Biology subjects

Calahorro, F.

Publications and source records attributed to Calahorro, F..

7 recordsLinked to original sources

bioRxiv2026

Potential nematicides identified through targeted in vivo screening of a 5-HT-gated chloride channel, MOD-1

New approaches to mitigate the reduction of crop yields by plant parasitic nematodes are needed in the face of increasing concerns of the impact of nematicides on precious ecosystems. One approach is to target receptors in the parasitic nematode that are vital for their survival that less widely expressed in non-target organisms. Nematodes express a phylogenetically restricted 5-HT-gated chloride channel, MOD-1, activation of which causes paralysis in Caenorhabditis elegans. We show that MOD-1 is expressed in the motor nervous system of the plant parasitic nematode Globodera pallida and its functional characterisation is validated by 5-HT activation when reconstituted in Xenopus laevis oocytes. To evaluate MOD-1 as a nematicide target we utilised a previously described platform called PhaGeM4 for PharmacoGenetic targeting of M4 neurone in which MOD-1 is expressed in transgenic C. elegans and nematode development in the face of MOD-1 chemical modulation is tracked. We screened Pathogen Box, a chemical library of 400 diverse drug-like molecules, using PhaGeM4. This identified 10 putative hits for C. elegans MOD-1. These hits were pursued through a sequential, iterative pipeline encompassing mod-1 dependent C. elegans motility and G.pallida motility assays in combination with pharmacological interrogation of G. pallida mod-1 in PhaGeM4. This approach highlights 3 compounds with a mod-1 dependent action (quipazine, our benchmark compound; MMV687251, a vancomycin-like compound; MMV688774, an antifungal with common name posaconazole) and one compound that acts through an undetermined target (MMV002816, also known as the antifilarial drug, diethylcarbamazine). Each of these compounds had a significant inhibitory effect on G. pallida J2 root invasion. Overall, this lends confidence that the PhaGeM4 screening platform can delivery new chemical leads for crop protection and highlights four new chemistries of interest. More generally, this approach could be applied to other ligand-gated ion channels of interest as targets.

pharmacology and toxicology↗

bioRxiv2025

Binding Affinity Ranking at the Molecular Initiating Event (BARMIE): An open-source computational pipeline for ecological hazard ranking of endocrine disrupting chemicals.

A challenge in ecological risk assessment is identifying the chemicals that pose the greatest threat and determining which species are most vulnerable to them. To help address this, this study has developed an in-silico open-source tool called BARMIE (Binding Affinity Ranking at the Molecular Initiating Event) to rapidly predict the chemical binding affinity of steroid receptor proteins to synthetic steroids to identify potentially vulnerable species and chemicals of concern. BARMIE was used to screen 163 teleost fish glucocorticoid receptors (GRs) for binding to the natural ligand cortisol and to 10 synthetic glucocorticoid drugs (GCs) designed to interact within the ligand-binding pocket (LBP) of GRs. BARMIE identified species from the superorder Protacanthopterygii with high-affinity GRs to synthetic GCs (e.g. vulnerable species). . BARMIE was also used to screen binding profiles of compounds in the Medicine for Malaria Venture Global Health Priority Box to rainbow trout GRs (rtGR1 and rtGR2). Of the 178 compounds, 24 and 36 bind within the LBP of rtGR1 and rtGR2, respectively. For 30 of these compounds, transactivation activity was assessed at 1{micro}M in the presence or absence of 1{micro}M cortisol and confirmed 2 compounds with agonistic properties (e.g. chemicals of concern) that would require further in vitro and/or in vivo studies to assess the environmental risk. BARMIE can rapidly generate predicted binding affinities for 100s of species and chemicals as a first screen in environmental risk assessment to provide information on which substances to prioritise in downstream tests.

pharmacology and toxicology↗

bioRxiv2021

Confounds of using the unc-58 selection marker highlights the importance of genotyping co-CRISPR genes

