Development of peptide aptamers as alternatives for antibody in the detection of amyloid-beta 42 aggregates.

Alzheimer's disease (AD) causes cognitive impairment and serious social isolation. However, there are no effective treatments and even no established confirmatory diagnostic tools for the disease. Amyloid beta (Aß) aggregation in the brain is the best-known pathognomonic mechanism of AD, so various methods for Aß detection have been developed for the diagnosis of this disease. We synthesized two novel, ultra-sensitive peptide probes specialized in detecting Aß aggregates, and examined their potential for future diagnostic application. The peptides are produced through phage high-throughput screening (HTS) and amplified through a serial process called biopanning, which is a repeating method of elution and amplification of probes. We picked phages specific for amyloid from two kinds of phage display. The synthesized peptides were confirmed to have excellent binding affinity to Aß aggregates, by immunohistochemical staining and western blotting using the brains of 3X transgenic (Tg) AD mice at different stages (5-7, 12-17 months old) of AD severity. In the present study, it was confirmed that newly developed amyloid-binding peptides could be used as novel probes for the detection of Aß aggregates, which can be used for clinical diagnosis of AD in the future.

Ultrasensitive Fluorescence Detection of Alzheimer's Disease Based on Polyvalent Directed Peptide Polymer Coupled to a Nanoporous ZnO Nanoplatform.

Amyloid-beta 42 (Aß42), the key biomarker of Alzheimer's disease (AD), aggregates to form neurotoxic amyloid plaques. In this work, we modified two fluorescein isothiocyanate-labeled Aß42-targeting peptides and designed an Aß42-specific ultrasensitive polyvalent-directed peptide polymer (PDPP) to enhance AD diagnosis sensitivity. The dissociation constant of Aß42 by PDPP was 103-fold higher than the single-site-directed peptide. The improved binding was due to the ability of PDPP to detect multiple receptors on the target. The power of the PDPP diagnostic probe was verified in its application to detect Aß42 in cerebrospinal fluid (CSF), which showed a lower limit of detection (LOD) in the fg mL-1 range that is more sensitive than detection by antibodies or single peptides. In addition, we present a novel ultrasensitive diagnostic system using an array of nanoporous ZnO nanoparticles, which play a role in fluorescence signal amplification, to further improve AD diagnosis sensitivity. We enhanced the signal on the basis of the properties of nanoporous ZnO nanoparticles and measured and quantified an ultralow concentration (ag mL-1 range) of Aß42. This PDPP coupled to the nanoporous ZnO-based system is a novel approach to AD diagnosis that might also be useful for the detection of other target biomarkers and clinical applications.

Novel Peptide-Based Inhibitors for Microtubule Polymerization in Phytophthora capsici.

The plant disease Phytophthora blight, caused by the oomycete pathogen Phytophthora capsici, is responsible for major economic losses in pepper production. Microtubules have been an attractive target for many antifungal agents as they are involved in key cellular events such as cell proliferation, signaling, and migration in eukaryotic cells. In order to design a novel biocompatible inhibitor, we screened and identified inhibitory peptides against alpha- and beta-tubulin of P. capsici using a phage display method. The identified peptides displayed a higher binding affinity (nanomolar range) and improved specificity toward P. capsici alpha- and beta-tubulin in comparison to Homo sapiens tubulin as evaluated by fluorometric analysis. One peptide demonstrated the high inhibitory effect on microtubule formation with a nanomolar range of IC50 values, which were much lower than a well-known chemical inhibitor-benomyl (IC50 = 500 µM). Based on these results, this peptide can be employed to further develop promising candidates for novel antifungal agents against Phytophthora blight.

Dynamic Chloroplast Genome Rearrangement and DNA Barcoding for Three Apiaceae Species Known as the Medicinal Herb "Bang-Poong".

