PCR- and wash-free detection of serum miRNA via signaling probe hybridization.

Detection of microRNAs (miRNAs) in the serum is an effective liquid biopsy technique for cancer diagnosis. However, conventional diagnostic methods are time-consuming and complex. Therefore, in this study, we established a signaling probe-based DNA microarray system for miRNA detection. PCR, fluorescence labeling, and washing are not necessary for signaling probes. Four probes were designed using different miRNAs as diagnostic cancer markers. The developed system is useful for various miRNAs, regardless of their target lengths (18-26-mer) and GC content (36%-89%). Here, all the assays were performed within 40 min. Overall, our signaling probe-based DNA hybridization system facilitates the simple and rapid detection of serum miRNAs without the need for gene amplification, fluorescence labeling and washing.

Measurement of changes in serum-based inflammatory indicators to monitor response to nivolumab monotherapy in advanced gastric cancer: a multicenter retrospective study.

BACKGROUND: Nonresectable gastric cancer develops rapidly; thus, monitoring disease progression especially in patients receiving nivolumab as late-line therapy is important. Biomarkers may facilitate the evaluation of nivolumab treatment response. Herein, we assessed the utility of serum-based inflammatory indicators for evaluating tumor response to nivolumab. METHODS: This multicenter retrospective cohort study included 111 patients treated with nivolumab monotherapy for nonresectable advanced or recurrent gastric cancer from October 2017 to October 2021. We measured changes in the C-reactive protein (CRP)-to-albumin ratio (CAR), platelet-to-lymphocyte ratio (PLR), and neutrophil-to-lymphocyte ratio (NLR) in serum from baseline to after the fourth administration of nivolumab. Furthermore, we calculated the area under the receiver operating characteristic curves (AUC ROCs) for CAR, PLR, and NLR to identify the optimal cutoff values for treatment response. We also investigated the relationship between clinicopathologic factors and disease control (complete response, partial response, and stable disease) using the chi-squared test. RESULTS: The overall response rate (complete and partial response) was 11.7%, and the disease control rate was 44.1%. The median overall survival (OS) was 14.0 (95% CI 10.7‒19.2) months, and the median progression-free survival (PFS) was 4.1 (95% CI 3.0‒5.9) months. The AUC ROCs for CAR, PLR, and NLR before nivolumab monotherapy for patients with progressive disease (PD) were 0.574 (95% CI, 0.461‒0.687), 0.528 (95% CI, 0.418‒0.637), and 0.511 (95% CI, 0.401‒0.620), respectively. The values for changes in CAR, PLR, and NLR were 0.766 (95% CI, 0.666‒0.865), 0.707 (95% CI, 0.607‒0.807), and 0.660 (95% CI 0.556‒0.765), respectively. The cutoff values for the treatment response were 3.0, 1.3, and 1.4 for CAR, PLR, and NLR, respectively. The PFS and OS were significantly longer when the treatment response values for changes in CAR, PLR, and NLR were below these cutoff values (CAR: OS, p < 0.0001 and PFS, p < 0.0001; PLR: OS, p = 0.0289 and PFS, p = 0.0302; and NLR: OS, p = 0.0077 and PFS, p = 0.0044). CONCLUSIONS: Measurement of the changes in CAR, PLR, and NLR could provide a simple, prompt, noninvasive method to evaluate response to nivolumab monotherapy. TRIAL REGISTRATION: This study is registered with number K2023006.

Nonplanar Nanographene: A Hydrocarbon Hole-Transporting Material That Competes with Triarylamines.

