Elegans N2 Research Articles

Cyclic di-GMP incorporates the transcriptional factor FleQ03 in Pseudomonas syringae MB03 to elicit biofilm-dependent resistance in response to Caenorhabditis elegans predation

Bacteria usually form biofilms as a defense mechanism against predation by bacterivorous nematodes. In this context, the second messenger c-di-GMP from the wild-type Pseudomonas syringae MB03 actuates the transcriptional factor FleQ03 to elicit biofilm-dependent nematicidal activity against Caenorhabditis elegans N2. P. syringae MB03 cells exhibited nematicidal activity and c-di-GMP content in P. syringae MB03 cells was increased after feeding to nematodes. Expression of a diguanylate cyclase (DGC) gene in P. syringae MB03 resulted in an increased c-di-GMP content, biofilm yield and nematicidal activity, whereas converse effects were obtained when expressing a phosphodiesterase (PDE) gene. Molecular docking and isothermal titration calorimetry assays verified the affinity activity between c-di-GMP and the FleQ03 protein. The disruption of the fleQ03 gene in P. syringae MB03, while increasing c-di-GMP content, significantly diminished both biofilm formation and nematicidal activity. Interestingly, P. syringae MB03 formed a full-body biofilm around the worms against predation, probably extending from the tail to the head, whereas it was not observed in the fleQ03 gene disrupted cells. Thus, we hypothesized that c-di-GMP incorporated FleQ03 to reinforce bacterial biofilm and biofilm-dependent pathogenicity in response to C. elegans predation, providing insights into a possible means of resisting bacterivorous nematodes by bacteria in natural ecosystems.

Components in SLPE Alleviate AD Model Nematodes by Up-Regulating Gene gst-5.

Salvia leucantha is a perennial herb of the genus Salvia in the family Labiatae, which has a wide range of biological activities, mainly including inhibition of acetylcholinesterase, antibacterial, and anti-inflammatory activity. To explore the protective effects and mechanism of action of S. leucantha on Alzheimer's disease (AD), the anti-AD activity of SLE (extracts of S. leucantha) was determined by using a transgenic Caenorhabditis elegans (C. elegans) model (CL4176). Analyses included paralysis assay, phenotypic experiments, transcriptome sequencing, RNA interference (RNAi), heat shock assays, and gas chromatography-mass spectrometry (GC-MS). SLPE (S. leucantha petroleum ether extract) could significantly delay CL4176 paralysis and extend the longevity of C. elegans N2 without harmful effects. A total of 927 genes were significantly changed by SLPE treatment in C. elegans, mainly involving longevity regulatory pathways-nematodes, drug metabolism-cytochrome P450, and glutathione metabolic pathways. RNAi showed that SLPE exerted its anti-AD activity through up-regulation of the gene gst-5; the most abundant compound in SLPE analyzed by GC-MS was 2,4-Di-tert-butylphenol (2,4-DTBP), and the compound delayed nematode paralysis. The present study suggests that active components in S. leucantha may serve as new-type anti-AD candidates and provide some insights into their biological functions.

Design, Synthesis, and Biological Evaluation of the Quorum-Sensing Inhibitors of Pseudomonas aeruginosa PAO1.

Due to the resistance of Gram-negative bacteria Pseudomonas aeruginosa PAO1 to most clinically relevant antimicrobials, the use of traditional antibiotic treatments in hospitals is challenging. The formation of biofilms, which is regulated by the quorum-sensing (QS) system of Pseudomonas aeruginosa (PA), is an important cause of drug resistance. There are three main QS systems in P. aeruginosa: the las system, the rhl system, and the pqs system. The inhibitors of the las system are the most studied. Previously, the compound AOZ-1 was found to have a certain inhibitory effect on the las system when screened. In this study, twenty-four compounds were designed and synthesized by modifying the Linker and Rings of AOZ-1. Using C. violaceum CV026 as a reporter strain, this study first assessed the inhibitory effects of new compounds against QS, and their SAR was investigated. Then, based on the SAR analysis of compound AOZ-1 derivatives, the parent core of AOZ-1 was replaced to explore the structural diversity. Then, nine new compounds were designed and synthesized with a new nucleus core component of 3-amino-tetrahydro-l,3-oxazin-2-one. The compound Y-31 (IC50 = 91.55 ± 3.35 µM) was found to inhibit the QS of C. violaceum CV026. Its inhibitory effect on C. violaceum CV026 was better than that of compound AOZ-1 (IC50 > 200 µM). Furthermore, biofilm formation is one of the important causes of Pseudomonas aeruginosa PAO1 resistance. In this study, it was found that compound Y-31, with a new nucleus core component of 3-amino-tetrahydro-l,3-oxazin-2-one, had the highest biofilm inhibition rate (40.44%). The compound Y-31 has a certain inhibitory effect on the production of PAO1 virulence factors (pyocyanin, rhamnolipid, and elastase) and swarming. When the concentration of compound Y-31 was 162.5 µM, the inhibition rates of pyocyanin, rhamnolipid, and elastase were 22.48%, 6.13%, and 22.67%, respectively. In vivo, the lifetime of wildtype Caenorhabditis elegans N2 infected with P. aeruginosa PAO1 was markedly extended by the new parent nucleus Y-31. This study also performed cytotoxicity experiments and in vivo pharmacokinetics experiments on the compound Y-31. In conclusion, this study identified a compound, Y-31, with a new nucleus core component of 3-amino-tetrahydro-l,3-oxazin-2-one, which is a potential agent for treating P. aeruginosa PAO1 that is resistant to antibiotics and offers a way to discover novel antibacterial medications.

