Search

Plasmodium, the malaria parasite, undergoes a complex life cycle alternating between a vertebrate host and a mosquito vector of the genus Anopheles. In red blood cells of the vertebrate host, Plasmodium multiplies asexually or differentiates into gamete precursors, the male and female gametocytes, responsible for parasite transmission. Sexual stage maturation occurs in the midgut of the mosquito vector, where male and female gametes egress from the host erythrocytes to fuse and form a zygote. Gamete egress entails the successive rupture of two membranes surrounding the parasite, the parasitophorous vacuole membrane and the erythrocyte plasma membrane. In this study, we used the rodent model parasite Plasmodium berghei to design a label-free quantitative proteomic approach aimed at identifying gender-related proteins differentially released/secreted by purified mature gametocytes when activated to form gametes. We compared the abundance of molecules secreted by wild type gametocytes of both genders with that of a transgenic line defective in male gamete maturation and egress. This enabled us to provide a comprehensive data set of egress-related molecules and their gender specificity. Using specific antibodies, we validated eleven candidate molecules, predicted as either gender-specific or common to both male and female gametocytes. All of them localize to punctuate, vesicle-like structures that relocate to cell periphery upon activation, but only three of them localize to the gametocyte-specific secretory vesicles named osmiophilic bodies. Our results confirm that the egress process involves a tightly coordinated secretory apparatus that includes different types of vesicles and may put the basis for functional studies aimed at designing novel transmission-blocking molecules.

Background: Tracking the white matter principal tracts is routinely typically included during the pre-surgery planning examinations and has revealed to limit functional resection of low-grade gliomas (LGGs) in eloquent areas.Objective: We examined the integrity of the Superior Longitudinal Fasciculus (SLF) and Inferior Fronto-Occipital Fasciculus (IFOF), both known to be part of the language-related network in patients with LGGs involving the temporo-insular cortex. In a comparative approach, we contrasted the main quantitative fiber tracking values in the tumoral (T) and healthy (H) hemispheres to test whether or not this ratio could discriminate amongst patients with different post-operative outcomes.Methods: Twenty-six patients with LGGs were included. We obtained quantitative fiber tracking values in the tumoral and healthy hemispheres and calculated the ratio (HIFOF–TIFOF)/HIFOF and the ratio (HSLF–TSLF)/HSLF on the number of streamlines. We analyzed how these values varied between patients with and without post-operative neurological outcomes and between patients with different post-operative Engel classes.Results: The ratio for both IFOF and SLF significantly differed between patient with and without post-operative neurological language deficits. No associations were found between white matter structural changes and post-operative seizure outcomes.Conclusions: Calculating the ratio on the number of streamlines and fractional anisotropy between the tumoral and the healthy hemispheres resulted to be a useful approach, which can prove to be useful during the pre-operative planning examination, as it gives a glimpse on the potential clinical outcomes in patients with LGGs involving the left temporo-insular cortex.

Epoxiconazole (EPO) and fludioxonil (FLU) are two widely used fluorinated pesticides known to be highly persistent and with high ecotoxicological potential, turning them into pollutants of concern. This work aimed to optimize two degrading bacterial consortia, previously obtained from an agricultural soil through enrichment with EPO and FLU, by characterizing the contribution of their corresponding bacterial isolates to the biodegradation of these pesticides using both culture-dependent and independent methodologies. Results showed that a co-culture of the strains Hydrogenophaga eletricum 5AE and Methylobacillus sp. 8AE was the most efficient in biodegrading EPO, being able to defluorinate ca. 80% of this pesticide in 28 days. This catabolic performance is likely the result of a commensalistic cooperation, in which H. eletricum may be the defluorinating strain and Methylobacillus sp. may assume an accessory, yet pivotal, catabolic role. Furthermore, 16S rRNA metabarcoding analysis revealed that these strains represent a minority in their original consortium, showing that the biodegradation of EPO can be driven by less abundant phylotypes in the community. On the other hand, none of the tested combinations of bacterial strains showed potential to biodegrade FLU, indicating that the key degrading strains were not successfully isolated from the original enrichment culture. Overall, this work shows, for the first time, the direct involvement of two bacterial species, namely H. eletricum and Methylobacillus sp., in the biodegradation of EPO, while also offering insight on how they might cooperate to accomplish this process. Moreover, the importance of adequate culture-dependent approaches in the engineering of microbial consortia for bioremediation purposes is also emphasized.

Synthetic pyrethroids (SPs) are one of the most common pesticides used worldwide. Their use has greatly increased in the last decades and its' continuous application lead to added pesticides concentration in soil. Consequently, SPs may enter the food chain, affecting the environment and human health. The degradation over time of the pyrethroid pesticide deltamethrin applied to cabbages was monitored. The evolution was followed both on cabbages and the surrounding soils, and the soil microbial community characterized by next-generation sequencing of the 16S rRNA gene. The main shift in the microbial community structure was observed during the first 30 days after pesticides' application. The modification in the microbial community composition, where an increased abundance of Nocardioides sp. and Sphingomonas sp. were observed, was correlated respectively with the conversions of deltamethrin and its metabolite, 3-phenoxybenzoic acid (3-PBA). Although deltamethrin was not found in any of the tested samples (soil and cabbage) after 180 days, it caused an environmental impact much further than the 7 days security interval. These findings suggest that deltamethrin application can disturb soil microbial community and that natural biodegradation can have an important part in pesticides soil decontamination.

Fruitiness is a highly desirable aroma quality in white wines. This study aimed to investigate the fruitiness aroma perception of white wines using a rapid sensory method and to compare the sensory maps obtained from wine experts and trained consumers. A polarized projective mapping (PPM) method was adapted by using fruit standards as fixed poles. Twenty-one white wines were selected for this study. Ten wine experts and twelve trained consumers participated in the adapted PPM followed by ultra-flash profiling (UFP). While experts and trained consumers used different poles to characterize the Pinot Gris and Viogniers, both panels used the stone fruit pole for the Chardonnays. UFP revealed that the Pinot Gris and Viognier samples presented fruity and floral aromas and most Chardonnay wines presented non-fruity aromas. The white wines investigated presented a wide range of fruity aromas. The use of aroma standards instead of products as poles seems to be a reliable alternative to investigate fruitiness in white wines. This study helped us to understand the predominant aromas of varietal white wines from Oregon and emphasized the importance of adapting sensory methods to investigate fruitiness perception.

Landing data are the most basic information used to manage fisheries, although they are often unavailable or incomplete. The objective of this work was to reconstruct the national database of marine commercial landings for the Brazilian industrial and artisanal fisheries, from 1950 to 2015. Total landings increased strongly from 1950 to mid-1980s and suffered sharp decline in the early 1990s, mainly associated to the collapse of sardine fisheries. After that, another period of increasing landings was observed, but at a much lower rate. Industrial landings always surpassed artisanal landings in Brazilian waters, except for the beginning of the time series, when many industrial fleets had not started yet, and in the early 2000s, when a change in the methodology for collecting landing statistics was implemented in the state of Pará leading to an overestimation of artisanal landings. Artisanal fisheries have been declining since 2005, which is worrisome due to the social impact it may have on local income and food security. Regional differences were also observed, with industrial landings being always higher than artisanal landings in southeastern-southern Brazil, while the opposite was true for the northern-northeastern regions. Higher landings were observed in the southeastern-southern regions when both artisanal and industrial fleets were combined. Sardine and demersal fishes were the main resources landed by industrial fishers. Artisanal fishers caught more species than their industrial counterpart, featuringXiphopenaeus kroyeri,Cynoscion acoupa, andUcides cordatus. Although the fishing ofEpinephelus itajarawas banned in Brazil, it continues to be landed. Yet, catches of this species and others under some threat status are still not properly registered, including:Carcharhinus longimanus,Galeorhinus galeus,Sphyrna lewini,Sphyrna mokarran,Pristis pectinata, andPseudobatos horkelii. Fishing resources not identified in previous landing reconstruction efforts, such as sea urchins and sea cucumbers, have now been reported. The database presented here should be continuously updated and improved. It is of paramount importance to resume the collection of landing statistics, including information on fishing effort, to assess the relative impact of fisheries and environmental factors on the main Brazilian fishing stocks.

