Cre recombinase microinjection for single-cell tracing and localised gene targeting.

Tracing and manipulating cells in embryos are essential to understand development. Lipophilic dye microinjections, viral transfection and iontophoresis have been key to map the origin of the progenitor cells that form the different organs in the post-implantation mouse embryo. These techniques require advanced manipulation skills and only iontophoresis, a demanding approach of limited efficiency, has been used for single-cell labelling. Here, we perform lineage tracing and local gene ablation using cell-permeant Cre recombinase (TAT-Cre) microinjection. First, we map the fate of undifferentiated progenitors to the different heart chambers. Then, we achieve single-cell recombination by titrating the dose of TAT-Cre, which allows clonal analysis of nascent mesoderm progenitors. Finally, injecting TAT-Cre to Mycnflox/flox embryos in the primitive heart tube revealed that Mycn plays a cell-autonomous role in maintaining cardiomyocyte proliferation. This tool will help researchers identify the cell progenitors and gene networks involved in organ development, helping to understand the origin of congenital defects.

PATO: genome-wide prediction of lncRNA-DNA triple helices.

MOTIVATION: Long non-coding RNA (lncRNA) plays a key role in many biological processes. For instance, lncRNA regulates chromatin using different molecular mechanisms, including direct RNA-DNA hybridization via triplexes, cotranscriptional RNA-RNA interactions, and RNA-DNA binding mediated by protein complexes. While the functional annotation of lncRNA transcripts has been widely studied over the last 20 years, barely a handful of tools have been developed with the specific purpose of detecting and evaluating lncRNA-DNA triple helices. What is worse, some of these tools have nearly grown a decade old, making new triplex-centric pipelines depend on legacy software that cannot thoroughly process all the data made available by next-generation sequencing (NGS) technologies. RESULTS: We present PATO, a modern, fast, and efficient tool for the detection of lncRNA-DNA triplexes that matches NGS processing capabilities. PATO enables the prediction of triple helices at the genome scale and can process in as little as 1 h more than 60 GB of sequence data using a two-socket server. Moreover, PATO's efficiency allows a more exhaustive search of the triplex-forming solution space, and so PATO achieves higher levels of prediction accuracy in far less time than other tools in the state of the art. AVAILABILITY AND IMPLEMENTATION: Source code, user manual, and tests are freely available to download under the MIT License at https://github.com/UDC-GAC/pato.

The distribution of fitness effects of plasmid pOXA-48 in clinical enterobacteria.

Antimicrobial resistance (AMR) in bacteria is a major public health problem. The main route for AMR acquisition in clinically important bacteria is the horizontal transfer of plasmids carrying resistance genes. AMR plasmids allow bacteria to survive antibiotics, but they also entail physiological alterations in the host cell. Multiple studies over the last few years have indicated that these alterations can translate into a fitness cost when antibiotics are absent. However, due to technical limitations, most of these studies are based on analysing new associations between plasmids and bacteria generated in vitro, and we know very little about the effects of plasmids in their native bacterial hosts. In this study, we used a CRISPR-Cas9-tool to selectively cure plasmids from clinical enterobacteria to overcome this limitation. Using this approach, we were able to study the fitness effects of the carbapenem resistance plasmid pOXA-48 in 35 pOXA-48-carrying isolates recovered from hospitalized patients. Our results revealed that pOXA-48 produces variable effects across the collection of wild-type enterobacterial strains naturally carrying the plasmid, ranging from fitness costs to fitness benefits. Importantly, the plasmid was only associated with a significant fitness reduction in four out of 35 clones, and produced no significant changes in fitness in the great majority of isolates. Our results suggest that plasmids produce neutral fitness effects in most native bacterial hosts, helping to explain the great prevalence of plasmids in natural microbial communities.

