Purification of lysozyme from chicken egg white using diatom frustules.

A nontoxic chromatographic matrix, with low cost and high adsorption capacity, is always a major goal for therapeutic protein purification. In this study, the frustules from two cultured diatoms, Nitzschia bilobata (AQ1) and Psammodictyon panduriforme (NP), were investigated as cation exchange materials for lysozyme purification from chicken egg white. The surface area and cation exchange capacity of frustules were about 400 m2/g and 140 µmol/mL for AQ1, 390 m2/g and 130 µmol/mL for NP. The optimal pH was 9 for adsorption. Through batch purification, the lysozyme recovery was 86% with a purity of 95% by AQ1 frustules, which was higher than that by NP frustules (82% with a purity of 90%). In the flow-through system, the purity using AQ1 frustules notably increased to 99%, higher than the result of 91% using NP frustules. Diatom frustules from AQ1 are more effective and could be an alternative chromatographic matrix for lysozyme purification.

Coral lipid bodies as the relay center interconnecting diel-dependent lipidomic changes in different cellular compartments.

Lipid bodies (LBs) in the coral gastrodermal tissues are key organelles in the regulation of endosymbiosis and exhibit a diel rhythmicity. Using the scleractinian Euphyllia glabrescens collected across the diel cycle, we observed temporally dynamic lipid profiles in three cellular compartments: host coral gastrodermal cells, LBs, and in hospite Symbiodinium. Particularly, the lipidome varied over time, demonstrating the temporally variable nature of the coral-Symbiodinium endosymbiosis. The lipidome-scale data highlight the dynamic, light-driven metabolism of such associations and reveal that LBs are not only lipid storage organelles but also act as a relay center in metabolic trafficking. Furthermore, lipogenesis in LBs is significantly regulated by coral hosts and the lipid metabolites within holobionts featured predominantly triacylglycerols, sterol esters, and free fatty acids. Given these findings through a time-varied lipidome status, the present study provided valuable insights likely to be crucial to understand the cellular biology of the coral-Symbiodinium endosymbiosis.

Therapeutic microRNA Delivery Strategies with Special Emphasis on Cancer Therapy and Tumorigenesis: Current Trends and Future Challenges.

BACKGROUND: Over the decade, miRNAs are the most important molecules for the biopharmaceutical industry due to their relation with several human diseases. Presently, the phase-II clinical trial has been initiated for the first miRNA-based therapeutics ("Miravirsen") to treat HCV infection. It has been expected that many more miRNA-based therapeutics will enter the clinical trials. Therefore, it is important to develop different kinds of novel delivery systems with better efficacy and more efficiency, but fewer side effects. METHODS: We have undertaken a structured search of bibliographic databases for peer-reviewed research literature to solve our review question. Literature survey was performed widely to write this review article. RESULTS: In this review, we have discussed the various types of miRNA delivery systems such as viral vectors, lipid-based systems, nanocarriers, and LNA-customized DNA delivery without any delivery-mediated agent. Current status, technical support, and the future challenges for miRNA-based delivery are also discussed. CONCLUSION: Recent development and understanding of miRNA had shown the therapeutic potentiality of miRNA.

Pinnisterols A-C, New 9,11-Secosterols from a Gorgonian Pinnigorgia sp.

Three new 9,11-secosterols, pinnisterols A-C (1-3), were isolated from a gorgonian coral Pinnigorgia sp., collected off the waters of Taiwan. The structures of these compounds were elucidated on the basis of spectroscopic methods. The new sterols 1 and 3 displayed significant inhibitory effects on the generation of superoxide anions and the release of elastase by human neutrophils, and sterol 1 was found to show moderate cytotoxicity in hepatic stellate cells (HSCs).

Crystallization and X-ray diffraction of virus-like particles from a piscine betanodavirus.

