Leukemic infiltration presenting as myocardial hypertrophy after complete remission of acute myeloid leukemia.

Here, we report a rare case of isolated leukemic infiltrate of the myocardium (extramedullary involvement) presenting as restrictive cardiomyopathy in a patient in complete remission of acute myeloid leukemia. It was evaluated with multimodality imaging studies (echocardiography and cardiac MRI) and further confirmed by pathology. The present case highlights the importance of maintaining a high degree of clinical suspicion when evaluating patients with progressive ventricular hypertrophy of unknown cause, including recognition of the potential involvement by recurrent hematologic malignancy.

Structure-based investigation into the functional roles of the extended loop and substrate-recognition sites in an endo-ß-1,4-D-mannanase from the Antarctic springtail, Cryptopygus antarcticus.

Endo-ß-1,4-D-mannanase from the Antarctic springtail, Cryptopygus antarcticus (CaMan), is a cold-adapted ß-mannanase that has the lowest optimum temperature (30°C) of all known ß-mannanases. Here, we report the apo- and mannopentaose (M5) complex structures of CaMan. Structural comparison of CaMan with other ß-mannanases from the multicellular animals reveals that CaMan has an extended loop that alters topography of the active site. Structural and mutational analyses suggest that this extended loop is linked to the cold-adapted enzymatic activity. From the CaMan-M5 complex structure, we defined the mannose-recognition subsites and observed unreported M5 binding site on the surface of CaMan.

Expression at 279†K, purification, crystallization and preliminary X-ray crystallographic analysis of a novel cold-active ß-1,4-D-mannanase from the Antarctic springtail Cryptopygus antarcticus.

The CaMan gene product from Cryptopygus antarcticus, which belongs to the glycoside hydrolase family 5 type ß-1,4-D-mannanases, has been crystallized using a precipitant solution consisting of 0.1 M Tris-HCl pH 8.5, 25%(w/v) polyethylene glycol 3350 by the microbatch crystallization method at 295 K. The CaMan protein crystal belonged to space group P212121, with unit-cell parameters a = 73.40, b = 83.81, c = 163.55 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be about 61.29%. CaMan-mannopentaose (M5) complex crystals that were isomorphous to the CaMan crystals were obtained using the same mother liquor containing 1 mM M5.

Cultivation at 6-10°C is an effective strategy to overcome the insolubility of recombinant proteins in Escherichia coli.

Protein expression in Escherichia coli at 15-25°C is widely used to increase the solubility of recombinant proteins. However, many recombinant proteins are insolubly expressed even at those low temperatures. Here, we show that recombinant proteins can be expressed as soluble forms by simply lowering temperature to 6-10°C without cold adapted chaperon systems. By using E. coli Rosetta-gami2(DE3), we obtained 1.8 and 0.9mg of Cryptopygus antarticus mannanase (CaMan) and cellulase (CaCel) from 1l culture grown at 6 and 10°C, respectively. Cultivation at 10°C also led to successful expression of EM3L7 (a lipase isolated from a metagenomic library) in a soluble form in E. coli BL21(DE3). Consequently, E. coli cultivation at 6-10°C is an effective strategy for overcoming a major hurdle of the inclusion body formation.

Metabarcoding of Fish Larvae in the Merbok River Reveals Species Diversity and Distribution Along its Mangrove Environment.

