Y-SNP Haplogroup Hierarchy Finder: a web tool for Y-SNP haplogroup assignment.

The application of massively parallel sequencing (MPS) data from whole genomes has allowed very many more Y-SNP loci to be genotyped simultaneously than previously possible. Although this greatly increases the resolution of Y-SNP haplogroups to link common ancestors, it remains a great challenge to provide a phylogenetic tree to clearly display the relationship of varying haplogroups. Y-SNP Haplogroup Hierarchy Finder is a web tool to generate hierarchical haplogroups based on Y-SNP data with the derived allele at the terminal of a haplogroup tree. The input data can include that from whole-genome sequencing. Confidence in assignment using Y-SNP Haplogroup Hierarchy Finder was demonstrated using Y-SNP genotypes of 1233 samples, sourced from the 1000 genomes project phase 3, used to generate the expected haplogroups. The outcome includes 2 reports: a 'Haplogroup Report' lists mutation types from the submitted Y-SNPs and their corresponding haplogroups, and a 'Haplogroup Hierarchy Report' lists all possible hierarchical haplogroups and ranks the three most supported haplogroups. Each layer of the descending haplogroups from one step to the next is shown and the supporting numbers of Y-SNPs are also included in these reports. All haplogroups that exhibited a clear relationship between the ancestral through to the derived SNPs can be clustered into a hierarchy of haplogroups. The assigned 1233 haplogroups were compared with 2 other software programs designed to assemble haplogroups, which resulted in one where there were many differences and the other one where there was only minor difference. The advantage of this web-based tool is that it provides an easy way to assign Y-SNP haplogroup based on the visualized hierarchical pattern.

Population inference based on mitochondrial DNA control region data by the nearest neighbors algorithm.

Population and geographic assignment are frequently undertaken using DNA sequences on the mitochondrial genome. Assignment to broad continental populations is common, although finer resolution to subpopulations can be less accurate due to shared genetic ancestry at a local level and members of different ancestral subpopulations cohabiting the same geographic area. This study reports on the accuracy of population and subpopulation assignment by using the sequence data obtained from the 3070 mitochondrial genomes and applying the K-nearest neighbors (KNN) algorithm. These data also included training samples used for continental and population assignment comprised of 1105 Europeans (including Austria, France, Germany, Spain, and England and Caucasian countries), 374 Africans (including North and East Africa and non-specific area (Pan-Africa)), and 1591 Asians (including Japan, Philippines, and Taiwan). Subpopulations included in this study were 1153 mitochondrial DNA (mtDNA) control region sequences from 12 subpopulations in Taiwan (including Han, Hakka, Ami, Atayal, Bunun, Paiwan, Puyuma, Rukai, Saisiyat, Tsou, Tao, and Pingpu). Additionally, control region sequence data from a further 50 samples, obtained from the Sigma Company, were included after they were amplified and sequenced. These additional 50 samples acted as the "testing samples" to verify the accuracy of the population. In this study, based on genetic distances as genetic metric, we used the KNN algorithm and the K-weighted-nearest neighbors (KWNN) algorithm weighted by genetic distance to classify individuals into continental populations, and subpopulations within the same continent. Accuracy results of ethnic inferences at the level of continental populations and of subpopulations among KNN and KWNN algorithms were obtained. The training sample set achieved an overall accuracy of 99 to 82% for assignment to their continental populations with K values from 1 to 101. Population assignment for subpopulations with K assignments from 1 to 5 reached an accuracy of 77 to 54%. Four out of 12 Taiwanese populations returned an accuracy of assignment of over 60%, Ami (66%), Atayal (67%), Saisiyat (66%), and Tao (80%). For the testing sample set, results of ethnic prediction for continental populations with recommended K values as 5, 10, and 35, based on results of the training sample set, achieved overall an accuracy of 100 to 94%. This study provided an accurate method in population assignment for not only continental populations but also subpopulations, which can be useful in forensic and anthropological studies.

Evaluation of the virucidal effects of rosmarinic acid against enterovirus 71 infection via in vitro and in vivo study.

