Menu
April 21, 2020

Somatic proteome of Haemonchus contortus.

Currently, there is a dearth of proteomic data to underpin fundamental investigations of parasites and parasitism at the molecular level. Here, using a high throughput LC-MS/MS-based approach, we undertook the first reported comprehensive, large-scale proteomic investigation of the barber’s pole worm (Haemonchus contortus) – one of the most important parasitic nematodes of livestock animals worldwide. In total, 2487 unique H. contortus proteins representing different developmental stages/sexes (i.e. eggs, L3s and L4s, female (Af) and male (Am) adults) were identified and quantified with high confidence. Bioinformatic analyses of this proteome revealed substantial alterations in protein profiles during the life cycle, particularly in the transition from the free-living to the parasitic phase, and key groups of proteins involved specifically in feeding, digestion, metabolism, development, parasite-host interactions (including immunomodulation), structural remodelling of the body wall and adaptive processes during parasitism. This proteomic data set will facilitate future molecular, biochemical and physiological investigations of H. contortus and related nematodes, and the discovery of novel intervention targets against haemonchosis. Copyright © 2019 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.


April 21, 2020

Genomics and Experimental Analysis Reveal a Novel Factor Contributing to the Virulence of Cronobacter sakazakii Strains Associated With Neonate Infection.

Cronobacter sakazakii causes meningitis and necrotizing enterocolitis in premature infants. However, its virulence determinants, especially those specific for strains associated with neonate infections, remain largely unknown.In this study, we performed a comparative genomic analysis of 209 C. sakazakii genomes, and 8 clonal groups (CGs) were revealed.CG1 and CG2 were found to be significantly associated with neonate infections, and significantly prevalent genes in these 2 CGs were identified. Of these, a gene encoding the LysR-type regulator, CklR, was shown to contribute to bacterial pathogenicity based on animal experiments. We found that CklR directly binds and activates the suf Fe-S cluster biosynthesis operon, and high expression of the suf operon increases bacterial resistance to oxidative stress, which increases survival within the host. This leads to a high degree of bacteremia, which contributes to the development of meningitis.Our work revealed a novel virulence factor specific to predominant pathogenic C. sakazakii strains. © The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.


April 21, 2020

Function and Distribution of a Lantipeptide in Strawberry Fusarium Wilt Disease-Suppressive Soils.

Streptomyces griseus S4-7 is representative of strains responsible for the specific soil suppressiveness of Fusarium wilt of strawberry caused by Fusarium oxysporum f. sp. fragariae. Members of the genus Streptomyces secrete diverse secondary metabolites including lantipeptides, heat-stable lanthionine-containing compounds that can exhibit antibiotic activity. In this study, a class II lantipeptide provisionally named grisin, of previously unknown biological function, was shown to inhibit F. oxysporum. The inhibitory activity of grisin distinguishes it from other class II lantipeptides from Streptomyces spp. Results of quantitative reverse transcription-polymerase chain reaction with lanM-specific primers showed that the density of grisin-producing Streptomyces spp. in the rhizosphere of strawberry was positively correlated with the number of years of monoculture and a minimum of seven years was required for development of specific soil suppressiveness to Fusarium wilt disease. We suggest that lanM can be used as a diagnostic marker of whether a soil is conducive or suppressive to the disease.


April 21, 2020

Hybrid sequencing-based personal full-length transcriptomic analysis implicates proteostatic stress in metastatic ovarian cancer.

Comprehensive molecular characterization of myriad somatic alterations and aberrant gene expressions at personal level is key to precision cancer therapy, yet limited by current short-read sequencing technology, individualized catalog of complete genomic and transcriptomic features is thus far elusive. Here, we integrated second- and third-generation sequencing platforms to generate a multidimensional dataset on a patient affected by metastatic epithelial ovarian cancer. Whole-genome and hybrid transcriptome dissection captured global genetic and transcriptional variants at previously unparalleled resolution. Particularly, single-molecule mRNA sequencing identified a vast array of unannotated transcripts, novel long noncoding RNAs and gene chimeras, permitting accurate determination of transcription start, splice, polyadenylation and fusion sites. Phylogenetic and enrichment inference of isoform-level measurements implicated early functional divergence and cytosolic proteostatic stress in shaping ovarian tumorigenesis. A complementary imaging-based high-throughput drug screen was performed and subsequently validated, which consistently pinpointed proteasome inhibitors as an effective therapeutic regime by inducing protein aggregates in ovarian cancer cells. Therefore, our study suggests that clinical application of the emerging long-read full-length analysis for improving molecular diagnostics is feasible and informative. An in-depth understanding of the tumor transcriptome complexity allowed by leveraging the hybrid sequencing approach lays the basis to reveal novel and valid therapeutic vulnerabilities in advanced ovarian malignancies.


