Transcriptomic profiling and the first spatial expression analysis of candidate genes in the salivary gland of the East Asian medicinal leech, Hirudo nipponia.

Hirudo nipponia, a blood-sucking leech native to East Asia, possesses a rich repertoire of active ingredients in its saliva, showcasing significant medical potential due to its anticoagulant, anti-inflammatory, and antibacterial effects against human diseases. Despite previous studies on the transcriptomic and proteomic characteristics of leech saliva, which have identified medicinal compounds, our knowledge of tissue-specific transcriptomes and their spatial expression patterns remains incomplete. In this study, we conducted an extensive transcriptomic profiling of the salivary gland tissue in H. nipponia based on de novo assemblies of tissue-specific transcriptomes from the salivary gland, teeth, and general head region. Through gene ontology (GO) analysis and hierarchical clustering, we discovered a novel set of anti-coagulant factors-i.e., Hni-Antistasin, Hni-Ghilanten, Hni-Bdellin, Hni-Hirudin-as well as a previously unrecognized immune-related gene, Hni-GLIPR1 and uncharacterized salivary gland specific transcripts. By employing in situ hybridization, we provided the first visualization of gene expression sites within the salivary gland of H. nipponia. Our findings expand on our understanding of transcripts specifically expressed in the salivary gland of blood-sucking leeches, offering valuable resources for the exploration of previously unidentified substances with medicinal applications.

Draft genome of the sea cucumber Apostichopus japonicus and genetic polymorphism among color variants.

The Japanese sea cucumber (Apostichopus japonicus Selenka 1867) is an economically important species as a source of seafood and ingredient in traditional medicine. It is mainly found off the coasts of northeast Asia. Recently, substantial exploitation and widespread biotic diseases in A. japonicus have generated increasing conservation concern. However, the genomic knowledge base and resources available for researchers to use in managing this natural resource and to establish genetically based breeding systems for sea cucumber aquaculture are still in a nascent stage. A total of 312 Gb of raw sequences were generated using the Illumina HiSeq 2000 platform and assembled to a final size of 0.66 Gb, which is about 80.5% of the estimated genome size (0.82 Gb). We observed nucleotide-level heterozygosity within the assembled genome to be 0.986%. The resulting draft genome assembly comprising 132 607 scaffolds with an N50 value of 10.5 kb contains a total of 21 771 predicted protein-coding genes. We identified 6.6-14.5 million heterozygous single nucleotide polymorphisms in the assembled genome of the three natural color variants (green, red, and black), resulting in an estimated nucleotide diversity of 0.00146. We report the first draft genome of A. japonicus and provide a general overview of the genetic variation in the three major color variants of A. japonicus. These data will help provide a comprehensive view of the genetic, physiological, and evolutionary relationships among color variants in A. japonicus, and will be invaluable resources for sea cucumber genomic research.

Microbacterium rhizosphaerae sp. nov., isolated from a Ginseng field, South Korea.

A novel Gram-stain positive, aerobic, short rod-shaped, non-motile bacterium, designated strain CHO1T, was isolated from rhizosphere soil from a ginseng agriculture field. Strain CHO1T was observed to form yellow colonies on R2A agar medium. The cell wall peptidoglycan was found to contain alanine, glycine, glutamic acid, D-ornithine and serine. The cell wall sugars were identified as galactose, mannose, rhamnose and ribose. Strain CHO1T was found to contain MK-11, MK-12, MK-13 as the predominant menaquinones and anteiso-C15:0, iso-C16:0, and anteiso-C17:0 as the major fatty acids. Diphosphatidylglycerol, phosphatidylglycerol, phosphoglycolipid, an unidentified phospholipid and three unidentified glycolipids were found to be present in strain CHO1T. Based on 16S rRNA gene sequence analysis, strain CHO1T was found to be closely related to Microbacterium mangrovi DSM 28240T (97.81 % similarity), Microbacterium immunditiarum JCM 14034T (97.45 %), Microbacterium oryzae JCM 16837T (97.33 %) and Microbacterium ulmi KCTC 19363T (97.10 %) and to other species of the genus Microbacterium. The DNA G+C content of CHO1T was determined to be 70.1 mol %. The DNA-DNA hybridization values of CHO1T with M. mangrovi DSM 28240T, M. immunditiarum JCM 14034T, M. oryzae JCM 16837T and M. ulmi KCTC 19363T were 46.7 ± 2, 32.4 ± 2, 32.0 ± 2 and 29.2 ± 2 %, respectively. On the basis of genotypic, phenotypic and phylogenetic properties, it is concluded that strain CHO1T represents a novel species within the genus Microbacterium, for which the name Microbacterium rhizosphaerae sp. nov. is proposed. The type strain of M. rhizosphaerae is CHO1T (= KEMB 7306-513T = JCM 31396T).

