Dereplication of natural products from complex extracts by regression analysis and molecular networking: case study of redox-active compounds from <Emphasis Type="Italic">Viola alba</Emphasis> subsp. <Emphasis Type="Italic">dehnhardtii</Emphasis>

Introduction

In natural product research, bioassay-guided fractionation was previously widely employed but is now judged to be inadequate in terms of time and cost, particularly if only known compounds are ultimately isolated. The development of metabolomics, along with improvements in analytical tools, allows comprehensive metabolite profiling. This enables dereplication to target unknown active compounds early in the purification workflow.

Objectives

Starting from an ethanolic extract of violet leaves, this study aims to predict redox active compounds within a complex matrix through an untargeted metabolomics approach and correlation analysis.

Methods

Rapid fractionation of crude extracts was carried out followed by multivariate data analysis (MVA) of liquid chromatography–high resolution mass spectrometry (LC–HRMS) profiles. In parallel, redox active properties were evaluated by the capacity of the molecules to reduce 2,2-diphenyl-1-picrylhydrazyl (DPPH^·) and superoxide (O₂ ^·?) radicals using UV–Vis and electron spin resonance spectroscopies (ESR), respectively. A spectral similarity network (molecular networking) was used to highlight clusters involved in the observed redox activities.

Results

Dereplication on Viola alba subsp. dehnhardtii highlighted a reproducible pool of redox active molecules. Polyphenols, particularly O-glycosylated coumarins and C-glycosylated flavonoids, were identified and de novo dereplicated through molecular networking. Confirmatory analyses were undertaken by thin layer chromatography (TLC)–DPPH–MS assays and nuclear magnetic resonance (NMR) spectra of the most active compounds.

Conclusion

Our dereplication strategy allowed the screening of leaf extracts to highlight new biologically active metabolites in few steps with a limited amount of crude material and reduced time-consuming manipulations. This approach could be applied to any kind of natural extract for the study of various biological activities.

     

Four anti-protozoal and anti-bacterial compounds from Tapirira guianensis

Tapirira guianensis is a common tree used in traditional medicine in French Guiana against several infectious diseases (malaria, leishmaniasis, bacteria, etc.). The bioassay-guided purification of CH₂Cl₂ bark extract led to the isolation of four cyclic alkyl polyol derivatives: 4,6,2′-trihydroxy-6-[10′(Z)-heptadecenyl]-1-cyclohexen-2-one (1a), 1,4,6-trihydroxy-1,2′-epoxy-6-[10′(Z)-heptadecenyl]-2-cyclohexene (1b), 1,4,5,2′-tetrahydroxy-1-[10′(Z)-heptadecenyl]-2-cyclohexene (2), and 1,3,4,6-tetrahydroxy-1,2′-epoxy-6-[10′(Z)-heptadecenyl]-cyclohexane (3). The structures were established on the basis of 1D and 2D NMR analyses. The anti-leishmanial, anti-plasmodial, anti-bacterial (on Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli), and anti-fungal (on Candida albicans) activities of the extracts and of these original compounds were evaluated. Two showed medicinal interest supporting the traditional uses of the plant. The structures were established through spectral analyses of the isolates and their derivatives.     

IFNγ Response to Mycobacterium tuberculosis,Risk of Infection and Disease in Household Contacts of Tuberculosis Patients in Colombia

Objectives

Household contacts (HHCs) of pulmonary tuberculosis patients are at high risk of Mycobacterium tuberculosis infection and early disease development. Identification of individuals at risk of tuberculosis disease is a desirable goal for tuberculosis control. Interferon-gamma release assays (IGRAs) using specific M. tuberculosis antigens provide an alternative to tuberculin skin testing (TST) for infection detection. Additionally, the levels of IFNγ produced in response to these antigens may have prognostic value. We estimated the prevalence of M. tuberculosis infection by IGRA and TST in HHCs and their source population (SP), and assessed whether IFNγ levels in HHCs correlate with tuberculosis development.

Methods

A cohort of 2060 HHCs was followed for 2–3 years after exposure to a tuberculosis case. Besides TST, IFNγ responses to mycobacterial antigens: CFP, CFP-10, HspX and Ag85A were assessed in 7-days whole blood cultures and compared to 766 individuals from the SP in Medellín, Colombia. Isoniazid prophylaxis was not offered to child contacts because Colombian tuberculosis regulations consider it only in children under 5 years, TST positive without BCG vaccination.