Research on the emerging COVID-19 pandemic is demonstrating that wastewater infrastructures can be used as public health observatories of virus circulation in human communities. Important efforts are being organized worldwide to implement sewage-based surveillance of SARS-CoV-2 that can be used for preventive or early warning purposes, informing preparedness and response measures. However, its successful implementation requires important and iterative methodological improvements, as well as the establishment of standardized methods. The aim of this study was to develop a continuous monitoring protocol for SARS-CoV-2 in wastewater, that could be used to model virus circulation within the communities, complementing the current clinical surveillance. Specific objectives included (1) optimization and validation of a sensitive method for virus quantification; (2) monitoring the time-evolution of SARS-CoV-2 in wastewater from two wastewater treatment plants (WWTPs) in the city of Porto, Portugal. Untreated wastewater samples were collected weekly from the two WWTPs between May 2020 and March 2021, encompassing two COVID-19 incidence peaks in the region (mid-November 2020 and mid-January 2021). In the first stage of this study, we compared, optimized and selected a sampling and analysis protocol that included RNA virus concentration through centrifugation, RNA extraction from both liquid and solid fractions and quantification by reverse transcription quantitative PCR (RT-qPCR). In the second stage, we used the selected methodology to track SARS-CoV-2 in the collected wastewater over time. SARS-CoV-2 RNA was detected in 39 and 37 out of 48 liquid and solid fraction samples of untreated wastewater, respectively. The copy numbers varied throughout the study between 0 and 0.15 copies/ng RNA and a good fit was observed between the SARS-CoV-2 RNA concentration in the untreated wastewater and the COVID-19 temporal trends in the study region. In agreement with the recent literature, the results from this study support the use of wastewater-based surveillance to complement clinical testing and evaluate temporal and spatial trends of the current pandemic.

This study aims to investigate the effect of two different groups of phenolic compounds (the alkylphenols nonylphenol (NP) and octylphenol (OP), and the chlorophenol pentachlorophenol (PCP)) on constructed wetlands (CWs) performance, including on organic matter, nutrients and contaminants removal efficiency, and on microbial community structure in the plant bed substrate. CWs were assembled at lab scale simulating a vertical flow configuration and irrigated along eight weeks with Ribeira de Joane (an urban stream) water not doped (control) or doped with a mixture of NP and OP or with PCP (at a 100 μg L−1 concentration each). The presence of the phenolic contaminants did not interfere in the removal of organic matter or nutrients in CWs in the long term. Removals of NP and OP were &gt, 99%, whereas PCP removals varied between 87% and 98%, mainly due to biodegradation. Microbial richness, diversity and dominance in CWs substrate were generally not affected by phenolic compounds, with only PCP decreasing diversity. Microbial community structure, however, showed that there was an adaptation of the microbial community to the presence of each contaminant, with several specialist genera being enriched following exposure. The three more abundant specialist genera were Methylotenera and Methylophilus (methylophilaceae family) and Hyphomicrobium (hyphomicrobiaceae family) when the systems were exposed to a mixture of NP and OP. When exposed to PCP, the three more abundant genera were Denitromonas (Rhodocyclaceae family), Xenococcus_PCC_7305 (Xenococcaceae family) and Rhodocyclaceae_uncultured (Rhodocyclaceae family). To increase CWs efficiency in the elimination of phenolic compounds, namely PCP which was not totally removed, strategies to stimulate (namely biostimulation) or increase (namely bioaugmentation) the presence of these bacteria should be explore. This study clearly shows the potential of vertical flow CWs for the removal of phenolic compounds, a still little explored subject, contributing to promote the use of CWs as nature-based solutions to remediate water contaminated with different families of persistent and/or emergent contaminants.

Volatile compounds are responsible for driving the aroma of wine. Because of their low perception thresholds, norisoprenoids may play an important role in wine aroma. Studies have shown that β-damascenone may act as an aroma enhancing compound. However, the direct impact on wine aroma is unclear. Our study examined the direct impact of β-ionone and β-damascenone on the aroma sensory perception of Pinot noir wines. Triangle tests were used to determine if assessors could distinguish between wines with varying concentrations of β-ionone and β-damascenone in three different Pinot noir wine matrixes. Descriptive analysis was performed on these treatments, perceived as different in triangle tests. Results show that β-ionone acts as a significant contributor to aromas in Pinot noir wine, as individuals could differentiate both the low and high concentration wines from the control. How β-ionone impacted wine aroma depends on the wine matrix, as different aroma descriptors were affected based on the model wine used, resulting in floral, red berry or dark berry aromas. The effect of β-damascenone on Pinot noir aroma was less clear, as perception seems to be heavily influenced by wine matrix composition. This study contributes to our understanding of the complex chemical causation of fruity aromas in Pinot noir wine.

Highlights • A left temporal cavernoma caused focal epilepsy facilitated by drawing, in an artist. • Drawing triggered delta activity on left anterior temporal and orbito-frontal areas. • These areas were activated by drawing and the left uncinate fasciculus connecting them was affected. • Neuronal excessive discharges and hyper-synchrony may be facilitated by altered connectivity of different cortical areas., There are few studies in literature reporting drawing as a strong trigger of praxis-induced focal seizures. The aim of the present case report was describing a case of focal epilepsy with praxis induced EEG activation, due to a cavernoma, in the left middle anterior temporal lobe by using a multimodal approach. We combined video-EEG, showing that drawing increased a sustained monomorphic delta activity localized on left anterior temporal region (F7-T1a), diffusing to the vertex (Fz) and the fronto-polar electrodes (F3), with DTI data, showing that the left uncinate fasciculus, connecting the temporal pole to the orbitofrontal cortex, significantly differed from controls. fMRI confirmed that drawing increased activation in these areas. The congruence between findings supports the role of the left uncinated fasciculus linking the temporal lobe to the orbitofrontal cortex in the present focal epilepsy mainly facilitated by drawing.

Malolactic fermentation (MLF) is an important process in wine production due to the resulting reduction in acidity. MLF is typically induced by the addition of Oenococcus oeni after the completion of alcoholic fermentation (AF), but can occur concurrent with AF by co-inoculation of O. oeni with Saccharomyces cerevisiae. This study investigated the effect of MLF inoculation timing and temperature (15 &deg, C and 21 &deg, C) and the presence of the non-Saccharomyces yeast Torulaspora delbrueckii on Chardonnay wine aroma and mouthfeel. Aroma composition was measured using headspace solid-phase microextraction-gas chromatography mass spectrometry (HS-SPME-GCMS). Mouthfeel attributes of the wines produced were assessed by a winemaker panel, using Napping&reg, and Ultra-flash profiling. Significant differences in aroma composition and mouthfeel perception were found based on MLF timing and inoculation conditions, as well as between temperatures. Temperature had a greater impact on the aroma composition for sequential inoculations, while there were little differences based on the temperature of concurrent fermentations. Treatment type and temperature also affected the chemical composition of finished wines. Mouthfeel was impacted, although not as strongly as aroma composition. These findings demonstrate the usefulness of various MLF practices to influence the sensory qualities of a Chardonnay wine.