Multiple advances have been made to increase the efficiency of CRISPR/Cas9 editing using the model genetic organism Caenorhabditis elegans (C. elegans). Here we report on the use of co-CRISPR marker genes: worms in which co-CRISPR events have occurred have overt, visible phenotypes which facilitates the selection of worms that harbour CRISPR events in the target gene. Mutation in the co-CRISPR gene is then removed by outcrossing to wild type but this can be challenging if the CRISPR and co-CRISPR gene are hard to segregate. However, outcrossing can be avoided by selecting worms of wild type appearance from a jackpot brood. These are broods in which a high proportion of the progeny of a single injected worm display the co-CRISPR phenotype suggesting high CRISPR efficiency. This can deliver worms that harbour the desired mutation in the target gene locus without the co-CRISPR mutation. We have successfully generated a discrete mutation in the C. elegans nlg-1 gene using this method. However, in the process of sequencing to authenticate editing in the nlg-1 gene we discovered genomic rearrangements that arise at the co-CRISPR gene unc-58 that by visual observation were phenotypically silent but nonetheless resulted in a significant reduction in motility scored by thrashing behaviour. This highlights that careful consideration of the hidden consequences of co-CRISPR mediated genetic changes should be taken before downstream analysis of gene function. Given this, we suggest sequencing of co-CRISPR genes following CRISPR procedures that utilise phenotypic selection as part of the pipeline.

genetics↗

bioRxiv2020

Investigating autism associated genes in C. elegans reveals candidates with a role in social behaviour

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterised by a triad of behavioural impairments and includes disruption in social behaviour. ASD has a clear genetic underpinning and hundreds of genes are implicated in its aetiology. However, how single penetrant genes disrupt activity of neural circuits which lead to affected behaviours is only beginning to be understood and less is known about how low penetrant genes interact to disrupt emergent behaviours. Investigations are well served by experimental approaches that allow tractable investigation of the underpinning genetic basis of circuits that control behaviours that operate in the biological domains that are neuro-atypical in autism. The model organism C. elegans provides an experimental platform to investigate the effect of genetic mutations on behavioural outputs including those that impact social biology. Here we use progeny-derived social cues that modulate C. elegans food leaving to assay genetic determinants of social behaviour. We used the SAFRI Gene database to identify C. elegans orthologues of human ASD associated genes. We identified a number of mutants that displayed selective deficits in response to progeny. The genetic determinants of this complex social behaviour highlight the important contribution of synaptopathy and implicates genes within cell signalling, epigenetics and phospholipid metabolism functional domains. The approach overlaps with a growing number of studies that investigate potential molecular determinants of autism in C. elegans. However, our use of a complex, sensory integrative, emergent behaviour provides routes to enrich new or underexplored biology with the identification of novel candidate genes with a definable role in social behaviour.

animal behavior and cognition↗

bioRxiv2020

Neuroligin dependence of social behaviour in C. elegans provides a model to investigate an autism associated gene

Autism spectrum disorder (ASD) is characterised by a triad of behavioural impairments including social behaviour. Neuroligin, a trans-synaptic adhesion molecule, has emerged as a penetrant genetic determinant of behavioural traits that signature the neuroatypical behaviours of autism. However, the function of neuroligin in social circuitry and the impact of genetic variation to this gene is not fully understood. Indeed, in animal studies designed to model autism there remains controversy regarding the role of neuroligin dysfunction in the expression of disrupted social behaviours. The model organism, C. elegans, offers an informative experimental platform to investigate the impact of genetic variants on social behaviour. In a number of paradigms it has been shown that inter-organismal communication by chemical cues regulates C. elegans social behaviour. We utilise this social behaviour to investigate the effect of autism associated genetic variants within the social domain of the research domain criteria. We have identified neuroligin as an important regulator of social behaviour and segregate the importance of this gene to the recognition and/or processing of social cues. We also use CRISPR/Cas9 to edit an R-C mutation that mimics a highly penetrant human mutation associated with autism. C. elegans carrying this mutation phenocopy the behavioural dysfunction of a C. elegans neuroligin null mutant, thus confirming its significance in the regulation of animal social biology. This highlights that quantitative behaviour and precision genetic intervention can be used to manipulate discrete social circuits of the worm to provide further insight to complex social behaviour.