Identification of atrial-enriched lncRNA Walras linked to cardiomyocyte cytoarchitecture and atrial fibrillation.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia in humans. Genetic and genomic analyses have recently demonstrated that the homeobox transcription factor Pitx2 plays a fundamental role regulating expression of distinct growth factors, microRNAs and ion channels leading to morphological and molecular alterations that promote the onset of AF. Here we address the plausible contribution of long non-coding (lnc)RNAs within the Pitx2>Wnt>miRNA signaling pathway. In silico analyses of annotated lncRNAs in the vicinity of the Pitx2, Wnt8 and Wnt11 chromosomal loci identified five novel lncRNAs with differential expression during cardiac development. Importantly, three of them, Walaa, Walras, and Wallrd, are evolutionarily conserved in humans and displayed preferential atrial expression during embryogenesis. In addition, Walrad displayed moderate expression during embryogenesis but was more abundant in the right atrium. Walaa, Walras and Wallrd were distinctly regulated by Pitx2, Wnt8, and Wnt11, and Wallrd was severely elevated in conditional atrium-specific Pitx2-deficient mice. Furthermore, pro-arrhythmogenic and pro-hypertrophic substrate administration to primary cardiomyocyte cell cultures consistently modulate expression of these lncRNAs, supporting distinct modulatory roles of the AF cardiovascular risk factors in the regulation of these lncRNAs. Walras affinity pulldown assays revealed its association with distinct cytoplasmic and nuclear proteins previously involved in cardiac pathophysiology, while loss-of-function assays further support a pivotal role of this lncRNA in cytoskeletal organization. We propose that lncRNAs Walaa, Walras and Wallrd, distinctly regulated by Pitx2>Wnt>miRNA signaling and pro-arrhythmogenic and pro-hypertrophic factors, are implicated in atrial arrhythmogenesis, and Walras additionally in cardiomyocyte cytoarchitecture.

Full-mouth rehabilitation in a completely digital workflow using partially adhesive monolithic zirconia restorations.

OBJECTIVE: This clinical case describes a completely digital workflow using current digital technologies for a full-mouth adhesive rehabilitation with ultra-translucent multilayer zirconia restorations. CLINICAL CONSIDERATIONS: A healthy 60-year-old man with abfractions on all upper and lower molars and severe tooth wear underwent a full-mouth rehabilitation with laminate veneers and partial adhesive restorations. A proper zirconia bonding protocol was realized to provide a durable bond between the ultra-translucent zirconia and the resin cement. Furthermore, the implementation of a digital workflow enables the clinician to have an effective communication during treatment planning and simplify the clinical and laboratory procedures, providing the patient with long-term esthetic treatment and functional results. CONCLUSION: The implementation of a completely digital workflow and the use of ultra-translucent multilayer zirconia for indirect adhesive restorations can be an alternative with simplified and predictable procedures for patients with dental wear and teeth discolorations. CLINICAL SIGNIFICANCE: The digital workflow described is intended to facilitate the planning and execution of a full-mouth adhesive rehabilitation and demonstrates to clinicians a reliable zirconia bonding concept for minimally invasive anterior and posterior restorations.

Complexities underlying the breeding and deployment of Dutch elm disease resistant elms.

Dutch elm disease (DED) is a vascular wilt disease caused by the pathogens Ophiostoma ulmi and Ophiostoma novo-ulmi with multiple ecological phases including pathogenic (xylem), saprotrophic (bark) and vector (beetle flight and beetle feeding wound) phases. Due to the two DED pandemics during the twentieth century the use of elms in landscape and forest restoration has declined significantly. However new initiatives for elm breeding and restoration are now underway in Europe and North America. Here we discuss complexities in the DED 'system' that can lead to unintended consequences during elm breeding and some of the wider options for obtaining durability or 'field resistance' in released material, including (1) the phenotypic plasticity of disease levels in resistant cultivars infected by O. novo-ulmi; (2) shortcomings in test methods when selecting for resistance; (3) the implications of rapid evolutionary changes in current O. novo-ulmi populations for the choice of pathogen inoculum when screening; (4) the possibility of using active resistance to the pathogen in the beetle feeding wound, and low attractiveness of elm cultivars to feeding beetles, in addition to resistance in the xylem; (5) the risk that genes from susceptible and exotic elms be introgressed into resistant cultivars; (6) risks posed by unintentional changes in the host microbiome; and (7) the biosecurity risks posed by resistant elm deployment. In addition, attention needs to be paid to the disease pressures within which resistant elms will be released. In the future, biotechnology may further enhance our understanding of the various resistance processes in elms and our potential to deploy trees with highly durable resistance in elm restoration. Hopefully the different elm resistance processes will prove to be largely under durable, additive, multigenic control. Elm breeding programmes cannot afford to get into the host-pathogen arms races that characterise some agricultural host-pathogen systems.

PyToxo: a Python tool for calculating penetrance tables of high-order epistasis models.