Male and female Plasmodium gametocytes ingested by the Anopheles mosquitoes during a blood meal egress from the red blood cells by rupturing the two surrounding membranes, the parasitophorous vacuole and the red blood cell membranes. Proteins of the so‐called osmiophilic bodies, (OBs), secretory organelles resident in the cytoplasm, are important players in this process. Once gametes emerge, the female is ready to be fertilized while the male develops into motile flagellar gametes. Here, we describe the function(s) of PBANKA_1115200, which we named Gamete Egress Protein (GEP), a protein specific to malaria parasites. GEP is restricted to gametocytes, expressed in gametocytes of both genders and partly localizes to the OBs. A mutant lacking the protein shows aberrant rupture of the two surrounding membranes, while OBs discharge is delayed but not aborted. Moreover, we identified a second function of GEP during exflagellation since the axonemes of the male flagellar gametes were not motile. Genetic crossing experiments reveal that both genders are unable to establish infections in mosquitoes and thus the lack of GEP leads to a complete block in Plasmodium transmission from mice to mosquitoes. The combination of our results reveals essential and pleiotropic functions of GEP in Plasmodium gametogenesis., PBANKA_1115200, which we named Gamete Egress Protein (GEP), is a gametocyte protein found only in malaria parasites. GEP partly localizes to osmophilic bodies. A mutant lacking this protein showed aberrant rupture of the parasitophorous vacuole and the red blood cell membranes. Axonemes of the male flagellar gametes gep(‐) were not motile.

Membrane microdomains that include lipid rafts, are involved in key physiological and pathological processes and participate in the entry of endocellular pathogens. These assemblies, enriched in cholesterol and sphingolipids, form highly dynamic, liquid-ordered phases that can be separated from the bulk membranes thanks to their resistance to solubilization by nonionic detergents. To characterize complexity and dynamics of detergent-resistant membranes of sexual stages of the rodent malaria parasite Plasmodium berghei, here we propose an integrated study of raft components based on proteomics, lipid analysis and bioinformatics. This analysis revealed unexpected heterogeneity and unexplored pathways associated with these specialized assemblies. Protein-protein relationships and protein-lipid co-occurrence were described through multi-component networks. The proposed approach can be widely applied to virtually every cell type in different contexts and perturbations, under physiological and/or pathological conditions.

The malaria parasite Plasmodium falciparum is exposed, during its development, to major changes of ionic composition in its surrounding medium. We demonstrate that the P. falciparum serpentine-like receptor PfSR25 is a monovalent cation sensor capable of modulating Ca2+ signaling in the parasites. Changing from high (140 mM) to low (5.4 mM) KCl concentration triggers [Ca2+]cyt increase in isolated parasites and this Ca2+ rise is blocked either by phospholipase C (PLC) inhibition or by depleting the parasite’s internal Ca2+ pools. This response persists even in the absence of free extracellular Ca2+ and cannot be elicited by addition of Na+, Mg2+ or Ca2+. However, when the PfSR25 gene was deleted, no effect on [Ca2+]cyt was observed in response to changing KCl concentration in the knocked out (PfSR25−) parasite. Finally, we also demonstrate that: i) PfSR25 plays a role in parasite volume regulation, as hyperosmotic stress induces a significant decrease in parasite volume in wild type (wt), but not in PfSR25− parasites; ii) parasites lacking PfSR25 show decreased parasitemia and metacaspase gene expression on exposure to the nitric oxide donor sodium nitroprusside (SNP) and iii), compared to PfSR25− parasites, wt parasites showed a better survival in albumax-deprived condition.

We addressed the neuroanatomical correlates of 54 right-brain-damaged neurosurgical patients on visuo-spatial design fluency, which is a measure of the ability to generate/plan a series of new abstract combinations in a flexible way. 22.2% of the patients were impaired. They failed the task because they did not use strategic behavior, in particular they used rotational strategy to a significantly lower extent and produced a significantly higher rate of perseverative errors. Overall performance did not correlate with neuropsychological tests, suggesting that proficient performance was independent of other cognitive domains. Performance significantly correlated with use of rotational strategy. Tasks related to executive functions such as psychomotor speed and capacity to shift were positively correlated to the number of strategies used to solve the task. Lesion analysis showed that the maximum density of the patients' lesions-obtained by subtracting the overlap of lesions of spared patients from the overlap of lesions of impaired patients-overlaps with the precentral gyrus, rolandic operculum/insula, superior/middle temporal gyrus/hippocampus and, at subcortical level, with part of the superior longitudinal fasciculus, external capsule, retrolenticular part of the internal capsule and sagittal stratum (inferior longitudinal fasciculus and inferior fronto-occipital fasciculus). These areas are part of the fronto-parietal-temporal network known to be involved in top-down control of visuo-spatial attention, suggesting that the mechanisms and the strategies needed for proficient performance are essentially visuo-spatial in nature.

Scientific interest in the genus Taxus has recently increased worldwide, for both conservation and biotechnological purposes. In contrast with abundant studies on other gymnosperms, basic cytogenetic information on this genus is still very limited, likely due to difficulties in appropriate tissue handling and lack of efficient procedures for chromosome analyses. In the present study, chromosome pre-treatment protocols were established, and karyotype analyses successfully performed on meristematic tissue of root-tips derived from plant cuttings of English yew (Taxus baccata L.). Twelve homologous pairs were identified, including seven metacentric and five sub-metacentric chromosomes. No heteromorphic sex chromosomes were found. Multicolour fluorescence in situ hybridization (FISH) was successfully applied using the 18S and 5S rDNA probe sequences. Loci of both sequences turned out to be located on the same sub-metacentric chromosome pair. Microsatellite (simple sequence repeat) sequences were also ...

Commercial fisheries catches by countries are documented since 1950 by the Food and Agriculture Organization (FAO). Unfortunately, this does not hold for marine recreational catches, of which only few, if any, estimates are reported to FAO. We reconstructed preliminary estimates of likely marine recreational catches for 1950—2014, based on independent reconstructions for 125 countries. Our estimates of marine recreational catches that are retained and landed increased globally until the early-1980s, stabilized through the 1990s, and began increasing again thereafter, amounting to around 900,000 t·year−1 in 2014. Marine recreational catches thus account for slightly less than 1% of total global marine catches. Trends vary regionally, increasing in Asia, South America and Africa, while slightly decreasing in Europe and Oceania, and strongly decreasing in North America. The derived taxonomic composition indicates that recent catches were dominated by Sparidae (12% of total catches), followed by Scombridae (10%), Carangidae (6%), Gadidae (5%), and Sciaenidae (4%). The importance of Elasmobranchii (sharks and rays) in recreational fisheries in some regions is of concern, given the life-history traits of these taxa. Our preliminary catch reconstruction, despite high data uncertainty, should encourage efforts to improve national data reporting of recreational catches.

The Plasmodium subtilisin‐like serine protease SUB1 is expressed in hepatic and both asexual and sexual blood parasite stages. SUB1 is required for egress of invasive forms of the parasite from both erythrocytes and hepatocytes, but its subcellular localisation, function, and potential substrates in the sexual stages are unknown. Here, we have characterised the expression profile and subcellular localisation of SUB1 in Plasmodium berghei sexual stages. We show that the protease is selectively expressed in mature male gametocytes and localises to secretory organelles known to be involved in gamete egress, called male osmiophilic bodies. We have investigated PbSUB1 function in the sexual stages by generating P. berghei transgenic lines deficient in PbSUB1 expression or enzyme activity in gametocytes. Our results demonstrate that PbSUB1 plays a role in male gamete egress. We also show for the first time that the PbSUB1 substrate PbSERA3 is expressed in gametocytes and processed by PbSUB1 upon gametocyte activation. Taken together, our results strongly suggest that PbSUB1 is not only a promising drug target for asexual stages but could also be an attractive malaria transmission‐blocking target.

An untargeted lipidomic profiling approach based on ultra - performance liquid chromatography - time-of-flight tandem mass spectrometry (UPLC-TOF-MS/MS) was successfully used to study the origin of commercial Pinot noir wines. The total wine lipids were extracted using a modified Bligh-Dyer method. In all wine samples, the total lipids were less than 0.1% (w/w) of wine. The wines analyzed consisted of 222 lipids from 11 different classes. 48 commercial Pinot noir wine samples were collected from producers in Burgundy, California, Oregon, and New Zealand. Lipidomic data was studied using advanced multivariate analysis methods, random forest, k-nearest neighbor (k-NN), and linear discriminant analysis. The overall classification accuracy was 97.5% for random forest and 90% for k-NN. Wine lipids showed a strong potential for classifying wines by origin, with the top 58 lipids contributing to the discrimination. This information could potentially be used for further study of the impacts of lipids on wine characteristics and authenticity.

