RNAi dynamics in Juvenile Fasciola spp. Liver flukes reveals the persistence of gene silencing in vitro

Background: Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST).

Methodology/Principal Findings: Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200–320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively.

Conclusions/Significance: In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control target validation. RNAi persistence in fluke encourages in vivo studies on gene function using worms exposed to RNAi-triggers prior to infection.

Climatic signals in the life histories of insects:the distribution and abundance of heather psyllids (Strophingia spp.) in the UK

1. The population density and age structure of two species of heather psyllid Strophingia ericae and Strophingia cinereae, feeding on Calluna vulgaris and Erica cinerea, respectively, were sampled using standardized methods at locations throughout Britain. Locations were chosen to represent the full latitudinal and altitudinal range of the host plants.

2. The paper explains how spatial variation in thermal environment, insect life-history characteristics and physiology, and plant distribution, interact to provide the mechanisms that determine the range and abundance of Strophingia spp.

3. Strophingia ericae and S. cinereae, despite the similarity in the spatial distribution patterns of their host plants within Britain, display strongly contrasting geographical ranges and corresponding life-history strategies. Strophingia ericae is found on its host plant throughout Britain but S. cinereae is restricted to low elevation sites south of the Mersey-Humber line and occupies only part of the latitudinal and altitudinal range of its host plant. There is no evidence to suggest that S. ericae has reached its potential altitudinal or latitudinal limit in the UK, even though its host plant appears to reach its altitudinal limit.

4. There was little difference in the ability of the two Strophingia spp. to survive shortterm exposure to temperatures as low as - 15 degrees C and low winter temperatures probably do not limit distribution in S. cinereae.

5. Population density of S. ericae was not related to altitude but showed a weak correlation with latitude. The spread of larval instars present at a site, measured as an index of instar homogeneity, was significantly correlated with a range of temperature related variables, of which May mean temperature and length of growing season above 3 degrees C (calculated using the Lennon and Turner climatic model) were the most significant. Factor analysis did not improve the level of correlation significantly above those obtained for single climatic variables. The data confirmed that S. ericae has a I year life cycle at the lowest elevations and a 2 year life cycle at the higher elevations. However, there was no evidence, as previously suggested, for an abrupt change from a one to a 2 year life cycle in S. ericae with increasing altitudes or latitudes.

6. By contrast with S. ericae, S. cinereae had an obligatory 1 year life cycle, its population decreased with altitude and the index of instar homogeneity showed little correlation with single temperature variables. Moreover, it occupied only part of the range of its host plant and its spatial distribution in the UK could be predicted with 96% accuracy using selected variables in discriminant analysis.

7. The life histories of the congeneric heather psyllids reflect adaptations that allow them to exploit host plants with different distributions in climatic and thereby geographical space. Strophingia ericae has the flexible life history that enables it to exploit C. vulgaris throughout its European boreal temperate range. Strophingia cinereae has a less flexible life history and is adapted for living on an oceanic temperate host. While the geographic ranges of the two Strophingia spp. overlap within the UK, the psyllids appear to respond differently to variation in their thermal environment.

Two-Year Monitoring of Water Samples from Dam of Iskar and the Black Sea, Bulgaria, by Molecular Analysis: Focus on Mycobacterium spp

The coast of the Bulgarian Black Sea is a popular summer holiday destination. The Dam of Iskar is the largest artificial dam in Bulgaria, with a capacity of 675 million m3. It is the main source of tap water for the capital Sofia and for irrigating the surrounding valley. There is a close relationship between the quality of aquatic ecosystems and human health as many infections are waterborne. Rapid molecular methods for the analysis of highly pathogenic bacteria have been developed for monitoring quality. Mycobacterial species can be isolated from waste, surface, recreational, ground and tap waters and human pathogenicity of nontuberculose mycobacteria (NTM) is well recognized. The objective of our study was to perform molecular analysis for key-pathogens, with a focus on mycobacteria, in water samples collected from the Black Sea and the Dam of Iskar. In a two year period, 38 water samples were collected-24 from the Dam of Iskar and 14 from the Black Sea coastal zone. Fifty liter water samples were concentrated by ultrafiltration. Molecular analysis for 15 pathogens, including all species of genus Mycobacterium was performed. Our results showed presence of Vibrio spp. in the Black Sea. Rotavirus A was also identified in four samples from the Dam of Iskar. Toxigenic Escherichia coli was present in both locations, based on markers for stx1 and stx2 genes. No detectable amounts of Cryptosporidium were detected in either location using immunomagnetic separation and fluorescence microscopy. Furthermore, mass spectrometry analyses did not detect key cyanobacterial toxins. On the basis of the results obtained we can conclude that for the period 2012-2014 no Mycobacterium species were present in the water samples. During the study period no cases of waterborne infections were reported.

