Genome-wide identification, characterization and expression analyses of two TNFRs in Yesso scallop (Patinopecten yessoensis) provide insight into the disparity of responses to bacterial infections and heat stress in bivalves.

Tumor necrosis factors receptors (TNFRs) comprise a superfamily of proteins characterized by a unique cysteine-rich domain (CRD) and play important roles in diverse physiological and pathological processes in the innate immune system, including inflammation, apoptosis, autoimmunity and organogenesis. Although significant effects of TNFRs on immunity have been reported in most vertebrates as well as some invertebrates, the complete TNFR superfamily has not been systematically characterized in scallops. In this study, two different types of TNFR-like genes, including PyTNFR1 and PyTNFR2 genes were identified from Yesso scallop (Patinopecten yessoensis, Jay, 1857) through whole-genome scanning. Phylogenetic and protein structural analyses were carried out to determine the identities and evolutionary relationships of the two genes. The expression profiling of PyTNFRs was performed at different development stages, in healthy adult tissues and in hemocytes after bacterial infection and heat stress. Expression analysis revealed that both PyTNFRs were significantly induced during the acute phase (3 h) after infection with Gram-positive (Micrococcus luteus) and Gram-negative (Vibrio anguillarum) bacteria, though much more dramatic chronic-phase (24 h) changes were observed after V. anguillarum challenge. For heat stress, only PyTNFR2 displayed significant elevation at 12 h and 24 h, which suggests a functional difference in the two PyTNFRs. Collectively, this study provides novel insight into the PyTNFRs and the specific role and response of TNFR-involved pathways in host immune responses against different bacterial pathogens and heat stress in bivalves.

Characterizations and expression analyses of NF-κB and Rel genes in the Yesso scallop (Patinopecten yessoensis) suggest specific response patterns against Gram-negative infection in bivalves.

Rel/NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) genes are evolutionarily conserved and play a pivotal role in several physiological events. They have been extensively studied from various species, including both vertebrates and invertebrates. However, the Rel/NF-κB genes have not been systematically characterized in bivalves. In this study, we identified and characterized PyNF-κB and PyRel in the Yesso scallop (Patinopecten yessoensis). Phylogenetic and protein structural analyses were conducted to determine the identities and evolutionary relationships of Rel/NF-κB genes in Yesso scallop. Compared with the Rel/NF-κB genes from vertebrate species, the PyNF-κB and PyRel are relatively conserved in their structural features, but there were no paralogs found in P. yessoensis or other invertebrates. To gain insights into the roles of Rel/NF-κB genes during the innate immune response in scallop, quantitative real-time PCR was used to investigate the expression profiles of these genes at different developmental stages, in healthy adult tissues and in the hemolymph after bacterial infection with Micrococcus luteus and Vibrio anguillarum. The real-time PCR results indicated the abundance of PyNF-κB in the first four embryonic stages, including oocytes, fertilized eggs, morulae and blastulae. By contrast, PyRel was abundantly expressed in blastulae, trochophores and D-shaped larvae. In adult scallops, PyNF-κB and PyRel were ubiquitously expressed in most healthy tissues and highly expressed in most of the immune related tissues. Both genes were significantly up-regulated during the acute phase (3 h) after infection with Gram-positive (M. luteus) and negative (V. anguillarum) bacteria, while the much higher expression level of PyNF-κB suggested the involvement of the extra immune deficiency (IMD)-like pathway against the Gram-negative bacterial infection. The complex pattern of Rel/NF-κB induced expression suggested that PyNF-κB and PyRel both have specific and cooperative roles in the acute immune responses to bacterial infection.

Gamma-Glutamyltransferase Activity (GGT) Is a Long-Sought Biomarker of Redox Status in Blood Circulation: A Retrospective Clinical Study of 44 Types of Human Diseases.

