Antichagasic effect of hemocyanin derived from antimicrobial peptides of penaeus monodon shrimp.

Trypanosoma cruzi, the etiological agent of Chagas disease, is responsible for the infection of millions of people worldwide and it is a public health problem, without an effective cure. Four fragments with antimicrobial potential from the hemocyanin of Penaeus monodon shrimp were identified using a computer software AMPA. The present study aimed to evaluate the antichagasic effect of these four peptides (Hmc364-382, Hmc666-678, Hmc185-197 and Hmc476-498). The peptides were tested against the epimastigote, trypomastigote and amastigote forms of Trypanosoma cruzi Y strain (benznidazole-resistant strain) and cytotoxicity in mammalian cells was evaluated against LLC-MK2 lineage cells. Two fragments (Hmc364-382, Hmc666-678) showed activity against the epimastigote and trypomastigote forms and their selectivity index (SI) was calculated. The Hmc364-382 peptide was considered the most promising (SI > 50) one and it was used for further studies, using flow cytometry analyses with specific molecular probes and scanning electron microscopy (SEM). Hmc364-382 was able to induce cell death in T. cruzi through necrosis, observed by loss of membrane integrity in flow cytometry analyses and pore formation in SEM. Overall, Hmc364-382 open perspectives to the development of new antichagasic agents.

Novel neurotoxic peptides from Protopalythoa variabilis virtually interact with voltage-gated sodium channel and display anti-epilepsy and neuroprotective activities in zebrafish.

We previously reported a novel toxic peptide identified from the anthozoan Protopalythoa variabilis transcriptome which is homologous to a novel structural type of sodium channel toxin isolated from a parental species (Palythoa caribaeorum). The peptide was named, according to its homologous, as Pp V-shape α-helical peptide (PpVα) in the present study. Through molecular docking and dynamics simulation, linear and hairpin folded PpVα peptides were shown to be potential voltage-gated sodium channel blockers. Nowadays, sodium channel blockers have been the mainstream of the pharmacological management of epileptic seizures. Also, sodium channel blockers could promote neuronal survival by reducing sodium influx and reducing the likelihood of calcium importation resulting in suppressing microglial activation and protecting dopaminergic neurons from degeneration. The folded PpVα peptide could decrease pentylenetetrazol (PTZ)-induced c-fos and npas4a expression level leading to reverse PTZ-induced locomotor hyperactivity in zebrafish model. In vitro, the folded PpVα peptide protected PC12 cells against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity via activating heme oxygenase-1 (HO-1) and attenuating inducible nitric oxide synthase (iNOS) expression. In vivo, PpVα peptide suppressed the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish and, importantly, prevented the 6-OHDA-induced excessive ROS generation and subsequent dopaminergic neurons loss. This study indicates that the single S-S bond folded PpVα peptide arises as a new structural template to develop sodium channel blockers and provides an insight on the peptide discovery from cnidarian transcriptome to potentially manage epilepsy and neurodegenerative disorders.

The Place for Enzymes and Biologically Active Peptides from Marine Organisms for Application in Industrial and Pharmaceutical Biotechnology.

Since the beginning of written history, diverse texts have reported the use of enzymatic preparations in food processing and have described the medicinal properties of crude and fractionated venoms to treat various diseases and injuries. With the biochemical characterization of enzymes from distinct sources and bioactive polypeptides from animal venoms, the last sixty years have testified the advent of industrial enzymology and protein therapeutics, which are currently applicable in a wide variety of industrial processes, household products, and pharmaceuticals. Bioprospecting of novel biocatalysts and bioactive peptides is propelled by their unsurpassed properties that are applicable for current and future green industrial processes, biotechnology, and biomedicine. The demand for both novel enzymes with desired characteristics and novel peptides that lead to drug development, has experienced a steady increase in response to the expanding global market for industrial enzymes and peptidebased drugs. Moreover, although largely unexplored, oceans and marine realms, with their unique ecosystems inhabited by a large variety of species, including a considerable number of venomous animals, are recognized as untapped reservoirs of molecules and macromolecules (enzymes and bioactive venom-derived peptides) that can potentially be converted into highly valuable biopharmaceutical products. In this review, we have focused on enzymes and animal venom (poly)peptides that are presently in biotechnological use, and considering the state of prospection of marine resources, on the discovery of useful industrial biocatalysts and drug leads with novel structures exhibiting selectivity and improved performance.

