The root extract of the medicinal plant Pelargonium sidoides is a potent HIV-1 attachment inhibitor.

Global HIV-1 treatment would benefit greatly from safe herbal medicines with scientifically validated novel anti-HIV-1 activities. The root extract from the medicinal plant Pelargonium sidoides (PS) is licensed in Germany as the herbal medicine EPs®7630, with numerous clinical trials supporting its safety in humans. Here we provide evidence from multiple cell culture experiments that PS extract displays potent anti-HIV-1 activity. We show that PS extract protects peripheral blood mononuclear cells and macrophages from infection with various X4 and R5 tropic HIV-1 strains, including clinical isolates. Functional studies revealed that the extract from PS has a novel mode-of-action. It interferes directly with viral infectivity and blocks the attachment of HIV-1 particles to target cells, protecting them from virus entry. Analysis of the chemical footprint of anti-HIV activity indicates that HIV-1 inhibition is mediated by multiple polyphenolic compounds with low cytotoxicity and can be separated from other extract components with higher cytotoxicity. Based on our data and its excellent safety profile, we propose that PS extract represents a lead candidate for the development of a scientifically validated herbal medicine for anti-HIV-1 therapy with a mode-of-action different from and complementary to current single-molecule drugs

Metabolomic investigations in cerebrospinal fluid of Parkinson's disease

The underlying mechanisms of Parkinson's disease are not completely revealed. Especially, early diagnostic biomarkers are lacking. To characterize early pathophysiological events, research is focusing on metabolomics. In this case-control study we investigated the metabolic profile of 31 Parkinson's disease-patients in comparison to 95 neurologically healthy controls. The investigation of metabolites in CSF was performed by a 12 Tesla SolariX Fourier transform-ion cyclotron resonance-mass spectrometer (FT-ICR-MS). Multivariate statistical analysis sorted the most important biomarkers in relation to their ability to differentiate Parkinson versus control. The affected metabolites, their connection and their conversion pathways are described by means of network analysis. The metabolic profiling by FT-ICR-MS in CSF yielded in a good group separation, giving insights into the disease mechanisms. A total number of 243 metabolites showed an affected intensity in Parkinson's disease, whereas 15 of these metabolites seem to be the main biological contributors. The network analysis showed a connection to the tricarboxylic cycle (TCA cycle) and therefore to mitochondrial dysfunction and increased oxidative stress within mitochondria. The metabolomic analysis of CSF in Parkinson's disease showed an association to pathways which are involved in lipid/ fatty acid metabolism, energy metabolism, glutathione metabolism and mitochondrial dysfunction

Digging into the low molecular weight peptidome with the OligoNet web server

Abstract Bioactive peptides play critical roles in regulating many biological processes. Recently, natural short peptides biomarkers are drawing significant attention and are considered as “hidden treasure” of drug candidates. High resolution and high mass accuracy provided by mass spectrometry (MS)-based untargeted metabolomics would enable the rapid detection and wide coverage of the low-molecular-weight peptidome. However, translating unknown masses (<1 500 Da) into putative peptides is often limited due to the lack of automatic data processing tools and to the limit of peptide databases. The web server OligoNet responds to this challenge by attempting to decompose each individual mass into a combination of amino acids out of metabolomics datasets. It provides an additional network-based data interpretation named “Peptide degradation network” (PDN), which unravels interesting relations between annotated peptides and generates potential functional patterns. The ab initio PDN built from yeast metabolic profiling data shows a great similarity with well-known metabolic networks, and could aid biological interpretation. OligoNet allows also an easy evaluation and interpretation of annotated peptides in systems biology, and is freely accessible at https://daniellyz200608105.shinyapps.io/OligoNet/

New molecular evidence of wine yeast-bacteria interaction unraveled by non-targeted exometabolomic profiling