Three Apiaceae species Ledebouriella seseloides, Peucedanum japonicum, and Glehnia littoralis are used as Asian herbal medicines, with the confusingly similar common name "Bang-poong". We characterized the complete chloroplast (cp) genomes and 45S nuclear ribosomal DNA (45S nrDNA) sequences of two accessions for each species. The complete cp genomes of G. littoralis, L. seseloides, and P. japonicum were 147,467, 147,830, and 164,633 bp, respectively. Compared to the other species, the P. japonicum cp genome had a huge inverted repeat expansion and a segmental inversion. The 45S nrDNA cistron sequences of the three species were almost identical in size and structure. Despite the structural variation in the P. japonicum cp genome, phylogenetic analysis revealed that G. littoralis diverged 5-6 million years ago (Mya), while P. japonicum diverged from L. seseloides only 2-3 Mya. Abundant copy number variations including tandem repeats, insertion/deletions, and single nucleotide polymorphisms, were found at the interspecies level. Intraspecies-level polymorphism was also found for L. seseloides and G. littoralis. We developed nine PCR barcode markers to authenticate all three species. This study characterizes the genomic differences between L. seseloides, P. japonicum, and G. littoralis; provides a method of species identification; and sheds light on the evolutionary history of these three species.

Ginseng Genome Database: an open-access platform for genomics of Panax ginseng.

BACKGROUND: The ginseng (Panax ginseng C.A. Meyer) is a perennial herbaceous plant that has been used in traditional oriental medicine for thousands of years. Ginsenosides, which have significant pharmacological effects on human health, are the foremost bioactive constituents in this plant. Having realized the importance of this plant to humans, an integrated omics resource becomes indispensable to facilitate genomic research, molecular breeding and pharmacological study of this herb. DESCRIPTION: The first draft genome sequences of P. ginseng cultivar "Chunpoong" were reported recently. Here, using the draft genome, transcriptome, and functional annotation datasets of P. ginseng, we have constructed the Ginseng Genome Database http://ginsengdb.snu.ac.kr /, the first open-access platform to provide comprehensive genomic resources of P. ginseng. The current version of this database provides the most up-to-date draft genome sequence (of approximately 3000 Mbp of scaffold sequences) along with the structural and functional annotations for 59,352 genes and digital expression of genes based on transcriptome data from different tissues, growth stages and treatments. In addition, tools for visualization and the genomic data from various analyses are provided. All data in the database were manually curated and integrated within a user-friendly query page. CONCLUSION: This database provides valuable resources for a range of research fields related to P. ginseng and other species belonging to the Apiales order as well as for plant research communities in general. Ginseng genome database can be accessed at http://ginsengdb.snu.ac.kr /.

Genome and evolution of the shade-requiring medicinal herb Panax ginseng.

Panax ginseng C. A. Meyer, reputed as the king of medicinal herbs, has slow growth, long generation time, low seed production and complicated genome structure that hamper its study. Here, we unveil the genomic architecture of tetraploid P. ginseng by de novo genome assembly, representing 2.98 Gbp with 59 352 annotated genes. Resequencing data indicated that diploid Panax species diverged in association with global warming in Southern Asia, and two North American species evolved via two intercontinental migrations. Two whole genome duplications (WGD) occurred in the family Araliaceae (including Panax) after divergence with the Apiaceae, the more recent one contributing to the ability of P. ginseng to overwinter, enabling it to spread broadly through the Northern Hemisphere. Functional and evolutionary analyses suggest that production of pharmacologically important dammarane-type ginsenosides originated in Panax and are produced largely in shoot tissues and transported to roots; that newly evolved P. ginseng fatty acid desaturases increase freezing tolerance; and that unprecedented retention of chlorophyll a/b binding protein genes enables efficient photosynthesis under low light. A genome-scale metabolic network provides a holistic view of Panax ginsenoside biosynthesis. This study provides valuable resources for improving medicinal values of ginseng either through genomics-assisted breeding or metabolic engineering.

Inhibition of anthrax lethal factor by ssDNA aptamers.

