Molecular characterization of a possible progenitor sodium channel toxin from the Old World scorpion Mesobuthus martensii

AbstractToxins affecting sodium channels widely exist in the venoms of scorpions throughout the world. These molecules comprise an evolutionarily related peptide family with three shared features including conserved three-dimensional structure and gene organization, and similar function. Based on different pharmacological profiles and binding properties, scorpion sodium channel toxins are divided into α- and β-groups. However, their evolutionary relationship is not yet established. Here, we report a gene isolated from the venom gland of scorpion Mesobuthus martensii which encodes a novel sodium channel toxin-like peptide of 64 amino acids, named Mesotoxin. The Mesotoxin gene is organized into three exons and two introns with the second intron location conserved across the family. This peptide is unusual in that it has only three disulfides and a long cysteine-free tail with loop size and structural characteristics close to β-toxins. Evolutionary analysis favors its basal position in the origin of scorpion sodium channel toxins as a progenitor. The discovery of Mesotoxin will assist investigations into the key issue regarding the origin and evolution of scorpion toxins

Retrieval accuracy, statistical significance and compositional similarity in protein sequence database searches

Protein sequence database search programs may be evaluated both for their retrieval accuracy—the ability to separate meaningful from chance similarities—and for the accuracy of their statistical assessments of reported alignments. However, methods for improving statistical accuracy can degrade retrieval accuracy by discarding compositional evidence of sequence relatedness. This evidence may be preserved by combining essentially independent measures of alignment and compositional similarity into a unified measure of sequence similarity. A version of the BLAST protein database search program, modified to employ this new measure, outperforms the baseline program in both retrieval and statistical accuracy on ASTRAL, a SCOP-based test set

Analysis of Babesia rossi transcriptome in dogs diagnosed with canine babesiosis

Background: Canine babesiosis is a tick-borne disease causing detrimental health effects on the domestic dogs with huge economic impact on the owners. The most complicated form of canine babesiosis is caused by a pathogenic Babesia rossi parasite. Canine babesiosis induced by B. rossi still remains the cause of mortality and morbidity in South African dogs, yet, the transcriptomic and genomic information of this parasite species is still not available. The transcriptomic and genomic information is essential in the disease development and processes for the design of effective disease control strategies. Consequently, our understanding of the mechanisms underlying the pathogenesis of the different genotypes of B. rossi remains limited. A previous study suggested a relationship between the parasite genotype and the disease phenotype. To date, thirteen B. rossi genotypes have been identified and associated with diverse clinical signs in their hosts. Hence the aim of this study was to sequence RNA from samples representing B. rossi genotypes, 19, 29 and 31, in order to have insight on the overall transcriptome of this parasite and to establish if there would be significant differences among the genotypes. Methodology: To screen for B. rossi positive samples, total DNA was extracted from 20 blood samples collected from sick domestic dogs presented at the Onderstepoort Veterinary Academic Hospital (OVAH). Babesia rossi infections were confirmed using the PCR-Reverse Line Blot (RLB) hybridization assay. Further confirmation of infection status was done by amplification of the B. rossi Erythrocyte Membrane Antigen 1 (BrEMA1) gene in all the DNA samples using qualitative PCR (qPCR), followed by sequencing of PCR products. Subsequently, total RNA was extracted from the 20 B. rossi-infected blood samples collected from the same dogs in which DNA was extracted. Three samples representing B. rossi genotypes 19, 29 and 31 were selected for transcriptome analysis. RNA sequencing was performed using the Illumina HiSeq 2000 to allow transcriptome analysis. De novo assembly was performed independently for all three transcriptomes using the Trinity software. The unigenes generated from specific transcriptome assemblies were subjected to global functional annotation using Blast2GO version 2.8.0 software, followed by KEGG database for annotation of biological pathways, and DAVID version 6.7, for COG classification to predict and classify their functions. Results: The sample representing B. rossi genotype 31 was excluded in the transcriptome analysis due to low RNA mass, which usually compromises the quality of the library used in RNA sequencing.Thus, a total of 26 747 238 and 25 709 627 paired-end reads were obtained from B. rossi genotypes 19 and 29, respectively. De novo transcriptome assembly produced a total of 3019 unigenes, with an average length of 419 bp and N50 of 362 bp in B. rossi genotype 19, and 2727 unigenes with an average length of 441 bp and N50 of 362 in B. rossi genotype 29. A total of 1193 unigenes were common between B. rossi genotype 19 and 29, while 1828 unigenes were exclusively detected in B. rossi genotype 19; and 1534 were specific to B. rossi genotype 29. Between the two B. rossi genotypes, a total of 4553 unigenes were obtained, representing the overall B. rossi transcriptome. From the overall transcriptome, 12.3% (n=558) of the unigenes could be annotated with 53 different gene ontology (GO) functional categories. About 34% (n=1550) of the unigenes represented in the overall transcriptome mapped to 237 KEGG pathways and only 2.5% (114) could be annotated in the COG database. Conclusion: Although, there were no striking differences in the transcriptomes of B. rossi genotypes 19 and 29, this study presents the first transcriptomic resource for B. rossi, which will highly contribute to our genetic understanding of B. rossi and provide a platform for future gene expression studies. Hypothetical proteins identified in this study will require further characterization as they may have a critical role in the biology and pathogenicity of B. rossi parasite.Life and Consumer SciencesM. Sc. (Life Sciences