Several diatom species produce resting stages as part of their life cycle. These resting stages accumulate in the sediments where they can remain for a long time before being re-suspended in the water column and switching to active growth. Until now, the abundance and diversity of viable diatom resting stages have been assessed using the Serial Dilution Culture (SDC) method. In the present study, surface sediment samples from the Gulf of Naples were used to compare results obtained with the SDC method with those provided by HTS metabarcoding based on DNA extracted from the same sediment sample; the marker used was the V4 region of 18S rDNA. HTS metabarcoding showed a marked dominance of polar centric diatoms, among which Chaetoceros species were the most represented, in terms of both sequence and ribotype number. Almost all the most abundant ribotypes identified with metabarcoding matched records of species observed in SDCs. In some cases, however, this marker region could not distinguish between morphologically and phylogenetically distinct species, e.g., Skeletonema pseudocostatum and S. tropicum. As expected, molecular analysis provided a higher number of ribotypes as compared to the number of taxa recorded by SDC. Despite the well-known biases of both methodologies for quantitative assessments, our results show that DNA metabarcoding via HTS sequencing is a promising approach to explore the diversity of diatom resting stages. This study also confirms the importance of curated reference sequences to fully interpret the diversity stored in environmental sediment samples.

The sporozoite, the stage of the malaria parasite transmitted by the mosquito, first develops for ∼2 weeks in an oocyst. Rupture of the oocyst capsule is required for release of sporozoites, which then transfer to the salivary gland where they are injected into a new host. Here we identify two parasite proteins that we call oocyst rupture proteins 1 (ORP1) and ORP2. These proteins have a histone-fold domain (HFD) that promotes heterodimer formation in the oocyst capsule at the time of rupture. Oocyst rupture is prevented in mutants lacking either protein. Mutational analysis confirms the HFD as essential for ORP1 and ORP2 function, and heterodimer formation was verified in vitro. These two proteins are potential targets for blocking transmission of the parasite in the mosquito., Oocyst rupture and release of malaria sporozoites is needed for transmission of parasites from vector to humans. Here the authors identify two proteins, which they name ORP1 and ORP2, that form heterodimers and are required for oocyst rupture.

Brown marmorated stink bug contamination in grape clusters results in the addition of an aroma compound, trans-2-decenal (T2D), to wine. Described as a green, musty aroma, it is considered detrimental to wine quality. The main focus of this study was to estimate the detection threshold (DT) and consumer rejection threshold (CRT) of T2D in Pinot noir, determine T2D’s impact on wine quality, and investigate potential consumer segmentation. The two thresholds were measured using an ascending forced choice method of limits applied to a series of triangle and paired comparison tests, and were estimated with a psychometric function whose significance was based on a binomial distribution as well as on d′ values based on Thurstonian models. The method of quantitation resulted in different T2D threshold levels. The DT of the panel was estimated to be 0.51 μg/L T2D from a psychometric fit and between 1.92 and 4.80 μg/L T2D based on Thurstonian scaled values. Similarly, for CRT, the psychometric function resulted in a threshold of 13.0 and 0.05 μg/L and d′ values between 4.80 and 12.00 μg/L. Wines containing T2D above the CRT were described as green, musty, and less fruity by a professional wine-tasting panel. When potential consumer segmentation was examined on the basis of the DT and CRT data, there was no direct link between sensitivity and preference. Based on these findings, the use of the CRT is recommended when establishing consumer tolerance levels of T2D in wine. Additionally, the use of d′, which provides a more sensitive method of threshold estimation, seems more appropriate for compounds, such as T2D, that negatively affect wine quality.

Floating wetland islands (FWI) are considered nature-based solutions with great potential to promote several ecosystem services, such as biodiversity and water quality enhancement through phytoremediation processes. To our knowledge, the present work is the first to scientifically document the in-situ establishment of an FWI in a seawater port marina. The establishment and performance of a cork floating platform with a polyculture (Sarcocornia perennis, Juncus maritimus, Phragmites australis, Halimione portulacoides, Spartina maritima, Limonium vulgare) was evaluated. The diversity of organisms present in the FWI was undertaken based on the macrofauna assessment, taking into consideration marine water characterization, with a focus on hydrocarbons. Microbial communities were assessed based on metabarcoding approach to study 16S rRNA gene from environmental DNA retrieved from biofilm (from the planting media), marine biofouling (from the submerged platform) and surface marina water. S. perennis was the species with the highest survival rate and growth. The structure of the microbial community showed clear differences between those established in the FWI and those in the surrounding water, showing the presence of some bacterial groups that can be relevant for bioremediation processes (e.g., Saprospiraceae family). Concerning the macrofauna analysis, Mytilus sp. was the predominant taxa. To be of relevance, total petroleum hydrocarbons were detected at the marina up to ca. 6 mg/L. This study gives new insights into broadening FWI application to the saline environments of port marinas and to supporting a management strategy to promote several ecosystem services such biodiversity, species habitat, water quality enhancement and added aesthetic value to the marina landscape.

One of the most prominent manifestations of climate change is the changing Arctic sea-ice regime with a reduction in the summer sea-ice extent and a shift from thicker, perennial multiyear ice towards thinner, first-year ice. These changes in the physical environment are likely to impact microbial communities, a key component of Arctic marine food webs and biogeochemical cycles. During the Norwegian young sea ICE expedition (N-ICE2015) north of Svalbard, seawater samples were collected at the surface (5 m), subsurface (20 or 50 m), and mesopelagic (250 m) depths on 9 March, 27 April, and 16 June 2015. In addition, several physical and biogeochemical data were recorded to contextualize the collected microbial communities. Through the massively parallel sequencing of the small subunit ribosomal RNA amplicon and metagenomic data, this work allows studying the Arctic’s microbial community structure during the late winter to early summer transition. Results showed that, at compositional level, Alpha- (30.7%) and Gammaproteobacteria (28.6%) are the most frequent taxa across the prokaryotic N-ICE2015 collection, and also the most phylogenetically diverse. Winter to early summer trends were quite evident since there was a high relative abundance of thaumarchaeotes in the under-ice water column in late winter while this group was nearly absent during early summer. Moreover, the emergence of Flavobacteria and the SAR92 clade in early summer might be associated with the degradation of a spring bloom of Phaeocystis. High relative abundance of hydrocarbonoclastic bacteria, particularly Alcanivorax (54.3%) and Marinobacter (6.3%), was also found. Richness showed different patterns along the depth gradient for prokaryotic (highest at mesopelagic depth) and protistan communities (higher at subsurface depths). The microbial N-ICE2015 collection analyzed in the present study provides comprehensive new knowledge about the pelagic microbiota below drifting Arctic sea-ice. The higher microbial diversity found in late winter/early spring communities reinforces the need to continue with further studies to properly characterize the winter microbial communities under the pack-ice.

Sporozoites are the infective form of malaria parasites which are transmitted from the mosquito salivary glands to a new host in a mosquito blood meal. The sporozoites develop inside the sporogonic oocyst and it is crucial for the continuation of the life cycle that the oocyst ruptures to release sporozoites. We recently described two Plasmodium Oocyst Rupture Proteins (ORP1 and ORP2), localized at the oocyst capsule, that are each essential for rupture of the oocysts. Both ORPs contain a histone fold domain implicated in the mechanism of oocyst rupture, possibly through the formation of a heterodimer between the two histone fold domains. To gain an understanding of the function of the different regions of the ORP2 protein, we generated deletion mutants. We monitored oocyst formation and rupture as well as sporozoites in the salivary gland. Our results show that different regions of ORP2 play independent roles in sporozoite egress. Deleting the N-terminal histone fold domain of ORP2 blocked sporozoite egress from the oocyst. Progressive deletions from the C-terminal resulted in no or significantly impaired sporozoite egress.