Dragon grouper nervous necrosis virus (DGNNV), a member of the genus Betanodavirus, causes high mortality of larvae and juveniles of the grouper fish Epinephelus lanceolatus. Currently, there is no reported crystal structure of a fish nodavirus. The DGNNV virion capsid is derived from a single open reading frame that encodes a 338-amino-acid protein of approximately 37 kDa. The capsid protein of DGNNV was expressed to form virus-like particles (VLPs) in Escherichia coli. The VLP shape is T = 3 quasi-symmetric with a diameter of ∼38 nm in cryo-electron microscopy images and is highly similar to the native virion. In this report, crystals of DGNNV VLPs were grown to a size of 0.27 mm within two weeks by the hanging-drop vapour-diffusion method at 283 K and diffracted X-rays to ∼7.5 Šresolution. In-house X-ray diffraction data of the DGNNV VLP crystals showed that the crystals belonged to space group R32, with unit-cell parameters a = b = 353.00, c = 800.40 Å, α = ß = 90, γ = 120°. 23 268 unique reflections were acquired with an overall Rmerge of 18.2% and a completeness of 93.2%. Self-rotation function maps confirmed the fivefold, threefold and twofold symmetries of the icosahedron of DGNNV VLPs.

Genome sequences characterizing five mutations in RNA polymerase and major capsid of phages ϕA318 and ϕAs51 of Vibrio alginolyticus with different burst efficiencies.

BACKGROUND: The burst size of a phage is important prior to phage therapy and probiotic usage. The efficiency for a phage to burst its host bacterium can result from molecular domino effects of the phage gene expressions which dominate to control host machinery after infection. We found two Podoviridae phages, ϕA318 and ϕAs51, burst a common host V. alginolyticus with different efficiencies of 72 and 10 PFU/bacterium, respectively. Presumably, the genome sequences can be compared to explain their differences in burst sizes. RESULTS: Among genes in 42.5 kb genomes with a GC content of 43.5%, 16 out of 47 open-reading frames (ORFs) were annotated to known functions, including RNA polymerase (RNAP) and phage structure proteins. 11 strong phage promoters and three terminators were found. The consensus sequence for the new vibriophage promoters is AATAAAGTTGCCCTATA, where the AGTTG bases of -8 through -12 are important for the vibriophage specificity, especially a consensus T at -9 position eliminating RNAP of K1E, T7 and SP6 phages to transcribe the genes. ϕA318 and ϕAs51 RNAP shared their own specific promoters. In comparing ϕAs51 with ϕA318 genomes, only two nucleotides were deleted in the RNAP gene and three mutating nucleotides were found in the major capsid genes. CONCLUSION: Subtle analyses on the residue alterations uncovered the effects of five nucleotide mutations on the functions of the RNAP and capsid proteins, which account for the host-bursting efficiency. The deletion of two nucleotides in RNAP gene truncates the primary translation due to early stop codon, while a second translational peptide starting from GTG just at deletion point can remediate the polymerase activity. Out of three nucleotide mutations in major capsid gene, H53N mutation weakens the subunit assembly between capsomeres for the phage head; E313K reduces the fold binding between ß-sheet and Spine Helix inside the peptide.

Nanoparticles as 'smart' pharmaceutical delivery.

Pharmaceuticals in conjunction with nanoparticle delivery systems are growing towards new heights. The aim of this review is to gain a thorough understanding of different types and characteristics of nanoparticle based delivery systems, important properties of delivery systems, pharmaceutical ingredient loading and release in the nanoparticle delivery systems. In this review, we have also highlighted about the promising pharmaceutical deliveries like brain targeted delivery, ocular delivery, oral delivery, dermal and transdermal delivery, cancer chemotherapy, vaccine delivery, nucleic acids delivery and delivery system coupling to implants. A snapshot of the nanoparticle mediated drug deliveries which are commercially available and ongoing clinical trials have been provided.

Genome-wide characterization of Vibrio phage φpp2 with unique arrangements of the mob-like genes.

