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Biodegradable polylactic acid (PLA) mulch has been developed to replace conventional polyethylene (PE) mulch in agriculture as a response to growing concerns about recalcitrant plastic pollution and the accumulation of microplastics (MPs) in soil. Cadmium is a significant soil pollutant in China. MPs have been shown to adsorb metals. In this study the earthworm Lumbricus terrestris was exposed to either Cd (1.0-100 mg / kg) or MPs (PE and PLA, 0.1-3 % w / w), or a combination of the two, for 28 days. Cd bioavailability significantly decreased in the presence of MPs. In particular, at the end of the experiment, PLA treatments had lower measured Cd concentrations in both earthworms (2.127-29.24 mg / kg) and pore water (below detection limits - 0.1384 mg /L) relative to PE treatments (2.720-33.77 mg / kg and below detection limits - 0.2489 mg / L). In our adsorption experiment PLA MPs adsorbed significantly more Cd than PE MPs with maximum adsorption capacities of 126.0 and 23.2 mg / kg respectively. These results suggest that the PLA MPs reduce earthworm exposure to Cd relative to PE by removing it from solution and reducing its bioavailability.

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INTRODUCTION: The demand for red blood cells (RBCs) is on the rise due to the increasing diagnosis of chronic diseases such as sickle cell anemia, malaria, and thalassemia. Despite many commercial attempts, there are no U.S. FDA-approved artificial RBCs for use in humans. Existing RBC substitutes have employed various strategies to transport oxygen, extend the circulation time, and reduce organ toxicity, but none have replicated the natural protective mechanisms of RBCs, which prevent hemoglobin (Hb) dimerization and heme iron oxidation. Lumbricus terrestris (earthworm) erythrocruorin (LtEc) is a naturally occurring extracellular hemoglobin (Hb) with promising attributes: large molecular diameter (30 nm), high molecular weight (3.6 MDa), low auto-oxidation rate, and limited nitric oxide-scavenging properties. These characteristics make LtEc an ideal candidate as an RBC substitute. However, LtEc has a significant drawback, its short circulatory half-life. To address this issue, we explored thiol-mediated surface PEGylation of LtEc (PEG-LtEc) at varying polyethylene glycol (PEG) surface coverages. Increasing PEG surface coverage beyond 40% destabilizes LtEc into smaller subunits that are 1/12th the size of LtEc. Therefore, we evaluated two PEG surface coverage options: PEG-LtEc-0.2 (20% PEGylation) and PEG-LtEc-1.0 (100% PEGylation). METHODS: We conducted experiments using golden Syrian hamsters with dorsal window chambers and catheters to assess the efficacy of these solutions. We measured microvascular parameters, organ function, cerebral blood flow, circulation time, mean arterial pressure, heart rate, and blood gases and performed histology to screen for toxicity. CONCLUSION: Our findings indicate that both PEG-LtEc molecules offer significant benefits in restoring microvascular parameters, organ function, cerebral blood flow, and circulation time compared to LtEc alone. Notably, PEG-LtEc-1.0 showed superior microvascular perfusion, although it exhibited a higher rate of auto-oxidation compared to PEG-LtEc-0.2. These results underscore the advantages of PEGylation in terms of tissue perfusion and organ health while highlighting its limitations.