Recreational fisheries in Brazil have increased in importance and attracted many foreign recreational fishers. The objectives of this article were to summarise the available data on Brazilian recreational fisheries, to discuss some of their features and to analyse how they are performing in different regions compared with international trends. A review of published and unpublished sources together with data from recreational fishing licences was used. The participation rate was low (0.9%). Overall, there was a high diversity of fish species, with the following species/group of species repeatedly reported: Menticirrhus spp., Centropomus spp., Cynoscion spp., and Micropogonias furnieri (marine/estuarine water), and Cichla spp., Hoplias malabaricus, and Piaractus mesopotamicus, together with the introduced Oreochromis niloticus and Coptodon rendalli (fresh water). The north-eastern region differs from the other regions: fishing occurs mostly in marine waters and fishers acquire mainly shore-based licences, have minimum fishing expenditures and rarely release fishes. There is no estimate of the total harvest or economic value. Conflicts include catch-and-release-oriented freshwater and marine recreational fishers vs consumption-oriented coastal fishers, tournament participants vs non-participants, commercial fishers and other leisure activities, pollution, ports, species introductions and translocations, protected areas, and federal and state laws. Cases of smooth shifts from artisanal to recreational fishing were detected, possibly associated with changes in the societal relationship with natural resources.

Background and Aims Relationships between the chemical composition and the sensory perception of red wines are generally poorly understood. In New Zealand, the vineyard area of Pinot Noir has increased markedly and wines from this red cultivar now constitute a significant proportion of total production. The aim of this study was to identify aroma compounds of major sensory significance in New Zealand Pinot Noir wines. Methods and Results The concentration of 34 aroma chemicals in 32 New Zealand Pinot Noir wines was quantified using three separate solid-phase microextraction-GC-MS methods. Relationships between these chemical data and sensory data obtained from the same wines were investigated with canonical correlation analysis. Subsequently, correlation results were validated with addition/omissions tests. Ethyl octanoate was found to increase red cherry aroma, the combination of ethyl octanoate and ethyl decanoate enhanced black cherry aroma, and 2-phenyl ethanol increased violet aroma. Benzaldehyde altered Pinot Noir aroma but its effect on specific attributes was unclear. Conclusions The influence of benzaldehyde, ethyl octanoate and 2-phenyl ethanol on the aroma of Pinot Noir wine was consistent with previous studies conducted on Merlot and Cabernet Sauvignon wines. This indicated that the contribution of these compounds is similar in a range of red wines. Ethyl decanoate appeared to act as an aroma enhancer compound when in combination with ethyl octanoate. Significance of the Study Improved knowledge of key aroma and flavour components and the factors that influence their concentration is required to allow producers to manipulate better sensory characteristics to meet the preferences of specific markets. This study provides new information on important aroma compounds in New Zealand Pinot Noir which can lead to future research on modulating factors.

Lactose within whey can be fermented and distilled to produce a potable distilled spirit. The aim of this study was to determine if acid and sweet whey types can be fermented and distilled using similar processes and to investigate differences in volatile aroma compounds for the 2 distillates. Fermentation and distillation of the 2 whey types progressed in a similar manner, using Kluyveromyces marxianus for the initial fermentation and a glass still fitted with a Vigreux column for the subsequent distillation. Ethanol content of the wash (fermented whey) varied considerably following each fermentation and ranged from 1.2 and 2.0% (wt/wt) with no clear trend between acid and sweet whey samples. Volatile aroma compounds were extracted using headspace solid-phase microextraction and identified via gas chromatography-mass spectrometry. Acid and sweet whey distillates contained unique volatile aromatic compounds, and significant differences in compound peak areas were observed. These differences may have an effect upon the organoleptic qualities of spirits produced from whey; therefore, whey source may be an important factor when fermenting and distilling whey.

A pressing clinical need exists for 63% to 65% of combat-wounded service members and 11% to 20% of civilians who develop heterotopic ossification (HO) after blast-related extremity injury and traumatic injuries, respectively. The mammalian target of rapamycin pathway is a central cellular sensor of injury. We evaluated the prophylactic effects of rapamycin, a selective inhibitor of mammalian target of rapamycin signaling, on HO formation in a rat model of blast-related, polytraumatic extremity injury. Rapamycin was administered intraperitoneally daily for 14 days at 0.5 mg/kg or 2.5 mg/kg. Ectopic bone formation was monitored by micro-computed tomography and confirmed by histologic examination. Connective tissue progenitor cells, platelet-derived growth factor receptor-α–positive cells, and α-smooth muscle actin–positive blood vessels were assayed at postoperative day 7 by colony formation and immunofluorescence. Early gene expression changes were determined by low-density microarray. There was significant attenuation of 1) total new bone and soft tissue ectopic bone with 0.5 mg/kg (38.5% and 14.7%) and 2.5 mg/kg rapamycin (90.3% and 82.9%), respectively, 2) connective tissue progenitor cells, 3) platelet-derived growth factor receptor-α–positive cells, 4) α-smooth muscle actin–positive blood vessels, and 5) of key extracellular matrix remodeling ( CD44 , Col1a1 , integrins), osteogenesis ( Sp7 , Runx2 , Bmp2 ), inflammation ( Cxcl5 , 10 , IL6 , Ccl2 ), and angiogenesis ( Angpt2 ) genes. No wound healing complications were noted. Our data demonstrate the efficacy of rapamycin in inhibiting blast trauma-induced HO by a multipronged mechanism.

© 2014 John Wiley & Sons Ltd. Summary: Gametogenesis is the earliest event after uptake of malaria parasites by the mosquito vector, with a decisive impact on colonization of the mosquito midgut. This process is triggered by a drop in temperature and contact with mosquito molecules. In a few minutes, male and female gametocytes escape from the host erythrocyte by rupturing the parasitophorous vacuole and the erythrocyte membranes. Electron-dense, oval-shaped organelles, the osmiophilic bodies (OB), have been implicated in the egress of female gametocytes. By comparative electron microscopy and electron tomography analyses combined with immunolocalization experiments, we here define the morphological features distinctive of male secretory organelles, hereafter named MOB (male osmiophilic bodies). These organelles appear as club-shaped, electron-dense vesicles, smaller than female OB. We found that a drop in temperature triggers MOB clustering, independently of exposure to other stimuli. MDV1/PEG3, a protein associated with OB in Plasmodium berghei females, localizes to both non-clustered and clustered MOB, suggesting that clustering precedes vesicle discharge. A P.berghei mutant lacking the OB-resident female-specific protein Pbg377 displays a dramatic reduction in size of the OB, accompanied by a delay in female gamete egress efficiency, while female gamete fertility is not affected. Immunolocalization experiments indicated that MDV1/PEG3 is still recruited to OB-remnant structures.

We describe involuntary language switching from L2 to L1 evoked by electro-stimulation in the superior temporal gyrus in a 30-year-old right-handed Serbian (L1) speaker who was also a late Italian learner (L2). The patient underwent awake brain surgery. Stimulation of other portions of the exposed cortex did not cause language switching as did not stimulation of the left inferior frontal gyrus, where we evoked a speech arrest. Stimulation effects on language switching were selective, namely, interfered with counting behaviour but not with object naming. The coordinates of the positive site were combined with functional and fibre tracking (DTI) data. Results showed that the language switching site belonged to a significant fMRI cluster in the left superior temporal gyrus/supramarginal gyrus found activated for both L1 and L2, and for both the patient and controls, and did not overlap with the inferior fronto-occipital fasciculus (IFOF), the inferior longitudinal fasciculus (ILF) and the superior longitudinal fasciculus (SLF). This area, also known as Stp, has a role in phonological processing. Language switching phenomenon we observed can be partly explained by transient dysfunction of the feed-forward control mechanism hypothesized by the DIVA (Directions Into Velocities of Articulators) model (Golfinopoulos, E., Tourville, J. A., & Guenther, F. H. (2010). The integration of large-scale neural network modeling and functional brain imaging in speech motor control.