Hole-transporting materials (HTMs) are essential for optoelectronic devices, such as organic light-emitting diodes (OLEDs), dye-sensitized solar cells, and perovskite solar cells. Triarylamines have been employed as HTMs since they were introduced in 1987. However, heteroatoms or side chains embedded in the core skeleton of triarylamines can cause thermal and chemical stability problems. Herein, we report that hexabenzo[a,c,fg,j,l,op]tetracene (HBT), a small nonplanar nanographene, functions as a hydrocarbon HTM with hole transport properties that match those of triarylamine-based HTMs. X-ray structural analysis and theoretical calculations revealed effective multidirectional orbital interactions and transfer integrals for HBT. In-depth experimental and theoretical analyses revealed that the nonplanarity-inducing annulative π-extension can achieve not only a stable amorphous state in bulk films, but also a higher increase in the highest occupied molecular orbital level than conventional linear or cyclic π-extension. Furthermore, an in-house manufactured HBT-based OLED exhibited excellent performance, featuring superior curves for current density-voltage, external quantum efficiency-luminance, and lifetime compared to those of representative triarylamine-based OLEDs. A notable improvement in device lifetime was observed for the HBT-based OLED, highlighting the advantages of the hydrocarbon HTM. This study demonstrates the immense potential of small nonplanar nanographenes for optoelectronic device applications.

Robust Pixel Design Methodologies for a Vertical Avalanche Photodiode (VAPD)-Based CMOS Image Sensor.

We present robust pixel design methodologies for a vertical avalanche photodiode-based CMOS image sensor, taking account of three critical practical factors: (i) "guard-ring-free" pixel isolation layout, (ii) device characteristics "insensitive" to applied voltage and temperature, and (iii) stable operation subject to intense light exposure. The "guard-ring-free" pixel design is established by resolving the tradeoff relationship between electric field concentration and pixel isolation. The effectiveness of the optimization strategy is validated both by simulation and experiment. To realize insensitivity to voltage and temperature variations, a global feedback resistor is shown to effectively suppress variations in device characteristics such as photon detection efficiency and dark count rate. An in-pixel overflow transistor is also introduced to enhance the resistance to strong illumination. The robustness of the fabricated VAPD-CIS is verified by characterization of 122 different chips and through a high-temperature and intense-light-illumination operation test with 5 chips, conducted at 125 °C for 1000 h subject to 940 nm light exposure equivalent to 10 kLux.

Impact of the endoscopic surgical skill qualification system on the oncological safety of laparoscopic gastrectomy for gastric cancer: A single-center retrospective cohort study.

BACKGROUND: This study aimed to investigate the laparoscopic gastrectomy (LG) performance of non-Endoscopic Surgical Skill Qualification System (ESSQS)-qualified surgeons under the ESSQS-qualified surgeon guidance and compare oncological outcomes of gastric cancer to LG performed by the ESSQS-qualified surgeons. METHODS: This study enrolled 1,030 patients diagnosed with both clinical and pathological stage ≤ III gastric cancer and undergoing LG from January 2009 to June 2019. ESSQS-qualified surgeons served as the operator or the instructive assistant in all LG procedures involving them. A propensity score-matched analysis was used to retrospectively compare the long-term outcomes between the ESSQS-qualified and non-ESSQS-qualified surgeons. RESULTS: Each group included 315 pairs after propensity score matching. The 3-year recurrence-free survival rates were 84.4% and 81.7% in the non-ESSQS and ESSQS groups, respectively. The difference was 2.7% (95% confidence interval: - 3.20%-8.44%, P < 0.001), and the non-ESSQS group statistically demonstrated noninferiority as the lower 95% confidence limit was greater than the prespecified margin of -10%, indicating the achieved primary endpoint. No significant differences in 5-year recurrence-free survival (non-ESSQS: 78.5% vs. ESSQS: 77.4%, P = 0.627) and 5-year overall survival (non-ESSQS: 80.9% vs. ESSQS: 79.3%, P = 0.475) were found between the two groups. The oncological outcomes stratified according to the presence of pathological stage I, II, and III disease did not significantly differ between the two groups. CONCLUSIONS: LG performed by non-ESSQS-qualified surgeons achieved comparable oncological outcomes to the ESSQS-qualified surgeons, as long as ESSQS-qualified surgeons provided intraoperative instructions, in a high-volume center.