April 21, 2020

Symbiotic organs shaped by distinct modes of genome evolution in cephalopods.

Microbes have been critical drivers of evolutionary innovation in animals. To understand the processes that influence the origin of specialized symbiotic organs, we report the sequencing and analysis of the genome of Euprymna scolopes, a model cephalopod with richly characterized host-microbe interactions. We identified large-scale genomic reorganization shared between E. scolopes and Octopus bimaculoides and posit that this reorganization has contributed to the evolution of cephalopod complexity. To reveal genomic signatures of host-symbiont interactions, we focused on two specialized organs of E. scolopes: the light organ, which harbors a monoculture of Vibrio fischeri, and the accessory nidamental gland (ANG), a reproductive organ containing a bacterial consortium. Our findings suggest that the two symbiotic organs within E. scolopes originated by different evolutionary mechanisms. Transcripts expressed in these microbe-associated tissues displayed their own unique signatures in both coding sequences and the surrounding regulatory regions. Compared with other tissues, the light organ showed an abundance of genes associated with immunity and mediating light, whereas the ANG was enriched in orphan genes known only from E. scolopes Together, these analyses provide evidence for different patterns of genomic evolution of symbiotic organs within a single host. Copyright © 2019 the Author(s). Published by PNAS.


April 21, 2020

Oenococcus sicerae sp. nov., isolated from French cider.

Two Gram-stain-positive, small ellipsoidal cocci, non-motile, oxidase- and catalase-negative, and facultative anaerobic strains (UCMA15228T and UCMA17102) were isolated in France, from fermented apple juices (ciders). The 16S rRNA gene sequence was identical between the two isolates and showed 97 % similarity with respect to the closest related species Oenococcus oeni and O. kitaharae. Therefore, the two isolates were classified within the genus Oenococcus. The phylogeny based on the pheS gene sequences also confirmed the position of the new taxon. DNA-DNA hybridizations based on in silico genome-to-genome comparisons (GGDC) and Average Nucleotide Identity (ANI) values, as well as species-specific PCR, validated the novelty of the taxon. Various phenotypic characteristics such as the optimum temperature and pH for growth, the ability to metabolise sugars, the aptitude to perform the malolactic fermentation, and the resistance to ethanol and NaCl, revealed that the two strains are distinguishable from the other members of the Oenococcus genus. The combined genotypic and phenotypic data support the classification of strains UCMA15228T and UCMA17102 into a novel species of Oenococcus, for which the name O. sicerae sp. nov. is proposed. The type strain is UCMA15228T (=DSM107163T=CIRM-BIA2288T).Copyright © 2018 Elsevier GmbH. All rights reserved.


April 21, 2020

The complexity of alternative splicing and landscape of tissue-specific expression in lotus (Nelumbo nucifera) unveiled by Illumina- and single-molecule real-time-based RNA-sequencing.

Alternative splicing (AS) plays a critical role in regulating different physiological and developmental processes in eukaryotes, by dramatically increasing the diversity of the transcriptome and the proteome. However, the saturation and complexity of AS remain unclear in lotus due to its limitation of rare obtainment of full-length multiple-splice isoforms. In this study, we apply a hybrid assembly strategy by combining single-molecule real-time sequencing and Illumina RNA-seq to get a comprehensive insight into the lotus transcriptomic landscape. We identified 211,802 high-quality full-length non-chimeric reads, with 192,690 non-redundant isoforms, and updated the lotus reference gene model. Moreover, our analysis identified a total of 104,288 AS events from 16,543 genes, with alternative 3′ splice-site being the predominant model, following by intron retention. By exploring tissue datasets, 370 tissue-specific AS events were identified among 12 tissues. Both the tissue-specific genes and isoforms might play important roles in tissue or organ development, and are suitable for ‘ABCE’ model partly in floral tissues. A large number of AS events and isoform variants identified in our study enhance the understanding of transcriptional diversity in lotus, and provide valuable resource for further functional genomic studies. © The Author(s) 2019. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.


