Four scenarios of environmental risk of diclofenac in European groundwater ecosystems.

Groundwater is the largest source of liquid freshwater on Earth. Groundwater ecosystems harbor a rich biodiversity, mainly consisting of microbes and invertebrates that provide substantial ecological services. Despite its importance, groundwater is affected by several anthropic pressures, including pollution from pharmaceutical compounds. Diclofenac is the non-steroidal drug most widely detected in freshwaters, both in surface waters (e.g., rivers, streams, lakes etc.) and groundwaters. Unlike surface waters, the environmental risk of diclofenac in European groundwaters has not yet been assessed by the competent Authorities. The environmental risk assessment refers to the analysis of the potential risk that a chemical compound poses to a given environment by comparing its measured environmental concentrations to its predicted no-effect concentration. In this study, we explored four environmental risk scenarios in European groundwaters using different methodologies. We obtained diverse risk expectations, some indicative of a moderately diffuse environmental risk for concentrations of diclofenac ≥42 ng/L and others indicative of a widespread environmental risk for concentrations ≥5 ng/L. The difference among the four scenarios mainly related to the methods of calculating the predicted no-effect concentration of diclofenac. We discussed the four scenarios in order to identify the most realistic risk expectations posed by diclofenac to European groundwater ecosystems.

Investigation of mechanisms underlying chaotic genetic patchiness in the intertidal marbled crab Pachygrapsus marmoratus (Brachyura: Grapsidae) across the Ligurian Sea.

BACKGROUND: Studies on marine community dynamics and population structures are limited by the lack of exhaustive knowledge on the larval dispersal component of connectivity. Genetic data represents a powerful tool in understanding such processes in the marine realm. When dealing with dispersion and connectivity in marine ecosystems, many evidences show patterns of genetic structure that cannot be explained by any clear geographic trend and may show temporal instability. This scenario is usually referred to as chaotic genetic patchiness, whose driving mechanisms are recognized to be selection, temporal shifts in local population dynamics, sweepstakes reproductive success and collective dispersal. In this study we focused on the marbled crab Pachygrapsus marmoratus that inhabits the rocky shores of the Mediterranean Sea, Black Sea and East Atlantic Ocean, and disperses through planktonic larvae for about 1 month. P. marmoratus exhibits unexpectedly low connectivity levels at local scale, although well-defined phylogeographic patterns across the species' distribution range were described. This has been explained as an effect of subtle geographic barriers or due to sweepstake reproductive success. In order to verify a chaotic genetic patchiness scenario, and to explore mechanisms underlying it, we planned our investigation within the Ligurian Sea, an isolated basin of the western Mediterranean Sea, and we genotyped 321 individuals at 11 microsatellite loci. RESULTS: We recorded genetic heterogeneity among our Ligurian Sea samples with the occurrence of genetic clusters not matching the original populations and a slight inter-population divergence, with the geographically most distant populations being the genetically most similar ones. Moreover, individuals from each site were assigned to all the genetic clusters. We also recorded evidences of self-recruitment and a higher than expected within-site kinship. CONCLUSIONS: Overall, our results suggest that the chaotic genetic patchiness we found in P. marmoratus Ligurian Sea populations is the result of a combination of differences in reproductive success, en masse larval dispersion and local larval retention. This study defines P. marmoratus as an example of marine spawner whose genetic pool is not homogenous at population level, but rather split in a chaotic mosaic of slightly differentiated genetic patches derived from complex and dynamic ecological processes.

Atlantic and Mediterranean populations of the widespread serpulid Ficopomatus enigmaticus: Developmental responses to carbon nanotubes.