Standardized procedure for preventing late intestinal complications following minimally invasive total gastrectomy for gastric cancer: a single-center retrospective cohort study.

BACKGROUND: Although minimally invasive total gastrectomy for gastric cancer is commonly performed, reports regarding late complications are limited. We have made several improvements each time we experienced severe late complications since 2009. This study aimed to evaluate the clinical efficacy of these improved procedures in preventing late complications. METHODS: Between January 2009 and December 2019, 302 patients who underwent laparoscopic or robotic total gastrectomy for gastric cancer were enrolled. The patients were divided into two groups: Period-I (2009-2013, before established standardization of procedure, 166 patients) and Period-II (2014-2019, after established standardization of procedure, 136 patients). The standardized procedure comprised four major steps, including closure of the mesentery defects and diaphragm crus, circumferential fixation of the anastomotic site into the diaphragm, and linearization around the anastomotic site of esophagojejunostomy. The incidence of late complications was retrospectively compared between the two groups. RESULTS: Late overall complications that occurred over 30 days after surgery were observed in 19 (6.3%) patients. In all, 14 of 24 (58.3%) patients admitted due to late intestinal complications eventually required reoperation for treatment. The most frequent complication was nonstenotic outlet obstruction of the distal jejunal limb. The incidence of late overall complications was significantly lower in Period-II than in Period-I (2.9 vs 9.0%, p = 0.030). Intestinal complications were reduced considerably in Period-II. The 3-year cumulative incidence rate of late overall complications was significantly lower in Period-II than in Period-I (0.03 vs 0.10, p = 0.035). Period-I as the only independent risk factor for the development of late intestinal complications. CONCLUSION: Late complications after laparoscopic total gastrectomy sometimes occurred, and more than half of the patients with intestinal complications required reoperation. Our standardized procedure was associated with a lower risk of late intestinal complications after minimally invasive total gastrectomy followed by intracorporeal esophagojejunostomy using linear staplers in a cohort of patients with gastric cancer.

Expansion of omega-3 polyunsaturated fatty acid metabolism of the oleaginous diatom Fistulifera solaris by genetic engineering.

Omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA; C20:5n-3) and docosahexaenoic acid (DHA; C22:6n-3) are widely used as additives in fish feed in the aquaculture sector. To date, the supply of omega-3 PUFAs have heavily depended upon fish oil production. As the need for omega-3 PUFAs supply for the growing population increases, a more sustainable approach is required to keep up with the demand. The oleaginous diatom Fistulifera solaris is known to synthesize EPA with the highest level among autotrophically cultured microalgae, however, this species does not accumulate significant amounts of DHA, which, in some cases, is required in aquaculture rather than EPA. This is likely due to the lack of expression of essential enzymes namely Δ5 elongase (Δ5ELO) and Δ4 desaturase. In this study, we identified endogenous Δ5ELO genes in F. solaris and introduced recombinant expression cassettes harboring Δ5ELO into F. solaris through bacterial conjugation. As a result, it managed to induce the synthesis of docosapentaenoic acid (DPA; C22:5n-3), a direct precursor of DHA. This study paves the way for expanding our understanding of the omega-3 PUFAs pathway using endogenous genes in the oleaginous diatom.

Anthocyanin glucosylation mediated by a glycoside hydrolase family 3 protein in purple carrot.

Purple carrot accumulates anthocyanins modified with galactose, xylose, glucose, and sinapic acid. Most of the genes associated with anthocyanin biosynthesis have been identified, except for the glucosyltransferase genes involved in the step before the acylation in purple carrot. Anthocyanins are commonly glycosylated in reactions catalyzed by UDP-sugar-dependent glycosyltransferases (UGTs). Although many studies have been conducted on UGTs, the glucosylation of carrot anthocyanins remains unknown. Acyl-glucose-dependent glucosyltransferase activity modifying cyanidin 3-xylosylgalactoside was detected in the crude protein extract prepared from purple carrot cultured cells. In addition, the corresponding enzyme was purified. The cDNA encoding this glucosyltransferase was isolated based on the partial amino acid sequence of the purified protein. The recombinant protein produced in Nicotiana benthamiana leaves via agroinfiltration exhibited anthocyanin glucosyltransferase activity. This glucosyltransferase belongs to the glycoside hydrolase family 3 (GH3). The expression pattern of the gene encoding this GH3-type anthocyanin glucosyltransferase was consistent with anthocyanin accumulation in carrot tissues and cultured cells.