Comparative transcriptome analysis of three color variants of the sea cucumber Apostichopus japonicus.

The sea cucumber Apostichopus japonicus Selenka 1867 represents an important resource in biomedical research, traditional medicine, and the seafood industry. Much of the commercial value of A. japonicus is determined by dorsal/ventral color variation (red, green, and black), yet the taxonomic relationships between these color variants are not clearly understood. We performed the first comparative analysis of de novo assembled transcriptome data from three color variants of A. japonicus. Using the Illumina platform, we sequenced nearly 177,596,774 clean reads representing a total of 18.2Gbp of sea cucumber transcriptome. A comparison of over 0.3 million transcript scaffolds against the Uniprot/Swiss-Prot database yielded 8513, 8602, and 8588 positive matches for green, red, and black body color transcriptomes, respectively. Using the Panther gene classification system, we assessed an extensive and diverse set of expressed genes in three color variants and found that (1) among the three color variants of A. japonicus, genes associated with RNA binding protein, oxidoreductase, nucleic acid binding, transferase, and KRAB box transcription factor were most commonly expressed; and (2) the main protein functional classes are differently regulated in all three color variants (extracellular matrix protein and phosphatase for green color, transporter and potassium channel for red color, and G-protein modulator and enzyme modulator for black color). This work will assist in the discovery and annotation of novel genes that play significant morphological and physiological roles in color variants of A. japonicus, and these sequence data will provide a useful set of resources for the rapidly growing sea cucumber aquaculture industry.

The Green Tea Component (-)-Epigallocatechin-3-Gallate Sensitizes Primary Endothelial Cells to Arsenite-Induced Apoptosis by Decreasing c-Jun N-Terminal Kinase-Mediated Catalase Activity.

The green tea component (-)-epigallocatechin-3-gallate (EGCG) has been shown to sensitize many different types of cancer cells to anticancer drug-induced apoptosis, although it protects against non-cancerous primary cells against toxicity from certain conditions such as exposure to arsenic (As) or ultraviolet irradiation. Here, we found that EGCG promotes As-induced toxicity of primary-cultured bovine aortic endothelial cells (BAEC) at doses in which treatment with each chemical alone had no such effect. Increased cell toxicity was accompanied by an increased condensed chromatin pattern and fragmented nuclei, cleaved poly(ADP-ribose) polymerase (PARP), activity of the pro-apoptotic enzymes caspases 3, 8 and 9, and Bax translocation into mitochondria, suggesting the involvement of an apoptotic signaling pathway. Fluorescence activated cell sorting analysis revealed that compared with EGCG or As alone, combined EGCG and As (EGCG/As) treatment significantly induced production of reactive oxygen species (ROS), which was accompanied by decreased catalase activity and increased lipid peroxidation. Pretreatment with N-acetyl-L-cysteine or catalase reversed EGCG/As-induced caspase activation and EC toxicity. EGCG/As also increased the phosphorylation of c-Jun N-terminal kinase (JNK), which was not reversed by catalase. However, pretreatment with the JNK inhibitor SP600125 reversed all of the observed effects of EGCG/As, suggesting that JNK may be the most upstream protein examined in this study. Finally, we also found that all the observed effects by EGCG/As are true for other types of EC tested. In conclusion, this is firstly to show that EGCG sensitizes non-cancerous EC to As-induced toxicity through ROS-mediated apoptosis, which was attributed at least in part to a JNK-activated decrease in catalase activity.

Synthesis and antimicrobial activities of 3-O-alkyl analogues of (+)-catechin: improvement of stability and proposed action mechanism.

We report here the synthesis and biological properties of 3-O-alkyl analogues of (+)-catechin (5), which itself is one of the major natural polyphenols found in green tea and has several physiological activities. Starting from 5, a series of 3-O-alkyl-(+)-catechin derivatives were investigated as potent antimicrobial agents. The presence of an alkyl chain rather than acyl on 3-O- showed an increase in antimicrobial activity which may be due to stability in standard culture condition. The most promising compound is 8e, 3-O-decyl analogue, with the MIC of 0.5-2, 32-128 and 2-4 microg/mL against Gram-positive bacteria, Gram-negative bacteria and human pathogenic fungi, respectively. Regarding action mechanism, the antimicrobial activity is possibly due to the lipophilicity and disrupting ability of the analogues to the liposome membrane.

