Repellent efficacy and safety evaluation of IR3535 derivative against Aedes albopictus,Culex pipiens pallens and Aedes togoi

The IR3535 derivative (LJH158), in which the ethyl ester of IR3535 was converted to methyl ester, was synthesized and studied as a new mosquito repellent. The repellent efficacy of LJH158 was compared with that of DEET against Aedes albopictus, Culex pipiens pallens and Aedes togoi. Also, the aromatic repellent tests were conducted with mixtures of repellents and the essential oils of cinnamon, which were obtained by supercritical fluid extraction. In addition, the safety issues of LJH158 were monitored using single oral dose safety methods and eye irritation, and skin irritation tests. The results of repellent efficacy in both biting and aromatic tests and safety tests demonstrate that LJH158 has high potential to be used as a new repellent or in combination with other repellents.     

Lethal and behavioural effects of three synthetic repellents (DEET, IR3535 and KBR 3023) on Aedes aegypti mosquitoes in laboratory assays

The knock-down, mortality and 'irritancy' effects of three synthetic repellents (DEET, IR3535 and KBR 3023) on Aedes aegypti (L) (Diptera: Culicidae) were evaluated in the laboratory in the absence of animal bait. Filter paper tests were carried out to assess the knock-down effect (KDt(50) and KDt(95)) and mortality (LC(50) and LC(95)) induced by each repellent. 'Irritancy' tests were carried out to compare the flight response (time to first take-off, or FT) to increasing concentrations of repellents (2-7%) and at five distances from the treated surface (0-40 mm). DEET had an insecticidal effect (KDt(50) = 9.7 min at 7%; CL(50)= 1165 mg/m(2)), whereas IR3535 and KBR 3023 did not. Relative to an untreated control, IR3535 was an irritant (relative irritancy or RI > 1) at doses of 5% and 7% (RI = 17.7 and 9.9, respectively), whereas DEET was an irritant at lower concentrations (RI = 12.3 at 2% DEET). KBR 3023 was the weakest irritant over the same range of concentrations (RI(max) = 3.6 at 6%). DEET was more of an irritant (RI(20) = 9.4) than IR3535 (RI(20) = 2.9) over a range of distances (0-20 mm), and KBR 3023 was not an irritant unless mosquitoes made contact with the treated surface. All three repellents had a significant effect on mosquitoes, but DEET exhibited a more complex mode of action than the others due to its insecticidal properties. The repellents do not behave as a single class of compounds with a common mode of action, but most probably affect different physiological systems in insects. The physiological and molecular mechanisms of repellents, especially DEET, should be investigated to ensure a better use of these molecules for skin applications and/or for treating materials against mosquitoes.     

A new effective assay to detect antimicrobial activity of filamentous fungi

The search for new antimicrobial compounds and the optimization of production methods turn the use of antimicrobial susceptibility tests a routine. The most frequently used methods are based on agar diffusion assays or on dilution in agar or broth. For filamentous fungi, the most common antimicrobial activity detection methods comprise the co-culture of two filamentous fungal strains or the use of fungal extracts to test against single-cell microorganisms. Here we report a rapid, effective and reproducible assay to detect fungal antimicrobial activity against single-cell microorganisms. This method allows an easy way of performing a fast antimicrobial screening of actively growing fungi directly against yeast. Because it makes use of an actively growing mycelium, this bioassay also provides a way for studying the production dynamics of antimicrobial compounds by filamentous fungi. The proposed assay is less time consuming and introduces the innovation of allowing the direct detection of fungal antimicrobial properties against single cell microorganisms without the prior isolation of the active substance(s). This is particularly useful when performing large screenings for fungal antimicrobial activity. With this bioassay, antimicrobial activity of Hypholoma fasciculare against yeast species was observed for the first time.     