Low-pathogenicity Mycoplasma spp. alter human monocyte and macrophage function and are highly prevalent among patients with ventilator-acquired pneumonia

Background
Ventilator-acquired pneumonia (VAP) remains a significant problem within intensive care units (ICUs). There is a growing recognition of the impact of critical-illness-induced immunoparesis on the pathogenesis of VAP, but the mechanisms remain incompletely understood. We hypothesised that, because of limitations in their routine detection, Mycoplasmataceae are more prevalent among patients with VAP than previously recognised, and that these organisms potentially impair immune cell function.
Methods and setting
159 patients were recruited from 12 UK ICUs. All patients had suspected VAP and underwent bronchoscopy and bronchoalveolar lavage (BAL). VAP was defined as growth of organisms at >104 colony forming units per ml of BAL fluid on conventional culture. Samples were tested for Mycoplasmataceae (Mycoplasma and Ureaplasma spp.) by PCR, and positive samples underwent sequencing for speciation. 36 healthy donors underwent BAL for comparison. Additionally, healthy donor monocytes and macrophages were exposed to Mycoplasma salivarium and their ability to respond to lipopolysaccharide and undertake phagocytosis was assessed.

Results
Mycoplasmataceaewerefoundin49%(95%CI 33% to 65%) of patients with VAP, compared with 14% (95% CI 9% to 25%) of patients without VAP. Patients with sterile BAL fluid had a similar prevalence to healthy donor BAL fluid (10% (95% CI 4% to 20%) vs 8% (95% CI 2% to 22%)). The most common organism identified was M. salivarium. Blood monocytes from healthy volunteers incubated with M. salivarium displayed an impaired TNF-α response to lipopolysaccharide ( p=0.0003), as did monocyte-derived macrophages (MDMs) (p=0.024). MDM exposed to M. salivarium demonstrated impaired phagocytosis ( p=0.005).

Discussion and conclusions
This study demonstrates a high prevalence of Mycoplasmataceae among patients with VAP, with a markedly lower prevalence among patients with suspected VAP in whom subsequent cultures refuted the diagnosis. The most common organism found, M. salivarium, is able to alter the functions of key immune cells. Mycoplasmataceae may contribute to VAP pathogenesis.

Chemical composition and pharmacological activities of essential oils of Lavandula spp.

Lavandula spp. belong to the family Lamiatae and some species are often used in popular medicine and have been used for centuries in a large number of medical applications and in aromatherapy. Although similar ethnobotanical properties of Lavandula spp., its essential oils, general chemical composition and therapeutic applications differ from different species. Lavandula stoechas L. subsps. luisieri (Rozeira) Rozeira and L. viridis L’Hér are endemic to the Iberian Peninsula, widespread in the South of Portugal, namely in Southern Alentejo and Algarve. The aim of our study was evaluate the chemical composition and toxicological and pharmacological activities of leaves essential oils of spontaneous plants of L. stoechas L. subsps. luisieri (Alentejo) and L. viridis (Algarve). The essential oils of these wild plants, collected in spring, were obtained by hydrodistillation in a Clevenger-type apparatus and its chemical composition was evaluated by GC/FID. The acute toxicity of essential oils was evaluated "in vitro" using brine shrimp (LC50) and "in vivo" using Swiss mice (DL50). The analgesic and anti-inflammatory pharmacological properties of L. stoechas subsp. luisieri essential oil were evaluated in mouse or rats by the Amour-Smith and carrageen-induced paw edema tests, respectively. Results showed important differences in chemical composition of essential oils from two species analyzed either to diversity and proportion of its constituents. The essentials oils showed citotoxicity against Artemia salina and a DL50 higher than 2000 mg/kg for mice. The analgesic and anti-inflammatory activities of essential oils were exhibit for the doses of 100 and 200 mg/kg.

CHEMICAL COMPOSITION AND ANTIMICROBIAL ACTIVITY OF ESSENTIAL OILS OF LAVANDULA SPP.

Lavenders belong to the family Labiatae and represent some of the most popular medicinal plants of great economic importance. Their essential oils are important for the perfume, cosmetic, flavouring and pharmaceutical industries. However, despite its popularity, and the long tradition of use, biological properties of the various Lavandula species are not yet been well sustained by scientific or clinical studies and some available data being inconclusive and controversial [1]. Although Lavandula spp. have similar ethnobotanical properties, however, chemical composition and therapeutic uses differ from different species and main composition of essential oils showed differences with species and with the region were they grow [1,2,3]. L. stoechas L. subsps. luisieri (Rozeira) Rozeira. L. pedunculata (Mill.) Cav. and L. viridis L’Hér are endemic to the Iberian Peninsula, widespread in the South of Portugal, namely in Alentejo and Algarve. In our work, essential oils from the stems or leaves from wild grown plants of L. luisieri (Alentejo), L. pedunculata (Alentejo) and L. viridis (Algarve), were extracted by hydrodistillation and analyzed by GC-FID. Antimicrobial activity was evaluated by solid diffusion disk assay and minimal inhibitory concentration (MIC) against pathogenic Gram-positive and Gram-negative bacteria and food spoilage fungi.