Ookinetes, one of the motile and invasive forms of the malaria parasite, rely on gliding motility in order to establish an infection in the mosquito host. Here we characterize the protein PBANKA_0407300 which is conserved in the Plasmodium genus but lacks significant similarity to proteins of other eukaryotes. It is expressed in gametocytes and throughout the invasive mosquito stages of P. berghei, but is absent from asexual blood stages. Mutants lacking the protein developed morphologically normal ookinetes that were devoid of productive motility although some stretching movement could be detected. We therefore named the protein Ookinete Motility Deficient (OMD). Several key factors known to be involved in motility however were normally expressed and localized in the mutant. Importantly, the mutant failed to establish an infection in the mosquito which resulted in a total malaria transmission blockade.

Seabob shrimp ( Xiphopenaeus kroyeri ) fishery is operated all over the coast off Sao Paulo State. For that reason the management and supervision of those catches is necessary in order to assure the maintenance of the stocks and the stability of that activity. This paper aims to contribute to the management of the resource through the analysis of the landing dynamics of the species caught off the coast of S a o Paulo State from 2000 to 2011. The activity is operated by a fleet considered industrial, that comprises 15% of the productivity units and contributes with 47% of the landing production, and by an artisanal fleet that corresponds to 85% of all the productivity units and 53% of the landed shrimp. The CPUE (Catch per Unit Effort) analysis shows stability through the abundance indicator in the last years. The decrease on the number of units from the industrial fleet, probably due to the fisheries low profitability, was the most probable cause for the CPUE stability, or the fishing effort has not exceeded the sustainability limit of the seabob shrimp stock off Sao Paulo State. The observed stability in the abundance indicator is a very promising factor, and it can modify the vision of the status of the resource in different sectors of the fisheries management. It can also influence management measures that must be established with the participation of the artisanal sector, due to the contribution that the artisanal fleet presents in numbers of productivity units and in the percentage of the total of shrimp landing. That participation, although necessary, has not occurred yet.

Pinot noir is the most widely planted red grape variety in New Zealand and is considered a premium product based on the price per volume. To date no studies have attempted to characterize the different styles of the four main Pinot noir producing regions: Central Otago, Marlborough, Martinborough, and Waipara. The intensities of aroma, flavor, and mouthfeel attributes of commercial regional wines from two vintages were investigated. Descriptive analysis was carried out by a panel consisting of experienced but untrained wine professionals. Canonical variate analysis showed that the four wine regions were differentiated according to aroma (barnyard, black cherry, herbal, raspberry, red cherry, oak, spice and violet), in-mouth flavor (fruit density/concentration and red fruit), and mouthfeel (balance, body, and finish length) attributes. Results show that Pinot noir wines from the four regions of New Zealand are stylistically different and that experienced but untrained wine professionals produce reliable results for this type of sensory analysis.

Brown marmorated stink bugs (BMSB) release stress compounds, tridecane and (E)-2-decenal, that affect final wine quality. This study focuses on determining the effect of wine processing on (E)-2-decenal and tridecane release in both red and white wines. Wines were produced by adding live BMSB to grape clusters at densities of 0, 0.3, 1, and 3 bugs/cluster. Compound concentrations were measured using headspace solid phase microextraction with multidimensional gas chromatography and mass spectrometry. For red wines, the highest levels of stress compounds were found using 3 BMSB/cluster [tridecane, 614 μg/L; (E)-2-decenal, 2.0 μg/L]. Pressing was found to be the critical process point for stress compound release, and additional pressing processes, press types, and press fractions were investigated. BMSB taint for white wines was not found to be problematic with respect to wine quality. An action control of 3 BMSB/cluster is recommended as this was related to the known consumption rejection threshold for (E)-2-decenal.

The p16INK4a gene, localized within chromosome 9p21, has been identified as a cyclin-dependent kinase inhibitor and may negatively regulate the cell cycle acting as a tumor suppressor. Genetic alterations involving the 9p21 region are common in human cancers. A consecutive series of 64 untreated patients (median of follow up 53 months) undergoing surgical resection for locally advanced laryngeal squamous-cell carcinomas (LSCCs) has been studied prospectively. Our purpose was to investigate p16 alterations (9p21 allelic loss, hypermethylation and point mutations) and their possible association with clinico-pathological data and flow cytometric variables (DNA-ploidy and S-phase fraction (SPF)), and to determine the possible prognostic role of this gene in these tumors. PCR-based techniques were used for investigating 9p21 loss of heterozygosity (LOH) and methylation promoter status of the p16 gene. p16 mutations were detected by PCR-SSCP (single strand conformation polymorphism) and sequencing. 9p21 LOH was detected in 16/62 (26%) informative tumors, point mutations in 5% (3/64) and hypermethylation in 9% (6/64) of the cases. p16 alterations were significantly associated with high SPF and DNA-aneuploidy. By univariate analysis, poor histologic differentiation, stage IV, DNA-aneuploidy and p16 point mutations proved to be significantly related to quicker relapse, whereas these same factors, and in addition high SPF, 9p21 LOH and any p16 alterations were significantly related to shorter overall survival. By Cox proportional hazards analysis only histologic grade (G3) and p16 point mutations were independently related to both disease relapse and death. Our study has identified p16 point mutations as important biomolecular indicators in LSCCs. © 2002 Wiley-Liss, Inc.

The malaria parasite Plasmodium largely modifies the infected erythrocyte through the export of proteins to multiple sites within the host cell. This remodeling is crucial for pathology and translocation of virulence factors to the erythrocyte surface. In this study, we investigated localization and export of small exported proteins/early transcribed membrane proteins (SEP/ETRAMPs), conserved within Plasmodium genus. This protein family is characterized by a predicted signal peptide, a short lysine-rich stretch, an internal transmembrane domain and a highly charged C-terminal region of variable length. We show here that members of the rodent Plasmodium berghei family are components of the parasitophorous vacuole membrane (PVM), which surrounds the parasite throughout the erythrocytic cycle. During P. berghei development, vesicle-like structures containing these proteins detach from the PVM en route to the host cytosol. These SEP-containing vesicles remain associated with the infected erythrocyte ghosts most probably anchored to the membrane skeleton. Transgenic lines expressing the green fluorescent protein appended to different portions of sep-coding region allowed us to define motifs required for protein export. The highly charged terminal region appears to be involved in protein-protein interactions.

The malaria parasite invades the terminally differentiated erythrocytes, where it grows and multiplies surrounded by a parasitophorous vacuole. Plasmodium blood stages translocate newly synthesized proteins outside the parasitophorous vacuole and direct them to various erythrocyte compartments, including the cytoskeleton and the plasma membrane. Here, we show that the remodeling of the host cell directed by the parasite also includes the recruitment of dematin, an actin-binding protein of the erythrocyte membrane skeleton and its repositioning to the parasite. Internalized dematin was found associated with Plasmodium 14-3-3, which belongs to a family of conserved multitask molecules. We also show that, in vitro, the dematin-14-3-3 interaction is strictly dependent on phosphorylation of dematin at Ser(124) and Ser(333), belonging to two 14-3-3 putative binding motifs. This study is the first report showing that a component of the erythrocyte spectrin-based membrane skeleton is recruited by the malaria parasite following erythrocyte infection.

