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Parks R, Peng L, Crimmins S, Wilson S, Schneider L, Standaert DG, Shacka JJ, Zhou Y, Caldwell GA, Lu Y, Walls KC, Crabtree D, Uchiyama Y, Liang Q, Yacoubian TA, Xie ZL, Zhang J, Caldwell KA, Speake LD, Iwatsubo T, Qiao L, Hamamichi S, Roth KA
[
Mol Brain,
2008]
-synuclein (-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, including Parkinson's disease (PD), dementia with LB (DLB) and multi-system atrophy. -syn mutations or amplifications are responsible for a subset of autosomal dominant familial PD cases, and overexpression causes neurodegeneration and motor disturbances in animals. To investigate mechanisms for -syn accumulation and toxicity, we studied a mouse model of lysosomal enzyme cathepsin D (CD) deficiency, and found extensive accumulation of endogenous -syn in neurons without overabundance of -syn mRNA. In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for -syn metabolism. Conversely, CD overexpression reduces -syn aggregation and is neuroprotective against -syn overexpression-induced cell death in vitro. In a C. elegans model, CD deficiency exacerbates -syn accumulation while its overexpression is protective against -syn-induced dopaminergic neurodegeneration. Mutated CD with diminished enzymatic activity or overexpression of cathepsins B (CB) or L (CL) is not protective in the worm model, indicating a unique requirement for enzymatically active CD. Our data identify a conserved CD function in -syn degradation and identify CD as a novel target for LB disease therapeutics.
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Kunzler M, Wohlschlager T, Grassi P, Dell A, Butschi A, Schmieder SS, Gauss R, Titz A, Haslam SM, Sutov G, Aebi M, Hauck D, Hengartner MO, Knobel M
[
Proc Natl Acad Sci U S A,
2014]
Effector proteins of innate immune systems recognize specific non-self epitopes. Tectonins are a family of -propeller lectins conserved from bacteria to mammals that have been shown to bind bacterial lipopolysaccharide (LPS). We present experimental evidence that two Tectonins of fungal and animal origin have a specificity for O-methylated glycans. We show that Tectonin 2 of the mushroom Laccaria bicolor (Lb-Tec2) agglutinates Gram-negative bacteria and exerts toxicity toward the model nematode Caenorhabditis elegans, suggesting a role in fungal defense against bacteria and nematodes. Biochemical and genetic analysis of these interactions revealed that both bacterial agglutination and nematotoxicity of Lb-Tec2 depend on the recognition of methylated glycans, namely O-methylated mannose and fucose residues, as part of bacterial LPS and nematode cell-surface glycans. In addition, a C. elegans gene, termed
samt-1, coding for a candidate membrane transport protein for the presumptive donor substrate of glycan methylation, S-adenosyl-methionine, from the cytoplasm to the Golgi was identified. Intriguingly, limulus lectin L6, a structurally related antibacterial protein of the Japanese horseshoe crab Tachypleus tridentatus, showed properties identical to the mushroom lectin. These results suggest that O-methylated glycans constitute a conserved target of the fungal and animal innate immune system. The broad phylogenetic distribution of O-methylated glycans increases the spectrum of potential antagonists recognized by Tectonins, rendering this conserved protein family a universal defense armor.
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[
PLoS One,
2012]
Burkholderia pseudomallei, a Gram-negative saprophytic bacterium, is the causative agent of the potentially fatal melioidosis disease in humans. In this study, environmental parameters including temperature, nutrient content, pH and the presence of glucose were shown to play a role in in vitro biofilm formation by 28 B. pseudomallei clinical isolates, including four isolates with large colony variants (LCVs) and small colony variants (SCVs) morphotypes. Enhanced biofilm formation was observed when the isolates were tested in LB medium, at 30 C, at pH 7.2, and in the presence of as little as 2 mM glucose respectively. It was also shown that all SVCs displayed significantly greater capacity to form biofilms than the corresponding LCVs when cultured in LB at 37 C. In addition, octanoyl-homoserine lactone (C(8)-HSL), a quorum sensing molecule, was identified by mass spectrometry analysis in bacterial isolates referred to as LCV CTH, LCV VIT, SCV TOM, SCV CTH, 1 and 3, and the presence of other AHL's with higher masses; decanoyl-homoserine lactone (C(10)-HSL) and dodecanoyl-homoserine lactone (C(12)-HSL) were also found in all tested strain in this study. Last but not least, we had successfully acquired two Bacillus sp. soil isolates, termed KW and SA respectively, which possessed strong AHLs degradation activity. Biofilm formation of B. pseudomallei isolates was significantly decreased after treated with culture supernatants of KW and SA strains, demonstrating that AHLs may play a role in B. pseudomallei biofilm formation.