The Merbok River (north-west of Peninsular Malaysia) is a mangrove estuary that provides habitat for over 100 species of fish, which are economically and ecologically important. Threats such as habitat loss and overfishing are becoming a great concern for fisheries conservation and management. The identification of larval fish in this estuarine system is important to complement information on the adults. This is because the data could inform the spawning behaviour, reproductive biology, selection of nursery grounds and migration route of fish. Such information is invaluable for fisheries and aquatic environmental monitoring, and thus for their conservation and management. However, identifying fish larvae is a challenging task based only on morphology and even traditional DNA barcoding. To address this, DNA metabarcoding was utilised to detect the diversity of fish in the Merbok River. To complete the study, the fish larvae were collected at six sampling sites of the river. The extracted larval DNA was amplified for the Cytochrome Oxidase subunit 1 (COI) and 12S ribosomal RNA (12S rRNA) genes based on the metabarcoding approach using shotgun sequencing on the next-generation sequencing (NGS) Illumina MiSeq platform. Eighty-nine species from 65 genera and 41 families were detected, with Oryzias javanicus, Oryzias dancena, Lutjanus argentimaculatus and Lutjanus malabaricus among the most common species. The lower diversity observed from previous morphological studies is suggested to be mainly due to seasonal variation over the sampling period between the two methods and limited 12S rRNA sequences in current databases. The metabarcode data and a validation Sanger sequencing step using 15 species-specific primer pairs detected three species in common: Oryzias javanicus, Decapterus maruadsi and Pennahia macrocephalus. Several discrepancies observed between the two molecular approaches could be attributed to contaminants during sampling and DNA extraction, which could mask the presence of target species, especially when DNA from the contaminants is more abundant than the target organisms. In conclusion, this rapid and cost-effective identification method using DNA metabarcoding allowed the detection of numerous fish species from bulk larval samples in the Merbok River. This method can be applied to other sites and other organisms of interest.

DNA barcoding of brackish and marine water fishes and shellfishes of Sundarbans, the world's largest mangrove ecosystem.

The present study aims to apply a DNA barcoding tool through amplifying two mitochondrial candidate genes i.e., COI and 16S rRNA for accurate identification of fish, aquatic molluscs and crustaceans of Sundarbans mangrove wetland, to build a reference library of fish and shellfishes of this unique ecosystems. A total of 185 mitochondrial COI barcode sequences and 59 partial sequences of the 16S rRNA gene were obtained from 120 genera, 65 families and 21 orders of fish, crustaceans and molluscs. The collected samples were first identified by examining morphometric characteristics and then assessed by DNA barcoding. The COI and 16S rRNA sequences of fishes and crustaceans were clearly discriminated among genera in their phylogenies. The average Kimura two-parameter (K2P) distances of COI barcode sequences within species, genera, and families of fishes are 1.57±0.06%, 15.16±0.23%, and 17.79±0.02%, respectively, and for 16S rRNA sequences, these values are 1.74±.8%, 0.97±.8%, and 4.29±1.3%, respectively. The minimum and maximum K2P distance based divergences in COI sequences of fishes are 0.19% and 36.27%, respectively. In crustaceans, the K2P distances within genera, families, and orders are 1.4±0.03%, 17.73±0.15%, and 22.81±0.02%, respectively and the minimum and maximum divergences are 0.2% and 33.93%, respectively. Additionally, the present study resolves the misidentification of the mud crab species of the Sundarbans as Scylla olivacea which was previously stated as Scylla serrata. In case of molluscs, values of interspecific divergence ranges from 17.43% to 66.3% in the barcoded species. The present study describes the development of a molecular and morphometric cross-referenced inventory of fish and shellfish of the Sundarbans. This inventory will be useful in future biodiversity studies and in forming future conservation plan.

Zooplankton diversity monitoring strategy for the urban coastal region using metabarcoding analysis.

Marine ecosystems in urban coastal areas are exposed to many risks due to human activity. Thus, long-term and continuous monitoring of zooplankton diversity is necessary. High-throughput DNA metabarcoding has gained recognition as an efficient and highly sensitive approach to accurately describing the species diversity of marine zooplankton assemblages. In this study, we collected 30 zooplankton samples at about 2-week intervals for 1 year. Zooplankton diversity showing a typical four season pattern. Of the "total" and "common" zooplankton, we assigned 267 and 64 taxa. The cluster structure and seasonal diversity pattern were rough when only the "common" zooplankton was used. Our study examined how to maximize the benefits of metabarcoding for monitoring zooplankton diversity in urban coastal areas. The results suggest that to take full advantage of metabarcoding when monitoring a zooplankton community, it is necessary to carefully investigate potential ecosystem threats (non-indigenous species) through sufficient curation rather than disregarding low-abundance operational taxonomic units.

