvant chemotherapy followed by 3 stage esophagectomy between 2010 and 2019. Predictive probabilities of pN, pM in AJCC-ypTNM 8th and JPN-CT-pTNM 11th systems were evaluated to propose novel system. RESULTS: In training data from 234 patients, the overall survival rate was statistically better for ypStage IIIA than ypStage II (P = 0.040) resulting in staging inversion in AJCC-ypTNM 8th. Predictive probability of pathological N status in AJCC-ypTNM 8th (Akaike Information Criterion: AIC = 979.53) was superior to that in JPN-CT-pTNM 11th (AIC = 999.07). In AJCC-ypTNM 8th, 71% (15/21) of ypM1 diseases were supraclavicular lymph nodes (No. 104 L/N as regional in JPN-CT-pTNM 11th) metastases with considerably good prognosis. The predictive probability of the novel AJCC-CT-pTNM 8th [unification of ypStage II and IIIA, conversion of supraclavicular L/Ns metastases from ypM to ypN] (AIC = 1054.24) was superior to that of the existing AJCC-ypTNM 8th (AIC = 1070.74). The feasibility of novel system was validated using test data from 70 patients. CONCLUSIONS: Unification of the AJCC and Japanese systems yields a simpler and more precise predictive system after neoadjuvant chemotherapy.

[Long-Term Survival of a Patient with Hepatocellular Carcinoma after Surgical Resection of Metachronous Hilar Lymph Node Metastases].

The recurrence of hepatocellular carcinoma(HCC)is primarily due to intrahepatic metastases. Additionally, extrahepatic HCC metastases most commonly occurs in the lungs, lymph nodes, adrenal glands, and bones. Systemic chemotherapy is the standard treatment for extrahepatic metastases. Although several reports on surgical resection of lymph node metastases (LNM) in patients with HCC have been published, its clinical benefits remain controversial. We report a case in which surgical resection of LNM was performed in a patient with HCC. The patient was a 74-year-old woman diagnosed with HCC and non-B non-C chronic hepatitis, for which she underwent a laparoscopic partial hepatectomy. The pathological diagnosis was St-A, 1.6×1.4 cm, confluent multinodular type, pT1N0M0, fStage Ⅰ. Nine months later, 2 LNM on the liver hilum were detected and managed with sorafenib. Sorafenib was discontinued after 2 months due to the development of Grade 3 hand-foot syndrome. Since no new lesions were detected on follow-up, lymph node resection was performed. The patient remains disease-free 4.5 years postoperatively.

Salvage hepatectomy for local recurrence after particle therapy using proton and carbon ion beams for liver cancer.

AIM: With the increased use of particle therapy for liver cancer, local recurrence after particle therapy increased. Salvage hepatectomy is an acceptable treatment option for local recurrence following particle therapy; however, its safety and effectiveness remain unclear. Therefore, this multi-center study aimed to verify the feasibility and efficacy of salvage hepatectomy and assess clinical issues associated with its application. METHODS: We retrospectively assessed the perioperative outcomes, prognosis, and pathological characteristics of 15 patients who underwent salvage hepatectomy for local recurrence after particle therapy between 2006 and 2019. RESULTS: Hepatocellular carcinoma and metastatic liver tumors were noted in eight and seven patients, respectively. The mean total dose and number of fractions were 66.5 Gy and 12, respectively, and the mean interval between particle therapy and surgery was 30.1 months. Major hepatectomy was performed in seven cases. Moreover, the mortality rate was 0%, and surgical complications of Clavien-Dindo grade IIIa or higher were observed in four cases (27%)-two bile leakages, one pleural effusion, and one refractory skin fistula. The median overall survival time and 5-year overall survival rate after salvage hepatectomy were 29.9 months and 43.1%, respectively. Histological examination of the irradiated liver tissue surrounding the tumor showed sinusoidal dilatation, loss of hepatocyte, and fibrosis in most cases. CONCLUSION: Salvage hepatectomy after particle therapy is a feasible therapy; however, the risk of refractory complications associated with particle therapy is relatively high. Therefore, the first-line treatment for resectable liver cancer should be carefully determined considering second-line treatment after local recurrence.

Constitutive cell surface expression of ZZ domain for the easy preparation of yeast-based immunosorbents.

We describe a novel expression cassette that enables efficient and constitutive expression of the ZZ domain derived from Staphylococcus aureus protein A on the yeast cell surface to easily prepare yeast-based immunosorbents. Using this expression cassette containing the PGK1 promoter, a secretion signal derived from α-factor, and a Flo1-derived anchor protein, we successfully created a yeast-based immunosorbent for human serum albumin.

