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Phytoplasma strains were sporadically found in specimens of Hishimonus hamatus, Japananus hyalinus and Graphocephala fennahi caught during an entomological survey conducted in Southern Switzerland on the edges and woods surrounding cultivated vineyards in 2017. The subsequent molecular characterization of the pathogens allowed to identify them as strains strictly associated to Flavescence doree, a serious European grapevine disease, transmitted from vine to vine by the leafhopper Scaphoideus titanus. In particular, M12 and M50 genotypes were found in H. hamatus and J. hyalinus, respectively, while G. fennahi harboured mixed phytoplasma genotypes with at least two different Flavescence doree isolates. The potential importance of these findings in the frame of the Flavescence doree epidemics in Europe is discussed.

The Nearctic leafhopper species Gyponana (Gyponana) mali DeLong, 1942 is reported from Europe for the fi rst time and represents the fi rst record of the tribe Gyponini Stål, 1870 (Hemiptera: Cicadellidae: Iassinae: Gyponini) for the Palearctic Region. Specimens were collected in southern Switzerland (Ticino) and two regions of northern Italy (Lombardy and Veneto) in 2015–2019. The preferred host plant in these areas appears to be Cornus sanguinea L. Phylogenetic analysis of the COI barcode sequences grouped one of the European specimens with three individuals of G. (G.) mali from Ontario, Canada. Morphological study indicated that the male genitalia of the European population are intermediate between G. (G.) mali and G. (G.) extenda DeLong, 1942.

Limited information is available on the distribution of grapevine red blotch virus (GRBV) in grape-growing regions outside North America. In this study, the presence of GRBV was investigated in 596 grapevine samples coming from different sources in Northern Italy, including rootstock and table grape accessions from ampelographic collections, and commercial varieties with red blotch-like symptoms. Only ‘Queen’ and ‘Incrocio Dalmasso VIII-5’ accessions, collected from the CREA-Viticulture and Enology repository of table grape varieties, tested positive for GRBV. Sequencing of fragments from replication and coat protein genes revealed that the two accessions were infected with GRBV isolates belonging to two distinct lineages. While the American origin of ‘Queen’ could explain the GRBV presence in this accession, the viral infection of the Italian ‘Incrocio Dalmasso VIII-5’ remains an unsolved mystery.

ObjectivesThe WHO Access, Watch and Reserve (AWaRe) classification has been developed to support countries and hospitals in promoting rational use of antibiotics while improving access to these essential medicines. We aimed to describe patterns of worldwide antibiotic use according to the AWaRe classification in the adult inpatient population.MethodsThe Global Point Prevalence Survey on Antimicrobial Consumption and Resistance (Global-PPS) collects hospital antibiotic use data using a standardized PPS methodology. Global-PPS 2015, 2017 and 2018 data, collected by 664 hospitals in 69 countries, were categorized into AWaRe groups to calculate proportional AWaRe use, Access-to-Watch ratios and the most common indications for treatment with selected Watch antibiotics. Only prescriptions for systemic antibiotics on adult inpatient wards were analysed.ResultsRegional Access use ranged from 28.4% in West and Central Asia to 57.7% in Oceania, whereas Watch use was lowest in Oceania (41.3%) and highest in West and Central Asia (66.1%). Reserve use ranged from 0.03% in sub-Saharan Africa to 4.7% in Latin America. There were large differences in AWaRe prescribing at country level. Watch antibiotics were prescribed for a range of very different indications worldwide, both for therapeutic and prophylactic use.ConclusionsWe observed considerable variations in AWaRe prescribing and high use of Watch antibiotics, particularly in lower- and upper-middle-income countries, followed by high-income countries. The WHO AWaRe classification has an instrumental role to play in local and national stewardship activities to assess prescribing patterns and to inform and evaluate stewardship activities.

Acanthamoeba spp. are the causative agents of a sight-threatening infection of the cornea known as Acanthamoeba keratitis (AK). Amphotericin B - deoxycholate (AB) is used in the treatment of infectious keratitis, however, its topical administration has side effects as blepharitis, iritis, and painful instillation. In this context, the preheating of AB can decrease its toxicity by the formation of super aggregates (hAB). hAB associated with a thermoreversible in situ gelling ophthalmic system is a promising option due to the latter biocompatibility, low toxicity, and high residence time on the ocular surface. Our objective was to develop a topical ocular formulation of hAB for the treatment of AK. After heating at 70°C for 20 min, hAB was incorporated into a thermoreversible gelling system. The amebicidal activity of AB and hAB was evaluated against trophozoites and cysts of A. castellanii (ATCC 50492) and a regional clinical isolate (IC01). The results showed that the preheating of AB did not change the pharmacological action of the drug, with the amebicidal effect of AB and hAB under trophozoites and cysts of Acanthamoeba spp. The thermoreversible system remained stable, allowing the increase of drug retention time. For assessment of cytotoxicity, HUVEC (ATCC® CRL-1730) cells were challenged with AB and hAB for 48h. Cell viability was assessed, and hAB did not show cytotoxicity for HUVEC cells. As far as we know this was the first study that showed the preheated AB associated with a thermoreversible in situ gelling ophthalmic system as a promising system for topical ocular topical administration of hAB for AK therapy.

Phytoplasmas (Mollicutes, Acholeplasmataceae), vector‐borne obligate bacterial plant parasites, infect nearly 1,000 plant species and unknown numbers of insects, mainly leafhoppers (Hemiptera, Deltocephalinae), which play a key role in transmission and epidemiology. Although the plant–phytoplasma–insect association has been evolving for >300 million years, nearly all known phytoplasmas have been discovered as a result of the damage inflicted by phytoplasma diseases on crops. Few efforts have been made to study phytoplasmas occurring in noneconomically important plants in natural habitats. In this study, a subsample of leafhopper specimens preserved in a large museum biorepository was analyzed to unveil potential new associations. PCR screening for phytoplasmas performed on 227 phloem‐feeding leafhoppers collected worldwide from natural habitats revealed the presence of 6 different previously unknown phytoplasma strains. This indicates that museum collections of herbivorous insects represent a rich and largely untapped resource for discovery of new plant pathogens, that natural areas worldwide harbor a diverse but largely undiscovered diversity of phytoplasmas and potential insect vectors, and that independent epidemiological cycles occur in such habitats, posing a potential threat of disease spillover into agricultural systems. Larger‐scale future investigations will contribute to a better understanding of phytoplasma genetic diversity, insect host range, and insect‐borne phytoplasma transmission and provide an early warning for the emergence of new phytoplasma diseases across global agroecosystems., Phytoplasmas are a diverse group of obligate intracellular bacterial parasites, and diseases associated with these bacteria are among the most important problems affecting agriculture worldwide. However, most knowledge of the diversity and ecology of phytoplasmas and their hosts has been accumulated through studies of phytoplasma disease epidemiology in agroecosystems. In light of recent attention to the importance of wildlife as reservoirs of emergent diseases, we screened potential insect vectors collected in natural areas and preserved in museum biorepositories, 6 of them were positive for the presence of phytoplasmas.