Anthrax is caused by Bacillus anthracis, a bacterium that is able to secrete the toxins protective antigen, edema factor and lethal factor. Due to the high level of secretion from the bacteria and its severe virulence, lethal factor (LF) has been sought as a biomarker for detecting bacterial infection and as an effective target to neutralize toxicity. In this study, we found three aptamers, and binding affinity was determined by fluorescently labeled aptamers. One of the aptamers exhibited high affinity, with a Kd value of 11.0 ±â€¯2.7 nM, along with low cross reactivity relative to bovine serum albumin and protective antigen. The therapeutic functionality of the aptamer was examined by assessing the inhibition of LF protease activity against a mitogen-activated protein kinase kinase. The aptamer appears to be an effective inhibitor of LF with an IC50 value of 15 ±â€¯1.5 µM and approximately 85% cell viability, suggesting that this aptamer provides a potential clue for not only development of a sensitive diagnostic device of B. anthracis infection but also the design of novel inhibitors of LF.

Development of ssDNA aptamers as potent inhibitors of Mycobacterium tuberculosis acetohydroxyacid synthase.

Acetohydroxyacid synthase (AHAS) from Mycobacterium tuberculosis (Mtb) is a promising potential drug target for an emerging class of new anti-tuberculosis agents. In this study, we identify short (30-mer) single-stranded DNA aptamers as a novel class of potent inhibitors of Mtb-AHAS through an in vitro DNA-SELEX method. Among all tested aptamers, two candidate aptamers (Mtb-Apt1 and Mtb-Apt6) demonstrated the greatest inhibitory potential against Mtb-AHAS activity with IC50 values in the low nanomolar range (28.94±0.002 and 22.35±0.001 nM respectively). Interestingly, inhibition kinetics analysis of these aptamers showed different modes of enzyme inhibition (competitive and mixed type of inhibition respectively). Secondary structure-guided mutational modification analysis of Mtb-Apt1 and Mtb-Apt6 identified the minimal region responsible for their inhibitory action and consequently led to 17-mer and 20-mer shortened aptamers that retained equivalent or greater inhibitory potential. Notably, a modeling and docking exercise investigated the binding site of these two potent inhibitory aptamers on the target protein and showed possible involvement of some key catalytic dimer interface residues of AHAS in the DNA-protein interactions that lead to its potent inhibition. Importantly, these two short candidate aptamers, Mtb-Apt1 (17-mer) and Mtb-Apt6 (20-mer), also demonstrated significant growth inhibition against multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of tuberculosis with very low MIC of 5.36 µg/ml and 6.24 µg/ml, respectively and no significant cytotoxicity against mammalian cell line. This is the first report of functional inhibitory aptamers against Mtb-AHAS and provides the basis for development of these aptamers as novel and strong anti-tuberculosis agents.

Genome-wide characterization of long intergenic non-coding RNAs (lincRNAs) provides new insight into viral diseases in honey bees Apis cerana and Apis mellifera.

BACKGROUND: Long non-coding RNAs (lncRNAs) are a class of RNAs that do not encode proteins. Recently, lncRNAs have gained special attention for their roles in various biological process and diseases. RESULTS: In an attempt to identify long intergenic non-coding RNAs (lincRNAs) and their possible involvement in honey bee development and diseases, we analyzed RNA-seq datasets generated from Asian honey bee (Apis cerana) and western honey bee (Apis mellifera). We identified 2470 lincRNAs with an average length of 1011 bp from A. cerana and 1514 lincRNAs with an average length of 790 bp in A. mellifera. Comparative analysis revealed that 5 % of the total lincRNAs derived from both species are unique in each species. Our comparative digital gene expression analysis revealed a high degree of tissue-specific expression among the seven major tissues of honey bee, different from mRNA expression patterns. A total of 863 (57 %) and 464 (18 %) lincRNAs showed tissue-dependent expression in A. mellifera and A. cerana, respectively, most preferentially in ovary and fat body tissues. Importantly, we identified 11 lincRNAs that are specifically regulated upon viral infection in honey bees, and 10 of them appear to play roles during infection with various viruses. CONCLUSIONS: This study provides the first comprehensive set of lincRNAs for honey bees and opens the door to discover lincRNAs associated with biological and hormone signaling pathways as well as various diseases of honey bee.