BACKGROUND: Although enterovirus 71 (EV71) is an important public health threat, especially in the Asia-Pacific region, there are still no effective drugs or vaccines to treat and prevent EV71 infection. Therefore, it is critical to develop prophylactic and therapeutic agents against EV71. Rosmarinic acid (RA), a phytochemical, has been discovered to possess a broad spectrum of biological activities. METHODS: The virucidal effects of RA on EV71 were determined by MTT, western blot, median cell culture infectious dose, apoptosis detection, plaque reduction, semi-quantitative real-time polymerase chain reaction, immunofluorescence detection, molecular docking analysis, and mouse protection assay. RESULTS: RA showed a strong protective effect against EV71 infection in human rhabdomyosarcoma cells when the multiplicity of infection was 1, with a low IC50 value (4.33 ± 0.18 µM) and high therapeutic index (340). RA not only protected cells from EV71-induced cytopathic effects, but also from EV71-induced apoptosis. The results of time-of-addition analysis demonstrated that the inhibitory activity of RA was highest at the early stage of viral infection. Consistent with this, the infectivity of EV71 in the early stage of viral infection also was observed to be limited in neonatal mice treated with RA. Further, molecular docking predicts that RA could replace the natural pocket factor within the VP1 capsid-binding hydrophobic pocket. CONCLUSIONS: This study suggests that RA has the potential to be developed as an antiviral agent against initial EV71 infection to prevent or reduce EV71-induced pathogenesis and complications, since RA can effectively reduce EV71 infection in the early stages of viral infection.

Establishment of 11 linked X-STR loci within 1.1 Mb to assist with kinship testing.

This report identifies and characterizes 10 novel short tandem repeat (STR) loci on the human X chromosome, all of which are within a range of 1.1 Mb. These newly characterized loci were developed to aid in kinship assignment when the X chromosome is specifically required. The repeat DNA sequences were identified initially using data in GenBank and are located immediately upstream and downstream from the previously described locus DXS6807. Only those loci with seven or more observed alleles were used for further study resulting in the identification of 10 new loci. The distance between each pair of loci ranged from 24,998 to 244,701 bp with an average of approximately 110.8 kb. The number of observed alleles ranged from 7 to 30 for these 10 loci with a polymorphic information content ranging from 0.593 to 0.930. The LOD score from a pairwise linkage study ranged from 4.40 to 23.73, indicating that these 11 loci were highly linked, as expected. In line with standard forensic practice, all 11 loci can be amplified in one multiplex reaction, and comprehensive allelic ladders for all the loci have been constructed. These newly established 11 linked STR loci on the human X chromosome were found to be highly polymorphic and have the potential to aid in kinship testing where the X chromosome loci currently plays a role.

Structural basis of antizyme-mediated regulation of polyamine homeostasis.

Polyamines are organic polycations essential for cell growth and differentiation; their aberrant accumulation is often associated with diseases, including many types of cancer. To maintain polyamine homeostasis, the catalytic activity and protein abundance of ornithine decarboxylase (ODC), the committed enzyme for polyamine biosynthesis, are reciprocally controlled by the regulatory proteins antizyme isoform 1 (Az1) and antizyme inhibitor (AzIN). Az1 suppresses polyamine production by inhibiting the assembly of the functional ODC homodimer and, most uniquely, by targeting ODC for ubiquitin-independent proteolytic destruction by the 26S proteasome. In contrast, AzIN positively regulates polyamine levels by competing with ODC for Az1 binding. The structural basis of the Az1-mediated regulation of polyamine homeostasis has remained elusive. Here we report crystal structures of human Az1 complexed with either ODC or AzIN. Structural analysis revealed that Az1 sterically blocks ODC homodimerization. Moreover, Az1 binding triggers ODC degradation by inducing the exposure of a cryptic proteasome-interacting surface of ODC, which illustrates how a substrate protein may be primed upon association with Az1 for ubiquitin-independent proteasome recognition. Dynamic and functional analyses further indicated that the Az1-induced binding and degradation of ODC by proteasome can be decoupled, with the intrinsically disordered C-terminal tail fragment of ODC being required only for degradation but not binding. Finally, the AzIN-Az1 structure suggests how AzIN may effectively compete with ODC for Az1 to restore polyamine production. Taken together, our findings offer structural insights into the Az-mediated regulation of polyamine homeostasis and proteasomal degradation.

