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This paper reports the use of near infrared and mid-infrared spectroscopy to detect the presence and quantity of eggs of the gastrointestinal nematode Haemonchus contortus in sheep faeces. Haemonchus contortus eggs were quantified in dried, finely ground sheep faeces and in moist, coarsely ground faeces using near infrared and mid-infrared bench top spectrometers and a portable near infrared spectrometer. When Haemonchus contortus eggs were presented without faecal medium, it was found that the wavelength region of 1880–2100 nm was most important for detection. Broad classes of chemical properties found in the near infrared region were identified for dried Haemonchus contortus eggs using a mid-infrared spectrometer. However, when Haemonchus contortus eggs were mixed into the complex matrix of sheep faeces, the development of a robust calibration model for egg detection proved to be challenging (R2

Funding: We wish to thank all the collaborators (administrative staff, nurses, etc.) of the surgery block, as well as the doctors of the vascular surgery and transplantation departments of the Hospital of Santa Maria for the availability and assistance in the collection of the samples. We also thank Sociedade Portuguesa de Reumatologia for funding with two fellowships: Fundo de Apoio à Investigação 2014 and SPR/MSD 2015. DC-F received funding from a PhD grant from Fundação para a Ciência e a Tecnologia (SFRH/BD/80940/2011). Background and Aims: Atherosclerosis and osteoporosis share common risk factors, as well as inflammatory mechanisms. Our aim was to understand how atherosclerotic lesions are related with disturbances in bone. Methods: Gene expression of pro-inflammatory and bone metabolism related proteins (IL-1β, IL-6, IL-17A, TNF, RANKL, OPG, COL1, CTSK, OCL, TRAP, CBFA1, DKK1, SOST, ADIPOQ, and ADIPOR1) were analyzed in arteries and bones from 45 deceased donors and adipose tissue was used as control. Additionally, in 139 patients with advanced atherosclerosis submitted to carotid endarterectomy we compared calcium content (Alizarin red) and plaque inflammatory scores (CD3+, CD68+, and adiponectin) of patients with normal bone mineral density (BMD) with those with low BMD and explored the associations between gene expression in atherosclerotic plaques and BMD. Serum levels of pro-inflammatory and bone related proteins were measured both in donors and patients. Associations were investigated by the Pearson or Spearman correlation tests, and multivariate regression analyzes were performed when justified. Results: Gene expression of bone remodeling and pro-inflammatory proteins correlated positively in bone and aorta, independently of age and sex of donors, but not in adipose tissue. The expression of bone formation genes was significantly higher in atheroma plaques from endarterectomized patients with normal vs. low BMD as well as inflammatory CD68+ scores, regardless of patients' age and sex, but not of body mass index. No relationship was observed between serum levels and gene expression levels of pro-inflammatory or bone remodeling proteins. Conclusions: Our results suggest that the relationship between bones and vessels in the context of atherosclerotic disease and osteoporosis may rely on the intrinsic connection between the tissues involved, independently of disease stage. Serum measurements of pro-inflammatory and bone-remodeling proteins do not accurately translate tissue pathologic processes. publishersversion published

Winning the war against resistant bacteria will require a change of paradigm in antibiotic discovery. A promising new direction is the targeting of non-essential pathways required for successful infection, such as quorum-sensing, virulence, and biofilm formation. Similarly important will be strategies to prevent or revert antibiotic resistance. Here, we argue that the (p)ppGpp signaling pathway should be a prime target of this effort, since its inactivation could potentially achieve all these goals simultaneously. The hyperphosphorylated guanine nucleotide (p)ppGpp is an ancient and universal second messenger of bacteria that has pleotropic effects on the physiology of these organisms. Initially described as a stress signal—an alarmone—it is now clear that (p)ppGpp plays a more general and fundamental role in bacterial adaptation, by integrating multiple internal and environmental signals to establish the optimal balance between growth and maintenance functions at any given time. Given such centrality, perturbation of the (p)ppGpp pathway will affect bacteria in multiple ways, from the ability to adjust metabolism to the available nutrients to the capacity to differentiate into developmental forms adapted to colonize different niches. Here, we provide an overview of the (p)ppGpp pathway, how it affects bacterial growth, survival and virulence, and its connection with antibiotic tolerance and persistence. We will emphasize the dysfunctions of cells living without (p)ppGpp and finalize by reviewing the efforts and prospects of developing inhibitors of this pathway, and how these could be employed to improve current antibiotic therapy.

Snap and common beans (Phaseolus vulgaris L.) are severely affected by Bean golden mosaic virus (BGMV) infection, so far the only begomovirus reported on these crops in Brazil (1). Samples of snap and common beans colonized by the whitefly Bemisia tabaci biotype B and displaying golden mosaic, chlorotic spots, and leaf distortion were collected in three production regions in Goiás State (Goianápolis, Luziânia, and Itaberaí) between 2003 and 2007. Total DNA extracted from leaf samples was used as template in PCR assays using universal primers targeting conserved regions of the DNA-A and DNA-B genomes (3). Begomovirus-specific amplicons were observed only with DNA template from symptomatic plants. Two single amplicons were observed for both genomic segments, indicating the presence of bipartite species in all samples. Sequence analysis of four isolates (named as GO-176, GO-260, GO-354, and GO-368) obtained from common bean samples indicated identity levels of approximately 95% with the DNA-A segment of BGMV (GenBank Accession No. FJ665283). However, the complete DNA-A sequence (GenBank Accession No. HM357459.1) of the GO-060 isolate (from a symptomatic snap bean plant collected in Goianápolis) displayed 76% identity with BGMV (GenBank Accession No. FJ665283) and 95% identity with the DNA-A of a Sida micrantha mosaic virus (SimMV) isolate (GenBank Accession No. EU908733.1) reported to be infecting okra (Abelmoschus esculentus L.) and 94.8% with a SimMV isolate reported to be infecting soybean (GenBank Accession No. FJ686693) in Brazil (2). Koch's postulates were fulfilled for the isolate GO-060 by inoculating a set of soybean and bean accessions via a biolistic approach. The ratio of positive PCR amplicons per total of inoculated plants were 15 of 16 for snap bean cv. Trepador, 9 of 10 for snap bean cv. Fartura, 18 of 24 for common bean cv. Olate Pinto, and 19 of 25 for common bean cv. Carioca. The isolate was also able to infect eight of nine soybean ‘Doko’ plants. Sequence analysis using symptomatic leaf samples (15 days after inoculation) confirmed SimMV as the causal agent. To our knowledge, this is the first report of a SimMV isolate infecting P. vulgaris. This virus is apparently fast expanding its host range from Malvaceae to Solanaceae species and leguminous hosts after the introduction of B. tabaci biotype B (2). More extensive surveys are necessary to access the current epidemiological importance of SimMV in both snap and common beans in Brazil. References: (1) J.C. Faria and D. P. Maxwell. Phytopathology 89:262, 1999. (2) F. R. Fernandes et al. Arch. Virol. 154:1567, 2009. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