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Microplastic (MP) pollution constitutes an emerging type of pollution threatening both aquatic and terrestrial ecosystems. The impact on aquatic ecosystems has been extensively studied, but the effect on terrestrial ecosystems and their inhabitants is mostly underexplored. In this study, we explored the effect of MP pollution on gut bacterial microbiome of endogeic (Aporrectodea caliginosa) and anecic (Lumbricus terrestris) earthworms. The experiments were performed in sandy soil with 0.2% of low-density polyethylene MPs (LDPE MPs). We observed that the endogeic earthworms had 100% survival, while anecic earthworms survived 25 days in the control (i.e. in absence of MPs) and 21 days in the treatment with LDPE MPs. The main driver of shifts in the diversity and composition of the bacterial communities in the gut of tested earthworms was the lifestyle of the worms, followed by the presence of MPs. The bacterial microbiome diversity was significantly different among the two types of earthworms, and the highest bacterial diversity was found in the gut of the endogeic earthworms. The effect of MPs on gut bacterial microbiome was clearly observed in the changes in the relative abundance of several phyla and families of the bacterial communities in both types of earthworms, although it was most evident in the anecic earthworms. The Actinobacteriota, Proteobacteria, and Firmicutes were the main groups enhanced in the MP treatments, suggesting enrichment of the bacterial communities with potential plastic degraders.

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Haemorrhagic shock is a leading cause of death worldwide. Blood transfusions can be used to treat patients suffering severe blood loss but donated red blood cells (RBCs) have several limitations that limit their availability and use. To solve the problems associated with donated RBCs, several acellular haemoglobin-based oxygen carriers (HBOCs) have been developed to restore the most important function of blood: oxygen transport. One promising HBOC is the naturally extracellular haemoglobin (i.e. erythrocruorin) of Lumbricus terrestris (LtEc). The goal of this study was to maximise the portability of LtEc by lyophilising it and then testing its stability at elevated temperatures. To prevent oxidation, several cryoprotectants were screened to determine the optimum formulation for lyophilisation that could minimise oxidation of the haem iron and maximise recovery. Furthermore, samples were also deoxygenated prior to storage to decrease auto-oxidation, while resuspension in a solution containing ascorbic acid was shown to partially reduce LtEc that had oxidised during storage (e.g. from 42% Fe3+ to 11% Fe3+). Analysis of the oxygen equilibria and size of the resuspended LtEc showed that the lyophilisation, storage, and resuspension processes did not affect the oxygen transport properties or the structure of the LtEc, even after 6 months of storage at 40 °C. Altogether, these efforts have yielded a shelf-stable LtEc powder that can be stored for long periods at high temperatures, but future animal studies will be necessary to prove that the resuspended product is a safe and effective oxygen transporter in vivo.

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BACKGROUND: Artificial light at night, also referred to as light pollution (LP), has been shown to affect many organisms. However, little is known about the extent to which ecological interactions between earthworms and plants are altered by LP. We investigated the effects of LP on anecic earthworms (Lumbricus terrestris) that come to the surface at night to forage and mate, and on the germination and growth of the invasive and allergenic ragweed (Ambrosia artemisiifolia). In a full factorial pot experiment in the greenhouse, we tested four factors and their interactions: LP (5 lux vs. 0 lux at night), earthworms (two individuals vs. none), plant species (seeding of ragweed only vs. mixed with Phacelia seeds) and sowing depth (seed placed at the surface vs. in 5 cm depth). Data were analysed using Generalized Linear (Mixed) Models and multifactorial ANOVAs with soil parameters as covariates. RESULTS: Light pollution reduced earthworm surface activity by 76% as measured by casting activity and toothpick index; 85% of mating earthworms were observed in the absence of LP. Light pollution in interaction with earthworms reduced ragweed germination by 33%. However, LP increased ragweed height growth by 104%. Earthworms reduced ragweed germination especially when seeds were placed on the soil surface, suggesting seed consumption by earthworms. CONCLUSIONS: Our data suggest that anecic earthworms are negatively affected by LP because reduced surface activity limits their ability to forage and mate. The extent to which earthworm-induced ecosystem services or community interactions are also affected by LP remains to be investigated. If the increased height growth of ragweed leads to increased pollen and seed production, it is likely that the competition of ragweed with field crops and the risks to human health will also increase under LP.

