Global warming-related response after bacterial challenge in Astroides calycularis, a Mediterranean thermophilic coral.

A worldwide increase in the prevalence of coral diseases and mortality has been linked to ocean warming due to changes in coral-associated bacterial communities, pathogen virulence, and immune system function. In the Mediterranean basin, the worrying upward temperature trend has already caused recurrent mass mortality events in recent decades. To evaluate how elevated seawater temperatures affect the immune response of a thermophilic coral species, colonies of Astroides calycularis were exposed to environmental (23 °C) or elevated (28 °C) temperatures, and subsequently challenged with bacterial lipopolysaccharides (LPS). Using immunolabeling with specific antibodies, we detected the production of Toll-like receptor 4 (TLR4) and nuclear factor kappa B (NF-kB), molecules involved in coral immune responses, and heat shock protein 70 (HSP70) activity, involved in general responses to thermal stress. A histological approach allowed us to characterize the tissue sites of activation (epithelium and/or gastroderm) under different experimental conditions. The activity patterns of the examined markers after 6 h of LPS stimulation revealed an up-modulation at environmental temperature. Under warmer conditions plus LPS-challenge, TLR4-NF-kB activation was almost completely suppressed, while constituent elevated values were recorded under thermal stress only. An HSP70 up-regulation appeared in both treatments at elevated temperature, with a significantly higher activation in LPS-challenge colonies. Such an approach is useful for further understanding the molecular pathogen-defense mechanisms in corals in order to disentangle the complex interactive effects on the health of these ecologically relevant organisms related to global climate change.

Sea anemones, methylmercury, and bacterial infection: A closer look at multiple stressors.

Specimens of the Mediterranean sea anemone Anemonia viridis were exposed to methylmercury (MeHg) and bacterial infection to study their immune responses to a well-known toxic pollutant. Anemones were housed in laboratory conditions and divided into five experimental groups: 1. control (no microinjection); 2. filtered seawater + buffer injection; 3. filtered seawater + Escherichia coli injection; 4. MeHg + buffer injection; 5. MeHg + E. coli injection. Data showed an increase in antioxidant enzyme production compared to the constitutive condition, while methylmercury inhibited lysozyme production. The buffer inoculation had no statistically significant effects on the animals. In addition, electrophoretic and protease analyses revealed differences in the type of proteins produced, as well as a modulation of proteases depending on the treatment. The study demonstrated the immunomodulatory effect of the organic pollutant on A. viridis, validating its use as a model organism for marine coastal biomonitoring programmes and multiple stress studies.

AIF-1 and RNASET2 are involved in the inflammatory response in the Mediterranean mussel Mytilus galloprovincialis following Vibrio infection.

Filter-feeding bivalves, such as the Mytilus species, are exposed to different types of bacteria in the surrounding waters, in particular of the Vibrio genus. Mussels lack an adaptive immune system and hemocytes can recognize pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) to activate intracellular signaling pathways to trigger the antimicrobial effectors synthesis. Among the areas of bivalve immunity that deserve study include the role of hemocyte subpopulations. Since little information are available on immune responses at the tissue level to human pathogenic vibrios commonly detected in coastal waters involved in seafood-borne diseases, in this work, immunological parameters of the hemocytes from the Mediterranean mussel M. galloprovincialis were evaluated in response to in vivo challenge with Vibrio splendidus. The histological approach has been first used in order to identify the hemocytes recruitment at the infection site and the morphological change of muscular fibers. In addition, using immunolabeling with specific antibody we detected the production of molecules involved in the inflammatory activated cascade: Toll-like receptors 4 (TLR4), the myeloid differentiation factor 88 (MyD88), the Allograft inflammatory factor-1 (AIF-1) and the ribonucleases RNASET2, belonging to the T2 family, that in vertebrates are involved in the recruitment and activation of macrophages. Our results indicate the activation of TLR4 during bacterial infection preparatory to the recruitment of the MyD88 adapter with a putative role in recognition and intracellular signalling. Furthermore, the data presented in this work suggest that challenging with Gram-negative bacteria causes a massive migration of AIF-1+ hemocytes and that the ribonuclease RNASET2 could play a key role in the recruitment of these activated hemocytes. Our approach is useful for further understanding the complex molecular defence mechanisms of the host in invertebrates, especially in relation to the need to develop methods to evaluate the immunological response of bivalve molluscs used in aquaculture.

Sabella spallanzanii mucus bacterial agglutinating activity after arsenic exposure. The equilibrium between predation safety and immune response stability.