BackgroundExisting diagnostic methods for the parasitic gastrointestinal nematode,Haemonchus contortus, are time consuming and require specialised expertise, limiting their utility in the field. A practical, on-farm diagnostic tool could facilitate timely treatment decisions, thereby preventing losses in production and flock welfare. We previously demonstrated the ability of visible–near-infrared (Vis–NIR) spectroscopy to detect and quantify blood in sheep faeces with high accuracy. Here we report our investigation of whether variation in sheep type and environment affect the prediction accuracy of Vis–NIR spectroscopy in quantifying blood in faeces.MethodsVisible–NIR spectra were obtained from worm-free sheep faeces collected from different environments and sheep types in South Australia (SA) and New South Wales, Australia and spiked with various sheep blood concentrations. Spectra were analysed using principal component analysis (PCA), and calibration models were built around the haemoglobin (Hb) wavelength region (387–609 nm) using partial least squares regression. Models were used to predict Hb concentrations in spiked faeces from SA and naturally infected sheep faeces from Queensland (QLD). Samples from QLD were quantified using Hemastix® test strip and FAMACHA© diagnostic test scores.ResultsPrincipal component analysis showed that location, class of sheep and pooled versus individual samples were factors affecting the Hb predictions. The models successfully differentiated ‘healthy’ SA samples from those requiring anthelmintic treatment with moderate to good prediction accuracy (sensitivity 57–94%, specificity 44–79%). The models were not predictive for blood in the naturally infected QLD samples, which may be due in part to variability of faecal background and blood chemistry between samples, or the difference in validation methods used for blood quantification. PCA of the QLD samples, however, identified a difference between samples containing high and low quantities of blood.ConclusionThis study demonstrates the potential of Vis–NIR spectroscopy for estimating blood concentration in faeces from various types of sheep and environmental backgrounds. However, the calibration models developed here did not capture sufficient environmental variation to accurately predict Hb in faeces collected from environments different to those used in the calibration model. Consequently, it will be necessary to establish models that incorporate samples that are more representative of areas whereH. contortusis endemic.Graphical Abstract

Wolbachia are maternally transmitted intracellular bacteria that naturally infect over 40% of all insect species. Wolbachia have become promising biocontrol agents against mosquito-borne diseases due to their unique effects on mosquito reproduction and immunity. However, the phenotypes mediated by Wolbachia vary between strains and host-pathogen systems. Discovery of novel Wolbachia strains is essential for broadening the application of Wolbachia for mosquito control. In this study, I identified and characterized the anti-pathogen effects of natural Wolbachia infections in Australian mosquitoes. I identified three previously uncharacterized Wolbachia strains based on detection and sequencing of wsp, 16S rRNA and ftsZ genes. Wolbachia sequences were also detected from Coquillettidia xanthogaster, however, it remained unclear if the detection was resulted from a genuine infection. I established colonies of two local species that are naturally infected with Wolbachia; Aedes notoscriptus and Culex sitiens, and discovered a unique pattern of infection rates fluctuating between 15% and 60% in both colonies. Study on the maternal transmission of Wolbachia in Cx. sitiens revealed high transmission rates (99.7%) but low cytoplasmic incompatibility (9.9% mortality). I orally inoculated Wolbachia positive and negative Ae. notoscriptus and Cx. sitiens with the alphavirus Ross River virus (RRV; 103.31 – 6.48 and 105.41 – 6.36 CCID50 per mosquito, respectively). RRV infection rates varied between 17% - 85% for Ae. notoscriptus and 0% - 18% for Cx. sitiens with no significant differences between infection rates for Wol+ and Wol- mosquitoes. However, mean virus load in Wol+ Cx. sitiens was 1000× lower than in Wol- mosquitoes, suggesting that natural Wolbachia infection suppresses virus proliferation in that species. I report on progress towards transinfecting cells (Aag2 and Aa20) and mosquitoes (Aedes aegypti) with Wolbachia, including oral ingestion trials. Inoculations using the Shell vial technique resulted in temporal in vitro infections for up to three passages, however, stable infections were not yet achieved. I observed evidence that Wolbachia infected cells within the epithelial lining of the crop and potentially the midgut by fluorescence microscopy, however, the infection was transient. These discoveries broadened the current understanding on Wolbachia-host-pathogen interactions and shed light on future mosquito control strategies. The behaviour of Wolbachia in new hosts can be difficult to predict, therefore transinfection and further characterization is required to determine if these strains can induce anti-pathogen effects in the context of mosquito control.

Heavy infestations of the Barber’s pole worm, Haemonchus contortus, can cause severe wasting, morbidity and mortality in animals if not promptly treated. The current detection methods for this blood-sucking parasite involve faecal worm egg counts and diagnosis of anaemia, both of which are time consuming and require expertise. As blood is detected in sheep faeces sooner during infection than worm eggs, quantitative evaluation of blood may serve as a sensitive indicator of H. contortus infection. Here we investigated the feasibility of rapid estimation of haemoglobin (Hb) in sheep faeces using visible near-infrared spectroscopy. Haemoglobin (Hb) was assessed at various concentrations in moist sheep faeces using portable visible near-infrared (vis-NIR) spectrometers. Calibration models were developed for the region of 400–600 nm, where Hb absorption bands can be found. Within this wavelength region, Hb in sheep faeces can be estimated with minimal interference from background moisture (970 nm) or chlorophyll (670 nm), suggesting that difference in diets in sheep will have minimal effect on prediction accuracy. Predictions for sheep faeces based on a drenching threshold of 3 µg Hb/mg faeces showed high levels of accuracy with minimal sample preparation (Sensitivity = 89%, specificity = 80%). The success in detecting Hb in sheep faeces indicates the potential of vis-NIR spectroscopy as a rapid, on-farm diagnostic method for predicting blood in sheep faeces, and timing treatment of H. contortus infections.

