Exosome proteomes reveal glycolysis-related enzyme enrichment in primary canine mammary gland tumor compared to metastases.

OBJECTIVE: Numerous evidence has highlighted the differences between primary tumors and metastases. Nonetheless, the differences in exosomal proteins derived from primary tumor and metastases remain elusive. Here, we aimed to identify differentially expressed exosomal proteins from primary canine mammary gland tumor and metastases to understand how they shape their own tumor microenvironment. METHODS: We clearly distinguished primary canine mammary gland tumors (CHMp) from metastases (CHMm) and profiled the proteins within their secreted exosomes using LC-MS/MS. Moreover, the abundance of glycolysis enzymes (GPI, LDHA) in CHMp exosome was verified with Western blotting, To broaden the scope, we extended to human colorectal cancer-derived exosomes (SW480 vs. SW620) for comparison. RESULTS: We identified significant differences in 87 and 65 proteins derived from CHMp and CHMm, respectively. Notably, glycolysis enzymes (GPI, LDHA, LDHB, TPI1, and ALDOA) showed specific enrichment in exosomes from the primary tumor. CONCLUSION: We observed significant differences in the cellular proteome between primary tumors and metastases, and intriguingly, we identified a parallel heterogeneity the protein composition of exosomes. Specifically, we reported that glycolysis enzymes were significantly enriched in CHMp exosomes compared to CHMm exosomes. We further demonstrated that this quantitative difference in glycolysis enzymes persisted across primary and metastases, extending to human colorectal cancer-derived exosomes (SW480 vs. SW620). Our findings of the specific enrichment of glycolysis enzymes in primary tumor-derived exosomes contribute to a better understanding of tumor microenvironment modulation and heterogeneity between primary tumors and metastases.

ARID3C Acts as a Regulator of Monocyte-to-Macrophage Differentiation Interacting with NPM1.

ARID3C is a protein located on human chromosome 9 and expressed at low levels in various organs, yet its biological function has not been elucidated. In this study, we investigated both the cellular localization and function of ARID3C. Employing a combination of LC-MS/MS and deep learning techniques, we identified NPM1 as a binding partner for ARID3C's nuclear shuttling. ARID3C was found to predominantly localize with the nucleus, where it functioned as a transcription factor for genes STAT3, STAT1, and JUNB, thereby facilitating monocyte-to-macrophage differentiation. The precise binding sites between ARID3C and NPM1 were predicted by AlphaFold2. Mutating this binding site prevented ARID3C from interacting with NPM1, resulting in its retention in the cytoplasm instead of translocation to the nucleus. Consequently, ARID3C lost its ability to bind to the promoters of target genes, leading to a loss of monocyte-to-macrophage differentiation. Collectively, our findings indicate that ARID3C forms a complex with NPM1 to translocate to the nucleus, acting as a transcription factor that promotes the expression of the genes involved in monocyte-to-macrophage differentiation.

Chromosome-Level Genome Assembly of the Butter Clam Saxidomus purpuratus.

Herein, we provide the first whole-genome sequence of the purple butter clam (Saxidomus purpuratus), an economically important bivalve shellfish. Specifically, we sequenced and de novo assembled the genome of Sa. purpuratus based on PromethION long reads and Hi-C data. The 978-Mb genome of Sa. purpuratus comprises 19 chromosomes with 36,591 predicted protein-coding genes. The N50 length of Sa. purpuratus genome is 52 Mb, showing the highest continuous assembly among bivalve genomes. The Benchmarking by Universal Single-Copy Orthologs assessment indicated that 95.07% of complete metazoan universal single-copy orthologs (n = 954) were present in the assembly. Approximately 51% of Sa. purpuratus genome comprises repetitive sequences. Based on the high-quality Sa. purpuratus genome, we resolved half of the immune-associated genes, namely, scavenger receptor (SR) proteins, which are collinear to those in the closely related Cyclina sinensis genome. This finding suggested a high degree of conservation among immune-associated genes. Twenty-two (19%) SR proteins are tandemly duplicated in Sa. purpuratus genome, suggesting putative convergence evolution. Overall, Sa. purpuratus genome provides a new resource for the discovery of economically important traits and immune-response genes.

KOREF_S1: phased, parental trio-binned Korean reference genome using long reads and Hi-C sequencing methods.

