Can Virus-like Particles Be Used as Synergistic Agent in Pest Management?

Among novel strategies proposed in pest management, synergistic agents are used to improve insecticide efficacy through an elevation of intracellular calcium concentration that activates the calcium-dependent intracellular pathway. This leads to a changed target site conformation and to increased sensitivity to insecticides while reducing their concentrations. Because virus-like particles (VLPs) increase the intracellular calcium concentration, they can be used as a synergistic agent to synergize the effect of insecticides. VLPs are self-assembled viral protein complexes, and by contrast to entomopathogen viruses, they are devoid of genetic material, which makes them non-infectious and safer than viruses. Although VLPs are well-known to be used in human health, we propose in this study the development of a promising strategy based on the use of VLPs as synergistic agents in pest management. This will lead to increased insecticides efficacy while reducing their concentrations.

Drug Delivery Systems for the Oral Administration of Antimicrobial Peptides: Promising Tools to Treat Infectious Diseases.

Antimicrobial peptides (AMPs) have a great potential to face the global expansion of antimicrobial resistance (AMR) associated to the development of multidrug-resistant (MDR) pathogens. AMPs are usually composed of 10-50 amino acids with a broad structural diversity and present a range of antimicrobial activities. Unfortunately, even if the oral route is the most convenient one, currently approved therapeutic AMPs are mostly administrated by the intravenous route. Thus, the development of novel drug delivery systems (DDSs) represents a promising opportunity to protect AMPs from chemical and enzymatic degradation through the gastrointestinal tract and to increase intestinal permeability leading to high bioavailability. In this review, the classification and properties as well as mechanisms of the AMPs used in infectiology are first described. Then, the different pharmaceutical forms existing in the market for oral administration are presented. Finally, the formulation technologies, including microparticle- and nanoparticle-based DDSs, used to improve the oral bioavailability of AMPs are reviewed.

[Virological aspects and diagnosis of SARS-CoV-2 coronavirus]. / Aspects virologiques et diagnostic du coronavirus Sars-CoV-2.

Since the emergence of the new Sars-CoV-2 coronavirus in China at the end of December 2019 and its spread around the world, the scientific community has been mobilized to study its phylogeny, virological aspects, and to understand viral and immune kinetics. In order to propose the best diagnosis, the use of direct diagnosis, by reverse transcriptase-polymerase chain reaction, or indirect diagnosis, by serology, needs to be clarified.

Orthosteric muscarinic receptor activation by the insect repellent IR3535 opens new prospects in insecticide-based vector control.

The insect repellent IR3535 is one of the important alternative in the fight against mosquito-borne disease such as malaria, dengue, chikungunya, yellow fever and Zika. Using a multidisciplinary approach, we propose the development of an innovative insecticide-based vector control strategy using an unexplored property of IR3535. We have demonstrated that in insect neurosecretory cells, very low concentration of IR3535 induces intracellular calcium rise through cellular mechanisms involving orthosteric/allosteric sites of the M1-muscarinic receptor subtype, G protein ßγ subunits, background potassium channel inhibition generating depolarization, which induces voltage-gated calcium channel activation. The resulting internal calcium concentration elevation increases nicotinic receptor sensitivity to the neonicotinoid insecticide thiacloprid. The synergistic interaction between IR3535 and thiacloprid contributes to significantly increase the efficacy of the treatment while reducing concentrations. In this context, IR3535, used as a synergistic agent, seems to promise a new approach in the optimization of the integrated vector management for vector control.

Nanoencapsulated deltamethrin as synergistic agent potentiates insecticide effect of indoxacarb through an unusual neuronal calcium-dependent mechanism.