BACKGROUND: Vibrio parahaemolyticus is associated with gastroenteritis, wound infections, and septicemia in human and animals. Phages can control the population of the pathogen. So far, the only one reported genome among giant vibriophages is KVP40: 244,835 bp with 26% coding regions that have T4 homologs. Putative homing endonucleases (HE) were found in Vibrio phage KVP40 bearing one segD and Vibrio cholerae phage ICP1 carrying one mobC/E and one segG. RESULTS: A newly isolated Vibrio phage φpp2, which was specific to the hosts of V. parahaemolyticus and V. alginolyticus, featured a long nonenveloped head of ~90 × 50 nm and tail of ~110 nm. The phage can survive at 50°C for more than one hour. The genome of the phage φpp2 was sequenced to be 246,421 bp, which is 1587 bp larger than KVP40. 383 protein-encoding genes (PEGs) and 30 tRNAs were found in the phage φpp2. Between the genomes of φpp2 and KVP40, 254 genes including 29 PEGs for viral structure were of high similarity, whereas 17 PEGs of KVP40 and 21 PEGs of φpp2 were unmatched. In both genomes, the capsid and tail genes have been identified, as well as the extensive representation of the DNA replication, recombination, and repair enzymes. In addition to the three giant indels of 1098, 1143 and 3330 nt, ϕpp2 possessed unique proteins involved in potassium channel, gp2 (DNA end protector), tRNA nucleotidyltransferase, and mob-type HEs, which were not reported in KVP40. The φpp2 PEG274, with strong promoters and translational initiation, was identified to be a mobE type, flanked by NrdA and NrdB/C homologs. Coincidently, several pairs of HE-flanking homologs with empty center were found in the phages of Vibrio phages φpp2 and KVP40, as well as in Aeromonas phages (Aeh1 and Ae65), and cyanophage P-SSM2. CONCLUSIONS: Vibrio phage φpp2 was characterized by morphology, growth, and genomics with three giant indels and different types of HEs. The gene analysis on the required elements for transcription and translation suggested that the φpp2 PEG274 was an active mobE gene. The phage was signified to be a new species of T4-related, differing from KVP40.

In silico analyses of COMT, an important signaling cascade of dopaminergic neurotransmission pathway, for drug development of Parkinson's disease.

Catechol-O-methyltransferase (COMT) has a vital role for degradation of dopamine, a neurotransmitter, and this dopamine performs an important function in our mental and physical health. The scarcity of dopamine may lead to Parkinson's disease, and inhibition of COMT can stop dopamine metabolism. Here, we have carried out genomics and proteomics analyses of COMT in order to facilitate new inhibitors of COMT. For genomics analyses, we performed codon composition investigation of COMT gene which shows A+T content which is 53.3 %. For proteomics analyses, conservation patterns and residues (highly conserved amino acids GLU64, LEU65, GLY66, CYS69, GLY70, ALA77, GLU90, THR99, SER119, ASP136, LEU140, ASP141, THR164, ASN170, VAL171, and ILE172), binding grooves, binding pockets, binding and conformation with substrate, evaluation of amino acid composition (15 % leucine rich), high scoring hydrophobic segments, high scoring transmembrane segments, tandem and periodic repeats, and disulfide bonds (three numbers), sequence logos (maximum stack height of 3 b and minimum stack height of <0.5 b) have been investigated for COMT protein. Furthermore, using COMT sequences of different species (class Mammalia, class Amphibia, and class Pisces), a phylogenetic tree has been constructed to examine the evolutionary relationship among different species.

Characterization of a new phage, termed ϕA318, which is specific for Vibrio alginolyticus.