Laparoscopic repair of concurrent direct and indirect inguinal, femoral, and obturator hernias on the same side: A case report.

Concurrent direct and indirect inguinal, femoral, and obturator hernias are rare. This case report describes a rare case treated using the laparoscopic approach. A 68-year-old female patient presented with a moving left inguinal lump and pain. Physical examination and abdominal computed tomography scan revealed the coexistence of a left inguinal hernia or Nuck canal hydrocele and a left femoral hernia. The patient underwent laparoscopic transabdominal preperitoneal repair, and all four orifices were covered with one mesh. The patient was discharged on the second postoperative day without any complications. The concurrent presence of four hernias on the same side is rare and has not been previously reported. The laparoscopic approach is useful in such cases because it allows visualization of multiple hernia orifices from the intra-abdominal cavity.

A materials informatics driven fine-tuning of triazine-based electron-transport layer for organic light-emitting devices.

Materials informatics in the development of organic light-emitting diode (OLED) related materials have been performed and exhibited the effectiveness for finding promising compounds with a desired property. However, the molecular structure optimization of the promising compounds through the conventional approach, namely the fine-tuning of molecules, still involves a significant amount of trial and error. This is because it is challenging to endow a single molecule with all the properties required for practical applications. The present work focused on fine-tuning triazine-based electron-transport materials using machine learning (ML) techniques. The prediction models based on localized datasets containing only triazine derivatives showed high prediction accuracy. The descriptors from density functional theory calculations enhanced the prediction of the glass transition temperature. The proposed multistep virtual screening approach extracted the promising triazine derivatives with the coexistence of higher electron mobility and glass transition temperature. Nine selected triazine compounds from 3,670,000 of the initial search space were synthesized and used as the electron transport layer for practical OLED devices. Their observed properties matched the predicted properties, and they enhanced the current efficiency and lifetime of the device. This paper provides a successful model for the ML assisted fine-tuning that effectively accelerates the development of practical materials.

Safety and feasibility of minimally invasive gastrectomy following preoperative chemotherapy for highly advanced gastric cancer.

BACKGROUND: This study aimed to determine the safety and feasibility of minimally invasive gastrectomy in patients who underwent preoperative chemotherapy for highly advanced gastric cancer. METHODS: Preoperative chemotherapy was indicated for patients with advanced large tumors (≥ cT3 and ≥ 5 cm) and/or bulky node metastasis (≥ 3 cm × 1 or ≥ 1.5 cm × 2). Between January 2009 and March 2022, 150 patients underwent preoperative chemotherapy followed by gastrectomy with R0 resection, including conversion surgery (robotic, 62; laparoscopic, 88). The outcomes of these patients were retrospectively examined. RESULTS: Among them, 41 and 47 patients had stage IV disease and underwent splenectomy, respectively. Regarding operative outcomes, operative time was 475 min, blood loss was 72 g, morbidity (grade ≥ 3a) rate was 12%, local complication rate was 10.7%, and postoperative hospital stay was 14 days (Interquartile range: 11-18 days). Fifty patients (33.3%) achieved grade ≥ 2 histological responses. Regarding resection types, total/proximal gastrectomy plus splenectomy (29.8%) was associated with significantly higher morbidity than other types (distal gastrectomy, 3.2%; total/proximal gastrectomy, 4.9%; P < 0.001). Specifically, among splenectomy cases, the rate of postoperative complications associated with the laparoscopic approach was significantly higher than that associated with the robotic approach (40.0% vs. 0%, P = 0.009). In the multivariate analysis, splenectomy was an independent risk factor for postoperative complications [odds ratio, 8.574; 95% confidence interval (CI), 2.584-28.443; P < 0.001]. CONCLUSIONS: Minimally invasive gastrectomy following preoperative chemotherapy was feasible and safe for patients with highly advanced gastric cancer. Robotic gastrectomy may improve surgical safety, particularly in the case of total/proximal gastrectomy combined with splenectomy.

