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.

Salvage reconstruction of the esophagus using the left colon with microscopic supercharge following failed ileocolic reconstruction: A case report.

INTRODUCTION: Ileocolic interposition is often used for the reconstruction of patients with esophageal cancer with a history of gastrectomy. However, graft failure due to conduit necrosis has been reported in 0-5 % of patients. Salvage reconstruction surgery for this situation is considered challenging, and only a few cases of successful salvage operations following failure of ileocolic interposition have been reported. PRESENTATION OF CASE: A 70s year-old male patient with a history of distal and total gastrectomy underwent subtotal esophagectomy for esophageal cancer. Reconstruction using a pedicled ileocolic interposition was performed; however, the ileocolic graft failed. After recovery of the nutritional status, salvage reconstruction was planned. Due to a history of Roux-en-Y reconstruction for gastric cancer, jejunal reconstruction was not considered feasible. Therefore, salvage reconstruction was performed using left colon interposition with microscopic supercharge and superdrainage anastomosis. The graft was pedicled by the left colic artery and the inferior mesenteric vein, and microscopic anastomosis was performed between the intrathoracic and middle colic vessels. The patient recovered without major complications and retained the ability to consume normal food. DISCUSSION: Microscopic supercharge and superdrainage vascular anastomosis have been reported to ensure augmented blood flow. This is the first case report of successful salvage reconstruction using the left colon interposition technique following failure of ileocolic interposition for esophageal cancer. CONCLUSION: We report a case of salvage reconstruction using left colon interposition with microscopic supercharge and superdrainage anastomosis following failure of ileocolic reconstruction for esophageal cancer.

The cadmium tolerance and bioaccumulation mechanism of Tetratostichococcus sp. P1: insight from transcriptomics analysis.

Cadmium (Cd) has become a severe issue in relatively low concentration and attracts expert attention due to its toxicity, accumulation, and biomagnification in living organisms. Cd does not have a biological role and causes serious health issues. Therefore, Cd pollutants should be reduced and removed from the environment. Microalgae have great potential for Cd absorption for waste treatment since they are more environmentally friendly than existing treatment methods and have strong metal sorption selectivity. This study evaluated the tolerance and ability of the microalga Tetratostichococcus sp. P1 to remove Cd ions under acidic conditions and reveal mechanisms based on transcriptomics analysis. The results showed that Tetratostichococcus sp. P1 had a high Cd tolerance that survived under the presence of Cd up to 100 µM, and IC50, the half-maximal inhibitory concentration value, was 57.0 µM, calculated from the change in growth rate based on the chlorophyll content. Long-term Cd exposure affected the algal morphology and photosynthetic pigments of the alga. Tetratostichococcus sp. P1 removed Cd with a maximum uptake of 1.55 mg g-1 dry weight. Transcriptomic analysis revealed the upregulation of the expression of genes related to metal binding, such as metallothionein. Group A, Group B transporters and glutathione, were also found upregulated. While the downregulation of the genes were related to photosynthesis, mitochondria electron transport, ABC-2 transporter, polysaccharide metabolic process, and cell division. This research is the first study on heavy metal bioremediation using Tetratostichococcus sp. P1 and provides a new potential microalga strain for heavy metal removal in wastewater.[Figure: see text]Abbreviations:BP: Biological process; bZIP: Basic Leucine Zipper; CC: Cellular component; ccc1: Ca (II)-sensitive cross complementary 1; Cd: Cadmium; CDF: Cation diffusion facilitator; Chl: Chlorophyll; CTR: Cu TRansporter families; DAGs: Directed acyclic graphs; DEGs: Differentially expressed genes; DVR: Divinyl chlorophyllide, an 8-vinyl-reductase; FPN: FerroportinN; FTIR: Fourier transform infrared; FTR: Fe TRansporter; GO: Gene Ontology; IC50: Growth half maximal inhibitory concentration; ICP: Inductively coupled plasma; MF: molecular function; NRAMPs: Natural resistance-associated aacrophage proteins; OD: Optical density; RPKM: Reads Per Kilobase of Exon Per Million Reads Mapped; VIT1: Vacuolar iron transporter 1 families; ZIPs: Zrt-, Irt-like proteins.

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.

Genetic Modification of Aurantiochytrium sp. 18W-13a for Enhancement of Proteolytic Activity by Heterologous Expression of Extracellular Proteases.