International audienceIntroduction Bacterial malolactic fermentation (MLF) has a considerable impact on wine quality. The yeast strain used for primary fermentation can systematically stimulate (MLF+ phenotype) or inhibit (MLF-) bacteria and the MLF process as a function of numerous winemaking practices, but the underlying molecular evidence still remains a mystery.Objectives The goal of the study was to elucidate such evidence by the direct comparison of extracellular metabolic profiles of MLF? and MLF-phenotypes.Methods We have applied a non-targeted metabolomic approach combining ultrahigh-resolution FT-ICR-MS analysis, powerful statistical tools and a comprehensive wine metabolite database.Results We discovered around 2500 unknown masses and 800 putative biomarkers involved in phenotypic distinction. For the putative biomarkers, we also developed a biomarker identification workflow and elucidated the exact structure (by UPLC-Q-ToF-MS2) and/or exact physiological impact (by in vitro tests) of several novel biomarkers, such as D-gluconic acid, citric acid, trehalose and tripeptide Pro-Phe-Val. In addition to valid biomarkers, molecular evidence was reflected by unprecedented chemical diversity (around 3000 discriminant masses) that characterized both the yeast phenotypes. While distinct chemical families such as phenolic compounds, carbohydrates, amino acids and peptides characterize the extracellular metabolic profiles of the MLF? phenotype, the MLF-phenotype is associated with sulphur-containing peptides.Conclusion The non-targeted approach used in this study played an important role in finding new and unexpected molecular evidence

Evaluation of anti-HIV-1 activity of <i>Pelargonium sidoides</i> (PS) aqueous root extract with various HIV-1 target cells and HIV-1 isolates.

<p>Panels A-C show representative results for dose-dependent inhibition of HIV-1 infection of LC5-RIC indicator cells, human peripheral blood mononuclear cells (PBMC) and human monocyte-derived macrophages (MDM) by PS extract. Data for infection of PS-treated cultures are expressed in percentage of values measured for untreated cultures ( = 100% infection). The graph in D shows the EC₅₀ values calculated from multiple independent infection experiments (n = number of independent infection experiments) for each cell type. Panel E shows HIV-1 inhibitory activity (EC₅₀) of PS extract against clinical HIV-1 isolates. <i>A</i>, HIV-1 reporter cells (LC5-RIC) were exposed to HIV-1_LAI and serial dilutions of PS extract and infection assayed by measuring reporter gene expression (step 1) and production of infectious virus (step 2). Mean values and standard deviation of the mean are indicated for each extract dilution. <i>B</i>, IL-2 stimulated PBMCs were exposed to HIV-1_LAI and different concentrations of PS extract and levels of infectious virus in culture supernatants of PBMCs assayed with LC5-RIC cells. Results are shown for 4 independent infection experiments using PBMCs that were pooled from 4 different donors, with each extract concentration assayed in triplicate in each experiment. <i>C</i>, MDMs were exposed to the HIV-1 reporter virus R5 HIV-1NL4-3 IRES-eGFP that coexpresses Nef and EGFP from a bicistronic mRNA via an IRES element. Three days later, proportions (%) of GFP positive cells were determined by flow cytometry. Data were normalized to values for untreated cells. Results are shown for three independent infection experiments with MDMs from different donors. <i>D</i>, The graph shows EC₅₀ values (effective concentration for half-maximal inhibition) for each cell type, calculated from the compiled results of multiple independent infection experiments. n = number of independent infection experiments with triplicate analysis of each extract concentration. Mean values and standard deviations of the mean are indicated for each cell type. <i>E</i>, HIV-1 reporter cells (LC5-RIC-R5) were exposed to the indicated clinical HIV-1 isolates and serial dilutions of PS extract in triplicate wells and reporter gene expression was measured 5 days post infection. Mean values and standard deviations of the mean are indicated for each HIV-1 isolate.</p

PS extracts inhibit attachment of virus particles to host cells.

<p>The effects of PS extract (PS), polyphenols enriched from PS extract (PSPP) by adsorption to polyvinylpolypyrrolidone and the reference compound Griffithsin (GRFT) on virus attachment were determined by fluorescent imaging of virus particles associated with host cells. Controls consisted of cells without virus inoculum (background) and cells exposed to virus particles in the absence of inhibitory compounds (attachment control). LC5-RIC-R5 cells were exposed to fluorescently tagged R5 virus particles (R5 HIV-1 NL4-3 Gag-iGFP) in the presence of the fusion inhibitor T20. Cells were stained with DiD (membrane stain; pink) and DAPI (DNA stain; blue). <i>A</i>, Representative images are shown for each sample. Scale bars: 35 µm. <i>B</i>, Quantitative analysis of cell-associated fluorescent signals. Quantification of cell-associated GFP signals was performed with Volocity 6.2.1 imaging software. Cells were identified manually. 160-180 cells from at least 5 images were analyzed for each sample. Mean values and standard deviations are shown. Statistical significances were determined by one-way Anova with Bonferroni post-hoc test. **** p<0.0001.</p

Effects of pre-incubation of virus preparations or target cells with PS extracts or reference compounds on inhibition of HIV-1 infection.