Efficient remediation of di-(2-ethylhexyl) phthalate and plant-growth promotion with the application of a phosphate-solubilizing compound microbial agent

The ecotoxic endocrine-disrupting chemical di-(2-ethylhexyl) phthalate (DEHP) is ubiquitous in agricultural soil, posing a serious threat to human health. Here, we report efficient soil-borne DEHP degradation and plant growth promotion by a microbial organic fertilizer GK-PPB prepared by combining a recycled garden waste-kitchen waste compost product with ternary compound microbial agent PPB-MA, composed of Penicillium oxalic MB08F, Pseudomonas simiae MB751, and Bacillus tequilensis MB05B. The combination of MB08F and MB751 provided synergistic phosphorus solubilization, and MB05B enhanced the DEHP degradation capacity of MB08F via bioemulsification. Under optimal conditions (25.70 °C and pH 7.62), PPB-MA achieved a 96.81 % degradation percentage for 1000 mg L−1 DEHP within 5 days. The degradation curve followed first-order kinetics with a half-life of 18.24 to 24.76 h. A complete mineralization pathway was constructed after identifying the degradation intermediates of 2H-labeled DEHP. Evaluation in Caenorhabditis elegans N2 showed that PPB-MA eliminated the ecological toxicity of DEHP. A pakchoi (Brassica chinensis L.) pot experiment demonstrated that GK-PPB promoted phosphorus solubilization and plant growth, reduced soil DEHP residue, and decreased DEHP accumulation in pakchoi, suggesting its potential practical utility in environmentally responsible and safe cultivation of vegetables.

Биофункциональная активность in vivo хлорогеновой кислоты и биоханина А, выделенных из экстрактов каллусной культуры Trifolium pratense L.

Polyphenols are potential neuroprotectors that increase lifespan and slow down aging. Red clover (Trifolium pratense L.) is a promising source of biologically active substances. Its extracts contain biochanin A and chlorogenic acid. This research used Caenorhabditis elegans to study the effect of polyphenols extracted from red clover callus cultures on SOD-3 and HSP-16.2 genes, as well as their anti-amyloid potential. 
 The chlorogenic acid and biochanin A (200, 100, 50, and 10 µM) with a purity of 95% were isolated from callus extracts of T. pratense L. The effect of polyphenols on SOD-3 and HSP-16.2 was assessed after 5 and 2 h of heat stress (35°C), respectively, using C. elegans N2 Bristol as model organism. The neuroprotective potential was measured by counting paralyzed nematodes after 18, 40, and 62 h of incubation.
 The research established a dose-dependent effect between the concentration of biologically active substances and the percentage of paralyzed nematodes after 18 h of cultivation. The lowest paralysis phenotype count occurred at a concentration of 200 μM. The activity of 200 μM biochnin A was 1.18 times as high as that of a 200 μM chlorogenic acid solution. Biochanin A solutions increased SOD-3 expression by 3.7 times, compared to the control.
 The biologically active substances exhibited relative neuroprotective activity and affected the expression of antioxidant defense gene in C. elegans.