A marine thraustochytrid, Aurantiochytrium, is a promising organism to produce docosahexaenoic acid (DHA) and squalene. Utilization of inexpensive substances such as proteins in wastes and by-products from the food industry for cultivation is a considerable option to reduce production cost; however, the proteolytic ability of Aurantiochytrium spp. is low compared to taxonomically close Shizochytrium aggregatum. We previously identified extracellular protease (extracellular protease 1, EP1) in S. aggregatum ATCC 28209 from the supernatant of the culture and found that a similar protease gene (EP2) was located downstream of the EP1 gene. In the present study, we created the transformants expressing SaEP1 and/or SaEP2 to enhance the proteolytic ability of Aurantiochytrium sp. 18W-13a strain and cultivated them in the medium containing casein as a test protein substrate. Through SDS-PAGE analysis, we confirmed that casein in the supernatant was more efficiently degraded by the transformants than the wild type, suggesting that the expressed protease(s) were properly expressed and excreted. After 4-day cultivation in the casein medium, the value of optical density at 660 nm and the cell number in the culture of the transformant that expressed both SaEP1 and SaEP2 (designated as EP12 strain) showed 1.48- and 1.38-fold higher than those of wild type, respectively. The DHA and squalene yield of the EP12 strain were respectively 158.3 and 0.23 mg L-1, and these values were 1.42- and 2.01-fold higher than those of wild type, respectively, suggesting that the EP12 created in the present study is a favorable strain for the cultivation using protein-containing medium.

Chromosome-scale genome assembly of an acidophilic microalga Tetratostichococcus sp. P1 isolated from a tropical peatland in Malaysia.

Tetratostichococcus sp. P1 shows an acidophilic phenotype which could allow mass-scale monoculture of this green microalga without severe contamination by environmental microorganisms. In this study, we report a chromosome-scale genome assembly for Tetratostichococcus sp. P1.

[A Long-Term Survival Case of Multiple Liver Metastases from Colon Cancer Treated with Intensive Multimodality Therapy].

A 60-year-old man diagnosed with sigmoid colon cancer was admitted to our hospital. A CT scan revealed multiple liver metastases. The patient was administered 15 courses of FOLFIRI chemotherapy and 15 courses of FOLFIRI plus Cmab chemotherapy. After this treatment, multiple liver metastases disappeared, and laparoscopic resection of the sigmoid colon was performed. Two months later, a recurrent lesion was found in the liver segment(S1), and 5 courses of FOLFIRI plus Cmab chemotherapy were performed. Although the CEA level decreased, the tumor size remained unchanged. Therefore, partial resection of the liver was performed, followed by 18 courses of FOLFIRI chemotherapy. After that, the patient was followed for a year without chemotherapy. However, about 1 year later, recurrence was observed in liver segments S5 and S6. A right lobectomy was performed for these 2 lesions, and then 16 more courses of FOLFIRI chemotherapy were performed. The chemotherapy was discontinued, and the patient was then followed up as an outpatient without chemotherapy; there has been no recurrence.

Characterization of oil body-associated proteins obtained from oil bodies with different sizes in oleaginous diatom Fistulifera solaris.

Oil body-associated proteins from the oleaginous diatom Fistulifera solaris were identified by proteomic analysis of oil bodies of various sizes (small, middle, and large) by time-dependent culturing upon nutrient-starvation at 36, 96 and 168 h. This diatom strain has the capability to accumulate neutral lipids and triacylglycerol. Liquid chromatography-tandem mass spectrometry analysis revealed 662 proteins in all oil body sizes. Among these, 132 proteins were predicted to be localized to the endoplasmic reticulum. Seventeen proteins that exhibited a positive correlation with gene expression and the oil body size were selected as novel candidates for oil body-associated proteins. Among the 17 protein candidates, two proteins encoded by fso:g8246 and fso:g10200 were confirmed to be localized on the surface of the oil body and endoplasmic reticulum. A protein encoded by fso:g2514, which is involved in sterol biosynthesis, was also identified. This protein was likely to localize to mitochondria; however, inhibitor assays suggested that it might play a role in lipid degradation. Our work provides new insights into the proteomics of microalgae and provides a valuable strategy for boosting lipid productivity in microalgae.

Highly Efficient Genetic Transformation Methods for the Marine Oleaginous Diatom Fistulifera solaris.