Os manejos florestais constituem-se como uma alternativa de extracao florestal sustentavel. O objetivo deste artigo e demonstrar uma analise financeira para a implantacao de um manejo florestal no Estado de Mato Grosso. Metodologicamente foram utilizadas ferramentas financeiras, tais como: VPL (Valor Presente Liquido), TIR (Taxa Interna de Retorno), PAYBACK, alem de um levantamento de custos (fixos e variaveis). Para tanto, foi realizada uma pesquisa de campo e uma revisao bibliografica, ambas desenvolvidas criteriosamente, envolvendo custos relativos a implantacao de manejos florestais. Os resultados demonstram que os custos de implantacao de um manejo florestal com area de 917,88 hectares em Mato Grosso representam 6,95% do total das receitas obtidas com a comercializacao da madeira extraida, sendo que os custos estao estimados em R$ 1.257.974,47 e as receitas em R$ 18.091.564,41. Ao longo de 30 anos de UPA (Unidade de producao anual) 100%, em que o valor anual foi de R$ 603.052,15, tornando-se necessario um pouco mais de 2 dois anos para as receitas igualarem-se aos custos de implantacao demonstrando que o manejo florestal e uma alternativa rentavel e ao mesmo tempo que contribui para a reducao dos indices de desmatamento e de extracao ilegal. Os resultados dos indicadores financeiros demonstraram que a TIR foi de 48%, esta taxa e considerada elevada segundo a literatura economica. Em relacao ao VPL, levando em conta os fluxos de caixa em uma taxa de 10%, resultou em um valor de R$ 4.392.413,52, sendo este considerado positivo ao investimento.

Arachis hypogaea L. (peanut) leaf is traditionally used for the treatment of insomnia in Asia. However, studies describing the safety and toxicity profile for this plant preparation are limited. Thus, the goal of this study was to investigate the toxicity of peanut leaf hydroalcoholic extract (PLHE) repeated treatment. The extract was administered orally (100, 300 or 1000 mg/kg) in male and female Wistar rats for 28 days (OECD guideline 407). PLHE treatment did not cause mortality or weight variation in the animals. Also, there was no alteration on locomotor activity (open field test), motor coordination (rotarod test), or anxiety behaviour (elevated plus-maze test). Male rats had a reduction in relative liver weight (100 mg/kg) and an increase in total kidney weight (1000 mg/kg), but there was no change in biochemical and haematological parameters after PLHE treatment. Free extracellular double-stranded DNA (dsDNA) levels was also evaluated, but PLHE treatment did not increase this parameter in rat organs. Also, the dose of 1000 mg/kg of PLHE significantly increased the total thiols in the liver of females compared with the control animals. Thus, PLHE did not induce toxicity after repeated exposure for 28 days in rats.

Essential oils are considered an alternative for replacement of conventional insecticides that have provoked an increasing number of resistant species and damages to health. The objective of this work was to investigate the insecticidal activity of Lavandula dentata L. (Lamiales: Lamiaceae) oil against the species Musca domestica L. (Diptera: Muscidae) and Chrysomya albiceps Wiedemann (Diptera: Calliphoridae). Extraction of the essential oil from the aerial parts of the plant was carried out using hydro-distillation. Its principal compounds (1,8-cineol, camphor, and linalool oxide) were identified. Insecticidal activity was determined by evaluating adulticidal effect using topical application methods and exposure to impregnated paper; larvicidal effect was determined using immersion method. The essential oil presented toxicity in M. domestica and C. albiceps adults; the lethal concentration values (LC50) in the superficial application method were respectively 3.13 ± 0.64 and 1.39 ± 0.19% live weight (l/v). Toxicity was also found in the oil impregnated paper exposure test; the LC50 results for M. domestica and C. albiceps were respectively 4.15 ± 0.64 and 5.14 ± 0.81%. Larvicidal effect was observed on third stage M. domestica larvae when exposed to an oil concentration of 2.5% (m/v). In addition, the oil was evaluated for cytotoxicity, mutagenicity, and genotoxicity in human cells, and the in vitro safety of this oil in human cells was verified. Thus, L. dentata oil presented insecticidal activity in M. domestica and C. albiceps and can be used as an alternative for control of these dipterans.

Hydrophilic matrices are of utmost interest for oral prolonged release of drugs. However, they show decreasing release rate over time, mainly due to lengthening of the diffusional pathway across the gel formed upon glass-rubber transition of the polymer. Therefore, achievement of zero-order release kinetics, which could reflect in constant drug plasma levels, is still an open issue. With the aim of improving the release performance of hydroxypropyl methylcellulose (HPMC) systems, the use of cellulolytic enzymes was proposed to aid erosion of the swollen matrix, thereby counteracting the release rate decrease particularly toward the end of the process. The effectiveness of this strategy was evaluated by studying the mass loss and drug tracer release from tableted matrices consisting of high-viscosity HPMC (Methocel® K4M), Acetaminophen and increasing amounts (0.5–10% on HPMC) of a cellulolytic product (Sternzym® C13030). A faster erosion and progressive shift to linearity of the overall release profiles were observed as a function of the enzyme concentration. Release was markedly linear from matrices containing 5 and 10% Sternzym® C13030. In partially coated matrices with these cellulase concentrations, such results were in agreement with data of erosion and swelling front movement, which exhibited early and long-lasting synchronization.

Background: The point prevalence survey of healthcare-associated infections (HAIs) and antimicrobial use organized by the European Centre for Disease Prevention and Control (ECDC-PPS) and the Global Point Prevalence Survey of antimicrobial consumption (Global-PPS) were simultaneously performed in Belgian acute care hospitals in 2017. Methods: Belgian acute care hospitals were invited to participate in either the ECDC or Global-PPS. Hospital/ward/patient-level data were collected between September-December 2017. All patients present in the wards at 8 a.m. on the day of the PPS were included. The data of the ECDC and Global-PPS on antimicrobial consumption were pooled. Detailed data on HAIs were analysed for ECDC-PPS. Results: Overall, 110 Belgian acute care hospital sites participated in the ECDC and Global-PPS (countrywide participation rate: 81.4%, 28,007 patients). Overall, a crude prevalence of patients with at least one antimicrobial of 27.1% (95% confidence interval (CI) 26.5-27.6%) was found. The most frequently reported indications were pneumonia (23.2%), urinary tract infections (15.2%) and skin and soft tissue infections (11.9%). The reason for antimicrobial use was recorded for 81.9% of the prescriptions, a stop/review date for 40.8% and compliance with local antibiotic guidelines for 76.6%. In the ECDC-PPS, the crude prevalence of patients with at least one HAI was 7.3% (95%CI 6.8-7.7%). Most frequently reported HAIs were pneumonia (21.6%) and urinary tract infections (21.3%). Conclusions: HAI and antimicrobial use prevalence remained stable in comparison with the previous PPS (7.1% and 27.4% in 2011 and 2015, respectively). Belgian hospitals should be further stimulated to set local targets to improve antibiotic prescribing and reduce HAI., SCOPUS: ar.j, info:eu-repo/semantics/published

The involvement of the immunodominant membrane protein Imp of 16SrV-D phytoplasma in the interaction with insect proteins was investigated. The His-tagged partial Imp protein and a rabbit polyclonal antibody were produced and used in Far-Western dot Blot (FWdB) assays. Total native proteins were extracted from batches of six insect species (Scaphoideus titanus, Euscelidius variegatus, Macrosteles quadripunctulatus, Ricania speculum, Metcalfa pruinosa and Zyginidia pullula). FWdB showed interaction of Imp fusion protein with total proteins of S. titanus, E. variegatus (known as efficient vectors) and M. quadripuncltulatus (potential vector), while no interaction signal was detected with the other three species that are reported as non-phytoplasma vectors.

