Papers by Camila Esguerra
Natural products still represent an important source of new molecules for drug discovery. Neverth... more Natural products still represent an important source of new molecules for drug discovery. Nevertheless, isolation procedures are time-consuming and high-throughput activity tests are often not adapted for the screening of natural products. The use of microfractionation and sensitive NMR methods as microflow NMR shortens the isolation process and reduces sample amounts needed. Recently, zebrafish have been established as a powerful bioassay platform for a number of indication areas. The methanolic extract of Rhynchosia viscosa - traditionally used by Tanzanian healers to treat skin rashes and insect bites - revealed anti-inflammatory as well as anti-angiogenic activity during the zebrafish-based screening of East African medicinal plants. With UHPLC-TOFMS profiling, the extract was dereplicated and 20 mg of sample were subjected to microfractionation using semi-preparative LC-UV-MS. One tenth of each fraction was tested in zebrafish for bioactivity, revealing four fractions with anti...
Epilepsy & behavior : E&B, Jan 2, 2015
Treatment-resistant seizures affect about a third of patients suffering from epilepsy. To fulfill... more Treatment-resistant seizures affect about a third of patients suffering from epilepsy. To fulfill the need for new medications targeting treatment-resistant seizures, a number of rodent models offer the opportunity to assess a variety of potential treatment approaches. The use of such models, however, has proven to be time-consuming and labor-intensive. In this study, we performed pharmacological characterization of the allylglycine (AG) seizure model, a simple in vivo model for which we demonstrated a high level of treatment resistance. (d,l)-Allylglycine inhibits glutamic acid decarboxylase (GAD) - the key enzyme in γ-aminobutyric acid (GABA) biosynthesis - leading to GABA depletion, seizures, and neuronal damage. We performed a side-by-side comparison of mouse and zebrafish acute AG treatments including biochemical, electrographic, and behavioral assessments. Interestingly, seizure progression rate and GABA depletion kinetics were comparable in both species. Five mechanistically ...
Developmental dynamics : an official publication of the American Association of Anatomists, 2014
SMOC2 is a member of the BM-40 (SPARC) family of matricellular proteins, reported to influence si... more SMOC2 is a member of the BM-40 (SPARC) family of matricellular proteins, reported to influence signaling in the extracellular compartment. In mice, Smoc2 is expressed in many different tissues and was shown to enhance the response to angiogenic growth factors, mediate cell adhesion, keratinocyte migration, and metastasis. Additionally, SMOC2 is associated with vitiligo and craniofacial and dental defects. The function of Smoc2 during early zebrafish development has not been determined to date. In pregastrula zebrafish embryos, smoc2 is expressed ubiquitously. As development progresses, the expression pattern becomes more anteriorly restricted. At the onset of blood cell circulation, smoc2 morphants presented a mild ventralization of posterior structures. Molecular analysis of the smoc2 morphants indicated myelopoietic defects in the rostral blood islands during segmentation stages. Hemangioblast development and further specification of the myeloid progenitor cells were shown to be i...
Journal of Applied Toxicology, 2015
Epilepsy & behavior : E&B, 2012
Turmeric, obtained from the rhizomes of Curcuma longa, is used in South Asia as a traditional med... more Turmeric, obtained from the rhizomes of Curcuma longa, is used in South Asia as a traditional medicine for the treatment of epilepsy. To date, in vivo studies on the anticonvulsant activity of turmeric have focused on its principal curcuminoid, curcumin. However, poor absorption and rapid metabolism have limited the therapeutic application of curcumin in humans. To explore the therapeutic potential of turmeric for epilepsy further, we analyzed its anticonvulsant activity in a larval zebrafish seizure assay. Initial experiments revealed that the anticonvulsant activity of turmeric in zebrafish larvae cannot be explained solely by the effects of curcumin. Zebrafish bioassay-guided fractionation of turmeric identified bisabolene sesquiterpenoids as additional anticonvulsants that inhibit PTZ-induced seizures in both zebrafish and mice. Here, we present the first report of the anticonvulsant properties of bisabolene sesquiterpenoids and provide evidence which warrants further investigat...