The Holo-Transcriptome of the Zoantharian Protopalythoa variabilis (Cnidaria: Anthozoa): A Plentiful Source of Enzymes for Potential Application in Green Chemistry, Industrial and Pharmaceutical Biotechnology.

Marine invertebrates, such as sponges, tunicates and cnidarians (zoantharians and scleractinian corals), form functional assemblages, known as holobionts, with numerous microbes. This type of species-specific symbiotic association can be a repository of myriad valuable low molecular weight organic compounds, bioactive peptides and enzymes. The zoantharian Protopalythoa variabilis (Cnidaria: Anthozoa) is one such example of a marine holobiont that inhabits the coastal reefs of the tropical Atlantic coast and is an interesting source of secondary metabolites and biologically active polypeptides. In the present study, we analyzed the entire holo-transcriptome of P. variabilis, looking for enzyme precursors expressed in the zoantharian-microbiota assemblage that are potentially useful as industrial biocatalysts and biopharmaceuticals. In addition to hundreds of predicted enzymes that fit into the classes of hydrolases, oxidoreductases and transferases that were found, novel enzyme precursors with multiple activities in single structures and enzymes with incomplete Enzyme Commission numbers were revealed. Our results indicated the predictive expression of thirteen multifunctional enzymes and 694 enzyme sequences with partially characterized activities, distributed in 23 sub-subclasses. These predicted enzyme structures and activities can prospectively be harnessed for applications in diverse areas of industrial and pharmaceutical biotechnology.

Novel Kunitz-like Peptides Discovered in the Zoanthid Palythoa caribaeorum through Transcriptome Sequencing.

Palythoa caribaeorum (class Anthozoa) is a zoanthid that together jellyfishes, hydra, and sea anemones, which are venomous and predatory, belongs to the Phyllum Cnidaria. The distinguished feature in these marine animals is the cnidocytes in the body tissues, responsible for toxin production and injection that are used majorly for prey capture and defense. With exception for other anthozoans, the toxin cocktails of zoanthids have been scarcely studied and are poorly known. Here, on the basis of the analysis of P. caribaeorum transcriptome, numerous predicted venom-featured polypeptides were identified including allergens, neurotoxins, membrane-active, and Kunitz-like peptides (PcKuz). The three predicted PcKuz isotoxins (1-3) were selected for functional studies. Through computational processing comprising structural phylogenetic analysis, molecular docking, and dynamics simulation, PcKuz3 was shown to be a potential voltage gated potassium-channel inhibitor. PcKuz3 fitted well as new functional Kunitz-type toxins with strong antilocomotor activity as in vivo assessed in zebrafish larvae, with weak inhibitory effect toward proteases, as evaluated in vitro. Notably, PcKuz3 can suppress, at low concentration, the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish, which indicated PcKuz3 may have a neuroprotective effect. Taken together, PcKuz3 figures as a novel neurotoxin structure, which differs from known homologous peptides expressed in sea anemone. Moreover, the novel PcKuz3 provides an insightful hint for biodrug development for prospective neurodegenerative disease treatment.

The dinoponeratoxin peptides from the giant ant Dinoponera quadriceps display in vitro antitrypanosomal activity.

