Radial spoke protein 9 is necessary for axoneme assembly in <i>Plasmodium</i> but not in trypanosomatid parasites

Flagella are important for eukaryote cell motility, including in sperm, and are vital for life cycle progression of many unicellular eukaryotic pathogens. The ‘9+2’ axoneme in most motile flagella comprises nine outer doublet and two central-pair singlet microtubules. T-shaped radial spokes protrude from the outer doublets towards the central pair and are necessary for effective beating. We asked whether there were radial spoke adaptations associated with parasite lineage-specific properties in apicomplexans and trypanosomatids. Following an orthologue search for experimentally uncharacterised radial spoke proteins (RSPs), we identified and analysed RSP9. Trypanosoma brucei and Leishmania mexicana have an extensive RSP complement, including two divergent RSP9 orthologues, necessary for flagellar beating and swimming. Detailed structural analysis showed that neither orthologue is needed for axoneme assembly in Leishmania. In contrast, Plasmodium has a reduced set of RSPs including a single RSP9 orthologue, deletion of which in Plasmodium berghei leads to failure of axoneme formation, failed male gamete release, greatly reduced fertilisation and inefficient life cycle progression in the mosquito. This indicates contrasting selection pressures on axoneme complexity, likely linked to the different mode of assembly of trypanosomatid versus Plasmodium flagella

Regenerative potential of human muscle stem cells in chronic inflammation

International audienceABSTRACT: INTRODUCTION: Chronic inflammation is a profound systemic modification of the cellular microenvironment which could affect survival, repair and maintenance of muscle stem cells. The aim of this study was to define the role of chronic inflammation on the regenerative potential of satellite cells in human muscle. METHODS: As a model for chronic inflammation, 11 patients suffering from rheumatoid arthritis (RA) were included together with 16 patients with osteoarthritis (OA) as controls. The mean age of both groups was 64 years, with more females in the RA group compared to the OA group. During elective knee replacement surgery, a muscle biopsy was taken from the distal musculus vastus medialis. Cell populations from four RA and eight OA patients were used for extensive phenotyping because these cell populations showed no spontaneous differentiation and myogenic purity greater than 75% after explantation. RESULTS: After mononuclear cell explantation, myogenic purity, viability, proliferation index, number of colonies, myogenic colonies, growth speed, maximum number of population doublings and fusion index were not different between RA and OA patients. Furthermore, the expression of proteins involved in replicative and stress-induced premature senescence and apoptosis, including p16, p21, p53, hTERT and cleaved caspase-3, was not different between RA and OA patients. Mean telomere length was shorter in the RA group compared to the OA group. CONCLUSIONS: In the present study we found evidence that chronic inflammation in RA does not affect the in vitro regenerative potential of human satellite cells. Identification of mechanisms influencing muscle regeneration by modulation of its microenvironment may, therefore, be more appropriate

Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders

<p>Abstract</p> <p>Background</p> <p>Investigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies.</p> <p>Methods</p> <p>Using transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders.</p> <p>Results</p> <p>The immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both <it>in vitro </it>and <it>in vivo </it>after transplantation into regenerating muscle of immunodeficient mice.</p> <p>Conclusions</p> <p>Dystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess <it>in vivo </it>the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies.</p

Deconjugated bile salts produced by extracellular bile-salt hydrolase-like activities from the probiotic Lactobacillus johnsonii La1 inhibit Giardia duodenalis in vitro growth

Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro. We provide evidence that the supernatant of this strain contains active principle(s) not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia. By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting, or releasing BSH-like activity(ies) in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present

Heat Shock Transcriptional Responses in an MC-Producing Cyanobacterium (<i>Planktothrix agardhii</i>) and Its MC-Deficient Mutant under High Light Conditions

<div><p>Microcystins (MCs) are the most commonly-reported hepatotoxins produced by various cyanobacterial taxa in fresh waters to constitute a potential threat to human and animal health. The biological role of MCs in the producer organisms is not known, and it would be very useful to understand the driving force behind the toxin production. Recent studies have suggested that MCs may have a protective function in cells facing environmental stress. Following this starting premise, we speculate that under adverse conditions the expression of stress-related genes coding for Heat Shock Proteins (Hsp) might be different in an MC-producing strain and its MC-deficient mutant. We therefore used RT-qPCR to compare the expression of 13 <i>hsp</i> genes of an MC-producing strain of <i>Planktothrix agardhii</i> (CYA126/8) and its MC-deficient <i>ΔmcyD</i> mutant over different periods of exposure to high light stress (HL). Three reference genes (RGs) were selected from six candidates to normalize the RT-qPCR data. Of these three RGs (<i>rsh</i>, <i>rpoD,</i> and <i>gltA</i>), <i>gltA</i> is used here for the first time as an RG in prokaryotes. Under HL stress, five genes were found to be strongly up-regulated in both strains (<i>htpG</i>, <i>dnaK</i>, <i>hspA</i>, <i>groES,</i> and <i>groEL</i>). Unexpectedly, we found that the MC-producing wild type strain accumulated higher levels of <i>htpG</i> and <i>dnaK</i> transcripts in response to HL stress than the MC-deficient mutant. In addition, a significant increase in the <i>mcy</i>E transcript was detected in the mutant, suggesting that MCs are required under HL conditions. We discuss several possible roles of MCs in the response to HL stress through their possible involvement in the protective mechanisms of the cells.</p></div

Ranking of candidate reference genes by three different algorithms.

<p>GeNorm (75), NormFinder (2) and BestKeeper (51) used to identify the most stably expressed genes in control and HL conditions. Wild type (WT) and mutant (M) strains of <i>Planktothrix agardhii</i>.</p

Real-time PCR primers used in this study.

<p>Real-time PCR primers used in this study.</p

Real-time PCR C_T values in the samples collected.

<p>The distribution of the expression levels of candidate reference genes is shown by the median (lines), the lower and upper quartiles (boxes), and ranges (whiskers) (n = 20). The genes were divided into three groups by the arbitrary lines at C_T 15 and 23, on the basis of their different expression levels.</p

Relative expression levels of the <i>hsp</i> genes and <i>mcyE</i> gene of <i>P</i>. <i>agardhii</i> from control (T₀) to high light stress (1 h to 24 h).

<p>Relative mRNA expression of <i>hsp</i> genes was normalized against three RGs: <i>rsh</i>, <i>rpoD</i> and <i>gltA</i>. (A): unchanged expression; (B): <4 fold up-regulated; (C): >4 fold up-regulated. Error bars correspond to the standard deviation, including two technical replicates for two independent biological samples. An asterisk indicates a significant difference <i>versus</i> control (T₀) *: <i>p</i><0.05. Circles indicate a significant difference in the expression level between the WT and the mutant strains; °°: <i>p</i><0.01; °°°: <i>p</i><0.001.</p

Information about the genes investigated in this study.

*<p>Reference gene candidates;</p>**<p>Target genes.</p