An Approach for the Control of Caenorhabditis elegans N2 via the Regulation of Growth Conditions and Pleurotus ostreatus Po4

Food resources are essential for the survival and growth of the population. Soil phytopathogenic nematodes cause great damage to agricultural crops, endangering food supplies and resources in general. Different methods have been used to control them. However, this issue still requires a more effective solution. Caenorhabditis elegans (CGC strain wild-type N2) was applied as a model with an Escherichia coli OP50 feeding substrate for nematodes. Our approach was based on the thermodynamically substantiated creation of growth conditions that are unfavorable for nematodes to suppress them irreversibly. The thermodynamic calculations showed that obligate anaerobic conditions, namely the absence of oxygen and a low redox potential (−100 mV and below), were potentially unacceptable for nematodes. Anaerobic conditions were created using both abiogenic (physicochemical) and biological methods. Abiogenic anaerobic conditions were achieved by preventing oxygen access and adding low-potential sodium sulfide (Eh = −250...−200 mV) to the cultivation medium. By applying biological methods, Pleurotus ostreatus Po4 and E. coli O2 was completely removed and the redox potential was decreased from +100…+200 mV to −100...−200 mV (in particular, due to the synthesis of H2S). Even the short-term exposure (1–2 days) of nematodes under anaerobic conditions led to their suppression and death. Thus, the short-term creation of anaerobic conditions in the soil may be an effective method to control, e.g., phytopathogenic aerobic nematodes. This research contributes to the development of foundations to preserve agricultural plants and increase crop yield as well as the development of an approach for the environmentally friendly control of phytopathogens.

Research on the anti-aging efficacy of fermented lysate VHProbi® MixA as the functional skincare ingredient.

This study aimed to investigate the efficacy of a cream containing VHProbi® MixA for improving skin aging. In vitro studies demonstrated that the lysate produced from Lacticaseibacillus paracasei E12 (E12) exhibited immunoregulatory effects in a 3D skin model, with significant reductions in levels of interleukin (IL)-1α, IL-1β, and IL-8 (P<0.05) compared with the control group. In addition, the lysate of E12 mitigated the hydrogen peroxide-induced mortality of 3D skin cells and enhanced the transepithelial electrical resistance to show significant differences in comparison with control (P<0.05), suggesting favorable antioxidant effects. The antioxidant capacity of the lysate of E12 was also confirmed using the Caenorhabditis elegans N2 model. C. elegans N2 fed the E12 strain showed a significantly higher % survival than those fed Escherichia coli OP50 (P<0.05). Subsequently, VHProbi® MixA was formulated using the fermented lysates of E12, Lactiplantibacillus plantarum E15, and Limosilactobacillus reuteri E18. In a clinical study to ascertain if a cream containing VHProbi® MixA could improve the skin aging trends, participants were asked to use the investigational products for 60 days, and six indicators, transepidermal water loss (TEWL), hydration, elasticity, wrinkles, skin texture (roughness), and pores were measured at baseline and the endpoint of the study. A self-evaluation questionnaire analysis was also provided. TEWL, wrinkles, skin texture, and thickness of pores decreased significantly after treatment with the cream for 60 days (P<0.01), whereas hydration and elasticity increased significantly (P<0.01), in comparison to the baseline measurements. We hypothesize that the use of the cream containing VHProbi® MixA could be favorable for skin anti-aging management.

Investigation of anti-aging and anti-infection properties of Jingfang Granules using the Caenorhabditis elegans model.

Jingfang Granule (JFG), a traditional Chinese medicine, is frequently employed in clinical settings for the treatment of infectious diseases. Nevertheless, the anti-aging and anti-infection effects of JFG remain uncertain. In the present study, these effects were evaluated using the Caenorhabditis elegans (C. elegans) N2 as a model organism. The results demonstrated that JFG significantly increased the median lifespan of C. elegans by 31.2% at a dosage of 10mg/mL, without any discernible adverse effects, such as alterations in the pharyngeal pumping rate or nematode motility. Moreover, JFG notably increased oviposition by 11.3%. Subsequent investigations revealed that JFG enhanced oxidative stress resistance in C. elegans by reducing reactive oxygen species levels and significantly improved survival rates in nematodes infected with Pseudomonas aeruginosa ATCC 9027. These findings suggest that JFG delays reproductive senescence in C. elegans and protects them from oxidative stress, thereby extending their lifespan. Additionally, JFG improves the survival of P. aeruginosa-infected nematodes. Consequently, JFG has potential as a candidate for the development of anti-aging and anti-infection functional medicines.