BACKGROUND: KOREF is the Korean reference genome, which was constructed with various sequencing technologies including long reads, short reads, and optical mapping methods. It is also the first East Asian multiomic reference genome accompanied by extensive clinical information, time-series and multiomic data, and parental sequencing data. However, it was still not a chromosome-scale reference. Here, we updated the previous KOREF assembly to a new chromosome-level haploid assembly of KOREF, KOREF_S1v2.1. Oxford Nanopore Technologies (ONT) PromethION, Pacific Biosciences HiFi-CCS, and Hi-C technology were used to build the most accurate East Asian reference assembled so far. RESULTS: We produced 705 Gb ONT reads and 114 Gb Pacific Biosciences HiFi reads, and corrected ONT reads by Pacific Biosciences reads. The corrected ultra-long reads reached higher accuracy of 1.4% base errors than the previous KOREF_S1v1.0, which was mainly built with short reads. KOREF has parental genome information, and we successfully phased it using a trio-binning method, acquiring a near-complete haploid-assembly. The final assembly resulted in total length of 2.9 Gb with an N50 of 150 Mb, and the longest scaffold covered 97.3% of GRCh38's chromosome 2. In addition, the final assembly showed high base accuracy, with <0.01% base errors. CONCLUSIONS: KOREF_S1v2.1 is the first chromosome-scale haploid assembly of the Korean reference genome with high contiguity and accuracy. Our study provides useful resources of the Korean reference genome and demonstrates a new strategy of hybrid assembly that combines ONT's PromethION and PacBio's HiFi-CCS.

LT1, an ONT long-read-based assembly scaffolded with Hi-C data and polished with short reads.

We present LT1, the first high-quality human reference genome from the Baltic States. LT1 is a female de novo human reference genome assembly, constructed using 57× nanopore long reads and polished using 47× short paired-end reads. We utilized 72 GB of Hi-C chromosomal mapping data for scaffolding, to maximize assembly contiguity and accuracy. The contig assembly of LT1 was 2.73 Gbp in length, comprising 4490 contigs with an NG50 value of 12.0 Mbp. After scaffolding with Hi-C data and manual curation, the final assembly has an NG50 value of 137 Mbp and 4699 scaffolds. Assessment of gene prediction quality using Benchmarking Universal Single-Copy Orthologs (BUSCO) identified 89.3% of the single-copy orthologous genes included in the benchmark. Detailed characterization of LT1 suggests it has 73,744 predicted transcripts, 4.2 million autosomal SNPs, 974,616 short indels, and 12,079 large structural variants. These data may be used as a benchmark for further in-depth genomic analyses of Baltic populations.

Real-Time Longitudinal Evaluation of Tumor Blood Vessels Using a Compact Preclinical Fluorescence Imaging System.

Tumor angiogenesis is enhanced in all types of tumors to supply oxygen and nutrients for their growth and metastasis. With the development of anti-angiogenic drugs, the importance of technology that closely monitors tumor angiogenesis has also been emerging. However, to date, the technology for observing blood vessels requires specialized skills with expensive equipment, thereby limiting its applicability only to the laboratory setting. Here, we used a preclinical optical imaging system for small animals and, for the first time, observed, in real time, the entire process of blood vessel development in tumor-bearing mice injected with indocyanine green. Time-lapse sequential imaging revealed blood vessel volume and blood flow dynamics on a microscopic scale. Upon analyzing fluorescence dynamics at each stage of tumor progression, vessel volume and blood flow were found to increase as the tumor developed. Conversely, these vascular parameters decreased when the mice were treated with angiogenesis inhibitors, which suggests that the effects of drugs targeting angiogenesis can be rapidly and easily screened. The results of this study may help evaluate the efficacy of angiogenesis-targeting drugs by facilitating the observation of tumor blood vessels easily in a laboratory unit without large and complex equipment.

METTL8 mRNA Methyltransferase Enhances Cancer Cell Migration via Direct Binding to ARID1A.