Methods: The clinical GGT data from 168,858 patients with 44 diseases and 132,357 healthy control in the clinical laboratory of our hospital over the past five years were retrieved. All data were analyzed with SPSS, RStudio V.1.3.1073, and python libraries 3.8. Results: Thirty-eight out of 44 diseases had significantly increased (p < 0.001) circulating GGT activities, whereas gastric cancer, anemia, renal cyst, cervical cancer, preeclampsia, and knee-joint degenerative diseases had significantly decreased (p < 0.001) GGT activities compared to the healthy control. ROC analyses showed that GGT was an excellent biomarker for liver cancer (AUC = 0.86), pancreatitis (AUC = 0.84), or hepatic encephalopathy (AUC = 0.80). All pancreas-related diseases had more than 8-fold increases in GGT activity span than the healthy control, while pancreatic cancer had a 12-fold increase (1021 U/L vs. 82 U/L). The knee-joint degenerative disease had the lowest median and narrowest GGT activity range (63 U/L). Furthermore, most diseases' lowest to highest GGT activities were beyond the healthy control in both directions. Conclusions: Thirty-eight out of 44 diseases were in overall oxidative states defined by the increased GGT median values. In contrast, knee-joint degenerative disease, gastric cancer, anemia, renal cyst, cervical cancer, and preeclampsia were in overall antioxidative states. Moreover, most diseases swing between oxidative and antioxidative states, evidenced by the increased lowest to highest GGT activity ranges than the healthy control. Liver- and pancreas-related abnormalities were responsible for significantly increased GGT activities. Our overall results suggested that circulating GGT was a redox status biomarker.

Circulating Glycan Monosaccharide Composite-Based Biomarker Diagnoses Colorectal Cancer at Early Stages and Predicts Prognosis.

Introduction: Early diagnosis could lead to a cure of colorectal cancer (CRC). Since CRC is related to aging and lifestyles, we tested if the environmental information-enriched monosaccharide composite (MC) of circulating glycans could serve as an early diagnostic biomarker for CRC. Meanwhile, we evaluated its role in predicting prognosis. Methods: HPAEC-PAD was used to quantify glycan monosaccharide compositions from a total of 467 serum samples including CRC patients, colorectal adenoma (CRA) patients and healthy individuals. Two diagnostic model was constructed by logistic regression analysis. The diagnostic performance of the two models was verified in the retrospective validation group and the prospective validation group. The prognostic performance of the model was assessed by survival analysis. Results: The concentrations of monosaccharides in serum were significantly higher in CRA and CRC patients than in healthy individuals. Two diagnostic models were constructed: MC1 was used to distinguish between healthy individuals and CRC; MC2 was used to distinguish between healthy individuals and CRA. Area under receptor operating characteristic curve (AUC) of MC2 and MC1 was 0.8025 and 0.9403 respectively. However, the AUC of CEA between healthy individuals and CRC was 0.7384. Moreover, in early stage of CRC (without lymph node metastasis), the positive rates of CEA and MC1 were 28% and 80%, respectively. The follow-up data showed that the increased MC1 value was associated with poor survival in patients with CRC (p=0.0010, HR=5.30). Discussion: The MC1 model is superior to CEA in the diagnosis of CRC, especially in the early diagnosis. MC1 can be used for predicting prognosis of CRC patients, and elevated MC1 values indicate poor survival.

Coriolus versicolor polysaccharopeptide as an immunotherapeutic in China.

Coriolus versicoloris is one of the well-known traditional medicinal mushrooms used in China for over 2000 years. Polysaccharopeptide (PSP) is identified as the major bioactive component, which can be obtained from the mycelium or fermentation broth of Coriolus versicolor. The polysaccharide content in PSP is ~60% and the peptide content in PSP is ~10-30%. The main monosaccharides found in PSP include glucose, mannose, and a small amount of galactose, xylose, and fucose. ß-Glucan is one of the identified components in PSP with the established immunomodulatory function. PSP was approved by the authority and has been used clinically in Japan and China since 1970s. PSP is helpful in improving the survival and quality of life in patients suffering cancers, hepatopathy, hyperlipidemia, chronic bronchitis, and other complex diseases. In this article, the preclinical and clinical studies of PSP are summarized over the past 41 years based on a literature search covering the CNKI, VIP, and Wanfang databases. Current studies support PSP as an immunotherapeutic. PSP activates and enhances the function and recognition ability of immune cells, strengthens the phagocytosis of macrophages, increases the expressions of cytokines and chemokines such as tumor necrosis factor-α (TNF-α), interleukins (IL-1ß and IL-6), histamine, and prostaglandin E, stimulates the filtration of both dendritic cells and T-cells into tumors, and ameliorates the adverse events associated with chemotherapy. In recent years, immunotherapy has been widely used in cancer treatment. However, to use PSP as an immunotherapeutic at world stage, further chemical, biochemical and pharmacological studies of PSP are needed.