Comparison of the effects of acylation and amidation on the antimicrobial and antiviral properties of lactoferrin

AIMS: To compare amidation and acylation of lactoferrin (LF) from bovine milk, as a means of enhancing its antimicrobial and antiviral properties. METHODS AND RESULTS: LF was chemically modified by amidation with a 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC) in the presence of ammonium ions or by acylation with either succinic or acetic anhydride. In the test systems used, amidation substantially enhanced the activity of LF against Pseudomonas fluorescens in comparison with native LF. However, increasing the net negative charge of LF by acylation had no effect on the activity of LF against P. fluorescens, and abrogated the antimicrobial activity of LF against Bacillus subtilis and Saccharomyces cerevisiae. Increasing the net negative charges of LF by acylation eliminated its antimicrobial and antiviral effects against poliovirus and feline calicivirus (nonenveloped viruses). CONCLUSIONS: The addition of positive charges to LF via amidation enhanced antimicrobial properties in contrast to increasing the negative charges by acylation, which abolished both the antimicrobial and antiviral properties of LF. SIGNIFICANCE AND IMPACT OF THE STUDY: The effects of charge alteration of LF determined in this study provides a basis for further development of LF formulations with enhanced antimicrobial effectiveness for use in food process hygiene, veterinary and health-care applications.     

Generic insect repellent detector from the fruit fly Drosophila melanogaster

Background

Insect repellents are prophylactic tools against a number of vector-borne diseases. There is growing demand for repellents outperforming DEET in cost and safety, but with the current technologies R&D of a new product takes almost 10 years, with a prohibitive cost of $30 million dollar in part due to the demand for large-scale synthesis of thousands of test compounds of which only 1 may reach the market. R&D could be expedited and cost dramatically reduced with a molecular/physiological target to streamline putative repellents for final efficacy and toxicological tests.

Methodology

Using olfactory-based choice assay we show here that the fruit fly is repelled by not only DEET, but also IR3535 and picaridin thus suggesting they might have “generic repellent detector(s),” which may be of practical applications in new repellent screenings. We performed single unit recordings from all olfactory sensilla in the antennae and maxillary palps. Although the ab3A neuron in the wild type flies responded to picaridin, it was unresponsive to DEET and IR3535. By contrast, a neuron housed in the palp basiconic sensilla pb1 responded to DEET, IR3535, and picaridin, with apparent sensitivity higher than that of the DEET detectors in the mosquitoes Culex quinquefasciatus and Aedes aegypti. DmOr42a was transplanted from pb1 to the “empty neuron” and showed to be sensitive to the three insect repellents.

Conclusions

For the first time we have demonstrated that the fruit fly avoids not only DEET but also IR3535 and picaridin, and identified an olfactory receptor neuron (ORN), which is sensitive to these three major insect repellents. We have also identified the insect repellent-sensitive receptor, DmOr42a. This generic detector fulfils the requirements for a simplified bioassay for early screening of test insect repellents.     

The skin as interface in the transmission of arthropod-borne pathogens

Animal skin separates the inner world of the body from the largely hostile outside world and is actively involved in the defence against microbes. However, the skin is no perfect defence barrier and many microorganisms have managed to live on or within the skin as harmless passengers or as disease-causing pathogens. Microbes have evolved numerous strategies that allow them to gain access to the layers underneath the epidermis where they either multiply within the dermis or move to distant destinations within the body for replication. A number of viruses, bacteria and parasites use arthropod vectors, like ticks or mosquitoes, to deliver them into the dermis while taking their blood meal. Within the dermis, successful pathogens subvert the function of a variety of skin resident cells or cells of the innate immune system that rush to the site of infection. In this review several interactions with cells of the skin by medically relevant vector-borne pathogens are discussed to highlight the different ways in which these pathogens have come to survive within the skin and to usurp the defence mechanisms of the host for their own ends.     

Determination of antimicrobial properties of Picaridin and DEET against a broad range of microorganisms

Insect repellents are topically applied to the skin and clothing of human and pet to keep flies, mosquitoes, and ticks away. Two important repellents, N,N-diethyl-meta-toluamide (DEET) and Picaridin, have been widely used since their discovery. Although repellency and toxicological effects of DEET and Picaridin on human being and insects are well documented without understanding molecular mechanisms, there have been no attempts to study their effects on microorganisms up to now. In the current study, DEET and Picaridin have been investigated for their antimicrobial characteristics against a broad range of microorganisms including bacteria, yeast, and fungi for the first time. They exhibited considerable antibacterial, anticandidal and antifungal properties. Even though bacteria was found to be more sensitive to Picaridin, yeast and fungi were more susceptible to DEET. Antimicrobial properties of these two repellents will increase the usage and application areas of the products containing DEET and Picaridin.     