Penguins are the only extant family of flightless diving birds. They currently comprise at least 18 species, distributed from polar to tropical environments in the Southern Hemisphere. The history of their diversification and adaptation to these diverse environments remains controversial. We used 22 new genomes from 18 penguin species to reconstruct the order, timing, and location of their diversification, to track changes in their thermal niches through time, and to test for associated adaptation across the genome. Our results indicate that the penguin crown-group originated during the Miocene in New Zealand and Australia, not in Antarctica as previously thought, and that Aptenodytes is the sister group to all other extant penguin species. We show that lineage diversification in penguins was largely driven by changing climatic conditions and by the opening of the Drake Passage and associated intensification of the Antarctic Circumpolar Current (ACC). Penguin species have introgressed throughout much of their evolutionary history, following the direction of the ACC, which might have promoted dispersal and admixture. Changes in thermal niches were accompanied by adaptations in genes that govern thermoregulation and oxygen metabolism. Estimates of ancestral effective population sizes (Ne ) confirm that penguins are sensitive to climate shifts, as represented by three different demographic trajectories in deeper time, the most common (in 11 of 18 penguin species) being an increased Ne between 40 and 70 kya, followed by a precipitous decline during the Last Glacial Maximum. The latter effect is most likely a consequence of the overall decline in marine productivity following the last glaciation., Financial support for this work was provided by Instituto Antártico Chileno (INACH RT_12–14), Fondecyt Project 1150517, Genomics Antarctic Biodiversity/Programa de Investigación Asociativa/Comisión Nacional de Investigación Científica y Tecnológica (GAB PIA CONICYT ACT172065), NSF DEB-1441652, French Polar Institute Paul-Emile Victor (IPEV; Progs. 137 and 354), a Lakeside grant from the California Academy of Sciences, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Brazil and Instituto Nacional de Ciência e Tecnologia - Ecologia, Evolução e Conservação da Biodiversidade (INCT-EECBio) Brazil.

Nutrition education is one of the factors that may help to promote behavior change and therefore may improve the dietary habits of adolescent soccer players. However, information about the relationship between nutrition knowledge (NK) and the dietary behavior of these athletes is scarce. The purpose of this study was to evaluate the eating habits of adolescent soccer players and analyse the correlations among dietary intake and NK. Seventy-three Brazilian adolescent soccer players (aged 14–19 years), from four professional clubs, underwent anthropometric evaluation and completed 3-day food records. Misreporting of energy intake was evaluated and the dietary intake data were energy-adjusted and compared with recommendations for athletes and dietary reference intakes. The athletes also answered a questionnaire about barriers for healthy eating and a nutrition knowledge test divided into three sections: Basic Nutrition Knowledge (BNK), Sports Nutrition Knowledge (SNK), and Food Pyramid Nutrition Knowledge (FPNK). The participants showed a low NK (54.6%) and an inadequate intake of fruits, vegetables, dairy, carbohydrates, and micronutrients. A positive correlation was found between the ingestion of phosphorus and FPNK as well as among calcium and both SNK and Total NK (p<0.05). Sodium intake was negatively correlated with all categories of the NK test (p<0.05). The adolescents reported that the principal barriers for adopting a healthy diet were the lack of willpower and a busy lifestyle. In this context, nutrition education is recommended and should also provide practicable healthy eating goals according to athletes´ lifestyle as well as target motivational barriers to increase adherence.

Recruitment of the limpetPatella ulyssiponensiswas investigated in relation to the presence of living crustose coralline algae (CCA) in rocky-shore habitats. Juvenile limpets (≤10 mm maximum shell length) were counted in CCA-present and CCA-absent habitats, on three shores in SW Portugal during summer 2007 and winter 2009. Furthermore, the settling response of laboratory-reared larvae ofP. ulyssiponensisto CCA-covered substratum, and bare-rock, was examined. Across the intertidal zone, we found a clear association between the distribution and abundance of juveniles and the presence of CCA. Although the presence of CCA was not an absolute requisite for juvenile occurrence, null juvenile densities were mostly recorded in CCA-absent areas. The highest juvenile densities (maximum of 64 individuals in 15 × 15 cm) were consistently found in CCA-dominated habitats, namely steep wave-exposed areas at low-shore and rock-pools. The hypothesis of CCA-enhanced settlement was not supported, as settlement intensities of laboratory-reared larvae were similar between chips of rock encrusted by CCA and chips of bare-rock. From the overall number of settlers onto CCA-encrusted rock chips, 51% were found in tiny pits lacking CCA. This was the first study of the settlement patterns of larvae of the genusPatellausing naturally occurring rocky substrata. These results are preliminary and should be confirmed with choice-experiments and improved monitoring of the position of settlers. We suggest that CCA plays a role in the recruitment ofP. ulyssiponensis, potentially promoting survivorship of early benthic stages, but possibly not enhancing settlement.

Adolfo Lutz Institute in Sao Paolo State performs mycobacterial identification for many healthcare units, and in 2008 identified a possible outbreak involving patients submitted to bronchoscopy at the same hospital. This study aimed to analyse the clonality of isolates. Mycobacterium abscessus subsp. massiliense isolated from 28 patients, water from one bronchoscope and water from four automated endoscope reprocessing machines presented high similarity by pulsed-field gel electrophoresis. This strain was not found in the water supply, and it was hypothesized that an infected patient contaminated the bronchoscope, with further false-positive cultures from subsequent patients.