The association of RNA modification in cancer has recently been highlighted. Methyltransferase like 8 (METTL8) is an enzyme and its role in mRNA m3C modification has barely been studied. In this study, we found that METTL8 expression was significantly up-regulated in canine mammary tumor and investigated its functional roles in the tumor process, including cancer cell proliferation and migration. METTL8 expression was up-regulated in most human breast cancer cell lines tested and decreased by Yin Yang 1 (YY1) transcription factor knockdown, suggesting that YY1 is a regulating transcription factor. The knockdown of METTL8 attenuated tumor cell growth and strongly blocked tumor cell migration. AT-rich interactive domain-containing protein 1A (ARID1A) was identified as a candidate mRNA by METTL8. ARID1A mRNA binds to METTL8 protein. ARID1A mRNA expression was not changed by METTL8 knockdown, but ARID1A protein level was significantly increased. Collectively, our study indicates that METTL8 up-regulated by YY1 in breast cancer plays an important role in cancer cell migration through the mRNA modification of ARID1A, resulting in the attenuation of its translation.

Comparative analysis of 7 short-read sequencing platforms using the Korean Reference Genome: MGI and Illumina sequencing benchmark for whole-genome sequencing.

BACKGROUND: DNBSEQ-T7 is a new whole-genome sequencer developed by Complete Genomics and MGI using DNA nanoball and combinatorial probe anchor synthesis technologies to generate short reads at a very large scale-up to 60 human genomes per day. However, it has not been objectively and systematically compared against Illumina short-read sequencers. FINDINGS: By using the same KOREF sample, the Korean Reference Genome, we have compared 7 sequencing platforms including BGISEQ-500, DNBSEQ-T7, HiSeq2000, HiSeq2500, HiSeq4000, HiSeqX10, and NovaSeq6000. We measured sequencing quality by comparing sequencing statistics (base quality, duplication rate, and random error rate), mapping statistics (mapping rate, depth distribution, and percent GC coverage), and variant statistics (transition/transversion ratio, dbSNP annotation rate, and concordance rate with single-nucleotide polymorphism [SNP] genotyping chip) across the 7 sequencing platforms. We found that MGI platforms showed a higher concordance rate for SNP genotyping than HiSeq2000 and HiSeq4000. The similarity matrix of variant calls confirmed that the 2 MGI platforms have the most similar characteristics to the HiSeq2500 platform. CONCLUSIONS: Overall, MGI and Illumina sequencing platforms showed comparable levels of sequencing quality, uniformity of coverage, percent GC coverage, and variant accuracy; thus we conclude that the MGI platforms can be used for a wide range of genomics research fields at a lower cost than the Illumina platforms.

Common plasma protein marker LCAT in aggressive human breast cancer and canine mammary tumor.

Breast cancer is one of the most frequently diagnosed cancers. Although biomarkers are continuously being discovered, few specific markers, rather than classification markers, representing the aggressiveness and invasiveness of breast cancer are known. In this study, we used samples from canine mammary tumors in a comparative approach. We subjected 36 fractions of both canine normal and mammary tumor plasmas to highperformance quantitative proteomics analysis. Among the identified proteins, LCAT was selectively expressed in mixed tumor samples. With further MRM and Western blot validation, we discovered that the LCAT protein is an indicator of aggressive mammary tumors, an advanced stage of cancer, possibly highly metastatic. Interestingly, we also found that LCAT is overexpressed in high-grade and lymph-node-positive breast cancer in silico data. We also demonstrated that LCAT is highly expressed in the sera of advanced-stage human breast cancers within the same classification. In conclusion, we identified a possible common plasma protein biomarker, LCAT, that is highly expressed in aggressive human breast cancer and canine mammary tumor. [BMB Reports 2020; 53(12): 664-669].

Analysis of opposing histone modifications H3K4me3 and H3K27me3 reveals candidate diagnostic biomarkers for TNBC and gene set prediction combination.

Breast cancer encompasses a major portion of human cancers and must be carefully monitored for appropriate diagnoses and treatments. Among the many types of breast cancers, triple negative breast cancer (TNBC) has the worst prognosis and the least cases reported. To gain a better understanding and a more decisive precursor for TNBC, two major histone modifications, an activating modification H3K4me3 and a repressive modification H3K27me3, were analyzed using data from normal breast cell lines against TNBC cell lines. The combination of these two histone markers on the gene promoter regions showed a great correlation with gene expression. A list of signature genes was defined as active (highly enriched H3K4me3), including NOVA1, NAT8L, and MMP16, and repressive genes (highly enriched H3K27me3), IRX2 and ADRB2, according to the distribution of these histone modifications on the promoter regions. To further enhance the investigation, potential candidates were also compared with other types of breast cancer to identify signs specific to TNBC. RNA-seq data was implemented to confirm and verify gene regulation governed by the histone modifications. Combinations of the biomarkers based on H3K4me3 and H3K27me3 showed the diagnostic value AUC 93.28% with P-value of 1.16e-226. The results of this study suggest that histone modification analysis of opposing histone modifications may be valuable toward developing biomarkers and targets for TNBC. [BMB Reports 2020; 53(5): 266-271].