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[
PLoS One,
2013]
BACKGROUND: Caenorhbditis elegans has being vigorously used as a model organism in many research fields and often accompanied by administrating with various drugs. The methods of delivering drugs to worms are varied from one study to another, which make difficult in comparing results between studies. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the drug absorption efficiency in C. elegans using five frequently used methods with resveratrol with low aqueous solubility and water-soluble 5-Fluoro-2'-deoxyuridine (FUDR) as positive compounds. The drugs were either applied to the LB medium with bacteria OP50, before spreading onto Nematode Growth Medium (NGM) plates (LB medium method), or to the NGM with live (NGM live method) or dead bacteria (NGM dead method), or spotting the drug solution to the surface of plates directly (spot dead method), or growing the worms in liquid medium (liquid growing method). The concentration of resveratrol and FUDR increased gradually within C. elegans and reached the highest during 12 hours to one day and then decreased slowly. At the same time point, the higher the drug concentration, the higher the metabolism rate. The drug concentrations in worms fed with dead bacteria were higher than with live bacteria at the same time point. Consistently, the drug concentration in medium with live bacteria decreased much faster than in medium with dead bacteria, reach to about half of the original concentration within 12 hours. CONCLUSION: Resveratrol with low aqueous solubility and water-soluble FUDR have the same absorption and metabolism pattern. The drug metabolism rate in worms was both dosage and time dependent. NGM dead method and liquid growing method achieved the best absorption efficiency in worms. The drug concentration within worms was comparable with that in mice, providing a bridge for dose translation from worms to mammals.
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[
J Ind Microbiol Biotechnol,
2010]
Abundant larval transcript (ALT), a novel filarial protein, has been shown to have great potential as a vaccine in the prevention of human lymphatic filariasis. In this study, we report a method for the production of recombinant ALT-2 protein, expressed in the cytoplasm of bacterium Escherichia coli in soluble form and purification in a single step using hydrophobic interaction chromatography (HIC). Fermentation was done by continuous fed-batch methodology with dissolved oxygen (DO)-controlled feed addition. The culture was induced with 1mM isopropyl--D: -thiogalactopyranoside (IPTG). Up to 9g/l dry cell weight (DCW) of biomass was obtained from 1.6l of Luria-Bertani (LB) broth in a bench-scale reactor. Around 200mg/l of purified ALT-2 with a yield of about 60% was obtained. This is almost a 2.5-fold increase in final protein yield compared to purification using immobilized metal affinity chromatography (IMAC).
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[
PLoS One,
2008]
It was recently suggested that specific antidepressants of the serotonin-antagonist type, namely mianserin and methiothepin, may exert anti-aging properties and specifically extend lifespan of the nematode C.elegans by causing a state of perceived calorie restriction (Petrascheck M, Ye X, Buck LB: An antidepressant that extends lifespan in adult Caenorhabditis elegans; Nature, Nov 22, 2007;450(7169):553-6, PMID 18033297). Using the same model organism, we instead observe a reduction of life expectancy when employing the commonly used, standardized agar-based solid-phase assay while applying the same or lower concentrations of the same antidepressants. Consistent with a well-known side-effect of these compounds in humans, antidepressants not only reduced lifespan but also increased body fat accumulation in C. elegans reflecting the mammalian phenotype. Taken together and in conflict with previously published findings, we find that antidepressants of the serotonin-antagonist type not only promote obesity, but also decrease nematode lifespan.