Transcriptome analysis reveals in vitro cultured Withania somnifera leaf and root tissues as a promising source for targeted withanolide biosynthesis.

BACKGROUND: The production of metabolites via in vitro culture is promoted by the availability of fully defined metabolic pathways. Withanolides, the major bioactive phytochemicals of Withania somnifera, have been well studied for their pharmacological activities. However, only a few attempts have been made to identify key candidate genes involved in withanolide biosynthesis. Understanding the steps involved in withanolide biosynthesis is essential for metabolic engineering of this plant to increase withanolide production. RESULTS: Transcriptome sequencing was performed on in vitro adventitious root and leaf tissues using the Illumina platform. We obtained a total of 177,156 assembled transcripts with an average unigene length of 1,033 bp. About 13% of the transcripts were unique to in vitro adventitious roots but no unique transcripts were observed in in vitro-grown leaves. A putative withanolide biosynthetic pathway was deduced by mapping the assembled transcripts to the KEGG database, and the expression of candidate withanolide biosynthesis genes -were validated by qRT PCR. The accumulation pattern of withaferin A and withanolide A varied according to the type of tissue and the culture period. Further, we demonstrated that in vitro leaf extracts exhibit anticancer activity against human gastric adenocarcinoma cell lines at sub G1 phase. CONCLUSIONS: We report here a validated large-scale transcriptome data set and the potential biological activity of in vitro cultures of W. somnifera. This study provides important information to enhance tissue-specific expression and accumulation of secondary metabolites, paving the way for industrialization of in vitro cultures of W. somnifera.

Comprehensive analysis of Panax ginseng root transcriptomes.

BACKGROUND: Korean ginseng (Panax ginseng C.A. Meyer) is a highly effective medicinal plant containing ginsenosides with various pharmacological activities, whose roots are produced commercially for crude drugs. RESULTS: Here, we used the Illumina platform to generate over 232 million RNA sequencing reads from four root samples, including whole roots from one-year-old plants and three types of root tissue from six-year-old plants (i.e., main root bodies, rhizomes, and lateral roots). Through de novo assembly and reference-assisted selection, we obtained a non-redundant unigene set consisting of 55,949 transcripts with an average length of 1,250 bp. Among transcripts in the unigene set, 94 % were functionally annotated via similarity searches against protein databases. Approximately 28.6 % of the transcripts represent novel gene sequences that have not previously been reported for P. ginseng. Digital expression profiling revealed 364 genes showing differential expression patterns among the four root samples. Additionally, 32 were uniquely expressed in one-year-old roots, while seven were uniquely expressed in six-year-old root tissues. We identified 38 transcripts encoding enzymes involved in ginsenoside biosynthesis pathways and 189 encoding UDP-glycosyltransferases. CONCLUSION: Our analysis provides new insights into the role of the root transcriptome in development and secondary metabolite biosynthesis in P. ginseng.

Genome-wide SNP identification and QTL mapping for black rot resistance in cabbage.

BACKGROUND: Black rot is a destructive bacterial disease causing large yield and quality losses in Brassica oleracea. To detect quantitative trait loci (QTL) for black rot resistance, we performed whole-genome resequencing of two cabbage parental lines and genome-wide SNP identification using the recently published B. oleracea genome sequences as reference. RESULTS: Approximately 11.5 Gb of sequencing data was produced from each parental line. Reference genome-guided mapping and SNP calling revealed 674,521 SNPs between the two cabbage lines, with an average of one SNP per 662.5 bp. Among 167 dCAPS markers derived from candidate SNPs, 117 (70.1%) were validated as bona fide SNPs showing polymorphism between the parental lines. We then improved the resolution of a previous genetic map by adding 103 markers including 87 SNP-based dCAPS markers. The new map composed of 368 markers and covers 1467.3 cM with an average interval of 3.88 cM between adjacent markers. We evaluated black rot resistance in the mapping population in three independent inoculation tests using F2:3 progenies and identified one major QTL and three minor QTLs. CONCLUSION: We report successful utilization of whole-genome resequencing for large-scale SNP identification and development of molecular markers for genetic map construction. In addition, we identified novel QTLs for black rot resistance. The high-density genetic map will promote QTL analysis for other important agricultural traits and marker-assisted breeding of B. oleracea.