Chromosome-scale assembly comparison of the Korean Reference Genome KOREF from PromethION and PacBio with Hi-C mapping information.

BACKGROUND: Long DNA reads produced by single-molecule and pore-based sequencers are more suitable for assembly and structural variation discovery than short-read DNA fragments. For de novo assembly, Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) are the favorite options. However, PacBio's SMRT sequencing is expensive for a full human genome assembly and costs more than $40,000 US for 30× coverage as of 2019. ONT PromethION sequencing, on the other hand, is 1/12 the price of PacBio for the same coverage. This study aimed to compare the cost-effectiveness of ONT PromethION and PacBio's SMRT sequencing in relation to the quality. FINDINGS: We performed whole-genome de novo assemblies and comparison to construct an improved version of KOREF, the Korean reference genome, using sequencing data produced by PromethION and PacBio. With PromethION, an assembly using sequenced reads with 64× coverage (193 Gb, 3 flowcell sequencing) resulted in 3,725 contigs with N50s of 16.7 Mb and a total genome length of 2.8 Gb. It was comparable to a KOREF assembly constructed using PacBio at 62× coverage (188 Gb, 2,695 contigs, and N50s of 17.9 Mb). When we applied Hi-C-derived long-range mapping data, an even higher quality assembly for the 64× coverage was achieved, resulting in 3,179 scaffolds with an N50 of 56.4 Mb. CONCLUSION: The pore-based PromethION approach provided a high-quality chromosome-scale human genome assembly at a low cost with long maximum contig and scaffold lengths and was more cost-effective than PacBio at comparable quality measurements.

Exploring the key communicator role of exosomes in cancer microenvironment through proteomics.

There have been many attempts to fully understand the mechanism of cancer behavior. Yet, how cancers develop and metastasize still remain elusive. Emerging concepts of cancer biology in recent years have focused on the communication of cancer with its microenvironment, since cancer cannot grow and live alone. Cancer needs to communicate with other cells for survival, and thus they secrete various messengers, including exosomes that contain many proteins, miRNAs, mRNAs, etc., for construction of the tumor microenvironment. Moreover, these intercellular communications between cancer and its microenvironment, including stromal cells or distant cells, can promote tumor growth, metastasis, and escape from immune surveillance. In this review, we summarized the role of proteins in the exosome as communicators between cancer and its microenvironment. Consequently, we present cancer specific exosome proteins and their unique roles in the interaction between cancer and its microenvironment. Clinically, these exosomes might provide useful biomarkers for cancer diagnosis and therapeutic tools for cancer treatment.

The automatic frequency control based on artificial intelligence for compact particle accelerator.

In this work, an advantage actor critic (A2C) based intelligent automatic frequency control (AFC) system was developed for X-band linear accelerator (LINAC). A2C is one type of reinforcement learning, which indicates how software agents should perform actions in an environment. In this paper, the A2C based AFC algorithm and its environment design, simulation result, and controller hardware and software processes are described. The objective of our design is to match LINAC and magnetron frequency, and it is implemented via reward shaping using comparison with the reflected power in the adjacent time. The simulation with the A2C algorithm was implemented in two modes, namely, periodic and White Gaussian Noise (WGN) waves, for analysis with temperature and random disturbance, respectively. In order to create artificial disturbance in the experiment, the magnetron shaft was shifted randomly every 0.5 s by using WGN with 18° angle of the step motor. The standard deviation of the reflected power was 5.63 kW, and the average power was 130.9 kW. To obtain maximum reward at the beginning of the A2C training, the adjacent frequency is needed. The measured average reflected power and standard deviation were 122.8 kW and 1.75 kW, respectively, after 2000 iterations. The results show that the reflected power and standard deviation of the AFC with A2C were lower compared to open-loop with artificial disturbance. The RF station of a medical X-band LINAC was used as the test bench, and the performance was confirmed by the results of an experiment conducted at Sungkyunkwan University in Korea.