Investigation into length heteroplasmy in the mitochondrial DNA control region after treatment with bisulfite.

We report on a method to analyze length heteroplasmy within the human mitochondrial genome in which there are polycytosine [poly(C)] stretches. These poly(C) tracts induce heteroplasmy with the resultant inherent problems of accurate sequence designations. In this study, 20 samples that exhibited length heteroplasmy due to variation in the C-tracts within hypervariable region I (HVI) were treated with bisulfite, and one or more cytosine bases in these C-tracts were converted randomly to uracil. This resulted in an accurate sequence designation for nearly all samples. The only exceptions in which the DNA sequence could still not be determined occurred when there was total conversion, or a lack of conversion, of the cytosine bases. Replicate tests on the same samples showed that individual cytosine bases were randomly converted to uracil. This simple method was useful for investigating length heteroplasmy due to 16189C and 310C transitions in the mitochondrial-DNA control region. It is valuable for medical and forensic investigations.

DNA identification of monozygotic twins.

This study details the differentiation of identical twins based on single mutational base differences. There were three pairs of male monozygotic (MZ) twins in this study. DNA samples from blood, a buccal swab or saliva from each individual were all initially genotyped using 22 autosomal STR and 27 Y-STR loci. Preliminary screening confirmed there were no differences in the STR data between each pair of MZ twins. Whole Genome Sequence (WGS) data were generated from DNA extracted from the three body fluids from each individual. Kinship coefficients with 0.4254, 0.4557 and 0.4543 from 3 twins were generated based on WGS data to further confirm that their relationship was that of MZ twins. The fastq data generated by the Illumina Hiseq 2000 between MZ twins were then treated as "normal" as opposed to "tumor" using commercially available software tools to identify mutational single base changes. Sanger DNA sequencing confirmed there were 1, 5 and 9 single base changes found in WGS data from each of the three MZ twin sets. There was individual variation in the mutational base changes when comparing data from the three body fluids. The methods used in this study to differentiate MZ twins based on WGS data can readily be performed in many operational forensic DNA laboratories using user friendly software.

Novel c-di-GMP recognition modes of the mouse innate immune adaptor protein STING.

The mammalian ER protein STING (stimulator of interferon genes; also known as MITA, ERIS, MPYS or TMEM173) is an adaptor protein that links the detection of cytosolic dsDNA to the activation of TANK-binding kinase 1 (TBK1) and its downstream transcription factor interferon regulatory factor 3 (IFN3). Recently, STING itself has been found to be the direct receptor of bacterial c-di-GMP, and crystal structures of several human STING C-terminal domain (STING-CTD) dimers in the apo form or in complex with c-di-GMP have been published. Here, a novel set of structures of mouse STING-CTD (mSTING(137-344)) in apo and complex forms determined from crystals obtained under different crystallization conditions are reported. These novel closed-form structures exhibited considerable differences from previously reported open-form human STING-CTD structures. The novel mSTING structures feature extensive interactions between the two monomers, a unique asymmetric c-di-GMP molecule with one guanine base in an unusual syn conformation that is well accommodated in the dimeric interface with many direct specific interactions and two unexpected equivalent secondary peripheral c-di-GMP binding sites. Replacement of the amino acids crucial for specific c-di-GMP binding in mSTING significantly changes the ITC titration profiles and reduces the IFN-ß reporter luciferase activity. Taken together, these results reveal a more stable c-di-GMP binding mode of STING proteins that could serve as a template for rational drug design to stimulate interferon production by mammalian cells.

The risk of false inclusion of a relative in parentage testing - an in silico population study.