A case of intraductal papillary neoplasm of the bile duct accompanied by intraductal papillary mucinous neoplasm of the pancreas and hepatocellular carcinoma.

A 73-year-old man with mixed-type intraductal papillary mucinous neoplasm of the pancreas body was followed up for 14 years. Based on imaging findings, the intraductal papillary mucinous neoplasm of the pancreas met the high-risk stigmata, and new hepatic masses were suspected to be intraductal papillary neoplasms of the bile duct. With a diagnosis of intraductal papillary mucinous neoplasm of the pancreas and intraductal papillary neoplasm of the bile duct, the patient had undergone left lateral hepatectomy and distal pancreatectomy. Based on pathology, the pancreatic specimen was diagnosed as a high-grade intraductal papillary mucinous neoplasm of the pancreas, and the hepatic specimen was diagnosed as an intraductal papillary neoplasm of the bile duct and hepatocellular carcinoma. The intraductal papillary neoplasms of the bile duct and hepatocellular carcinoma were adjacent to each other. Fifteen months after surgery, recurrence in the remnant pancreas was detected. The patient had undergone residual total pancreatectomy, with no recurrence thirty months after the second resection. This case demonstrates that second surgery for metachronous high-risk lesions in the remnant pancreas of patients with intraductal papillary mucinous neoplasm of the pancreas and intraductal papillary neoplasm of the bile duct may also be considered to improve survival.

Hepatocellular carcinoma recurrence in the extrahepatic bile duct wall: A case report.

We should know that hepatocellular carcinoma can progress as if it replaces the bile duct wall itself.

Robust and flexible platform for directed evolution of yeast genetic switches.

A wide repertoire of genetic switches has accelerated prokaryotic synthetic biology, while eukaryotic synthetic biology has lagged in the model organism Saccharomyces cerevisiae. Eukaryotic genetic switches are larger and more complex than prokaryotic ones, complicating the rational design and evolution of them. Here, we present a robust workflow for the creation and evolution of yeast genetic switches. The selector system was designed so that both ON- and OFF-state selection of genetic switches is completed solely by liquid handling, and it enabled parallel screen/selection of different motifs with different selection conditions. Because selection threshold of both ON- and OFF-state selection can be flexibly tuned, the desired selection conditions can be rapidly pinned down for individual directed evolution experiments without a prior knowledge either on the library population. The system's utility was demonstrated using 20 independent directed evolution experiments, yielding genetic switches with elevated inducer sensitivities, inverted switching behaviours, sensory functions, and improved signal-to-noise ratio (>100-fold induction). The resulting yeast genetic switches were readily integrated, in a plug-and-play manner, into an AND-gated carotenoid biosynthesis pathway.

Randomized phase II study comparing the efficacy and safety of SOX versus mFOLFOX6 as neoadjuvant chemotherapy without radiotherapy for locally advanced rectal cancer (KSCC1301).

BACKGROUND: Preoperative chemoradiotherapy (CRT), the current standard of care for locally advanced rectal cancer (LARC), is associated with many radiotherapy (RT)-related side effects. We aimed to evaluate whether S-1 and oxaliplatin (SOX) or folinic acid, 5-FU, and oxaliplatin (mFOLFOX6) can be as effective as neoadjuvant chemotherapy (NAC) regimens for LARC without RT. METHODS: Patients with untreated resectable LARC were randomly assigned to receive SOX or mFOLFOX6. The NAC protocol period was 3 months. The primary endpoint was 3-year disease-free survival (DFS), and the secondary endpoints included pathological effects, surgical completion rate, 3-year survival, and safety. RESULTS: From September 2013 to October 2015, 56 and 54 patients were enrolled in the SOX and mFOLFOX6 arms, respectively. The 3-year DFS rates were 69.4% (95% confidence interval [CI] 54.9-83.6) and 73.4% (95% CI 58.7-83.6) in the SOX and mFOLFOX6 arms, respectively; no significant differences were found between the arms (log-rank test; P = 0.5315, hazard ratio: 0.808, 95% CI 0.414-1.578). The 3-year survival rates were 92.3 and 91.8% in the SOX and mFOLFOX6 arms, respectively. The surgical completion rate was 98.1% overall, 100% in the SOX arm, and 96.0% in the mFOLFOX6 arm. The incidences of pathological response rates ≥grade 1b were 41.5 and 43.8% in the SOX and mFOLFOX6 arms, respectively. Both treatments were manageable and tolerable. CONCLUSION: We demonstrated the effectiveness and safety of SOX and mFOLFOX6, both of which may be new neoadjuvant treatment candidates in previously untreated LARC cases. TRIAL REGISTRATION: Date of enrolment of the first participant to the trial: 3rd Oct 2013; This study was registered in the UMIN clinical trials registry on 14th Aug, 2013. (Prospectively registered, UMIN-CTR number UMIN000011486). https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=brows&recptno=R000013441&language=J.