The genome of the marine monogonont rotifer Brachionus plicatilis: Genome-wide expression profiles of 28 cytochrome P450 genes in response to chlorpyrifos and 2-ethyl-phenanthrene.

Brachionus spp. (Rotifera: Monogononta) are globally distributed in aquatic environments and play important roles in the aquatic ecosystem. The marine monogonont rotifer Brachionus plicatilis is considered a suitable model organism for ecology, evolution, and ecotoxicology. In this study, we assembled and characterized the B. plicatilis genome. The total length of the assembled genome was 106.9 Mb and the number of final scaffolds was 716 with an N50 value of 1.15 Mb and a GC content of 26.75%. A total of 20,154 genes were annotated after manual curation. To demonstrate the use of whole genome data, we targeted one of the main detoxifying enzyme of phase I detoxification system and identified in a total of 28 cytochrome P450 s (CYPs). Based on the phylogenetic analysis using the maximum likelihood, 28 B. plicatilis-CYPs were apparently separated into five different clans, namely, 2, 3, 4, mitochondrial (MT), and 46 clans. To better understand the CYPs-mediated xenobiotic detoxification, we measured the mRNA expression levels of 28 B. plicatilis CYPs in response to chlorpyrifos and 2-ethyl-phenanthrene. Most B. plicatilis CYPs were significantly modulated (P < 0.05) in response to chlorpyrifos and 2-ethyl-phenanthrene. In addition, xenobiotic-sensing nuclear receptor (XNR) response element sequences were identified in the 5 kb upstream of promoter regions of 28 CYPs from the genome of B. plicatilis, indicating that these XNR can be associated with detoxification of xenobiotics. Overall, the assembled B. plicatilis genome presented here will be a useful resource for a better understanding the molecular ecotoxicology in the view of molecular mechanisms underlying toxicological responses, particularly on xenobiotic detoxification in this species.

The Draft Genome of an Octocoral, Dendronephthya gigantea.

Coral reefs composed of stony corals are threatened by global marine environmental changes. However, soft coral communities of octocorallian species, appear more resilient. The genomes of several cnidarians species have been published, including from stony corals, sea anemones, and hydra. To fill the phylogenetic gap for octocoral species of cnidarians, we sequenced the octocoral, Dendronephthya gigantea, a nonsymbiotic soft coral, commonly known as the carnation coral. The D. gigantea genome size is ∼276 Mb. A high-quality genome assembly was constructed from PacBio long reads (29.85 Gb with 108× coverage) and Illumina short paired-end reads (35.54 Gb with 128× coverage) resulting in the highest N50 value (1.4 Mb) reported thus far among cnidarian genomes. About 12% of the genome is repetitive elements and contained 28,879 predicted protein-coding genes. This gene set is composed of 94% complete BUSCO ortholog benchmark genes, which is the second highest value among the cnidarians, indicating high quality. Based on molecular phylogenetic analysis, octocoral and hexacoral divergence times were estimated at 544 MYA. There is a clear difference in Hox gene composition between these species: unlike hexacorals, the Antp superclass Evx gene was absent in D. gigantea. Here, we present the first genome assembly of a nonsymbiotic octocoral, D. gigantea to aid in the comparative genomic analysis of cnidarians, including stony and soft corals, both symbiotic and nonsymbiotic. The D. gigantea genome may also provide clues to mechanisms of differential coping between the soft and stony corals in response to scenarios of global warming.

The complete mitochondrial genome of a Dokdo shrimp, Lebbeus groenlandicus.

Lebbeus groenlandicus is a shrimp species indigenous to the Dokdo islands in the East Sea of Korea. We report the 17,399 bp mitochondrial genome (mitogenome) of the species that consists of 13 protein-coding genes, 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), and a control region (CR). A maximum-likelihood tree, constructed with 18 prawn and 45 shrimp mitogenomes, confirmed that L. groenlandicus occupies the most basal position within the Caridea infra-order and is closely related to Pandalidae shrimps.

The genome of the marine medaka Oryzias melastigma.