The oleaginous diatom Fistulifera solaris is a promising producer of biofuel owing to the high content of the lipids. A genetic transformation technique by microparticle bombardment for this diatom was already established. However, the transformation efficiency was significantly lower than those of other diatoms. Devoting efforts to advance the genetic modifications of this diatom is crucial to unlock its full potential. In this study, we optimized the microparticle bombardment protocol, and newly established a multi-pulse electroporation protocol for this diatom. The nutrient-rich medium in the pre-culture stage played an essential role to increase the transformation efficiency of the bombardment method. On the other hand, use of the nutrient-rich medium in the electroporation experiments resulted in decreasing the efficiency because excess nutrient salts could hamper to establish the best conductivity condition. Adjustments on the number and voltage of the poring pulses were also critical to obtain the best balance between cell viability and efficient pore formation. Under the optimized conditions, the transformation efficiencies of microparticle bombardment and multi-pulse electroporation were 111 and 82 per 108 cells, respectively (37 and 27 times higher than the conventional bombardment method). With the aid of the optimized protocol, we successfully developed the transformant clone over-expressing the endogenous fat storage-inducing transmembrane protein (FIT)-like protein, which was previously found in the genome of the oleaginous diatom F. solaris and the oleaginous eustigmatophyte Nannochloropsis gaditana. This study provides powerful techniques to investigate and further enhance the metabolic functions of F. solaris by genetic engineering.

Chromosome-Scale Genome Assembly of the Marine Oleaginous Diatom Fistulifera solaris.

Microalgae including diatoms are of interest for environmentally friendly manufacturing such as production of biofuels, chemicals, and materials. The highly oil-accumulating marine diatom Fistulifera solaris has been studied as a promising host organism to be employed for these applications. Recently reported large-scale genetic engineering based on episomal vectors for diatoms could be useful to further enhance the potential of F. solaris, whereas we need to understand more the mode-of-action of diatom centromeres to rationally design the episomal vectors for stable extrachromosomal maintenance. Our previous genome analysis with pyrosequencing (short read sequencing) had generated the fragmented scaffolds which were not useful to predict centromeres on each chromosome. Here, we report the almost complete chromosomal structure of the genome of F. solaris using a long-read nanopore sequencing platform MinION. From just one single run using a MinION flow-cell, the chromosome-scale assembly with telomere-to-telomere resolution was achieved for 41 out of 44 chromosomes. Putative centromere regions were predicted from the 16 chromosomes, and we discovered putative consensus motifs in the predicted centromeres. Similar motif search had been performed in model diatoms, but no consensus motif was found. Therefore, this is the first study to successfully estimate consensus motifs in diatom centromeres. The chromosome-scale assembly also suggests the potential existence of multi-copy mini-chromosomes and tandemly repeated lipogenesis genes related to the oleaginous phenotype of F. solaris. Findings of this study are useful to understand and further engineer the oleaginous phenotype of F. solaris.

Molecular Insights into Lipoxygenases in Diatoms Based on Structure Prediction: a Pioneering Study on Lipoxygenases Found in Pseudo-nitzschia arenysensis and Fragilariopsis cylindrus.

Diatoms produce a variety of oxylipins which are oxygenated polyunsaturated fatty acids and are involved in chemical defense and intercellular communication, among other roles. Although the chemistry of diatom oxylipins has long been studied, the enzymes involved in their production, in particular lipoxygenase (LOX), which catalyzes the initial reaction of the synthesis, have not been discovered in diatom genomes. Recently, diatom LOXs were found in two species, Pseudo-nitzschia arenysensis (PaLOX) and Fragilariopsis cylindrus (FcLOX); however, the enzymology of these LOXs is largely unknown. In this review article, we discuss the potential functions of the diatom LOXs based on previously reported structures of LOXs derived from various organisms other than diatoms. Since the structures of PaLOX and FcLOX have not yet been solved, we discussed their functions, such as regio- and stereospecificities, on the basis of their structures predicted using a computational tool based on deep learning technology. Both diatom LOXs were predicted to conserve common core domains with relatively wide substrate-binding pockets. The stereo-determinant residues in PaLOX and FcLOX suggest S specificity. We assume that the highly conserved common core domain can be a clue to reveal unidentified lox genes from the accumulated diatom genome information with the aid of high-throughput structure prediction tools and structure-based alignment tools in the near future.

Giant ovarian tumor with colorectal cancer: suggestion concerning the need for colonoscopy screening in cases with large ovarian tumor-a report of three cases.