Ficopomatus enigmaticus was adopted as model species for ecotoxicological bioassay, with its larval development as endpoint. Two different populations of the same species, collected in areas far from each other (Mediterranean Sea and Atlantic Ocean), were exposed to multi-walled carbon nanotubes, a class of emerging pollutants with a constantly increasing relevance in the landscape of nanomaterials production. Moreover, a molecular analysis based on Cyt b amplification and sequencing, was carried out to confirm that both populations belong to the same species. The aim of the present work was to strengthen existing results about F. enigmaticus relevance in ecotoxicological bioassays, adding the variable of population effect. For both populations the concentration-response curve of effect at different toxicant concentrations was similar and, at certain concentrations, overlapping, confirming the ecological relevance of the assay. These results posed an interesting acceptance on the introduction of this species as model in ecotoxicological bioassay scenery, underlining the relevance of a widespread wild species to compare effects of chemicals and environmental samples over large distances using the same bioassay.

Engineering the surface of prostate tumor cells and hyaluronan/chitosan multilayer films to modulate cell-substrate adhesion properties.

This paper explores different film assembly conditions of the polyelectrolyte solutions of hyaluronan (HA) and chitosan (CHI), as well as both substrate and cell surface modifications, to investigate PC3 cells adhesion properties. UV-Visible, AFM-IR and Zeta potential techniques indicate that the solution ionic strength is a relevant parameter to modulate the free carboxylic groups of HA on the film surface. In addition, capacitive coupling measurements suggest that assembly conditions that favor surface charge mobility inhibit cell adhesion due to polymer rearrangements that support non-specific electrostatic interactions of positively charged CHI residues and the negatively charged cell moieties, rather than specific CD44-hyaluronan interactions. Moreover, the PC3 cells treatment with hyaluronidase and anti-CD44 antibody also highlighted the importance of CD44 binding site availability on the tumor cell adhesion properties. Finally, the conjugation of wheat germ agglutinin on the film surface proved to be a suitable strategy to boost the PC3 cell adhesion properties. Our results reveal the remarkable capacity of HA/CHI films to modulate cell-substrate properties, which pave the road for the development of surfaces suitable for several applications based on biosensing.

Effects of copper on larvae of the marbled crab Pachygrapsus marmoratus (Decapoda, Grapsidae): Toxicity test and biochemical marker responses.

The importance of trace elements in ecotoxicological investigations is a well-known issue when monitoring polluted areas such as commercial harbors. Copper represents one of the most common metal contaminants, often detected in these areas as it is widely employed in various fields and has many sources of inflow in the marine environment. Pachygrapsus marmoratus is a widespread intertidal crab species that has been extensively studied in ecology, ethology and population genetics. Ecotoxicological studies have also been performed, exclusively on the adult stage. In the present study we investigated the mortality and biochemical (oxidative stress and neurotoxicity) responses of P. marmoratus larvae exposure to environmental relevant concentration of copper. Results showed dose-dependent responses in terms of larval mortality, with a calculated LC50 value of 0.5 mg/L of Cu2+. The LC50 concentration was used as the starting point for subsequent biochemical response evaluation. Results also demonstrated dose-dependent activation of antioxidant systems assuming a compensatory antioxidant activity to prevent higher cellular damage when larvae were exposed to the highest concentrations of copper. Moreover, a significant enhancement of neurotransmitter activities was observed, assuming a possible direct interaction of copper with the enzymes or an increase of free copper ion aliquot into the cells.

Multimodal and non-linear optical microscopy applications in reproductive biology.

A plethora of optical techniques is currently available to obtain non-destructive, contactless, real time information with subcellular spatial resolution to observe cell processes. Each technique has its own unique features for imaging and for obtaining certain biological information. However none of the available techniques can be of universal use. For a comprehensive investigation of biological specimens and events, one needs to use a combination of bioimaging methods, often at the same time. Some modern confocal/multiphoton microscopes provide simultaneous fluorescence, fluorescence lifetime imaging, and four-dimensional imaging. Some of them can also easily be adapted for harmonic generation imaging, and to permit cell manipulation technique. In this work we present a multimodal optical workstation that extends a commercially available confocal microscope to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools. The nonlinear microscopy capabilities were added to the commercial confocal microscope by exploiting all the flexibility offered by the manufacturer. The various capabilities of this workstation as applied directly to reproductive biology are discussed. Microsc. Res. Tech. 79:567-582, 2016. © 2016 Wiley Periodicals, Inc.