DNA barcoding of coral reef fishes from Chuuk State, Micronesia.

The fish diversity of Chuuk Micronesia is currently under threat due to rapid changes in the coral reef ecosystem. Thus, accurate fish identification using DNA barcodes is fundamental for exploring species biodiversity and resource protection. In this study, we analyzed 162 fish mitochondrial DNA cytochrome c oxidase I (COI) barcodes from Chuuk Micronesia. Consequently, we identified 95 species from 53 genera in 26 families and seven orders. The average Kimura 2-parameter genetic distances within species, genera, families, and orders were calculated as 0.17%, 11.78%, 15.63%, and 21.90%, respectively. Also, we have utilized DNA barcodes to perform genetic divergence and phylogenetic analysis of families recognized as dominant groups in Chuuk State. Our findings confirm that DNA barcodes using COI are an effective approach in identifying coral reef fish species. We anticipate that the results of this study will provide baseline data for the protection of coral reef fish biodiversity at Chuuk Micronesia.

Effects of Balneotherapy in Jeju Magma-Seawater on Knee Osteoarthritis Model.

Balneotherapy is a common non-pharmacological treatment for osteoarthritis (OA), however, the efficacy is controversial in knee OA. Jeju magma-seawater (JMS) has high contents of various minerals, which has anti-inflammatory and antioxidant properties via an oral route. Thus, we examined the effects of JMS bathing on knee OA and the combination effects with diclofenac sodium as an anti-inflammatory drug. Knee OA was induced by transection of the anterior cruciate ligament and the partial meniscectomy in rat. The rats were administered subcutaneously saline or diclofenac sodium in saline, followed by bathing in thermal distilled water or JMS for 8 weeks. The model represented the characteristic changes of the cartilage degradation, osteophyte formation and synovial inflammation, and the relevant symptoms of the joint swelling and stiffness. However, the JMS bathing reduced the joint thickness and improved the mobility. It also contributed to a well-preserved tissue supported by increases in bone mineral density of the joint and decreases in Mankin scores in the cartilages. The effects involved anti-inflammation, chondroprotection, anti-apoptosis, and chondrogenesis. Overall, the JMS bathing in combination with diclofenac sodium showed a similar trend associated with synergic effects. It suggests that JMS bathing can be promising for a clinical use in knee OA.

Molecular cloning, characterization and expression analysis of peroxiredoxin 6 from disk abalone Haliotis discus discus and the antioxidant activity of its recombinant protein.

Peroxiredoxins (Prxs) play an important role against various oxidative stresses and intra-cellular signal transduction. Peroxiredoxin 6 (PrxVI) was identified from the disk abalone Haliotis discus discus cDNA library and named HdPrxVI. The full length cDNA of HdPrxVI was 1457 bp with a 654 bp open reading frame (ORF) encoding 218 amino acids. The predicted molecular mass and estimated isoelectric point (pI) of HdPrxVI were 24 kDa and 7.3, respectively. The deduced amino acid sequence demonstrated the greatest degree (72.4%) of identity with Crassostrea gigas PrxVI. The conserved peroxidase catalytic center (42PVCTTE47) with a conserved cysteine residue (Cys44) and a catalytic center for PLA2 activity (27GGSWA31) were observed in the sequence, indicating that it is a member of 1-Cys Prx. Real time PCR results revealed that HdPrxVI mRNA is constitutively expressed in all tissues in a tissue-specific manner. During exposure to haemorrhagic septicaemia virus (VHSV), HdPrxVI mRNA transcription was down-regulated in the gill, suggesting that the abalone responded to the viral infection quickly, and HdPrxVI played a physiological role against virus-induced oxidative stress. The purified recombinant HdPrxVI, together with dithiothreitol (DTT), was shown to scavenge H2O2 in human leukemia THP-1 cells and provided protection against H2O2-induced apoptosis.

Structure, evolution and phylogenetic informativeness of eelpouts (Cottoidei: Zoarcales) mitochondrial control region sequences.