PLoS ONE, 2012
Zebrafish are rapidly growing in popularity as an in vivo model system for chemical genetics, dru... more Zebrafish are rapidly growing in popularity as an in vivo model system for chemical genetics, drug discovery, and toxicology, and more recently also for natural product discovery. Experiments involving the pharmacological evaluation of small molecules or natural product extracts in zebrafish bioassays require the effective delivery of these compounds to embryos and larvae. While most samples to be screened are first solubilized in dimethyl sulfoxide (DMSO), which is then diluted in the embryo medium, often this method is not sufficient to prevent the immediate or eventual precipitation of the sample. Certain compounds and extracts are also not highly soluble in DMSO. In such instances the use of carriers and/or other solvents might offer an alternative means to achieve the required sample concentration. Towards this end, we determined the maximum tolerated concentration (MTC) of several commonly used solvents and carriers in zebrafish embryos and larvae at various developmental stages. Solvents evaluated for this study included acetone, acetonitrile, butanone, dimethyl formamide, DMSO, ethanol, glycerol, isopropanol, methanol, polyethylene glycol (PEG-400), propylene glycol, and solketal, and carriers included albumin (BSA) and cyclodextrin (2-hydroxypropyl-beta-cyclodextrin, or HPBCD). This study resulted in the identification of polyethylene glycol (PEG400), propylene glycol, and methanol as solvents that were relatively welltolerated over a range of developmental stages. In addition, our results showed that acetone was well-tolerated by embryos but not by larvae, and 1% cyclodextrin (HPBCD) was well-tolerated by both embryos and larvae, indicating the utility of this carrier for compound screening in zebrafish. However, given the relatively small differences (2-3 fold) between concentrations that are apparently safe and those that are clearly toxic, further studies -e.g. omics analyses -should be carried out to determine which cellular processes and signalling pathways are affected by any solvents and carriers that are used for small-molecule screens in zebrafish.
ACS Chemical Neuroscience, 2013
Danshen or Chinese red sage (Salvia miltiorrhiza, Bunge) is used by traditional Chinese medicine ... more Danshen or Chinese red sage (Salvia miltiorrhiza, Bunge) is used by traditional Chinese medicine (TCM) practitioners to treat neurological, cardiovascular, and cerebrovascular disorders and is included in some TCM formulations to control epileptic seizures. In this study, acetonic crude extracts of danshen inhibited pentylenetetrazol (PTZ)-induced seizure activity in zebrafish larvae. Subsequent zebrafish bioassay-guided fractionation of the extract resulted in the isolation of four major tanshinones, which suppressed PTZ-induced activity to varying degrees. One of the active tanshinones, tanshinone IIA, also reduced c-fos expression in the brains of PTZ-exposed zebrafish larvae. In rodent seizure models, tanshinone IIA showed anticonvulsive activity in the mouse 6-Hz psychomotor seizure test in a biphasic manner and modified seizure thresholds in a complex manner for the mouse i.v. PTZ seizure assay. Interestingly, tanshinone IIA is used as a prescription drug in China to address cerebral ischemia in patients. Here, we provide the first in vivo evidence demonstrating that tanshinone IIA has anticonvulsant properties as well.
Pharmaceutical companies are under increasing pressure to develop safe and effective drugs, while... more Pharmaceutical companies are under increasing pressure to develop safe and effective drugs, while at the same time controlling R&D expenditures. One partial solution to this challenge may be new technologies capable of generating in vivo data on the safety and efficacy of large numbers of compounds as early as possible in the drug discovery process, thereby reducing the number of drug candidates that fail in clinical development. Zebrafish offer the unique possibility to carry out high-throughput in vivo screens for compounds at the hit to lead stage, to detect both therapeutically relevant bioactivities and to assess potential toxicities. Here, we briefly highlight zebrafish assays for cardiotoxicity and hepatotoxicity.