The Cratylia mollis seed lectin induces membrane permeability transition in isolated rat liver mitochondria and a cyclosporine a-insensitive permeability transition in Trypanosoma cruzi mitochondria.

Previous results provided evidence that Cratylia mollis seed lectin (Cramoll 1,4) promotes Trypanosoma cruzi epimastigotes death by necrosis via a mechanism involving plasma membrane permeabilization to Ca(2+) and mitochondrial dysfunction due to matrix Ca(2+) overload. In order to investigate the mechanism of Ca(2+) -induced mitochondrial impairment, experiments were performed analyzing the effects of this lectin on T. cruzi mitochondrial fraction and in isolated rat liver mitochondria (RLM), as a control. Confocal microscopy of T. cruzi whole cell revealed that Cramoll 1,4 binding to the plasma membrane glycoconjugates is followed by its internalization and binding to the mitochondrion. Electrical membrane potential (∆Ψm ) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10 µM Ca(2+) was significantly decreased by 50 µg/ml Cramoll 1,4 via a mechanism insensitive to cyclosporine A (CsA, membrane permeability transition (MPT) inhibitor), but sensitive to catalase or 125 mM glucose. In RLM suspended in a medium containing 10 µM Ca(2+) this lectin, at 50 µg/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and ∆Ψm disruption. All these mitochondrial alterations were sensitive to CsA, catalase, and EGTA. These results indicate that Cramoll 1, 4 leads to inner mitochondrial membrane permeabilization through Ca(2+) dependent mechanisms in both mitochondria. The sensitivity to CsA in RLM characterizes this lectin as a MPT inducer and the lack of CsA effect identifies a CsA-insensitive MPT in T. cruzi mitochondria.

Sensitivity of hypogean and epigean freshwater copepods to agricultural pollutants.

Widespread pollution from agriculture is one of the major causes of the poor freshwater quality currently observed across Europe. Several studies have addressed the direct impact of agricultural pollutants on freshwater biota by means of laboratory bioassays; however, as far as copepod crustaceans are concerned, the ecotoxicological research is scarce for freshwater species and almost nonexistent for the hypogean ones. In this study, we conducted a comparative analysis of the available literature data on the sensitivity of freshwater copepods to agricultural pollutants. We also assessed the acute and chronic sensitivity of a hypogean and an epigean species, both belonging to the Crustacea Copepoda Cyclopoida Cyclopidae, to two N-fertilizers (urea and ammonium nitrate) and two herbicides (ARIANE(TM) II from Dow AgroSciences LLC, and Imazamox), widely used for cereal agriculture in Europe. According to the literature review, freshwater copepods are sensitive to a range of pesticides and N-fertilizers. Ecotoxicological studies on hypogean species of copepods account only one study. There are no standardized protocols available for acute and chronic toxicity tests for freshwater copepods, making comparisons about sensitivity difficult. From our experiments, ionized ammonia proved to be more toxic than the herbicide Imazamox, in both short and chronic bioassays. Urea was the less toxic chemical for both species. The hypogean species was more sensitive than the epigean one to all chemicals. For both species and for all tested chemicals, acute lethality and chronic lethality were induced at concentrations higher than the law limits of good water body quality in Europe, except for ionized ammonia, which provoked the chronic lethality of the hypogean species at a lower concentration. The hazardous concentration (HC) of un-ionized ammonia for 5 % of freshwater copepods, obtained by a species sensitivity distribution, was 92 µg l(-1), significantly lower than the HC computed for traditional test species from freshwater environments.

MHC genotype predicts mate choice in the ring-necked pheasant Phasianus colchicus.

