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Barmah Forest virus (BFV) is a mosquito-borne virus that causes arthralgia with accompanying rash, fever, and myalgia in humans. The virus is mainly found in Australia and has caused outbreaks associated with significant health concerns. As the sole representative of the Barmah Forest complex within the genus Alphavirus , BFV is not closely related genetically to other alphaviruses. Notably, basic knowledge of BFV molecular virology has not been well studied due to a lack of critical investigative tools such as an infectious clone. Here we describe the construction of an infectious BFV cDNA clone based on Genbank sequence and demonstrate that the clone-derived virus has in vitro and in vivo properties similar to naturally occurring virus, BFV field isolate 2193 (BFV2193-FI). A substitution in nsP4, V1911D, which was identified in the Genbank reference sequence, was found to inhibit virus rescue and replication. T1325P substitution in nsP2 selected during virus passaging was shown to be an adaptive mutation, compensating for the inhibitory effect of nsP4-V1911D. The two mutations were associated with changes in viral non-structural polyprotein processing and type I interferon (IFN) induction. Interestingly, a nuclear localization signal, active in mammalian but not mosquito cells, was identified in nsP3. A point mutation abolishing nsP3 nuclear localization attenuated BFV replication. This effect was more prominent in the presence of type I interferon signaling, suggesting nsP3 nuclear localization might be associated with IFN antagonism. Furthermore, abolishing nsP3 nuclear localization reduced virus replication in mice but did not significantly affect disease.IMPORTANCEBarmah Forest virus (BFV) is Australia's second most prevalent arbovirus, with approximately 1,000 cases reported annually. The clinical symptoms of BFV infection include rash, polyarthritis, arthralgia, and myalgia. As BFV is not closely related to other pathogenic alphaviruses or well-studied model viruses, our understanding of its molecular virology and mechanisms of pathogenesis is limited. There is also a lack of molecular tools essential for corresponding studies. Here we describe the construction of an infectious clone of BFV, variants harboring point mutations, and sequences encoding marker protein. In infected mammalian cells, nsP3 of BFV was located in the nuclei. This finding extends our understanding of the diverse mechanisms used by alphavirus replicase proteins to interact with host cells. Our novel observations highlight the complex synergy through which the viral replication machinery evolves to correct mutation errors within the viral genome., Competing Interests: The authors declare no conflict of interest.

Lyme disease, caused by Borrelia burgdorferi sensu lato infection, is the most widespread vector-borne illness in the Northern Hemisphere. Unfortunately, using targeted antibiotic therapy is often an ineffective cure. The antibiotic resistance and recurring symptoms of Lyme disease are associated with the formation of biofilm-like aggregates of B. burgdorferi . Plant extracts could provide an effective alternative solution as many of them exhibit antibacterial or biofilm inhibiting activities. This study demonstrates the therapeutic potential of Plantago major and Plantago lanceolata as B. burgdorferi inhibitors. Hydroalcoholic extracts from three different samples of each plant were first characterised based on their total concentrations of polyphenolics, flavonoids, iridoids, and antioxidant capacity. Both plants contained substantial amounts of named phytochemicals and showed considerable antioxidant properties. The major non-volatile constituents were then quantified using HPLC-DAD-MS analyses, and volatile constituents were quantified using HS-SPME-GC-MS. The most prevalent non-volatiles were found to be plantamajoside and acteoside, and the most prevalent volatiles were β-caryophyllene, D-limonene, and α-caryophyllene. The B. burgdorferi inhibiting activity of the extracts was tested on stationary-phase B. burgdorferi culture and its biofilm fraction. All extracts showed antibacterial activity, with the most effective lowering the residual bacterial viability down to 15%. Moreover, the extracts prepared from the leaves of each plant additionally demonstrated biofilm inhibiting properties, reducing its formation by 30%.