BACKGROUND: Epistasis is the interaction between different genes when expressing a certain phenotype. If epistasis involves more than two loci it is called high-order epistasis. High-order epistasis is an area under active research because it could be the cause of many complex traits. The most common way to specify an epistasis interaction is through a penetrance table. RESULTS: This paper presents PyToxo, a Python tool for generating penetrance tables from any-order epistasis models. Unlike other tools available in the bibliography, PyToxo is able to work with high-order models and realistic penetrance and heritability values, achieving high-precision results in a short time. In addition, PyToxo is distributed as open-source software and includes several interfaces to ease its use. CONCLUSIONS: PyToxo provides the scientific community with a useful tool to evaluate algorithms and methods that can detect high-order epistasis to continue advancing in the discovery of the causes behind complex diseases.

Regulation of Epicardial Cell Fate during Cardiac Development and Disease: An Overview.

The epicardium is the outermost cell layer in the vertebrate heart that originates during development from mesothelial precursors located in the proepicardium and septum transversum. The epicardial layer plays a key role during cardiogenesis since a subset of epicardial-derived cells (EPDCs) undergo an epithelial-mesenchymal transition (EMT); migrate into the myocardium; and differentiate into distinct cell types, such as coronary vascular smooth muscle cells, cardiac fibroblasts, endothelial cells, and presumably a subpopulation of cardiomyocytes, thus contributing to complete heart formation. Furthermore, the epicardium is a source of paracrine factors that support cardiac growth at the last stages of cardiogenesis. Although several lineage trace studies have provided some evidence about epicardial cell fate determination, the molecular mechanisms underlying epicardial cell heterogeneity remain not fully understood. Interestingly, seminal works during the last decade have pointed out that the adult epicardium is reactivated after heart damage, re-expressing some embryonic genes and contributing to cardiac remodeling. Therefore, the epicardium has been proposed as a potential target in the treatment of cardiovascular disease. In this review, we summarize the previous knowledge regarding the regulation of epicardial cell contribution during development and the control of epicardial reactivation in cardiac repair after damage.

Influence of earthworms on the microbial properties and extracellular enzyme activities during vermicomposting of raw and distilled grape marc.

The treatment of winery wastes by using appropriate management technologies is of utmost need in order to reduce to a minimum their disposal and avoid negative environmental impacts. This is of particular interest for grape marc, the main solid by-product of the winery industry. However, comparative studies on a pilot-scale dealing with the impact of earthworms on marc derived from both red and white grape varieties during vermicomposting are still scarce. The present study sought to evaluate the changes in the biochemical and microbiological properties of red and white raw marc in the presence and the absence of the earthworm species Eisenia andrei. The distilled marc obtained through distillation of the red grape marc was also considered under this scenario. Samples were taken after 14, 28, 42, and 63 days of vermicomposting. On day 14 earthworms led to a pronounced increase in most of the enzymatic activities, but only in those vermireactors fed with raw marc from the red grape variety. Alfa- and beta-glucosidase as well as chitinase and leucine-aminopeptidase activities were between 3 to 5-times higher relative to the control, while alkaline phosphomonoesterase was even up to 14-fold higher with earthworm presence. From day 28 onwards the magnitude of earthworms' effect on the studied enzymes was also dependent on the type of grape marc. Reduced values of basal respiration, ranging between 200 and 350 mg CO2 kg OM h-1 and indicative of stabilized materials were found in the resulting vermicomposts. Moreover, the content of macro- and micronutrients in the end products matched with those considered to have the quality criteria of a good vermicompost. Altogether, these findings reinforce the effectiveness of vermicomposting for the biological stabilization of grape marc with the dual purpose of fertilizer production and environmental protection.

Changes in the microbiota during biological treatment of palm oil mill waste: A critical review.

Palm oil mill waste has a complex cellulosic structure, is rich in nutrients, and provides a habitat for diverse microbial communities. Current research focuses on how the microbiota and organic components interact during the degradation of this type of waste. Some recent studies have described the microbial communities present in different biodegradation processes of palm oil mill waste, identifying the dominant bacteria/fungi responsible for breaking down the cellulosic components. However, understanding the degradation process's mechanisms is vital to eliminating the need for further pretreatment of lignocellulosic compounds in the waste mixture and facilitating the commercialization of palm oil mill waste treatment technology. Thus, the present work aims to review microbial community dynamics via three biological treatment systems comprehensively: composting, vermicomposting, and dark fermentation, to understand how inspiration from nature can further enhance existing degradation processes. The information presented could be used as an umbrella to current research on biological treatment processes and specific research on the bioaugmentation of indigenous microbial consortia isolated during the biological degradation of palm oil mill waste.