Caracterização química e actividade biológica dos óleos essenciais e extractos aquosos de algumas Lavandula spp. do Sul de Portugal

No presente trabalho estudaram-se três espécies do género Lavandula, do sul de Portugal: L. luisieri e L. pedunculata (Alentejo) e L.viridis (Algarve), com vista caraterizar a composição química dos óleos essenciais das diferentes espécies, parte vegetativa (folha) e espiga floral, e dos respetivos extratos de hidrodestilação, e avaliar as propriedades antioxidante, antimicrobiana, toxicológica, analgésica e antiinflamatória de alguns dos seus óleos essenciais e extratos aquosos, mais promissores. Os resultados mostraram importantes diferenças na composição química dos óleos essenciais, quanto à diversidade e à proporção dos seus constituintes. Os óleos essenciais e hidrolatos, em estudo, apresentaram importantes propriedades antioxidantes e antimicrobianas. Os estudos toxicológicos e farmacológicos mostraram que os óleos apresentaram citotoxicidade em Artemia salina, apresentaram valores de DL50 muito superiores a 2000 mg/kg em ratinhos Swiss, e que possuem importantes propriedades analgésicas e anti-inflamatória. Estes resultados sugerem o seu potencial uso para aplicações farmacológicas como agentes nutracêuticos e/ou fitoterapêuticos; ABSTRACT:The aim of present work was to develop a set of studies of three species of the genera Lavandula, at the South of Portugal: L. luisieri e L. pedunculata (Alentejo) e L.viridis (Algarve), in order to characterize the chemical composition of the essential oils (leaves and flowers) and to evaluate the antioxidant, antimicrobial, toxicological and pharmacological properties of selected essential oils and aqueous extracts. Results show important differences in chemical composition of essential oils, both in diversity as the proportion of their constituents. Essential oils and aqueous extracts of different Lavandula spp. showed important antimicrobial and antioxidant properties. Pharmacological studies have shown that essential oils showed cytotoxicity against Artemia salina, low acute toxicity, with LD50 >> 2000 mg/kg for mice, and important analgesic and anti-inflammatory properties. These results suggest their potential use for pharmacological applications as nutraceutical and/or phytotherapeutic agents.

Inactivation of Pseudomonas spp. biofilms with natural and synthetic photossensitizers

Cationic porphyrins have been widely used as photosensitizers (PSs) in the inactivation of microorganisms, both in biofilms and in planktonic forms. However, the application of curcumin, a natural PS, in the inactivation of biofilms, is poorly studied. The objectives of this study were (1) to evaluate and compare the efficiency of a cationic porphyrin tetra (Tetra-Py+-Me) and curcumin in the photodynamic inactivation of biofilms of Pseudomonas spp and the corresponding planktonic form; (2) to evaluate the effect of these PSs in cell adhesion and biofilm maturation. In eradication assays, biofilms of Pseudomonas spp adherent to silicone tubes were subjected to irradiation with white light (180 J cm-2) in presence of different concentrations (5 and 10 μM) of PS. In colonization experiments, solid supports were immersed in cell suspensions, PS was added and the mixture experimental setup was irradiated (864 J cm-2) during the adhesion phase. After transference solid supports to new PS-containing medium, irradiation (2592 J cm-2) was resumed during biofilm maturation. The assays of inactivation of planktonic cells were conducted in cell suspensions added of PS concentrations equivalent to those used in experiments with biofilms. The inactivation of planktonic cells and biofilms (eradication and colonization assays) was assessed by quantification of viable cells after plating in solid medium, at the beginning and at the end of the experiments. The results show that porphyrin Tetra-Py+-Me effectively inactivated planktonic cells (3.7 and 3.0 log) and biofilms of Pseudomonas spp (3.2 and 3.6 log). In colonization assays, the adhesion of cells was attenuated in 2.2 log, and during the maturation phase, a 5.2 log reduction in the concentration of viable cells was observed. Curcumin failed to cause significant inactivation in planktonic cells (0.7 and 0.9 log) and for that reason it was not tested in biofilm eradication assays. In colonization assays, curcumin did not affect the adhesion of cells to the solid support and caused a very modest reduction (1.0 log) in the concentration of viable cells during the maturation phase. The results confirm that the photodynamic inactivation is a promising strategy to control installed biofilms and in preventing colonization. Curcumin, however, does not represent an advantageous alternative to porphyrins in the case of biofilms of Pseudomonas spp.

Ecology of the early life-history stages of shad Alosa fallax fallax (Lacépède, 1803) in the River Mira, with a note on Alosa spp. larvae in the River Guadiana

Tese dout., Ecologia, Universidade do Algarve, 2006