Complex Biophysical and Computational Analyses of G-Quadruplex Ligands: The Porphyrin Stacks Back.

G-quadruplexes (G4 s), as non-canonical DNA structures, attract a great deal of research interest in the molecular biology as well as in the material science fields. The use of small molecules as ligands for G-quadruplexes has emerged as a tool to regulate gene expression and telomeres maintenance. Meso-tetrakis-(N-methyl-4-pyridyl) porphyrin (TMPyP4) was shown as one of the first ligands for G-quadruplexes and it is still widely used. We report an investigation comprising molecular docking and dynamics, synthesis and multiple spectroscopic and spectrometric determinations on simple cationic porphyrins and their interaction with different DNA sequences. This study enabled the synthesis of tetracationic porphyrin derivatives that exhibited binding and stabilizing capacity against G-quadruplex structures; the detailed characterization has shown that the presence of amide groups at the periphery improves selectivity for parallel G4 s binding over other structures. Taking into account the ease of synthesis, 5,10,15,20-tetrakis-(1-acetamido-4-pyridyl) porphyrin bromide could be considered a better alternative to TMPyP4 in studies involving G4 binding.

Electronic Effects in a Green Protocol for (Hetero)Aryl-S Coupling.

Aryl and heteroaryl iodides have been efficiently converted into the corresponding thioacetates in cyclopentyl methyl ether (CPME), a green solvent, under Cu catalysis. The chemoselectivity of the reaction is mainly controlled by electronic factors, enabling the conversion of both electron-rich and electron-deficient substrates into the corresponding thioacetates in good to excellent yields. The products can be easily deprotected to the corresponding thiolates to carry out additional synthetic transformations in situ. Surprisingly, despite CPME's relatively low dielectric constant, the reaction rate significantly increased when conducted under microwave irradiation conditions. This synthetic methodology exhibits a remarkable tolerance to functional groups, mild reaction conditions, and a wide substrate scope, utilizing a safe and inexpensive CuI pre-catalyst in the green solvent CPME. A non-aqueous workup allowing for the complete recovery of both catalyst and solvent makes this approach an environmentally sustainable protocol for C(sp2) sulfur functionalization. Additionally, the reaction shows selective cross-coupling with iodides in competition with chlorides and bromides, allowing its use in multistep syntheses. To demonstrate the potential of this methodology, it was applied to the high-yield synthesis of a photochromic dithienylethene, where a selective synthesis had not been reported before.

Experimental infection of Aedes (Stegomyia) albopictus and Culex pipiens mosquitoes with Bluetongue virus.

Bluetongue disease (BT), caused by Bluetongue virus (BTV), infects wild and domestic ruminants, causing severe economic damage in the cattle and sheep industry. Proven vectors of BTV are biting midges belonging to the Culicoides genus, but other arthropods are considered potential vectors, such as ticks, mosquitoes, wingless flies, and sand flies. The present study represents the first attempt to evaluate the vectorial capacity of Culex pipiens and Aedes albopictus for BTV. Mosquitoes were artificially fed with blood containing BTV serotype 1. Infection, dissemination and transmission rates were evaluated at 0, 3, 7, 14 and 21 days after an infected blood meal. Viral RNA was only detected up to 3 days post infection in the bodies of both species. This study indicates that the two Italian populations of Cx. pipiens and Ae. albopictus are not susceptible to BTV infection.

Pseudomonas protegens Affects Mosquito Survival and Development.

This study investigated the pathogenic potential of Pseudomonas protegens on mosquito larvae of the two species Culex pipiens and Aedes albopictus, representing major threats for disease transmission in the Mediterranean area and worldwide. The bacterium achieved to kill over 90% of the mosquito larvae within 72 h after exposition to a bacterial concentration of 100 million CFU/ml. These lethal effects were concentration dependent and a significantly higher susceptibility was associated with younger larvae of both mosquito species. Significant slowdown of immature (larval and pupal) development and decrease in adult emergence rate after treatment with sub-lethal doses of the bacterium were also detected. This study reports for the first time the insecticidal activity of a root-associated biocontrol bacterium against aquatic mosquito larvae.

Culicoides species responsible for the transmission of Epizootic Haemorrhagic Disease virus (EHDV) serotype 8 in Italy.