Differential Gene Expression Induced by Acute Exposure to Water Accommodated Fraction (WAF) and Chemically Enhanced WAF (CEWAF) of Light Crude Oil and Nokomis 3-F4 in Limulus polyphemus Larvae.

In 2018 we evaluated at 48 h and 96 h, the gene expression profile of larvae of Limulus polyphemus exposed to 10% and 100% of water-accommodated fraction (WAF) of light crude oil (API 35), and 10% and 100% of a chemically enhanced WAF (CEWAF) with the dispersant Nokomis 3-F4® in a static-acute (96 h) bioassay. Alkanes and PAHs concentrations were higher in CEWAF than in WAF stock solutions. Under the proved conditions, the expression profile of genes associated to detoxification processes (glutathione S-transferase and glutathione peroxidase), stress (heat shock protein), innate immunity (tumor necrosis factor receptor-associated factor 4 traf4), cell death (apoptosis inhibitor 5) and DNA repairing (E3 ubiquitin protein ligase), showed a deregulation at 48 h followed by an upregulation at 96 h, with exception of glutathione peroxidase, heat shock protein and innate immunity that remained low in CEWAF. In conclusion, by using genes that have been proposed as biomarkers to pollutants exposure, L. polyphemus larvae showed an early activation of genes related to the immune system, antioxidant, heat shock and NER.

Therapeutic Values of Earthworm Species Extract from Azad Kashmir as Anticoagulant, Antibacterial, and Antioxidant Agents.

AIMS: Current research aimed to explore the therapeutic values of different earthworms as antibacterial, anticoagulant, and antioxidant agents. METHODS: Ten different earthworms, i.e., Amynthas corticis, Amynthas gracilis, Pheretima posthuma, Eisenia fetida, Aporrectodea rosea, Allolobophora chlorotica, Aporrectodea trapezoides, Polypheretima elongata, Aporrectodea caliginosa, and Pheretima hawayana, were collected and screened for biological activities. Antibacterial effect analysis of earthworm species was done against fourteen bacterial pathogens, i.e., Escherichia coli, Serratia marcescens, Streptococcus pyogenes, Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa (1), Salmonella typhimurium, Shigella flexneri, Enterobacter amnigenus, Serratia odorifera, Pseudomonas aeruginosa (2), Staphylococcus warneri, and Lactobacillus curvatus, via agar well diffusion, crystal violet, MTT, agar disc diffusion, and direct bioautography assays. Antioxidant potential was evaluated through ABTS and DPPH assays. Lipolytic, proteolytic, and amylolytic assays were done for lipase, protease, and amylase enzymes confirmation. In vitro anticoagulant effects were examined in the blood samples by measuring prothrombin time. RESULTS: Results revealed that all earthworm extracts showed the inhibition of all tested bacterial pathogens except P. aeruginosa (1), P. aeruginosa (2), S. warneri, and L. curvatus. The maximum zone of inhibition of E. coli was recorded as 14.66 ± 0.57 mm by A. corticis, 25.0 ± 0.0 mm by P. posthuma, 20.0 ± 0.0 mm by E. fetida, and 20.0 ± 0.0 mm by A. trapezoid. Cell proliferation, biofilm inhibition, the synergistic effect of extracts along with antibiotics, and direct bioautography supported the results of agar well diffusion assay. Similarly, P. hawayana, A. corticis, A. caliginosa, and A. trapezoids increase the prothrombin time more efficiently compared to other earthworms. A. corticis, A. gracilis, A. rosea, A. chlorotica, P. elongata, and A. trapezoides showed maximum DPPH scavenging potential effect. CONCLUSIONS: The coelomic fluid of earthworms possessed several bioactive compounds/enzymes/antioxidants that play an important role in the bacterial inhibition and act as anticoagulant agents. Therefore, the development of new therapeutic drugs from invertebrates could be effective and potential for the prevention of the emergence of multidrug-resistant bacteria.

Embryonic Tbx3+ cardiomyocytes form the mature cardiac conduction system by progressive fate restriction.