We report the Sabella spallanzanii mucus bacterial agglutination response after inorganic arsenic (As) exposure. As is actively adsorbed from the surrounding environment and accumulated at high concentrations in tissues as an anti-predatory strategy. Here we investigated the effect of high As concentrations on its immunobiological response. It may act on mucus lectins and on its ability to agglutinate bacteria. We concluded that As at high concentrations leads to the inhibition of pathogen recognition. Nevertheless, although its biological activity is significant reduced in winter, responses to As concentrations are very similar, and below a certain threshold do not induce alterations, supporting the hypothesis of adaptation to high As concentrations related to involvement in predation defence.

The entangled multi-level responses of Mytilus galloprovincialis (Lamarck, 1819) to environmental stressors as detected by an integrated approach.

Anthropogenic pressure adds up and interacts with the effects of climate change with a varying magnitude and potential changes depend on species' Life History (LH) traits, local environmental conditions and co-occurrence of several stressors. Stressors exert negative effects on marine biota when acting as a single factor, but the effects may be amplified when more than one stressor work in combination, producing interacting effects on biodiversity and ecosystem functioning. The impairment of individual functional traits (FT) leads to strong rebounds on LH traits and this may have ecological consequences. No studies actually relate FT and antioxidant enzymes to multiple environmental stressors. In this paper we investigate the effects of food concentration, temperature and hypoxia on metabolic traits as expressed by a proxy such as respiration rate and feeding behaviour and on antioxidant enzymes (Catalase, Superoxide dismutase, Glutathione S-Transferase, Glutathione peroxidase) for the bivalve Mytilus galloprovincialis. Mussels were exposed to three temperatures (12, 20 and 28 °C) under normoxic (8 mg O2 l-1) and hypoxic (~2 mg O2 l-1) conditions, with varying food concentrations ranging from 0.9 to 3.5 µg of chlorophyll l-1. The results show that FTs and antioxidant enzymes were affected by temperature, hypoxia and food availability, and outcome allowed us to emphasise that a multi-scalar integrated approach is suitable to detect and monitor effects of anthropogenic disturbance on ecosystem functioning.

Antimicrobial activity of an aspartic protease from Salpichroa origanifolia fruits.

Plant proteases play a fundamental role in several processes like growth, development and in response to biotic and abiotic stress. In particular, aspartic proteases (AP) are expressed in different plant organs and have antimicrobial activity. Previously, we purified an AP from Salpichroa origanifolia fruits called salpichroin. The aim of this work was to determine the cytotoxic activity of this enzyme on selected plant and human pathogens. For this purpose, the growth of the selected pathogens was analysed after exposure to different concentrations of salpichroin. The results showed that the enzyme was capable of inhibiting Fusarium solani and Staphylococcus aureus in a dose-dependent manner. It was determined that 1·2 µmol l-1 of salpichroin was necessary to inhibit 50% of conidial germination, and the minimal bactericidal concentration was between 1·9 and 2·5 µmol l-1 . Using SYTOX Green dye we were able to demonstrate that salpichroin cause membrane permeabilization. Moreover, the enzyme treated with its specific inhibitor pepstatin A did not lose its antibacterial activity. This finding demonstrates that the cytotoxic activity of salpichroin is due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of the AP could represent a potential alternative for the control of pathogens that affect humans or crops of economic interest. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides insights into the antimicrobial activity of an aspartic protease isolated from Salpichroa origanifolia fruits on plant and human pathogens. The proteinase inhibited Fusarium solani and Staphylococcus aureus in a dose-dependent manner due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of salpichroin suggests its potential applications as an important tool for the control of pathogenic micro-organisms affecting humans and crops of economic interest. Therefore, it would represent a new alternative to avoid the problems of environmental pollution and antimicrobial resistance.

Rare earths, zirconium and hafnium distribution in coastal areas: The example of Sabella spallanzanii (Gmelin, 1791).

