Biology subjects

Ons, S.

Publications and source records attributed to Ons, S..

7 recordsLinked to original sources

bioRxiv2025

Magnetic Affinity-Based Purification of His-Tagged Proteins Using Ni2+-Functionalized Nanoparticles

Recombinant protein purification is essential in many applications across industry, research, and biotechnology. The use of a histidine tag (His6) in affinity chromatography is the gold standard for purification. However, magnetic nanoparticles (MNPs) offer an excellent alternative due to their unique properties, such as rapid separation, superparamagnetism, and a high surface-to-volume ratio that facilitates nanoscale functionalization. MNPs provide a more efficient, faster, cost-effective, and sustainable method compared to traditional chromatography. In this work, we report the development and validation of a magnetic nanoparticle-based platform for the efficient, selective, and scalable purification of His6 recombinant proteins. The synthesized Ni2+-functionalized MNPs demonstrated high specificity for histidine-tagged proteins, as shown by the selective retention and imidazole-mediated elution of target proteins both from pre-purified samples and complex E. coli lysates. Comparative assays with bovine serum albumin and His6 protein A confirmed the selective affinity of the MNPs for His6. Furthermore, a scaled-up protocol using 4 mL of lysate yielded approximately 0.6 mg/mL of purified protein, doubling the output obtained via conventional immobilized metal affinity chromatography, while preserving purity. The simplicity, efficiency, and cost-effectiveness of this method render it a powerful tool for both analytical and preparative-scale protein purification, particularly in low-resource or high-throughput settings.

molecular biology↗

bioRxiv2022

Food recognition in a blood-feeding insect: characterization of the pharyngeal taste organ

BackgroundObligate blood-feeding insects obtain the nutrients and water necessary to ensure survival from the vertebrate blood. The internal taste sensilla, situated in the pharynx, evaluate the suitability of the ingested food. Here, through multiple approaches, we characterized the pharyngeal organ (PO) of the hematophagous kissing bug Rhodnius prolixus to determine its role in food assessment. The PO, located antero-dorsally in the pharynx, comprises 8 taste sensilla that become bathed with the incoming blood. ResultsWe showed that these taste sensilla house gustatory receptor neurons projecting their axons through the labral nerves to reach the subesophageal zone in the brain. We found that these neurons are electrically activated by relevant appetitive and aversive gustatory stimuli such as NaCl, ATP and caffeine. Using RNA-Seq, we examined the expression of sensory-related gene families in the PO. We identified gustatory receptors, ionotropic receptors, transient receptor potential channels, pickpocket channels, opsins, takeouts, neuropeptide precursors, neuropeptide receptors and biogenic amine receptors. RNA interference assays demonstrated that the pickpocket channel Rproppk014276 is necessary for salt detection during feeding. ConclusionWe provide evidence of the role of the pharyngeal organ in food evaluation. This work shows the first comprehensive characterization of a pharyngeal taste organ in a hematophagous insect.

neuroscience↗

bioRxiv2022

Multiplex high resolution melting PCR for simultaneous genotyping of pyrethroid-resistance associated mutations in Aedes aegypti. First report on kdr mutations in wild populations from Argentina