The use of neurotoxic chemical insecticides has led to consequences against the environment, insect resistances and side-effects on non-target organisms. In this context, we developed a novel strategy to optimize insecticide efficacy while reducing doses. It is based on nanoencapsulation of a pyrethroid insecticide, deltamethrin, used as synergistic agent, combined with a non-encapsulated oxadiazine (indoxacarb). In this case, the synergistic agent is used to increase insecticide efficacy by activation of calcium-dependant intracellular signaling pathways involved in the regulation of the membrane target of insecticides. In contrast to permethrin (pyrethroid type I), we report that deltamethrin (pyrethroid type II) produces an increase in intracellular calcium concentration in insect neurons through the reverse Na/Ca exchanger. The resulting intracellular calcium rise rendered voltage-gated sodium channels more sensitive to lower concentration of the indoxacarb metabolite DCJW. Based on these findings, in vivo studies were performed on the cockroach Periplaneta americana and mortality rates were measured at 24 h, 48 h and 72 h after treatments. Comparative studies of the toxicity between indoxacarb alone and indoxacarb combined with deltamethrin or nanoencapsulated deltamethrin (LNC-deltamethrin), indicated that LNC-deltamethrin potentiated the effect of indoxacarb. We also demonstrated that nanoencapsulation protected deltamethrin from esterase-induced enzymatic degradation and led to optimize indoxacarb efficacy while reducing doses. Moreover, our results clearly showed the benefit of using LNC-deltamethrin rather than piperonyl butoxide and deltamethrin in combination commonly used in formulation. This innovative strategy offers promise for increasing insecticide efficacy while reducing both doses and side effects on non-target organisms.

Synergistic agent and intracellular calcium, a successful partnership in the optimization of insecticide efficacy.

Integrated Pest Management and Integrated Vector Management worldwide are developed in agriculture and public health to counteract and limit the exponential increasing development of insect resistance to insecticides. However, facing the predominance of some resistant populations, new strategies are urgently needed to target resistant insects. An innovative approach lies in the optimization of commonly used insecticides when combined with chemical or biological synergistic agents. By an increase of intracellular calcium concentration followed by activation of calcium-dependant signalling pathways, the synergistic agents are able to indirectly increase target sites sensitivity to insecticide by inducing conformational change. The synergistic agents are of great interest in optimizing the efficacy of insecticides and in overcoming resistance mechanisms.

Unusual modes of action of the repellent DEET in insects highlight some human side effects.

N,N-diethyl-m-toluamide (DEET) induces favorable repellency against insects by acting on the sensory nervous system. According to emerging literature reports, DEET side effects in humans involve new molecular targets including the cholinergic system, acetylcholinesterase (AChE), muscarinic M1 and M3 receptor and the participation of the second messenger nitric oxide (NO). Most of these molecular events targeted by DEET have previously been characterized in insects while they have been considered as marginal compared to classical repellent properties. Despite these uncommon actions in insects, there is no consensus on the effects in human. Based on these data, this review provides new insights on side effects in human and more largely in mammals by identifying the unusual properties of DEET in insects, which seem to be correlated with adverse effects in mammals. These data will be very helpful to understand better the toxicological effects observed in order to protect non-target organisms from the toxicity.

Microbial Pest Control Agents: Are they a Specific And Safe Tool for Insect Pest Management?

Microorganisms (viruses, bacteria and fungi) or their bioactive agents can be used as active substances and therefore are referred as Microbial Pest Control Agents (MPCA). They are used as alternative strategies to chemical insecticides to counteract the development of resistances and to reduce adverse effects on both environment and human health. These natural entomopathogenic agents, which have specific modes of action, are generally considered safer as compared to conventional chemical insecticides. Baculoviruses are the only viruses being used as the safest biological control agents. They infect insects and have narrow host ranges. Bacillus thuringiensis (Bt) is the most widely and successfully used bioinsecticide in the integrated pest management programs in the world. Bt mainly produces crystal delta-endotoxins and secreted toxins. However, the Bt toxins are not stable for a very long time and are highly sensitive to solar UV. So genetically modified plants that express toxins have been developed and represent a large part of the phytosanitary biological products. Finally, entomopathogenic fungi and particularly, Beauveria bassiana and Metarhizium anisopliae, are also used for their insecticidal properties. Most studies on various aspects of the safety of MPCA to human, non-target organisms and environment have only reported acute but not chronic toxicity. This paper reviews the modes of action of MPCA, their toxicological risks to human health and ecotoxicological profiles together with their environmental persistence. This review is part of the special issue "Insecticide Mode of Action: From Insect to Mammalian Toxicity".

The insect repellent N,N-diethyl-m-toluamide (DEET) induces angiogenesis via allosteric modulation of the M3 muscarinic receptor in endothelial cells.