Correcting BLAST e-values for low-complexity segments

The statistical estimates of BLAST and PSI-BLAST are of extreme importance to determine the biological relevance of sequence matches. While being very effective in evaluating most matches, these estimates usually overestimate the significance of matches in the presence of low complexity segments. In this paper we present a model, based on divergence measures and statistics of the alignment structure, that corrects BLAST e-values for low complexity sequences without filtering or excluding them. We evaluate our method and compare it to other known methods using the Gene Ontology (GO)knowledge resource as a benchmark. Various performance measures, including ROC analysis, indicate that the new model improves over the state of the art. The program is available at biozon.org/ftp/ and www.cs.technion.ac.il/~itaish/lowcomp

Verifikasi Payena acuminata (Blume) Pierre koleksi Kebun Raya Bogor melalui pendekatan morfologi dan molekuler

Payena acuminata (Blume) Pierre merupakan salah satu spesies dari marga Payena yang hanya terdapat satu nomor koleksi dan satu individu di Kebun Raya Bogor. Payena acuminata koleksi Kebun Raya Bogor memiliki karakteristik yang berbeda dengan P. acuminata (KF686292 dan HF542856) pada umumnya yang memiliki permukaan abaksial daun berwarna coklat – kuning. Hal ini berbeda dengan morfologi P. acuminata koleksi Kebun Raya Bogor yang memiliki permukaan abaksial berwarna putih keperakan. Penelitian ini bertujuan untuk memverifikasi P. acuminata koleksi Kebun Raya Bogor menggunakan pendekatan morfologi dan pendekatan molekuler. Hasil penelitian pendekatan morfologi menunjukkan bahwa P. acuminata koleksi Kebun Raya Bogor memiliki karakteristik morfologi yang sama dengan P. acuminata var. acuminata, yang membedakan hanya dari warna abaksial daun putih keperakan. Hasil dendogram menunjukkan bahwa P. acuminata koleksi Kebun Raya Bogor mengelompok dengan P. acuminata var. acuminata. Hasil penelitian pendekatan molekuler mendukung pendekatan morfologi, yang ditandai dengan mengelompoknya sekuen P. acuminata disertai nilai bootstrap tinggi pada rekonstruksi pohon filogenetik. Hasil analisis menggunakan MultAlin dan jarak genetik menunjukkan bahwa P. acuminata koleksi Kebun Raya Bogor memiliki sekuen yang sama dengan P. acuminata, diperkuat dengan hasil jarak genetik 0,000 dengan P. acuminata. Hasil analisis menggunakan pendekatan morfologi dan pendekatan molekuler dengan region ITS dapat memverifikasi P. acuminata koleksi Kebun Raya Bogor. ABSTRACT: Payena acuminata (Blume) Pierre is one of the species from the Payena genus, which only has one collection number and one individual in the Bogor Botanical Gardens. Payena acuminata Bogor Botanical Gardens collection has different characteristics from P. acuminata (KF686292 and HF542856) generally, which has a brown-yellow leaf abaxial surface. This differs from the morphology of P. acuminata in the Bogor Botanical Gardens collection, which has a silvery white abaxial surface. This study aims to verify the P. acuminata Bogor Botanical Garden collection using a morphological approach and a molecular approach. The results of the morphological approach showed that P. acuminata in the Bogor Botanical Garden collection had the same morphological characteristics as P. acuminata var. acuminata, which differed only from the abaxial color of the silvery white leaves. The dendogram results showed that P. acuminata in the Bogor Botanical Gardens collection was grouped with P. acuminata var. acuminata. The results of the molecular approach research support the morphological approach, which is characterized by the grouping of P. acuminata sequences accompanied by high bootstrap values in the phylogenetic tree reconstruction. The analysis using MultAlin and genetic distance showed that the Bogor Botanical Gardens collection of P. acuminata had the same sequence as P. acuminata, reinforced by the genetic distance of 0.000 with P. acuminata. The analysis results using a morphological approach and a molecular approach with the ITS region can verify P. acuminata in the Bogor Botanical Gardens collection

Evaluation of Justicia gendarussa crude leaf extract for enhancement of flavonoids production via adventitious root culture and genetic modifications