Control region (CR) is a major non-coding domain of mitochondrial DNA in vertebrates which contains the promoters for replication and transcription of mitochondrial genome along with the binding sites for metabolic machinery and, hence, is a vital element for the integrity of mitochondrial genome as a biological replicator. The origin and diversity of structural elements within CR have been intensively studied in recent years with the involvement of new diverse taxa. In this paper, we provide new data on the nucleotide and structural patterns of CR evolution and phylogenetic suitability among eelpouts (Cottoidei: Zoarcales). To achieve this, we carried out a comparative phylogenetic and structural analysis of 29 CR sequences belonging to the long shanny Stichaeus grigorjewi together with nine sequences of other eelpouts taxa representing four families in contrast to mitochondrial protein-coding fragments. The CR organization within S. grigorjewi, as well as in all other eelpouts, is consistent with the common three-domain structure known from most vertebrates. We found a hidden CR variation constrains on the landscape level and a lack of nucleotide saturation. Finally, our results demonstrate the advantage of the length variation in CR sequences for phylogenetic reconstructions among eelpouts.

Recombinant perlucin nucleates the growth of calcium carbonate crystals: molecular cloning and characterization of perlucin from disk abalone, Haliotis discus discus.

Perlucin is well known as an important functional protein regulating pearl formation and shell biomineralization. In this study, we cloned the perlucin gene from the abalone Haliotis discus discus cDNA library. The full-length cDNA of the abalone H. discus discus perlucin gene consisted of 1038 bp nucleotides, encoding a putative signal peptide of 22 amino acids and a mature protein of 129 amino acids, which shared 55% identity with the homologous protein in greenlip abalone. The mature protein coding sequence was inserted into pMal-c2X expression vector and it expressed the recombinant protein in E. coli (Rosetta-gammi DE3). The maltose binding protein (MBP) fusion perlucin successfully promoted calcium carbonate precipitation and directed calcite crystal morphological modification. The successful expression of active recombinant perlucin suggested that recombinant perlucin gene transfer has the capability by color modification to improve the pearl's value. In the view of molecular structure, perlucin was a typical C-type lectin, which contained one highly conserved carbohydrate recognition domain. Reverse transcription polymerase chain reaction (RT-PCR) results showed that perlucin gene was expressed not only in the mantle, but also in the gill and digestive tract. Further characterization of perlucin in abalone non-self recognition and disease resistance is promising and anticipated.

Molecular cloning and characterization of a novel cold-active beta-1,4-D-mannanase from the Antarctic springtail, Cryptopygus antarcticus.

A cDNA encoding a beta-1,4-d-mannanase (CaMan) was identified among the expressed sequence tags (ESTs) of the Antarctic springtail, Cryptopygus antarcticus. The open reading frame consisted of 1149 bp encoding 382 amino acids with a putative signal peptide. Amino acid sequence comparison with other mannanases indicated that CaMan likely belongs to subfamily 10 of the glycoside hydrolase family 5, together with mollusc beta-mannanases. CaMan shows typical features of a cold-active enzyme: it has a high frequency of polar residues such as Asn, Gln, and Ser, and a low frequency of hydrophobic residues as well as a low ratio of Arg/(Arg+Lys) compared to the mesophilic beta-mannanases. When CaMan was fused with the thioredoxin gene in pET-32a(+), expressed in E. coli Rosetta-gami (DE3), and purified after thrombin treatment, catalytically active enzyme was obtained. CaMan has high specific activity (416.3 U/mg) toward locust bean gum at an optimal temperature of 30 degrees C and an optimal pH of 3.5. Its optimal temperature is the lowest among those of the known mannanases and the optimal pH is also the lowest except those of fungi. Even at 0-5 degrees C, this enzyme retained 20-40% of its maximum activity. Divalent metal ions such as Ca(2+), Mg(2+), Cu(2+), and Zn(2+) enhanced the enzyme activity, but Mn(2+), Hg(2+), and Ag(+) inhibited activity. This study represents the first record of a beta-mannanase from an arthropod and provides a new source of carbohydrate hydrolysis enzyme with novel characteristics.