Females of several vertebrate species selectively mate with males on the basis of the major histocompatibility complex (MHC) genes. As androgen-mediated maternal effects have long-lasting consequences for the adult phenotype, both mating and reproductive success may depend on the combined effect of MHC genotype and exposure to androgens during early ontogeny. We studied how MHC-based mate choice in ring-necked pheasants (Phasianus colchicus) was influenced by an experimental in ovo testosterone (T) increase. There was no conclusive evidence of in ovo T treatment differentially affecting mate choice in relation to MHC genotype. However, females avoided mating with males with a wholly different MHC genotype compared with males sharing at least one MHC allele. Females also tended to avoid mating with MHC-identical males, though not significantly so. These findings suggest that female pheasants preferred males with intermediate MHC dissimilarity. Male MHC heterozygosity or diversity did not predict the expression of ornaments or male dominance rank. Thus, MHC-based mating preferences in the ring-necked pheasant do not seem to be mediated by ornaments' expression and may have evolved mainly to reduce the costs of high heterozygosity at MHC loci for the progeny, such as increased risk of autoimmune diseases or disruption of coadapted gene pools.

Argas (Persicargas) persicus (Oken, 1818) (Ixodida: Argasidae) in Sicily with considerations about its Italian and West-Mediterranean distribution.

Recently, in the province of Trapani (Western Sicily), some overwintering specimens of the argasid tick Argos (Persicargas) persicus (Oken, 1818) were observed and collected. Morphological and genetic analysis were utilized in order to reach a definitive identification. The species was found in two semi-natural sites where, having been found repeatedly, its presence does not appear accidental. Moreover the characteristics of the Sicilian findings seem to exclude a human-induced spread. This record, the first regarding Sicily and South Italy, is discussed together with the previous doubtful citations for Italy. These findings revalue not only all the old citations for Italy but also the hypothesis that the Mediterranean distribution of this argasid is of a natural origin.

Introgression of chukar genes into a reintroduced red-legged partridge (Alectoris rufa) population in central Italy.

Insight regarding the genetic origin and composition of the studied population of the red-legged partridge (Alectoris rufa) is likely to provide general and critical information for the appropriate management and possible conservation of the species. The reintroduced population of red-legged partridges living in Pianosa Island (National Park Tuscany Archipelago) has proven to be sustainable: captive-bred individuals, morphologically assigned to the taxon A. rufa, were released to the island approximately 20 years ago, establishing an apparently well-adapted population. We have investigated this population by means of 10 microsatellite loci in order to shed light on its genetic structure. Considering that A. rufa is known to crossbreed with A. chukar, the Pianosa Island population was compared at the molecular level with a red-legged partridge breeding stock (Aulla, MS) as well as with a population of pure A. chukar. Our results indicate that the red-legged partridge population from Pianosa, morphologically identified as A. rufa, is actually partly introgressed with A. chukar, questioning its genetic purity and the possible use of this population as a starting stock for future reintroductions elsewhere.

Polymorphic microsatellites developed by cross-species amplifications in common pheasant breeds.

Genetic variability was analysed in two common breeds of pheasant (Phasianus colchicus L. 1758) by means of cross-species amplifications of microsatellite loci: 154 chicken, Gallus gallus and 32 turkey, Meleagris gallopavo, primers were tested for amplification of pheasant DNA. Thirty-six primers (25 specific for chicken and 11 for turkey) amplified pheasant DNA. Fifteen markers yielded specific products and were tested for polymorphism. Eight of them (55%) were polymorphic, with an average polymorphism of two alleles per locus. Specific polymerase chain reaction (PCR) products were sequenced; repeats were found in 11 of the 15 markers, although only two loci showed the same repeat and could be homologous to chicken ones.

Sympathetic skin response in patients with Duchenne muscular dystrophy.