Justicia gendarussa extract possesses various bioactivities associated with the availability of flavonoids. Low availability of flavonoids could limit or even hinder the bioactivities effects. Therefore, attempts to enhance the flavonoids production via tissue culture approaches are being studied. This study aimed to optimize flavonoids contents in J. gendarussa using different tissue culture systems (in vitro plant regeneration and adventitious root culture) and genetic transformation methods. The cytotoxicity of plant extracts against various cancer cell lines was also evaluated. Detection and quantification of naringenin and kaempferol were performed using GC-FID. Cytotoxicity tests of crude extract against cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-468, HT-29, HeLa and BxPC-3) were determined by MTT assay. The optimization of elicitors used including yeast extracts (YE), casein hydrolysate (CH) and proline (P) at various concentrations (0, 0.2, 0.4, 0.6, 0.8 or 1.0 mg/L) were examined using nodal explants from in vitro plants. Adventitious roots were inoculated into MS liquid medium supplemented with IBA (2.0-4.0 mg/L). For genetic transformation studies, plasmids pCAMBIA 1305.2, which harbour the PKS gene and plant selectable marker, HPT for hygromycin resistance was used to transform nodal explants of J. gendarussa under the optimized transformation protocol using biolistic and Agrobacterium tumefaciens-mediated transformation. Results showed that mature leaves extract, JG1 had the highest naringenin (444.35 ± 81.43 mg/kg) and kaempferol (1591.80 ± 94.91 mg/kg), while the cytotoxicity against BxPC-3 cell was the strongest (IC50~16 μg/mL). The highest naringenin and kaempferol contents were obtained in leaf crude extracts when treated with 0.6 g/L of CH (1180.30 ± 50.23 mg/kg) and 0.6 g/L of P (385.01 ± 13.10 mg/kg), respectively. Adventitious root culture produced high naringenin (97.54 ± 5.47 mg/kg) and kaempferol (853.82 ± 56.52 mg/kg) when treated with 2.0 mg/L IBA. The optimal parameters for biolistic method were established at 1100 psi helium pressure and 12 cm target distance with 95% of transformation efficiency. Meanwhile, the optimal transformation condition of A. tumefaciens method was bacterial concentration at OD600nm ~ 0.8, 20 minutes of inoculation time, 500 μM AS and 1 cm explant size with 90% transformation efficiency. Even though A. tumefaciens method produced lower percentage of transient GUS expression than biolistic method, a few transformed explants were successfully produced. The integration of the PKS gene with band size of 1200 bp into the genome of transgenic plants were verified by PCR, sequencing and subsequently confirmed by Southern blot analysis. The content of kaempferol were found to be higher in stem extracts of transgenic plants (450.40 ± 7.82 mg/kg) than non-transgenic plants (197.13 ± 2.29 mg/kg). In conclusion, addition of elicitors, establishment of adventitious root culture and A. tumefaciens-mediated transformation could enhance flavonoid contents in J. gendarussa

Mining a Chinese hyperthermophilic metagenome

Philosophiae Doctor - PhDMetagenomic sequencing of environmental samples provide direct access to genomic information of organisms within the respective environments. This sequence information represents a significant resource for the identification and subsequent characterization of potentially novel genes, or known genes with acquired novel characteristics. Within this context, the thermophilic environments are of particular interest due to its potential for deriving novel thermostable enzymes with biotechnological and industrial applications. In this work metagenomic library construction, random sequencing and sequence analysis strategies were employed to enhance identification and characterisation of potentially novel genes, from a thermophilic soil sample. High molecular weight metagenomic DNA was extracted from two Chinese hydrothermal soil samples. This was used as source material for the construction of four genomic DNA libraries. The combined libraries were estimated to contain in the order of 1.3 million genes, which provides a rich resource for gene identification. Approximately 70 kbp of sequence data was generated from one of the libraries as a resource for sequence-based analysis. Initial BLAST analysis predicted the presence of 53 ORFs/partial ORFs. The BLAST similarity scores for the investigated ORFs were sufficiently high (>40%) to infer homology with database proteins while also being indicative of novel sequence variants of these database matches. In an attempt to enhance the potential for deriving more full length ORFs a novel strategy, based on WGA technology, was employed. This resulted in the recovery of the near complete sequence of partial ORF5, directly from the WGA DNA of the environmental sample. While the full length ORF5 could not be recovered, the feasibility of this novel approach, for enhanced metagenomic sequence recovery was proved in principle. The implementation of multiple insilico strategies resulted in the identification of two ORFs, classified as homologs of the DUF29 and Usp protein families respectively. The functional inference obtained from the integrated in-silico predictions was furthermore highly suggestive of a putative nucleotide binding/interaction role for both ORFs. A putative novel DNA polymerase gene (denoted TC11pol) was identified from the sequence data. Expression and characterization of the full length TC11pol did however not result in detectable polymerase activity. The implementation of a homology modeling approach proved succesfull for deriving a structural model of the polymerase that was used for: (i) deriving functional inferences of the potential activities of the polymerase and (ii) deriving a 5’ exonuclease deletion mutant for functional analysis. Expression and subsequent functional characterization of the putative 5’exo- TC11pol mutant resulted in detectable polymerase and 3’-5’ exonuclease activity at 37 and 45 oC, following a heat denaturation step at 55 oC for 1 hour. It was, therefore concluded that the putative 5’exo- TC11pol mutant was functionally equivalent to the Klenow fragment of E. coli, while exhibiting increased thermostability.South Afric