First clinical experiences of robotic gastrectomy for gastric cancer using the hinotori™ surgical robot system.

BACKGROUND: Although the da Vinci™ Surgical System is the most predominantly used surgical robot worldwide, other surgical robots are being developed. The Japanese surgical robot hinotori™ Surgical Robot System was launched and approved for clinical use in Japan in November 2022. We performed the first robotic gastrectomy for gastric cancer using hinotori in the world. Here, we report our initial experience and evaluation of the feasibility and safety of robotic gastrectomy for gastric cancer using hinotori. METHODS: A single-institution retrospective study was conducted. Between November 2022 and October 2023, 24 patients with gastric cancer underwent robotic gastrectomy with hinotori. Five ports, including one for an assistant, were placed in the upper abdomen, and gastric resection with standard lymphadenectomy and intracorporeal reconstruction were performed. The primary endpoint was the postoperative complication rate within 30 days after surgery. The secondary outcomes were surgical outcomes, including intraoperative adverse events, operative time, blood loss, and the number of dissected nodes. RESULTS: Of the 24 patients, 16 (66.7%) were male. The median age and body mass index were 73.5 years and 22.9 kg/m2, respectively. Twenty-three patients (95.8%) had tumors in the middle to lower stomach. Sixteen (66.7%) and seven (29.2%) patients had clinical stage I and II diseases, respectively. Twenty-three (95.8%) patients underwent distal gastrectomy. No patient had postoperative complications of Clavien-Dindo classification IIIa or higher, whereas two (8.3%) had the grade II complications (enteritis and pneumonia). No intraoperative adverse events, including conversion to other approaches, were observed. All patients received R0 resection. The median operative and console times were 400 and 305 min, respectively. The median blood loss was 14.5 mL, and the number of lymph nodes dissected was 51.5. CONCLUSIONS: This study found that robotic gastrectomy with standard lymphadenectomy for gastric cancer using hinotori can be safely performed.

Line image sensor-based colony fingerprinting system for rapid pathogenic bacteria identification.

The rapid identification of pathogenic bacteria is crucial across various industries, including food or beverage manufacturing. Bacterial microcolony image-based classification has emerged as a promising approach to expedite identification, automate inspections, and reduce costs. However, conventional imaging methods have significant practical limitations, namely low throughput caused by the limited imaging range and slow imaging speed. To address these challenges, we developed an imaging system based on a line image sensor for rapid and wide-field imaging compared to existing colony imaging methods. This system can image a standard Petri dish (92 mm in diameter) completely within 22 s, successfully acquiring bacterial microcolony images. This process yielded a set of discrimination parameters termed as colony fingerprints, which were employed for machine learning. We demonstrated the performance of our system by identifying Staphylococcus aureus in food products using a machine learning model trained on a colony fingerprint dataset of 15 species from 9 genera, including foodborne pathogens. While conventional mass spectrometry-based methods require 24 h of incubation, our colony fingerprinting approach achieved 96% accuracy in just 10 h of incubation. Line image sensor offer high imaging speeds and scalability, allowing for swift and straightforward microbiological testing, eliminating the need for specialized expertise and overcoming the limitations of conventional methods. This innovation marks a transformative shift in industrial applications.

The safe implementation of peripherally inserted central catheters by nurse practitioners for patients with gastroenterological diseases in Japan: a single-center retrospective study.