High‐throughput screening method for evaluating spatial repellency and vapour toxicity to mosquitoes

Spatial repellents are an essential tool for personal protection against mosquitoes that bite and transmit disease pathogens to humans. Current repellent screening methods, such as olfactometers and alternative choice tests, are complex systems that require a relatively large quantity of compound (mg). The present study validates a high‐throughput spatial repellent screening method using a glass tube that has the ends covered with netting, in addition to treated filters and plastic end caps. The apparatus occupies relatively little space, is easy to decontaminate, and requires small amounts of compound (μg). In a horizontal tube orientation, DEET (N,N‐diethyl‐meta‐toluamide), citronella oil and IR3535 had 1 h half repellent concentration (EC₅₀) values of 32, 32 and 298 μg/cm², respectively, against the Orlando strain of Aedes aegypti (L.) (Diptera: Culicidae). Vertical tube orientation increased EC₅₀ values by approximately two‐fold, except IR3535, which remained essentially unchanged. Transfluthrin showed concentration‐dependent spatial repellency (1 h EC₅₀ = 0.5 μg/cm²) without any knockdown, although only in vertical tubes. Transfluthrin showed 50% knockdown in 1 h at 0.5 μg/cm² and 50% mortality at 0.15 μg/cm² in horizontal tubes. In conclusion, this high‐throughput screening method is useful for assessing vapour toxicity and the spatial repellency of candidate molecules prior to semi‐field and field studies.     

Repellencies and toxicities of five ant-derived defensive compounds against the lone star tick, <Emphasis Type="Italic">Amblyomma americanum</Emphasis> (Acari: Ixodidae)

The lone star tick, Amblyomma americanum, is a vector of several important human and animal diseases. This tick species has rapidly expanded in its geographic distribution, and its aggressive behavior has increased the risk of tick-borne diseases in these new areas. Repellents are recommended by the Centers for Disease Control and Prevention (CDC) for protection against tick bites. DEET is the most common repellent, but public concerns over its safety have increased the need for alternative safe and efficacious tick repellents. Several naturally derived animal compounds have been tested against other species of ticks or other arthropod pests, but not against A. americanum. Based on EC₅₀ values obtained using a vertical paper bioassay, decylamine and MT-710 (a 2-tridecanone formulation) were found to be as repellent as DEET. Those two substances along with 2-tridecanone were also found to be as repellent as DEET when their EC₉₅ values were compared. Lone star ticks were more susceptible to the toxic effects of DEET in glass vial assays than all of the ant-derived defensive compounds/formulations. These results suggest that the ant-derived defensive compounds are likely more effective lone star tick repellents than DEET, but they are not as toxic as DEET towards the ticks. The suitability of these compounds for use as personal repellents, as well as at the landscape scale, should be explored.     

Purification of antimicrobial factor from granules of channel catfish peripheral blood leucocytes

The channel catfish (Ictalurus punctatus) is extensively used in aquaculture in the Southeast US and is susceptible to many bacterial infections acquired from its pond environment. Research is needed to better understand the defensive response and innate immunity of channel catfish against fish pathogens like Edwardsiella ictaluri and Aeromonas hydrophila. The main objectives were purification and characterization of an innate antimicrobial factor isolated from catfish leucocytes that has both bactericidal and antiviral activities. Oxygen-independent mechanisms of innate immunity for killing microorganisms have not been identified in leucocytes of channel catfish. Leucocytes were separated from catfish blood, and granule extracts were obtained by homogenization, centrifugation, and extraction with 10% acetic acid. The granule extracts were further purified by gel filtration chromatography. Bactericidal assays against the two fish pathogens and SDS-PAGE analysis were done on the isolated antimicrobial factor. Determination of antiviral activity of the factor was done by in vitro tissue culture using herpes simplex virus-type 1. Mass spectrometry analyses were done for molecular weight (655 Da), purity, and structural characterization of the innate non-peptide antimicrobial factor.     