Aim of this study was to evaluate the responsiveness of red mullet (Mullus barbatus) liver detoxification enzymes to PAHs at transcriptional and post-transcriptional levels in the field. Fish were captured in the north-eastern Adriatic Sea, close to an oil refinery. Sixteen PAHs (EPA) were determined in sediments and fish fillets; transcription levels of cyp1a, cyp3a and abcc2 genes and EROD, BROD, B(a)PMO, BFCOD, GST and UDPGT enzymatic activities were measured. Levels of PAHs in sediments reflect the oil pollution gradient of the area, with weak correspondence in fish fillets. cyp1a gene transcription and EROD, B(a)PMO and BFCOD activities were significantly induced in the oil refinery site, and a slight up-regulation of cyp3a and abcc2 was also observed. GST and UDPGT remained unchanged. The present study provides the first data on detoxification responses at transcriptional levels in the liver of red mullet and confirms phase I enzymes as suitable biomarkers of exposure to PAHs in field studies.

Malaria parasites invade erythrocytes of their host both for asexual multiplication and for differentiation to male and female gametocytes - the precursor cells of Plasmodium gametes. For further development the parasite is dependent on efficient release of the asexual daughter cells and of the gametes from the host erythrocyte. How malarial parasites exit their host cells remains largely unknown. We here report the characterization of a Plasmodium berghei protein that is involved in egress of both male and female gametes from the host erythrocyte. Protein MDV-1/PEG3, like its Plasmodium falciparum orthologue, is present in gametocytes of both sexes, but more abundant in the female, where it is associated with dense granular organelles, the osmiophilic bodies. Deltamdv-1/peg3 parasites in which MDV-1/PEG3 production was abolished by gene disruption had a strongly reduced capacity to form zygotes resulting from a reduced capability of both the male and female gametes to disrupt the surrounding parasitophorous vacuole and to egress from the host erythrocyte. These data demonstrate that emergence from the host cell of male and female gametes relies on a common, MDV-1/PEG3-dependent mechanism that is distinct from mechanisms used by asexual parasites.

There are many interactions that occur between taste and aroma that may impact perception. The main objective of this study was to ascertain whether the aroma fraction of wine should be considered when investigating relationships between chemical composition and sensory perception of mouthfeel. Chardonnay wines with different mouthfeels were produced by altering the fermentation temperature (15 °C and 21 °C) of the alcoholic and malolactic fermentations (MLF) as well as the timing of MLF and the presence of a non-Saccharomyces yeast during alcoholic fermentation. Napping® and Ultra-flash-profiling were conducted using a panel of white winemakers. Each procedure was conducted twice: once with retro-nasal aroma (+R) and once without retronasal aroma (−R). Napping® results showed that retronasal aroma impacted mouthfeel perception. Ultra-flash profiling of +R and −R displayed similar descriptive terms used. Several terms appear to be related to retronasal aroma as they were used in +R and not in −R. It is unclear if these terms are due to interactions or due to associated learning. These results suggest that for some mouthfeel terms the volatile fraction plays a role and, to establish relationships between chemical composition and mouthfeel perception, it is important to consider both the volatile and nonvolatile wine fractions.

It is well documented that blueberry phenolic compositions, such as phenolic acids and flavonoids differ based on species and cultivars. However, phenolic compositions in Florida blueberries have been little explored. Information on Florida blueberries are of interest as they are harvested earlier than other areas of the United States, which may result in compositional differences due to shorter daylight hours and cooler temperatures. Samples were harvested from University of Florida grower-cooperator farm near Gainesville, FL. After liquid-liquid extraction and C-18 SPE cartridge purification, concentrations of phenolic acids and flavonoids of twenty two blueberry cultivars (20 highbush and 2 rabbiteye) were measured using HPLC. Five phenolic acids (gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid and ferulic acid), two flavan-3-ols (catechin and epicatechin) and five flavonols (quercetin-3-galactoside or quercetin-3-glucoside, quercetin-3-arabinoside, myricetin, quercetin and kaempferol) were identified and quantified. Chlorogenic acid was the major component in highbush blueberries. Flavonoid and phenolic acid composition were profoundly different among Florida blueberries and these differences have not been previously reported. The first two components of PCA explained 95% of the variation totally; it showed clear differentiation of blueberry cultivars based on phenolic composition. The majority of the variation between the cultivars was due to variation in quercetin-3-galactoside or glucoside, quercetin-3-arabinoside and chlorogenic acid, which are most likely due to genetic and maturity differences. Complete-linkage clustering analysis displayed five significantly different (p

AOAC Method 966.04, the Sporicidal Activity of Disinfectants Test, is a carrier-based test that provides a qualitative measure of product efficacy against spores of Bacillus subtilis and Clostridium sporogenes. For regulatory purposes, Method 966.04 is accepted by the U.S. Environmental Protection Agency (EPA) and the U.S. Food and Drug Administration (FDA) for the generation of product performance data for sporicides and sterilants. In this study, we report on findings associated with proposed improvements (modifications) to the Clostridium component of the method. Egg meat medium (EMM), the culture medium for C. sporogenes currently specified in the method, is no longer commercially available and finding a suitable replacement is critical. In addition, the use of a nonstandardized extract of raw soil as an amendment to EMM, as stipulated in the current method, may result in a highly variable spore suspension. The primary focus of this study was to find replacements for EMM and soil extract. A carrier count procedure, the establishment of target carrier counts (spores/carrier), and a neutralization confirmation procedure were also evaluated. The study was limited to liquid products tested against Clostridium on a hard surface carrier (porcelain penicylinder). Spore suspensions of C. sporogenes were generated using: (1) EMM with soil extract (EMM/SE), (2) cooked meat medium with soil extract (CMM/SE), and (3) cooked meat medium with 5 g/mL manganese sulfate (CMM/MnSO4). The titer of the spore suspension, carrier counts, resistance to hydrochloric acid (HCl), and efficacy against 3 liquid sporicidal agents were used to evaluate the potential of CMM and MnSO4 as replacements. The study was performed by the EPA Office of Pesticide Programs Microbiology Laboratory, Fort Meade, MD. Use of CMM/SE and CMM/MnSO4 resulted in comparable results for titer of spore suspensions (approximately 108 spores/mL) and carrier counts (approximately 3 106 spores/carrier). The carrier counts for the EMM/SE were approximately 1 log lower than CMM-based treatments; however, no attempt was made to dilute the CMM spore suspensions prior to carrier inoculation to reduce the carrier counts for CMM. Resistance of spores to 2.5 M HCl was acceptable across the 3 media types. Treatments for comparative efficacy testing were designed to provide a range of sporicidal activity, i.e., high and low efficacy treatments. Sodium hypochlorite (bleach), hydrogen peroxide/peracetic acid, and glutaraldehyde were used as test chemicals. The number of carriers resulting in growth (positive) for the low treatments for all 3 chemicals ranged from 9 to 59 out of 60 across the 3 media typesEMM exhibited fewer positives overall. The high efficacy treatments for sodium hypochlorite and hydrogen peroxide/peracetic acid yielded a range of 0 to 2 positives out of 60 across the 3 media. However, the high glutaraldehyde treatment generated 3, 20, and 20 positives out of 60 for the EMM/SE, CMM/SE, and CMM/MnSO4, respectively. The lower number of positive carriers for EMM/SE may be due to the reduced carrier counts. CMM, either with SE or MnSO4, appears to be a suitable replacement for EMM/SE. On the basis of the results of this study, the Study Director recommends that CMM/MnSO4 and the spore enumeration target carrier count and neutralization procedures be considered for collaborative study to officially modify the Clostridium porcelain component of Method 966.04.