April 21, 2020

Penicillium purpurogenum Produces a Set of Endoxylanases: Identification, Heterologous Expression, and Characterization of a Fourth Xylanase, XynD, a Novel Enzyme Belonging to Glycoside Hydrolase Family 10.

The fungus Penicillium purpurogenum grows on a variety of natural carbon sources and secretes a large number of enzymes which degrade the polysaccharides present in lignocellulose. In this work, the gene coding for a novel endoxylanase has been identified in the genome of the fungus. This gene (xynd) possesses four introns. The cDNA has been expressed in Pichia pastoris and characterized. The enzyme, XynD, belongs to family 10 of the glycoside hydrolases. Mature XynD has a calculated molecular weight of 40,997. It consists of 387 amino acid residues with an N-terminal catalytic module, a linker rich in ser and thr residues, and a C-terminal family 1 carbohydrate-binding module. XynD shows the highest identity (97%) to a putative endoxylanase from Penicillium subrubescens but its highest identity to a biochemically characterized xylanase (XYND from Penicillium funiculosum) is only 68%. The enzyme has a temperature optimum of 60 °C, and it is highly stable in its pH optimum range of 6.5-8.5. XynD is the fourth biochemically characterized endoxylanase from P. purpurogenum, confirming the rich potential of this fungus for lignocellulose biodegradation. XynD, due to its wide pH optimum and stability, may be a useful enzyme in biotechnological procedures related to this biodegradation process.


April 21, 2020

PacBio full-length cDNA sequencing integrated with RNA-seq reads drastically improves the discovery of splicing transcripts in rice.

In eukaryotes, alternative splicing (AS) greatly expands the diversity of transcripts. However, it is challenging to accurately determine full-length splicing isoforms. Recently, more studies have taken advantage of Pacific Bioscience (PacBio) long-read sequencing to identify full-length transcripts. Nevertheless, the high error rate of PacBio reads seriously offsets the advantages of long reads, especially for accurately identifying splicing junctions. To best capitalize on the features of long reads, we used Illumina RNA-seq reads to improve PacBio circular consensus sequence (CCS) quality and to validate splicing patterns in the rice transcriptome. We evaluated the impact of CCS accuracy on the number and the validation rate of splicing isoforms, and integrated a comprehensive pipeline of splicing transcripts analysis by Iso-Seq and RNA-seq (STAIR) to identify the full-length multi-exon isoforms in rice seedling transcriptome (Oryza sativa L. ssp. japonica). STAIR discovered 11 733 full-length multi-exon isoforms, 6599 more than the SMRT Portal RS_IsoSeq pipeline did. Of these splicing isoforms identified, 4453 (37.9%) were missed in assembled transcripts from RNA-seq reads, and 5204 (44.4%), including 268 multi-exon long non-coding RNAs (lncRNAs), were not reported in the MSU_osa1r7 annotation. Some randomly selected unreported splicing junctions were verified by polymerase chain reaction (PCR) amplification. In addition, we investigated alternative polyadenylation (APA) events in transcripts and identified 829 major polyadenylation [poly(A)] site clusters (PACs). The analysis of splicing isoforms and APA events will facilitate the annotation of the rice genome and studies on the expression and polyadenylation of AS genes in different developmental stages or growth conditions of rice. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.


April 21, 2020

Structure elucidation and biosynthetic gene cluster analysis of caniferolides A-D, new bioactive 36-membered macrolides from the marine-derived Streptomyces caniferus CA-271066.

Bioassay-guided isolation based on the antifungal activity of a culture broth of the marine-derived actinomycete Streptomyces caniferus CA-271066 led to the discovery of new 36-membered polyol macrolides, caniferolides A-D (1-4). Their connectivity was determined by spectroscopic methods including ESITOF-MS and 1D/2D NMR. The relative stereochemistry of each stereocluster in these compounds was established using NOE analysis, the universal database method and J-based configuration analysis, further assisted by comparisons with NMR data of structurally related macrolides. Genome sequencing followed by detailed bioinformatics analysis led to the identification of the corresponding biosynthetic gene cluster and allowed the prediction of the stereochemical outcome of their biosynthesis, confirming the relative stereochemistry of each stereocluster already determined by NMR and establishing their stereochemical relationship, ultimately rendering the absolute configuration of all chiral centers. Furthermore, based on our results and already published data, it has been possible to derive the complete absolute configuration of the related macrolides PM100117 and PM100118, astolides A and B, and deplelides A and B. Caniferolides A-D have shown pronounced antifungal activity against Candida albicans and Aspergillus fumigatus alongside antiproliferative activity against five human tumoral cell lines.