The crude venom of the giant ant Dinoponera quadriceps is a cocktail of polypeptides and organic compounds that shows antiparasitic effects against Trypanosoma cruzi, the causative agent of Chagas disease. In order to investigate the venom-derived components responsible for such antitrypanosomal activity, four dinoponeratoxins (DnTxs) were identified, namely M-PONTX-Dq3a, -Dq3b, -Dq3c and -Dq4e, that are diverse in size, net charge, hydrophobicity and propensity to interact with eukaryote cell membranes. These peptides were tested against epimastigote, trypomastigote and amastigote forms of benznidazole (Bz)-resistant Y strain of T. cruzi and in mammalian host cells. The M-PONTX-Dq3a and -Dq4e inhibited all developmental forms of T. cruzi, including amastigotes, the responsible form for the maintenance of infection on chronic phase of the disease. The M-PONTX-Dq3a showed the highest selectivity index (SI) (80) and caused morphological alterations in T. cruzi, as observed by scanning electron microscopy (SEM), and induced cell death through necrosis, as seen by multiparametric flow cytometry analysis with specific biochemical markers. Altogether, the D. quadriceps venom appears as a source for the prospection of trypanocidal peptides and the M-PONTX-Dq3a arises as a candidate among the dinoponeratoxin-related peptides in the development of compounds against Chagas disease.

The holo-transcriptome of the zoantharian Protopalythoa variabilis (Cnidaria: Anthozoa): a plentiful source of enzymes for potential application in green chemistry, industrial and pharmaceutical biotechnology

Marine invertebrates, such as sponges, tunicates and cnidarians (zoantharians and scleractinian corals), form functional assemblages, known as holobionts, with numerous microbes. This type of species-specific symbiotic association can be a repository of myriad valuable low molecular weight organic compounds, bioactive peptides and enzymes. The zoantharian Protopalythoa variabilis (Cnidaria: Anthozoa) is one such example of a marine holobiont that inhabits the coastal reefs of the tropical Atlantic coast and is an interesting source of secondary metabolites and biologically active polypeptides. In the present study, we analyzed the entire holo-transcriptome of P. variabilis, looking for enzyme precursors expressed in the zoantharian-microbiota assemblage that are potentially useful as industrial biocatalysts and biopharmaceuticals. In addition to hundreds of predicted enzymes that fit into the classes of hydrolases, oxidoreductases and transferases that were found, novel enzyme precursors with multiple activities in single structures and enzymes with incomplete Enzyme Commission numbers were revealed. Our results indicated the predictive expression of thirteen multifunctional enzymes and 694 enzyme sequences with partially characterized activities, distributed in 23 sub-subclasses. These predicted enzyme structures and activities can prospectively be harnessed for applications in diverse areas of industrial and pharmaceutical biotechnology.

The holo-transcriptome of the zoantharian Protopalythoa variabilis (Cnidaria: Anthozoa): a plentiful source of enzymes for potential application in green chemistry, industrial and pharmaceutical biotechnology

Marine invertebrates, such as sponges, tunicates and cnidarians (zoantharians and scleractinian corals), form functional assemblages, known as holobionts, with numerous microbes. This type of species-specific symbiotic association can be a repository of myriad valuable low molecular weight organic compounds, bioactive peptides and enzymes. The zoantharian Protopalythoa variabilis (Cnidaria: Anthozoa) is one such example of a marine holobiont that inhabits the coastal reefs of the tropical Atlantic coast and is an interesting source of secondary metabolites and biologically active polypeptides. In the present study, we analyzed the entire holo-transcriptome of P. variabilis, looking for enzyme precursors expressed in the zoantharian-microbiota assemblage that are potentially useful as industrial biocatalysts and biopharmaceuticals. In addition to hundreds of predicted enzymes that fit into the classes of hydrolases, oxidoreductases and transferases that were found, novel enzyme precursors with multiple activities in single structures and enzymes with incomplete Enzyme Commission numbers were revealed. Our results indicated the predictive expression of thirteen multifunctional enzymes and 694 enzyme sequences with partially characterized activities, distributed in 23 sub-subclasses. These predicted enzyme structures and activities can prospectively be harnessed for applications in diverse areas of industrial and pharmaceutical biotechnology.

Identification of long non-coding RNAs in two anthozoan species and their possible implications for coral bleaching.