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The erythrocruorin of Lumbricus terrestris (LtEc) is a relatively large macromolecular assembly that consists of at least four different hemoglobin subunits (A, B, C, and D) and four linker subunits (L1, L2, L3, and L4). The complexity and stability of this large structure make LtEc an attractive hemoglobin-based oxygen carrier that could potentially be used as a substitute for donated red blood cells. However, the sequences of the LtEc subunit sequences must be determined before a scalable recombinant expression platform can be developed. The goal of this study was to sequence the L. terrestris genome to identify the complete sequences of the LtEc subunit genes. Our results revealed multiple homologous genes for each subunit (e.g., two homologous A globin genes; A1 and A2), with the exception of the L4 linker. Some of the homologous genes encoded identical peptide sequences (C1 and C2, L1a and L1b), while cDNA and mass spectrometry experiments revealed that some of the homologs are not expressed (e.g., A2). In contrast, multiple sequences for the B, D, L2, and L4 subunits were detected in LtEc samples. These observations reveal novel degeneracy in LtEc and other annelids, along with some new revisions to its previously published peptide sequences.

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Microorganisms from L. terrestris gut previously exposed to different types of plastic (PET, LDPE, LLDPE, and PS) were studied to be used as probiotics of earthworms in plastic-contaminated soils (LDPE, LLDPE and recycled mulching film) at mesocosm-scale trials. The most abundant morphotypes with enzymatic capacities of interest were identified. Pseudomonas alkylphenolica (PL4) and Pseudomonas putida (PL5) strains were selected to be used as inoculants using Morus alba leaves as carriers to strengthen the intestinal microbiota of earthworms. Culture (selective cetrimide agar medium) and molecular (qPCR) techniques were used to trace the presence of the inoculum in the intestine of the earthworms. Additionally, a metataxonomic analysis was carried out to study the biodiversity and functionality of the earthworm microbiome, and their measure of survival and weight. Probiotics improved the survival rates of earthworms exposed to plastics, which also increased the abundance of microbial groups of interest in plastic bioremediation tasks.

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Earthworms are attracting the attention of bioremediation research because of their short-term impact on pollutant fate. However, earthworm-assisted bioremediation largely depends on the earthworm sensitivity to target pollutants and its metabolic capacity to break down contaminants. The most studied species in soil bioremediation has been Eisenia fetida, which inhabits the soil surface feeding on decomposing organic residues. Therefore, its bioremediation potential may be limited to organic matter-rich topsoil. We compared the detoxification potential against organophosphate (OP) pesticides of three earthworm species representative of the main ecotypes: epigeic, anecic, and endogeic. Selected biomarkers of pesticide detoxification (esterases, cytochrome P450-dependent monooxygenase, and glutathione S-transferase) and oxidative homeostasis (total antioxidant capacity, glutathione levels, and glutathione reductase [GR] and catalase activities) were measured in the muscle wall and gastrointestinal tract of E. fetida (epigeic), Lumbricus terrestris (anecic) and Aporrectodea caliginosa (endogeic). Our results show that L. terrestris was the most suitable species to bioremediate OP-contaminated soil for the following reasons: 1) Gut carboxylesterase (CbE) activity of L. terrestris was higher than that of E. fetida, whereas muscle CbE activity was more sensitivity to OP inhibition than that of E. fetida, which means a high capacity to inactivate the toxic oxon metabolites of OPs. 2) Muscle and gut phosphotriesterase activities were significantly higher in L. terrestris than in the other species. 3) Enzymatic (catalase and GR) and molecular mechanisms of free radical inactivation (glutathione) were 3- to 4-fold higher in L. terrestris concerning E. fetida and A. caliginosa, which reveals a higher potential to keep the cellular oxidative homeostasis against reactive metabolites formed during OP metabolism. Together with biological and ecological traits, these toxicological traits suggest L. terrestris a better candidate for soil bioremediation than epigeic earthworms.

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We present a genome assembly from an individual Lumbricus terrestris (the common earthworm; Annelida; Clitellata; Haplotaxida; Lumbricidae). The genome sequence is 1,056.5 megabases in span. Most of the assembly is scaffolded into 18 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 15.93 kilobases in length.