Genetic identification and molecular phylogeny of Pseudodiaptomus species (Calanoida, Pseudodiaptomidae) in Korean waters.

Five species of the genus Pseudodiaptomus, P. inopinus, P. poplesia, P. marinus, P. nihonkaiensis, and P. sp. occur in Korea nearshore waters. Although the four species except for P. sp. have been classified into Lobus and Ramosus groups, two for each group, based on morphological characters, this classification had yet to be confirmed by molecular characters. Here, we determined molecular characters and phylogenetic relationships of the five species in order to evaluate the morphology-based groupings and the species identifications. For this, a 625-bp DNA region of the mitochondrial gene cytochrome oxidase subunit I (mtCOI) was sequenced and compared among the species. Intraspecific variation of the sequences is less than 0.6%, while interspecific variation ranges from 17.6-26.7%, indicating every species, including P. sp., is a genetically distinct, valid species. Phylogenetic trees of the mtCOI DNA reveal that the Lobus-group species including P. inopinus and P. poplesia form a well-supported clade and that P. sp. belongs to this group. On the other hand, the Ramosus group clade consisting of P. marinus and P. nihonkaiensis is not well supported by bootstrap analyses, suggesting that further evaluation of the validity of this group assignment is needed.

O-Methylation of flavonoids using DnrK based on molecular docking.

Omicron-Methylation is a common substitution reaction found in microbes as well as in mammalians. Some of the Omicron-methyltransferases (OMTs) have broad substrate specificity and could be used to methylate various compounds. DnrK from Streptomyces peucetius encodes an anthracycline 4-Omicron-methyltransferase, which uses carminomycin as a substrate, and its crystal structure has been determined. Molecular docking experiments with DnrK using various flavonoids were successfully conducted, and some of the flavonoids such as apigenin and genistein were predicted to serve as substrates. Based on these results, Omicron-methylations of various flavonoids with the DnrK were successfully carried out. The methylation position was determined to be at the hydroxyl group of C7. Important amino acid residues for the enzymatic reaction of DnrK with apigenin could be identified using site-directed mutagenesis. Molecular docking could be useful to predict the substrate specificity ranges of other OMTs.

Genetic and Structural Characterization of a Thermo-Tolerant, Cold-Active, and Acidic Endo-ß-1,4-glucanase from Antarctic Springtail, Cryptopygus antarcticus.

The CaCel gene from Antarctic springtail Cryptopygus antarcticus codes for a cellulase belonging to the glycosyl hydrolase family 45 (GHF45). Phylogenetic, biochemical, and structural analyses revealed that the CaCel gene product (CaCel) is closely related to fungal GHF45 endo-ß-1,4-glucanases. The organization of five introns within the open reading frame of the CaCel gene indicates its endogenous origin in the genome of the species, which suggests the horizontal transfer of the gene from fungi to the springtail. CaCel exhibited optimal activity at pH 3.5, retained 80% of its activity at 0-10 °C, and maintained a half-life of 4 h at 70 °C. Based on the structural comparison between CaCel and a fungal homologue, we deduced the structural basis for the unusual characteristics of CaCel. Under acidic conditions at 50 °C, CaCel was effective to digest the green algae (Ulva pertusa), suggesting that it could be exploited for biofuel production from seaweeds.

Bathing effects of east saline groundwater concentrates on allergic (atopic) dermatitis-like skin lesions induced by 2,4-dinitrochlorobenzene in hairless mice.