We report the first use of near-infrared spectroscopy for rapid, low-cost, noninvasive detection of Zika virus in Aedes aegypti., The accelerating global spread of arboviruses, such as Zika virus (ZIKV), highlights the need for more proactive mosquito surveillance. However, a major challenge during arbovirus outbreaks has been the lack of rapid and affordable tests for pathogen detection in mosquitoes. We show for the first time that near-infrared spectroscopy (NIRS) is a rapid, reagent-free, and cost-effective tool that can be used to noninvasively detect ZIKV in heads and thoraces of intact Aedes aegypti mosquitoes with prediction accuracies of 94.2 to 99.3% relative to quantitative reverse transcription polymerase chain reaction (RT-qPCR). NIRS involves simply shining a beam of light on a mosquito to collect a diagnostic spectrum. We estimated in this study that NIRS is 18 times faster and 110 times cheaper than RT-qPCR. We anticipate that NIRS will be expanded upon for identifying potential arbovirus hotspots to guide the spatial prioritization of vector control.

The global approach to mosquito-borne diseases (MBDs) is in need of critical re-evaluation. Although there have been dramatic reductions in malaria incidence since 2000, malaria elimination from high-transmission settings remains problematic. At the same time, arbovirus outbreaks have increased in their frequency and impact. The 2015-2016 Zika virus epidemic exposed the dire state of MBD control in many countries, calling for united global action. Despite international resolve to prevent future epidemics, current practices in MBD control are mostly reactive and of limited efficacy. In this Opinion article, we summarize the views of 25 international mosquito experts about the current state of MBD control and highlight the issues that must be addressed in order to tackle emerging threats on the horizon.

This study evaluates the contaminants in water and their bioaccumulation in the gills and liver of two ecologically distinct fish species, Astyanax fasciatus and Pimelodus maculatus, living in the reservoir of the Furnas hydroelectric power station located in Minas Gerais in the southeastern Brazil. The histological alterations in these organs are also examined. Water and fish were collected in June and December from five sites (site 1: FU10, site 2: FU20, site 3: FU30, site 4: FU40 and site 5: FU50) in the reservoir, and agrochemicals and metals selected based on their use in the field crops surrounding the reservoir were analyzed in the water and in the fish gills and livers. The concentrations of the organochlorines aldrin/dieldrin, endosulfan and heptachlor/heptachlor epoxide as well as the metals copper, chromium, iron and zinc in the gills and livers of both fish species were higher in June than in December; the liver accumulated higher concentrations of contaminants than the gills. The organochlorine metolachlor was detected only in the liver. The histological pattern of changes was similar in both species with regard to contaminant accumulation in the gills and liver. Fish from FU10, the least contaminated site, exhibited normal gill structure and moderate to heavy liver damage. Fish collected at FU20 to FU50, which were contaminated with organochlorines and metals, showed slight to moderate gill damage in June and irreparable liver damage in the livers in June and December. The histological changes in the gills and liver were suitable to distinguishing contaminated field sites and are therefore useful biomarkers for environmental contamination representing a biological end-point of exposure.

Leonurus sibiricus L. (Lamiaceae) is a subtropical weed frequently found with golden mosaic symptoms. Leonurus mosaic virus (LeMV) was the first begomovirus reported on L. sibiricus in Brazil (3). Later, a new bipartite species (Tomato yellow spot virus, ToYSV) was reported affecting tomatoes, beans, and also L. sibiricus (1,2). A survey of begomovirus isolates was conducted within tomato fields also displaying high incidence of plants with begomovirus-induced symptoms. Thirty L. sibiricus and 33 tomato samples were collected (2007 to 2012) in nine districts in Paraná State, Brazil. Two L. sibiricus isolates were also obtained within citrus orchards in Major Otaño, Itapúa, Paraguay. Total DNA was extracted from all 65 isolates and PCR assays were conducted with primers for conserved DNA-A (PAL1v1978/PAR1c496) and DNA-B (PBL1v2040/PCRc1) regions (3). Nucleotide sequence identity of the 1,193-bp DNA-A amplicons of our L. sibiricus isolates ranged from 93.4 to 98.2% with LeMV (GenBank Accession No. U925321) and from 92.4 to 94.8% with ToYSV isolates from tomato (DQ336350.1) and bean (FJ538207). None of the 33 tomato samples was found to be infected by ToYSV, with all having high nucleotide sequence identity (92 to 99%) only with Tomato severe rugose virus (GU358449). Complete DNA-A genome sequence was obtained via a rolling circle amplification-based strategy for one Brazilian L. sibiricus isolate (PR-088) and one isolate from Paraguay (PAR-07). The entire DNA-A genome of PR-088 (JQ429791) had 96.8% nucleotide sequence identity with PAR-07 (KC683374) and ranged from 95.6 to 96.3% with ToYSV isolates from bean, tomato, and L. sibiricus (JX513952). The nucleotide sequence identity of the 487-bp DNA-B amplicon ranged from 87 to 92% among PR-088 (KC 683374); PAR-07 (KC740619) and ToYSV isolates from tomato (DQ336351.1) and L. sibiricus (JX513953.1). Leonurus cuttings infected with the ToYSV (PR-088) were caged together with healthy L. sibiricus and tomato ‘Alambra’ seedlings. Hybridization assays with ToYSV-specific probes (2) and sequencing of PCR amplicons indicated that Bemisia tabaci biotype B adults were able to transmit ToYSV to both hosts as reported (1). Our results suggest that L. sibiricus is the main ToYSV reservoir under natural conditions and tomato seems to be an occasional alternative host. In fact, ToYSV has not often detected in tomatoes as observed in a number of extensive surveys (4). So far, the complete LeMV genome is not available for comparison (3). However, our analyses with a DNA-A segment indicated that LeMV and ToYSV isolates might represent strains of single virus at the current threshold of 89% nucleotide sequence identity for Begomovirus species discrimination (4). Thus, a reappraisal of the taxonomic status of ToYSV is necessary to clarify its genetic relationship with LeMV. This is the first report of ToYSV on L. sibiricus in Paraguay. References: (1) J. C. Barbosa et al. Plant Dis. 97:289, 2013. (2) R. F. Calegario et al. Pesq. Agrop. Bras. 42:1335, 2007. (3) J. C. Faria and D. P. Maxwell, Phytopathology 89:262, 1999. (4) F. R. Fernandes et al. Virus Genes 36:251, 2008.