The insect repellent N,N-diethyl-m-toluamide (DEET) has been reported to inhibit AChE (acetylcholinesterase) and to possess potential carcinogenic properties with excessive vascularization. In the present paper, we demonstrate that DEET specifically stimulates endothelial cells that promote angiogenesis which increases tumor growth. DEET activates cellular processes that lead to angiogenesis including proliferation, migration and adhesion. This is associated with an enhancement of NO production and VEGF expression in endothelial cells. M3 silencing or the use of a pharmacological M3 inhibitor abrogates all of these effects which reveals that DEET-induced angiogenesis is M3 sensitive. The experiments involving calcium signals in both endothelial and HEK cells overexpressing M3 receptors, as well as binding and docking studies demonstrate that DEET acts as an allosteric modulator of the M3 receptor. In addition, DEET inhibited AChE which increased acetylcholine bioavailability and binding to M3 receptors and also strengthened proangiogenic effects by an allosteric modulation.

Virus and calcium: an unexpected tandem to optimize insecticide efficacy.

The effective control of insect pests is based on the rational use of the most efficient and safe insecticide treatments. To increase the effects of classical insecticides and to avoid the ability of certain pest insects to develop resistance, it is essential to propose novel strategies. Previous studies have shown that calcium-dependent phosphorylation/dephosphorylation is now considered as a new cellular mechanism for increasing the target sensitivity to insecticides. Because it is known that virus entry is correlated with intracellular calcium concentration rise, this report attempts to present the most important data relevant to the feasibility of combining an insect virus such as baculovirus or densovirus with an insecticide. In this case, the insect virus is not used as a bioinsecticide but acts as a synergistic agent able to trigger calcium rise and to activate calcium-dependent intracellular signalling pathways involved in the increase of the membrane receptors and/or ion channels sensitivity to insecticides. This virus-insecticide mixture represents a promising alternative to optimize the efficacy of insecticides against insect pests while reducing the doses.

The Repellent DEET Potentiates Carbamate Effects via Insect Muscarinic Receptor Interactions: An Alternative Strategy to Control Insect Vector-Borne Diseases.

Insect vector-borne diseases remain one of the principal causes of human mortality. In addition to conventional measures of insect control, repellents continue to be the mainstay for personal protection. Because of the increasing pyrethroid-resistant mosquito populations, alternative strategies to reconstitute pyrethroid repellency and knock-down effects have been proposed by mixing the repellent DEET (N,N-Diethyl-3-methylbenzamide) with non-pyrethroid insecticide to better control resistant insect vector-borne diseases. By using electrophysiological, biochemichal, in vivo toxicological techniques together with calcium imaging, binding studies and in silico docking, we have shown that DEET, at low concentrations, interacts with high affinity with insect M1/M3 mAChR allosteric site potentiating agonist effects on mAChRs coupled to phospholipase C second messenger pathway. This increases the anticholinesterase activity of the carbamate propoxur through calcium-dependent regulation of acetylcholinesterase. At high concentrations, DEET interacts with low affinity on distinct M1/M3 mAChR site, counteracting the potentiation. Similar dose-dependent dual effects of DEET have also been observed at synaptic mAChR level. Additionally, binding and in silico docking studies performed on human M1 and M3 mAChR subtypes indicate that DEET only displays a low affinity antagonist profile on these M1/M3 mAChRs. These results reveal a selective high affinity positive allosteric site for DEET in insect mAChRs. Finally, bioassays conducted on Aedes aegypti confirm the synergistic interaction between DEET and propoxur observed in vitro, resulting in a higher mortality of mosquitoes. Our findings reveal an unusual allosterically potentiating action of the repellent DEET, which involves a selective site in insect. These results open exciting research areas in public health particularly in the control of the pyrethroid-resistant insect-vector borne diseases. Mixing low doses of DEET and a non-pyrethroid insecticide will lead to improvement in the efficiency treatments thus reducing both the concentration of active ingredients and side effects for non-target organisms. The discovery of this insect specific site may pave the way for the development of new strategies essential in the management of chemical use against resistant mosquitoes.

A novel method using Autographa californica multiple nucleopolyhedrovirus for increasing the sensitivity of insecticide through calcium influx in insect cell line.