Ceratitis capitata is responsible for significant economic losses in the fruit production industry, and the market lacks biopesticides that are effective but also cheaper and less contaminating, with fewer negative impacts on the environment. In this regard, the present study suggests as potential options ethanolic extracts from several Macaronesian plants, which inhibit the oviposition and are toxic to C. capitata , and whose preparation involve a non-toxic solvent (i.e., ethanol), low energy expenditure and cheap apparatus (i.e., maceration at room temperature). Among the evaluated species, the extracts of Hedychium gardnerianum , Cistus symphytifolius and Salvia canariensis are the most active (50 mg/mL), revealing an increase in C. capitata adults' mortality from 21.15% to 27.41% after 72 h, a value statistically identical to azadirachtin (25.93%) at the recommended concentration (0.88 mg/mL). Considering the quantity and biomass available to prepare a biopesticide in the future, and the level of activity, the ethanolic extract of H. gardnerianum was fractionated and each fraction tested. The water fraction at 50 mg/mL proved to be more effective than the original extract, both in terms of mortality (57.69%), with LT 50 = 72.5 h, and oviposition deterrence (83.43%), values statistically higher than those obtained by azadirachtin at 0.88 mg/mL. Analysis of this fraction by HPLC-MS/MS showed that it is mainly composed of glycosylated derivatives of quercetin and myricetin in addition to some triterpenes. These findings highlight some Macaronesian species, and in particular, the more polar fraction of H. gardnerianum ethanolic extract, as promising and ecological alternatives to conventional insecticides, for use in the integrated management of the C. capitata pest.

The aim of the present study was to examine three different Galium species from the native population of Estonia, Galium verum , Galium aparine , and Galium mollugo , to characterise their non-volatile and volatile phytochemical composition and antioxidant activity. The main groups of bioactive compounds in the plants were quantified by colorimetric tests, showing high concentrations of polyphenols (up to 27.2 ± 1.5 mg GAE/g), flavonoids (up to 7.3 ± 0.5 mg QE/g) and iridoids (up to 40.8 ± 2.9 mg AE/g). The species were compared using HPLC-DAD-MS/MS, revealing some key differences in the phytochemical makeup of the extracts. The most abundant compound in the extracts of Galium verum blossoms and herb was found to be asperuloside, in Galium aparine herb, asperulosidic acid, and in Galium mollugo herb, chlorogenic acid. Additionally, the composition of volatile compounds was analysed by SPME-GC-MS. The degree of variability between the samples was high, but three volatiles, hexanal, anethole, and β -caryophyllene, were quantified (≥1%) in all analysed samples. The antioxidative activity of all extracts was evaluated using the ORAC FL method, demonstrating that the Galium species from Estonia all exhibit strong antioxidant capacity (up to 9.3 ± 1.2 mg TE/g). Out of the extracts studied, Galium verum blossoms contained the highest amounts of bioactives and had the strongest antioxidant capacity.

In this study, deep eutectic solvent (DES) based systems were evaluated for selective extraction and optimized for increased recovery of chlorogenic acid derivatives, flavone glycosides and iridoid glycosides from Dipsacus fullonum L. leaves. Bioactives from Dipsacus plants has shown great antioxidant and antimicrobial activities as well as effectiveness against several cancer strains and a source for anti-Borrelia compounds. Twelve different hydrophilic and hydrophobic DESs were tested to find the best solvent composition. Choline chloride and betaine were used as hydrogen bond acceptors (HBA) for the preparation of hydrophilic DESs and menthol for hydrophobic DESs. The tested hydrogen bond donors (HBD) were various organic acids and glycerol. The composition of most effective DES was optimized using the Box-Behnken design for each of the three main group of analytes from D. fullonum L. to evaluate possible selectivity and highest recovery. HPLC-DAD-MS was used to identify and quantify the main bioactive compounds extracted from plant material. The optimal extraction for highest overall recovery was achieved using a molar ratio of choline chloride and lactic acid of 1:2.4 with 35% water and 27 mL of the solvent per one gram of dry material. The optimized DES extract gave concentrations 1.8 to 2.2 times higher than traditional organic solvent extracts depending on the group of analytes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

The aim of this study was to investigate the influence of organic solvents on the electrophoretic separation of phenylethylamines. The background electrolyte composition was adjusted with different protic (methanol, ethanol, and 2-propanol) and aprotic (dimethyl sulfoxide and acetonitrile) solvents. Two groups of analytes were studied. The first group contained phenylethylamines with an amine group only, DL-1-phenylethylamine and amphetamine. The second group represented phenylethylamines with both amine and phenolic hydroxyl groups, dopamine and tyramine. Experiments revealed no or minor influence of the organic modifiers on the electromigration behavior of the analytes from the first group (containing only the amine group) and a drastic effect on the second group (containing the additional phenolic hydroxyl group). Dopamine and tyramine showed various electrostatic, hydrophobic, and hydrogen-bonding interactions with both protic and aprotic organic solvents. The dependence of the electrophoretic mobility of dopamine and tyramine on the concentration of the organic solvents provided direct evidence of the formation of hydrogen bonds between dopamine or tyramine and the organic solvent. The baseline separation was achieved by the addition of at least 20% v/v of organic solvent (protic or aprotic) to the background electrolyte. The analyte migration time repeatabilities were within 0.7-4.1% for absolute and 0.2-1.9% for normalised migration times. The proposed bonding mechanism and behavior of phenylethylamines were examined and confirmed by NMR spectroscopy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Jekaterina Mazina-Sinkar reports financial support was provided by Estonian Research Council., (Copyright © 2022. Published by Elsevier B.V.)