Epizootic haemorrhagic disease (EHD) is a viral disease transmitted by Culicoides biting midges that affects wild and domestic ruminants. The causative agent, EHD virus (EHDV), belongs to the family Sedoreoviridae, genus Orbivirus. The virus has never been reported in Europe until October 2022, when the virus was for the first time detected in Sicily and Sardinia. After the first clinical cases, an intensive entomological field activity was carried out in five EHD affected farms located in Sardinia, with the aim of assessing the EHDV vector competence in European species of Culicoides. EHDV­8 was detected in C. imicola, C. obsoletus/scoticus, C. newsteadi, C. pulicaris ss, and C. bysta. The first 4 species have also been demonstrated to be able to transmit bluetongue virus (BTV). According to these results, it is likely that EHDV­8, sharing the same transmission patterns of BTV, can also spread to Europe.

Aquivion perfluorosulfonic superacid as an effective catalyst for selective epoxidation of vegetable oils.

The acid-promoted epoxidation of vegetable oils was studied using a variety of acidic ion exchange resins as heterogeneous acid catalysts. Quantitative and selective epoxidation of a series of vegetable oils with different composition of saturated, mono-, di- and tri-unsaturated fatty acids was obtained upon identification of the more efficient catalyst and experimental conditions. Furthermore, optimized reaction conditions were successfully applied to the epoxidation of a waste cooking oil, thus extending our procedure to the valorization of a biowaste, an area of increasing importance within a more sustainable society. The use of quantitative 1HNMR besides making accurate evaluation of the amounts of reagents to be employed and of the selectivity, allowed facile and rapid quantification of mono-, di- and tri-epoxides, thus providing an indirect indication on the fatty acid composition of the vegetable oils, even in the presence of very low quantities of linolenic acid.

Bluetongue virus detection in new Culicoides species in Sardinia, Italy.

Bluetongue is an infectious disease transmitted by Culicoides biting midges. Culicoides imicola is considered the main vector in the Mediterranean basin but other species have been implicated in the Bluetongue virus (BTV) transmission. During 2017, BTV serotype 4 re-occurred in Sardinia causing outbreaks in sheep farms. A survey was carried out on affected farms with the aim to detect the virus in field-collected Culicoides Biting midges were morphologically identified, pooled and then assayed with a real time RT-PCR. To evaluate BTV dissemination, some Culicoides were dissected and head, thorax and abdomen were tested singly by PCR. A total of 173,738 Culicoides adults were collected. Viral RNA was detected in 68 out of 77 pools and all species analysed resulted positive. Detection of BTV in parous female body regions (head, thorax and abdomen) confirmed the full dissemination of BTV in all species analysed. During this study, the vector competence of C imicola, C newsteadi s.l. and Obsoletus complex was confirmed. The authors found two new Culicoides species BTV positive, C paolae never associated with BTV transmission and C circumscriptus only recently found BTV positive in Turkey, which could be considered potential vectors.

Vector Competence of Italian Populations of Culicoides for Some Bluetongue Virus Strains Responsible for Recent Northern African and European Outbreaks.

The distribution of Bluetongue virus (BTV) in Europe can be represented by two distinct and interconnected epidemiological systems (episystems), each characterized by different ecological characteristics and vector species. This study investigated the vector competence of Italian populations of Culicoides imicola and Culicoides obsoletus/scoticus to some representative BTV strains after artificial oral infection. The BTV strains were selected according to their ability to spread to one or both episystems and included BTV-4 ITA, responsible of the recent Italian and French BTV-4 outbreaks; the BTV-2 strain which caused the first BTV incursion in Italy, Corsica, and Balearic Islands; BTV-4 MOR, responsible for the epidemic in Morocco; and BTV-8, the strain which spread through Europe between 2006 and 2008. Blood-soaked cotton pledgets and Hemotek membrane feeder using Parafilm® membrane were used to artificially feed midges. For each population/strain, recovery rates (positive/tested heads) were evaluated using serogroup- and serotype-specific RT-PCR. The trial demonstrated that, except for the Abruzzo population of C. obsoletus/C. scoticus, which was refractory to BTV-4 MOR infection, all the investigated Culicoides populations are susceptible to the selected BTV strains and that, if prompt vaccination programs and restriction measures had not been implemented, BTV-2 and BTV-4 MOR could have spread all over Europe.