Vibrio alginolyticus is an opportunistic pathogen of animals and humans; its related strains can also produce tetrodotoxin and hemolysins. A new phage, ϕA318, which lysed its host V. alginolyticus with high efficiency, was characterized. The burst size of ϕA318 in V. alginolyticus was 72 PFU/bacterium at an MOI of 1 at room temperature; the plaque size was as large as 5 mm in diameter. Electron microscopy (EM) of the phage particles revealed a 50- to 55-nm isomorphous icosahedral head with a 12-nm non-contractile tail, similar to the T7-like phages of the family Podoviridae. Phylogenetic analysis based on complete sequences of the DNA-directed RNA polymerase gene revealed that ϕA318 had 28-47% amino acid identity to enterobacteria phages T7 and SP6, and other Vibrio phages, and the phylogenetic distance suggested that ϕA318 could be classified as a new T7-like bacteriophage. Nevertheless, several motifs in the ϕA318 phage RNA polymerase were highly conserved, including DFRGR (T7-421 motif), DG (T7-537 motif), PSEKPQDIYGAVS (T7-563 motif), RSMTKKPVMTL PYGS (T7-627 motif), and HDS (T7-811 motif). Genetic analysis indicated that phage ϕA318 is not a thermostable direct hemolysin producer. The results suggest that the MOI should be higher than 0.1 to prevent the chance of hemolysin production by the bacteria before they are lysed by the phage.

Briaviodiol A, a new Cembranoid from a soft coral Briareum violacea.

A new cembrane diterpenoid, briaviodiol A (1), has been isolated from a soft coral Briareum violacea. The structure of 1 was determined by spectroscopic methods and further confirmed by a single-crystal X-ray diffraction analysis.

Excavatoids O and P, new 12-hydroxybriaranes from the octocoral Briareum excavatum.

Two new 12-hydroxybriarane diterpenoids, designated as excavatoids O (1) and P (2), were isolated from the octocoral Briareum excavatum. The structures of briaranes 1 and 2 were established on the basis of extensive spectral data analysis. Excavatoid P (2) is the first metabolite which possesses a 6ß -chlorine atom in briarane analogues.

Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles.

The virus-like particle (VLP) assembled from capsid subunits of the dragon grouper nervous necrosis virus (DGNNV) is very similar to its native T = 3 virion. In order to investigate the effects of four cysteine residues in the capsid polypeptide on the assembly/dissociation pathways of DGNNV virions, we recombinantly cloned mutant VLPs by mutating each cysteine to destroy the specific disulfide linkage as compared with thiol reduction to destroy all S-S bonds. The mutant VLPs of C187A and C331A mutations were similar to wild-type VLPs (WT-VLPs); hence, the effects of Cys187 and Cys331 on the particle formation and thermostability were presumably negligible. Electron microscopy showed that either C115A or C201A mutation disrupted de novo VLP formation significantly. As shown in micrographs and thermal decay curves, beta-mercaptoethanol-treated WT-VLPs remained intact, merely resulting in lower tolerance to thermal disruption than native WT-VLPs. This thiol reduction broke disulfide linkages inside the pre-fabricated VLPs, but it did not disrupt the appearance of icosahedrons. Small dissociated capsomers from EGTA-treated VLPs were able to reassemble back to icosahedrons in the presence of calcium ions, but additional treatment with beta-mercaptoethanol during EGTA dissociation resulted in inability of the capsomers to reassemble into the icosahedral form. These results indicated that Cys115 and Cys201 were essential for capsid formation of DGNNV icosahedron structure in de novo assembly and reassembly pathways, as well as for the thermal stability of pre-fabricated particles.

Involvement of the residues of GSKIP, AxinGID, and FRATtide in their binding with GSK3beta to unravel a novel C-terminal scaffold-binding region.