April 21, 2020

Hypoglycemic activity and gut microbiota regulation of a novel polysaccharide from Grifola frondosa in type 2 diabetic mice.

GFP-N, a novel heteropolysaccharide with a molecular weight of 1.26?×?107?Da, was isolated from maitake mushroom and purified by anion-exchange chromatography on a DEAE cellulose-52 column and gel-filtration chromatography on a Sephadex G-100 column. Its structure was characterized by Fourier transform infrared spectroscopy and one-dimensional (1H- and 13C-) NMR spectra, 1H1H correlation spectroscopy, and 1H13C heteronuclear single-quantum coherence spectroscopy. The structure of GFP-N consisted of L-arabinose, D-mannose and D-glucose and mainly contained three kinds of linkage type units as ?2,6)-a-D-Manp-(1???4, a-L-Araf-C1?, and ?3,6)-ß-D-Glcp-(1???. GFP-N could activate insulin receptor substrate 1, phosphatidylinositol-3-kinase, and glucose transporter 4 and inhibit c-Jun N-terminal kinase 1/2 for hypoglycemic effects in diabetic mouse livers. This is also the first report of the regulatory efficacy of Grifola frondosa polysaccharide on intestinal microflora in vivo using single-molecule real-time sequencing. These results indicated that polysaccharide from maitake mushroom could be as an enhancer to improve type 2 diabetes and a healthy food option to help regulate gut microbiota in diabetic individuals. Copyright © 2019 Elsevier Ltd. All rights reserved.


April 21, 2020

Fudania jinshanensis gen. nov., sp. nov., isolated from faeces of the Tibetan antelope (Pantholops hodgsonii) in China.

Two hitherto unknown bacteria (strains 313T and 352) were recovered from the faeces of Tibetan antelopes on the Tibet-Qinghai Plateau, PR China. Cells were rod-shaped and Gram-stain-positive. The optimal growth conditions were at 37?°C and pH 7. The isolates were closely related to Actinotignum sanguinis (92.6?% 16S rRNA gene sequence similarity), Arcanobacterium haemolyticum (92.5?%), Actinotignum schaalii (92.4?%), Actinobaculum massiliense (92.2?%) and Flaviflexus huanghaiensis (91.6?%). Phylogenetic analyses showed that strains 313T and 352 clustered independently in the vicinity of the genera Actinotignum, Actinobaculum and Flaviflexus, but could not be classified clearly as a member of any of these genera. Phylogenomic analysis also indicated that strains 313T and 352 formed an independent branch in the family Actinomycetaceae. The major cellular fatty acids of the strains were C16?:?0 and C18?:?1?9c. The polar lipids comprised diphosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylglycerol, phosphatidylinositol and five unidentified components. The peptidoglycan contained lysine, alanine and glutamic acid. The respiratory quinone was absent. The whole-cell sugars included glucose and rhamnose. The DNA G+C?content of strain 313T was 60.6?mol%. Based on the low 16S rRNA gene sequence similarities, its taxonomic position in the phylogenetic and phylogenomic trees and its unique lipid pattern, we propose that strains 313T and 352 represent members of a novel species in a new genus, for which the name Fudania jinshanensis gen. nov., sp. nov. is proposed. The type strain is 313T (=CGMCC 4.7453T=DSM 106216T).


April 21, 2020

NOVA1 directs PTBP1 to hTERT pre-mRNA and promotes telomerase activity in cancer cells.