BACKGROUND: Patients with giant ovarian tumor often have severe symptoms, such as abdominal distention, and the tumor tends to grow rapidly; therefore, sufficient preoperative assessments are difficult to perform. It is not always easy to differentiate between primary and metastatic ovarian cancer, especially when the ovarian tumor is huge, since a precise diagnosis of ovarian tumor depends on the histopathological findings of the excised specimen. Although metastatic ovarian tumors account for over 20% of all malignant ovarian tumors, preoperative colonoscopy is not considered a routine examination before surgery for giant ovarian tumor. CASE PRESENTATION: We herein report 3 cases of giant (> 25 cm) ovarian tumor with colorectal cancer. All three patients visited the clinic with progressing abdominal distention, and were referred with primary ovarian malignancy. Case 1: Rectal tumor was suspected by a digital examination at the outpatient clinic, and rectal cancer was diagnosed preoperatively by colonoscopy. Computed tomography revealed a single-nodule liver tumor. Ovariectomy, rectal resection, and partial hepatectomy were performed. A histological examination revealed both primary mucinous ovarian carcinoma and rectal carcinoma with liver metastasis. Case 2: Initially, the ovarian tumor was diagnosed as primary carcinoma based on the histological findings of an incision biopsy at the previous hospital. Chemotherapy for ovarian cancer was administered without remission, and subsequently, the patient was referred to our hospital. Since the CEA level was high (142 ng/ml), colonoscopy was performed and cecal cancer was diagnosed. Ovariectomy and right colectomy were performed, and the ovarian tumor was histologically diagnosed as metastatic adenocarcinoma. Case 3: Initial ovariectomy was performed, and rectal cancer was suspected at intra-operative surveillance. Colonoscopy was performed after surgery, and rectal cancer was diagnosed. The ovarian tumor was diagnosed as metastatic adenocarcinoma. After six cycles of FOLFOX, rectal resection was performed. CONCLUSION: Regrettably, two of three cases in the current series were not diagnosed with colorectal cancer at the start of treatment. This experience suggests that screening colonoscopy should be considered before treatment for every case of giant ovarian tumor.

Single-cell genotyping of phytoplankton from ocean water by gel-based cell manipulation.

A comprehensive understanding of phytoplankton diversity is valuable for assessing an environment of interest as phytoplankton are primary producers in various aquatic food webs. Microscopic analyses are useful for diversity assessment based on characteristic cell morphologies. However, phylogenetic classification based solely on morphology requires an extremely high level of expertise. The genetic approach is another option for evaluating phytoplankton diversity; however, it cannot reveal morphological information. To integrate these two approaches, an original technology was developed, that is referred to as microcavity array (MCA)/gel-based cell manipulation (GCM). The model experiments using monocultures of various phytoplankton indicated that the efficiencies of cell recovery and isolation of single-cell plankton were dependent on cell size and shape. Cells with widths larger than the cavity width showed high level of recovery and isolation efficiency. Subsequent whole-genome amplification (WGA) of isolated single-cell plankton provided a sufficient amount (≈30 µg) of WGA products for genetic analyses. Furthermore, it is showed that MCA/GCM could directly analyze phytoplankton in ocean water obtained from Suruga Bay, Japan, without any cumbersome pretreatment. These results indicate that MCA/GCM technology is a powerful tool for elucidating the phytoplankton diversity in marine environment.

Effects of fatty acid synthase-inhibitors on polyunsaturated fatty acid production in marine diatom Fistulifera solaris JPCC DA0580.

Omega-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA, C20:5ω-3) are essential for human health and fish growth especially in aquaculture sector. However, with the growing of aquaculture, the demand of PUFA supply also has been increasing. Fistulifera solaris, a marine diatom, is known for its ability to accumulate 65% of lipid content per dry cell weight, and can produce the high content of EPA. Thus, this diatom shows a great potential to be a feedstock of omega-3 PUFAs for fish feeds. In this study, in order to further understand and enhance the metabolism of PUFA biosynthesis in the diatom, the impacts of ketoacyl-ACP synthase (KAS) and ketoacyl-CoA synthase (KCS) inhibition on the PUFA production were analyzed by adding the specific inhibitors. KAS and KCS enzymes both play a role in the fatty acid elongation. As a result, the inhibition of KAS showed an increase in EPA content without arresting the cell growth. On the other hand, inhibition of KCS did not show a significant impact on the PUFA content in F. solaris. Our finding suggests that the specific suppression of KAS function can be a promising way to enhance the omega-3 PUFA production in F. solaris.