The specificity and regulation of GSK3beta are thought to involve in the docking interactions at core kinase domain because of the particular amino acid residues. Recent X-ray diffraction studies illuminated the relative binding residues on AxinGID and FRATtide for GSK3beta docking and appeared that GSK3beta Val267Gly (V267G) and Tyr288Phe (Y288F) could distinguish the direct interaction between AxinGID and FRATtide. In order to explore the mode that involved the binding of GSKIP to GSK3beta and compare it with that of AxinGID and FRATtide, we pinpointed the binding sites of GSKIP to GSK3beta through the single-point mutation of four corresponding sites within GSK3beta (residues 260-300) as scaffold-binding region I (designated SBR-I(260-300)). Our data showed that these three binding proteins shared similar binding sites on GSK3beta. We also found that the binding of GSK3beta V267G mutant to GSKIP and AxinGID, but not that of Y288F mutant (effect on FRATtide), was affected. Further, based on the simulation data, the electron-density map of GSKIPtide bore closer similarity to the map AxinGID than to that of FRATtide. Interestingly, many C-terminal helix region point-mutants of GSK3beta L359P, F362A, E366K, and L367P were able to eliminate the binding with FRATtide, but not AxinGID or GSKIP. In addition, CABYR exhibited a unique mode in binding to C-terminal helix region of GSK3beta. Taken together, our data revealed that in addition to the core kinase domain, SBR-I(260-300), another novel C-terminus helix region, designated SBR-II(339-383), also appeared to participate in the recognition and specificity of GSK3beta in binding to other specific proteins.

Ammonium-dependent sodium uptake in mitochondrion-rich cells of medaka (Oryzias latipes) larvae.

In this study, a scanning ion-selective electrode technique (SIET) was applied to measure H(+), Na(+), and NH(4)(+) gradients and apparent fluxes at specific cells on the skin of medaka larvae. Na(+) uptake and NH(3)/NH(4)(+) excretion were detected at most mitochondrion-rich cells (MRCs). H(+) probing at MRCs revealed two group of MRCs, i.e., acid-secreting and base-secreting MRCs. Treatment with EIPA (100 muM) blocked 35% of the NH(3)/NH(4)(+) secretion and 54% of the Na(+) uptake, suggesting that the Na(+)/H(+) exchanger (NHE) is involved in Na(+) and NH(3)/NH(4)(+) transport. Low-Na(+) water (<0.001 mM) or high-NH(4)(+) (5 mM) acclimation simultaneously increased Na(+) uptake and NH(3)/NH(4)(+) excretion but decreased or even reversed the H(+) gradient at the skin and MRCs. The correlation between NH(4)(+) production and H(+) consumption at the skin surface suggests that MRCs excrete nonionic NH(3) (base) by an acid-trapping mechanism. Raising the external NH(4)(+) significantly blocked NH(3)/NH(4)(+) excretion and Na(+) uptake. In contrast, raising the acidity of the water (pH 7 to pH 6) enhanced NH(3)/NH(4)(+) excretion and Na(+) uptake by MRCs. In situ hybridization and real-time PCR showed that the mRNAs of the Na(+)/H(+) exchanger (slc9a3) and Rhesus glycoproteins (Rhcg1 and Rhbg) were colocalized in MRCs of medaka, and their expressions were induced by low-Na(+) acclimation. This study suggests a novel Na(+)/NH(4)(+) exchange pathway in apical membranes of MRCs, in which a coupled NHE and Rh glycoprotein is involved and the Rh glycoprotein may drive the NHE by generating H(+) gradients across apical membranes of MRCs.

Anticancer drugs discovery and development from marine organism.

The chemical and biological diversity of the different marine evolutionary group is endless and therefore, this is an amazing resource for the discovery of new anticancer drugs. Comprising 34 of the 36 Phyla of life, marine ecosystems are indeed our last genetic diversity and biotechnological boundary; terrestrial systems possess only 17 Phyla. Sponges, coelenterates and microorganisms are the foremost resources of therapeutic compounds. Algae, echinoderms, tunicates, mollusks, bryozoans are also the sources of anticancer drugs from marine resources. We highlight the past and current status of marine anticancer pharmacology using different marine groups.

Recent advances of fluorescent technologies for drug discovery and development.

Recent progresses in the development of fluorescent technologies become a reliable device for drug discovery research. The fluorescence tools offer attractive options for an opportunity to visualize the effects of drug candidates in the cells. The fluorescent tools, such as fluorescent protein, are regularly used in a range of drug discovery processes. A better understanding and use of fluorescent technologies facilitate drug discovery research faster and can open up new applications. Therefore, we have provided information about some new generation fluorescent reagents (GFP and fluorophores). This review illustrates how fluorescent technologies and fluorescent tools are contributing to the drug discovery process mainly high-throughput screening (HTS), disease mechanism based target discovery, disease-genes-based target discovery, 'target classes' based target candidate discovery, physiology-based drug discovery, genomics-based drug discovery, target validation and their future perspectives.