The Zr, Hf, Y and lanthanide (REE) distribution in biological tissues of Sabella spallanzanii and Styela plicata species collected from two harbours from the northern Sicily is studied for providing information regarding the Zr, Hf and REE uptake from the environment. Previous studies determined the fractionation of dissolved REE scavenged on binding sites onto biological surfaces. By comparing the recognised shale-normalised REE patterns of studied samples with evidence from reference data, the observed behaviour of these elements in biological tissues of Sabella spallanzanii and Styela plicata is interpreted to result from the preferential uptake of intermediate REE onto carboxylic sites. Moreover, the relationship observed between the Fe content and Zr/Hf ratio suggests that preferential Hf accumulation occurs via siderophore-like binding sites. Features of the REE bioaccumulation factors (BAF), in addition to the absolute La, Ce and Sm contents and Zr-Hf fractionation, allow definition of the different origins of studied elements in the investigated localities. Higher BAF values for La and Ce associated with larger REE contents and lower Zr/Hf values strongly suggest that the environmental REE distribution in the Termini Imerese harbour is influenced by the delivery of particles from industrial sources and power plants. On the contrary, the REE contents of biological tissues collected in the Cala tourist harbour are affected by the dust dissolution from automotive traffic. These results suggest that the geochemical behaviour of REE and Zr/Hf signature can be used in environmental studies of biological tissues for reconstructing the nature of anthropogenic contaminations.

Temperature increases, hypoxia, and changes in food availability affect immunological biomarkers in the marine mussel Mytilus galloprovincialis.

Temperature increases, hypoxia, and changes in food availability are predicted to occur in the future. There is growing concern for the health status of wild and farmed organisms, since environmental stressors alter organism functions, and elicit coordinated physiological responses for homeostasis. Mussels are good bioindicators of environmental conditions. Their ability to maintain unaltered immunosurveillance under adverse environmental conditions may enhance their survival capability. Few studies are currently concerned with the relationships and feedback among multiple stressors. Here, food concentration, temperature, and oxygenation treatments were evaluated for their effects on immune enzymatic parameters of Mytilus galloprovincialis detected in the digestive gland and the lysosomal viability by neutral red uptake. Mussels were exposed to three temperatures (12, 20, and 28 °C) under normoxic (8 mg O2l-1) and anoxic conditions and specimens were fed with six food concentrations, ranging 0.2-5 g chlorophyll l-1. Temperature increases affected esterase and alkaline phophatase enzyme functionality, and addition of food buffered detrimental effects generated by harsh conditions, such as those provided by low oxygen concentrations. Kinetics of the phenoloxidase was negatively correlated with increasing temperature. In this case, food had a buffering effect that counteracted the limiting temperature only under normoxic conditions. In addition, the stability of the lysosomal membrane was altered under conditions of thermal stress and food change, under normoxic and anoxic conditions. Overall, environmental stress factors affected immune biomarkers of Mediterranean mussels, and the level of food acted as a buffer, increasing the thermal resistance of the specimens.

The ascidian Styela plicata hemocytes as a potential biomarker of marine pollution: In vitro effects of seawater and organic mercury.

Toxic metals, such as mercury, contribute substantially to anthropogenic pollution in many estuarine environments. Animals living in those environments, particularly invertebrate filter feeders like tunicates, can be used as bioindicators. In an attempt to identify cellular markers for revealing pollution, this study examined in vitro the effects of different concentrations of methyl mercury on Styela plicata hemocytes. The harvested hemocytes from S. plicata that were exposed to the metal had a significant mortality, cellular count and morphometric alterations. These findings provided evidence of MeHg immunotoxic effects on S. plicata, resulting in hemocyte death and morphological changes induced by cytoskeleton alterations. Thus, a morphometric cellular parameter, such as spreading ability, was used as a complementary method for differentiation between hemocytes treated with a marine solution (as a negative control) and hemocytes incubated with methylmercury and/or Sicilian seawater samples.

Specific inflammatory response of Anemonia sulcata (Cnidaria) after bacterial injection causes tissue reaction and enzymatic activity alteration.

The evolution of multicellular organisms was marked by adaptations to protect against pathogens. The mechanisms for discriminating the ''self'' from ''non-self" have evolved into a long history of cellular and molecular strategies, from damage repair to the co-evolution of host-pathogen interactions. We investigated the inflammatory response in Anemonia sulcata (Cnidaria: Anthozoa) following injection of substances that varied in type and dimension, and observed clear, strong and specific reactions, especially after injection of Escherichia coli and Vibrio alginolyticus. Moreover, we analyzed enzymatic activity of protease, phosphatase and esterase, showing how the injection of different bacterial strains alters the expression of these enzymes and suggesting a correlation between the appearance of the inflammatory reaction and the modification of enzymatic activities. Our study shows for the first time, a specific reaction and enzymatic responses following injection of bacteria in a cnidarian.

Sea bass Dicentrarchus labrax (L.) bacterial infection and confinement stress acts on F-type lectin (DlFBL) serum modulation.