Results

Using TST 65.9% of HHCs and 42.7% subjects from the SP were positive (OR 2.60, p<0.0001). IFNγ response to CFP-10, a biomarker of M. tuberculosis infection, tested positive in 66.3% HHCs and 24.3% from the SP (OR = 6.07, p<0.0001). Tuberculosis incidence rate was 7.0/1000 person years. Children <5 years accounted for 21.6% of incident cases. No significant difference was found between positive and negative IFNγ responders to CFP-10 (HR 1.82 95% CI 0.79–4.20 p = 0.16). However, a significant trend for tuberculosis development amongst high HHC IFNγ producers was observed (trend Log rank p = 0.007).

Discussion

CFP-10-induced IFNγ production is useful to establish tuberculosis infection prevalence amongst HHC and identify those at highest risk of disease. The high tuberculosis incidence amongst children supports administration of chemoprohylaxis to child contacts regardless of BCG vaccination.     

Combined Mutation And Rearrangement Screening by Quantitative PCR High-Resolution Melting: Is It Relevant for Hereditary Recurrent Fever Genes?

The recent identification of genes implicated in hereditary recurrent fevers has allowed their specific diagnosis. So far however, only punctual mutations have been identified and a significant number of patients remain with no genetic confirmation of their disease after routine molecular approaches such as sequencing. The possible involvement of sequence rearrangements in these patients has only been examined in familial Mediterranean fever and was found to be unlikely. To assess the existence of larger genetic alterations in 3 other concerned genes, MVK (Mevalonate kinase), NLRP3 (Nod like receptor family, pyrin domain containing 3) and TNFRSF1A (TNF receptor superfamily 1A), we adapted the qPCR-HRM method to study possible intragenic deletions and duplications. This single-tube approach, combining both qualitative (mutations) and quantitative (rearrangement) screening, has proven effective in Lynch syndrome diagnosis. Using this approach, we studied 113 unselected (prospective group) and 88 selected (retrospective group) patients and identified no intragenic rearrangements in the 3 genes. Only qualitative alterations were found with a sensitivity similar to that obtained using classical molecular techniques for screening punctual mutations. Our results support that deleterious copy number alterations in MVK, NLRP3 and TNFRSF1A are rare or absent from the mutational spectrum of hereditary recurrent fevers, and demonstrate that a routine combined method such as qPCR-HRM provides no further help in genetic diagnosis. However, quantitative approaches such as qPCR or SQF-PCR did prove to be quick and effective and could still be useful after non contributory punctual mutation screening in the presence of clinically evocative signs.     

Application of the major capsid protein as a marker of the phylogenetic diversity of Emiliania huxleyi viruses

Studies of the Phycodnaviridae have traditionally relied on the DNA polymerase (pol) gene as a biomarker. However, recent investigations have suggested that the major capsid protein (MCP) gene may be a reliable phylogenetic biomarker. We used MCP gene amplicons gathered across the North Atlantic to assess the diversity of Emiliania huxleyi-infecting Phycodnaviridae. Nucleotide sequences were examined across >6000 km of open ocean, with comparisons between concentrates of the virus-size fraction of seawater and of lysates generated by exposing host strains to these same virus concentrates. Analyses revealed that many sequences were only sampled once, while several were over-represented. Analyses also revealed nucleotide sequences distinct from previous coastal isolates. Examination of lysed cultures revealed a new richness in phylogeny, as MCP sequences previously unrepresented within the existing collection of E. huxleyi viruses (EhV) were associated with viruses lysing cultures. Sequences were compared with previously described EhV MCP sequences from the North Sea and a Norwegian Fjord, as well as from the Gulf of Maine. Principal component analysis indicates that location-specific distinctions exist despite the presence of sequences common across these environments. Overall, this investigation provides new sequence data and an assessment on the use of the MCP gene.     

The Streamlined Genome of Phytomonas spp. Relative to Human Pathogenic Kinetoplastids Reveals a Parasite Tailored for Plants

Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the wide biological and pathological diversity of these protists. While some Phytomonas spp. multiply in the latex of plants, or in fruit or seeds without apparent pathogenicity, others colonize the phloem sap and afflict plants of substantial economic value, including the coffee tree, coconut and oil palms. Plant trypanosomes have not been studied extensively at the genome level, a major gap in understanding and controlling pathogenesis. We describe the genome sequences of two plant trypanosomatids, one pathogenic isolate from a Guianan coconut and one non-symptomatic isolate from Euphorbia collected in France. Although these parasites have extremely distinct pathogenic impacts, very few genes are unique to either, with the vast majority of genes shared by both isolates. Significantly, both Phytomonas spp. genomes consist essentially of single copy genes for the bulk of their metabolic enzymes, whereas other trypanosomatids e.g. Leishmania and Trypanosoma possess multiple paralogous genes or families. Indeed, comparison with other trypanosomatid genomes revealed a highly streamlined genome, encoding for a minimized metabolic system while conserving the major pathways, and with retention of a full complement of endomembrane organelles, but with no evidence for functional complexity. Identification of the metabolic genes of Phytomonas provides opportunities for establishing in vitro culturing of these fastidious parasites and new tools for the control of agricultural plant disease.     