A small network of spontaneously active Tbx3+ cardiomyocytes forms the cardiac conduction system (CCS) in adults. Understanding the origin and mechanism of development of the CCS network are important steps towards disease modeling and the development of biological pacemakers to treat arrhythmias. We found that Tbx3 expression in the embryonic mouse heart is associated with automaticity. Genetic inducible fate mapping revealed that Tbx3+ cells in the early heart tube are fated to form the definitive CCS components, except the Purkinje fiber network. At mid-fetal stages, contribution of Tbx3+ cells was restricted to the definitive CCS. We identified a Tbx3+ population in the outflow tract of the early heart tube that formed the atrioventricular bundle. Whereas Tbx3+ cardiomyocytes also contributed to the adjacent Gja5+ atrial and ventricular chamber myocardium, embryonic Gja5+ chamber cardiomyocytes did not contribute to the Tbx3+ sinus node or to atrioventricular ring bundles. In conclusion, the CCS is established by progressive fate restriction of a Tbx3+ cell population in the early developing heart, which implicates Tbx3 as a useful tool for developing strategies to study and treat CCS diseases.

Gut microbiome and telomere length in gull hatchlings.

In many animals, recent evidence indicates that the gut microbiome may be acquired during early development, with possible consequences on newborns' health. Thus, it has been hypothesized that a healthy microbiome protects telomeres and genomic integrity against cellular stress. However, the link between the early acquired microbiome and telomere dynamics has not hitherto been investigated. In birds, this link may also be potentially modulated by the transfer of maternal glucocorticoids, since these substances dysregulate microbiome composition during postnatal development. Here, we examined the effect of the interplay between the microbiome and stress hormones on the telomere length of yellow-legged gull hatchlings by using a field experiment in which we manipulated the corticosterone content in eggs. We found that the hatchling telomere length was related to microbiome composition, but this relationship was not affected by the corticosterone treatment. Hatchlings with a microbiome dominated by potential commensal bacteria (i.e. Catellicoccus and Cetobacterium) had larger telomeres, suggesting that an early establishment of the species-specific microbiome during development may have important consequences on offspring health and survival.

Toxo: a library for calculating penetrance tables of high-order epistasis models.

BACKGROUND: Epistasis is defined as the interaction between different genes when expressing a specific phenotype. The most common way to characterize an epistatic relationship is using a penetrance table, which contains the probability of expressing the phenotype under study given a particular allele combination. Available simulators can only create penetrance tables for well-known epistasis models involving a small number of genes and under a large number of limitations. RESULTS: Toxo is a MATLAB library designed to calculate penetrance tables of epistasis models of any interaction order which resemble real data more closely. The user specifies the desired heritability (or prevalence) and the program maximizes the table's prevalence (or heritability) according to the input epistatic model boundaries. CONCLUSIONS: Toxo extends the capabilities of existing simulators that define epistasis using penetrance tables. These tables can be directly used as input for software simulators such as GAMETES so that they are able to generate data samples with larger interactions and more realistic prevalences/heritabilities.

Local adaptation fuels cryptic speciation in terrestrial annelids.

Uncovering the genetic and evolutionary basis of cryptic speciation is a major focus of evolutionary biology. Next Generation Sequencing (NGS) allows the identification of genome-wide local adaptation signatures, but has rarely been applied to cryptic complexes - particularly in the soil milieu - as it is the case with integrative taxonomy. The earthworm genus Carpetania, comprising six previously suggested putative cryptic lineages, is a promising model to study the evolutionary phenomena shaping cryptic speciation in soil-dwelling lineages. Genotyping-By-Sequencing (GBS) was used to provide genome-wide information about genetic variability between 17 populations, and geometric morphometrics analyses of genital chaetae were performed to investigate unexplored cryptic morphological evolution. Genomic analyses revealed the existence of three cryptic species, with half of the previously-identified potential cryptic lineages clustering within them. Local adaptation was detected in more than 800 genes putatively involved in a plethora of biological functions (most notably reproduction, metabolism, immunological response and morphogenesis). Several genes with selection signatures showed shared mutations for each of the cryptic species, and genes under selection were enriched in functions related to regulation of transcription, including SNPs located in UTR regions. Finally, geometric morphometrics approaches partially confirmed the phylogenetic signal of relevant morphological characters such as genital chaetae. Our study therefore unveils that local adaptation and regulatory divergence are key evolutionary forces orchestrating genome evolution in soil fauna.

Non-coding RNAs and Atrial Fibrillation.