Synthetic cannabinoids (SCs) are the largest group of illicit compounds currently monitored in Europe. Reliable analytical methods for the differentiation and quantification of SCs and phytocannabinoids (PCs) from plant-based products are needed to reduce possible public health risks. The objective of this research was to develop a new method for the detection of four SCs (JWH-018, JWH-073, JWH-200, JWH-250) and two PCs (THC, CBD) from plant materials by using micellar electrokinetic chromatography with UV-absorbance detection. A novel method with a water-based background electrolyte containing cholate micelles was developed and optimized using two sets of the Box-Behnken experimental design. The method enables the quantification of JWH-018, JWH-073, JWH-200, JWH-250, and detection of THC and CBD. The selectivity studies revealed that several plants that are prevalently used as carrier matrixes for SCs could be analyzed without an added sample purification procedure. A basic method validation was performed. The detection and quantification limits were 3.0 and 5.0 for SCs, and 3.7 and 6.2 mg/L for PCs. For all analytes, the method intra-day precision was under 8%, the inter-day precision under 13%, and recoveries up to 100%., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Currently used methods for in-field determination of illegal drugs involve various test kits based mainly on the immunoassay technique, where the presence of a compound of interest is assessed by antibody-antigen reaction and manifested by observable color change. Despite being accepted and widely used by police forces to test the presence of illegal drugs in a suspect person, these tests often suffer from unreliable results (high level of false-positive and/or false-negative) due to the cross-reactivity and difficulties with quantification. Therefore, we have developed a portable capillary electrophoresis instrument to determine illegal drugs in oral fluid collected from a suspected person. However, this drug analyzer has still required manual sample preparation. Therefore, this research aimed to develop, test, and validate a fully automated sample pretreatment (purification, extraction, pre-concentration) prototype compatible with the capillary electrophoresis drug of abuse analyzer and suitable for confirmatory analysis by mass spectrometry. The cotton swab from Salivette® oral fluid collector was examined and integrated into the fully automated extractor prototype. The recoveries for the automated extractor were between 18 and 20%, with repeatabilities within 5-11% for 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), cocaine (COC), and cocaethylene (COET). The developed extraction device was easy to use even for unskilled persons, required minimal liquid handling, and was applicable to use in field conditions., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Lyme disease (LD) is a tick-borne bacterial disease that is caused by Borrelia burgdorferi . Although acute LD is treated with antibiotics, it can develop into relapsing chronic form caused by latent forms of B. burgdorferi . This leads to the search for phytochemicals against resistant LD. Therefore, this study aimed to evaluate the activity of Dipsacus fullonum L. leaves extract (DE) and its fractions against stationary phase B. burgdorferi in vitro. DE showed high activity against stationary phase B. burgdorferi (residual viability 19.8 ± 4.7%); however, it exhibited a noticeable cytotoxicity on NIH cells (viability 20.2 ± 5.2%). The iridoid-glycoside fraction showed a remarkable anti- Borrelia effect and reduced cytotoxicity. The iridoid-glycoside fraction was, therefore, further purified and showed to contain two main bioactives-sylvestrosides III and IV, that showed a considerable anti- Borrelia activity being the least toxic to murine fibroblast NIH/3T3 cells. Moreover, the concentration of sylvestrosides was about 15% of DE, endorsing the feasibility of purification of the compounds from D. fullonum L. leaves.

Flow analysis is the science of performing quantitative analytical chemistry in flowing streams. Because of its efficiency and speed of analysis, capillary electrophoresis (CE) is a prospective method for the monitoring of a flow composition withdrawn from various processes (e.g., occurring in bioreactors, fermentations, enzymatic assays, and microdialysis samples). However, interfacing CE to a various flow of interest requires further study. In this paper, several ingenious approaches on interfacing flow from various chemical or bioprocesses to a capillary electrophoresis instrument are reviewed. Most of these interfaces can be described as computer-controlled autosamplers. Even though most of the described interfaces waste too many samples, many interesting and important applications of the devices are reported. However, the lack of commercially available devices prevents the wide application of CE for flow analysis. On the contrary, this fact opens up a potential avenue for future research in the field of flow sampling by CE.