New influenza A Virus Entry Inhibitors Derived from the Viral Fusion Peptides

Influenza A viral (IAV) fusion peptides are known for their important role in viral-cell fusion process and membrane destabilization potential which are compatible with those of antimicrobial peptides. Thus, by replacing the negatively or neutrally charged residues of FPs with positively charged lysines, we synthesized several potent antimicrobial peptides derived from the fusogenic peptides (FPs) of hemagglutinin glycoproteins (HAs) of IAV. The biological screening identified that in addition to the potent antibacterial activities, these positively charged fusion peptides (pFPs) effectively inhibited the replication of influenza A viruses including oseltamivir-resistant strain. By employing pseudovirus-based entry inhibition assays including H5N1 influenza A virus (IAV), and VSV-G, the mechanism study indicated that the antiviral activity may be associated with the interactions between the HA2 subunit and pFP, of which, the nascent pFP exerted a strong effect to interrupt the conformational changes of HA2, thereby blocking the entry of viruses into host cells. In addition to providing new peptide “entry blockers”, these data also demonstrate a useful strategy in designing potent antibacterial agents, as well as effective viral entry inhibitors. It would be meaningful in treatment of bacterial co-infection during influenza pandemic periods, as well as in our current war against those emerging pathogenic microorganisms such as IAV and HIV.     

Lambda interferon (IFN-lambda), a type III IFN, is induced by viruses and IFNs and displays potent antiviral activity against select virus infections in vivo

Type III interferons (IFNs) (interleukin-28/29 or lambda interferon [IFN-lambda]) are cytokines with IFN-like activities. Here we show that several classes of viruses induce expression of IFN-lambda1 and -lambda2/3 in similar patterns. The IFN-lambdas were-unlike alpha/beta interferon (IFN-alpha/beta)-induced directly by stimulation with IFN-alpha or -lambda, thus identifying type III IFNs as IFN-stimulated genes. In vitro assays revealed that IFN-lambdas have appreciable antiviral activity against encephalomyocarditis virus (EMCV) but limited activity against herpes simplex virus type 2 (HSV-2), whereas IFN-alpha potently restricted both viruses. Using three murine models for generalized virus infections, we found that while recombinant IFN-alpha reduced the viral load after infection with EMCV, lymphocytic choriomeningitis virus (LCMV), and HSV-2, treatment with recombinant IFN-lambda in vivo did not affect viral load after infection with EMCV or LCMV but did reduce the hepatic viral titer of HSV-2. In a model for a localized HSV-2 infection, we further found that IFN-lambda completely blocked virus replication in the vaginal mucosa and totally prevented development of disease, in contrast to IFN-alpha, which had a more modest antiviral activity. Finally, pretreatment with IFN-lambda enhanced the levels of IFN-gamma in serum after HSV-2 infection. Thus, type III IFNs are expressed in response to most viruses and display potent antiviral activity in vivo against select viruses. The discrepancy between the observed antiviral activity in vitro and in vivo may suggest that IFN-lambda exerts a significant portion of its antiviral activity in vivo via stimulation of the immune system rather than through induction of the antiviral state.     

Repellency of deet and SS220 applied to skin involves olfactory sensing by two species of ticks

Responses of host-seeking nymphs of the blacklegged tick, Ixodes scapularis Say and lone star tick, Amblyomma americanum (Linnaeus) (Acari: Ixodidae) to the repellents N,N-diethyl-3-methylbenzamide (deet) and (1S, 2'S)-2-methylpiperidinyl-3-cyclohexene-1-carboxamide (SS220) were studied using fingertip laboratory bioassays. Ethanol solutions of both compounds applied to the skin strongly repelled both species of ticks at 0.8 and 1.6 micromole of compound/cm2 skin. The ticks were also repelled when two layers of organdie cloth covered the portion of a finger treated with either deet or SS220. Gas chromatographic analyses of the outer layer of cloth that had covered skin treated with 1.6 micromole compound/cm2 skin revealed only 0.1 nmole SS220/cm2 cloth and 2.8 nmole deet/cm2 cloth. However, in bioassays in which a single layer of cloth was treated with a dose of deet or SS220 equivalent to the amount found in the outer layer of cloth, ticks were not repelled. Results unequivocally demonstrated that these ticks responded to the repellents in the vapour phase when repellent treated skin was covered with cloth to obviate tactile contact with them, and made it clear that the ticks detect the repellents by olfactory sensing. Heretofore, the mode of action of deet and SS220 was unclear.     