The nematode Caenorhabditis elegans and diverse potential invertebrate vectors predominantly interact opportunistically

Some small animals migrate with the help of other, more mobile animals (phoresy) to leave short-lived and resource-poor habitats. The nematode Caenorhabditis elegans lives in ephemeral habitats such as compost, but has also been found associated with various potential invertebrate vectors. Little research has been done to determine if C. elegans is directly attracted to these invertebrates. To determine whether C. elegans is attracted to compounds and volatile odorants of invertebrates, we conducted chemotaxis experiments with the isopods Porcellio scaber, Oniscus asellus, and Armadillidium sp. and with Lithobius sp. myriapods, Drosophila melanogaster fruit flies, and Arion sp. slugs as representatives of natural vectors. Because phoresy is an important escape strategy in nature, especially for dauer larvae of C. elegans, we examined the attraction of the natural C. elegans isolate MY2079 in addition to the laboratory-adapted strain N2 at the dauer and L4 stage. We found that DMSO washing solution of Lithobius sp. and the odor of live D. melanogaster attracted C. elegans N2 L4 larvae. Surprisingly, the natural isolate MY2079 was not attracted to any invertebrate during either the dauer or L4 life stages and both C. elegans strains were repelled by various compounds from O. asellus, P. scaber, Armadillidium sp., Lithobius sp., and Arion sp. feces. We hypothesize that this is due to defense chemicals released by the invertebrates. Although compounds from Lithobius sp. and D. melanogaster odorants were mildly attractive, the lack of attraction to most invertebrates suggests a predominantly opportunistic association between C. elegans and invertebrate vectors.

EVALUATION OF THE GEROPROTECTIVE EFFECT OF SQUALENE ON THE CAENORHABDITIS ELEGANS MODEL

Background. Increasing healthy life expectancy is a priority in modern healthcare. Squalene, a triterpene exhibiting antioxidant, anti-inflammatory and other properties, is a promising candidate for geroprotectors.&#x0D; Purpose. Assess the activity of squalene, that is, the possibility of its further use as a geroprotector.&#x0D; Materials and methods. Squalene was purchased from Sigma-Aldrich (USA) for research. Squalene solutions were prepared with concentrations of 10, 50, 100, 200, 400, 800, and 1600 µmol/L (solvent dimethyl sulfoxide—DMSO). The work was carried out on the basis of the Laboratory for Biotesting of Natural Nutraceuticals (KemGU, Russia). Antioxidant activity was assessed by capturing ABTS radical cations in comparison with the activity of ascorbic acid. The model organism Caenorhabditis elegans N2 Bristol was purchased from the Laboratory for the Development of Innovative Medicines and Agrobiotechnologies of the Moscow Institute of Physics and Technology. To assess the stress resistance of nematodes during cultivation, conditions of thermal (33°C) and oxidative (adding 15 μl of 1 M paraquat, 20°C) stress were created for 24 and 48 h. conditions (20 °C) for 61 days. The statistical significance of the results was assessed using Student’s t-test (in Microsoft Office Excel 2007). Lifespan data were assessed using a Kaplan-Meier survival plot combined with a log-rank test (Mantel-Cox). Data processing was carried out using the OASIS online application (https://sbi.postech.ac.kr/oasis/).&#x0D; Results. The results showed that squalene solutions did not exhibit antioxidant properties; did not trap radical cations with ABTS. A solution of squalene at a concentration of 10 μM contributed to an increase in the stress resistance of nematodes. Under conditions of oxidative stress, the survival of nematodes during cultivation for 24 hours is 3.1% higher than in the control. Under conditions of heat stress during cultivation for 48 hours, the survival rate is higher by 11.3%. Squalene solutions did not affect the lifespan of nematodes.&#x0D; Conclusion. Presumably, the antioxidant activity of squalene is expressed not in the capture of free radicals, but in the ability to act on biomolecules that lead to oxidative stress. A squalene solution with a concentration of 10 μM can reduce the negative impact of one of the factors causing premature aging. In order to assess the possibility of using squalene as a geroprotector without relying on cation-radical capture, further studies are needed to study the impact of the substance on other targets, mechanisms of action related to the occurrence of age-associated diseases. For example, analysis of the effect of squalene solutions on the growth rate of C. elegans larvae, the ability to influence the expression of antioxidant defense genes, the presence of anti-inflammatory and other properties.

Ligusticum chuanxiong Hort as a medicinal and edible plant foods: Antioxidant, anti-aging and neuroprotective properties in Caenorhabditis elegans.