neuroscience↗

bioRxiv2018

Identification and characterisation of serotonin signalling in the potato cyst nematode Globodera pallida reveals new targets for crop protection

Plant parasitic nematodes are microscopic pests that invade plant roots and cause extensive damage to crops worldwide. To investigate mechanisms underpinning their parasitic behaviour we used a chemical biology approach: We discovered that reserpine, a plant alkaloid known for its antagonism of the mammalian vesicular monoamine transporter VMAT and ability to impart a global depletion of synaptic biogenic amines in the nervous system, potently impairs the ability of the potato cyst nematode Globodera pallida to enter the host plant root. We show that this effect of reserpine is mediated by an inhibition of serotonergic signalling that is essential for activation of the stylet, a lance-like organ that protrudes from the mouth of the worm and which is used to pierce the host root to gain access. Prompted by this we identified core molecular components of G. pallida serotonin signalling encompassing the target of reserpine, VMAT; the synthetic enzyme for serotonin, tryptophan hydroxylase; the G protein coupled receptor SER-7 and the serotonin-gated chloride channel MOD-1. We found that inhibitors of tryptophan hydroxylase, SER-7 and MOD-1 phenocopy the plant protecting action of reserpine. Thus targeting the serotonin signalling pathway presents a promising new route to control plant parasitic nematodes.\n\nSummaryIndian snakeroot, an herbal medicine prepared from the roots of the shrub Rauwolfia serpentina, has been used for centuries for its calming action. The major active constituent is reserpine which works by depleting a specific class of mood regulating chemical in the brain, the biogenic amines. We have discovered a remarkable effect of reserpine on a pest of global concern, the plant parasitic nematodes. These microscopic worms invade the roots of crops presenting a severe threat to food production. We show that reserpine disables serotonin signalling in the worms brain that regulates the rhythmic thrusting of the stylet: a lance-like structure that protrudes from its mouth to pierce the plant root and which is essential to its parasitic lifecycle. Thus, reserpine joins nicotine as another intriguing example of Nature evolving its own protection against pests. We have identified key components of the serotonin signalling pathway in the potato cyst nematode Globodera pallida and show that chemicals that target these sites inhibit the ability of the nematode to invade its host plant. We conclude that biogenic amine transmitters are intimately involved in the worms parasitic behaviour and provide a new discrete route to crop protection.

pharmacology and toxicology↗

bioRxiv2018

Neuroligin dependence of pharyngeal pumping reveals an extrapharyngeal modulation of Caenorhabditis elegans feeding.

The integration of distinct sensory modalities is essential for behavioural decision making. In C. elegans this process is coordinated by neural circuits that integrate sensory cues from the environment to generate an appropriate behaviour at the appropriate output muscles. Food is a multimodal cue that impacts on the microcircuits to modulating feeding and foraging drivers at the level of the pharyngeal and body wall muscle respectively. When food triggers an upregulation in pharyngeal pumping it allows the effective ingestion of food. Here we show that a C. elegans mutant in the single orthologous gene of human neuroligins, nlg-1 are defective in food induced pumping. This is not explained by an inability to sense food, as nlg-1 mutants are not defective in chemotaxis towards bacteria. In addition, we show that neuroligin is widely expressed in the nervous system including AIY, ADE, ALA, URX and HSN neurones. Interestingly, despite the deficit in pharyngeal pumping neuroligin is not expressed within the pharyngeal neuromuscular network, which suggests an extrapharyngeal regulation of this circuit. We resolve electrophysiologically the neuroligin contribution to the pharyngeal circuit by mimicking a food-dependent pumping, and show that the nlg-1 phenotype is similar to mutants impaired in GABAergic and/or glutamatergic signalling. We suggest that neuroligin organizes extrapharyngeal circuits that regulate the pharynx. These observations based on the molecular and cellular determinants of feeding are consistent with the emerging role of neuroligin in discretely impacting functional circuits underpinning complex behaviours.

neuroscience↗