Long non-coding RNAs (lncRNAs) have been shown to play regulatory roles in a diverse range of biological processes and are associated with the outcomes of various diseases. The majority of studies about lncRNAs focus on model organisms, with lessened investigation in non-model organisms to date. Herein, we have undertaken an investigation on lncRNA in two zoanthids (cnidarian): Protolpalythoa varibilis and Palythoa caribaeorum. A total of 11,206 and 13,240 lncRNAs were detected in P. variabilis and P. caribaeorum transcriptome, respectively. Comparison using NONCODE database indicated that the majority of these lncRNAs is taxonomically species-restricted with no identifiable orthologs. Even so, we found cases in which short regions of P. caribaeorum's lncRNAs were similar to vertebrate species' lncRNAs, and could be associated with lncRNA conserved regulatory functions. Consequently, some high-confidence lncRNA-mRNA interactions were predicted based on such conserved regions, therefore revealing possible involvement of lncRNAs in posttranscriptional processing and regulation in anthozoans. Moreover, investigation of differentially expressed lncRNAs, in healthy colonies and colonial individuals undergoing natural bleaching, indicated that some up-regulated lncRNAs in P. caribaeorum could posttranscriptionally regulate the mRNAs encoding proteins of Ras-mediated signal transduction pathway and components of innate immune-system, which could contribute to the molecular response of coral bleaching.

Cell-penetrating peptides (CPPs): From delivery of nucleic acids and antigens to transduction of engineered nucleases for application in transgenesis.

Cell-penetrating peptides (CPPs) have been studied for their capacity to translocate across the lipid membrane of several cell types. In membrane translocation, these peptides can remarkably transport biologically active hydrophilic molecules, such as pharmaceuticals, nucleic acids (DNA and RNA) and even high-molecular-weight proteins, Fig. 3 into the cell cytoplasm and organelles. The development of CPPs as transduction agents includes the modification of gene and protein expression, the reprogramming and differentiation of induced pluripotent stem cells and the preparation of cellular vaccines. A relatively recent field of CPP application is the transduction of plasmid DNA vectors and CPP-fusion proteins to modify genomes and introduce new traits in cells and organisms. CPP-mediated transduction of components for genome editing is an advantageous alternative to viral DNA vectors. Engineered site-specific nucleases, such as Cre recombinase, ZFN, TALENs and CRISPR associated protein (Cas), have been coupled to CPPs, and the fused proteins have been used to permeate targeted cells and tissues. The functionally active fusion CPP-nucleases subsequently home to the nucleus, incise genomic DNA at specific sites and induce repair and recombination. This review has the objective of discussing CPPs and elucidating the prospective use of CPP-mediated transduction technology, particularly in genome modification and transgenesis.

NanoCAGE: A Method for the Analysis of Coding and Noncoding 5'-Capped Transcriptomes.

Transcripts in all eukaryotes are characterized by the 5'-end specific cap structure in mRNAs. Cap Analysis Gene Expression or CAGE makes use of these caps to specifically obtain cDNA fragments from the 5'-end of RNA and sequences those at high throughput for transcript identification and genome-wide mapping of transcription start sites for coding and noncoding genes. Here, we provide an improved version of our nanoCAGE protocol that has been developed for preparing CAGE libraries from as little as 50 ng of total RNA within three standard working days. Key steps in library preparation have been improved over our previously published protocol to obtain libraries having a good 5'-end selection and a more equal size distribution for higher sequencing efficiency on Illumina MiSeq and HiSeq sequencers. We recommend nanoCAGE as the method of choice for transcriptome profiling projects even from limited amounts of RNA, and as the best approach for genome-wide mapping of transcription start sites within promoter regions.

The Transcriptome of the Zoanthid Protopalythoa variabilis (Cnidaria, Anthozoa) Predicts a Basal Repertoire of Toxin-like and Venom-Auxiliary Polypeptides.