Impact of Lymph Node Ratio on Survival Outcome in Esophageal Squamous Cell Carcinoma After Minimally Invasive Esophagectomy.

BACKGROUND: Esophageal squamous cell cancer (ESCC) is one of the deadliest cancers in the world. Esophagectomy remains the principal treatment, and minimally invasive esophagectomy (MIE) has been performed worldwide. This study aimed to clarify whether the lymph node ratio (LNR), defined as the ratio of metastatic lymph nodes (LNs) to examined, is a prognostic factor for ESCC after MIE. METHODS: This study included 327 MIEs with the patient in the prone position at two institutions from 2010 to 2015. Cox proportional hazards regression analyses using clinicopathologic characteristics and the LNR were performed for the pN1 patients and the whole cohort. RESULTS: In the multivariate analysis for all stages, independent prognostic factors were depth of tumor invasion (P < 0.0001), LNR (P = 0.014), operative time (P = 0.003), and pneumonia (P = 0.012). In the analysis of the pN1 subgroup, the optimum LNR cutoff level for overall survival (OS) was 9 based on receiver operation characteristic analysis. The LNR was significantly associated with depth of tumor invasion (P = 0.004) and number of metastatic LNs (P < 0.0001). The OS curve for the group with an LNR of 9 or higher was significantly worse than for the group with an LNR lower than 9 (P < 0.001). Multivariate analyses demonstrated that the LNR is a unique independent prognostic factor for the pN1 subgroup (hazard ratio, 6.811; 95% confidence interval, 2.009-23.087; P = 0.002). CONCLUSIONS: The LNR is an independent prognostic factor in ESCC after MIE. Especially for patients with pN1 status, the LNR is more useful than the number of metastatic LNs for predicting survival outcome.

A case of laparoscopic anterior resection for rectal cancer with duplication of the inferior vena cava using preoperative 3D computed tomography angiography.

We report the case of a patient with duplication of the inferior vena cava (DIVC) who underwent anterior laparoscopic resection for rectal cancer. A 66-year-old woman presented with abnormal lung shadows on a chest x-ray during a routine health checkup. She was diagnosed with rectal cancer and lung metastasis using colonoscopy and thoracoabdominal computed tomography (CT). In addition, a 3D CT angiography revealed double inferior vena cava, one on either side of the aorta. The preoperative diagnosis was rectal cancer cT3N0M1a(Lung) cStage IVA with DIVC, and a two-stage surgery was planned. The first stage was high anterior laparoscopic resection. This was safely performed because the pre-hypogastric nerve fascia was preserved and the left inferior vena cava was not visualized during the surgery. During the second stage of the surgery, video-assisted thoracoscopic left lower lobectomy was performed and no recurrence was observed for >6 months after the second surgery.

Correction to: Thoracic Duct Resection During Esophagectomy Does Not Contribute to Improved Prognosis in Esophageal Squamous Cell Carcinoma: A Propensity Score Matched-Cohort Study.

In the original article, there are errors in Fig. 1. Following is the corrected figure.

Laparoscopic hepatectomy for liver metastasis of lung large-cell neuroendocrine carcinoma: A case report.

INTRODUCTION: Lung large-cell neuroendocrine carcinoma (LCNEC) is an aggressive and a rare type of lung cancer, and the prognosis of LCNEC with distant metastasis is extremely poor, with a five-year survival rate of 0%. Here, we report a case of laparoscopic hepatectomy for liver metastasis of lung LCNEC. PRESENTATION OF CASE: A 63-year-old man received a routine physical examination, and abnormal chest radiographic findings were observed; chest computed tomography (CT) in our hospital revealed that the patient had left pneumothorax and a lesion measuring 18 mm in the inferior lingular segment of the lung. The patient underwent thoracoscopic lobectomy, and the final pathological diagnosis was lung LCNEC. Four years after surgery, abdominal CT revealed a mass measuring 27 mm in the liver. The patient underwent laparoscopic partial hepatectomy, and postoperative pathological examination showed liver metastasis of LCNEC. There was no sign of recurrence 6 months after hepatectomy. DISCUSSION: LCNEC with distant metastasis has a poor response to systemic chemotherapy, and the median survival time of patients with distant metastasis is estimated to be approximately 6 months, with a five-year survival rate of 0%. Although the common site of metastasis from LCNEC is the liver, there are no previous reports of hepatectomy for liver metastasis of LCNEC. CONCLUSION: We report a case of laparoscopic hepatectomy for liver metastasis of lung LCNEC. It is suggested that surgical resection for solitary distant metastasis of LCNEC may improve prognosis.