The coelomic fluid of the sea urchin Tripneustes depressus shows antiviral activity against Suid herpesvirus type 1 (SHV-1) and rabies virus (RV)

Several studies have reported that molecules extracted from invertebrates have activity against different viruses, even against those that do not infect these organisms in their environment. One of the main mechanisms against pathogens in these organisms is the production of antimicrobial peptides. The objective of this study was to determine whether the coelomic fluid (CF) of the sea urchin Tripneustes depressus has activity against Suid herpesvirus type 1 (SHV-1) and/or rabies virus (RV). We tested the antiviral activity of CF in neutralizing assays and observed 50% inhibition against SHV-1 lytic plaque formation using 33 μg of CF, whereas 21 μg CF was sufficient to obtain more than 90% inhibition for RV. Cytotoxicity to MDBK and BHK-21 cells was found with whole CF yet was eliminated by heating at 56 or 72 °C (even when using 50 μg of heat-inactivated CF supernatant [SN or thermostable fraction]), and SN retained the antiviral effect. In both cases, the antiviral effect was direct and thermostable (SN 56 and 72 °C), and the best inhibition was observed when CF + virus was incubated prior to the addition of the cells. Therefore, the coelomic fluid of T. depressus has antiviral activity against SHV-1 and RV that is direct and stable at 72 °C. We suggest that further assays should be performed using more accurate methods to characterize new molecules with antiviral activity that may result in new drugs.     

SAR analysis of a series of acylthiourea derivatives possessing broad-spectrum antiviral activity

A series of acylthiourea derivatives were designed, synthesized, and evaluated for broad-spectrum antiviral activity with selected viruses from Poxviridae (vaccinia virus) and two different genera of the family Bunyaviridae (Rift Valley fever and La Crosse viruses). A compound selected from a library screen, compound 1, displayed submicromolar antiviral activity against both vaccinia virus (EC(50)=0.25 μM) and La Crosse virus (EC(50)=0.27 μM) in cytopathic effect (CPE) assays. SAR analysis was performed to further improve antiviral potency and to optimize drug-like properties of the initial hits. During our analysis, we identified 26, which was found to be nearly fourfold more potent than 1 against both vaccinia and La Crosse viruses. Selected compounds were further tested to more fully characterize the spectrum of antiviral activity. Many of these possessed single digit micromolar and sub-micromolar antiviral activity against a diverse array of targets, including influenza virus (Orthomyxoviridae), Tacaribe virus (Arenaviridae), and dengue virus (Flaviviridae).     

Characterization of antiviral and antibacterial activity of Bombyx mori seroin proteins

Lepidopterans as other insects have a very potent innate immune system, which basically comprises cellular and humoral defence mechanisms against bacterial and fungal infections. In lepidopterans, not much is known about the defence mechanisms against viral pathogens, such as baculoviruses. Here we show that small silk proteins of the domesticated silkworm, Bombyx mori, called seroins, act as antiviral agents against a baculovirus pathogen, Bombyx mori nucleopolyhedrovirus (BmNPV). Involvement of these proteins in the inhibition of baculovirus infection was revealed by estimating the viral load upon their dsRNA‐mediated knockdown. Additionally, we found through antimicrobial assays that seroins are potent inhibitors of bacterial growth. Binding competition assays followed by antimicrobial assays showed that seroins bind to peptidoglycan, a cell wall component of bacteria. Analysis of bacterial load upon knockdown of seroins resulted in higher proliferation of bacteria. Phylogenetic analysis showed the recent origin of seroins in a few moth species and duplication only in Bombycids. The antiviral and antibacterial activity of seroins shown in this study using several biochemical and molecular biological assays provide strong evidence to characterize them as antimicrobial proteins. Hence, we hypothesize that seroins are potent candidates for use in development of transgene‐based disease resistant silkworm strains.     