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Agronomic factors can affect mycotoxin contamination of maize, one of the most produced cereals. Maize is usually harvested at 18% moisture, but it is not microbiologically stable until it reaches 14% moisture at the drying plants. We studied how three agronomic factors (crop diversification, tillage system and nitrogen fertilization rate) can affect fungal and mycotoxin contamination (deoxynivalenol and fumonisins B1 and B2) in maize at harvest. In addition, changes in maize during a simulated harvest-till-drying period were studied. DON content at harvest was higher for maize under intensive tillage than using direct drilling (2695 and 474 µg kg-1, respectively). We found two reasons for this: (i) soil crusting in intensive tillage plots caused the formation of pools of water that created high air humidity conditions, favouring the development of DON-producing moulds; (ii) the population of Lumbricus terrestris, an earthworm that would indirectly minimize fungal infection and mycotoxin production on maize kernels, is reduced in intensive tillage plots. Therefore, direct drilling is a better approach than intensive tillage for both preventing DON contamination and preserving soil quality. Concerning the simulated harvest-till-drying period, DON significantly increased between storage days 0 and 5. Water activity dropped on the 4th day, below the threshold for DON production (around 0.91). From our perspective, this study constitutes a step forward towards understanding the relationships between agronomic factors and mycotoxin contamination in maize, and towards improving food safety.

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Accurate prediction of organic contaminant bioavailability for risk assessment in ecological applications is hindered by limited validation on relevant bioassay species. Here, six in-vitro chemical extraction methods (butanol, non-buffered and buffered hydroxypropyl-ß-cyclodextrin (HPCD, Buf-HPCD), Tenax, potassium persulfate oxidation, polyoxymethylene solid phase extraction (POM)) were tested for PAH bioaccumulation prediction in three earthworm ecotypes with dissimilar exposures, Amynthas sp., Eisenia fetida, and Lumbricus terrestris, in historically contaminated soils from manufactured gas plant (MGP) sites. Extractions were compared directly and modelled in a calculation approach using equilibrium partitioning theory (EqPT) with a novel combination of different organic carbon/octanol-water partitioning parameters (KOC and KOW). In the direct comparison approach Buf-HPCD showed the closest prediction of accumulation for burrowing Amynthas sp. and L. terrestris (within 1.5 and 3.1, respectively), but Tenax and POM showed the closest approximation for E. fetida (within 1.1 and 0.9, respectively). The optimum method for predicting PAH bioaccumulation in the calculation approach depended on earthworm species and the partitioning parameters used in equations of the four models, but overall POM, which was independent of KOC, showed the closest approximation of accumulation, within a factor of 2.5 across all species. This work effectively identifies the optimum in-vitro based approaches for PAH bioavailability prediction in earthworms as a model soil health indicator for ecological risk assessment within regulatory and remediation decision frameworks.

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Microplastic fibres (MPFs) and nanoplastics (NPs) have the potential to be hazardous to soil organisms. Understanding uptake into organisms is key in assessing these effects, but this is often limited by the analytical challenges to quantify smaller-sized plastics in complex matrices. This study used MPFs and NPs containing inorganic tracers (In, Pd) to quantify uptake in the earthworm Lumbricus terrestris. Following seven days exposure, tracer concentrations were measured in earthworms and faeces. Earthworms exposed to 500 µg MPFs/g soil retained an estimated 32 MPFs in their tissues, while at 5000 µg MPFs/g earthworms retained between 2 and 593 MPFs. High variation in body burdens of MPFs was linked to soil retention in earthworms and reduced faeces production, suggesting egestion was being affected by MPFs. NPs uptake and elimination was also assessed over a more extended time-period of 42 days. After 1 day, NPs were no longer detectable in faeces during the elimination phase. However, some retention of NPs in the earthworms was estimated, not linked to retained soil, indicating not all NPs were eliminated. MPFs and NPs uptake can be quantified in earthworms and both particle types can be retained beyond the depuration period, suggesting the potential for longer-term accumulation.