PLoS ONE, 2013
Over the past decade, zebrafish (Danio rerio) have emerged as an attractive model for in vivo dru... more Over the past decade, zebrafish (Danio rerio) have emerged as an attractive model for in vivo drug discovery. In this study, we explore the suitability of zebrafish larvae to rapidly evaluate the anti-inflammatory activity of natural products (NPs) and medicinal plants used in traditional medicine for the treatment of inflammatory disorders. First, we optimized a zebrafish assay for leukocyte migration. Inflammation was induced in four days post-fertilization (dpf) zebrafish larvae by tail transection and co-incubation with bacterial lipopolysaccharides (LPS), resulting in a robust recruitment of leukocytes to the zone of injury. Migrating zebrafish leukocytes were detected in situ by myeloperoxidase (MPO) staining, and antiinflammatory activity was semi-quantitatively scored using a standardized scale of relative leukocyte migration (RLM). Pharmacological validation of this optimized assay was performed with a panel of anti-inflammatory drugs, demonstrating a concentration-responsive inhibition of leukocyte migration for both steroidal and non-steroidal anti-inflammatory drugs (SAIDs and NSAIDs). Subsequently, we evaluated the bioactivity of structurally diverse NPs with well-documented antiinflammatory properties. Finally, we further used this zebrafish-based assay to quantify the anti-inflammatory activity in the aqueous and methanolic extracts of several medicinal plants. Our results indicate the suitability of this LPS-enhanced leukocyte migration assay in zebrafish larvae as a front-line screening platform in NP discovery, including for the bioassayguided isolation of anti-inflammatory secondary metabolites from complex NP extracts.
PLoS ONE, 2013
Zebrafish have recently emerged as an attractive in vivo model for epilepsy. Seven-day-old zebraf... more Zebrafish have recently emerged as an attractive in vivo model for epilepsy. Seven-day-old zebrafish larvae exposed to the GABA A antagonist pentylenetetrazol (PTZ) exhibit increased locomotor activity, seizure-like behavior, and epileptiform electrographic activity. A previous study showed that 12 out of 13 antiepileptic drugs (AEDs) suppressed PTZ-mediated increases in larval movement, indicating the potential utility of zebrafish as a high-throughput in vivo model for AED discovery. However, a question remained as to whether an AED-induced decrease in locomotion is truly indicative of anticonvulsant activity, as some drugs may impair larval movement through other mechanisms such as general toxicity or sedation. We therefore carried out a study in PTZ-treated zebrafish larvae, to directly compare the ability of AEDs to inhibit seizure-like behavioral manifestations with their capacity to suppress epileptiform electrographic activity. We re-tested the 13 AEDs of which 12 were previously reported to inhibit convulsions in the larval movement tracking assay, administering concentrations that did not, on their own, impair locomotion. In parallel, we carried out open-field recordings on larval brains after treatment with each AED. For the majority of AEDs we obtained the same response in both the behavioral and electrographic assays. Overall our data correlate well with those reported in the literature for acute rodent PTZ tests, indicating that the larval zebrafish brain is more discriminatory than previously thought in its response to AEDs with different modes of action. Our results underscore the validity of using the zebrafish larval locomotor assay as a rapid firstpass screening tool in assessing the anticonvulsant and/or proconvulsant activity of compounds, but also highlight the importance of performing adequate validation when using in vivo models.
Journal of Natural Products, 2013
Chemicals targeting the liver stage (LS) of the malaria parasite are useful for causal prophylaxi... more Chemicals targeting the liver stage (LS) of the malaria parasite are useful for causal prophylaxis of malaria. In this study, four lichen metabolites, evernic acid (1), vulpic acid (2), psoromic acid (3), and (+)-usnic acid (4), were evaluated against LS parasites of Plasmodium berghei. Inhibition of P. falciparum blood stage (BS) parasites was also assessed to determine stage specificity. Compound 4 displayed the highest LS activity and stage specificity (LS IC50 value 2.3 μM, BS IC50 value 47.3 μM). The compounds 1-3 inhibited one or more enzymes (PfFabI, PfFabG, and PfFabZ) from the plasmodial fatty acid biosynthesis (FAS-II) pathway, a potential drug target for LS activity. To determine species specificity and to clarify the mechanism of reported antibacterial effects, 1-4 were also evaluated against FabI homologues and whole cells of various pathogens (S. aureus, E. coli, M. tuberculosis). Molecular modeling studies suggest that lichen acids act indirectly via binding to allosteric sites on the protein surface of the FAS-II enzymes. Potential toxicity of compounds was assessed in human hepatocyte and cancer cells (in vitro) as well as in a zebrafish model (in vivo). This study indicates the therapeutic and prophylactic potential of lichen metabolites as antibacterial and antiplasmodial agents.