BackgroundAedes aegypti is an urban mosquito vector of Dengue and other arboviruses. During epidemic periods, pyrethroid insecticides are used for the control of adult mosquitoes; the worldwide distributed resistance to these insecticides is a cause of failures in vector control campaigns. The primary target of pyrethroids is the voltage-gated sodium channel; point mutations on this channel, called kdr mutations, are associated with pyrethroid resistance. Two kdr mutations, called V1016I and F1534C, augmented in frequency in natural populations of Ae. aegypti from the Americas in the last decade. The diagnostic of kdr polymorphisms allows an early detection of insecticide resistance spreading, which is critical for timely decisions on vector management. Given the relevance of resistance management, high-throughput methods for kdr genotyping are invaluable tools for resistance monitoring programs. These methods should also be cost-effective, to allow regional-scale surveys. Despite the extended presence of Ae. aegypti and the incidence of dengue in Argentina, the presence, abundance and distribution of kdr mutations were not reported in this country up to date. Methodology and findingsWe report a multiplex high-throughput assay based in High Resolution Melting PCR for the simultaneous genotyping of 1016 and 1534 sites in voltage-gated sodium channel gene. We used this method for the study of individual mosquito samples collected in localities which received different selection pressure with pyrethroids. Compared to other genotyping methods, multiplex High Resolution Melting was high-throughput, cost-efficient, sensitive and specific. We demonstrate for the first time the presence of kdr mutations in Argentina in regions under different selection pressure with pyrethroids. Conclusions and SignificanceWe have developed a high-throughput method for the genotyping of alleles associated with pyrethroid resistance in Ae. aegypti from the American continent. The method developed here is comparable in its sensitivity and reliability with other genotyping methods, but reduces costs and running time. It could be incorporated in control campaigns for control the presence and spreading of resistance-associated alleles. We report here for the first time the presence of kdr mutations in distant populations from Argentina, with different epidemiological situations and different history of mosquito control efforts. Authors summaryAedes aegypti is a mosquito vector of viruses such as dengue, causing millions of infections yearly worldwide. Emergence and distribution of insecticide resistance in this mosquito is a challenge for control campaigns. In this context, the implementation of resistance management strategies became a requisite for successful and sustainable mosquito management. To achieve this objective, it is important to count with early genetic markers of resistance, such as kdr mutations, which are associated with pyrethroid resistance phenotype. Kdr markers can be detected by molecular genotyping diagnostic at early stages in the emergence of resistance; this information should be considered in rational design of control campaigns. Here we present a high-throughput cost-effective method to detect kdr mutations in Ae. aegypti. Also, we demonstrate for the first time the presence of kdr mutations in Argentina.

genetics↗

bioRxiv2022

eIF3 subunit M regulates blood meal digestion in Rhodnius prolixus affecting ecdysis, reproduction and survival.

In triatomines, blood-feeding triggers many physiological processes, including post-embryonic development and reproduction. Different feeding habits, such as hematophagy, can shape gene functions to meet the challenges of each type of diet. A comparison of transcriptomic and proteomic data indicates that post-transcriptional regulation of gene expression is crucial in triatomines, so we evaluated the impact of RNAi silencing of the eukaryotic translation initiation factors 3 subunit m (eIF3m) in R. prolixus physiology. We showed that eIF3m is essential for correct digestion, affecting the processes triggered by a blood meal. The silencing of this gene inhibited moulting and caused the premature death of nymphs, while in adult females inhibited oviposition and increased resistance to starvation. Male survival was not affected by eIF3m knockdown. The information regarding the eIF3m function in insects is scarce. The phenotypes observed in R. prolixus upon eIF3m gene silencing are different and more severe than those described in Drosophila melanogaster, pointing to the particular importance of this gene in triatomines. Summary statementThe information provided here indicates the importance of mRNA translation in modulating growth, reproduction, lifespan and starvation resistance in triatomine vectors.

molecular biology↗

bioRxiv2022

The role of neuropeptides in regulating ecdysis and reproduction in the hemimetabolous insect Rhodnius prolixus

In ecdysozoan animals, moulting entails the production of a new exoskeleton and the shedding of the old one during ecdysis. It is induced by a pulse of ecdysone that regulates the expression of different hormonal receptors and activates a peptide-mediated signalling cascade. In Holometabola, the peptidergic cascade regulating ecdysis has been well described. However, very little functional information regarding the neuroendocrine regulation of ecdysis is available for Hemimetabola, which displays an incomplete metamorphosis. Here, we studied neuropeptides related to ecdysis regulation in the hemi-metabolous insect Rhodnius prolixus. The RNA interference-mediated reduction of ETH expression in fourth instar nymphs resulted in lethality at the expected time of ecdysis, thereby showing its crucial role in this process. Furthermore, the results revealed the involvement of ETH in the regulation of reproductive fitness. Different from holometabolous, the knockdown of ETH in adult females led to failures in egg hatching without affecting the oviposition. Most of the first instar nymphs hatched from the eggs laid by females injected with dsEH, dsCCAP and dsOKA died at the expected time of ecdysis, indicating the crucial involvement of these genes for post-embryonic development. No phenotypes were observed upon CZ knockdown in nymphs or adult females. The conservation of the role of these neuropeptides in regulating ecdysis and reproduction throughout the class Insecta is discussed. Summary statementThe information provided here is of interest for evolutive studies on the neuroendocrine regulation of ecdysis and reproduction in insects, and the research for new targets to control pest insects.