Comparison of total versus subtotal gastrectomy for remnant gastric cancer.

PURPOSE: Completion gastrectomy (CG) is a common procedure for remnant gastric cancer (RGC). However, partial gastrectomy for gastric cancer has several benefits compared to total gastrectomy in terms of the quality of life. In this study, we evaluated the feasibility and advantage of subtotal resection of the remnant stomach (SR) for clinical stage IA RGC. METHODS: A total of 43 patients who underwent gastrectomy for clinical stage IA RGC were included. CG and SR were performed on 27 (62.8%) and 16 patients (37.2%), respectively. The short- and long-term outcomes, including the nutritional status, after CG and SR for clinical stage IA RGC were compared between the two groups. RESULTS: There were no significant differences in pathological stage or incidence of postoperative complications between the two groups. The decrease in body weight, body mass index, and serum albumin level was significantly lower in the SR group than in the CG group (P < 0.001, P = 0.025, and 0.008). In the SR group, there was no recurrence at the remaining lymph nodes or gastric stump. The 5-year overall survival rate was 87.8% in the CG group and 86.1% in the SR group, without a significant difference between the two groups (P = 0.959). CONCLUSIONS: The present study showed the noninferiority of SR to CG based on surgical and oncological outcomes for clinical stage IA RGC. Furthermore, SR has an advantage over CG in terms of postoperative nutritional status. Therefore, SR could be an alternative elective treatment option for early RGC located around the anastomotic site.

A rare case of metastatic solitary fibrous tumor of the pancreas manifesting as a cystic neoplasm: a case report.

BACKGROUND: Solitary fibrous tumor (SFT) is a rare mesenchymal tumor that typically arises from the pleura. Although it may appear in other organs, it rarely develops in the pancreas. We report herein a rare case of metastatic SFT of the pancreas originating from an intracranial tumor and subsequently identified as a cystic neoplasm of the pancreas. CASE PRESENTATION: A 58-year-old woman with a past medical history of brain tumor visited the hospital for further investigation of a cystic tumor in the pancreas tail. Abdominal imaging showed a heterogeneously enhancing mass that was initially suspected as a neuroendocrine neoplasm, solid pseudopapillary neoplasm, or mucinous cystic neoplasm of the pancreas. Distal pancreatectomy was performed without any intraoperative and postoperative complications. Pathological findings confirmed a diagnosis of malignant SFT of the pancreas with hyperproliferative potential. A histopathological review of her brain tumor revealed that the pancreatic tumor was derived from her brain lesion. The patient developed recurrent brain disease 4 years after the pancreatectomy, but no recurrence has been observed in the abdominal cavity. CONCLUSIONS: SFT should be considered in the differential diagnosis of untypical hypervascular pancreatic mass, particularly in patients with a history of an intrathoracic or intracranial mesenchymal tumor. Immunohistochemical analysis is crucial in detecting this tumor entity. Hyperproliferative status indicates a malignant disease and requires careful postoperative observation.

Laparoscopic anatomical segment 3 segmentectomy for hepatocellular carcinoma accompanied by hypoplasia of the right hepatic lobe.

We report a case of laparoscopic anatomical segment 3 segmentectomy for hepatocellular carcinoma (HCC) accompanied by hypoplasia of the right hepatic lobe. An 80-year-old man was admitted with a suspicion of HCC diagnosed by computed tomography during follow-up for thyroid cancer. Dynamic computed tomography showed 40-mm HCC in segment 3 and hypoplasia of the right hepatic lobe with the Chilaiditi sign. We performed laparoscopic anatomical segment 3 segmentectomy. There were no postoperative complications, and the patient was discharged 6 days postoperatively. This procedure can be performed safely and is technically feasible, but special attention should be paid to anatomical alterations to avoid fatal surgical complications.