The relevance of bryophytes in the macrophyte-based reference conditions in Portuguese rivers

The identification of macrophyte reference communities represents a significant challenge in Mediterranean zones, where few aquatic taxa exist compared to other regions of the world. In the highly seasonal rivers of Portugal, we explored the contribution of bryophytes to enhance the compositional and ecological characterization of reference conditions. We performed partitioning, classification, and ordination analyses and obtained eight groups of bryophytic assemblages explained by water availability and quality. Groups A, B, and H assemblages are type-specific of the inland areas of the northern eco-region, reflecting the neutral minerotrophic currents with summer low currents. Groups C and D are type-specific of acidic oligotrophic shifting currents dependent of precipitation events in the mountainous streams of the North Atlantic eco-region. Groups E, F, and G are widespread in continental northern, central, and southern zones in neutral and highly mineralized watercourses with seasonal dryness. We propose bryophytes as a tool in the characterization of the reference conditions for two main reasons: (i) many of the recorded taxa are capable of withstanding the natural seasonal desiccation, and (ii) the type-specific bryophytes have perennial life-strategies and can be assessed at any time of year as a support for other type-specific macrophytes in compositional assessments of reference conditions.     

A Fluorescence‐Based Sensor Assay that Monitors General Protein Aggregation in Human Cells

Protein conformational disorders are characterized by disruption of protein folding and toxic accumulation of protein aggregates. Here we describe a sensitive and simple method to follow and monitor general protein aggregation in human cells. Heat shock protein 27 (HSP27) is an oligomeric small heat shock protein that binds and keeps unfolded proteins in a folding competent state. This high specificity of HSP27 for aggregated proteins can be explored to monitor aggregation in living cells by fusing it to a fluorescent protein as Green Fluorescent Protein (GFP). We have constructed a HeLa stable cell line expressing a HSP27:GFP chimeric reporter protein and after validation, this stable cell line is exposed to different agents that interfere with proteostasis, namely Arsenite, MG132, and Aβ‐peptide. Exposure to proteome destabilizers lead to re‐localization of HSP27:GFP fluorescence to foci, confirming that our reporter system is functional and can be used to detect and follow protein aggregation in living cells. This reporter is a valuable tool to setup wide‐genetic screens to identify genes and pathways involved in protein misfolding and aggregation.     

Headspace gas chromatography with capillary column for urine alcohol determination

A headspace gas chromatographic method using a fused-silica capillary column Poraplot Q has been developed and validated for the detection and quantification of ethanol in urine. Under optimized conditions, ethanol was properly separated from acetaldehyde, acetone, isopropanol, methanol and n-propanol. Limits of detection (LODs) and quantification (LOQs) were 0.008 and 0.010 g/l, respectively. The precision studies within-run and between-run, using spiked urine samples (0.08, 0.8 and 2.0 g/l) showed maximum coefficients of variation 5.9 and 6.5%, respectively. Results of ethanol recovery varied from 91.6±0.8 to 103.3±1.8% over the concentration range from 0.01 to 3.20 g/l. The method was appropriate for the detection of ethanol in urine samples. This matrix can be used for monitoring alcohol abuse in the workplace and used in alcohol rehabilitation programs.     

The impact of genetically modified crops on soil microbial communities.

Genetically modified (GM) plants represent a potential benefit for environmentally friendly agriculture and human health. Though, poor knowledge is available on potential hazards posed by unintended modifications occurring during genetic manipulation. The increasing amount of reports on ecological risks and benefits of GM plants stresses the need for experimental works aimed at evaluating the impact of GM crops on natural and agro-ecosystems. Major environmental risks associated with GM crops include their potential impact on non-target soil microorganisms playing a fundamental role in crop residues degradation and in biogeochemical cycles. Recent works assessed the effects of GM crops on soil microbial communities on the basis of case-by-case studies, using multimodal experimental approaches involving different target and non-target organisms. Experimental evidences discussed in this review confirm that a precautionary approach should be adopted, by taking into account the risks associated with the unpredictability of transformation events, of their pleiotropic effects and of the fate of transgenes in natural and agro-ecosystems, weighing benefits against costs.     

Characterisation of the serotonin efflux induced by cytosolic Ca2+ and Na+ concentration increase in human platelets.

BACKGROUND/AIM: The present study aimed at elucidating the mechanism(s) of serotonin (5-HT) efflux induced by thapsigargin from human platelets in the absence of extra-cellular Ca2+. METHODS: Efflux of pre-loaded radiolabeled serotonin was generally determined by filtration techniques. Cytosolic concentrations of Ca2+, Na+ and H+ were measured with appropriate fluorescent probes. RESULTS: 5-HT efflux from control or reserpine-treated platelets--where reserpine prevents 5-HT transport into the dense granules--was proportional to thapsigargin evoked cytosolic [Ca2+]c increase. Accordingly factors as prostacyclin, aspirin and calyculin which reduced [Ca2+]c-increase also inhibited the 5-HT efflux. Thapsigargin, which also caused a remarkable increase in cytosolic [Na+]c, promoted less 5-HT release, in parallel to lower [Na+]c and [Ca2+]c increase, when added to platelet suspensions containing low [Na+]. The Na+/H+ exchanger monensin increased the [Na+]c and induced 5-HT efflux without affecting the Ca2+ level. The 5-HT efflux induced by both [Ca2+] or [Na+]c increase did not depend on pH or membrane potential changes, whereas it decreased in the absence of extra-cellular K+, and increased in the absence of Cl- or Na+. CONCLUSION: Increases in [Ca2+]c and [Na+]c independently induce serotonin efflux through the outward directed plasma membrane serotonin transporter SERT. This event might be physiologically important at the level of capillaries or narrowed arteries where platelets are subjected to high shear stress which causes [Ca2+]c increase followed by 5-HT release which might exert vasodilatation.     

ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules

Correct orientation of cell division is considered an important factor for the achievement of normal brain size, as mutations in genes that affect this process are among the leading causes of microcephaly. Abnormal spindle orientation is associated with reduction of the neuronal progenitor symmetric divisions, premature cell cycle exit, and reduced neurogenesis. This mechanism has been involved in microcephaly resulting from mutation of ASPM, the most frequently affected gene in autosomal recessive human primary microcephaly (MCPH), but it is presently unknown how ASPM regulates spindle orientation. In this report, we show that ASPM may control spindle positioning by interacting with citron kinase (CITK), a protein whose loss is also responsible for severe microcephaly in mammals. We show that the absence of CITK leads to abnormal spindle orientation in mammals and insects. In mouse cortical development, this phenotype correlates with increased production of basal progenitors. ASPM is required to recruit CITK at the spindle, and CITK overexpression rescues ASPM phenotype. ASPM and CITK affect the organization of astral microtubules (MT), and low doses of MT‐stabilizing drug revert the spindle orientation phenotype produced by their knockdown. Finally, CITK regulates both astral‐MT nucleation and stability. Our results provide a functional link between two established microcephaly proteins.     

Comparison of expression of the fragile site at Xq27 in T and B lymphocytes

Summary We compared the fragile X (fraX) expression in T and B lymphocytes from four hemizygous males with fraX. Blood cultures were stimulated with a T cell mitogen (phytohemagglutinin: PHA) and with a B cell mitogen (pokeweed mitogen: PWM). A significant decrease in fraX expression was observed in cultures stimulated with PWM when compared to PHA-stimulated ones.