Antimicrobial activity of AOT-isooctane reverse micelle as a bioseparation and biocatalysis tool

Abstract

The antimicrobial activity of different reverse micelles on microorganisms is been compared using the disc diffusion method. The bis (2-ethylhexyl) sodium sulfosuccinate (AOT) reverse micelle showed a more significant inhibitory effect than do other reverse. micelles, and it had an antimicrobial activity against a broad range of microorganisms. Results from an antimicrobial activity test of isooctane and a forward extraction containing soybean protein suggest that the surfactant was chiefly responsible for inhibiting microbes in AOT/isooctane reverse micelle, while isooctane hardly inhibited the microbial growth. The properties of S. aureus, cultured in the TSB with AOT reverse micellar solution, were identified by the SEM and SDS-PAGE fingerprinting of cell-wall proteins. It is concluded that the cell-wall of the S. aureus decreased in the TSB with AOT reverse micellar solution, and some cell protein subunits of the S. aureus did not occurr, especially between 14.4 and 42.7 kDa, while one new protein subunit at near 97.4 kDa occurred     

Antimicrobial activity of the resins obtained from the roots and stems of Cedrus libani and Abies cilicica

The antimicrobial activity of the ethanol extract of resins obtained from the roots and stems of Cedrus libani and Abies cilicica was investigated using a disc diffusion method. Results revealed that crude extracts of the resins of both plants are highly effective against tested microorganisms by preventing their growth to a greater extent.     

Chemical composition and antimicrobial activity of the essential oils from Chloranthus japonicus Sieb. and Chloranthus multistachys Pei

We examined the composition and antimicrobial activity of two essential oils from Chloranthus japonicus Sieb. and Chloranthus multistachys Pei. GC-FID and GC-MS analyses identified 48 and 39 compounds, which represented 95.56% and 94.58%, respectively, of all components in these oils. Of these, 28 compounds were common to both, with a relatively high amount of oxygenated monoterpenes (50.95% and 39.97%). Antimicrobial properties were evaluated in vitro via disc diffusion and microbroth dilution assays. Activities were strong against most tested microorganisms, with inhibition zones ranging from 8.1 to 22.2 mm. For both species, minimum values for inhibitory and bactericidal concentrations were 0.39 to 12.50 mg/mL and 0.78 to 50.00 mg/mL, respectively. These results suggest that these essential oils are potent natural sources of antimicrobial agents for the medicinal and pharmaceutical industries.     

RNase 7, a novel innate immune defense antimicrobial protein of healthy human skin

We analyzed healthy human skin for the presence of endogenous antimicrobial proteins that might explain the unusually high resistance of human skin against infections. A novel 14.5-kDa antimicrobial ribonuclease, termed RNase 7, was isolated from skin-derived stratum corneum. RNase 7 exhibited potent ribonuclease activity and thus may contribute to the well known ribonuclease activity of human skin. RNase 7 revealed broad spectrum antimicrobial activity against many pathogenic microorganisms and remarkably potent activity (lethal dose of 90% < 30 nm) against a vancomycin-resistant Enterococcus faecium. Molecular cloning from skin-derived primary keratinocytes and purification of RNase 7 from supernatants of cultured primary keratinocytes indicate that keratinocytes represent the major cellular source in skin and that RNase 7 is secreted. RNase 7 mRNA expression was detected in various epithelial tissues including skin, respiratory tract, genitourinary tract, and at a low level, in the gut. In addition to a constitutive expression, RNase 7 mRNA was induced in cultured primary keratinocytes by interleukin-1beta, interferon-gamma, and bacterial challenge. This is the first report demonstrating RNases as a novel class of epithelial inducible antimicrobial proteins, which may play an important role in the innate immune defense system of human epithelia.