Background The incidence of nosocomial infections due to carbapenem-resistant Klebsiella pneumoniae is increasing worldwide. Whole-genome sequencing (WGS) can help elucidate the transmission route of nosocomial pathogens. Methods We combined WGS and epidemiological data to analyze an outbreak of New Delhi metallo-β-lactamase (NDM)-producing K. pneumoniae that occurred in 2 Belgian hospitals situated about 50 miles apart. We characterized 74 NDM-producing K. pneumoniae isolates (9 from hospital A, 24 from hospital B, and 41 contemporary isolates from 15 other Belgian hospitals) using pulsed-field gel electrophoresis and WGS. Results A K. pneumoniae sequence type 716 clone was identified as being responsible for the outbreak with all 9 strains from hospital A and 20 of 24 from hospital B sharing a unique pulsotype and being clustered together at WGS (compared with 1 of 41 isolates from other Belgian hospitals). We identified the outpatient clinic of hospital B as the probable bridging site between the hospitals after combining epidemiological, phylogenetic, and resistome data. We also identified the patient who probably caused the transmission. In fact, all but 1 strain from hospital A carried a Tn1331-like transposon, whereas none of the hospital B isolates did. The patient from hospital A who did not have the Tn1331-like transposon was treated at the outpatient clinic of hospital B on the same day as the first NDM-producing K. pneumoniae-positive patient from hospital B. Conclusions The results from our WGS-guided investigation highlight the importance of implementing adequate infection control measures in outpatient settings, especially when healthcare delivery moves from acute care facilities to outpatient clinics., SCOPUS: ar.j, info:eu-repo/semantics/published

This test performance study (TPS) was carried out on DNA samples from grapevine, clematis, fungi and bacteria to compare and validate loop-mediated isothermal amplification (LAMP) tests for detection of Grapevine flavescence dorée phytoplasma and 'Candidatus Phytoplasma solani' (Grapevine Bois noir phytoplasma). Two LAMP tests, for Grapevine flavescence dorée phytoplasma and 'Candidatus Phytoplasma solani' (as developed by Kogov?ek and colleagues), with proven applicability for rapid laboratory or on-site detection were included in this study. They were performed in 10 laboratories. In addition, the commercial Qualiplante/Hyris isothermal amplification test for Grapevine flavescence dorée phytoplasma was performed in three laboratories. The accuracy of the three tests was shown to be over 98%. Moreover, the high accuracy of these tests, which used different devices across different laboratories, confirmed their reproducibility.

In light of the evidence that in contrast to most healthy tissues, several neoplasms overexpress fatty acid synthase (FASN) upon their dependence on increased lipogenesis; targeting of this protein is being considered as a valuable strategy in anticancer drug development. This can be particularly relevant for aggressive tumors such as melanoma in which FASN overexpression has been associated with increased depth of invasion and worse prognosis. We have previously shown that a sub-class of cyclic imides, the N-phenylmaleimides, presented antitumor activity against L1210 leukemia and B16F10 melanoma with evidences of interference in the energetic metabolism. Here, we aimed to investigate if some selected N-phenylmaleimides (M1 and M5) interfere with fatty acids metabolism and its relation with cancer. For that, a model of pre-adipocytes differentiation (3T3-L1 cells) and also human melanoma cells (SK-Mel-147) were used. As results, when 3T3-L1 cells were exposed to non-cytotoxic concentrations of M1 and M5 in the presence of an adipogenic cocktail, intracellular lipid content decreased by 26-36%, marking the inhibition of adipocyte differentiation. High selectivity indexes were obtained for both compounds for tumoral cells. Cell cycle phases analysis revealed a remarkable proportion of cells with DNA fragmentation after their exposure to M1 and M5. This was correlated to both apoptosis and necrosis, showed by Annexin-V/PI assay. Furthermore, M1 and M5 reduced FASN expression by 19-39%, respectively. In conclusion, M1 and M5 presented antiadipogenic and antitumoral activities. The antitumoral activity that was associated to apoptosis and necrosis is a possible consequence of the FASN reduction, which in turn, might result in a fuel decrease to cell proliferation. As it happens with antiangiogenic activity, reduction of fatty acid synthesis might be a potential target for cancer treatment in a strategy of hunger-strike, which valorizes these N-phenylmaleimides as candidates for drug development.

Aims:In this study, binding between the immunodominant membrane protein Imp of the 16SrV-D phytoplasma associated with Flavescence dorée disease (FD-Dp) and insect proteins of vectors and non-vectors of FD-Dp was tested. Methods and results:Six Auchenorrhyncha species, from distantly related groups were selected: Scaphoideus titanus, Euscelidius variegatus, Macrosteles quadripunctulatus, Zyginidia pullula (Cicadomorpha), Ricania speculum and Metcalfa pruinosa (Fulgoromorpha). The vector status of each species was retrieved from the literature or determined by transmission trials in this study. A His-tagged partial Imp protein and a rabbit polyclonal antibody were synthesized and used for Western and Far-Western dot Blot (FWdB) experiments. Total native and membrane proteins (MP) were extracted from entire bodies and organs (gut and salivary glands) of each insect species. FWdB showed decreasing interaction intensities of Imp fusion protein with total proteins from entire bodies of S. titanus, E. variegatus (competent vectors) and M. quadripunctulatus (non-vector), while no interaction signal was detected with the other three species (non-vectors). A strong signal detected upon interaction of FD-D Imp and MP from guts of closely related insects supports the role of this organ as the first barrier to ensure successful transmission. Conclusions:Our results showed that specific Imp binding, correlated with vector status, is involved in interactions between FD-Dp and insect proteins. Significance and impact of the study:Integrating knowledge on host-pathogen protein-protein interactions and on insect phylogeny would help to identify the actual range of vectors of phytoplasma strains of economic importance.