PURPOSE: As a safe and reliable alternative to central venous catheters (CVCs), peripherally inserted central catheters (PICCs) are commonly used in clinical practice. However, the insertion of PICCs by nurse practitioners (NPs), especially in Japan, has not been reported extensively. Thus, we investigated the safety and efficiency of PICC insertions by NPs. METHODS: The participants were 1322 patients who underwent PICC insertion by NPs at Fujita Health University Hospital (FNPs). The basilic vein in the brachium was the preferred vein for insertion; the brachial vein was the alternative. Patients were monitored from the time of PICC insertion until its removal. Ultrasonography-guided puncture was used for all catheter insertions, and the catheter tip was replaced into the superior vena cava under fluoroscopic imaging with maximal sterile barrier precautions. The outcomes of the PICC insertions by the FNPs were evaluated retrospectively. RESULTS: Overall, 23 FNPs inserted a collective total of 1322 PICCs, which remained in place for a collective total of 23,619 catheter days. The rate of successful PICC insertion was 99% (1310 patients). The median time taken for PICC insertion was 12 min (interquartile range, 10-15 min). Intraoperative complications occurred in two patients (0.2%). The confirmed incidence of central line-associated bloodstream infection was 3.4% (45 patients), and these infections occurred on 1.9 per 1000 catheter days. The median duration of PICC placement was 15 days (range, 10-23 days). CONCLUSION: PICC insertion by NPs is safe and a potential alternative to CVC insertion by surgeons.

Laparoscopic gastrectomy for remnant gastric cancer: A single-center retrospective study.

BACKGROUND: Laparoscopic gastrectomy (LG) for remnant gastric cancer (RGC) remains controversial because of its rarity and heterogeneity of clinical characteristics. Based on our experience, we posited that our established methodology in LG could be applied to the laparoscopic procedure for RGC surgery and introduced LG for RGC at our institution in 2004. METHODS: This study enrolled 46 patients who underwent LG for RGC between January 2004 and December 2017. Data were obtained through a review of our prospectively maintained database. Laparoscopic total gastrectomy (LTG) was the standard surgical procedure for RGC. Laparoscopic subtotal gastrectomy (LsTG) was performed as an alternative procedure for patients with RGC located near the anastomotic site after primary gastrectomy. The technical and oncological feasibility and safety of LG for RGC were evaluated. RESULTS: LTG for RGC was performed on 36 patients. LsTG for RGC was performed on 10 patients. All patients completed LG procedure and succeeded R0 resection. Complications of Clavien-Dindo classification grade ≥ IIIa occurred in 4 (8.7%) patients. The retrospective video reviews showed that the time for adhesiotomy around the suprapancreatic area and the lesser curvature of the remnant stomach was significantly shorter in the primary-benign group than in the primary-malignant group. With the median follow-up period of 40 months, the 3-year recurrence-free survival and 3-year overall survival rates were 72.3% and 80.2%, respectively. CONCLUSION: LG for RGC represents a safe and feasible surgical option with favorable short-term and long-term outcomes in patients with RGC.

Robotic esophagectomy with outermost layer-oriented dissection for esophageal cancer: technical aspects and a retrospective review of a single-institution database.