CA242 as a biomarker for pancreatic cancer and other diseases.

CA242 is a sialic acid-containing carbohydrate antigen attached to core proteins/lipids detected on the cell surface or in serum. Increased serum levels of CA242 have been used clinically as diagnostic biomarkers for pancreatic, colorectal, and other cancers. Since CA242 is overexpressed in malignant tumor tissues compared to that in normal tissues, it is believed that serum CA242 is a product of cancer cells. Thus far, the serum CA242 levels in patients suffering from different types of cancer and noncancerous diseases have not been systematically compared. In our current study, we acquired data of serum CA242 levels from 34,680 patients with 27 clinically defined diseases and from healthy controls (1317) in the clinical laboratory of our hospital over the past 5 years. The mean, median, and -log10p values were calculated. Our data showed that patients with pancreatic cancer, cervical cancer, and lymphoma had the highest median values of serum CA242, which were followed by esophagus cancer, colon cancer, ovarian cancer, type 2 diabetes mellitus, rectal cancer, coronary heart disease, breast cancer, diabetic nephropathy, acute myocardial infarction, and 15 other types of diseases compared to that of healthy controls. In contrast, patients suffering from sequela of brain damage and anemia had statistically lower median values than healthy controls. Based on the -log10p values, the increased serum CA242 levels could be used not only for diagnosis of pancreatic and other cancers, but also for diagnosis of type 2 diabetes mellitus and coronary heart disease, suggesting CA242 might be a systemic malfunction biomarker associated with cancers and other chronic diseases.

Heparinoids Danaparoid and Sulodexide as clinically used drugs.

Heparin is the first glycosaminoglycan ever identified. All the heparin-like glycosaminoglycans that are also isolated from animal tissues or any polysaccharides that mimic the biological activities of heparin are called heparinoids. Heparin is the mostly sulfated glycosaminoglycan made by mast cells and an essential anticoagulant drug in modern medicine. Heparin inhibits both thrombin generation and thrombin activity, releases tissue factor pathway inhibitor, and possesses anti-inflammatory, anti-viral, anti-angiogenesis, anti-neoplastic, and anti-metastatic properties though high affinity interactions with a variety of proteins in the blood circulation. The multi-pharmacological effects of heparin are both sequence- and sulfation degree dependent. Less sulfated heparinoids have been indicated to have more physiological functions than heparin. Since the anticoagulant heparin is associated with severe side effects, such as bleeding and heparin-induced thrombocytopenia and thrombosis, it is expected that the less sulfated heparinoids might serve as alternative drugs for patients who cannot use heparin. The crude heparin isolated from animal tissues contains ~50% heparin and ~50% less sulfated heparinoids. Indeed, the less sulfated waste heparinoids 1 during heparin production is chemically degraded and developed into the clinical drug Danaparoid and the more sulfated waste heparinoids 2 during heparin production is chemically degraded and developed into the clinical drug Sulodexide. Moreover, clinical studies indicate that Danaparoid and Sulodexide have the expected pharmacological activities. We will provide an update on the chemical characteristics and clinical use of the heparinoids Danaparoid and Sulodexide. In addition, the potential clinical applications of Danaparoid and Sulodexide in other therapeutic area will also be discussed.

Fucosylation in cancer biology and its clinical applications.