Protopalythoa is a zoanthid that, together with thousands of predominantly marine species, such as hydra, jellyfish, and sea anemones, composes the oldest eumetazoan phylum, i.e., the Cnidaria. Some of these species, such as sea wasps and sea anemones, are highly venomous organisms that can produce deadly toxins for preying, for defense or for territorial disputes. Despite the fact that hundreds of organic and polypeptide toxins have been characterized from sea anemones and jellyfish, practically nothing is known about the toxin repertoire in zoanthids. Here, based on a transcriptome analysis of the zoanthid Protopalythoa variabilis, numerous predicted polypeptides with canonical venom protein features are identified. These polypeptides comprise putative proteins from different toxin families: neurotoxic peptides, hemostatic and hemorrhagic toxins, membrane-active (pore-forming) proteins, protease inhibitors, mixed-function venom enzymes, and venom auxiliary proteins. The synthesis and functional analysis of two of these predicted toxin products, one related to the ShK/Aurelin family and the other to a recently discovered anthozoan toxin, displayed potent in vivo neurotoxicity that impaired swimming in larval zebrafish. Altogether, the complex array of venom-related transcripts that are identified in P. variabilis, some of which are first reported in Cnidaria, provides novel insight into the toxin distribution among species and might contribute to the understanding of composition and evolution of venom polypeptides in toxiferous animals.

One-step detection of the 2009 pandemic influenza A(H1N1) virus by the RT-SmartAmp assay and its clinical validation.

BACKGROUND: In 2009, a pandemic (pdm) influenza A(H1N1) virus infection quickly circulated globally resulting in about 18,000 deaths around the world. In Japan, infected patients accounted for 16% of the total population. The possibility of human-to-human transmission of highly pathogenic novel influenza viruses is becoming a fear for human health and society. METHODOLOGY: To address the clinical need for rapid diagnosis, we have developed a new method, the "RT-SmartAmp assay", to rapidly detect the 2009 pandemic influenza A(H1N1) virus from patient swab samples. The RT-SmartAmp assay comprises both reverse transcriptase (RT) and isothermal DNA amplification reactions in one step, where RNA extraction and PCR reaction are not required. We used an exciton-controlled hybridization-sensitive fluorescent primer to specifically detect the HA segment of the 2009 pdm influenza A(H1N1) virus within 40 minutes without cross-reacting with the seasonal A(H1N1), A(H3N2), or B-type (Victoria) viruses. RESULTS AND CONCLUSIONS: We evaluated the RT-SmartAmp method in clinical research carried out in Japan during a pandemic period of October 2009 to January 2010. A total of 255 swab samples were collected from outpatients with influenza-like illness at three hospitals and eleven clinics located in the Tokyo and Chiba areas in Japan. The 2009 pdm influenza A(H1N1) virus was detected by the RT-SmartAmp assay, and the detection results were subsequently compared with data of current influenza diagnostic tests (lateral flow immuno-chromatographic tests) and viral genome sequence analysis. In conclusion, by the RT-SmartAmp assay we could detect the 2009 pdm influenza A(H1N1) virus in patients' swab samples even in early stages after the initial onset of influenza symptoms. Thus, the RT-SmartAmp assay is considered to provide a simple and practical tool to rapidly detect the 2009 pdm influenza A(H1N1) virus.

DNA amplification techniques in pharmacogenomics.

The variable predisposition of patients, both to disease susceptibility and drug response, is well established. It is largely attributed to genetic, as well as epigenetic variations between individuals, which may be inherited or acquired. The most common variation in the human genome is the SNP, which occurs throughout the genome, both within coding and noncoding regions. Characterization of SNPs in the context of both inherited and acquired conditions, such as cancer, are a main focus of many genotyping procedures. The demand for identifying (diagnosing) targeted SNPs or other variations, as well as the application of genome-wide screens, is continuously directing the development of new technologies. In general, most methods require a DNA amplification step to provide the amounts of DNA needed for the SNP detection step. In addition, DNA amplification is an important step when investigating other types of genomic information, for instance when addressing repeat, deletion, copy number variation or epigenetic regulation by DNA methylation. Besides the widely used PCR technique, there are several alternative approaches for genomic DNA amplification suitable for supporting the detection of genomic variation. In this article, we describe and evaluate a number of techniques, and discuss possible future prospects of DNA amplification in the fields of pharmacogenetics and pharmacogenomics.