A novel peptide-based recognition probe for the sensitive detection of CD44 on breast cancer stem cells.

Metastasis and recurrence of breast cancer remain significant clinical problems. The expression level of CD44 protein is higher in breast cancer-initiating cancer stem cells; therefore, the early detection of CD44 using a sensitive diagnostic probe is important for breast cancer diagnosis and therapeutic purposes. In this study, we fabricated a polyvalent directed peptide polymer (PDPP) that specifically recognized the CD44 biomarker, as confirmed by immunocytochemistry tests and fluorescence-activated cell sorting assessment. Our results indicate that PDPP is useful as a novel tool for the sensitive detection of breast cancer stem cells.

Association of molecular markers derived from the BrCRTISO1 gene with prolycopene-enriched orange-colored leaves in Brassica rapa [corrected]..

KEY MESSAGE: Sequence polymorphism in BrCRTISO1, encoding carotenoid isomerase, is identified in orange-colored B. rapa , and three resulting gene-based markers will be useful for marker-assisted breeding of OC cultivars. Carotenoids are color pigments that are important for protection against excess light in plants and essential sources of retinols and vitamin A for animals. We identified a single recessive gene that might cause orange-colored (OC) inner leaves in Brassica rapa. The inner leaves of the OC cultivar were enriched in lycopene-like compounds, specifically prolycopene and its isomers, which can be a useful functional trait for Kimchi cabbage. We used a candidate gene approach based on the 21 genes in the carotenoid pathway to identify a candidate gene responsible for the orange color. Among them, we focused on two carotenoid isomerase (CRTISO) genes, BrCRTISO1 and BrCRTISO2. The expression of BrCRTISO1 was higher than that of BrCRTISO2 in a normal yellow-colored (YE) cultivar, but full-length BrCRTISO1 transcripts were not detected in the OC cultivar. Genomic sequence analysis revealed that BrCRTISO1 of the OC cultivar had many sequence variations, including single nucleotide polymorphisms (SNPs) and insertions and deletions (InDels), compared to that of the YE cultivar. We developed molecular makers for the identification of OC phenotype based on the polymorphic regions within BrCRTISO1 in B. rapa breeding. The BrCRTISO1 gene and its markers identified in this study are novel genetic resources and will be useful for studying the carotenoid biosynthesis pathway as well as developing new cultivars with unique carotenoid contents in Brassica species.

Characterization of a new high copy Stowaway family MITE, BRAMI-1 in Brassica genome.

BACKGROUND: Miniature inverted-repeat transposable elements (MITEs) are expected to play important roles in evolution of genes and genome in plants, especially in the highly duplicated plant genomes. Various MITE families and their roles in plants have been characterized. However, there have been fewer studies of MITE families and their potential roles in evolution of the recently triplicated Brassica genome. RESULTS: We identified a new MITE family, BRAMI-1, belonging to the Stowaway super-family in the Brassica genome. In silico mapping revealed that 697 members are dispersed throughout the euchromatic regions of the B. rapa pseudo-chromosomes. Among them, 548 members (78.6%) are located in gene-rich regions, less than 3 kb from genes. In addition, we identified 516 and 15 members in the 470 Mb and 15 Mb genomic shotgun sequences currently available for B. oleracea and B. napus, respectively. The resulting estimated copy numbers for the entire genomes were 1440, 1464 and 2490 in B. rapa, B. oleracea and B. napus, respectively. Concurrently, only 70 members of the related Arabidopsis ATTIRTA-1 MITE family were identified in the Arabidopsis genome. Phylogenetic analysis revealed that BRAMI-1 elements proliferated in the Brassica genus after divergence from the Arabidopsis lineage. MITE insertion polymorphism (MIP) was inspected for 50 BRAMI-1 members, revealing high levels of insertion polymorphism between and within species of Brassica that clarify BRAMI-1 activation periods up to the present. Comparative analysis of the 71 genes harbouring the BRAMI-1 elements with their non-insertion paralogs (NIPs) showed that the BRAMI-1 insertions mainly reside in non-coding sequences and that the expression levels of genes with the elements differ from those of their NIPs. CONCLUSION: A Stowaway family MITE, named as BRAMI-1, was gradually amplified and remained present in over than 1400 copies in each of three Brassica species. Overall, 78% of the members were identified in gene-rich regions, and it is assumed that they may contribute to the evolution of duplicated genes in the highly duplicated Brassica genome. The resulting MIPs can serve as a good source of DNA markers for Brassica crops because the insertions are highly dispersed in the gene-rich euchromatin region and are polymorphic between or within species.