Ligusticum chuanxiong Hort. (CX) is a medicinal and edible plant including a variety of active substances, which may be an available resource for the treatment of related diseases. To expand the medicinal uses of CX, this study aims to explore the antioxidant, anti-aging and neuroprotective effects of the Ligusticum chuanxiong leaves (CXL) and rhizome (CXR) extracts. We first characterize CX phytochemical spectrum by LC-MS as well as antioxidant capacity. Acute toxicity, anti-oxidative stress capacity, lifespan and healthspan was evaluated in C elegans N2. Neuroprotective effect was evaluated in vitro and in vivo (C elegans CL4176 and CL2355). In this study, we detected 74 and 78 compounds from CXR and CXL, respectively, including phthalides, alkaloids, organic acids, terpenes, polyphenols and others. Furthermore, we found that CXs not only protect against oxidative stress, but also prolong the lifespan, alleviate lipofuscin, malondialdehyde (MDA) and reactive oxygen species (ROS) accumulation, and improve movement level, antioxidant enzyme activity in C elegans N2. However, only CXR reduced the β-amyloid peptide (Aβ)-induced paralysis phenotype in CL4176s and alleviated chemosensory behavior dysfunction in CL2355s. In addition, CXR treatment reduced the production of Aβ and ROS, enhanced SOD activity in CL4176s. The possible mechanism of anti-aging of CXL and CXR is to promote the expression of related antioxidant pathway genes, increase the activity of antioxidant enzymes, and reduce the accumulation of ROS, which is dependent on DAF-16 and HSF-1 (only in CXR). CXR was able to activate antioxidase-related (sod-3 and sod-5) and heat shock protein genes (hsp-16.1 and hsp-70) expression, consequently ameliorating proteotoxicity related to Aβ aggregation. In summary, these findings demonstrate the antioxidant, anti-aging and neuroprotective (only in CXR) activities of the CX, which provide an important pharmacological basis for developing functional foods and drugs to relieve the symptoms of aging and AD. However, the material basis of neuroprotective activity and antiaging effects need to be elucidated, and the relationship between these activities should also be clarified in future studies.

Discovery and Biosynthesis of Antimicrobial Phenethylamine Alkaloids from the Marine Flavobacterium Tenacibaculum discolor sv11.

The bacterial genus Tenacibaculum has been associated with various ecological roles in marine environments. Members of this genus can act, for example, as pathogens, predators, or episymbionts. However, natural products produced by these bacteria are still unknown. In the present work, we investigated a Tenacibaculum strain for the production of antimicrobial metabolites. Six new phenethylamine (PEA)-containing alkaloids, discolins A and B (1 and 2), dispyridine (3), dispyrrolopyridine A and B (4 and 5), and dispyrrole (6), were isolated from media produced by the predatory bacterium Tenacibaculum discolor sv11. Chemical structures were elucidated by analysis of spectroscopic data. Alkaloids 4 and 5 exhibited strong activity against Gram-positive Bacillus subtilis DSM10, Mycobacterium smegmatis ATCC607, Listeria monocytogenes DSM20600, and Staphylococcus aureus ATCC25923, with minimum inhibitory concentration (MIC) values ranging from 0.5 to 4 μg/mL, and moderate activity against Candida albicans FH2173 and Aspergillus flavus ATCC9170. Compound 6 displayed moderate antibacterial activities against Gram-positive bacteria. Dispyrrolopyridine A (4) was active against efflux pump deficient Escherichia coli ATCC25922 ΔtolC, with an MIC value of 8 μg/mL, as well as against Caenorhabditis elegans N2 with an MIC value of 32 μg/mL. Other compounds were inactive against these microorganisms. The biosynthetic route toward discolins A and B (1 and 2) was investigated using in vivo and in vitro experiments. It comprises an enzymatic decarboxylation of phenylalanine to PEA catalyzed by DisA, followed by a nonenzymatic condensation to form the central imidazolium ring. This spontaneous formation of the imidazolium core was verified by means of a synthetic one-pot reaction using the respective building blocks. Six additional strains belonging to three Tenacibaculum species were able to produce discolins, and several DisA analogues were identified in various marine flavobacterial genera, suggesting the widespread presence of PEA-derived compounds in marine ecosystems.

Effect of Autoinducer-2 Quorum Sensing Inhibitor on Interspecies Quorum Sensing.