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[
Elife,
2023]
The causality and mechanism of dietary effects on brain aging are still unclear due to the long time scales of aging. The nematode <i>Caenorhabditis elegans</i> has contributed to aging research because of its short lifespan and easy genetic manipulation. When fed the standard laboratory diet, <i>Escherichia coli</i>, <i>C. elegans</i> experiences an age-dependent decline in temperature-food associative learning, called thermotaxis. To address if diet affects this decline, we screened 35 lactic acid bacteria as alternative diet and found that animals maintained high thermotaxis ability when fed a clade of <i>Lactobacilli</i> enriched with heterofermentative bacteria. Among them, <i>Lactobacill</i>us <i>reuteri</i> maintained the thermotaxis of aged animals without affecting their lifespan and motility. The effect of <i>Lb. reuteri</i> depends on the DAF-16 transcription factor functioning in neurons. Furthermore, RNA sequencing analysis revealed that differentially expressed genes between aged animals fed different bacteria were enriched with DAF-16 targets. Our results demonstrate that diet can impact brain aging in a <i>
daf-16</i>-dependent manner without changing the lifespan.
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[
Biol Direct,
2010]
BACKGROUND: The success of invertebrates throughout evolution is an excellent illustration of the efficiency of their defence strategies. Caenorhabditis elegans has proven to be an appropriate model for transcriptome studies of host-pathogen interactions. The aim of this paper is to complement this knowledge by investigating the worm's response to a Staphylococcus aureus infection through a 2-dimensional differential proteomics approach. RESULTS: Different types of growth media in combination with either E. coli OP50 or Staphylococcus aureus were tested for an effect on the worm's lifespan. LB agar was chosen and C. elegans samples were collected 1 h, 4 h, 8 h and 24 h post S. aureus infection or E. coli incubation. Proteomics analyses resulted in the identification of 130 spots corresponding to a total of 108 differentially expressed proteins. CONCLUSIONS: Exploring four time-points discloses a dynamic insight of the reaction against a gram-positive infection at the level of the whole organism. The remarkable upregulation after 8 h and 24 h of many enzymes involved in the citric acid cycle might illustrate the cost of fighting off an infection. Intriguing is the downregulation of chaperone molecules, which are presumed to serve a protective role. A comparison with a similar experiment in which C. elegans was infected with the gram-negative Aeromonas hydrophila reveals that merely 9% of the identified spots, some of which even exhibiting an opposite regulation, are present in both studies. Hence, our findings emphasise the complexity and pathogen-specificity of the worm's immune response and form a firm basis for future functional research. REVIEWERS: This article was reviewed by Itai Yanai, Dieter Wolf and Torben Luebke (nominated by Walter Lutz).
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[
Parasit Vectors,
2015]
BACKGROUND: Lectins are carbohydrate-binding proteins that are involved in fundamental intra- and extracellular biological processes. They occur ubiquitously in nature and are especially abundant in plants and fungi. It has been well established that certain higher fungi produce lectins in their fruiting bodies and/or sclerotia as a part of their natural resistance against free-living fungivorous nematodes and other pests. Despite relatively high diversity of the glycan structures in nature, many of the glycans targeted by fungal lectins are conserved among organisms of the same taxon and sometimes even among different taxa. Such conservation of glycans between free-living and parasitic nematodes is providing us with a useful tool for discovery of novel chemotherapeutic and vaccine targets. In our study, a subset of fungal lectins emanating from toxicity screens on Caenorhabditis elegans was tested for their potential to inhibit larval development of Haemonchus contortus. METHODS: The effect of Coprinopsis cinerea lectins - CCL2, CGL2, CGL3; Aleuria aurantia lectin - AAL; Marasmius oreades agglutinin - MOA; and Laccaria bicolor lectin - Lb-Tec2, on cultivated Haemonchus contortus larval stages was investigated using a larval development test (LDT). To validate the results of the toxicity assay and determine lectin binding capacity to the nematode digestive tract, biotinylated versions of lectins were fed to pre-infective larval stages of H. contortus and visualized by fluorescent microscopy. Lectin histochemistry on fixed adult worms was performed to investigate the presence and localisation of lectin binding sites in the disease-relevant developmental stage. RESULTS: Using an improved larval development test we found that four of the six tested lectins: AAL, CCL2, MOA and CGL2, exhibited a dose-dependent toxicity in LDT, as measured by the number of larvae developing to the L3 stage. In the case of AAL, CGL2 and MOA lectin, doses as low as 5g/ml caused >95% inhibition of larval development while 40g/ml were needed to achieve the same inhibition by CCL2 lectin. MOA was the only lectin tested that caused larval death while other toxic lectins had larvistatic effect manifesting as L1 growth arrest. Using lectin histochemistry we demonstrate that of all lectins tested, only the four toxic ones displayed binding to the larvae's gut and likewise were found to interact with glycans localized to the gastrodermal tissue of adults. CONCLUSION: The results of our study suggest a correlation between the presence of target glycans of lectins in the digestive tract and the lectin-mediated toxicity in Haemonchus contortus. We demonstrate that binding to the structurally conserved glycan structures found in H. contortus gastrodermal tissue by the set of fungal lectins has detrimental effect on larval development. Some of these glycan structures might represent antigens which are not exposed to the host immune system (hidden antigens) and thus have a potential for vaccine or drug development. Nematotoxic fungal lectins prove to be a useful tool to identify such targets in parasitic nematodes.