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Earthworms are ecological engineers that can contribute to the displacement of biological control agents such as the entomopathogenic nematodes (EPNs) and fungi (EPF). However, a previous study showed that the presence of cutaneous excreta (CEx) and feeding behavior of the earthworm species Eisenia fetida (Haplotaxida: Lumbricidae) compromise the biocontrol efficacy of certain EPN species by reducing, for example, their reproductive capability. Whether this phenomenon is a general pattern for the interaction of earthworms-entomopathogens is still unknown. We hypothesized that diverse earthworm species might differentially affect EPN and EPF infectivity and reproductive capability. Here we investigated the interaction of different earthworm species (Eisenia fetida, Lumbricus terrestris, and Perionyx excavatus) (Haplotaxida) and EPN species (Steinernema feltiae, S. riojaense, and Heterorhabditis bacteriophora) (Rhabditida) or EPF species (Beauveria bassiana and Metarhizium anisopliae) (Hypocreales), in two independent experiments. First, we evaluated the application of each entomopathogen combined with earthworms or their CEx in autoclaved soil. Hereafter, we studied the impact of the earthworms' CEx on entomopathogens applied at two different concentrations in autoclaved sand. Overall, we found that the effect of earthworms on entomopathogens was species-specific. For example, E. fetida reduced the virulence of S. feltiae, resulted in neutral effects for S. riojaense, and increased H. bacteriophora virulence. However, the earthworm P. excavates increased the virulence of S. feltiae, reduced the activity of H. bacteriophora, at least at specific timings, while S. riojaense remained unaffected. Finally, none of the EPN species were affected by the presence of L. terrestris. Also, the exposure to earthworm CEx resulted in a positive, negative or neutral effect on the virulence and reproduction capability depending on the earthworm-EPN species interaction. Concerning EPF, the impact of earthworms was also differential among species. Thus, E. fetida was detrimental to M. anisopliae and B. bassiana after eight days post-exposure, whereas Lumbricus terrestris resulted only detrimental to B. bassiana. In addition, most of the CEx treatments of both earthworm species decreased B. bassiana virulence and growth. However, the EPF M. anisopliae was unaffected when exposed to L. terrestris CEx, while the exposure to E. fetida CEx produced contrasting results. We conclude that earthworms and their CEx can have positive, deleterious, or neutral impacts on entomopathogens that often coinhabit soils, and that we must consider the species specificity of these interactions for mutual uses in biological control programs. Additional studies are needed to verify these interactions under natural conditions.

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We assessed the enzymatic activation of four different biochars produced from pyrolyzing swine manure and poultry litter, and by co-pyrolyzing these livestock residues with agricultural spent mulch plastic film wastes (plastichars). Enzymatic activation consisted of incubating biochars in soil inoculated with earthworms (Lumbricus terrestris), which acted as biological vectors to facilitate retention of extracellular enzymes onto biochar surface. The activity of carboxylesterase ‒a pesticide-detoxifying enzyme‒ was measured in non-bioturbed soils (reference), linings of the burrows created by earthworms, casts (feces) and biochar particles recovered from the soil. Our results revealed that: 1) biochar increased soil carboxylesterase activity respect to biochar-free (control) soils, which was more prominent in the presence of earthworms. 2) The maximum enzyme activity was found in soils amended with plastichars. 3) The plastichars showed higher enzyme binding capacities than that of the biochars produced from animal manure alone, corroborating the pattern of enzyme distribution found in soil. 4) The presence of earthworms in soil significantly increased the potential of the plastichars for enzymatic activation. These findings suggest that the plastichars are suitable for increasing and stabilizing soil enzyme activities with no toxicity on earthworms.