Fucosylation is the process of transferring fucose from GDP-fucose to their substrates, which includes certain proteins, N- and O-linked glycans in glycoprotein or glycolipids, by fucosyltransferases in all mammalian cells. Fucosylated glycans play vital role in selectin-mediated leukocyte extravasation, lymphocyte homing, and pathogen-host interactions, whereas fucosylated proteins are essential for signaling transduction in numerous ontogenic events. Aberrant fucosylation due to the availability of high energy donor GDP-fucose, abnormal expression of FUTs and/or α-fucosidase, and the availability of their substrates leads to different fucosylated glycan or protein structures. Accumulating evidence demonstrates that aberrant fucosylation plays important role in all aspects of cancer biology. In this review, we will summarize the current knowledge about fucosylation in different physiological and pathological processes with a focus on their roles not only in cancer cell proliferation, invasion, and metastasis but also in tumor immune surveillance. Furthermore, the clinical potential and applications of fucosylation in cancer diagnosis and treatment will also be discussed.

Retrospective analysis of glycan-related biomarkers based on clinical laboratory data in two medical centers during the past 6 years.

Most of clinically used cancer biomarkers are either specific glycan structures or glycoproteins. Although the high serum levels of the cancer biomarkers are also present in certain patients suffering noncancer diseases, systematic measurement and comparison of the serum levels of all cancer biomarkers among cancer and noncancer patients have not been reported. In this study, the serum levels of 17 glucose and glycan-related biomarkers including 10 cancer biomarkers SCCA, CA724, CA50, CA242, CA125, CA199, CA153, AFP, CEA, and PSA were retrospectively investigated based on clinical laboratory data in two medical centers during the past 6 years (2012-2018). The data included a total of 1,477,309 clinical lab test results of 17 biomarkers from healthy controls and patients suffering 64 different types of cancer and noncancer diseases. We found that the median serum levels of CA724, CEA, CA153, SCCA, and CA125 were highest not in cancer patients but in patients suffering gout, lung fibrosis, nephrotic syndrome, uremia, and cirrhosis, respectively. Consistently, the classical ovarian cancer biomarker CA125 had better overall sensitivity and specificity as biomarker for cirrhosis (67% and 92%, respectively) than that for ovarian cancer (41% and 97%, respectively). Furthermore, the information shown as heatmap or waterfall built on the -Log10p values of the 17 glycan-related biomarkers in different clinically defined diseases suggested that all glycan-related biomarkers had cancer-, aging-, and disease-relevant characteristics and cancers were systems disease. The detailed presentation of the data for each of the 17 biomarkers will be deliberated in chapters 6-23 in this book series.

Optimizing microwave-assisted hydrolysis conditions for monosaccharide composition analyses of different polysaccharides.

Releasing all monosaccharides during acid hydrolysis for composition analysis of polysaccharides has been a time consuming process. In current study, an efficient (10 µL sample + 10 µL acid), sensitive, and quick monosaccharide composition analysis of polysaccharides was accomplished by using microwave-assisted HCl hydrolysis (10 min) of the polysaccharides followed by high-performance anion-exchange chromatography (HPAEC) combined with pulsed amperometric detection (PAD) analysis. Compared to the conventional hydrolysis procedure, this method is an efficient approach for monosaccharide composition analysis of acidic, basic, and neutral polysaccharides and particularly suited to polysaccharides that are difficult to hydrolyse fully such as chitosan, heparin and chondroitin sulfates.

Whole-Genome Restriction Mapping by "Subhaploid"-Based RAD Sequencing: An Efficient and Flexible Approach for Physical Mapping and Genome Scaffolding.

Assembly of complex genomes using short reads remains a major challenge, which usually yields highly fragmented assemblies. Generation of ultradense linkage maps is promising for anchoring such assemblies, but traditional linkage mapping methods are hindered by the infrequency and unevenness of meiotic recombination that limit attainable map resolution. Here we develop a sequencing-based "in vitro" linkage mapping approach (called RadMap), where chromosome breakage and segregation are realized by generating hundreds of "subhaploid" fosmid/bacterial-artificial-chromosome clone pools, and by restriction site-associated DNA sequencing of these clone pools to produce an ultradense whole-genome restriction map to facilitate genome scaffolding. A bootstrap-based minimum spanning tree algorithm is developed for grouping and ordering of genome-wide markers and is implemented in a user-friendly, integrated software package (AMMO). We perform extensive analyses to validate the power and accuracy of our approach in the model plant Arabidopsis thaliana and human. We also demonstrate the utility of RadMap for enhancing the contiguity of a variety of whole-genome shotgun assemblies generated using either short Illumina reads (300 bp) or long PacBio reads (6-14 kb), with up to 15-fold improvement of N50 (∼816 kb-3.7 Mb) and high scaffolding accuracy (98.1-98.5%). RadMap outperforms BioNano and Hi-C when input assembly is highly fragmented (contig N50 = 54 kb). RadMap can capture wide-range contiguity information and provide an efficient and flexible tool for high-resolution physical mapping and scaffolding of highly fragmented assemblies.