BACKGROUND: Systematic lymph node dissection in patients with gastric cancer could be sufficiently and reproducibly achieved along the outermost layer of the autonomic nerves and similar concept has been extensively used for robotic esophagectomy (RE) since 2018. This study aimed to determine the surgical and oncological safety of RE using the outermost layer-oriented approach for esophageal cancer (EC). METHODS: Sixty-six patients who underwent RE with total mediastinal lymphadenectomy for primary EC between April 2018 and December 2021 were retrospectively reviewed. All underwent the outermost layer-oriented approach with intraoperative nerve monitoring (IONM). Postoperative complications within 30 days were analyzed. RESULTS: Among the patients, 51 (77.3%) were male. The median age was 64 years, and the body mass index was 21.8 kg/m2. Furthermore, 58 (87.9%) patients had squamous cell carcinoma and eight (12.1%) patients had adenocarcinoma. Clinical stages I, II, and III were seen in 23 (34.8%), 23 (34.8%), and 16 (24.2%) patients, respectively. Thirty-four (51.5%) patients received preoperative treatment. No patient shifted to conventional thoracoscopic or open procedure intraoperatively. The median operative time was 716 min with 119 mL of blood loss. Additionally, 64 (97%) patients underwent R0 resection. The morbidity rates based on Clavien-Dindo grades ≥ II and ≥ IIIa were 30.3% and 10.6%, respectively, within 30 postoperative days. None died within 90 days postoperatively. Three (4.5%) patients exhibited recurrent laryngeal nerve (RLN) palsy (CD grade ≥ II). The sensitivity and specificity of IONM for RLN palsy were 50% and 98.3% at the right RLN and 33.3% and 98.0% at the left RLN, respectively. CONCLUSION: RE with the outermost layer-oriented approach can provide safe short-term outcomes.

Detection of Transcription Factors Related to Axillary Bud Development after Exposure to Cold Conditions in Hexaploid Chrysanthemum morifolium Using Arabidopsis Information.

Chrysanthemum is one of the most commercially used ornamental flowering plants in the world. As chrysanthemum is self-incompatible, the propagation of identical varieties is carried out through cuttings rather than through seed. Axillary bud development can be controlled by changing the temperature; for instance, axillary bud development in some varieties is suppressed at high temperatures. In this study, we focused on the simultaneous axillary bud growth from multiple lines of chrysanthemum upon changing conditions from low to normal temperature. Transcriptome analysis was conducted on the Chrysanthemum morifolium cultivar 'Jinba' to identify the important genes for axillary bud development seen when moved from low-temperature treatment to normal cultivation temperature. We performed RNA-Seq analysis on plants after cold conditions in two-day time-course experiments. Under these settings, we constructed a transcriptome of 415,923 C. morifolium and extracted 7357 differentially expressed genes. Our understanding of Arabidopsis axillary meristem development and growth showed that at least 101 genes in our dataset were homologous to transcription factors involved in the biological process. In addition, six genes exhibited statistically significant variations in expression throughout conditions. We hypothesized that these genes were involved in the formation of axillary buds in C. morifolium after cold conditions.

Extracellular oil production by Rhodotorula paludigena BS15 for biorefinery without complex downstream processes.

To realize biomass refinery without complex downstream processes, we extensively screened for microbial strains that efficiently produce extracellular oil from sugars. Rhodotorula paludigena (formerly Rhodosporidium paludigenum) BS15 was found to efficiently produce polyol esters of fatty acids (PEFAs), which mainly comprised of 3-acetoxypalmitic acid and partially acetylated mannitol/arabinitol. To evaluate the performance of this strain, fed-batch fermentation was demonstrated on a flask scale, and 110 g/L PEFA and 103 g/L dry cells were produced in 12 days. To the best of our knowledge, the strain BS15 exhibited the highest PEFA titer (g/L) ever to be reported so far. Because the PEFA precipitated at the bottom of the culture broth, it could be easily recovered by simply discarding the upper phase. Various carbon sources can be utilized for cell growth and/or PEFA production, which signifies the potential for converting diverse biomass sources. Two different types of next-generation sequencers, Illumina HiSeq and Oxford Nanopore PromethION, were used to analyze the whole-genome sequence of the strain BS15. The integrative data analysis generated a high-quality and reliable reference genome for PEFA-producing R. paludigena. The 22.5-M base genome sequence and the estimated genes were registered in Genbank (accession numbers BQKY01000001-BQKY01000019). KEY POINTS: • R. paludigena BS15 was isolated after an extensive screening of extracellular oil producers from natural sources. • Fed-batch fermentation of R. paludigena BS15 yielded 110 g/L of PEFA, which is the highest titer ever reported to date. • Combined analysis using Illumina and Oxford Nanopore sequencers produced the near-complete genome sequence.