To verify an eventual disfunction of the palmar sympathetic skin activity in Duchenne Muscular Dystrophy, we studied the sympathetic skin response (SSR) with endogenous, Deep Breathing (DB-SSR), exogenous Startle (SE-SSR) and Electric type stimuli (EE-SSR) in a group of patients and a control group. At the same time as the SSR, the heart rate (HR) and the basic R-R interval variation (RRIV) as well as the EMG diaphragmatic activity were checked. In this way, significant differences in the latency (p = 0.02) and amplitude (p = 0.009) values of the EE-SSR and in the amplitude values of the SE-SSR (p = 0.001) and of the DB-SSR (p = 0.02) were noted in the DMD group compared to the control group. In the group of patients the rise in latency of the EE-SSR correlates with the age (p = 0.0001) and with the reduction of its amplitude (p = 0.03). This last parameter correlates with the stage of the illness (p = 0.02). In the control group the mean amplitude of the EE-SSR is approximately 30% greater than that of startle and 50% of deep breathing. Although with amplitudes homogeneously reduced, these ratios are also conserved in the DMD group. Moreover the HR is greater (p = 0.0001) whilst the RRIV is reduced (p = 0.02) in the group of the patients. However these parameters do not correlated either to the cardiac involvement or to the stage of the illness. The presence of the SSR for all the modalities of stimulation indicates the substantial integrity of the afferent and the efferent paths of the ANS. The alterations in latency and amplitude of the SSR in the patients could be the consequence of a lack of dystrophin at the level of the sudoriparous myopithelium in analogy to what has been demonstrated in the mdx mouse.

[Lyme disease. Case contribution and review of the literature]. / Malattia di Lyme. Contributo casistico e revisione della letteratura.

Lyme borreliosis is a systemic disease. The etiologic agent is a tick-bite borne spirochete (Borrelia burgdorferi) of which men are casual hosts. Although the illness is reported worldwide, it is more frequent in the northern parts of Countries. Some isolated clinical pictures are reported in the past, but only in 1975 the illness was well described at Old Lyme (Connecticut, Usa) and then well defined in its pathogenesis and clinical course in three stages. Lyme disease, involving skin, heart, nervous system and joints, is frequently protean and misdiagnosed, so that it was called "the new great imitator". In Italy the first case is reported in 1985. The authors report three (3) new cases, mostly with neuro-articular signs and symptoms. Literature is partially reviewed, for a better definition and outline of some epidemiological, etiopathogenetic, historical and clinico-therapeutic aspects of Lyme disease.

Incidence of stroke in young adults in the Reggio Emilia area, northern Italy.

A retrospective epidemiological study on the first episode of stroke in young adults aged 15-44 years was carried out in the territory of the Local Health Unit No. 9 in Reggio Emilia (46,491 km2), Italy, from 1987 to 1989. 29 patients were identified: 17 were affected with cerebral infarction and 12 with hemorrhage. All young patients were discharged with diagnostic codes 430-438 according to the International Classification of Disease, i.e. the criteria of the World Health Organisation for stroke definition. All patients had computed tomography or necropsy. The average annual incidence rate per 100,000 population aged 15-44 for all strokes was 13.6 and the 95% confidence interval (CI 95%) was 9.1-19.6. The general population of the same age on January 1, 1987, was 69,845 and 71,920 on December 31, 1989; the incidence rate of stroke was 14.0 for males (CI 95% 7.9-2.3) and 13.2 (CI 95% 7.1-22.2) for females. The average annual incidence rates were 8.0 (CI 95% 4.7-12.2) for cerebral infarction (8.4, CI 95% 3.9-16 for males, 7.6, CI 95% 3.3-14.9 for females), 5.6 (CI 95% 2.9-9.9) for cerebral hemorrhage and 2.8 (CI 95% 1.0-6.1) for both subarachnoid (SAH) and intracerebral hemorrhage (ICH). Based on angiography or necropsy findings, aneurysms or arteriovenous malformations were present in 83% of the patients with SAH and in 66% of the patients with ICH. The 1-month fatality ratio was 0 for cerebral infarction, 50% for SAH and 33% for ICH.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of propranolol in head tremor: quantitative study following single-dose and sustained drug administration.