Although inflammation is a biological phenomenon that exists to protect the host against infections and/or related problems, its unceasing activation results in the aggravation of several medical conditions. Imidazoles, whether natural or synthetic, are molecules related to a broad spectrum of biological effects, including anti-inflammatory properties. In this study, we screened eight novel small molecules of the imidazole class synthesized by our research group for their in vitro anti-inflammatory activity. The effect of the selected molecules was confirmed in an in vivo inflammatory model. We also analyzed whether the effects were caused by inhibition of nuclear factor kappa B (NF-κB) transcription factor transmigration. Of the eight imidazoles tested, methyl 1-allyl-2-(4-fluorophenyl)-5-phenyl-1H-imidazole-4-acetate (8) inhibited nitric oxide metabolites and pro-inflammatory cytokine (TNF-α, IL-6, and IL-1β) secretion in J774 macrophages stimulated with LPS. It also attenuated leukocyte migration and exudate formation in the pleural cavity of mice challenged with carrageenan. Furthermore, imidazole 8 reverted the oxidative stress pattern triggered by carrageenan in the pleural cavity by diminishing myeloperoxidase, superoxide dismutase, catalase, and glutathione S-transferase activities and reducing the production of nitric oxide metabolites and thiobarbituric acid-reactive substances. Finally, these effects can be attributed, at least in part, to the ability of this compound to prevent NF-κB transmigration. In this context, our results demonstrate that imidazole 8 has promising potential as a prototype for the development of a new anti-inflammatory drug to treat inflammatory conditions in which NF-κB and oxidative stress play a prominent role. Graphical Abstract ᅟ.

The most important Grapevine Yellows (GY) phytoplasma diseases in Europe are Flavescence doree (FD) and Bois noir (BN); they are spread in vineyard by two proven vectors, Scaphoideus titanus Ball and Hyalesthes obsoletus Signoret, respectively. Other potential vectors of GY have been identified, which are thought to play a secondary role. The GY control strategies are not always effective and an in-depth study on the ecological cycle of the pathogens at regional scale would be of paramount importance. This study was carried out in 48 representative sites of wine-growing area South of Swiss Alps, with the aim to identify known and potential vectors and to characterize the FD and BN phytoplasmas isolates. Out of 167 Auchenorrhyncha species recorded, 27 were known or potential vectors of phytoplasmas and five of those tested positive for phytoplasmas. S. titanus was infected by 16SrV-D subgroup phytoplasma and no clear relationship between its population density and disease outbreaks was observed. Orientus ishidae harboured 16SrV-C and 16SrV-D subgroups suggesting its potential role in spreading 16SrV-C phytoplasma isolates from arboreal plants to grapevine and FD-D from grapevine to grapevine. H. obsoletus was infected by BN phytoplasmas, tuf-types a and b, however it was collected with relatively low abundance. Reptalus panzeri and R. cuspidatus tested positive to tuf-type b, but only R. cuspidatus was common and abundant in the investigated vineyards. To define the range of alternative vectors using a detailed approach on regional scale provides background information to get a more clear vision on the spreading of phytoplasmas in the vineyards.

Grapevine Pinot gris virus (GPGV), a member of the genus Trichovirus in the family Betaflexiviridae, was recently discovered in Italy and subsequently in other European countries and in Korea. In this study, we assessed the occurrence of GPGV in 441 samples from Western and Eastern Europe collected over the period 2002-2014. The results suggest that the virus had recently appeared in the Veneto region (Northeast Italy) and had been present in some Eastern European countries for at least 10 years. The molecular characterization of the 5'-terminal genomic region of several GPGV isolates from Italy and other European countries showed low polymorphism, with a maximum nucleotide sequence divergence of 3.2%.

In this study, we used an experimental model of congenital hypothyroidism to show that deficient thyroid hormones (TH) disrupt different neurochemical, morphological and functional aspects in the cerebral cortex of 15-day-old offspring. Our results showing decreased glutamine synthetase (GS) activity and Ca2+ overload in the cerebral cortex of hypothyroid pups suggest misregulated glutamate metabolism associated with developmentally induced TH deficiency. The 14C-MeAIB accumulation indicates upregulated System A activity and glutamine uptake by neurons. Energy metabolism in hypothyroid cortical slices was preserved, as demonstrated by unaltered glucose metabolism. We also found upregulated acetylcholinesterase activity, depleting acetylcholine from the synaptic cleft, pointing to disrupted cholinergic system. Increased reactive oxygen species (ROS) generation, lipid peroxidation, glutathione (GSH) depletion, which were associated with glutathione peroxidase, superoxide dismutase and gamma-glutamyltransferase downregulation suggest redox imbalance. Disrupted astrocyte cytoskeleton was evidenced by downregulated and hyperphosphorylated glial fibrillary acidic protein (GFAP). Morphological and structural characterization of the sensorimotor cerebral cortex (SCC) showed unaltered thickness of the SCC. However, decreased size of neurons on the layers II & III and IV in the right SCC and increased NeuN positive neurons in specific SCC layers, suggest that they are differently affected by the low TH levels during neurodevelopment. Hypothyroid pups presented increased number of foot-faults in the gridwalk test indicating affected motor functions. Taken together, our results show that congenital hypothyroidism disrupts glutamatergic and cholinergic neurotransmission, Ca2+ equilibrium, redox balance, cytoskeleton integrity, morphological and functional aspects in the cerebral cortex of young rats.

Ethnopharmacological relevance A prolonged estrogen deficiency alters lipid metabolism and increases risks of cardiovascular diseases. Phytoestrogens, naturally occurring compounds with estrogenic properties are reported to have cardiovascular protective effects. Millettia macrophylla used in the Cameroonian traditional system to treat physiological disorders related to menopause, was previously reported to have estrogenic effects. Aim We, therefore, proposed evaluating the in vitro and in vivo effects of M. macrophylla phenolic fraction on some risk factors for cardiovascular diseases. Material and methods In vitro, the ability of the M. macrophylla phenolic fraction (PF) as well as the 9 isolates to prevent the 3T3-L1 preadipocytes differentiation was assessed. Further, the preventive effects of PF on abdominal fat accumulation, body weight gain, lipid profile, nitric oxide level, superoxide dismutase (SOD) and catalase activities, reduced glutathione (GSH) and malondialdehyde (MDA) levels were assessed in a postmenopausal rat model. Results In vitro, PF and its isolate secundiferol I inhibited lipid accumulation in 3T3-L1 cells. Moreover, all the isolates except daidzein dimethylether prevented the interleukin IL-6 production in 3T3-L1 cells. In vivo, PF prevented ovariectomy-induced abdominal fat accumulation, body weight gain, dyslipidemia, glucose intolerance and decreased atherogenic index. In addition, it induced a vasorelaxant effect by preventing the low level of nitric oxide in the aorta. PF also exhibited antioxidant effects as it increased aorta GSH level, SOD, and catalase activities and decreased MDA level. Conclusions Taken together, our data suggest that PF prevents the increased risks of cardiovascular diseases in ovariectomized rats.

Phytochemical investigation of Pogonopus tubulosus trunk led to the isolation of isotubulosine and alangiside, tetrahydroisoquinoline indolic monoterpene alkaloids reported here for the first time for the family Rubiaceae and the genus Pogonopus, respectively. Isotubulosine proved cytotoxic against MCF-7, PC-3, 786-0, HT-29, and HL-60 human cancer cell lines, with GI50 values ranging from 5.26 to 20.61 μM, in addition to causing G2/M arrest, possibly by inhibiting DNA topoisomerase IIα. Alangiside showed weak cytotoxicity against MCF-7 and HL-60 and proved inactive against PC-3, HT-29, and 786-0 cell lines, with no sign of apoptosis. The alkaloid structures were established on the basis of 1D- and/or 2D-NMR, optical rotation, and HR ESIMS data. Complete 1H NMR assignments of isotubulosine were also performed, using 1H-1H COSY, HSQC, HMBC, NOESY, and 1H J-resolved techniques and employing experimental and calculated values of homonuclear coupling constants based on the lowest energy conformations. The foregoing results provide new information on the cytotoxicity and mechanism of action of tetrahydroisoquinoline indole monoterpene-type alkaloids and reveal isotubulosine to merit further studies as a potential anticancer agent.