Comparative analysis of mitochondrial genomes of Schisandra repanda and Kadsura japonica.

The family Schisandraceae is a basal angiosperm plant group distributed in East and Southeast Asia and includes many medicinal plant species such as Schisandra chinensis. In this study, mitochondrial genomes (mitogenomes) of two species, Schisandra repanda and Kadsura japonica, in the family were characterized through de novo assembly using sequencing data obtained with Oxford Nanopore and Illumina sequencing technologies. The mitogenomes of S. repanda were assembled into one circular contig (571,107 bp) and four linear contigs (10,898-607,430 bp), with a total of 60 genes: 38 protein-coding genes (PCGs), 19 tRNA genes, and 3 rRNA genes. The mitogenomes of K. japonica were assembled into five circular contigs (211,474-973,503 bp) and three linear contigs (8,010-72,712 bp), with a total of 66 genes: 44 PCGs, 19 tRNA genes, and 3 rRNA genes. The mitogenomes of the two species had complex structural features with high repeat numbers and chloroplast-derived sequences, as observed in other plant mitogenomes. Phylogenetic analysis based on PCGs revealed the taxonomical relationships of S. repanda and K. japonica with other species from Schisandraceae. Finally, molecular markers were developed to distinguish between S. repanda, K. japonica, and S. chinensis on the basis of InDel polymorphisms present in the mitogenomes. The mitogenomes of S. repanda and K. japonica will be valuable resources for molecular and taxonomic studies of plant species that belong to the family Schisandraceae.

Mitochondrial genome recombination in somatic hybrids of Solanum commersonii and S. tuberosum.

Interspecific somatic hybridization has been performed in potato breeding experiments to increase plant resistance against biotic and abiotic stress conditions. We analyzed the mitochondrial and plastid genomes and 45S nuclear ribosomal DNA (45S rDNA) for the cultivated potato (S. tuberosum, St), wild potato (S. commersonii, Sc), and their somatic hybrid (StSc). Complex genome components and structure, such as the hybrid form of 45S rDNA in StSc, unique plastome in Sc, and recombinant mitogenome were identified. However, the mitogenome exhibited dynamic multipartite structures in both species as well as in the somatic hybrid. In St, the mitogenome is 756,058 bp and is composed of five subgenomes ranging from 297,014 to 49,171 bp. In Sc, it is 552,103 bp long and is composed of two sub-genomes of 338,427 and 213,676 bp length. StSc has 447,645 bp long mitogenome with two subgenomes of length 398,439 and 49,206 bp. The mitogenome structure exhibited dynamic recombination mediated by tandem repeats; however, it contained highly conserved genes in the three species. Among the 35 protein-coding genes of the StSc mitogenome, 21 were identical for all the three species, and 12 and 2 were unique in Sc and St, respectively. The recombinant mitogenome might be derived from homologous recombination between both species during somatic hybrid development.