The effect of the beta-adrenoceptor antagonist propranolol has been investigated in nine patients suffering from isolated (six patients) or prominent (three patients) essential tremor of the head. In a double-blind, placebo-controlled study the tremorolytic efficacy of propranolol has been assessed by a quantitative accelerometric method after a single oral dose (120 mg) and following 2 weeks of sustained treatment with two different dosage regimens of the drug (120 and 240 mg daily). As compared with placebo, a significant reduction in tremor magnitude was found following a single oral dose but not on sustained administration of the beta-blocker at either dosage. The results suggest that the efficacy of sustained propranolol on isolated or prominent essential head tremor is less predictable and satisfactory than expected on the basis of the single-dose response, as compared with hand tremor.

Clinical and computer-based assessment of long-term therapeutic efficacy of propranolol in essential tremor.

Despite a large number of studies demonstrating the effectiveness of propranolol in relieving essential tremor (ET) the long-term therapeutic outcome of these patients remains poorly defined. The results of a one-year follow-up study in 18 patients with mild to severe ET performed by using clinical and computer-based methods of assessment indicate that the initial therapeutic benefit of propranolol is apparently retained. However, following 3-6 months of sustained treatment a proportion of patients required an increase in daily dosage of propranolol in order to maintain adequate symptomatic control of tremor, indicating a relative decrease in tremorolytic efficacy of the drug possibly due to long-term tolerance.

[Polycythemic chorea. Description of a clinical case]. / Corea policitemica. Descrizione di un caso clinico.

The authors report a 69-year-old man affected by polycythaemia rubra vera since the age of sixty, who presented persistent left hemichorea of acute onset, occurring ten months before the exacerbation of the haematologic abnormalities. Previously he had been suffering from a transient episode of chorea concomitant with relapsing of polycythaemia. CT scan of the brain showed diffuse cortico-subcortical atrophy, whereas Doppler velocimetry of carotid arteries demonstrated a rise in cerebral blood flow resistance and a stenosis of the right carotid artery in its first tract. Pathogenetic mechanisms of chorea complicating polycythaemia vera are discussed and a multifactorial origin of the syndrome is suggested.

Frequency/amplitude characteristics of postural tremor of the hands in a population of patients with bilateral essential tremor: implications for the classification and mechanism of essential tremor.

Amplitude/frequency characteristics of postural hand tremor in 59 patients with bilateral essential tremor of various degrees of severity were assessed using accelerometric recordings and spectral analysis. Intra-subject comparisons of tremor characteristics between the more and less affected hands were used to control for variability of tremor due to age factors and intersubject differences in amplitude and frequency. Statistical analysis distinguished three different patient groups. Some patients had low amplitude (less than 0.1-0.015 cm) tremor in the less affected limb (which tended to be 7 Hz or more in frequency in the young) and a larger amplitude tremor in the more affected hand which was 1 Hz or more lower in frequency. Other subjects had either bilaterally small or bilaterally large amplitude tremors of similar frequencies. These findings imply that there is a downwards step in frequency between symptomatic tremors of small and large amplitude. The amplitude and frequency of the small amplitude tremors were unrelated but frequency declined with age. The frequency of the large amplitude tremor was generally determined by amplitude but a wide range of amplitudes were compatible with similar frequencies. The frequency of large amplitude tremor also declined with age. It was concluded that there are two types of essential tremor, the smaller amplitude tremor probably derives from an exaggeration of some or all of the mechanisms of normal physiological tremor whereas the larger amplitude tremor probably arises from a separate "pathological" central nervous mechanism. It is not known if or how one may transform into, or be replaced by, the other during progression of the disease.