A five-year survey of tick species and identification of tick-borne bacteria in Sardinia, Italy.

Sardinia is a hotspot for studying tick-borne diseases in the Mediterranean region, where cases of notifiable tick-borne diseases are increasing. The aim of this study was to determine the presence of tick-borne bacteria of medical and veterinary importance in ixodid ticks collected from domestic and wild animals, humans, and vegetation from different collection sites in Sardinia. Using standard PCR and sequencing techniques, the presence of Rickettsia, Anaplasma, Ehrlichia, and Bartonella species, as well as Coxiella burnetii was evaluated. A total of 1619 ticks were morphologically identified as Rhipicephalus sanguineus sensu lato, R. bursa, R. annulatus, Dermacentor marginatus, Haemaphysalis punctata, Ha. sulcata, Hyalomma lusitanicum, H. marginatum, Ixodes festai (sometimes referred to erroneously as I. ventalloi), and Argas reflexus. Results indicated the presence of several circulating pathogens in Sardinian ticks. DNA of Rickettsia species was detected in 58 out of 1619 (4%) belonging to R. sanguineus s.l., D. marginatus, Ha. punctata, H. marginatum, and I. festai species. Ehrlichia canis DNA was detected in 33 out of 1619 ticks (2%) belonging to R. sanguineus s.l., R. bursa, and Ha. punctata species. A total of 61 out of 1619 (4%) ticks (R. sanguineus s. l., R. bursa, Ha. punctata, and I. festai) tested positive for Anaplasma spp. Coxiella burnetii was detected in 21 out of 1619 (1%) ticks belonging to R. sanguineus s.l., R. bursa, R. annulatus, and H. marginatum species. Five R. sanguineus s.l. and one R. bursa ticks were positive for the presence of Bartonella sp. 16S rRNA gene. Our findings expand the knowledge on tick-borne microorganism repertoires and tick distribution in Sardinia. Tick distribution should be monitored for effective control of these arthropods and the infections they transmit.

Entomological surveillance of Zika virus in Sardinia, Italy, 2016.

Zika Virus (ZIKV) is a RNA virus belonging to the genus Flavivirus, family Flaviviridae. This virus is transmitted through bite of Aedes mosquitoes, in particular Ae. aegypti. On February 1st 2016, the World Health Organization (WHO) has declared ZIKV a Public Health Emergency of International Concern. Successively, considering the establishment of Ae. albopictus, WHO has classified Italy as having a moderate likelihood of local transmission of ZIKV, preceded in Europe only by France. For this reason an entomological surveillance plan was been activated in Sardinia in 2016. BG Sentinel Mosquito Traps have been positioned in 29 sites, comprising urban areas and points of entry, as ports and airports. Mosquitoes were collected fortnightly from April to December. A total of 3,089 mosquitoes were collected belonging to 10 species. The most numerous species have been Cx. pipiens s.l. and Ae. albopictus. All mosquitoes sampled have been assayed by real time reverse transcriptase PCR for detection of ZIKV RNA. A total of 584 pool have been analyzed and have been reported no evidence of ZIKV. A permanent entomological surveillance should be implemented principally in the urban areas and points of entry, as ports and airports, because Ae. albopictus, susceptible to ZIKV, is established in Sardinia and also know the recent introduction of invasive mosquitoes species Ae. koericus and Ae. japonicus in Italy.

Combined larvicidal and adulticidal treatments to control Culicoides biting midges (Diptera: Ceratopogonidae): Results of a pilot study.

Culicoides biting midges can transmit viruses such as bluetongue virus and Schmallenberg virus to ruminants and African horse sickness virus to equines. The control of Culicoides population can be based on physical and/or chemical methods. This study aims to evaluate the efficacy of larvicides and adulticides combined treatments to control the vector Culicoides populations. Field trials have been conducted during 2014 on seven farms in northern Sardinia (Italy). Three insecticide treatments (larvicides and adulticides) have been carried out on three farms, whereas four farms remained unsprayed as untreated controls. For the larval control two commercial formulations, based on diflubenzuron and Bacillus thuringiensis var. israelensis (H-14), have been used. Against adults a deltamethrin based product has been sprayed on the walls and roofs of animal shelters. Adult Culicoides have been collected on each farm twice a week with an Onderstepoort blacklight suction trap. After each treatment a significant reduction in the abundance of total Culicoides, C. imicola and C. newsteadi s.l., has been observed. The Obsoletus complex density has been affected by the first two treatments. Our results suggest the possibility to control the Culicoides populations with a combination of adulticide and larvicide treatments.