developmental biology↗

bioRxiv2021

Transcriptomic modulation in response to an intoxication with deltamethrin in Triatoma infestans, vector of Chagas disease

BackgroundTriatoma infestans is the main vector of Chagas disease in the Southern Cone. The resistance to pyrethroid insecticides developed by populations of this species impairs the effectiveness of vector control campaigns in wide regions of Argentina. The study of the global transcriptomic response to pyrethroid insecticides is important to deepen the knowledge about detoxification in triatomines. Methodology and findingsWe used RNA-Seq to explore the early transcriptomic response of T. infestans after intoxication with deltamethrin. We were able to assemble a complete transcriptome of this vector and found evidence of differentially expressed genes belonging to diverse families such as chemosensory and odorant-binding proteins, ABC transporters and heat-shock proteins. Moreover, genes related to transcription and translation, energetic metabolism and cuticle rearrangements were also modulated. Finally, we characterized the repertoire of previously uncharacterized detoxification-related gene families in T. infestans and Rhodnius prolixus. Conclusions and significanceOur work contributes to the understanding of the detoxification response in vectors of Chagas disease. Given the absence of genomic information from T. infestans, the analysis presented here constitutes a resource for molecular and physiological studies in this species. The results increase the knowledge on detoxification processes in vectors of Chagas disease, and provide relevant information to explore new potential insecticide resistance mechanisms in these insects. Author summaryChagas disease affects millions of people worldwide. In the Southern Cone, the development of pyrethroid resistant populations from T. infestans is related to vector persistence and affects the efficiency of vector control campaigns. Several studies have explored the causes of insecticide resistance in T. infestans populations. However, the global transcriptomic response after insecticide treatment has not been analyzed in this species so far. In this study, we obtained transcriptomic information which allowed us to characterize important gene families despite the absence of genomic information. Furthermore, we performed a quantitative analysis of gene expression after deltamethrin intoxication. The results provided here increase the knowledge on detoxification processes in vectors of Chagas disease, which is essential for the design of new vector control strategies.

genomics↗

bioRxiv2019

Transcriptomics supports local sensory regulation in the antennae of the kissing bug Rhodnius prolixus

Rhodnius prolixus has become a model for revealing the molecular bases of insect sensory biology due to the publication of its genome, its well characterized behavioural repertoire and the advent of NGS technologies. Gene expression modulation underlies behaviour-triggering processes at peripheral and central levels. Still, the regulation of sensory-related gene transcription in sensory organs is poorly understood. Here we study the genetic bases of plasticity in antennal sensory function, using R. prolixus as an insect model. Antennal expression of neuromodulatory genes such as those coding for neuropeptides, neurohormones and their receptors was characterized by means of RNA-Seq. New nuclear receptor and takeout gene sequences were identified for this species, as well as those of enzymes involved in the biosynthesis and processing of neuropeptides and biogenic amines. We report a broad repertoire of neuromodulatory and endocrine genes expressed in antennae and suggest that they modulate sensory neuron function locally. Diverse neuropeptide-coding genes showed consistent expression in the antennae of all stages studied. Future studies should characterize the contribution of these modulatory components acting over antennal sensory processes to assess the relative contribution of peripheral and central regulatory systems on the plastic expression of insect behaviour.

neuroscience↗