The current study is focused on the in situ synthesis of a carbon aerogel (CA)-based solid-phase microextraction (SPME) fiber coating on stainless steel wire and evaluation of the suitability of CAs as SPME coating materials for the analysis of selected organophosphorus pesticides (OPPs) contained in environmental samples. A CA-based coating was obtained by pyrolyzing organic aerogels, which were prepared by the sol-gel polymerization of formaldehyde and 5-methylresorcinol, an oil shale processing by-product. The results demonstrated, for the first time, the in situ synthesis of a CA-based SPME fiber coating on stainless steel wire and its suitability for the extraction and preconcentration of six OPPs. Main parameters affecting the extraction efficiency were investigated and optimized. The direct immersion (DI)-SPME procedure combined with gas chromatography-mass spectrometry (GC-MS) for the simultaneous analysis of selected OPPs was successfully applied to the efficient and sensitive determination of analytes of interest in environmental matrices of honey and natural water samples. The developed CA-coated SPME fiber showed good linearity (R 2 = 0.981-0.994), low detection limits (0.11-0.83 μg L -1 ) and satisfactory single fiber and fiber-to-fiber reproducibilities (8.8-12.3%, n = 5 and 11.4-17.2%, n = 3). The performance of the CA-coating was compared with that of commercially available SPME fiber coatings.

The use of colored tubers of Solanum tuberosum L. is growing worldwide due to their health benefits and attractive color. The positive health effects of purple-fleshed tubers are a result of anthocyanins and various phenolic compounds. The aim of this study was to evaluate and compare variety Blue Congo and its cross-breeds of Desiree and Granola to yellow-fleshed tubers. The concentration of total phenols, anthocyanins, sugars, and mineral elements were evaluated in all tubers. The results showed differences between all tested materials, with largest differences in sugar content. Moreover, the results confirmed the preservation of health improving compounds of Blue Congo when cross-bred with yellow-fleshed tubers. The total phenolic content and anthocyanin concentrations of all analyzed tubers were above the comparison yellow ones.

The aim of the current study was to develop and validate an analytical method to determine whether drugs of abuse (DOA) were present in oral fluid (OF) using a newly-developed, portable capillary electrophoresis (CE) instrument coupled to a deep ultra-violet fluorescence detector (FD). The performance of this portable CE-FD DOA analyser was tested at the Weekend Festival Baltic (Pärnu, Estonia) between 2016 and 2018 as well as on the roadside OF samples collected by the police. The study reported 128 analysed cases in which persons were allegedly found to have been under the influence of DOA. The samples were analysed for amphetamine (AMP), methamphetamine (METH), 3,4-methylenedioxy-methamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), cocaine (COC) and cocaethylene (COET). Subsequent toxicological reports revealed that 26% cases were negative, and 74% were positive. The most frequently detected and quantified DOA was MDMA (68 cases, 62%). A comparative study was conducted to validate the accuracy of using the CE-FD DOA analyser versus classic high-performance liquid chromatography coupled to mass spectrometry (HPLC-ESI-MS). Diagnostic statistics for CE-FD DOA were also evaluated and were higher than 99.5%. In addition, all zeta-scores were lower than 2 when both methods were compared, showing that the CE-FD analyser can be implemented as a reliable, sensitive and convenient tool for roadside and workplace testing for DOA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In the present study, a capillary electrophoresis method, with a native fluorescence detection for the quantification of three amphetamine derivatives, methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-methamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) in an oral fluid is described. The reported CE method has made it possible to assess Ecstasy abuse in approximately 15 min, including a saliva sample collection, pretreatment procedures and capillary electrophoresis (CE) analysis. The proof of the principle that was demonstrated on a home-made lab scale instrument has had the potential to be easily translated onto a truly portable instrument for on-site measurements. The baseline CE separation of the three illegal drugs was achieved in 10 min, by applying an aqueous background electrolyte (BGE) that was composed of 40 mM phosphoric acid and 10 mM triethanolamine. The amphetamine derivatives were detected at their λ ex /λ em maximum (280/326 nm) with LOD values of about 3 ng/mL for each amphetamine. The recovery of the compounds from the collection pad was about 40% of the LOQ concentrations and the inter-day precision was less than 6% for all of the analytes. The procedure was applied to a quantitation of oral fluid (OF) samples that were collected during the Baltic Weekend Music Festival that was held in Estonia., (Copyright © 2019 Elsevier B.V. All rights reserved.)