Mutation analysis of 2009 pandemic influenza A(H1N1) viruses collected in Japan during the peak phase of the pandemic.

BACKGROUND: Pandemic influenza A(H1N1) virus infection quickly circulated worldwide in 2009. In Japan, the first case was reported in May 2009, one month after its outbreak in Mexico. Thereafter, A(H1N1) infection spread widely throughout the country. It is of great importance to profile and understand the situation regarding viral mutations and their circulation in Japan to accumulate a knowledge base and to prepare clinical response platforms before a second pandemic (pdm) wave emerges. METHODOLOGY: A total of 253 swab samples were collected from patients with influenza-like illness in the Osaka, Tokyo, and Chiba areas both in May 2009 and between October 2009 and January 2010. We analyzed partial sequences of the hemagglutinin (HA) and neuraminidase (NA) genes of the 2009 pdm influenza virus in the collected clinical samples. By phylogenetic analysis, we identified major variants of the 2009 pdm influenza virus and critical mutations associated with severe cases, including drug-resistance mutations. RESULTS AND CONCLUSIONS: Our sequence analysis has revealed that both HA-S220T and NA-N248D are major non-synonymous mutations that clearly discriminate the 2009 pdm influenza viruses identified in the very early phase (May 2009) from those found in the peak phase (October 2009 to January 2010) in Japan. By phylogenetic analysis, we found 14 micro-clades within the viruses collected during the peak phase. Among them, 12 were new micro-clades, while two were previously reported. Oseltamivir resistance-related mutations, i.e., NA-H275Y and NA-N295S, were also detected in sporadic cases in Osaka and Tokyo.

Tail variation of the folding primer affects the SmartAmp2 process differently.

Folding primer (FP), together with turn-back primer (TP) and boost primer (BP), is one of the major components of SmartAmp2, a rapid amplification-based method for SNP detection. FP has a unique design where the annealing region is combined with a tail that can fold back. FP tails can be classified as either "strong" or "weak", depending on the melting temperature and free energy of the hairpin structure. We report that FP tails affect the amplification process differently; by changing the FP concentration, we can increase the amplification reaction speed with "strong tails". Unlike "strong tails", concentration change of FP with "weak tails" did not show significant impact on the amplification speed. The comparative analyses using gel electrophoresis demonstrate that the FP type and FP ratio in the reaction change the amplification pattern. The above observations can be used to optimize the reaction and manipulate the reaction speed of SmartAmp2.

Determination of silencing potency of synthetic and RNase III-generated siRNA using a secreted luciferase assay.

RNA interference (RNAi) is an established tool for functional genomics studies that is also showing great potential for medical applications. Currently, one of the main goals in RNAi technology is the design and discovery of potent small interfering RNAs (siRNAs). Using a secreted luciferase from Gaussia princeps (GLuc), we developed a reporter assay, which allows for rapid potency assessment of siRNAs, by measuring luminescence activity in cell culture supernatants. The method was applied in microtiter plate format and validated by comparison to quantitative reverse transcription PCR (RT-PCR) and Western blot analysis. This reporter assay was used to evaluate in HeLa cells the potency of different siRNA mixtures generated by RNase III, or several synthetic siRNAs, all directed against human p53. The results show that all four siRNA mixtures generated by RNase III induce 50%-75% decrease of the reporter activity at less than 10 nM transfected concentration. In contrast, only one out of the five commercially available synthetic siRNAs showed comparable potency. These results suggest that one advantage of using enzymatic complex siRNA mixtures for RNAi is that, unlike single synthetic siRNAs, selecting a target region is not important to ensure potency.