Treatment of metastatic melanoma still remains a challenge, since in advanced stage it is refractory to conventional treatments. Most patients with melanoma have either B-RAF or N-RAS mutations, and these oncogenes lead to activation of the RAS-RAF-MEK-ERK and AKT signal pathway, keeping active the proliferation and survival pathways in the cell. Therefore, the identification of small molecules that block metastatic cell proliferation and induce cell death is needed. Violacein, a pigment produced by Chromobacterium violaceum found in Amazon River, has been used by our group as a biotool for scrutinizing signaling pathways associated with proliferation, survival, aggressiveness, and resistance of cancer cells. In the present study, we demonstrate that violacein diminished the viability of RAS- and RAF-mutated melanoma cells (IC50 value ∼500 nM), and more important, this effect was not abolished after treatment medium removal. Furthermore, violacein was able to reduce significantly the invasion capacity of metastatic melanoma cells in 3D culture. In the molecular context, we have shown for the first time that violacein causes a strong drop on histone deacetylase 6 expression, a proliferating activator, in melanoma cells. Besides, an inhibition of AXL and AKT was detected. All these molecular events propitiate an inhibition of autophagy, and consequently, melanoma cell death by apoptosis.

Molecular-level data are analyzed to produce a phylogeny for the four species of edible goose barnacles in the genus Pollicipes. Genetic distances among these species are calculated to determine relative times of genetic divergence. The four extant species of Pollicipes have a Tethyan relict distribution that geographically borders that of many of the fossil records for the genus. Three of the living species of Pollicipes exhibit an eastern boundary distribution in either the Pacific or the Atlantic Oceans. However, Pollicipes polymerus differs by having a range into the high latitudes of the northern Pacific. Pollicipes elegans inhabits the tropical eastern Pacific Ocean. The third and fourth species, Pollicipes pollicipes and an undescribed Pollicipes from the Cape Verde Islands, live in the eastern Atlantic Ocean. Pollicipes aboriginalis, a fossil species from western Australia, and the closest known living relatives of Pollicipes, Capitulum mitella and Calantica spinosa, two West Pacific species, are relicts in what was the far eastern region of the Tethys Sea. Bayesian analysis of concatenated DNA sequence data from mitochondrial genes cytochrome c oxidase subunit 1 (CO1) and ribosomal subunit 16s (16s), and nuclear gene histone subunit 3 (H3) fragments support the hypothesis, initially based on morphological similarities, that the two eastern Atlantic species, P. pollicipes and an undescribed species of Pollicipes from the Cape Verde Islands, and P. elegans, the amphitropical eastern tropical Pacific species, are the most recently diverged of the four species in the genus. In addition to the molecular-level evidence for earlier divergence from the other three species of Pollicipes, P. polymerus differs from them morphologically. A single phylogenetic relationship among these three most closely related species of Pollicipes is not conclusively supported by the trees generated from the individual gene or combined DNA sequence data. The CO1 and 16s sequence distance data among these three species suggest that they all emerged at about the same time during a species radiation event in the proto-Atlantic as the Tethys was closing. Compared to the mtDNA gene distance data, the nDNA H3 distances among all four species of Pollicipes are much lower than would be expected, less than 1.3%. The distances between each of the Pollicipes species and Capitulum H3 sequence data pairs are close to that expected, about 7%. Distance between Pollicipes spp. and Calantica H3 sequence pairs, about 17%, is more than expected from CO1 distance values.

Crotamines 1 and 2 differ only at residue 19 (which is Leu in 1, and Ile in 2), but 1 presents a greater myone- crotic activity. PM3 geometry optimizations of fragments of 1 (I17-C18-L19-P20-P21) and 2 (I17-C18-I19-P20-P21) yielded the minimum energy conformations I-a and II-a, respectively. The HF and DFT calculation of chemical proper- ties (atomic charge, orbital population, and MO energy) of I-a and II-a did not reveal significant differences that would explain the differences in biological activities of the corresponding crotamines. PM3 optimized geometries of full peptides 1 and 2 presented different globular spatial arrangements when disulfide bonds between cysteine residues were consid- ered. This fact may be related to the difference in biological activities observed for the two crotamines.

Members of the family Myrmecophagidae (i.e. anteaters) show a variety of anatomical and behavioural adaptations to deal with their low-energy diet; for example, they all have low body temperatures in comparison with other eutherian mammals. In this study, we investigated the tympanic membrane temperatures (Tmt) of two giant anteaters and three southern tamanduas, housed in captivity and exposed to natural climatic variations in temperature, using an infrared thermometer. Additionally, we measured external dorsal temperature (Td), air temperature (Ta), substrate temperature (Ts) and whether the subject was active or not. To understand the effect of time of day on these variables, we recorded them, on the hour, over four 24-h cycles for each animal during which the subjects were non-restrained within their enclosures. The results show that both giant anteaters and southern tamanduas allow their Tmt to reduce between 4.0 and 6.5 °C when they are sleeping. Furthermore, linear regressions between Tmt and Ta or Ts showed that the giant anteaters were much more affected by Ta and Ts than the southern tamanduas. Both species also showed higher Tmt when active (comparing subjects active and inactive at the same Ta). Both species appear to use shallow torpor during a normal 24-h cycle probably as a means to economize energy. The torpor in giant anteaters occurred during the night when asleep, whereas in the southern tamanduas it occurred at any time of day when asleep. The giant anteaters appeared to be more directly affected by environmental temperature than the southern tamanduas.

Biochemical biomarkers have shown to be useful toxicity tools in environmental assessment programmes. However, few studies involving freshwater invertebrate species have been performed and even fewer on tropical species. The aim of this paper was to evaluate cholinesterase (ChE) and glutathione-S-transferase (GST) activities in larvae of Chironomus xanthus (Chironomidae, Diptera) exposed to the organophosphate parathion (ethyl) and the metals copper and cadmium. Considering ChE, significant reduction in activity was observed following C. xanthus exposure to parathion and cadmium (48 and 96 h, respectively). Inhibition of ChE activity (IC50) related to parathion seemed to be a more consistent and sensitive effect criteria than the LC50. For GST, there was significant reduction in activity following parathion exposure (48 h). Copper did not elicit any change in ChE or GST activities in C. xanthus. The findings of this paper show the two biomarkers as promising tools for assessing exposure to contaminats in tropical regions.