It is important to understand the extent of transfer of explosive particles to different surfaces in order to better evaluate potential cross-contamination by explosives in crowded security controls such as those at airports. This work investigated the transfer of nine explosive residues (ANFO, dynamite, black powder, TNT, HMTD, PETN, NH 4 NO 3 , KNO 3 , NaClO 3 ) through fingerprints from one surface to another. First, the extent of adhesion of explosive residues from different surfaces to the bare finger, nitrile and latex gloves was studied. Then, the transfer of explosive residues from one surface to another through fingerprints was investigated. Cotton fabric (hereinafter referred to as cotton) as clothing material and polycarbonate plastic (hereinafter referred to as polycarbonate) as luggage material were chosen for the experiments. These surfaces containing explosive particles were imaged using a reflex camera before and after the particles were transferred. Afterwards the images were processed in MATLAB where pixels corresponding to explosive residues were quantified. Results demonstrated that transfer of explosive residues frequently occurred with certain differences among materials. Generally, the amount of explosive particles adhered to the finger decreased in the following order: skin>latex>nitrile, while the transfer of particles from the finger to another surface was the opposite. The adhesion of explosive residues from polycarbonate to the finger was found to be better compared to cotton, while the amount of particles transferred to cotton was higher., (Copyright © 2018 The Chartered Society of Forensic Sciences. Published by Elsevier B.V. All rights reserved.)

This novel investigation focused on studying the transfer of explosive residues (TNT, HMTD, PETN, ANFO, dynamite, black powder, NH 4 NO 3 , KNO 3 , NaClO 3 ) in ten consecutive fingerprints to two different surfaces - cotton fabric and polycarbonate plastic - by using multispectral imaging (MSI). Imaging was performed employing a reflex camera in a purpose-built photo studio. Images were processed in MATLAB to select the most discriminating frame - the one that provided the sharpest contrast between the explosive and the material in the red-green-blue (RGB) visible region. The amount of explosive residues transferred in each fingerprint was determined as the number of pixels containing explosive particles. First, the pattern of PETN transfer by ten different persons in successive fingerprints was studied. No significant differences in the pattern of transfer of PETN between subjects were observed, which was also confirmed by multivariate analysis of variance (MANOVA). Then, the transfer of traces of the nine above explosives in ten consecutive fingerprints to cotton fabric and polycarbonate plastic was investigated. The obtained results demonstrated that the amount of explosive residues deposited on successive fingerprints tended to undergo a power or exponential decrease, with the exception of inorganic salts (NH 4 NO 3 , KNO 3 , NaClO 3 ) and ANFO (consists of 90% NH 4 NO 3 )., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The present study demonstrates the potential of a portable capillary electrophoresis (CE) instrument, coupled to deep UV fluorescence detector (FD) with a 230-255 nm excitation wavelength range, for the determination of the abuse of illegal drugs in oral fluids in situ. CE was introduced in this study due its exceptional power of separation and resolution, short analysis time, and ability for miniaturization for on-site assessment of different substances. The deep UV fluorescence detector was equipped with five interchangeable emission filters, in the emission wavelength range from 278 to 600 nm, and was successfully employed for determination of natively fluorescing illegal drugs, such as cocaine, cocaethylene, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxeamphetamine (MDA), 3,4-methylenedioxy- N-ethylamphetamine (MDEA), para-methoxyamphetamine (PMA), para-methoxy- N-methylamphetamine (PMMA), amphetamine (AMP), methamphetamine (METH), tetrahydrocannabinol (THC) and cannabidiol (CBD). The developed FD showed impressive sensitivity. The instrumental detection limit was 0.5 μg/L for MDMA. It also showed broad linearity, up to 50 mg/L for MDMA. The noise CV% was 1.1% for an empty capillary and 0.6% for a capillary filled with acetonitrile. The portable CE-FD with developed electrophoretic methodologies was successfully utilized for the determination of illegal abuse of drugs during "Weekend" 2016 and 2017 Music Festivals (Estonia). Moreover, CE-FD can be employed for detection of other natively fluorescing compounds in the proposed range (e.g., for different phenolic compounds, BTEX, naphthalene derivatives, and others), significantly widening the applicability of developed CE-FD instrument.