Scallop genome reveals molecular adaptations to semi-sessile life and neurotoxins.

Bivalve molluscs are descendants of an early-Cambrian lineage superbly adapted to benthic filter feeding. Adaptations in form and behavior are well recognized, but the underlying molecular mechanisms are largely unknown. Here, we investigate the genome, various transcriptomes, and proteomes of the scallop Chlamys farreri, a semi-sessile bivalve with well-developed adductor muscle, sophisticated eyes, and remarkable neurotoxin resistance. The scallop's large striated muscle is energy-dynamic but not fully differentiated from smooth muscle. Its eyes are supported by highly diverse, intronless opsins expanded by retroposition for broadened spectral sensitivity. Rapid byssal secretion is enabled by a specialized foot and multiple proteins including expanded tyrosinases. The scallop uses hepatopancreas to accumulate neurotoxins and kidney to transform to high-toxicity forms through expanded sulfotransferases, probably as deterrence against predation, while it achieves neurotoxin resistance through point mutations in sodium channels. These findings suggest that expansion and mutation of those genes may have profound effects on scallop's phenotype and adaptation.

Scallop genome provides insights into evolution of bilaterian karyotype and development.

Reconstructing the genomes of bilaterian ancestors is central to our understanding of animal evolution, where knowledge from ancient and/or slow-evolving bilaterian lineages is critical. Here we report a high-quality, chromosome-anchored reference genome for the scallop Patinopecten yessoensis, a bivalve mollusc that has a slow-evolving genome with many ancestral features. Chromosome-based macrosynteny analysis reveals a striking correspondence between the 19 scallop chromosomes and the 17 presumed ancestral bilaterian linkage groups at a level of conservation previously unseen, suggesting that the scallop may have a karyotype close to that of the bilaterian ancestor. Scallop Hox gene expression follows a new mode of subcluster temporal co-linearity that is possibly ancestral and may provide great potential in supporting diverse bilaterian body plans. Transcriptome analysis of scallop mantle eyes finds unexpected diversity in phototransduction cascades and a potentially ancient Pax2/5/8-dependent pathway for noncephalic eyes. The outstanding preservation of ancestral karyotype and developmental control makes the scallop genome a valuable resource for understanding early bilaterian evolution and biology.

MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes.

Characterization of dynamic DNA methylomes in diverse phylogenetic groups has attracted growing interest for a better understanding of the evolution of DNA methylation as well as its function and biological significance in eukaryotes. Sequencing-based methods are promising in fulfilling this task. However, none of the currently available methods offers the 'perfect solution', and they have limitations that prevent their application in the less studied phylogenetic groups. The recently discovered Mrr-like enzymes are appealing for new method development, owing to their ability to collect 32-bp methylated DNA fragments from the whole genome for high-throughput sequencing. Here, we have developed a simple and scalable DNA methylation profiling method (called MethylRAD) using Mrr-like enzymes. MethylRAD allows for de novo (reference-free) methylation analysis, extremely low DNA input (e.g. 1 ng) and adjustment of tag density, all of which are still unattainable for most widely used methylation profiling methods such as RRBS and MeDIP. We performed extensive analyses to validate the power and accuracy of our method in both model (plant Arabidopsis thaliana) and non-model (scallop Patinopecten yessoensis) species. We further demonstrated its great utility in identification of a gene (LPCAT1) that is potentially crucial for carotenoid accumulation in scallop adductor muscle. MethylRAD has several advantages over existing tools and fills a void in the current epigenomic toolkit by providing a universal tool that can be used for diverse research applications, e.g. from model to non-model species, from ordinary to precious samples and from small to large genomes, but at an affordable cost.