Objectives To test the toxicity and antitumoral activity of the compound N-oleyl-daunorubicin (oDNR) with a cholesterol-rich nanoemulsion (LDE) formulation. Methods LDE-oDNR was prepared by high-pressure homogenisation of lipid mixtures. B16F10 melanoma cells and NIH/3T3 fibroblasts were used for cytotoxicity tests. The maximum tolerated dose (MTD) of both commercial and LDE-oDNR was determined in mice, and melanoma-bearing mice were used for the antitumoral activity tests. Key findings CC50 for LDE-oDNR and DNR in melanoma cells were 200 μm and 15 μm, respectively, but LDE-oDNR was less toxic against fibroblasts than DNR. MTD for LDE-oDNR was 65-fold higher than commercial DNR. In tumour-bearing mice, LDE-oDNR (7.5 μmol/kg) reduced tumour growth by 59 ± 2%, whereas the reduction by DNR was only 23 ± 2%. LDE-oDNR increased survival rates (P < 0.05), which was not achieved by DNR treatment. The number of mice with metastasis was only 30% in LDE-oDNR-treated mice, compared with 82% under DNR treatment. By flow cytometry, there were 9% viable cells in tumours of animals treated with LDE-oDNR compared with 27% in DNR-treated animals. Less haematological toxicity was observed in LDE-oDNR-treated mice. Conclusions Compared with DNR, LDE-oDNR improved tumour growth inhibition and survival rates with pronouncedly less toxicity, and thus may become a new tool for cancer treatment.

Acute lymphoblastic leukemia (ALL) is a malignant disorder caused by the proliferation of lymphoid progenitor cells and is the most common cancer in children. Cytotoxic nucleoside analogues are important chemotherapeutic agents, which are used in many cancers, including leukemias. In this study, we investigated the effects of the synthetic nucleoside analogue 1-(5,5,5-trichloro-2-methoxy-4-oxopenten-2-yl)-4-trichloromethyl-pyrimidin-2(1H)-one, named compound 3 or C3, on leukemia cell lines. The compound stimulated cell death by apoptosis, evidenced by DNA fragmentation, phosphatidylserine externalization, and caspase-3 activation. Compound 3 seemed to trigger several cell death pathways. The mitochondrial pathway was evidenced through a disturbance of mitochondrial membrane potential, strong cytochrome c liberation, decrease of antiapoptotic Bcl-2 protein expression, and caspase-9 activation. The C3 also induced caspase-8 and -12 activation, an increase in the intracellular calcium level, and an overproduction of reactive oxygen species. Increased caspase 8 activity suggests that the extrinsic pathway was activated and that the ROS production and enzyme activity alteration (glutathione S-transferase, glutathione peroxidase, catalase, and glutathione reductase) might be related to oxidative stress. Finally, the increase in calcium release, CHOP expression, and caspase-12 activity might characterize endoplasmic reticulum stress. Compound 3 was likewise cytotoxic to leukemic and melanoma human cell lines. Taken together, the results contribute to further understanding the new pyrimidine analogue as a potential chemotherapeutic drug or lead molecule.

2-Amino-1,3-thiazin-4-ones were subjected to acetylation followed by mild acid hydrolysis to give compounds containing the 1,3-thiazine-2,4-dione core. The potential of these S,N-containing heterocycles as antitumor agents against human cancer cell lines, among other types, was evaluated. The results show that phenyl- and naphthyl-substituted thiazinediones presented selective antitumoral activity against leukemia cells. These compounds caused cell death with DNA fragmentation and the mechanism of action seems to involve caspase cascade activation, imbalance in intracellular Ca(2+) and mitochondrial metabolism, and/or endoplasmic reticulum stress.

The mechanisms of muscle wasting and decreased mobility have a major functional effect in rheumatoid arthritis, but they have been poorly studied. The objective of our study is to describe muscular involvement and the pathways in an experimental model of arthritis compared to the pathways in disuse atrophy. Female Wistar rats were separated into three groups: control (CO), collagen-induced arthritis (CIA), and immobilized (IM). Spontaneous locomotion and weight were evaluated weekly. The gastrocnemius muscle was evaluated by histology and immunoblotting to measure the expression of myostatin (a negative regulator), LC3 (autophagy), MuRF-1 (proteasome-mediated proteolysis), MyoD, and myogenin (satellite-cell activation). The significance level was set at P

Hypoxia has been implicated as a possible cause of adipose tissue inflammation. Furthermore, the acute phase protein serum amyloid A (SAA) has been associated with the modulation of the adipogenic process, and it is well-known that obese individuals have increased levels of SAA. The effect of hypoxia in the expression and production of SAA was examined in murine 3T3-L1 adipocytes. Hypoxia leads to a substantial increase in SAA3 mRNA and protein level, apparently in a time-dependent manner (threefold in 48 h), in fully differentiated 3T3-L1, followed by reestablishment of gene expression to basal levels after 24 h of reoxygenation. Hypoxia-induced SAA may be one of the key molecules to the development of the inflammatory response in adipose tissue.

Over the last years we have utilised chimeras from aequorin and green fluorescent protein (GFP) to monitor the dynamics of second messenger levels in living cells. In this contribution we address two problems, i.e. the complexity of Ca2+ handling by mitochondria and the localization of cAMP signalling. As to the first, we here demonstrate that physiological increases in mitochondrial Ca2+, monitored with selectively localized recombinant aequorin, concern a sub-population of organelles that is stably and selectively associated with the endoplasmic reticulum. As to cAMP, we describe the use of a novel probe to monitor its changes in living cells, that takes advantage of the phenomenon of fluorescence resonance energy transfer (FRET) between suitable GFPs linked to the regulatory and catalytic subunits of protein kinase A (PKA). When cAMP is low the two fluorophores are in close proximity and generate FRET while increasing levels of cAMP determine progressive reduction of FRET as the two subunits (linked to the GFPs) diffuse apart. We also demonstrate that by using such cAMP sensor, localized increase of this second messenger can be observed upon selective stimulation of plasma membrane receptors.