Practical Asymmetric Synthesis of Chiral Sulfoximines via Sulfur-Selective Alkylation.

Chiral sulfoximines have recently been considered as promising bioisosteres in medicinal chemistry. However, methods for preparing chiral sulfoximines in a stereoselective manner are underdeveloped. Herein, we demonstrate an asymmetric synthesis of chiral sulfoximines through a stereospecific S-alkylation of readily accessible chiral sulfinamides under practical conditions. A key to establishing the practical conditions was the identification of the intermediate structure in our previously reported S-alkylation by X-ray crystallographic analysis.

Signaling probe design for amplification-free detection of bacterial genes using DNA microarray.

DNA microarrays are useful to detect microorganisms for various purposes including clinical testing and food safety. However, conventional DNA microarrays need complicated operations such as amplification, fluorescence labeling, and washing steps. To address this issue, we previously developed the signaling probe-based DNA microarray system that can eliminate these steps, and demonstrated a direct detection of bacterial genes. Nonetheless, this system requires well-designed probe sets due to the fluorescence resonance energy transfer (FRET)-based mode of action. Up to date, the probe design was highly dependent on the trial-and-error processes. In this study, we propose a strategy to rationally design the sequences of signaling probes based on the thermodynamic analysis. This analysis aided to improve the probe performance approximately 2.8 times, without experiments, by suppressing the secondary structure formation of the probes. We successfully demonstrated the specific and amplification-free detection of 5S rRNA from total RNA extracted from Escherichia coli within 30 min.

Lensless imaging-based discrimination between tumour cells and blood cells towards circulating tumour cell cultivation.

Circulating tumour cells (CTCs) are recognized as important markers for cancer research. Nonetheless, the extreme rarity of CTCs in blood samples limits their availability for multiple characterization. The cultivation of CTCs is still technically challenging due to the lack of information of CTC proliferation, and it is difficult for conventional microscopy to monitor CTC cultivation owing to low throughput. In addition, for precise monitoring, CTCs need to be distinguished from the blood cells which co-exist with CTCs. Lensless imaging is an emerging technique to visualize micro-objects over a wide field of view, and has been applied for various cytometry analyses including blood tests. However, discrimination between tumour cells and blood cells was not well studied. In this study, we evaluated the potential of the lensless imaging system as a tool for monitoring CTC cultivation. Cell division of model tumour cells was examined using the lensless imaging system composed of a simple setup. Subsequently, we confirmed that tumour cells, JM cells (model lymphocytes), and erythrocytes exhibited cell line-specific patterns on the lensless images. After several discriminative parameters were extracted, discrimination between the tumour cells and other blood cells was demonstrated based on linear discriminant analysis. We also combined the highly efficient CTC recovery device, termed microcavity array, with the lensless-imaging to demonstrate recovery, monitoring and discrimination of the tumour cells spiked into whole blood samples. This study indicates that lensless imaging can be a powerful tool to investigate CTC proliferation and cultivation.

Assessment on the oil accumulation by knockdown of triacylglycerol lipase in the oleaginous diatom Fistulifera solaris.

Microalgae are promising producers of biofuel due to higher accumulation of triacylglycerol (TAG). However, further improvement of the lipid metabolism is critical for feasible application of microalgae in industrial production of biofuel. Suppression of lipid degradation pathways is a promising way to remarkably increase the lipid production in model diatoms. In this study, we established an antisense-based knockdown (KD) technique in the marine oleaginous diatom, Fistulifera solaris. This species has a capability to accumulate high content of lipids. Tgl1 KD showed positive impact on cell growth and lipid accumulation in conventional culture in f/2 medium, resulting in higher oil contents compared to wild type strain. However, these impacts of Tgl1 KD were slight when the cells were subjected to the two-stage growth system. The Tgl1 KD resulted in slight change of fatty acid composition; increasing in C14:0, C16:0 and C16:1, and decreasing in C20:5. This study indicates that, although Tgl1 played a certain role in lipid degradation in F. solaris, suppression of only a single type of TAG lipase was not significantly effective to improve the lipid production. Comprehensive understanding of the lipid catabolism in this microalga is essential to further improve the lipid production.