AIM: To investigate the potential of false inclusion of a close genetic relative in paternity testing by using computer generated families. METHODS: 10000 computer-simulated families over three generations were generated based on genotypes using 15 short tandem repeat loci. These data were used in assessing the probability of inclusion or exclusion of paternity when the father is actually a sibling, grandparent, uncle, half sibling, cousin, or a random male. Further, we considered a duo case where the mother's DNA type was not available and a trio case including the mother's profile. RESULTS: The data showed that the duo scenario had the highest and lowest false inclusion rates when considering a sibling (19.03 ± 0.77%) and a cousin (0.51 ± 0.14%) as the father, respectively; and the rate when considering a random male was much lower (0.04 ± 0.04%). The situation altered slightly with a trio case where the highest rate (0.56 ± 0.15%) occurred when a paternal uncle was considered as the father, and the lowest rate (0.03 ± 0.03%) occurred when a cousin was considered as the father. We also report on the distribution of the numbers for non-conformity (non-matching loci) where the father is a close genetic relative. CONCLUSIONS: The results highlight the risk of false inclusion in parentage testing. These data provide a valuable reference when incorporating either a mutation in the father's DNA type or if a close relative is included as being the father; particularly when there are varying numbers of non-matching loci.

DNA identification from dental pulp and cementum.

Teeth are one of the body tissues remaining after severe decomposition from which a DNA profile can be obtained to aid in human identification. Currently, the standard approach to isolate DNA from teeth requires pulverizing the entire tooth. This destructive approach compromises any further morphological or anthropological study. We report on two methods of DNA isolation that minimizes destruction of the tooth when accessing the DNA within pulp and cementum. Forty-nine teeth, removed as part of normal dental procedures, were buried for up to 92 days, with a further nine teeth acting as unburied controls. Additionally, four teeth samples collected during a forensic examination were included in this study. The two processes were: using a fine drill to access the pulp from the crown and then using endodontic files to collect the biological material; and using a sterile blade to scrape the cementum. It was found that the samples collected from the cementum had greater DNA quality compared to those samples obtained from the pulp. Microbial activity was found to play a role in the degradation of the nuclear material, reducing DNA yields from pulp. DNA profiling data from 24 loci, including 22 STR markers, indicated that multi-rooted teeth provided better DNA quantity and quality than those with a single root. The DNA quantity obtained from pulp samples of teeth which exhibited cavities was adversely affected, although this DNA loss was not from samples collected from the cementum of teeth in similar condition. Obtaining samples from DNA profiling from the cementum was found to be ideal if the morphological preservation of the tooth is required. Obtaining pathogen DNA is of interest when an occlusal approach to retrieve pulp may serve as a good alternative to prepare DNA without destruction of the tooth structure.

Structural polymorphism of c-di-GMP bound to an EAL domain and in complex with a type II PilZ-domain protein.

Cyclic di-GMP (c-di-GMP) is a novel secondary-messenger molecule that is involved in regulating a plethora of important bacterial activities through binding to an unprecedented array of effectors. Proteins with a canonical PilZ domain that bind c-di-GMP play crucial roles in regulating flagellum-based motility. In contrast, noncanonical type II PilZ domains that do not effectively bind c-di-GMP regulate twitching motility, which is dependent on type IV pili (T4P). Recent data indicate that T4P biogenesis is initiated via the interaction of a noncanonical type II PilZ protein with the GGDEF/EAL-domain protein FimX and the pilus motor protein PilB at high c-di-GMP concentrations. However, the molecular details of such interactions remain to be elucidated. In this manuscript, the first hetero-complex crystal structure between a type II PilZ protein and the EAL domain of the FimX protein (FimX(EAL)) from Xanthomonas campestris pv. campestris (Xcc) in the presence of c-di-GMP is reported. This work reveals two novel conformations of monomeric c-di-GMP in the XccFimX(EAL)-c-di-GMP and XccFimX(EAL)-c-di-GMP-XccPilZ complexes, as well as a unique interaction mode of a type II PilZ domain with FimX(EAL). These findings indicate that c-di-GMP is sufficiently flexible to adjust its conformation to match the corresponding recognition motifs of different cognate effectors. Together, these results represent a first step towards an understanding of how T4P biogenesis is controlled by c-di-GMP at the molecular level and also of the ability of c-di-GMP to bind to a wide variety of effectors.