Alternative splicing is dysregulated in cancer cells, driving the production of isoforms that allow tumor cells to survive and continuously proliferate. Part of the reactivation of telomerase involves the splicing of hTERT transcripts to produce full-length (FL) TERT. Very few splicing factors to date have been described to interact with hTERT and promote the production of FL TERT. We recently described one such splicing factor, NOVA1, that acts as an enhancer of FL hTERT splicing, increases telomerase activity, and promotes telomere maintenance in cancer cells. NOVA1 is expressed primarily in neurons and is involved in neurogenesis. In the present studies, we describe that polypyrimidine-tract binding proteins (PTBPs), which are also typically involved in neurogenesis, are also participating in the splicing of hTERT to FL in cancer. Knockdown experiments of PTBP1 in cancer cells indicate that PTBP1 reduces hTERT FL splicing and telomerase activity. Stable knockdown of PTBP1 results in progressively shortened telomere length in H1299 and H920 lung cancer cells. RNA pulldown experiments reveal that PTBP1 interacts with hTERT pre-mRNA in a NOVA1 dependent fashion. Knockdown of PTBP1 increases the expression of PTBP2 which also interacts with NOVA1, potentially preventing the association of NOVA1 with hTERT pre-mRNA. These new data highlight that splicing in cancer cells is regulated by competition for splice sites and that combinations of splicing factors interact at cis regulatory sites on pre-mRNA transcripts. By employing hTERT as a model gene, we show the coordination of the splicing factors NOVA1 and PTBP1 in cancer by regulating telomerase that is expressed in the vast majority of cancer cell types.


April 21, 2020

MSC: a metagenomic sequence classification algorithm.

Metagenomics is the study of genetic materials directly sampled from natural habitats. It has the potential to reveal previously hidden diversity of microscopic life largely due to the existence of highly parallel and low-cost next-generation sequencing technology. Conventional approaches align metagenomic reads onto known reference genomes to identify microbes in the sample. Since such a collection of reference genomes is very large, the approach often needs high-end computing machines with large memory which is not often available to researchers. Alternative approaches follow an alignment-free methodology where the presence of a microbe is predicted using the information about the unique k-mers present in the microbial genomes. However, such approaches suffer from high false positives due to trading off the value of k with the computational resources. In this article, we propose a highly efficient metagenomic sequence classification (MSC) algorithm that is a hybrid of both approaches. Instead of aligning reads to the full genomes, MSC aligns reads onto a set of carefully chosen, shorter and highly discriminating model sequences built from the unique k-mers of each of the reference sequences.Microbiome researchers are generally interested in two objectives of a taxonomic classifier: (i) to detect prevalence, i.e. the taxa present in a sample, and (ii) to estimate their relative abundances. MSC is primarily designed to detect prevalence and experimental results show that MSC is indeed a more effective and efficient algorithm compared to the other state-of-the-art algorithms in terms of accuracy, memory and runtime. Moreover, MSC outputs an approximate estimate of the abundances.The implementations are freely available for non-commercial purposes. They can be downloaded from https://drive.google.com/open?id=1XirkAamkQ3ltWvI1W1igYQFusp9DHtVl. © The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.


April 21, 2020

Genetic and biochemical characterization of FRI-3, a novel variant of the Ambler class A carbapenemase FRI-1.

To characterize a new variant of the FRI class A carbapenemase isolated from an MDR clinical Enterobacter cloacae isolate.A carbapenem-resistant E. cloacae was isolated from a rectal swab from a patient in an ICU in Southern Germany. Various phenotypic tests confirmed production of a putative class A carbapenemase. The new bla gene variant, blaFRI-3, and its genetic environment were characterized by WGS. Biochemical characterization was performed by heterologous expression in Escherichia coli TOP10 and by purification of the enzyme with subsequent determination of its kinetic parameters.PCR and sequencing carried out for different class A carbapenemase genes confirmed the presence of a novel variant of blaFRI-1. The novel variant was named FRI-3 and exhibited 91%, 96% and 92% amino acid identity to FRI-1, FRI-2 and FRI-4, respectively. E. coli TOP10 expressing blaFRI-3 showed increased resistance to almost all ß-lactams. Comparing the catalytic behaviour of FRI-3 and FRI-1, it was shown that FRI-3 had the same substrate spectrum, but basically hydrolysed ß-lactams less efficiently than FRI-1. WGS data revealed that blaFRI-3 was located on a 111?kb plasmid.The biochemical characterization of FRI-3 illustrates that even a few differences in the amino acid sequence can lead to altered catalytic activities of ß-lactamases belonging to the same family. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.


Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.