This study was undertaken to address the current deficient knowledge of cellular response to solid lipid nanoparticles (SLNs) exposure. We investigated the cytotoxicity of several SLNs formulations in two fibroblast cell lineages, Vero and MDCK. Several methods were used to explore the mechanisms involved in this cytotoxic process, including cell viability assays, flow cytometry and ROS generation assessment. Among nanoparticles tested, two of them (F4 and F5) demonstrated more cytotoxic effects in both cell lineages. The cell viability assays suggested that F4 and F5 interfere in cell mitochondrial metabolism and in lysosomal activity. In addition, F5 decreased the percentage of MDCK cells in G0/G1 and G2/M phases, with a marked increase in the Sub/G1 population, suggesting DNA fragmentation. Regarding F4, although IC50 was higher (~ 700 μg/mL), this formulation affected mitochondrial membrane potential for Vero cells. However, the IC50 of F5 was around 250 μg/mL, suggesting the effect of SDS (sodium dodecyl sulfate) present in the formulation. In summary, the nanoparticles tested here appears to be biocompatible, with the exception of F5. Further studies are required to elucidate the in vivo effects of these nanoscale structures, in order to evaluate or predict the connotation of their increased and widespread use.

At present serological assays for the detection of “flavescence dorée” (FD), a quarantine disease of grapevine, are not available. The aim of this study was to produce polyclonal antibodies targeting a surface abundant protein of FD and related phytoplasmas, Imp (immunodominant membrane protein), and to verify its use for the detection of FD phytoplasmas in grapevine, other host plant species, and insect vectors. Two antisera were obtained using recombinant Imp proteins from two FD strains. Preliminary results confirmed the specificity and good ELISA performance in the detection of the target in grapevine, clematis and insect vectors tissues.

The constant, even if low, presence of grapevine “flavescence doree” (FD) in the vineyards of Ticino, Switzerland, challenged the stakeholders to investigate for other players involved in the FD dynamics. This study was aimed to explore the impact that the exotic leafhopper Orientus ishidae may have on FD epidemics in Ticino by monitoring its population density and level of infection with 16SrV group phytoplasmas. In 2018, 283 leafhoppers were collected from three FD infected areas, both from the vineyards and the surrounding landscape. Spatial density maps showed that the captures occurred mostly in the landscape, with a clear clustered distribution among the traps. Molecular analyses revealed a high infection percentage of O. ishidae samples in the three investigated plots, both inside and outside the vineyards.

A survey of genetic diversity of “flavescence doree” (FD)-related phytoplasmas in grapevines, alders and clematis as well as alder-feeding leafhoppers was conducted in France, Hungary, Germany, Italy and Serbia. Genotyping was based on the housekeeping gene map and on the vmp genes encoding surface variable membrane proteins. Transmission assays of the phytoplasmas were performed with alder and/or grapevine-feeding leafhoppers. The study demonstrated that European alders constitute an original reservoir of FD phytoplasma by hosting a high diversity of FD-related phytoplasma genotypes, also present in non viticultural areas. The alder phytoplasmas grouping in Vmp-I cluster were transmitted by the Macropsinae O. alni, but were not compatible with the FD phytoplasma vector on grapevine the Deltocephalinae Scaphoideus titanus. The alder phytoplasmas in Vmp-II and-III clusters were transmitted by the Deltocephalinae Allygus spp. and Orientus ishidae. Such pre-existing phytoplasmas were compatible with S. titanus transmissibility and can be responsible for the emergence of FD phytoplasma epidemics in grapevine. VmpA proteins of cluster II better adhered to Euscelidius variegatus and S. titanus insect cells and midguts than those of cluster I. Such adhesins might play a key role in the adaptation to new vectors.

Skeletal muscle repair can be understood as a balance between fibrosis and regeneration, the result of which may lead to complete recovery or loss of muscle function. To study the involvement of nitric oxide in post-trauma muscle repair, we used an experimental murine model of crush injury muscle. The animals were divided into four groups, (i) control (CO), (ii) sham trauma, (iii) trauma and (iv) trauma+l-NAME. The animals received a single dose of 100mg/kg of the l-NAME, an inhibitor of nitric oxide synthase, 2h after lesion, and the muscle tissue was analyzed in two time-points: 24h and 7days. Twenty-four hours after injury, the crushed muscle was characterized by an intense inflammatory cell infiltrate and edema demonstrated by histological analysis. These changes were accompanied by increased iNOS, MMP-2 and HGF mRNA transcription and protein expression of the iNOS and MMP-2 in the gastrocnemius muscle. Crushing injury also promoted cell proliferation and increase number satellite cell, responsible for the regeneration of the muscle fiber. Treatment with l-NAME blocking local NO production, greatly attenuated these histological and molecular findings at 24h. On the 7th day the molecular findings of both groups were comparable to the control (sham trauma) group. However, the l-NAME group showed increase deposition of collagen and decrease of SC expression. These findings demonstrate that activation of NO during muscle crush is critical in the early phases of the skeletal muscle repair process and indicate its possible role as a regulator of the balance between fibrosis and muscle regeneration.

We evaluated the performance of the automated Vitek 2 system against disk diffusion for susceptibility testing of Staphylococcus aureus strains showing various resistance mechanisms to macrolides and lincosamides (ML). The Vitek 2 system showed 100% concordance with the D-zone test in detection of the most common resistance mechanisms to ML, including methylase and efflux systems.

International audience; A survey was conducted over several years in Italy and the Balkans in order to gain an understanding of the relationship between the Flavescence doree (FD) phytoplasma isolates found in clematis and grapevine. A total of 399 clematis and 107 grapevine samples were analyzed. The results showed that 36% of the Clematis vitalba plant samples were infected by phytoplasmas which, in grapevine, are associated with FD, a quarantine disease in Europe. Infected clematis plants were also found in areas where FD phytoplasma had never previously been reported to infect grapevine, Such as Macedonia, Croatia and some areas of Italy and Serbia. Molecular data from three phytoplasma genomic fragments showed the presence of different FD phytoplasma isolates, all belonging to the 16SrV-C subgroup, including the Italian FD-C isolate, the isolate found in Serbia, an isolate similar to the French FD2000 and a new isolate typical of central Italy. A few clematis plants were infected with single nucleotide polymorphism, insertion or deletion mutants of the FD-C isolate. Of all the potential Hemipteran vector species surveyed in Italy and Serbia, only 18 of 527 Dictyophara europaea individuals tested proved to he infected with the FD phytoplasma. Preliminary transmission experiments showed that this species is able to transmit the FD phytoplasma from clematis to grapevine. The presence of FD-infected clematis and of D. europaea could, therefore, constitute a risk for FD epidemics in the European viticultural regions.

The present work was carried out in order to verify the possible association between a new grapevine disease, characterized by leaf mottling and deformation, and the genetic variability and concentration of Grapevine Pinot gris virus (GPGV), a recently identified virus tentatively associated with the pathology. After vineyard surveys and the establishment of real-time qPCR assays, characterization of GPGV isolates and evaluation of GPGV titre were assessed in more than 100 samples of grapevine Glera, collected from plants regardless of whether or not they showed the symptomatology. Results showed that there was an important association between the GPGV variants and manifestation of the symptoms, and that grapevines with symptoms harboured significantly higher GPGV titre than symptomless vines. Moreover, an interesting relationship among the phylogenetic clustering of the isolates originating from plants with symptoms and some epidemiological characteristics of the disease was found. The current study confirmed the role of GPGV in the emergent disease characterized by grapevine leaf mottling and deformation.