The F-lectin, a fucose-binding protein found from invertebrates to ectothermic vertebrates, is the last lectin family to be discovered. Here, we describe effects of two different types of stressors, bacterial infection and confinement stress, on the modulation of European sea bass Dicentrarchus labrax (L.) F-lectin (DlFBL), a well-characterized serum opsonin, using a specific antibody. The infection of the Vibrio alginolyticus bacterial strain increased the total haemagglutinating activity during the 16-day testing period. The DlFBL value showed an upward regulation on the first, second and last days and underwent a slight downward regulation 4 days post-challenge. In contrast, the effect of confinement and density stress showed a decrease in the plasma concentration of lectin, ranging from 50% to 60% compared with the control. The modulation of DlFBL is in line with the hypothesis that humoral lectins could be involved and recruited in the initial recognition step of the inflammation, which leads to agglutination, and the activation of mechanisms responsible for killing of the pathogens.

F-type lectin from the sea bass (Dicentrarchus labrax): purification, cDNA cloning, tissue expression and localization, and opsonic activity.

Recently described biochemical and structural aspects of fucose-binding lectins from the European eel (Anguilla anguilla) and striped bass (Morone saxatilis) led to the identification of a novel lectin family ("F-type" lectins) characterized by a unique sequence motif and a characteristic structural fold. The F-type fold is shared not only with other members of this lectin family, but also with apparently unrelated proteins ranging from prokaryotes to vertebrates. Here we describe the purification, biochemical and molecular properties, and the opsonic activity of an F-type lectin (DlFBL) isolated from sea bass (Dicentrarchus labrax) serum. DlFBL exhibits two tandemly arranged carbohydrate-recognition domains that display the F-type sequence motif. In situ hybridization and immunohistochemical analysis revealed that DlFBL is specifically expressed and localized in hepatocytes and intestinal cells. Exposure of formalin-killed Escherichia coli to DlFBL enhanced their phagocytosis by D. labrax peritoneal macrophages relative to the unexposed controls, suggesting that DlFBL may function as an opsonin in plasma and intestinal mucus.

Use of RAPD and 16S rDNA sequencing for the study of Lactobacillus population dynamics in natural whey culture.

The development of communities of the thermophilic microflora of natural whey culture for Parmigiano Reggiano cheese production was studied by means of molecular techniques. RAPD analysis facilitates the identification of the Lactobacillus strains involved in this microbial association and permitted the study of population dynamics during two cycles of whey fermentation. Analysis of RAPD fingerprints revealed the presence of four biotypes that dominate the whey fermentation process. Sequence analysis of 16S rDNA demonstrated that the strains isolated from whey belong to Lact. helveticus and Lact. delbrueckii ssp. lactis species.

[Indications for percutaneous atrioseptostomy. Comparison of echocardiographic and fluoroscopic monitoring]. / Indicazioni alla atriosettostomia percutanea. Confronto fra monitorizzazione ecocardiografica e fluoroscopica.

Balloon atrial septostomy (BAS) is of established value in the management of several congenital heart diseases in the neonatal period. This procedure, which leads to creation of a tear in the membrane of the Foramen Ovalis using a balloon catheter, may be undertaken under fluoroscopic monitoring in catheter laboratory as well as under echographic control, even bedside. In this study we present our experience and discuss the indications to these two techniques. 91 neonates underwent to BAS; in 14 of them this was carried out under two-dimensional echocardiographic control in the intensive care unit. In all the patients BAS had good result, with clinical improvement in the majority of cases (97%) and a low rate (6%) of minor complications (such as transient supraventricular arrhythmias), without differences between fluoroscopic and ultrasound monitoring. The higher rate of mortality in the fluoroscopic monitoring group (2/91 = 2.2%) was thought to depend on an extremely critical presentation of the neonates. The echographic monitoring does not seem to offer any real advantage from the technical point of view, but the necessity of a prompt treatment of very ill patients emphasizes the advantages of a quickly and easily feasible procedure. Thus, we recommend the use two-dimensional echocardiographic imaging only in the very ill neonates in whom the septostomy can be more safely performed in intensive care unit bedside.

[Epidemiological and clinical findings in congenital heart diseases during the neonatal period. Report on personal cases]. / Rilievi epidemiologici e clinici sulle cardiopatie congenite in epoca neonatale. Contributo casistico personale.