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Biochar is the degradation-resistant product generated by the pyrolysis of organic materials and is produced for the intended use of land application in order to promote carbon sequestration and soil improvement. However, despite the many potential benefits biochar application offers, it is important to quantify any ecological impacts that may result from soil amendment in order to avoid potentially causing negative effects upon soil biota which are crucial in the many ecosystem services provided by soil. Any impacts on earthworms in particular are important to evaluate because of their pivotal role in organic matter breakdown, nutrient cycling and soil formation. In this study, we conducted a series of ecotoxicological assays to determine lethal and sublethal (avoidance, mass change and moisture content) effects of heavy biochar applications that reflect levels that may be used in soil restoration efforts. Two earthworm species, Eisenia fetida, an epigeic species, and Lumbricus terrestris, an anecic species, were utilised as test organisms. Two types of biochar, produced from wheat straw and rice husk feedstocks, respectively, were applied to OECD artificial soil and to a natural soil (Kettering loam) at rates of up to 20% w/w. The influence of biochar application on soil porewater chloride, fluoride and phosphate concentrations was also assessed. The biochar applications induced only a subtle level of avoidance behaviour while effects on survival over a 4-week exposure period were inconsistent. However, death and physical damage to some individual earthworms at high biochar application rates were observed, the mechanisms and processes leading to which should be investigated further. Earthworm development (mean mass change over time) proved to be a more sensitive measure, revealing negative effects on L. terrestris at 10% and 20% (w/w) wheat biochar applications in OECD soil and at 20% (w/w) applications of both biochars in Kettering loam. The moisture content of E. fetida remained remarkably consistent across all treatments (~ 82%), indicating that this is not a sensitive measure of effects. The high rates of biochar application resulted in increased chloride (2 to 3-fold) and phosphate (100-fold) concentrations in simulated soil porewaters, which has important implications for soil fertility and production but also for environmental management.

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Asiaticoside-D (AD) was shown to efficacy of ganglionic degenerated Lumbricus terrestris as a pioneering observation in our earlier research. Though, extract molecular mechanisms of AD for degenerative diseases (DDs) remains largely unknown. We investigated the neuroprotective effects of AD against ROT in cerebral ganglions (CGs) of degenerative L. terrestris. Worms were exposed to 0.4 ppm ROT for 7 days were subjected to co- treatment with 15 ppm of AD. After, CGs was removed. The levels oxidant, non-antioxidant, antioxidant status, ganglioside, ceramide and ceramide glycanase (CGase) were estimated. The m-RNA levels of dopamine transporter (DAT), octopamine transporter (OAT), innexins-9 (inx-9), ionotropic glutamate receptor 3 (iGlu3), heat shock proteins (hsp70), XPRLamide neuropeptide precursor, tyramine beta-hydroxylase (tbh-1) and ß- adrenergic receptor kinase-2 (ß-ARK2-3) by semi-qRT- PCR. The expression pattern of tyramine beta hydroxylase (TBH), glutamate receptor (iGluR), serotonin transporter (SERT), dopamine transporters (DAT), nerve growth factors (NGF), cytochrome C oxidase (COC), NADH dehydogenase subunit-1 (ND-1), neurotrophin receptor p75 (p75NTR), neuronal nitric oxiside synthase (nNOs) interleukin 1- beta (IL1-ß) and tumor necrosis factor alpha (TNF-α) by western blotting. Glutaminergic, serotogenic and dopaminergic toxicity variations were also performed. The levels of oxidant, non-antioxidant, antioxidant status, lipids, proteins and m-RNAs were significantly altered (p < 0.001) on ROT-induced (group II) and their levels were significantly changes (p < 0.05) by ROT+AD in CGs. The sensitive study plan concluded the neuroprotective effects of AD against ROT induced degeneration in worms and suggest that the AD deserves future studies for its use as an effective alternative medicine that could minimize the morbidity of ganglionic degenerative diseases patients.