A novel strategy for sibship determination in trio sibling model.

AIM: To use a virtually simulated population, generated from published allele frequencies based on 15 short tandem repeats (STR), to evaluate the efficacy of trio sibship testing and sibling assignment for forensic purposes. METHODS: Virtual populations were generated using 15 STR loci to create a large number of related and unrelated genotypes (10,000 trio combinations). Using these virtual populations, the probability of related and unrelated profiles can be compared to determine the chance of inclusions of being siblings if they are true siblings and the chance of inclusion if they are unrelated. Two specific relationships were tested - two reference siblings were compared to a third true sibling (3S trio, sibling trio) and two reference siblings were compared to an unrelated individual (2S1U trio, non-sibling trio). RESULTS: When the likelihood ratio was greater than 1, 99.87% of siblings in the 3S trio population were considered as siblings (sensitivity); 99.88% of non-siblings in the 2S1U trio population were considered as non-siblings (specificity); 99.9% of both populations were identified correctly as siblings and non-siblings; and the accuracy of the test was 99.88%. CONCLUSIONS: The high sensitivity and specificity figures when using two known siblings compared to a putative sibling are significantly greater than when using only one known relative. The data also support the use of increasing number of loci allowing for greater confidence in genetic identification. The system established in this study could be used as the model for evaluating and simulating the cases with multiple relatives.

The strategies to DVI challenges in Typhoon Morakot.

Small village populations in which there is a high amount of kinship can cause complications in cases of disaster victim identification. This problem was highlighted by the loss of life after Typhoon Morakot struck Taiwan where over 500 people from small isolated communities lost their lives. Most of the victims were buried by landslides in the remote mountainous areas of southern Taiwan. Only 146 pieces of human remains were recovered after searching for 4 months. Most of the human remains were received for examination as severely damaged fragments prevented possible identification by morphological features. DNA testing using the traditional duo parent/child or sibling screening by STR data opens the possibility of including not only the actual victim but also false positives. Variable likelihood ratios were obtained when comparing DNA types from human remains to those from potential relatives; however, with the DNA typing of numerous members of the same living family, multiple matches to potential families were avoided. Of the 146 samples obtained and collapsed to 130 victims, they were linked to 124 individuals resulting in their identification when compared to a pool of 588 potential relatives. Six of the human remains could not be linked to any living relative and remain unknown.

Coexpression of omega subunit in E. coli is required for the maintenance of enzymatic activity of Xanthomonas campestris pv. campestris RNA polymerase.

In this study, plasmid pBBad22K was modulated to be able to coexpress the subunits of Xanthomonascampestris pv. campestris (Xcc) core RNA polymerase (RNAP) in an Escherichia coli host. The subunit-encoding genes of Xcc core RNAP were PCR-amplified respectively to convert into gene cassettes in which an intact subunit-encoding gene and a ribosome binding site (RBS) preceding the gene were contained, and were then cloned one by one into pBBad22K. In addition, a hexahistidine tag (His-tag) was introduced into the C-terminus of alpha subunit-encoded rpoA during PCR for facilitating purification of Xcc core RNAP. The resultant vectors, pBC-CBA and pBC-CBAZ, were used for overproduction of Xcc core RNAP lacking or containing omega, respectively. The assembly of Xcc core RNAP subunits that were coexpressed from these vectors was demonstrated after purification via two-step column chromatography. The yield of Xcc core RNAP containing omega had a 67% increase compared with that of one lacking omega, indicating that omega promotes the assembly of Xcc core RNAP. In addition, Xcc core RNAP lacking omega showed a 13-fold decrease in enzymatic activity in comparison with that containing omega. Promoter-specific transcription assays by recombinant Xcc core RNAP reconstituted with external added sigma factor showed that the absence of omega debilitates the transcriptional activity of Xcc RNAP. Our results demonstrated that omega is not only capable of strengthening the stability, but is also required for the maintenance of enzymatic activity of Xcc RNAP.