Bacterial drug resistance caused by overuse and misuse of antibiotics is common, especially in clinical multispecies infections. It is of great significance to discover novel agents to treat clinical bacterial infections. Studies have demonstrated that autoinducer-2 (AI-2), a signal molecule in quorum sensing (QS), plays an important role in communication among multiple bacterial species and bacterial drug-resistance. Previously, 14 AI-2 inhibited compounds were selected through virtual screening by using the AI-2 receptor protein LuxP as a target. Here, we used Vibrio harveyi BB170 as a reporter strain for the preliminary screening of 14 inhibitors and compound Str7410 had higher AI-2 QS inhibition activity (IC50 = 0.3724 ± 0.1091 μM). Then, co-culture of Pseudomonas aeruginosa PAO1 with Staphylococcus aureus ATCC 25923 was used to evaluate the inhibitory effects of Str7410 on multispecies infection in vitro and in vivo. In vitro, Str7410 significantly inhibited the formation of mixed bacterial biofilms. Meanwhile, the combination of Str7410 with meropenem trihydrate (MEPM) significantly improved the susceptibility of mixed-species-biofilm cells to the antibiotic. In vivo, Str7410 significantly increased the survival rate of wild-type Caenorhabditis elegans N2 co-infected by P. aeruginosa PAO1 and S. aureus ATCC 25923. Real-time quantitative PCR analysis showed that Str7410 reduced virulence factor (pyocyanin and elastase) production and swarming motility of P. aeruginosa PAO1 by downregulating the expression of QS-related genes in strain PAO1 in co-culture with S. aureus ATCC 25923. Compound Str7410 is a candidate agent for treating drug-resistant multispecies infections. The work described here provides a strategy for discovering novel antibacterial drugs.

Genomic architecture of 5S rDNA cluster and its variations within and between species

BackgroundRibosomal DNAs (rDNAs) are arranged in purely tandem repeats, preventing them from being reliably assembled onto chromosomes during generation of genome assembly. The uncertainty of rDNA genomic structure presents a significant barrier for studying their function and evolution.ResultsHere we generate ultra-long Oxford Nanopore Technologies (ONT) and short NGS reads to delineate the architecture and variation of the 5S rDNA cluster in the different strains of C. elegans and C. briggsae. We classify the individual rDNA’s repeating units into 25 types based on the unique sequence variations in each unit of C. elegans (N2). We next perform assembly of the cluster by taking advantage of the long reads that carry these units, which led to an assembly of 5S rDNA cluster consisting of up to 167 consecutive 5S rDNA units in the N2 strain. The ordering and copy number of various rDNA units are consistent with the separation time between strains. Surprisingly, we observed a drastically reduced level of variation in the unit composition in the 5S rDNA cluster in the C. elegans CB4856 and C. briggsae AF16 strains than in the C. elegans N2 strain, suggesting that N2, a widely used reference strain, is likely to be defective in maintaining the 5S rDNA cluster stability compared with other wild isolates of C. elegans or C. briggsae.ConclusionsThe results demonstrate that Nanopore DNA sequencing reads are capable of generating assembly of highly repetitive sequences, and rDNA units are highly dynamic both within and between population(s) of the same species in terms of sequence and copy number. The detailed structure and variation of the 5S rDNA units within the rDNA cluster pave the way for functional and evolutionary studies.

Explaining inter-lab variance in C. elegans N2 lifespan: Making a case for standardized reporting to enhance reproducibility.

The nematode Caenorhabditis elegans is a powerful model organism for studying cell development, apoptosis, neuronal circuits, and aging. The isolate N2 is recognized by the C. elegans community as the reference wild-type strain. Interestingly, the lifespan of presumably isogenic C. elegans N2 worms—even when grown under comparable conditions—varies significantly amongst distinct laboratories. This hinders the inter-laboratory comparability of C. elegans lifespan data and raises questions regarding data interpretation and reproducibility. Here, we hypothesized slight alterations in experimental design and worm handling could explain the observed discrepancies. To test this hypothesis, we collected and assessed data from over 1000 published C. elegans N2 lifespan assays as well as corresponding methodological meta-data. We find that mean N2 lifespans range from approximately 7 days to upwards of 35 days, despite laboratories disclosing seemingly comparable experimental conditions. We further demonstrate that, in addition to temperature, the use of the chemical sterilizer 5-fluoro-2′-deoxyuridine (FUDR) may change N2 lifespan. Additionally, we observed differences in average N2 lifespan from experiments originating from distinct geographic locations, indicating a potential effect of location-specific factors on experimental outcomes. Taken as a whole, our work indicates the sum of many small, rather than a few critical, differences in experimental conditions may account for the observed variance in N2 lifespan. We also find that the absence of standardized experimental methods and the insufficient disclosure of experiment details in the peer-reviewed literature limits the inter-lab comparability of published results. We thus propose the establishment of a succinct reporting standard for C. elegans lifespan experiments to increase the reliability and reproducibility, and thus scientific value, of these studies.