Acupuncture treatment for idiopathic Horner's syndrome in a dog.

A one-year-old female English Cocker Spaniel dog with idiopathic Horner's Syndrome is described. The specific clinical signs in this specimen were miosis, ptosis, enophthalmos, and prolapsed nictitans for 2 days following sudden onset. According to history taking, ophthalmic, neurological, and radiological examination, the patient was diagnosed with idiopathic Horner's syndrome. Manual acupuncture treatment was applied to the dog on local points two times in 2 days. The local acupoints were ST-4 (Di Chang) and GB-1 (Tong Zi Liao). The day after the initial acupuncture treatment, clinical signs related to idiopathic Horner's syndrome had almost disappeared. The day after the second treatment, specific clinical signs were completely absent. During this period, the dog did not receive any orthodox treatment. Thus, it is suggested that manual acupuncture might be an effective therapy for idiopathic Horner's syndrome.

Anticancer activity of 3-O-acyl and alkyl-(-)-epicatechin derivatives.

By changing the structure or replacing the gallate group of (-)-ECG, 3-O-acyl and alkyl-(-)-epicatechin derivatives were synthesized to be screen as anticancer agents using the MTT assay in vitro against cancer cell lines (PC3, SKOV3, U373MG). 3-O-Acyl and alkyl-(-)-epicatechin derivatives (4-25) exhibited better anticancer activity than (-)-ECG and specially, compounds 6-8, 17-19, which were modified aliphatic chains with moderate sizes (C8-C12) showed strong anticancer activity (IC50=6.4-31.2 microM). The introduction of an alkyloxy group on 3-O-hydroxyl instead of an acyloxy group significantly enhanced inhibitory activity. Consequently, the compound that showed the most potency as anticancer agents were 3-O-decyl-(-)-epicatechin (18) (IC50=8.9, 7.9, 6.4 microM against PC3, SKOV3, U373MG, respectively), which modified the appropriate lipophilic group on the C-3 hydroxyl as an alkyloxy group.

Antimicrobial activity of 3-O-acyl-(-)-epicatechin and 3-O-acyl-(+)-catechin derivatives.

As an exploratory investigation of antimicrobial promoting compounds, 3- O-acyl-(-)-epicatechins and 3- O-acyl-(+)-catechins possessing various aromatic groups and aliphatic chains of varying length from C4 to C16 for increasing lipophilicity were synthesized and tested for antimicrobial activities against Gram-positive, Gram-negative bacteria and fungi. The (-)-epicatechin and (+)-catechin derivatives comprised of aromatic groups increased activity and derivatives with acyl chain groups of carbon atoms in the close vicinity of C8 to C10 showed strong antimicrobial activity (MIC = 2 - 8 microg/ml) against Gram-positive bacteria and weak activity against fungi. However, the activity decreased when the carbon chain length of the substituents was too short (C4 to C6) or too long (C16). These results suggest that the presence of lipophilic substituents with moderate sizes might be crucial for the optimal antimicrobial activity.

Testing non-additivity of biological activity in a combinatorial library.

Combinatorial chemistry offers new opportunities to generate and analyze QSAR data. Traditional QSAR attempts to correlate activity with structure. With combinatorial chemistry, it is possible to correlate activity directly with the reagents used in a combinatorial library. If one can determine which reagents lead to the compounds of highest activities, it may then be possible to predict active compounds in virtual libraries of 10(6) to 10(10) compounds. This would greatly facilitate library design and provide confidence that the best compounds are being considered for synthesis. An important question is whether the activity of a product molecule can be considered as a sum of its components. This is referred to as additivity between reagents. If there is non-additivity, it is necessary to identify and include the non-additive terms in the model in order to improve QSAR models. Presented here are methods for developing QSAR models relating compound activity to reagents and a method for detecting the second effects of side-chain non-additivity. If the reagents in a library are shown to be additive in their contribution to activity, simple QSAR based on additive models can be applied confidently to reagents. Testing non-additivity can also guide the synthesis of the library. If the contributions are shown to be additive then the strategy for library synthesis may be shifted to include many reagents of a given type but not to make all combinations. The result is more efficient use of resources. In the analysis of percent inhibition data of a combinatorial library an additive model using reagents as descriptors yields a R(2) of 0.43. Application of this method is probably appropriate for HTS single point data while methods employing topological or pharmacophore based descriptors would be necessary to adequately model IC50 data.