Atrial fibrillation is the most frequent type of cardiac arrhythmia in humans, with an estimate incidence of 1-2% in the general population, rising up to 8-10% in the elderly. Cardiovascular risk factors such as diabetes, obesity, hypertension and hyperthyroidism can increase the occurrence of AF. The onset of AF triggers additional AF episodes, leading to structural and electrical remodeling of the diseased heart. Understanding the molecular bases of atrial fibrillation have greatly advance over the last decade demonstrating a pivotal role of distinct ion channels in AF pathophysiology. A new scenario has opened on the understanding of the molecular mechanisms underlying AF, with the discovery of non-coding RNAs and their wide implication in multiple disease states, including cardiac arrhythmogenic pathologies. microRNAs are small non-coding RNAs of 22-24 nucleotides that are capable of regulating gene expression by interacting with the mRNA transcript 3'UTRs and promoting mRNA degradation and/or protein translation blockage. Long non-coding RNAs are a more diverse group of non-coding RNAs, providing transcriptional and post-transcriptional roles and subclassified according to their functional properties. In this chapter we summarized current state-of-the-art knowledge on the functional of microRNAs and long non-coding RNAs as well as their cross-talk regulatory mechanisms in atrial fibrillation.

Unraveling the environmental impacts of bioactive compounds and organic amendment from grape marc.

In a society that produces large amounts of solid waste, the search for new methods of valorisation has led to the development of techniques that make it possible to obtain new products from waste. In the case of bio-waste, biological treatment such as anaerobic digestion or composting appear to be suitable options for producing bio-energy or bio-fertilizers respectively. Vermicomposting is a method of converting solid organic waste into resources through bio-oxidation and stabilization of the organic waste by earthworms. The purpose of this study is to establish the environmental impacts of a complete route for the valorisation of grape pomace in order to identify environmental hotspots. In this valorisation route, different value-added products are produced with potential application in the cosmetic, food and pharmaceutical sectors. Priority was given to the use of primary data in the elaboration of the data inventories needed to perform the life cycle assessment (LCA). The main findings from this study reported that the energy requirement of the distillation process is an important hot spot of the process. Although the valorisation route has some poor results in terms of the two environmental indicators (carbon footprint and normalised impact index), when economic revenues were included in this analysis, its environmental performance was better than that of other alternatives for bio-waste recovery.

Strategies for recycling and valorization of grape marc.

Grapes are one of the most cultivated fruit crops worldwide. Either for wine or juice production, grape processing generates a large amount of residues that must be treated, disposed of or reused properly to reduce their pollution load before being applied to the soil. In this review, a special focus is given to the treatment and valorization of the winemaking by-product like grape marc via anaerobic digestion, composting and vermicomposting at laboratory, pilot, and industrial scales. The impact of the final products (digestates, composts, and vermicomposts) on soil properties is briefly addressed. Moreover, the role of grape marc and seeds as a valuable source of natural phytochemicals that include polyphenols and other bioactive compounds of interest for pharmaceutical, cosmetic, and food industries is also discussed. This is of paramount importance given the fact that sustainability requires the use of management and valorization strategies that allow the recovery of valuable compounds (e.g. antioxidants) with minimum disposal of waste streams.

Exploring the potential enzymatic bioremediation of vermicompost through pesticide-detoxifying carboxylesterases.

Vermicompost is a known biofertilizer of potential use in soil bioremediation. This study was undertaken to explore the capacity of grape marc-derived vermicompost to inactivate methyl carbamate (MC) and organophosphorus (OP) pesticides via exploring the carboxylesterase (CE) activity level and its response to pesticide exposure. We first optimized the method for enzyme activity assay comparing the CE activity in two contrasting homogenization procedures (30-min mixing and mortar grinding). Thereafter, we assessed the sensitivity of the enzyme by both in vitro and vermicompost incubation trials with selected pesticides. The main findings can be summarized as follows: i) grinding the vermicompost in water (2% w/v) yielded maximum enzyme activity; ii) at concentrations around 10-4 M, highly toxic oxygen-analog metabolites of OPs strongly inhibited the CE activity (76-93% inhibition), but MC did not inhibit the enzyme activity; iii) liquid vermicompost was able to degrade chlorpyrifos and inactivate its highly toxic metabolite chlorpyrifos-oxon. Our results suggest that liquid vermicompost is the most appropriate preparation to increase the enzymatic potential of vermicompost in pesticide-contaminated soils.