In the present study, it was evaluated whether east saline groundwater concentration solution (ESGWc) exerted a favorable inhibitory effect on 2,4-dinitrochlorobenzene (DNCB)-induced allergic/atopic-like dermatitis (AD). AD was induced and boosted by sensitization with DNCB via topical application on the dorsal back skins. Mice with DNCB-induced AD were bathed in 100-, 200- and 400-fold diluted ESGWc. After 6 weeks bathing, changes to body weight, clinical skin severity scores, scratching behavior, serum total immunoglobulin (Ig)E levels, submandibular lymph node and spleen weights, splenic cytokine levels, skin cytokine mRNA expressions, antioxidant defense systems and superoxide anion productions were recorded to determine the effects of bathing on the histopathology of dorsal back skin tissues. All DNCB-induced mice demonstrated that the induction of AD through IgE-mediated hypersensitivities, oxidative stresses, activation of MMP and apoptosis of keratinocytes resulted in no significant differences in body weight between the different groups at each time point following initial sensitization. However, markers of DNCB-induced AD were significantly inhibited (P<0.05) in a concentration-dependent manner following bathing in all concentrations of ESGWc. The results obtained in the present study suggest that bathing in ESGWc may have favorable protective effects against DNCB-induced AD due to favorable systemic and local immunomodulatory effects, active cytoprotective anti-apoptotic effects, inhibitory effects of matrix metalloproteinase activity, and anti-inflammatory and antioxidative effects.

Molecular cloning and characterization of Mn-superoxide dismutase from disk abalone (Haliotis discus discus).

The mitochondrial enzyme manganese superoxide dismutase (mitMn-SOD) is one of the antioxidant enzymes involved in cellular defense against oxidative stress and catalyzes the conversion of O(2)(-) into the stabler H(2)O(2). In this study, a putative gene encoding Mn-SOD from disk abalone (Haliotis discus discus, aMn-SOD) was cloned, sequenced, expressed in Escherichia coli K12(TB1) and the protein was purified using pMAL protein purification system. Sequencing resulted ORF of 681 bp, which corresponded to 226 amino acids. The protein was expressed in soluble form with molecular weight of 68 kDa including maltose binding protein and pI value of 6.5. The fusion protein had 2781 U/mg activity. The optimum temperature of the enzyme was 37 degrees C and it was active in a range of acidic pH (from 3.5 to 6.5). The enzyme activity was reduced to 50% at 50 degrees C and completely heat inactivated at 80 degrees C. The alignment of aMn-SOD amino acid sequence with Mn-SODs available in NCBI revealed that the enzyme is conserved among animals with higher than 30% identity. In comparison with human mitMn-SOD, all manganese-binding sites are also conserved in aMn-SOD (H28, H100, D185 and H189). aMn-SOD amino acid sequence was closer to that of Biomphalaria glabrata in phylogenetic analysis.

The F type mitochondrial genome of hard-shelled mussel: Mytilus coruscus (Mytiloida, Mytilidae).

We determined the complete mitochondrial genome of the female hard-shelled mussel Mytilus coruscus (Gould, 1869) (F type). The F type genome is composed of 16,642 bp in length including 12 protein coding genes (PCGs), 2 rRNA and 23 tRNA with the same gene content and order as the other Mytilus species which is characterized by the absence of the ATPase8 gene and addition of tRNA(Met) (AUA). The nucleotide composition of the genome shows that the percentage of A+T (63.2%) is higher than those of M. edulis complex species (M. edulis, M. galloprovincialis, M. trossulus) but lower than that of M. californianus. The F type mitochondrial genome of M. coruscus will provide useful information on the evolutionary history of the Mytilus species in the family Mitilidae.

Complete mitochondrial genome of the Yellownose skate: Zearaja chilensis (Rajiformes, Rajidae).

The complete sequence of mitochondrial DNA of a Yellownose skate, Zearaja chilensis was determined for the first time. It is 16,909 bp in length covering 2 rRNA, 22 tRNA and 13 protein coding genes with the identical gene order and structure as those of other Rajidae species. The nucleotide of L-strand is composed of low G (14.3%), and slightly high A + T (58.9%) nucleotides. The strong codon usage bias against the use of G (6.0%) is found at the third codon positions. Twelve of the 13 protein coding genes use ATG as the start codon while COX1 starts with GTG. As for the stop codon, only ND4 shows an incomplete stop codon TA. This is the first report of the mitogenome for a species in the genus Zearaja, providing a valuable source of genetic information on the evolution of the family Rajidae and the genus Zearaja as well as for establishment of a sustainble fishery management plan of the species.