Overproduction of soluble recombinant transglutaminase from Streptomyces netropsis in Escherichia coli.

A novel microbial transglutaminase (TGase) from the cultural filtrate of Streptomyces netropsis BCRC 12429 (Sn) was purified. The specific activity of the purified TGase was 18.2 U/mg protein with an estimated molecular mass of 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The TGase gene of S. netropsis was cloned and an open reading frame of 1,242 bp encoding a protein of 413 amino acids was identified. The Sn TGase was synthesized as a precursor protein with a preproregion of 82 amino acid residues. The deduced amino acid sequence of the mature S. netropsis TGase shares 78.9-89.6% identities with TGases from Streptomyces spp. A high level of soluble Sn TGase with its N-terminal propeptide fused with thioredoxin was expressed in E. coli. A simple and efficient process was applied to convert the purified recombinant protein into an active enzyme and showed activity equivalent to the authentic mature TGase.

Structural insights into the gating of DNA passage by the topoisomerase II DNA-gate.

Type IIA topoisomerases (Top2s) manipulate the handedness of DNA crossovers by introducing a transient and protein-linked double-strand break in one DNA duplex, termed the DNA-gate, whose opening allows another DNA segment to be transported through to change the DNA topology. Despite the central importance of this gate-opening event to Top2 function, the DNA-gate in all reported structures of Top2-DNA complexes is in the closed state. Here we present the crystal structure of a human Top2 DNA-gate in an open conformation, which not only reveals structural characteristics of its DNA-conducting path, but also uncovers unexpected yet functionally significant conformational changes associated with gate-opening. This structure further implicates Top2's preference for a left-handed DNA braid and allows the construction of a model representing the initial entry of another DNA duplex into the DNA-gate. Steered molecular dynamics calculations suggests the Top2-catalyzed DNA passage may be achieved by a rocker-switch-type movement of the DNA-gate.

A novel restriction-modification system from Xanthomonas campestris pv. vesicatoria encodes a m4C-methyltransferase and a nonfunctional restriction endonuclease.

A novel restriction-modification (R-M) system, designated as xveIIRM, from chromosomal DNA of the Xanthomonas campestris pv. vesicatoria strain 7-1 (Xcv7-1) was cloned and characterized. The xveIIRM genes involved in this R-M system are aligned in a tail-to-tail orientation and overlapped by 12 base pairs. XveII methyltransferase gene could encode a 299-amino acid protein (M.XveII) with an estimated mass of 33.7 kDa and was classified to be a member of beta-class of m4C-MTase. M.XveII methylates the second cytosine of the 5'-CCCGGG-3' recognition sequence. The predicted amino acid sequence of the intact XveII endonuclease shared 41.9% identity with SmaI. However, a premature TAA translation termination codon was found in the open reading frame of xveIIR and expected to encode an 18.3 kDa truncated protein. The sequence data are consistent with observation of this study that no SmaI-like restriction activity could be detected in the cell extract of Xcv7-1.

Expression of Mastoparan B, a Venom Peptide, Via Escherichia coli C43 (DE3) Coupled with an Artificial Oil Body-Cyanogen Bromide Technology Platform.