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CONTEXT: Homology modeling plays role in determining the therapeutic targets dreadful for condition such as neurodegenerative diseases (NDD), which pose challenge in achieving the effective managements. The structures of the serotonin transporter (SERT), aquaporin (AQP), and tropomyosin receptor kinase (TrkA) which are implicated in NDD pathology are still unknown for Lumbricus terrestris, but the three-dimensional (3D) structure of the human counterpart for modeling. AIM: This study aims to generate and evaluate the 3D structure of TrkA, SERT, and AQP proteins and their interaction with the ligands, namely Asiaticoside-D (AD) and levodopa (L-DOPA) the anti-NDD agents. SUBJECTS AND METHODS: Homology modeling for SERT, AQP, and TrkA proteins of Lumbricus terrestris using SWISS-MODEL Server and the modeled structure was validated using Rampage Server. Wet-lab analysis of their correspondent m-RNA levels was also done to validate the in silico data. RESULTS: It was found that TrkA had moderately high homology (67%) to human while SERT and AQP could exhibit 58% and 42%, respectively. The reliability of the model was assessed by Ramachandran plot analysis. Interactions of AD with the SERT, AQP-4, and TrkA showed the binding energies as -9.93, 8.88, and -7.58 of Kcal/mol, respectively, while for L-DOPA did show -3.93, -5.13, and -6.0 Kcal/mol, respectively. The levels of SERT, TrkA, and AQP-4 were significantly reduced (P < 0.001) on ROT induced when compared to those of control worms. On ROT + AD supplementation group (III), m-RNA levels were significantly increased (P < 0.05) when compared to those of ROT induced worms (group II). CONCLUSION: Our pioneering docking data propose the possible of target which is proved useful for therapeutic investigations against the unconquered better of NDD.

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Herein, we examined whether synergistic effects of earthworms (Lumbricus terrestris) and pine needle-derived biochar result in biochar-coated burrows with enhanced carboxylesterase (CE) activity (a pesticide-detoxifying enzyme). Biochar was placed at the top of soil columns at two doses (2.5 and 5% w/w dry mass), with an additional biochar-free treatment as control. Carboxylesterase and dehydrogenase activities were measured in the burrow walls sampled at three depths (0-4, 8-12, and 18-22 cm). Biochar was recovered from these samples to confirm its vertical transport and enzymatic activation. We tested whether biochar protected CE activity against desiccation stress of burrow wall samples. Likewise, the role of earthworm mucus in stabilizing CE onto biochar surface was also investigated by measuring the enzyme activity in fresh biochar particles previously incubated in the presence of earthworm mucus and purified esterase. Finally, we checked for the sensitivity of biochar-bound CE activity against selected organophosphorus pesticides. The main results were: i) co-application of earthworms and biochar caused a significant increase of CE activity in the first 12 cm of the soil column, ii) recovered biochar particles displayed CE activity which was significantly higher in the particles collected from the bottom of columns, iii) soil desiccation decreased the enzymatic activity, although such effect depended on biochar treatment and column depth (22-77% inhibition). Nevertheless, CE activity still was higher in the samples from the 5%-biochar treatment than activity in control and 2.5%-biochar treatments, iv) earthworm mucus favored the retention of CE onto the biochar surface, and v) the activity of biochar-bound CE was sensitive to inhibition by chlorpyrifos-oxon. These results suggest that the joint application of L. terrestris and biochar may be a suitable vermiremediation strategy to inactivate OP pesticides.