Development of SNP Markers for White Immature Fruit Skin Color in Cucumber (Cucumis sativus L.) Using QTL-seq and Marker Analyses.

Despite various efforts in identifying the genes governing the white immature fruit skin color in cucumber, the genetic basis of the white immature fruit skin color is not well known. In the present study, genetic analysis showed that a recessive gene confers the white immature fruit skin-color phenotype over the light-green color of a Korean slicer cucumber. High-throughput QTL-seq combined with bulked segregation analysis of two pools with the extreme phenotypes (white and light-green fruit skin color) in an F2 population identified two significant genomic regions harboring QTLs for white fruit skin color within the genomic region between 34.1 and 41.67 Mb on chromosome 3, and the genomic region between 12.2 and 12.7 Mb on chromosome 5. Further, nonsynonymous SNPs were identified with a significance of p < 0.05 within the QTL regions, resulting in eight homozygous variants within the QTL region on chromosome 3. SNP marker analysis uncovered the novel missense mutations in Chr3CG52930 and Chr3CG53640 genes and showed consistent results with the phenotype of light-green and white fruit skin-colored F2 plants. These two genes were located 0.5 Mb apart on chromosome 3, which are considered strong candidate genes. Altogether, this study laid a solid foundation for understanding the genetic basis and marker-assisted breeding of immature fruit skin color in cucumber.

Inheritance of chloroplast and mitochondrial genomes in cucumber revealed by four reciprocal F1 hybrid combinations.

Both genomes in chloroplasts and mitochondria of plant cell are usually inherited from maternal parent, with rare exceptions. To characterize the inheritance patterns of the organelle genomes in cucumber (Cucumis sativus var. sativus), two inbred lines and their reciprocal F1 hybrids were analyzed using an next generation whole genome sequencing data. Their complete chloroplast genome sequences were de novo assembled, and a single SNP was identified between the parental lines. Two reciprocal F1 hybrids have the same chloroplast genomes with their maternal parents. Meanwhile, 292 polymorphic sites were identified between mitochondrial genomes of the two parental lines, which showed the same genotypes with their paternal parents in the two reciprocal F1 hybrids, without any recombination. The inheritance patterns of the chloroplast and mitochondria genomes were also confirmed in four additional cucumber accessions and their six reciprocal F1 hybrids using molecular markers derived from the identified polymorphic sites. Taken together, our results indicate that the cucumber chloroplast genome is maternally inherited, as is typically observed in other plant species, whereas the large cucumber mitochondrial genome is paternally inherited. The combination of DNA markers derived from the chloroplast and mitochondrial genomes will provide a convenient system for purity test of F1 hybrid seeds in cucumber breeding.

Development of a receptor-based inhibitory penta-unit-conjugated peptide to enhance anthrax toxin neutralization.

Anthrax toxin is a key virulence factor for Bacillus anthracis. The cell-binding component of anthrax toxin, protective antigen (PA), mediates the entry of the toxin into cells by first binding to the extracellular von Willebrand factor A (VWA) domain of the cellular anthrax toxin receptor (ATR). Herein, we targeted the VWA domain of the cellular receptor to develop a more effective antitoxin agent for neutralization of anthrax toxin. We selected ATR-binding peptides by using a phage display: among these, we identified two novel peptides binding to the ATR with high affinity and specificity, and that neutralized anthrax toxicity in cells. Furthermore, to enhance the functional efficiency of the probes, the peptides were modified and conjugated to three polyvalent probe backbones: a 17 amino-acid-based cyclic form penta-unit, poly-d-lysine (PDL), or the M13 bacteriophage. One of the functionally modified polyvalent peptide probes, the penta-unit-conjugated probe (PUCP) produced the most potent neutralization of anthrax toxin, with half-maximal inhibitory concentration (IC50) of 20 nM. The PUCP disrupted anthrax toxin binding to its receptor and reduced endocytosis of anthrax toxin. This peptide-based approach may, therefore, represent a promising strategy to combat anthrax toxicosis and other bacterial diseases and may be efficient for disease treatment.