Capnellene, a natural marine compound derived from soft coral, attenuates chronic constriction injury-induced neuropathic pain in rats.

BACKGROUND AND PURPOSE: Natural compounds obtained from marine organisms have received considerable attention as potential sources of novel drugs for treatment of human inflammatory diseases. Capnellene, isolated from the marine soft coral Capnella imbricate, 4,4,6a-trimethyl-3-methylene-decahydro-cyclopenta[]pentalene-2,3a-diol (GB9) exhibited anti-inflammatory actions on activated macrophages in vitro. Here we have assessed the anti-neuroinflammatory properties of GB9 and its acetylated derivative, acetic acid 3a-hydroxy-4,4,6a-trimethyl-3-methylene-decahydro-cyclopenta[]pentalen-2-yl ester (GB10). EXPERIMENTAL APPROACH: Effects of GB9 or GB10 on the expression of inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in interferon-gamma (IFN-gamma)-stimulated mouse microglial BV2 cells were measured by Western blot. The in vivo effects of these compounds were examined in the chronic constriction injury (CCI) rat model of neuropathic pain, measuring thermal hyperalgesia, and microglial activation and COX-2 protein in lumbar spinal cord, by immunohistochemistry. KEY RESULTS: In BV2 cells, GB9 and GB10 inhibited the expression of iNOS and COX-2, stimulated by IFN-gamma. Intrathecal administration of GB9 and GB10 inhibited CCI-induced nociceptive sensitization and thermal hyperalgesia in a dose-dependent manner. Intraperitoneal injection of GB9 inhibited CCI-induced thermal hyperalgesia and also inhibited CCI-induced activation of microglial cells and up-regulation of COX-2 in the dorsal horn of the lumbar spinal cord ipsilateral to the injury. CONCLUSION AND IMPLICATIONS: Taken together, these data indicate that the marine-derived capnellenes, GB9 and GB10, had anti-neuroinflammatory and anti-nociceptive properties in IFN-gamma-stimulated microglial cells and in neuropathic rats respectively. Therefore, capnellene may serve as a useful lead compound in the search for new therapeutic agents for treatment of neuroinflammatory diseases.

Chloride transport in mitochondrion-rich cells of euryhaline tilapia (Oreochromis mossambicus) larvae.

A noninvasive scanning ion-selective electrode technique (SIET) was applied to measure Cl- transport at individual mitochondrion-rich cells (MRCs) in the skin of euryhaline tilapia (Oreochromis mossambicus) larvae. In seawater (SW)-acclimated larvae, outward Cl- gradients (20-80 mM higher than the background) were measured at the surface, indicating a secretion of Cl- from the skin. By serial probing over the surface of MRCs and adjacent keratinocytes (KCs), a significant outward flux of Cl- was detected at the apical opening (membrane) of MRCs. Treatment with 100 microM ouabain or bumetanide inhibited the Cl- secretion by approximately 75%. In freshwater (FW)-acclimated larvae, a lower level of outward Cl- gradients (0.2-1 mM) was measured at the skin surface. Low-Cl- water (<0.005 mM) acclimation increased the apical Na+-Cl- cotransporter (NCC) immunoreactivity of MRCs in the larval skin. An inward flux of Cl- was detected when probing the exterior surface of a group of MRCs (convex-MRCs) that express the NCC. An NCC inhibitor (100 microM metolazone) reduced the flux by approximately 90%. This study provides direct and convincing evidence for Cl- transport by MRCs of SW- and FW-acclimated euryhaline tilapia and the involvement of an apical NCC in Cl- uptake of MRCs of FW-acclimated fish.