This comparative study of gill morphometrics in near-term embryos of freshwater stingray potamotrygonids examines gill dimensions in relation to neonatal lifestyle and habitat. In embryos of the potamotrygonids Paratrygon aiereba, Plesiotrygon iwamae, Potamotrygon motoro, Potamotrygon orbignyi, and cururu ray Potamotrygon sp. the number and length of filaments, total gill surface area, mass-specific surface area, water-blood diffusion distance, and anatomical diffusion factor were analysed. In all potamotrygonids, the 3rd branchial arch possessed a larger respiratory surface than the other gill arches. Larger embryos had more gill surface area and large spiracles, which are necessary to maintain the high oxygen uptake needed due to their larger body size. However, the higher mass-specific gill surface area observed in near-term embryos may be advantageous because neonates can use hypoxic environments as refuges against predators, as well as catch small prey that inhabit the same environment. As expected from their benthic mode of life, freshwater stingrays are sluggish animals compared to pelagic fishes. However, based on gill respiratory morphometry (such as gill area, mass-specific gill area, the water-blood diffusion barrier, anatomical diffusion factor, and relative opening of the spiracle), subtypes of lifestyles can be observed corresponding to: active, intermediate, and sluggish species according to Gray's scale. Este estudo realizado com embriões a termo de arraias de água doce (Potamotrygonidae) compara e analisa as dimensões branquiais em relação ao estilo de vida e habitat dos neonatos. Nos embriões de Paratrygon aiereba, Plesiotrygon iwamae, Potamotrygon motoro, Potamotrygon orbignyi e Potamotrygon sp. (arraia cururu) foram analisados número e comprimento dos filamentos, área branquial, área superficial branquial massa-específica, barreira de difusão água-sangue e fator de difusão anatômico. Em todos os potamotrigonídeos estudados, o 3º arco branquial possui uma superfície respiratória maior que os demais arcos. Embriões de espécies de maior porte possuem grandes espiráculos e maior área de superfície branquial. Isso ajuda a manter a taxa de absorção de oxigênio proporcional ao requerimento do animal. No entanto, a grande área de superfície branquial massa-específica observadas nos embriões a termo pode ser vantajosa, pois os neonatos podem usar ambientes hipóxicos como refúgios contra predadores, bem como capturar pequenas presas que habitam o mesmo ambiente. Devido ao modo de vida bentônico, as arraias de água doce são nadadoras lentas comparadas aos peixes pelágicos. No entanto, com base na morfometria branquial (área de superfície branquial, área branquial massa-específica, barreira de difusão água-sangue, fator de difusão anatômico e abertura relativa do espiráculo), subtipos de estilos de vida podem ser observados: ativas, intermediárias e lentas, conforme escala definida por Gray.

The stalked barnacle Pollicipes pollicipes is the most important intertidal economical resource in Portugal. The assessment of the state of the fishery, conservation and management of P. pollicipes in Portugal was made for the first time in three regions with different regulations regarding this fishery: two marine protected areas ("Reserva Natural das Berlengas", RNB; and "Parque Natural do Sudoeste Alentejano e Costa Vicentina", PNSACV); and the Center coast. Different approaches (independent observations, inquiries, logbooks) and sources of data (past and recent) were used. An overall negative tendency of the state of the fishery and conservation of this resource was observed in all regions, with the exception of the stable tendency detected in PNSACV when using the inquiries approach. A weak management was considered to be in practice at Center and at PNSACV, while an acceptable management was inferred for RNB. We recommend a change into a co-management system that should be tested in pilot regions as RNB and/or PNSACV.

The developmental requirements of ovarian follicles are dependent on the maturation stage of the follicle; in particular, elegant studies with genetic models have indicated that FSH is required for antral, but not preantral, follicle growth and maturation. To elucidate further the role of FSH and other regulatory molecules in preantral follicle development, in vitro culture systems are needed. We employed a biomaterials-based approach to follicle culture, in which follicles were encapsulated within matrices that were tailored to the specific developmental needs of the follicle. This three-dimensional system was used to examine the impact of increasing doses of FSH on follicle development for two-layered secondary (100-130 microm; two layers of granulosa cells surrounding the oocyte) and multilayered secondary (150-180 microm, several layers of granulosa cells surrounding the oocyte) follicles isolated from mice. Two-layered secondary follicles were FSH responsive when cultured in alginate-collagen I matrices, exhibiting FSH dose-dependent increases in follicle growth, lactate production, and steroid secretion. Multilayered secondary follicles were FSH dependent, with follicle survival, growth, steroid secretion, metabolism, and oocyte maturation all regulated by FSH. However, doses greater than 25 mIU/ml of FSH negatively impacted multilayered secondary follicle development (reduced follicle survival). The present results indicate that the hormonal and environmental needs of the follicular complex change during the maturation process. The culture system can be adapted to each stage of development, which will be especially critical for translation to human follicles that have a longer developmental period.

Myosin-V is involved in organelle and vesicle trafficking inSaccharomyces cerevisiae and in other eukaryotic cells from yeast to human. In the present study, we determined by FACS that the major subpopulations of the peripheral blood mononuclear cells express myosin-V with similar fluorescence intensity. Confocal microscopy showed intense labeling for myosin-V at the centrosomal region and a punctate staining throughout the cytoplasm, frequently associated with the central microtubule arrays and the actin-rich cortex. Some degree of overlap with an endolysosomal marker and dynein light-chain 8 k was found at the cell center. Striking colocalization was observed with the major histocompatibility complex (MHC) class II molecules near the cell surface. Treatment with phytohemagglutinin, which induces T-lymphocyte activation, associated with MHC class II expression, increased the levels of myosin-V protein and mRNA for the three members of class V myosins. These data suggest that class V myosins might be involved in relevant functions in the immune response.

Soil is usually carried to the mills, as an impurity in sugar cane, leading to economic drawbacks for the industry. The quantification of this dirt is important to identify its causes and for routine quality control. Several methods have been used for this purpose, however, no single one has been pointed out as an industrial standard. The use of a γ-ray emitting radionuclide of natural occurence was investigated and, after several soil and cane radioactivity analyses,212Pb was chosen as the best tracer. Calibration curves developed with the addition of soil in clean cane, from 0 to 10% (dry mass), demonstrated the linearity of the method. Analyses of eleven samples taken from consignments showed that the procedure was consistent and reliable when compared to the traditional ash method.