BACKGROUND: Mastoparan B (MPB) is a venom peptide isolated from Vespa basalis (black-bellied hornet), one of the dangerous vespine wasps found in Taiwan. MPB is a tetradecapeptide (LKLKSIVSWAKKVL), amphiphilic venom peptide, with a molecular mass of 1.6 kDa. MPB belongs to an evolutionarily conserved component of the innate immune response against microbes. In this study, we attempted to modify a reliable oleosin-based fusion expression strategy coupled with the artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce bioactive MPB. OBJECTIVES: The aim of this study was to develop an artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce the bioactive form of mastoparan B (MPB), which in a manner identical to that of its native counterpart. METHODS: The plasmid pET30-His6-rOle(127M→L)-MPB was constructed, and then four different E. coli strains- BL21(DE3), BL21(DE3)pLysS, C41(DE3), and C43(DE3) were tested to identify the most suitable host for the pET30-His6-rOle(127M→L)-MPB fusion protein expression. We optimized the expression conditions by testing different growth temperatures, isopropyl-ß-D-thiogalactoside (IPTG) concentrations, and post-induction collection times. Afterwards, the His6-rOle(127M→L)-MPB protein was purified by one-step nickel-chelated affinity chromatography (Ni2+-NTA) under denaturing conditions. The purified His6-rOle(127M→L)-MPB was selectively cleaved by thrombin protease to remove the His6-tag and the leader peptide from the N-terminus. Subsequently, rOle(127M→L)-MPB protein was constituted into AOB and incubated with CNBr for a cleavage reaction, which resulted in the release of the MPB from rOle(127M→L)-MPB protein via AOB. The purified MPB was identified by MALDI-MS and HPLC analysis, and its bioactivity was examined by antimicrobial testing. RESULTS: After a 2-h induction period, the E. coli C43(DE3) was found to be superior to BL21(DE3) and the other protease-deficient strains as an expression host. And, the optimal His6-rOle(127M→L)-MPB expression at 37°C for 2 h after induction with 5 µM IPTG. The purified MPB showed that a single major peak was detected by HPLC/UV detection with a retention time of 22.5 minutes, which was approximately 90% pure. The putative MPB, and over two-third of the peptide sequence was verified by the MALDI-MS analysis. Finally, the purified MPB was examined by a broth dilution-antimicrobial susceptibility test. These results indicated that the purified MPB was bioactive and very effective in anti-bacterial (E. coli J96) activity. Here, we successfully used the oleosin-based fusion expression strategy coupled with the artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce bioactive MPB peptide which, in a manner identical to that of its native counterpart. CONCLUSION: In this study, the recombinant oleosin based fusion strategy coupled with AOB-CNBr purification platform open a new avenue for the production of active MPB and facilitate the studies and applications of the peptide in the future for medicinal applications such as hypotension and antibacterial effect.

Investigation of length heteroplasmy in mitochondrial DNA control region by massively parallel sequencing.

Accurate sequencing of the control region of the mitochondrial genome is notoriously difficult due to the presence of polycytosine bases, termed C-tracts. The precise number of bases that constitute a C-tract and the bases beyond the poly cytosines may not be accurately defined when analyzing Sanger sequencing data separated by capillary electrophoresis. Massively parallel sequencing has the potential to resolve such poor definition and provides the opportunity to discover variants due to length heteroplasmy. In this study, the control region of mitochondrial genomes from 20 samples was sequenced using both standard Sanger methods with separation by capillary electrophoresis and also using massively parallel DNA sequencing technology. After comparison of the two sets of generated sequence, with the exception of the C-tracts where length heteroplasmy was observed, all sequences were concordant. Sequences of three segments 16184-16193, 303-315 and 568-573 with C-tracts in HVI, II and III can be clearly defined from the massively parallel sequencing data using the program SEQ Mapper. Multiple sequence variants were observed in the length of C-tracts longer than 7 bases. Our report illustrates the accurate designation of all the length variants leading to heteroplasmy in the control region of the mitochondrial genome that can be determined by SEQ Mapper based on data generated by massively parallel DNA sequencing.

Complete Genome Sequence of Xanthomonas campestris pv. campestris Strain 17 from Taiwan.

Xanthomonas campestris pv. campestris 17 is a Gram-negative bacterium that is phytopathogenic to cruciferous plants in Taiwan. The 4,994,426-bp-long genome consists of 24 contigs with 4,050 protein-coding genes, 1 noncoding RNA (ncRNA) gene, 6 rRNA genes, and 55 tRNA genes.