Endocrinology, 2014
Exposure to appropriate levels of thyroid hormones (THs) at the right time is of key importance f... more Exposure to appropriate levels of thyroid hormones (THs) at the right time is of key importance for normal development in all vertebrates. Type 3 iodothyronine deiodinase (D3) is the prime TH-inactivating enzyme, and its expression is highest in the early stages of vertebrate development, implying that it may be necessary to shield developing tissues from overexposure to THs. We used antisense morpholino knockdown to examine the role of D3 during early development in zebrafish. Zebrafish possess 2 D3 genes, dio3a and dio3b. Here, we show that both genes are expressed during development and both contribute to in vivo D3 activity. However, dio3b mRNA levels in embryos are higher, and the effects of dio3b knockdown on D3 activity and on the resulting phenotype are more severe. D3 knockdown induced an overall delay in development, as determined by measurements of otic vesicle length, eye and ear size, and body length. The time of hatching was also severely delayed in D3-knockdown embryos. Importantly, we also observed a severe disturbance of several aspects of development. Swim bladder development and inflation was aberrant as was the development of liver and intestine. Furthermore, D3-knockdown larvae spent significantly less time moving, and both embryos and larvae exhibited perturbed escape responses, suggesting that D3 knockdown affects muscle development and/or functioning. These data indicate that D3 is essential for normal zebrafish embryonic and early larval development and show the value of morpholino knockdown in this model to further elucidate the specific role of D3 in some aspects of vertebrate development.
American journal of human genetics, Jan 7, 2013
Dravet syndrome is a severe epilepsy syndrome characterized by infantile onset of therapy-resista... more Dravet syndrome is a severe epilepsy syndrome characterized by infantile onset of therapy-resistant, fever-sensitive seizures followed by cognitive decline. Mutations in SCN1A explain about 75% of cases with Dravet syndrome; 90% of these mutations arise de novo. We studied a cohort of nine Dravet-syndrome-affected individuals without an SCN1A mutation (these included some atypical cases with onset at up to 2 years of age) by using whole-exome sequencing in proband-parent trios. In two individuals, we identified a de novo loss-of-function mutation in CHD2 (encoding chromodomain helicase DNA binding protein 2). A third CHD2 mutation was identified in an epileptic proband of a second (stage 2) cohort. All three individuals with a CHD2 mutation had intellectual disability and fever-sensitive generalized seizures, as well as prominent myoclonic seizures starting in the second year of life or later. To explore the functional relevance of CHD2 haploinsufficiency in an in vivo model system,...
Signaling by the many ligands of the TGFβ family strongly converges towards only five receptor-ac... more Signaling by the many ligands of the TGFβ family strongly converges towards only five receptor-activated, intracellular Smad proteins, which fall into two classes i.e. Smad2/3 and Smad1/5/8, respectively. These Smads bind to a surprisingly high number of Smad-interacting proteins (SIPs), many of which are transcription factors (TFs) that co-operate in Smad-controlled target gene transcription in a cell type and context specific manner. A combination of functional analyses in vivo as well as in cell cultures and biochemical studies has revealed the enormous versatility of the Smad proteins. Smads and their SIPs regulate diverse molecular and cellular processes and are also directly relevant to development and disease. In this survey, we selected appropriate examples on the BMP-Smads, with emphasis on Smad1 and Smad5, and on a number of SIPs, i.e. the CPSF subunit Smicl, Ttrap (Tdp2) and Sip1 (Zeb2, Zfhx1b) from our own research carried out in three different vertebrate models.
Molecular and Cellular Neuroscience, 1998
Motoneuronal degenerative diseases are characterized by their progressivity; once affected, the m... more Motoneuronal degenerative diseases are characterized by their progressivity; once affected, the motoneurons remain in altered states during an intermediate phase of degeneration prior to their final disappearance. Whether this survival period coincides with active metabolic rearrangements in the affected neuron remains unknown. As a first step toward the elucidation of this question, we developed cDNA pooled samples obtained from degenerating and control motoneuron mRNA populations through cellular patch sampling and RT-PCR, using the murine wobbler mutant as a model of spinal atrophy. Hybridization of the cDNA pools to various markers of intact or degenerating motoneurons allowed us to verify the cellular specificity of the patch sampling and indicated conservation of the original mRNA population complexity. Exploration of transcriptional alterations of genes encoding growth factors thought to be involved in motoneuronal development revealed that gene expression of the neurotrophin BDNF was induced in affected motoneurons, while expression of neurotrophin-3 was present in both neuronal types. Likewise, expression of a member of the epidermal growth factor (EGF) family, the neuregulin transcript sensory motor neuron-derived factor, was detected in both control and degenerating motoneurons, while transforming growth factor alpha, the functional homolog of EGF, was present only in the affected motoneurons. Immunohistochemical detection of corresponding proteins corroborated these observations. These results demonstrate that, during the course of their degeneration, motoneurons can initiate expression of novel genes which lead to the production of molecules endowed with trophic and/or differentiative properties for the neurons themselves and their glial environment. They also validate the use of the developed cDNA pooled samples for further exploration of transcriptional alterations taking place in degenerating motoneurons.