Cardiovascular impairment is frequent in the neonatal period and can be linked to malformative and non malformative pathologies. The Authors performed a retrospective study on the incidence of these two types of pathologies in a population of 3326 newborns who underwent cardiological examination. Malformative cardiopathy was observed in 779 (23.4%) of them, while non malformative cardiopathy was present in 316 (9.5%). Acyanogen forms (86.7%) were more common than cyanogen forms (13.2%) in malformative, while cyanogen forms (64.8%) were more frequent than acyanogen ones (35.1%) in non malformative cardiopathies. The Authors reviewed the incidence of the single cardiac defects and report some clinical and physiopathological considerations that underline the need for prompt differential diagnosis and the validity of echocardiography in achieving this aim.

[THe echographic diagnosis of diaphragmatic eventration in newborn infants. 2 cases]. / Diagnosi ecografica di eventratio diaframmatica nel neonato. Due casi.

The authors report two cases of neonatal partial diaphragmatic eventration. The diagnosis in both two cases was echographic more than radiographic. The authors underline the role of B-mode echography in the diagnosis and follow-up of diaphragmatic anomalies, especially eventration, and suggest that examination of diaphragm and of its motion should complete any abdominal and thoracic echography.

[Heart involvement in progressive spinal muscular atrophy. A review of the literature and case histories in childhood]. / Il coinvolgimento cardiaco nell'atrofia muscolare spinale progressiva. Revisione della letteratura e contributo casistico in età pediatrica.

There are few cardiological studies in progressive spinal muscular atrophy and mainly concern subjects affected by the juvenile form (Kugelberg-Welander disease). The presence of a cardiomyopathy has been reported in these patients but the cardiac involvement is often secondary to the chronic respiratory insufficiency typical of the disease. We performed a retrospective study in our Institute on 43 patients, age range 3 months to 3 years, 37 of which presented type I (Werdnig-Hoffmann disease) and 6 type II (intermediate form) of the disease. No clinical nor instrumental signs of cardiomyopathy were observed. However, ECG revealed signs of right ventricular overload in 37.3% of the patients, probably provoked by pulmonary hypertension due to respiration anomalies. The authors underline the importance of correct respiratory assistance to prevent onset of cardiological alterations.

[A rare cause of a continuous murmur. Our experience with a newborn infant with a congenital coronary fistula]. / Una rara causa di soffio continuo. Nostra esperienza in un lattante con fistola coronarica congenita.

Congenital coronary artery fistulas are very rare. The most common connect one of the two coronary arteries with the right cardiac chambers, in particular the right ventricle. So, a left to right shunt appears; furthermore ischemic problems are possible. Notwithstanding in the pediatric age subset the patients are usually free of symptoms. The most important clinical sign is a continuous murmur in the precordium. We report a case of a newborn with a fistula between the right coronary artery and the right ventricle. He presented with the typical murmur; ECG and chest-ray were considered as normal. Echocardiography did not do the diagnosis, and the baby was submitted to a cardiac catheterization. There was a mild left to right shunt (QP/QS = 1.5/1), without pulmonary hypertension. The diagnosis was done by selective coronary angiography. We decided not to close the fistula, and the baby was discharged. Three months later he is still well. In this discussion we consider indications for surgery, and hypothesize a wider application in the future of transcutaneous embolization.

[Hypertrophic cardiomyopathy (probably secondary) in an infant with Stickler's syndrome. The physiopathological aspects]. / Cardiomiopatia ipertrofica (probabilmente secondaria) in un lattante con sindrome di Stickler. Aspetti fisiopatologici.

Stickler syndrome is a hereditary connective tissue disease transmitted by AD pattern. It is characterized by peculiar facies with bone and ocular anomalies. Heart anomalies (except mitral valve prolapse) are an uncommon features that may be associated. The authors report on a 4 months patient affected by this syndrome who had hypertrophic cardiomyopathy. Pathogenetic mechanism of this anomaly is discussed.

[Physiopathologic and therapeutic aspects of persistent fetal circulation. Review of the literature and personal histological observations]. / Aspetti fisiopatologici e terapeutici della persistenza della circolazione fetale. Revisione della letteratura ed osservazioni istologiche personali.

Persistence of the fetal circulation (PFC) is a syndrome characterized by failure of the cardiocirculatory system to adapt successfully to postnatal life. Its typical feature is persisting right-left shunt across fetal channels which determines cyanosis refractory to oxygen treatment. PFC can simulate cyanotic congenital cardiopathy. It has two forms: a primitive form and secondary one due to various causes especially perinatal asphyxia. Both forms have a common pathogenesis consisting of hypertension of the pulmonary arterial circulation. This article reviews the physiology of the main prenatal and postnatal circulatory characteristics and the factors which regulate the pulmonary circulation. It also reports the latest findings on PFC physiopathology and treatment indicating the prognostic factors and future perspectives.