Marine medaka (Oryzias melastigma) is considered to be a useful fish model for marine and estuarine ecotoxicology studies and has good potential for field-based population genomics because of its geographical distribution in Asian estuarine and coastal areas. In this study, we present the first whole-genome draft of O. melastigma. The genome assembly consists of 8,602 scaffolds (N50 = 23.737 Mb) and a total genome length of 779.4 Mb. A total of 23,528 genes were predicted, and 12,670 gene families shared with three teleost species (Japanese medaka, mangrove killifish and zebrafish) were identified. Genome analyses revealed that the O. melastigma genome is highly heterozygous and contains a large number of repeat sequences. This assembly represents a useful genomic resource for fish scientists.

The genome of the freshwater monogonont rotifer Brachionus calyciflorus.

Monogononta is the most speciose class of rotifers, with more than 2,000 species. The monogonont genus Brachionus is widely distributed at a global scale, and a few of its species are commonly used as ecological and evolutionary models to address questions related to aquatic ecology, cryptic speciation, evolutionary ecology, the evolution of sex and ecotoxicology. With the importance of Brachionus species in many areas of research, it is remarkable that the genome has not been characterized. This study aims to address this lacuna by presenting, for the first time, the whole-genome assembly of the freshwater species Brachionus calyciflorus. The total length of the assembled genome was 129.6 Mb, with 1,041 scaffolds. The N50 value was 786.6 kb, and the GC content was 24%. A total of 16,114 genes were annotated with repeat sequences, accounting for 21% of the assembled genome. This assembled genome may form a basis for future studies addressing key questions on the evolution of monogonont rotifers. It will also provide the necessary molecular resources to mechanistically investigate ecophysiological and ecotoxicological responses.

Identification and characterization of homeobox (Hox) genes and conservation of the single Hox cluster (324.6 kb) in the water flea Daphnia magna.

We report the complete sequence analysis of the entire complement of eight typical homeobox (Hox) genes (Lab, Pb, Dfd, Scr, Antp, Ubx, Abd-A, and Abd-B) and two other genes (Hox3 and Ftz) in a 324.6-kb region in the water flea Daphnia magna. In the cluster of D. magna Hox genes, we found one long interspersed nuclear element (LINE)/R2-NeSL between Ubx and Abd-A that was not present in Daphnia pulex Hox genes. In basal expression of Hox genes at different developmental stages, biothorax complex genes (Ubx, Abd-A, and Abd-B) and some antennapedia complex genes (Lab, Scr, Antp) were moderately expressed, but the Hox3 gene was barely expressed. Three homeobox genes (Antp, Ubx, Abd-A) were highly expressed at 6-7 days after release from the brood chamber and/or in the adult stage. The structural array and transcribed orientation of Dm-Hox genes were identical to those of the sister species D. pulex (∼340 kb), indicating that the Hox gene structure in daphnids is highly conserved. However, Dm- and Dp-Hox3, -deformed (Dfd), and -fushi tarazu (Ftz) genes varied from orthologous genes in pancrustacean species.

Identification of 74 cytochrome P450 genes and co-localized cytochrome P450 genes of the CYP2K, CYP5A, and CYP46A subfamilies in the mangrove killifish Kryptolebias marmoratus.

BACKGROUND: The mangrove killifish Kryptolebias marmoratus is the only vertebrate that reproduces by self-fertilizing and is an important model species in genetics and marine ecotoxicology. Using whole-genome and transcriptome sequences, we identified all members of the cytochrome P450 (CYP) family in this model teleost and compared them with those of other teleosts. RESULTS: A total of 74 cytochrome P450 genes and one pseudogene were identified in K. marmoratus. Phylogenetic analysis indicated that the CYP genes in clan 2 were most expanded, while synteny analysis with other species showed orthologous relationships of CYP subfamilies among teleosts. In addition to the CYP2K expansions, five tandem duplicated gene copies of CYP5A were observed. These features were unique to K. marmoratus. CONCLUSIONS: These results shed a light on CYP gene evolution, particularly the co-localized CYP2K, CYP5A, and CYP46A subfamilies in fish. Future studies of CYP expression could identify specific endogenous and exogenous environmental factors that triggered the evolution of tandem CYP duplication in K. marmoratus.