Antioxidant Activities of Commiphora leptophloeos (Mart.) J. B. Gillett) (Burseraceae) Leaf Extracts Using In Vitro and In Vivo Assays.

Commiphora leptophloeos is widely used in folk medicine without any scientific basis. Considering this, the aim of this study was to evaluate the chemical profile and the antioxidant activity of C. leptophloeos leaf extracts using in vitro and in vivo assays. Six extracts were obtained from fresh leaves using a serial extraction (nonpolar to polar solvents). These extracts were first evaluated with the presence of phytochemical compounds using the methods thin layer chromatography (TLC), ultrahigh performance liquid chromatography (UHPLC-DAD), and high performance liquid chromatography, both with diode array detection (HPLC-DAD). Based on the compounds identified, it was used some bioinformatics tools in order to identify possible pathway and gene targets. After that, the antioxidant capacity from these extracts was analysed by in vitro assays and in vivo assays using Caenorhabditis elegans model. Phytochemical analyses showed the presence of polyphenols, such as rutin, vitexin, and quercetin diglycosides in all extracts, especially in ethanol extract (EE) and methanol extract (EM). Bioinformatics analysis showed these polyphenols linked to antioxidant pathways. Furthermore, EE and EM displayed a high antioxidant capacity in DPPH and superoxide radical scavenging assays. They also had no effect on cell viability for 3T3 nontumour cell. However, for B16-F10 tumour cell lines, these extracts had toxicity effect. In vivo assays using C. elegans N2 showed that EE was not toxic, and it did not affect its viability nor its development. Besides, EE increased worm survival under oxidative stress and reduced intracellular reactive oxygen species (ROS) levels by 50%. Thus, C. leptophloeos EE displayed an important in vitro and in vivo antioxidant capacity. The EE extract has polyphenols, suggesting that these compounds may be responsible for a myriad of biological activities having this potential to be used in various biotechnological applications.

Ursolic acid ameliorates stress and reactive oxygen species in C. elegans knockout mutants by the dopamine Dop1 and Dop3 receptors

BackgroundDepression and stress-related disorders are leading causes of death worldwide. Standard treatments elevating serotonin or noradrenaline levels are not sufficiently effective and cause adverse side effects. A connection between dopamine pathways and stress-related disorders has been suggested. Compounds derived from herbal medicine could be a promising alternative. We examined the neuroprotective effects of ursolic acid (UA) by focusing on dopamine signalling. MethodsTrolox equivalent capacity assay was used to determine the antioxidant activities of UA in vitro. C. elegans N2 wildtype and dopamine receptor-knockout mutants (dop-1-deficient RB665 and dop-3-deficient LX703 strains) were used as in vivo models. H2DCFDA and acute juglone assays were applied to determine the antioxidant activity in dependency of dopamine pathways in vivo. Stress was assessed by heat and acute osmotic stress assays. The influence of UA on overall survival was analyzed by a life span assay. The dop-1 and dop-3 mRNA expression was determined by real time RT-PCR. We also examined the binding affinity of UA towards C. elegans Dop1 and Dop3 receptors as well as human dopamine receptors D1 and D3 by molecular docking. ResultsAntioxidant activity assays showed that UA exerts strong antioxidant activity. UA increased resistance towards oxidative, osmotic and heat stress. Additionally, UA increased life span of nematodes. Moreover, dop-1 and dop-3 gene expression was significantly enhanced upon UA treatment. Docking analysis revealed stronger binding affinity of UA to C. elegans and human dopamine receptors than the natural ligand, dopamine. Binding to Dop1 was stronger than to Dop3. ConclusionUA reduced stress-dependent ROS generation and acted through Dop1 and to a lesser extent through Dop3 to reduce stress and prolong life span in C. elegans. These results indicate that UA could be a promising lead compound for the development of new antidepressant medications.