Brain : a journal of neurology, Jan 17, 2015
Photosensitivity is a heritable abnormal cortical response to flickering light, manifesting as pa... more Photosensitivity is a heritable abnormal cortical response to flickering light, manifesting as particular electroencephalographic changes, with or without seizures. Photosensitivity is prominent in a very rare epileptic encephalopathy due to de novo CHD2 mutations, but is also seen in epileptic encephalopathies due to other gene mutations. We determined whether CHD2 variation underlies photosensitivity in common epilepsies, specific photosensitive epilepsies and individuals with photosensitivity without seizures. We studied 580 individuals with epilepsy and either photosensitive seizures or abnormal photoparoxysmal response on electroencephalography, or both, and 55 individuals with photoparoxysmal response but no seizures. We compared CHD2 sequence data to publicly available data from 34 427 individuals, not enriched for epilepsy. We investigated the role of unique variants seen only once in the entire data set. We sought CHD2 variants in 238 exomes from familial genetic generalize...
Brain : a journal of neurology, Jan 17, 2015
Photosensitivity is a heritable abnormal cortical response to flickering light, manifesting as pa... more Photosensitivity is a heritable abnormal cortical response to flickering light, manifesting as particular electroencephalographic changes, with or without seizures. Photosensitivity is prominent in a very rare epileptic encephalopathy due to de novo CHD2 mutations, but is also seen in epileptic encephalopathies due to other gene mutations. We determined whether CHD2 variation underlies photosensitivity in common epilepsies, specific photosensitive epilepsies and individuals with photosensitivity without seizures. We studied 580 individuals with epilepsy and either photosensitive seizures or abnormal photoparoxysmal response on electroencephalography, or both, and 55 individuals with photoparoxysmal response but no seizures. We compared CHD2 sequence data to publicly available data from 34 427 individuals, not enriched for epilepsy. We investigated the role of unique variants seen only once in the entire data set. We sought CHD2 variants in 238 exomes from familial genetic generalize...
PLOS ONE, 2015
Dravet syndrome (DS) is one of the most pharmacoresistant and devastating forms of childhood epil... more Dravet syndrome (DS) is one of the most pharmacoresistant and devastating forms of childhood epilepsy syndromes. Distinct de novo mutations in the SCN1A gene are responsible for over 80% of DS cases. While DS is largely resistant to treatment with existing anti-epileptic drugs, promising results have been obtained in clinical trials with human patients treated with the serotonin agonist fenfluramine as an add-on therapeutic. We developed a zebrafish model of DS using morpholino antisense oligomers (MOs) targeting scn1Lab, the zebrafish ortholog of SCN1A. Zebrafish larvae with an antisense knockdown of scn1Lab (scn1Lab morphants) were characterized by automated behavioral tracking and high-resolution video imaging, in addition to measuring brain activity through local field potential recordings. Our findings reveal that scn1Lab morphants display hyperactivity, convulsive seizure-like behavior, loss of posture, repetitive jerking and a myoclonic seizure-like pattern. The occurrence of spontaneous seizures was confirmed by local field potential recordings of the forebrain, measuring epileptiform discharges. Furthermore, we show that these larvae are remarkably sensitive to hyperthermia, similar to what has been described for mouse models of DS, as well as for human DS patients. Pharmacological evaluation revealed that sodium valproate and fenfluramine significantly reduce epileptiform discharges in scn1Lab morphants. Our findings for this zebrafish model of DS are in accordance with clinical data for human DS patients. To our knowledge, this is the first study demonstrating effective seizure inhibition of fenfluramine in an animal model of Dravet syndrome.
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Papers by Camila Esguerra