Role of different Culicoides vectors (Diptera: Ceratopogonidae) in bluetongue virus transmission and overwintering in Sardinia (Italy).

BACKGROUND: Bluetongue (BT) epidemics have affected the Mediterranean island of Sardinia since 2000. While Culicoides imicola represents the main bluetongue virus (BTV) vector, other European Culicoides biting midges, possibly implicated in virus transmission, have been detected here. Understanding their distribution, seasonal abundance, and infection rates is necessary to predict disease incidence and spread across coastal and inland areas, and to define their role in virus overwintering. METHODS: Biting midge abundance was determined by light traps on selected farms representing diverse climatic conditions of Sardinia. Livestock-associated Culicoides species were morphologically and molecularly identified. Infection rates in prevailing midge species captured in 2013 during a BTV-1 outbreak were determined using RT-qPCR based virus detection in insect body pools, supplemented by specific body region analyses. The seasonal infection prevalence in Culicoides samples collected in 2001 in a BTV-2 affected farm was also determined. RESULTS: The Newsteadi complex (C. newsteadi species A and species B) prevailed among all biting midge species (47.7 %), followed by C. imicola (27.8 %) and the Obsoletus complex (C. obsoletus and C. scoticus) (17.6 %). Whilst Culicoides imicola was more abundant along the coast, the Newsteadi complex was frequently collected at higher altitude and the Obsoletus complex was notably associated to cattle farms. Culicoides pulicaris and C. punctatus abundance was found to be marginal in all farms. BTV was detected in parous female samples of all these species, and the full dissemination of the virus within the body of C. imicola, C. obsoletus, C. scoticus, and Newsteadi complex species was confirmed by analyses of thorax and head, containing salivary glands. Higher infection rates were associated with C. scoticus, C. newsteadi species A and species B, compared to C. imicola. The virus was detected in C. newsteadi species A and C. obsoletus in winter and spring, whereas it was mainly found in summer and autumn in C. imicola. CONCLUSIONS: In Sardinia, bluetongue virus is transmitted by multiple Culicoides vectors, including C. imicola and the Newsteadi complex being the most important. The Newsteadi complex and other midge species can play an important role in internal areas and are likely to be directly involved in virus overwintering.

The emergence of Schmallenberg virus across Culicoides communities and ecosystems in Europe.

Schmallenberg virus (SBV), a novel arboviral pathogen, has emerged and spread across Europe since 2011 inflicting congenital deformities in the offspring of infected adult ruminants. Several species of Culicoides biting midges (Diptera: Ceratopogonidae) have been implicated in the transmission of SBV through studies conducted in northern Europe. In this study Culicoides from SBV outbreak areas of mainland France and Italy (Sardinia) were screened for viral RNA. The role of both C. obsoletus and the Obsoletus complex (C. obsoletus and C. scoticus) in transmission of SBV were confirmed in France and SBV was also discovered in a pool of C. nubeculosus for the first time, implicating this species as a potential vector. While collections in Sardinia were dominated by C. imicola, only relatively small quantities of SBV RNA were detected in pools of this species and conclusive evidence of its potential role in transmission is required. In addition to these field-based studies, infection rates in colony-derived individuals of C. nubeculosus and field-collected C. scoticus are also examined in the laboratory. Rates of infection in C. nubeculosus were low, confirming previous studies, while preliminary examination of C. scoticus demonstrated that while this species can replicate SBV to a potentially transmissible level, further work is required to fully define comparative competence between species in the region. Finally, the oral competence for SBV of two abundant and widespread mosquito vector species in the laboratory is assessed. Neither Aedes albopictus nor Culex pipiens were demonstrated to replicate SBV to transmissible levels and appear unlikely to play a major role in transmission. Other vector competence data produced from studies across Europe to date is then comprehensively reviewed and compared with that generated previously for bluetongue virus.