CRISPR Is an Optimal Target for the Design of Specific PCR Assays for Salmonella enterica Serotypes Typhi and Paratyphi A

Background

Serotype-specific PCR assays targeting Salmonella enterica serotypes Typhi and Paratyphi A, the causal agents of typhoid and paratyphoid fevers, are required to accelerate formal diagnosis and to overcome the lack of typing sera and, in some situations, the need for culture. However, the sensitivity and specificity of such assays must be demonstrated on large collections of strains representative of the targeted serotypes and all other bacterial populations producing similar clinical symptoms.

Methodology

Using a new family of repeated DNA sequences, CRISPR (clustered regularly interspaced short palindromic repeats), as a serotype-specific target, we developed a conventional multiplex PCR assay for the detection and differentiation of serotypes Typhi and Paratyphi A from cultured isolates. We also developed EvaGreen-based real-time singleplex PCR assays with the same two sets of primers.

Principal findings

We achieved 100% sensitivity and specificity for each protocol after validation of the assays on 188 serotype Typhi and 74 serotype Paratyphi A strains from diverse genetic groups, geographic origins and time periods and on 70 strains of bacteria frequently encountered in bloodstream infections, including 29 other Salmonella serotypes and 42 strains from 38 other bacterial species.

Conclusions

The performance and convenience of our serotype-specific PCR assays should facilitate the rapid and accurate identification of these two major serotypes in a large range of clinical and public health laboratories with access to PCR technology. These assays were developed for use with DNA from cultured isolates, but with modifications to the assay, the CRISPR targets could be used in the development of assays for use with clinical and other samples.     

Genotypic variation and chromosomal location of QTLs for somatic embryogenesis revealed by epidermal layers culture of recombinant inbred lines in the sunflower (Helianthus annuus L.)

The present study was conducted to identify the genetic factors controlling somatic embryogenesis in the sunflower. Two traits, the number of embryogenic explants per 40 explants plated (EE/40 E) and the number of embryos per 40 explants (E/40 E), were scored in 74 recombinant inbred lines (RILs) from a cross between ’PAC-2’ and ’RHA-266’. The experiment was designed as a randomized complete block with 76 genotypes (74 recombinant inbred lines and two parents) and three replications. Each replication consisted of three Erlenmeyer flasks with 40 epidermal layers (explants). Analyses of variance indicated the existence of highly significant differences among parental genotypes and their RILs. Heritabilities for the somatic embryogenesis traits studied, EE/40 E and E/40 E, were high (0.64 and 0.77 respectively) and the genetic gain, in percentage of the best parent for 10% of selected RILs, was significant. Four QTLs for EE/40 E (tee) and seven for E/40 E (ete) were detected using composite interval mapping and AFLP mapping. The QTLs for EE/40 E explained 48% of the phenotypic variation while the QTLs for E/40 E explained about 89% of the variation. Received:14 December 1999 / Accepted:18 May 2000     

Testing the antagonistic effect of follistatin on BMP family members in ovine granulosa cells

Follistatin was first demonstrated as an activin-binding protein, neutralizing its actions. However, there is emerging evidence that follistatin inhibits the action of other members of the transforming growth factor beta(TGFbeta) / bone morphogenetic protein (BMP) superfamily. Recently, numerous BMP factors have been shown to play important roles in regulating folliculogenesis and ovulation rate in mammals, and such a potential antagonistic role of follistatin is of particular interest in the context of ovarian function. Using a biological test based on progesterone production by ovine primary granulosa cells in culture, we show that follistatin was a strong antagonist of activin A, but not BMP-2 or BMP-4 actions. In contrast, noggin, a known specific BMP antagonist, had no effect on activin A but strongly neutralized BMP-2 and BMP-4 actions. BMP-6 action was only slightly reduced by both follistatin and noggin. Our data led to the conclusion that follistatin would not represent a determinant physiological modulator of the biological effect of BMP factors on granulosa cells.