Due to an intensive use of chemical insecticides, resistance mechanisms to insecticides together with adverse effects on non-target organisms have been largely reported. Improvement in pest control strategy represents an urgent need to optimize efficiency in the control of pest insects. In this context, a novel method based on the use of insect specific virus applied in combination with chemical insecticide, which could lead to sensitization of the insect target to insecticides is described. Insect virus, the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), applied onto Sf9 cells induces an increase of intracellular calcium concentration via extracellular calcium influx. Co-application of AcMNPV with chlorpyrifos-ethyl onto Sf9 cells expressing the key enzyme acetylcholinesterase (AChE), known to be targeted by organophosphate insecticides, increases 1.5-fold the sensitivity of AChE to the insecticide. This effect is correlated with intracellular calcium concentration rise since AcMNPV-induced potentiating insecticide effect is counteracted by pretreatment with the calcium channel blocker, cadmium chloride. Increasing insecticide target sensitivity through intracellular calcium modulation by using insect virus co-applied with a chemical insecticide is a very promising strategy allowing optimization of insecticide treatment while reducing the concentration of insecticides used.

Anopheles gambiae mosquito isolated neurons: a new biological model for optimizing insecticide/repellent efficacy.

To understand better the mode of action of insecticides and repellents used in vector-borne diseases control, we developed a new biological model based on mosquito neurons isolated from adults Anopheles gambiae heads. This cellular model is well adapted to multidisciplinary approaches: electrophysiology, pharmacology, molecular biology and biochemical assays. Using RT-PCR, we demonstrated that isolated neurons express the nicotinic acetylcholine receptor subunit α1 (Agα1 nAchR), two acetylcholinesterases (AChE-1 and AChE-2) and three voltage-gated ion channels required for membrane excitability (AgCav1, AgNav1 and AgKv1). In order to correlate the expression of the different transcripts, encoding functional AgNav channel, nAChR receptor and AChE enzymes detected by RT-PCR, with electrophysiological activity we used patch-clamp technique. We revealed that AgNav and AChE which are targeted by insecticide and/or repellent were sensitive to the pyrethroid permethrin and to the repellent DEET, respectively. In addition, using colorimetric method, we also showed that AChE was sensitive to the carbamate propoxur. These results indicated that this novel neuronal mosquito model will lead to molecular and functional characterization of insecticide/repellent targets and appears as a powerful tool to investigate the development of highly specific and effective strategies for disease vector control.

Disruption of the GPI protein-encoding gene IFF4 of Candida albicans results in decreased adherence and virulence.

Candida albicans is the most important cause of systemic fungal infection in immunocompromised humans. Candidiasis is often initiated by the adherence and the colonization of inert surfaces such as peripheral venous catheters, central catheters, prosthetic cardiac valves, and other prostheses. We have studied the early stage of adherence and have shown that the disruption of C. albicans IFF4 gene encoding a GPI-anchor protein, led to a decrease of adherence of the germ tubes to plastic. Here, we demonstrated the role of the IFF4 gene in adherence to silicone catheter, as well as in virulence using a murine model of disseminated candidiasis. The iff4 Delta null mutant showed both a decrease of adherence to silicone catheter and a reduction of virulence. This work presents evidence for the importance of the IFF4 gene in host-fungal interaction.

Innovative applications for insect viruses: towards insecticide sensitization.

The effective management of emerging insect-borne disease is dependent on the use of safe and efficacious chemical insecticides. Given the inherent ability of insects to develop resistance, it is essential to propose innovative strategies because insecticides remain the most important element of integrated approaches to vector control. Recently, intracellular phosphorylation and dephosphorylation of membrane receptors and ion channels targeted by insecticides have been described as new processes for increasing the sensitivity of insecticides. An efficient method might be to infect host insects with recombinant viruses overexpressing specific protein phosphatases/kinases known to regulate specific insecticide-sensitive targets. This attractive strategy could lead to sensitization of the insects, thus reducing the doses of insecticides and increasing the efficacy of treatments.

Development of an oligonucleotide array for direct detection of fungi in sputum samples from patients with cystic fibrosis.