When a Palearctic bacterium meets a Nearctic insect vector: genetic and ecological insights into the emergence of the grapevine ‘[i]Candidatus Phytoplasma vitis[/i]’ and ‘[i]Ca. P. vitialni[/i]’ epidemics in Europe. 25. International Congress of Entomology (ICE 2016)

This study investigated the apoptotic effect of electronic waste on fibroblast cell line. Cells were treated with different concentrations of the leachate for 24 h. Cell viability was detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, nuclear morphology of cells was explored by acridine orange (AO)/ethidium bromide (EB) double staining, mitochondrial membrane potential was evaluated using JC-1 probe while cell cycle analysis was conducted using flow cytometry. The oxidative status was detected using DCFH-DA (dichlorofluorescin diacetate) probe and the relationship between cell death and ROS (reactive oxygen species) was investigated using N -acetylcysteine. Results showed an increased cell death as detected by MTT assay and AO/EB staining. Cell cycle analysis indicated an induction of sub/G1 events while JC-1 probe showed significant disruption of mitochondrial membrane potential. There was significant induction of ROS, while N -acetylcysteine protected the cells from DNA damage. These suggest apoptotic pathway as a possible mechanism of e-waste induced cyto-genotoxicity.

Background and Objective To investigate the effects of low-level laser therapy (LLLT) on nuclear factor kappa B (NF-κB) activation and inducible nitric oxide synthase (iNOS) expression in an experimental model of muscle trauma. Study Design/Materials and Methods Injury to the gastrocnemius muscle in the rat was produced by a single impact blunt trauma. A low-level galium arsenide (Ga–As) laser (904 nm, 45 mW, and 5 J/cm2) was applied for 35 seconds duration, continuously. Results Histological abnormalities with increase in collagen concentration, and oxidative stress were observed after trauma. This was accompanied by activation of NF-κB and upregulation of iNOS expression, whereas protein concentration of IκBα decreased. These effects were blocked by LLLT. Conclusion LLLT reduced the inflammatory response induced by trauma and was able to block the effects of reactive oxygen species (ROS) release and the activation of NF-κB. The associated reduction of iNOS overexpression and collagen production suggest that the NF-κB pathway may be a signaling route involved in the pathogenesis of muscle trauma. Lasers Surg. Med. © 2006 Wiley-Liss, Inc.

Maleimides consist of an important class of compounds easily synthesized with multiple functional group modification that provides expressive pharmacological properties including, antitumoral activity, mediated mainly by oxidative stress. For this reason, the present study was designed to evaluate the cytotoxicity and the role of reactive oxygen species (ROS) in maleimide-induced cell death. Cell viability assays were performed to determine the cell death type in leukemia cell line induced by the compounds. The oxidative stress in maleimidetreated cells was characterized by antioxidant enzymes activities, intracellular ROS generation, and lipid peroxidation. In addition, we evaluated mitochondrial membrane potential and ATP level. Maleimide derivatives exhibited cytotoxic effects in leukemia cells with significantly increased ROS generation. However, cell viability was partly restored by catalase-treated cells. Caspases activities and caspase-independent key genes related to apoptosis were not altered by maleimides, suggesting necrosis as the main cell death process. Maleimide-induced necrosis was associated with oxidative stress, as an imbalance between ROS levels and glutathione reductase (GR) activity. This damage was also demonstrated by loss of mitochondrial membrane potential (MMP) and ATP depletion in cells treated with maleimide derivatives. These findings strongly confirmed that maleimide derivatives promoted cell death in leukemia cells triggered by oxidative stress, indicating that these compounds might be promising antitumor agents or lead molecules.

2-Amino-1,3-thiazin-4-ones were subjected to acetylation followed by mild acid hydrolysis to give compounds containing the 1,3-thiazine-2,4-dione core. The potential of these S,N-containing heterocycles as antitumor agents against human cancer cell lines, among other types, was evaluated. The results show that phenyl- and naphthyl-substituted thiazinediones presented selective antitumoral activity against leukemia cells. These compounds caused cell death with DNA fragmentation and the mechanism of action seems to involve caspase cascade activation, imbalance in intracellular Ca(2+) and mitochondrial metabolism, and/or endoplasmic reticulum stress.

The serum amyloid A (SAA) protein is known to function in the acute phase response and immunoregulation. Recently, SAA has been shown to be involved in cell proliferation, differentiation and migratory behavior in different cell types. Here, we evaluated whether exogenous SAA could influence trophoblast invasion and differentiation using both the trophoblast-like BeWo cell line and fully differentiated human extravillous trophoblast cells (EVT) isolated from term placentae. SAA stimulated BeWo cell invasion, as measured in Matrigel invasion assays, and induced metalloprotease mRNA expression and activity. Given that BeWo cells express Toll-like receptor 4 (TLR4), a known receptor for SAA, we examined the role of TLR4 in SAA-induced invasion using a TLR4 neutralizing antibody. We also tested whether SAA could affect markers of trophoblast syncytialization in BeWo cells. We observed that SAA decreased βhCG secretion and did not influence trophoblast syncytialization. Using EVT cells isolated from human term basal plates, we confirmed that SAA at 1 and 10 µg/mL doubled EVT invasion in a TLR4-dependent manner, but at 20 µg/mL inhibited EVT cells invasiveness. In addition, we observed that SAA was expressed in both BeWo cells and human term placentae, specifically in the syncytiotrophoblast, decidual cells and EVT. In conclusion, SAA was identified as a molecule that functions in the placental microenvironment to regulate metalloprotease activity and trophoblast invasion, which are key processes in placentation and placental homeostasis.

Considerando que a SAA é uma proteína de fase aguda e que uma concentração elevada desta proteína é observada em pacientes obesos e com resistência à insulina, estimulou-se pré-adipócitos murinos 3T3-L1 a baixas concentrações de rSAA durante o processo de proliferação e diferenciação. Ensaios de incorporação de [metil-3H]-timidina, ciclo e viabilidade celular por citometria de fluxo foram realizados, assim como genes adipogênicos foram determinados durante a fase de diferenciação. Ainda, investigou-se a participação da rSAA metabolismo da glicose, bem como a expressão do seu receptor GLUT4 e os perfis de lipólise. Como resultados, obteve-se que a rSAA causou um aumento na proliferação celular assim como na porcentagem de células na fase S. Este efeito foi dose dependente e mediado via sinalização da ERK1/2. Ainda, rSAA inibiu a diferenciação por meio da diminuição da expressão de fatores transcrição (PPARγ, C/EBPβ e C/EBPα) e proteínas adipogênicas (FABP4 e perilipina). Em relação à captação de 2-desoxi-[1,2-3H]-D-glicose, a rSAA preveniu este processo, corroborando com os resultados de expressão diminuída receptor GLUT4. Ainda, o aumento da lipólise provocada pela rSAA, favorece resistência à insulina no modelo estudado. Portanto, conclui-se que a SAA aumenta a proliferação mas inibe a diferenciação de células 3T3-L1 sugerindo papel importante desta proteína no processo de adipogênese. Considering that SAA is an acute phase protein and increased serum levels are associated with chronic hyperglycemia, insulin resistance and obesity, we first examined the possibility that rSAA could affect proliferation and differentiation 3T3-L1 preadipocytes. 3T3-L1 adipocytes were treated with recombinant human SAA and [methyl-3H]-thymidine incorporation, flow cytometric analysis of cell cycle and viability were performed. Also, gene expression profiles of adipogenic factor were performed during differentiation protocol as well as glucose uptake, GLUT4 gene expression and lipolysis assay. rSAA caused an increment in cell proliferation consisted with FACS analysis with a percentage of cells in the S phase. Cell proliferation was mediated by ERK1/2 signaling pathway and in dose-dependent manner. Also, SAA inhibited differentiation process by decreasing adipogenic genes PPARγ, C/EBPβ, C/EBPα, and proteins FABP4, and perilipin expression. Also, rSAA prevented 2-deoxy-[1,2-3H]-glucose uptake and GLUT4 expression. In addiction, lipolysis was increased favoring insulin resistance in 3T3L1 adipogenic model. In conclusion, it was demonstrated that rSAA enhances proliferation but prevents differentiation in 3T3-L1 adipocytes, supporting a even more complex participation, than previously expected, of inflammatory proteins the adipogenic process.