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[
Mikrobiyol Bul,
2018]
Poultry animals and poultry associated products are important risk sources for Salmonellosis. S.Kentucky and S.Infantis are among the serovars frequently isolated from retail chickens and were reported to be isolated in Turkey. In this study, the role of plasmids carried by S.Kentucky and S.Infantis isolates in antibiotic resistance profiles of the isolates and their pathogenicity on Caenorhabditis elegans nematode model system were investigated. The isolates used, 1 of Kentucky and 2 of Infantis serotypes, were selected among food-borne Salmonella isolated from chicken carcass in Edirne. All three isolates were previously shown to contain plasmids carrying multidrug resistance and were known to be pathogenic on C.elegans nematode model system. S.Kentucky A10 isolate was resistant to ampicillin, nalidixic acid, tetracycline, ciprofloxacin, trimethoprim and sulphonamide and carried one plasmid with 31.6 kb size. S.Infantis A15 isolate was resistant to ampicillin, streptomycine, nalidixic acid, tetracycline, neomycin, sulphonamide and kanamycin and carried a plasmid with 19.9 kb size while the other S.Infantis isolate (A16) was resistant to streptomycin, nalidixic acid, tetracycline, trimethoprim, neomycin, sulphonamide and kanamycin and carried 3 plasmids with 42.4, 1.5 and 1.2 kb sizes. Plasmid curing experiments were performed to investigate the role of plasmids in antibiotic resistance and pathogenicity in C.elegans. Ethidium bromide (EtBr) dye was used as the plasmid curing agent. Plasmids were isolated from cultures grown in LB broth with different concentrations of EtBr (50, 75, 100, 125 g/ml) according to the Kado-Liu method and the most effective EtBr concentration was determined as 125 g/ml. C.elegans pathogenicity assays were carried out using plasmid cured isolates. The time 50% of the nematode die (TD50) values of the nematode groups fed with plasmid cured isolates were compared with previously obtained TD50 values of the nematode groups fed with wild type Salmonella isolates. Studentos t-test (p< 0.05) was used to showthe level of significance between TD50 values of the two groups. TD50 values of the positive control group fed with S.Typhimurium ATCC 14028 and the negative control group fed with Escherichia coli OP50 were found as 4.0 +/- 0.4 and 8.0 +/- 0.02 days, respectively. The differences between TD50 values of nematode groups fed with wild type and plasmid cured isolates were statistically significant both for S.Kentucky (A10) (4.9 +/- 0.04-6.2 +/- 0.1) and S.Infantis (A16) (4.4 +/- 0.01-6.2 +/- 0.2) (p< 0.05) strains, but no significant difference was observed for the groups fed with wild type and plasmid cured S.Infantis (A15) (5.7 +/- 0.39-5.8 +/- 0.16) strain. Broth microdilution method was used to determine whether there was any change in minimal inhibitory concentrations (MIC) of the antibiotics for which the isolates were resistant before plasmid elimination. No significant difference was found between the MIC values of the resistant antibiotics among Salmonella isolates carrying plasmids and with cured plasmid. This study is important since the first in vivo results about the role of Kentucky and Infantis serovar plasmids on C.elegans nematode model system were presented.