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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

Metal accumulation in marine bivalves under various tributyltin burdens.

In the present study, a field survey was conducted to measure the accumulation of butyltin, Cu, Zn, and Cd in green mussels (Perna viridis) and Pacific oysters (Crassostrea gigas) at the regions along a tributyltin pollution gradient. A negative correlation was found between the tributyltin/total butyltin ratio (0.87-0.31) and tributyltin content (114-5,817 ng/g as tin dry wt) in oysters, while the Cu content (44.2-381 mg/kg dry wt) was positively correlated with the logarithm of tributyltin content during the summer and winter. This suggests that as the tributyltin burden increases, the rates of tributyltin metabolism may be elevated, leading to enhanced Cu accumulation. A similar accumulation pattern for Zn was also found in oysters. In mussels, however, the tributyltin/total butyltin ratio and the Cu and Zn contents remained relatively constant (~ 0.7, 12, and 100 mg/kg dry wt, respectively) regardless of the tributyltin burden. Clearly, the butyltin and Cu/Zn accumulation processes in oysters differ from those in mussels under tributyltin pollution. These observations provide valuable information for those who evaluate or compare tributyltin and/or Cu/Zn pollution using oysters and mussels as bioindicators.

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.

Inducible nitric oxide synthase and cyclooxygenase-2 participate in anti-inflammatory and analgesic effects of the natural marine compound lemnalol from Formosan soft coral Lemnalia cervicorni.

Lemnalol (8-isopropyl-5-methyl-4-methylene-decahydro-1,5-cyclo-naphthalen-3-ol) is a natural compound isolated from the marine soft coral Lemnalia cervicorni. In the present study, the anti-inflammatory and anti-nociceptive properties of lemnalol were investigated in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and carrageenan-injected rats, respectively. Our results demonstrate that lemnalol significantly inhibited the expression of the pro-inflammatory proteins, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in LPS-stimulated RAW 264.7 cells. An in vivo inflammation model was induced by intraplantar injection of carrageenan into rat hind paws. An intramuscular injection of lemnalol (15 mg/kg) 10 min before carrageenan injection resulted in significant inhibition of carrageenan-induced rat paw edema and thermal hyperalgesia behavior. Western blot experiments revealed that the carrageenan-induced expression of iNOS and COX-2 in paw tissue was significantly down-regulated by lemnalol. Moreover, post-intrathecal injection of lemnalol produced a dose-dependent anti-nociceptive effect in carrageenan-injected rats (1 and 5 microg). The present results indicate that the marine-derived compound lemnalol had anti-inflammatory and analgesic effects in LPS-stimulated RAW 264.7 cells and carrageenan-injected rats, respectively. In addition, inhibition of elevated iNOS and COX-2 protein expression as well as neurophil infiltration of carrageenan-injected paws may be involved in the beneficial effects of lemnalol.

The zebrafish model: use in studying cellular mechanisms for a spectrum of clinical disease entities.

Although the zebrafish model provides an important platform for the study of developmental biology, recent work with the zebrafish model has extended its application to a wide variety of experimental studies relevant to human disease. Currently, the zebrafish model is used for the study of human genetic disease, caveolin-associated muscle disease, homeostasis, kidney development and disease, cancer, cardiovascular disorders, oxidative stress, caloric restriction, insulin-like pathways, angiogenesis, neurological diseases, liver disease, hemophilia, bacterial pathogenesis, apoptosis, osteoporosis, immunological studies, germ cell study, Bardet-Biedl syndrome gene (BBS11), Alzheimer's disease, virology studies and vaccine development. Here we describe the essential use of the zebrafish model that applies to several clinical diseases. With increased understanding of the cellular mechanisms responsible for disease, we can use knowledge gained from the zebrafish model for the development of therapeutics.

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.

Removal of multiple nitrogenous wastes by Aspergillus niger in a continuous fixed-slab reactor.

A biofilter reactor, to which is attached a large variety of microorganisms, can be employed to treat circulating water in an intensive aquaculture system. Some nitrogen-containing wastes, such as ammonium and nitrite, are toxic to the aquatic organisms. The removal rates of the nitrogenous wastes are regarded as indices for the efficiency of treatment by biofilters. In this study, a fungus that was characterized as being able to remediate multiple nitrogenous wastes was identified as Aspergillus niger NBG5. In a continuous fixed-slab reactor, the heterotrophic fungus utilized ammonium, nitrite, protein, and glucose simultaneously. The fungus assimilated ammonium, nitrite and protein at rates of 0.247, 0.07 and 0.096 g-N/g-cell/day, respectively, at 22 degrees C. The remediation rates of ammonium nitrogenous wastes decreased by a factor of eight at 35 degrees C, while the specific growth rates slightly increased. For nitrogenous wastes, ammonium was a preferred substrate but its rate of consumption declined significantly as temperature increased. The nitrogen consumption rates were inconsistent with the cell yields at high temperature. Further analysis of consumption ratios of C/N revealed that cells grew predominantly from the carbon at high temperature. The A. niger NBG5 consumed glucose rapidly at specific rates of 2-2.5 g-C/g-cell/day at 35 degrees C in the presence of ammonium and nitrite; while sluggish consumption of glucose was observed in the protein substrate. The protein could serve as an alternative carbon source. Further ANOVA statistical analysis with P < 0.05 revealed no significant effects of temperature on the specific growth rates of A. niger on the SG-NH4 and milk-protein substrates, whereas significant effects on the C/N ratio at culture temperatures higher than 25 degrees C were observed. These findings indicated that the carbon utilization rate increased with high temperature, whereas nitrogen utilization increased as temperature declined. A suitable operational temperature was suggested, depending upon the amount of waste contents of C/N. A high temperature stimulates the use of carbon waste, while a low temperature favors remediation of all nitrogenous wastes.

Histological, ultrastructural, and in situ hybridization study on enlarged cells in grouper Epinephelus hybrids infected by grouper iridovirus in Taiwan (TGIV).

Grouper iridovirus in Taiwan (TGIV) infection in the Epinephelus hybrid is a major problem in the grouper industry. ATPase gene sequences indicate that this virus is closely related to cell hypertrophy iridoviruses. Histologically, the appearance of basophilic or eosinophilic enlarged cells in internal organs is the most characteristic feature of this disease. These cells are acid-phosphatase positive and are able to phagocytose injected carbon particles. In our study, TGIV infection inhibited normal phagocytic ability in these cells in vivo after 4 d post-infection (p.i.) but not before 2 d p.i. Their staining properties and phagocytic ability suggested a monocyte origin of enlarged cells, which appeared in high numbers in the trunk kidney, head kidney, spleen and gill. After infection, the enlarged cells first appeared in the spleen, with an abundance peak at 64 h p.i. (Peak 1); at 120 h p.i., a second peak (Peak 2) occurred in the spleen, head kidney, trunk kidney and gill. Lower numbers of enlarged cells were observed in the liver, muscle, heart, eye, intestine, but no enlarged cells were found in the brain. A TGIV-specific DNA probe labeled most of the basophilic but not eosinophilic enlarged cells. Nuclei of infected cells were labeled during an early stage of the infection; at later stages, both nuclei and cytoplasms were labeled. Ultrastructurally, heterochromatins of the infected cells were marginated or aggregated to one side of the nuclei during the early stages of infection. Damage and rupture of the nuclear membrane started before formation of the viromatrix. Capsids were assembled in ring-shaped or disc-shaped structures. Bullet-shaped electron-dense material was present near the incomplete virus particles, and is speculated to be inserted into the capsids later.

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.