<p>Inhibition of HIV-1 infection by PS extract was enhanced by pre-incubation of virus preparations with PS extract, compared to inhibition of infection without virus pre-incubation. HIV-1 target cells pre-incubated with PS extract prior to infection did not retain HIV-1 inhibitory activity. Virus pre-incubation (green curves): Inhibitory activity of PS extract was assayed with HIV-1_LAI virus preparations pre-incubated with different concentrations of PS extract for 4 hours before adding to LC5-RIC cells. Target cell pre-incubation (black curves): LC5-RIC cells were incubated with different concentrations of inhibitors and subsequently exposed to HIV-1_LAI in fresh medium in the absence of inhibitors. No pre-incubation (blue curves): Virus and inhibitors were added simultaneously to LC5-RIC cells. The entry inhibitors Griffithsin, AMD3100 and T20 and the reverse transcriptase inhibitor Efavirenz were analyzed as reference compounds. Mean values of triplicate wells and standard deviation of the mean are indicated for each extract concentration.</p

PS extracts inhibit entry of HIV-1 into the host cell.

<p><i>A</i>, Comparison of time-of-addition (TOA) profiles of PS extract and reference compounds that inhibit HIV-1 entry (i.e. Griffithsin and T-20) and reverse transcription (AZT), respectively. For TOA assays, virus preparations (HIV-1_LAI) were added to LC5-RIC cells and HIV-1 inhibitors added either simultaneously with the virus (time point 0) or at later time points (h.p.i.  =  hours post infection) at concentrations yielding maximum inhibition (i.e. >5× EC₅₀). Cultures were assayed for reporter expression 48 hours after virus addition and fluorescent signal intensities normalized to those of cultures exposed to virus in the absence of inhibitors. Each time point was measured in triplicate. Mean values and standard deviation of the mean are indicated for each time point. <i>B</i>, Reduction of input HIV-1 viral RNA levels by treatment with PS-extract. LC5-CD4 cells were exposed to HIV-1_LA1 and Efavirenz in the presence or absence of PS extract (50 µg/ml) in triplicate samples. After 4 hours exposure time, RNA was isolated from each sample, cDNA prepared and real-time PCR performed with HIV-1 specific primers to quantify viral RNA levels. HIV-1 transcript levels were normalized to RPII. Relative expression was calculated by the −2^−ΔΔCT method. The data is expressed as fold expression of HIV-1 RNA in HIV-1 exposed/treated samples compared to unexposed/untreated samples. Columns indicate mean values and error bars the standard deviations of the means. <i>C</i>, Reduction of HIV-1 DNA loads in HIV-1-exposed PBMCs by treatment with PS extract. HIV-1 DNA levels were determined by real-time PCR in peripheral blood derived mononuclear cells treated with PS extract during exposure to HIV-1 and in untreated infected cells (analysed in triplicate samples). Columns indicate mean values and error bars the standard deviations of the means. <i>D</i>, Influence of envelope proteins on the inhibition of virus infection by PS extract. LC5-RIC cells were exposed to HIV-1 variants with different HIV-1 envelope proteins representing different virus tropisms (HIV-1_LAI: X4-tropic; HIV-1-AD-8 and HIV-1ΔEnv_JRFL(R5): R5-tropic) or HIV-1 based virus particles pseudotyped with an envelope protein (G-protein) from a heterologous virus (<i>Vesicular Stomatitis Virus</i>; HIV-1ΔEnv_VSVG) in the presence of different concentrations of PS extract. Cultures were assayed for reporter expression 48 hours after virus addition and fluorescent signal intensities normalized to those of cultures exposed to virus without inhibitors. Mean values of triplicate wells and standard deviation of the mean are indicated for each extract concentration.</p