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BACKGROUND: Centella asiatica (CA) is one of the most valuable herbal medicines widely being used for the treatment of various neurological ailments that are challenging for health-care providers and also is deemed to be safe and effective. PURPOSE: Monoamines (MAs) are neurotransmitters and neuromodulators that play a significant role in the neural communication, regulation of motor and cognitive functions in the brain. Neurodegeneration is associated with elevated levels of MAO-B that can lead to damaging reactive oxygen species (ROS) in the brain. The current study, evaluated the effects of asiaticoside-D (AD) from neuroprotective CA, on the levels and activities of monoamine oxidase A and B (MAO-A and B), in addition to the behavioral analysis. METHODS: Qualitative and quantitative analyses of various solvent extracts of CA were performed. The extracts were screened for antioxidant potential using 1,1-diphenyl-2-picryl hydroxyl (DPPH), 2,2'-azinobis-3-ethylbenzothiozoline-6-sulfonic acid (ABTS), hydrogen peroxide (H2O2) radical, nitric oxide (NO) radical inhibition, lipid peroxidation (LPO) and ferric reducing antioxidant power (FRAP) assays. The purification of AD was done by column, thin layer and high-performance liquid chromatographies followed by structural elucidation using IR, HR-MS, 1H and 13C NMR spectra. Docking studies were performed to assess the impact of AD on MAO-A and B.In vivo, Lumbricus terrestris were exposed to 0.4 ppm rotenone (ROT) of medium for 7 days and were subjected to co-treatment along with 15 ppm of AD from CA. At the end of experiment period, the neuronal behavior of worms was assessed. Cerebral ganglions (CGs) were removed and the m-RNA levels of MAO-A and B were analyzed by Semi Q-PCR and their activities were also analyzed. RESULTS: The ethanolic extracts exhibited higher antiradical activity against DPPH, ABTS, H2O2, LPO, FRAP, NO and vitamin C with EC50 value of 20.2, 20.9, 20.4, 22.0, 24.9, 28.1, 25.5 and 22.0 µg/ml respectively. Structural analysis by IR, HR-MS, 1H and 13C NMR spectrum have shown the structure of the isolated compound as (2α, 3ß)-2,3-dihydroxyurs-12-en-28-oicacid-O-α-L-rhamnopyranosyl-(1→4)-O-ß-d-glucopyranosyl (1→6)-ß-copyranosyl ester and was represented as AD. In silico interaction of AD with MAO-A and B residues Lys312 at distances of 1.84 Šand 2.44 Šrespectively was found to exhibit high binding energy of -9.4 and 7.4 kcal. The neuronal behavior using L. terrestris showed significant improvement against (p < 0.001) ROT impaired behavior (group II) on AD supplementation (p < 0.05). Further, the m-RNA levels and activities of MAO-A and B which were significantly altered (p < 0.001) by ROT could be effectively maintained on AD supplementation. CONCLUSION: AD was found to exert its negative impact on the levels and activities of MAO-A and B in CGs of rotenone- induced changes in L. terrestris, the property which is considered to be crucial against ROT induced neurodegenerative pathology like -Parkinsonism.

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Rotenone (ROT) was shown to affect cerebral ganglions (CGs) of Lumbricus terrestris as a pioneering observation in our earlier investigation. Though ROT is a well-known neurotoxin causing neurodegeneration (ND), the precipitation of movement dysfunction remains largely unknown. We have designed the current study to analyze motor abnormalities in worms by exposing them to different concentrations (0.0-0.4 ppm) of ROT for 7 days. GABA, cholinergic receptor, serotonin transporter (SERT), acetylcholine esterase (AchE), and dopamine-ß-hydroxylase that are well known for their involvement in neuromuscular junctions were investigated by qRT-PCR. Further, neuronal mitochondrial genes (cytochrome C oxidase-2, NADH deydrogenase-1, cytochrome-b) and actin-1 that are essential for regeneration and calreticulin (phagocytosis) were investigated. The levels of neurotransmitters, lipids, ATPase, neuronal behavior analyses, and fluorescence analysis (lipid droplets) were performed in CGs which showed significant variations at 0.3 ppm. Ultrastructural changes in lipid droplet and neuromelatonin were prominent in 0.3 ppm. Dose-dependent effect of ROT on behavior alteration and expression of m-RNAs studied suggested that at 0.3 ppm, it could deteriorate motor and cognitive functions. We predict that perhaps, by virtue of its effect on cerebral ganglionic genes and their neurotransmitting potential, ROT may cause morbidities that resemble features characteristic of hemiparkinsonic degeneration.

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