Summary The time-course for the thermal acclimation of aerobic metabolism was determined for juvenile and adult P. scrqfa by following the modifications in oxygen consumption (O2). Fish previously acclimated at 15 and 25°C and transferred to water temperature 10°C and above, showed 3–4-fold increase in O2 in the first 6 h after the change in temperature, reaching a stable O2 level in 24 and 48 h (adult and juvenile fish, respectively). Fish acclimated at 35 and 25°C and transferred to water temperature 10°C below that to which they were previously acclimated decreased the O2 in the first 6 h after the change in water temperature (3.0-fold lower, from 35 to 25°C for both fish sizes and 6.5- and 3.0-fold lower from 25 to 15°C for juvenile and adult fish, respectively), reaching a stable O2 level in 48 h (from 25 to 15°C) and 24–72 h (from 35 to 25°C). Regardless of the direction of water temperature change, P. scrofa showed partial O2 compensation and short time-course for the adjustments in aerobic metabolism, reaching a stable rate at the new water temperature in 1–3 days. These results indicate an ecological advantage for this species, mainly for the adult fish, which migrate along rivers prior to reproduction and which may experience fluctuations in water temperature, including the heated effluents from electrical power stations.

The methylesterification status of cell wall pectins, mediated through the interplay of pectin methylesterases (PMEs) and pectin methylesterase inhibitors (PMEIs), influences the biophysical properties of plant cell walls. We found that the overexpression of a PMEI gene in Arabidopsis thaliana plants caused the stems to develop twists and loops, most strongly around points on the stem where leaves or inflorescences failed to separate from the main stem. Altered elasticity of the stem, underdevelopment of the leaf cuticle, and changes in the sugar composition of the cell walls of stems were evident in the PMEI overexpression lines. We discuss the mechanisms that potentially underlie the aberrant growth phenotypes.

The structural organization of the interbranchial septum of the gill arch of the air-breathing loricariid fish Hypostomus plecostomus was examined using light and electron microscopy. In the middle of the interbranchial septum, an extensive interconnection was found between the afferent primary arteries from successive and opposing primary lamellae. The blood circulates among numerous trabeculae consisting of connective tissue, smooth muscle cells, and collagen fibres. A sheet of smooth muscle cells is localized at the borders of these interconnected primary arteries and joins the cartilage rod from one primary lamella to the adjacent one on the same hemibranch. The adductor muscles are restricted to the distal end of the interbranchial septum and consist of transverse and oblique striated muscle fibres fixed to the cartilage rod from the primary lamella of opposite hemibranchs. The arrangement of these muscle fibres suggests a double movement of adduction: approximation of the tips of the primary lamellae of opposing hemibranchs and reduction of the space between adjacent primary lamellae of the same hemibranch. The action of both smooth and striated muscles reduces the interconnecting vascular septal space between the primary arteries, which may allow fine adjustment of vascular perfusion of the distal part of the filaments as an adaptation for better blood flow under hypoxic conditions.

7 pages, 1 table.-- PMID: 16762114 [PubMed]., An epidemiological survey was undertaken to explore human exposure to Rickettsia slovaca in two provinces of northern Spain. When IFAT were used to test 200 members of the general population for antibodies to rickettsiae of the spotted-fever group, six (3.3%) were found positive, presumably, since Dermacentor is one of the most common genera of human-biting tick in the study area, for antibodies to R. slovaca. Thirty-one (16.9%) of an additional 183 subjects who presented shortly after being bitten by ticks were also found seropositive. The difference in seroprevalence between the general and the tick-bitten populations was significant. Subject gender had no influence on seroprevalence in either population, although, in the tick-bitten group, age and occupation did have a significant influence on the prevalence recorded. Immunoblotting was used to confirm the presence of antibodies in the five subjects, all from the tick-bitten group, found to have acute infections. Three D. marginatus ticks obtained from three of these acute cases were found PCR-positive for R. slovaca DNA., The authors would like to thank all the physicians who provided serum samples, and the members of the EBATRAG (Enfermedades Bacterianas Transmitidas por Garrapatas) network in Castilla-León (G03/057).

In this work, anthers isolated from male flowers of carob tree (Ceratonia siliqua L.) at developmental phase II were cryopreserved by vitrification, using a fast freezing method. Different types of carbohydrates at different concentrations, namely sorbitol (0.5 M, 1 M, and 2 M), mannitol (0.5 M and 1 M), sucrose (0.5 M, 1 M, and 2 M), and glucose (0.5 M, 1 M, and 2 M), namely sorbitol, mannitol, sucrose and glucose, were compared with respect to their capacity of inducing freeze tolerance in pollen of carob tree. Carbohydrates were applied as a pre-treatment to anthers before cryostorage. It was also assessed if the presence of the cryoprotectant during the storage period was beneficial for pollen viability. The viability of cryopreserved pollen was evaluated after 5 and 8 months of storage. The application of the cryoprotectants generally increased pollen viability as compared with the control. The best results were obtained after 5 months storage, in the presence of the cryo-protector, with pollen pre-treated with sucrose 0.5 M. The viability of pollen decreased with increasing the duration of storage period.

Laminin-5, the major extracellular matrix protein produced by mammary epithelial cells, is composed of three chains (designated alpha3A, beta3, and gamma2), each encoded by a separate gene. Laminin-5 is markedly down-regulated in breast cancer cells. Little is known about the regulation of laminin gene transcription in normal breast cells, nor about the mechanism underlying the down-regulation seen in cancer. In the present study, we cloned the promoter of the gene for the human laminin alpha3A chain (LAMA3A) and investigated its regulation in functionally normal MCF10A breast epithelial cells and several breast cancer cell lines. Using site-directed mutagenesis of promoter-reporter constructs in transient transfection assays in MCF10A cells, we find that two binding sites for Kruppel-like factor 4 (KLF4/GKLF/EZF) are required for expression driven by the LAMA3A promoter. Electrophoretic mobility shift assays reveal absence of KLF4 binding activity in extracts from T47D, MDA-MB 231, ZR75-1, MDA-MB 436, and MCF7 breast cancer cells. Transient transfection of a plasmid expressing KLF4 activates transcription from the LAMA3A promoter in breast cancer cells. A reporter vector containing duplicate KLF4-binding sites in its promoter is expressed at high levels in MCF10A cells but at negligible levels in breast cancer cells. Thus, KLF4 is required for LAMA3A expression and absence of laminin alpha3A in breast cancer cells appears, at least in part, attributable to the lack of KLF4 activity.