Cystic fibrosis (CF) is the most common inherited genetic disease in Caucasian populations. Besides bacteria, many species of fungi may colonize the respiratory tract of these patients, sometimes leading to true respiratory infections. In this study, an oligonucleotide array capable of identifying 20 fungal species was developed to directly detect fungi in the sputum samples of CF patients. Species-specific oligonucleotide probes were designed from the internal transcribed spacer (ITS) regions of the rRNA operon and immobilized on a nylon membrane. The fungal ITS regions were amplified by PCR and hybridized to the array for species identification. The array was validated by testing 182 target strains (strains which we aimed to identify) and 141 nontarget strains (135 species), and a sensitivity of 100% and a specificity of 99.2% were obtained. The validated array was then used for direct detection of fungi in 57 sputum samples from 39 CF patients, and the results were compared to those obtained by culture. For 16 sputum samples, the results obtained by the array corresponded with those obtained by culture. For 33 samples, the array detected more fungal species than culture did, while the reverse was found for eight samples. The accuracy of the array for fungal detection in sputum samples was confirmed (or partially confirmed) in some samples by cloning and resequencing the amplified ITS fragments. The present array is a useful tool for both the simultaneous detection of multiple fungal species present in the sputa of CF patients and the identification of fungi isolated from these patients.

Evaluation of an immunomagnetic separation method to capture Candida yeasts cells in blood.

BACKGROUND: Candida species have become the fourth most-frequent cause of nosocomial bloodstream infections in immunocompromised patients. Therefore, rapid identification of pathogenic fungi to species level has been considered critical for treatment. Conventional diagnostic procedures such as blood culture or biochemical tests are lacking both sensitivity and species specificity, so development of rapid diagnostic is essential. RESULTS: An immunomagnetic method involving anti-Candida monoclonal antibodies was developed to capture and concentrate in human blood four different species of Candida cells responsible for invasive yeast infections. In comparison with an automated blood culture, processing time of immunomagnetic separation is shorter, saving at least 24 hours to obtain colonies before identification. CONCLUSION: Thus, this easy to use method provides a promising basis for concentrating all Candida species in blood to improve sensitivity before identification.

Disruption of Candida albicans IFF4 gene involves modifications of the cell electrical surface properties.

During the past two decades, the prevalence of candidiasis has increased markedly and Candida albicans has now become one of the most important causes of nosocomial infections, especially after colonization of inert surfaces such as catheters or prostheses. In a previous report, we demonstrated the overexpression of 35 unidentified genes in response to adherence of C. albicans germ tubes to plastic. Therefore, a bioinformatic analysis was performed searching for genes encoding surface proteins potentially involved in adherence. Nineteen genes were thus selected, and one of them, CaIFF4, was further investigated. The deduced protein of this CaIFF4 gene revealed a glycosylphosphatidylinositol (GPI)-anchored site as well as the presence of a N-terminal signal peptide. Disruption of both alleles of CaIFF4 gene from C. albicans parent strain BWP17 was performed by PCR method. Then investigations of properties of null mutant for CaIFF4 gene showed a decrease of adherence of germ tubes to plastic in comparison to the parent strain BWP17. Besides, electrophoretic mobilities of germ tubes of CaIFF4 null mutant and of parental strain BWP17 were measured. Data were then analysed with soft particles analysis theory. Results point out a less important electrophoretic mobility of germ tubes of CaIFF4 null mutant in comparison to germ tubes of BWP17 parental strain.

DNA array analysis of Candida albicans gene expression in response to adherence to polystyrene.

Candidiasis is often initiated by the colonization of inert surfaces. In order to elucidate the mechanisms involved in this adherence process, DNA macroarrays were used to analyze the transcriptome of Candida albicans, the main causative agent of this mycoses, in a simple adherence model using germ tubes produced in polystyrene Petri dishes. Non-adherent germ tubes produced on glass surface were used as a control. Analysis of gene expression displayed 77 genes identified as statistically overexpressed in adherent germ tubes. Among these genes, some encoded enzymes participating in metabolism of lipids (such as LIP6), of proteins (such as SAP1) or of carbohydrates (like PGI1, PMI40 and PSA1. Some of these genes have already been reported as playing a role in pathogenesis of C. albicans. However, functions were unknown for a large part (45.5%) of the overexpressed genes which will be analyzed further in order to define their relationship with adherence.