Can landscape metrics help determine the Culicoides imicola distribution in Italy?

Culicoides imicola is considered to be one of the main vectors of bluetongue disease in the Mediterranean Basin. However, local variations occur. For example, in Italy, C. imicola is a stable and abundant population in Sardinia and is widely distributed across the island, whilst in Tuscany on the Italian mainland, it ranges from low abundance in the west and coastal areas to absence in the eastern part of the region. Entomological surveillance data collected over 10 years were used to classify 52 sites as low to medium or high C. imicola abundance in Sardinia, and 59 sites as either positive or negative in Tuscany. The land cover was mapped from high-resolution remote sensing images using an object-based image analysis approach and a set of landscape metrics with 500 m buffers around each site. Multivariate analysis was used to test the statistical association of landscape metrics to C. imicola presence and abundance together with other eco-climatic and topographic variables. In Sardinia, 75% of the sites were correctly classified based on altitude alone and the inclusion of landscape- related variables did not improve the classification. In Tuscany, the mean annual temperature allowed classifying 70% of the positive/negative sites correctly. When landscape metrics was included in the multivariate model, an improvement up to 80% was obtained. The presence of riparian vegetation and water was found to be positively correlated with C. imicola presence, whilst forest (including the edge between the forest and cultivated areas) was found to be negatively related to the presence of C. imicola.

Rickettsia slovaca from Dermacentor marginatus ticks in Sardinia, Italy.

Nineteen ticks belonging to the species Dermacentor marginatus, Rhipicephalus bursa, and Haemaphysalis sulcata were collected from wild animals (wild boar, deer, and mouflon) in south-western Sardinia, Italy. Five D. marginatus ticks from wild boar were PCR-positive when analyzed using gltA-specific and ompA-specific primers, leading to the identification and first isolation in cell culture of Rickettsia slovaca, the causative agent of tick-borne lymphadenopathy (TIBOLA), on the island of Sardinia. This study confirms the detection of a new tick-borne rickettsia that can be added to the others already known to be present in Sardinia (Rickettsia aeschlimannii, R. massiliae, and Candidatus Rickettsia barbariae). These data increase our knowledge of tick-borne rickettsioses in Sardinia and, more generally, in the Mediterranean basin.

Pathogens and symbionts in ticks: a survey on tick species distribution and presence of tick-transmitted micro-organisms in Sardinia, Italy.

A total of 1485 adult ticks were collected from mammalian hosts in south-eastern Sardinia, Italy, during the years 2007-2008. Ticks were identified and tested by PCR analysis for presence of Rickettsia species of the spotted fever group, Ehrlichia canis, Anaplasma phagocytophilum, Coxiella burnetii, Bartonella species and Leishmania species. Among all tick species examined (Rhipicephalus sanguineus, Rhipicephalus turanicus, Rhipicephalus bursa, Rhipicephalus pusillus, Hyalomma marginatum marginatum, Haemaphysalis sulcata and Dermacentor marginatus), only Hyalomma marginatum marginatum produced negative results. A total of 22 pools belonging to the three tick species Rhipicephalus sanguineus (0.9 %), Rhipicephalus turanicus (4.5 %) and Rhipicephalus pusillus (100 %) were positive for Rickettsia species, while a total of five pools belonging to Rhipicephalus sanguineus (0.09 %), Haemaphysalis sulcata (16.7 %) and D. marginatus (7.8 %) were positive for E. canis. Five pools of Rhipicephalus turanicus (1.8 %) were positive for A. phagocytophilum. Positivity for C. burnetii was found in seven pools belonging to three tick species: Rhipicephalus sanguineus (0.5 %), Rhipicephalus turanicus (0.3 %) and Haemaphysalis sulcata (4.4 %). Finally, four pools belonging to Rhipicephalus sanguineus (0.09 %), Rhipicephalus turanicus (0.7 %) and Rhipicephalus bursa (1.1 %) were positive for Bartonella species. Leishmania species DNA was not detected in any of the tick pools examined. Data presented here increase our knowledge on tick-borne diseases in Sardinia, and provide a useful contribution to understanding their epidemiology.