Quantitative benzimidazole resistance and fitness effects of parasitic nematode beta-tubulin alleles

Infections by parasitic nematodes inflict a huge burden on the health of humans and livestock throughout the world. Anthelmintic drugs are the first line of defense against these infections. Unfortunately, resistance to these drugs is rampant and continues to spread. To improve treatment strategies, we must understand the genetics and molecular mechanisms that underlie resistance. Studies of the fungus Aspergillus nidulans and the free-living nematode Caenorhabditis elegans discovered that a beta-tubulin gene is mutated in benzimidazole (BZ) resistant strains. In parasitic nematode populations, three beta-tubulin alleles, F167Y, E198A, and F200Y, have long been correlated with resistance. Additionally, improvements in sequencing technologies have identified new alleles - E198V, E198L, E198K, E198I, and E198Stop - also correlated with BZ resistance. However, none of these alleles have been proven to cause resistance. To empirically demonstrate this point, we independently introduced the F167Y, E198A, and F200Y alleles as well as two of the newly identified alleles, E198V and E198L, into the BZ susceptible C. elegans N2 genetic background using the CRISPR-Cas9 system. These genome-edited strains were exposed to both albendazole and fenbendazole to quantitatively measure animal responses to BZs. We used a range of concentrations for each BZ compound to define response curves and found that all five of the alleles conferred resistance to BZ compounds equal to a loss of the entire beta-tubulin gene. These results prove that the parasite beta-tubulin alleles cause resistance. The E198V allele is found at low frequencies along with the E198L allele in natural parasite populations, suggesting that it could affect fitness. We performed competitive fitness assays and demonstrated that the E198V allele reduces animal health, supporting the hypothesis that this allele might be less fit in field populations. Overall, we present a powerful platform to quantitatively assess anthelmintic resistance and effects of specific resistance alleles on organismal fitness in the presence or absence of the drug.

Inhibitory effects of novel 1,4-disubstituted 1,2,3-triazole compounds on quorum-sensing of P. aeruginosa PAO1.

Quorum sensing (QS) inhibition is an essential strategy to combat bacterial infection. Previously, we have synthesized a series of thymidine derivatives bearing isoxazole and 1,2,3-triazole rings (TITL). Herein, the inhibitory effects of TITL on QS of Pseudomonas aeruginosa PAO1 were evaluated. In vitro results demonstrated that TITL effectively inhibited biofilm formation and reduced the virulence factors of P. aeruginosa PAO1. In combination with antibiotics, our TITL compounds significantly prolonged the lifespans of Caenorhabditis elegans N2 nematodes that were infected with P. aeruginosa PAO1 in vivo. In conclusion, TITL compounds are promising candidates for the treatment of antibiotic-resistant P. aeruginosa PAO1.

Design, synthesis, and biological evaluation of 3-amino-2-oxazolidinone derivatives as potent quorum-sensing inhibitors of Pseudomonas aeruginosa PAO1

Due to the increasing resistance of Pseudomonas aeruginosa to most clinically relevant antimicrobials, it is challenging to treat bacterial infection with traditional antibiotics. Quorum sensing can regulate the production of biofilms and virulence factors which are closely related to bacterial resistance. Previously we synthesized a series of oxazolidinone compounds targeting the quorum-sensing transcriptional regulatory protein CviR and ZS-12 showed good activity against Chromobacterium violaceum CV026 quorum-sensing. In this study, eighteen 3-amino-2-oxazolidinone compounds were designed and synthesized using ZS-12 as the lead compound. We initially evaluated the inhibitory activities of novel oxazolidinone compounds against QS using C. violaceum CV026 as a reporter strain. Thirteen compounds showed good activities (IC50 range 3.69–63.58 μM) and YXL-13 inhibition was the most significant (IC50 = 3.686 ± 0.5790 μM) against biofilm formation and virulence factors determination of P. aeruginosa PAO1. In vitro, YXL-13 significantly inhibited the formation of PAO1 biofilm (range 42.98%–17.67%), the production of virulence factors (pyocyanin, elastase, rhamnolipid, and protease), and bacterial motility. Moreover, the combination of YXL-13 with an antibiotic (meropenem trihydrate) could significantly improve the antibiotic susceptibility of biofilm P. aeruginosa PAO1 cells. In vivo, YXL-13 significantly prolonged the lifespan of wildtype Caenorhabditis elegans N2 infected by P. aeruginosa PAO1. In conclusion, YXL-13 is a candidate agent for antibiotic-resistant P. aeruginosa PAO1and provides a method for finding new antibacterial drugs.