3002 Background: AZD2171 is a highly potent and orally available inhibitor of VEGF receptor tyrosine kinase activity, and has shown antitumor activity in a wide range of tumor xenograft models. This Phase I trial was designed to assess the safety, tolerability and pharmacokinetics (PK) of ascending doses of AZD2171 in patients with advanced tumors with liver metastases. Methods: Eligibility criteria included solid tumors refractory to standard therapies and a WHO performance status of 0–2. Patients received a single oral dose of AZD2171 (0.5–60 mg) followed by a 7-day washout period, before continuing daily oral therapy at the same initial dose for 28 days. Further 28-day cycles were administered until a withdrawal criterion was met. Results: As of Sep 10, 2004, 36 patients (19–75 years) have received treatment with AZD2171 (0.5, 1, 2.5, 5, 10, 20, 30, 45 or 60 mg; n = 3, 3, 3, 3, 4, 3, 5, 4 and 8, respectively). AZD2171 was generally well tolerated at doses ≤45 mg/day. Overall, the most common adverse ev...

Intraperitoneal (i. p.) inoculation of several strains of mice with myocarditic strains of coxsackievirus B3 (CVB3) induces focal lesions in the myocardium [7, 9, 18, 41–43]. Within hours of virus inoculation, virus appears in the blood [24] and remains in the plasma phase over the next 2–3 days [1, 8]. Virus is also associated with circulating leukocytes [6, 13]. During this period, virus enters the myocardium and replicates in unidentified cells. Histologic examination of heart tissues during this prelesion period reveals single and multiple foci of necrotic myocytes [17]. Beginning on day 4 postinoculation (p. i.), lesion formation is detected in the myocardium of CVB3-inoculated CD-1 mice [11]. The initial cells detected in the nascent lesion were macrophages, T suppressor/cytotoxic lymphocytes, and natural killer cells. T helper lymphocytes were detected 1 day later. At no time during the 4–10 day (p. i.) period were cells producing viral antigens [11] or viral RNA (N. M. Chapman, S. M. Tracy, and C. J. Gauntt, unpublished data) detected in nascent or developing lesions. The data suggest that virus infection of a cell (or cells) in the myocardium initiates a series of events that induce a focal inflammatory response.

The frequency of electric organ discharges (EOD) of a gymnotiform fish of "pulse" frequency (40-100 Hz) from South America - Ramphicthys rostratuswas studied. The animals were settled in pairs in a aquarium and thus observed: variation in EOD frequency had at least two components: one more positively correlated with temperature, another less positively correlated due to social interaction. A frequência de descargas do órgão elétrico {DOE) de um peixe gymnotiforme de freqüência "em pulso" (40-100 Hz) da América do Sul - Rhamphicthys rostratus - foi estudada. Os animais foram colocados aos pares em aquários e depois observados: variação da freqüência do DOE tinha sido menor duas componentes: uma mais positivamente correlacionada devido a interação social.

Recently, genetic evidence supported the existence of a new species of the genus Pollicipes from the Cape Verde Islands, previously considered a population of P. pollicipes. However, P. pollicipes was not sampled at its southern limit of distribution (Dakar, Senegal), which is geographically separated from the Cape Verde Islands by about 500 km. Herein we describe Pollicipes caboverdensis sp. nov. from the Cape Verde Islands and compare its morphology with the other three species of Pollicipes: P. pollicipes, P. elegans and P. polymerus. Pollicipes pollicipes was sampled at both the middle (Portugal) and southern limit (Dakar, Senegal) of its geographical distribution. The genetic divergence among and within these two regions and Cape Verde was calculated through the analysis of partial mtDNA CO1 gene sequences. Pollicipes caboverdensis sp. nov. has a single whorl of capitular plates below the subrostrum, peduncular scales pointing up toward the capitulum and multi-articulate caudal appendages (all characters shared with P. pollicipes and P. elegans), reddish-orange capitular plates (large specimens), a single rostral median latus between the median latus and the rostrolatus (both characters shared with P. elegans), and uniquely possesses peduncular scales that are approximately the same width as height. The genetic distance between the Cape Verde population and the Senegal and Portugal populations is 13–14%, whilst between Senegal and Portugal it is < 1%.

The stalked barnacle Pollicipes pollicipes is a cirriped crustacean that lives on very exposed rocky shores. This barnacle is the most important economical resource on intertidal rocky shores of continental Portugal. It is highly prized as food and heavily exploited (professional and recreational fishery), but fishery data are scarce and do not estimate the real pressure upon this resource. Despite its socio-economic interest, specific regulations on this fishery are recent and different along the Portuguese coast. Four regions with different regulation can be identified: the marine reserve “Reserva Natural das Berlengas” (RNB) and the marine park “Parque Marinho Prof. Luiz Saldanha” (PMLS) (both in central Portugal); the natural park located in SW Portugal (“Parque Natural do Sudoeste Alentejano e Costa Vicentina”, PNSACV); and the rest of the coast. The main objective of the present study was to study the spatial patterns of percentage cover, biomass, density and size structure of P. pollicipes in areas with different exploitation regimes, including harvested areas and no-take areas. Additionally, variability between mid shore and low shore barnacles was also analysed. Seven areas were sampled with a variable number of sites (a total of 24) randomly sampled in each area during 2011. Photographs and image analysis (percentage cover) and destructive sampling (density, biomass and size) were used. In general, percentage cover, biomass and density were higher in mid shore when compared to low shore, namely in harvested areas. Low shore barnacles had a higher proportion of adults with moderate and high commercial value, while juveniles were relatively more abundant at mid shore. There were no consistent differences in the patterns of distribution and abundance of P. pollicipes among areas subject to different exploitation regimes. The most different area was the harvested area by professional fishers in RNB, where the highest biomass within the study was registered (mid shore, 7.7 kg·m − 2 ). Barnacles within this area presented a higher proportion of adults with commercial value, while recruits and juveniles were relatively more abundant in other areas. The hypothesis of a highest percentage cover, density and biomass in the low shore of no-take sites was not supported.