In an effort to improve AOAC Method 966.04, the Sporicidal Activity of Disinfectants Test, selected modifications to the procedure were evaluated in a collaborative study. Method 966.04 is used to generate efficacy data to support the product registration of sporicides and sterilants. The method is a carrier-based test that provides a qualitative measure of product efficacy against spores of Bacillus subtilis and Clostridium sporogenes. The use of garden soil extract and the lack of standard procedures for the enumeration of spores and neutralization of the test chemicals have been considered problematic for many years. The proposed modifications were limited to the B. subtilis and hard surface carrier (porcelain penicylinder) components of the method. The study included the evaluation of a replacement for soil extract nutrient broth and an establishment of a minimum spore titer per carrier, both considered crucial for the improvement and utilization of the method. Additionally, an alternative hard surface material and a neutralization confirmation procedure were evaluated. To determine the equivalence of the proposed alternatives to the standard method, 3 medium/carrier combinations, (1) soil extract nutrient broth/porcelain carrier (current method), (2) nutrient agar amended with 5 g/mL manganese sulfate/porcelain carrier, and (3) nutrient agar amended with 5 g/mL manganese sulfate/stainless steel carrier were analyzed for carrier counts, HCl resistance, efficacy, quantitative efficacy, and spore wash-off. The test chemicals used in the study represent 3 chemical classes and are commercially available antimicrobial liquid products: sodium hypochlorite (bleach), glutaraldehyde, and a combination of peracetic acid and hydrogen peroxide. Four laboratories participated in the study. The results of the spore titer per carrier, HCl resistance, efficacy, and wash-off studies demonstrate that amended nutrient agar in conjunction with the porcelain is comparable to the current method, soil extract nutrient broth/porcelain. The nutrient agar method is simple, inexpensive, reproducible, and provides an ample supply of high quality spores. Due to the current use of porcelain carriers for testing C. sporogenes, it is advisable to retain the use of porcelain carriers until stainless steel can be evaluated as a replacement carrier material for Clostridium. The evaluation of stainless steel for Clostridium has been initiated by the Study Director. Study Director recommendations for First Action revisions are provided in a modified method.

ToprospectivelyevaluatetheprognosticsignificanceofTP53,H-,K-,andN-Rasmutations,DNA-ploidyandS-phasefraction(SPF) in patients affected by locally advanced laryngeal squamous cell carcinoma (LSCC). Eight-one patients (median follow-up was 71 months) who underwent resective surgery for primary operable locally advanced LSCC were analyzed. Tumor DNA was screened for mutational analysis by PCR/SSCP and sequencing. DNA-ploidy and SPF were performed byflow cytometric analyses. Thirty-six patients (44%) had, at least, a mutation in the TP53 gene. Of them, 22% (8/36) had double mutations and 3% (1/36) had triplemutations.Intotal,46TP53mutationswereobserved.Themajority(41%)oftheseoccurinexon5(19/46),whilethemutations inexons6,7,and8wererepresentedin14,7,and6patients,respectively(31%,15%,and16%).FiveLSCCpatients(6%)showeda mutation in H-Ras gene. Sixty-three percent of the cases (51/81) were DNA aneuploidy, 14% of these (7/51) were multiclonal. Thirty-ninepatients(48%)hadanhighSPFvalue.AtUnivariateanalysis,theDNAaneuploidy,highSPF(>15.1%),TP53mutations and, in particular, the mutations that occur in exons 5 and 8 were significantly related to quicker disease relapse and short OS. At Multivariate analysis, the major significant predictors for both disease relapse and death were high SPF and any TP53 mutations. While histological grade G3 was an independent factor only for relapse. In conclusions, any TP53 mutations and high SPF are importantbiologicalindicatorstopredicttheoutcomeofLSCCpatients. J.Cell.Physiol.206:181‐188,2006.2005Wiley-Liss,Inc.

Trans-2-decenal and tridecane are compounds found in wine made from brown marmorated stink bug (BMSB)-contaminated grapes. The effectiveness of post-fermentation processes on reducing their concentration in finished wine and their longevity during wine aging was evaluated. Red wines containing trans-2-decenal were treated with fining agents and put through reverse osmosis filtration. The efficacy of these treatments was determined using chemical analysis (MDGC-MS) and sensory descriptive analysis. Tridecane and trans-2-decenal concentrations in red and white wine were determined at bottle aging durations of 0, 6, 12 and 24 months using MDGC-MS. Reverse osmosis was found to be partially successful in removing trans-2-decenal concentration from finished wine. While tridecane and trans-2-decenal concentrations decreased during bottle aging, post-fermentative fining treatments were not effective at removing these compounds. Although French oak did not alter the concentration of tridecane and trans-2-decenal in red wine, it did mask the expression of BMSB-related sensory characters. Because of the ineffectiveness of removing BMSB taint post-fermentation, BMSB densities in the grape clusters should be minimized so that the taint does not occur in the wine.

Receptor activator of NFκB ligand (RANKL), RANK, and osteoprotegerin (OPG) represent the key regulators of bone metabolism both in normal and pathological conditions, including bone metastases. To our knowledge, no previous studies investigated and compared RANK expression in primary tumors and in bone metastases from the same patient. We retrospectively examined RANK expression by immunohistochemistry in 74 bone metastases tissues from solid tumors, mostly breast, colorectal, renal, lung, and prostate cancer. For 40 cases, tissue from the corresponding primary tumor was also analyzed. Sixty-six (89%) of the 74 bone metastases were RANK-positive and, among these, 40 (59.5%) showed more than 50% of positive tumor cells. The median percentage of RANK-positive cells was 60% in primary tumors and metastases, without any statistically significant difference between the two groups (P = 0.194). The same percentage was obtained by considering only cases with availability of samples both from primary and metastasis. Our study shows that RANK is expressed by solid tumors, with high concordance between bone metastasis and corresponding primary tumor. These data highlight the central role of RANK/RANKL/OPG pathway as potential therapeutic target not only in bone metastasis management, but also in the adjuvant setting.

Objective: There are preclinical data showing that fluoxetine stimulated expression of Brain Derived Neurotrophic Factor (BDNF) and its specific tyrosine kinase receptor, and caused neuritic elongation and increased dendritic branching density of CA3 hippocampal pyramidal cell neurons in rodents. The latter effect of fluoxetine has been referred to as neuronal remodeling. In view of this preclinical data, we wondered if specific cognitive measures could serve as novel therapeutic targets for fluoxetine in head-injured patients. Theoretically, fluoxetine-induced “neuronal remodeling” might improve cognition, independently of a primary effect on mood. Method: In an open-label pilot investigation, fluoxetine hydrochloride (Prozac; 20–60 mg/day) was administered to a heterogeneous group of five head-injured patients with either no or moderate depression for a period of eight months. These patients had no histories of prior treatment with antidepressant medications. They were administered cognitive and memory tests at baseline and after eight months of treatment on fluoxetine. Results: The preliminary results showed that fluoxetine improved mood, in addition to improving performance on the Trail Making Test Part A, an attentional-motor speed task, and the letter-number sequencing subtest of the WAIS-III, a measure reflecting “working memory.” Conclusions: Although fluoxetine had beneficial effects on some measures of cognition, more work is needed to connect these improvements with neuronal remodeling.

p53 alterations are considered the most common genetic events in many types of neoplasms, including colorectal carcinoma (CRC). These alterations include mutations of the gene and/or overexpression of the protein. The aim of our study was to assess whether in 160 patients undergoing resective surgery for primary operable CRC there was an association between p53 mutations and protein overexpression and between these and other biological variables, such as cell DNA content (DNA-ploidy) and S-phase fraction (SPF), and the traditional clinicopathological variables. p53 mutations, identified by PCR-SSCP-sequencing analysis, were found in 68/160 patients (43%) and positive staining for p53 protein, detected with the monoclonal antibody DO-7, was present in 48% (77/160) of the cases, with agreement of 57% (91/160). In particular, a significant association was found between increased p53 expression and genetic alterations localized in the conserved regions of the gene or in the L3 DNA-binding domain and the specific type of mutation. Furthermore, both overexpression of p53 and mutations in the conserved areas of the gene were found more frequently in distal than in proximal CRCs, suggesting that they might be "biologically different diseases." Although p53 mutations in conserved areas were associated with flow cytometric variables, overexpression of p53 and mutations in its L3 domain were only related respectively to DNA-aneuploidy and high SPF. These data may reflect the complex involvement of p53 in the different pathways regulating cell-cycle progression. In conclusion, the combination of the mutational status and immunohistochemistry of p53, and flow cytometric data may provide an important insight into the biological features of CRCs.