In addition to the grapevine flavescence dorée phytoplasmas, other members of taxonomic group 16SrV phytoplasmas infect grapevines, alders and species of the genera Clematis and Rubus in Europe. In order to investigate which phytoplasmas constitute discrete, species-level taxa, several strains were analysed by comparing their 16S rRNA gene sequences and a set of five housekeeping genes. Whereas 16S rRNA gene sequence similarity values were >97.5 %, the proposed threshold to distinguish two ‘Candidatus Phytoplasma’ taxa, phylogenetic analysis of the combined sequences of the tuf, rplV-rpsC, rplF-rplR, map and uvrB-degV genetic loci showed that two discrete phylogenetic clusters could be clearly distinguished. The first cluster grouped flavescence dorée (FD) phytoplasmas, alder yellows (AldY) phytoplasmas, Clematis (CL) phytoplasmas and the Palatinate grapevine yellows (PGY) phytoplasmas. The second cluster comprised Rubus stunt (RS) phytoplasmas. In addition to the specificity of the insect vector, the Rubus stunt phytoplasma contained specific sequences in the 16S rRNA gene. Hence, the Rubus stunt phytoplasma 16S rRNA gene was sufficiently differentiated to represent a novel putative taxon: ‘Candidatus Phytoplasma rubi’.

Three real-time PCR systems for direct detection of phytoplasmas associated to Flavescence doree (FD), Bois noir (BN) and aster yellows (AY) diseases were developed. TaqMan probes and primers were designed on the 16S ribosomal RNA sequences of phytoplasma genome. A further TaqMan assay, targeting a grapevine gene encoding for the chloroplast chaperonin 21, was developed in order to check the DNA quality and to verify the absence of PCR inhibition. A comparison between real-time PCR and conventional nested-PCR methods for phytoplasma detection was carried out on several reference samples from grapevine, periwinkle, other host plants and insect species. Detection of FD, BN and AY phytoplasma DNA on infected specimens was rapid, specific and reproducible. Sensitivity was as high as nested-PCR assay. The two procedures were then used on about 450 samples collected from grapevines showing yellows symptoms. The results showed that real-time PCR approach for phytodiagnostic purposes was more advantageous than nested-PCR method with regard to rapidity of the assay and reduced risk of sample cross contamination. These new protocols represent an improvement of existing analytical methods and could be used as a reliable diagnostic procedure in certification and control programs.

The role of mitochondria in Ca2+ handling has acquired renewed interest in recent years in the field of cell signaling. Detailed studies of Ca2+ dynamics in this organelle at the single cell level have been hampered by technical problems in the available Ca2+ probes. Some of the latest generation GFP-based Ca2+ probes (Camgaroos, Cameleons and Pericams) show great potential to address this issue. Our data show that the choice of targeting sequence influences not only the overall efficiency of subcellular localization of the probes, but also their functional characteristics within the matrix. In particular, we here show that the use of a tandemly duplicated mitochondrial targeting sequence is capable of improving the delivery efficacy of all tested probes into the organelle's matrix, in particular that of Cameleon, a GFP-based Ca2+ probe that is otherwise largely mistargeted to the cytosol. The devised strategy should be generally applicable to other proteins that are characterized by poor targeting. Last, but not least, we also demonstrate that if the targeting sequence is not removed from the imported protein, the fluorescent properties and the Ca2+ affinity of the probe can be grossly affected.

We here describe a new molecularly engineered green fluorescent protein chimera that shows a high sensitivity to pH in the alkaline range. This probe was named mtAlpHi, for mitochondrial alkaline pH indicator, and possesses several key properties that render it optimal for studying the dynamics of mitochondrial matrix pH, e.g. it has an apparent pK(a) (pK(a)') around 8.5, it shows reversible and large changes in fluorescence in response to changes in pH (both in vitro and in intact cells), and it is selectively targeted to the mitochondrial matrix. Using mtAlpHi we could monitor pH changes that occur in the mitochondrial matrix in a variety of situations, e.g. treatment with uncouplers or Ca(2+) ionophores, addition of drugs that interfere with ATP synthesis or electron flow in the respiratory chain, weak bases or acids, and receptor activation. We observed heterogeneous pH increases in the mitochondrial matrix during Ca(2+) accumulation by this organelle. Finally, we demonstrate that Ca(2+) mobilization from internal stores induced by ionomycin and A23187 cause a dramatic acidification of the mitochondrial matrix.

To better understand the functional role of the mitochondrial network in shaping the Ca2+ signals in living cells, we took advantage both of the newest genetically engineered green fluorescent protein-based Ca2+ sensors ("Cameleons," "Camgaroos," and "Pericams") and of the classical Ca(2+)-sensitive photoprotein aequorin, all targeted to the mitochondrial matrix. The properties of the green fluorescent protein-based probes in terms of subcellular localization, photosensitivity, and Ca2+ affinity have been analyzed in detail. It is concluded that the ratiometric pericam is, at present, the most reliable mitochondrial Ca2+ probe for single cell studies, although this probe too is not devoid of problems. The results obtained with ratiometric pericam in single cells, combined with those obtained at the population level with aequorin, provide strong evidence demonstrating that the close vicinity of mitochondria to the Ca2+ release channels (and thus responsible for the fast uptake of Ca2+ by mitochondria upon receptor activation) are highly stable in time, suggesting the existence of specific interactions between mitochondria and the endoplasmic reticulum.

In 2011 the insect species were surveyed in 48 vineyards in Southern Switzerland to determine potential vectors for the spread of “flavescence doree” and “bois noir” diseases. Overall, 367 insect samples were analysed by nested PCR and RFLP analyses. Sixteen samples, from 8 vineyards, were positive for phytoplasmas. Orientus ishidae and Scaphoideus titanus harboured “flavescence doree”-related phytoplasmas. Hyalesthes obsoletus and Reptalus species harboured “stolbur” phytoplasmas. This screening allowed to gain more insight into species potentially involved in the epidemiology of these grapevine diseases in Southern Switzerland.