SARS-CoV-2 viability on different surfaces after gaseous ozone treatment: a preliminary evaluation.

COVID-19 is a global health threat with a huge number of confirmed cases and deaths all over the world. Human-to-human transmission via respiratory droplets and contact with aerosol-infected surfaces are the major routes of virus spread. Because SARS-CoV-2 can remain in the air and on surfaces from several hours to several days, disinfection of frequently touched surfaces and critical rooms, in addition to observing individual hygiene tips, is required to reduce the virus spreading. Here we report on an investigation into the use of gaseous ozone as a potentially effective sanitizing method against the new coronavirus.

Diversity and bioactivity of fungi associated with the marine sea cucumber Holothuria poli: disclosing the strains potential for biomedical applications.

AIMS: Identification of the mycobiota associated to the marine echinoderm Holothuria poli and investigation of cytotoxic and pro-osteogenic potential of isolated strains. METHODS AND RESULTS: Fungal strains were isolated from the animal's body-wall, intestine and faeces. The species identification was based on DNA barcoding and morphophysiological observations. Forty-seven species were identified, all are Ascomycota and mainly belonging to Aspergillus and Penicillium genera. Sixteen strains were grown on three media for chemical extraction. Cytotoxic activity was tested on a hepatic cancer cell line (HepG2), the cells viability was evaluated after treatment using a resazurin based assay (AlamarBlue). Pro-osteogenic activity was tested on human Mesenchymal stem cell, differentiation was measured as the alkaline phosphatase production through reaction with p-nitrophenylphosphate or as the cells ability to mineralize calcium using a colorimetric kit (StanBio). Cytotoxic activity was recorded for four fungal species while five of 48 extracts highlighted bioactivity towards human mesenchymal stem cells. CONCLUSIONS: The presence of relevant animal-associated mycobiota was observed in H. poli and selected strains showed cytotoxic potential and pro-osteogenic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Our work represents the first report of a Mediterranean Sea cucumber mycobiota and highlights the isolates potential to synthetize compounds of pharmaceutical interest for regenerative medicine.

Methylenetetrahydrofolate reductase polymorphism (MTHFR C677T) and headache in children: a retrospective study from a tertiary level outpatient service.

BACKGROUND: In adult studies the MTHFR C677T polymorphism has been associated with an increased risk of migraine, but little research has been done in this area in children. METHODS: A retrospective study of children referred with headache to a tertiary level Paediatric Neurology Service between 2008 and 2012. This study included only patients who had been genotyped for the MTHFR C677T polymorphism. An evaluation of homocysteine serum levels was necessary to exclude other types of migraine. CONCLUSION: Compared with the wild-type genotype, the T/T genotype was associated with an increased risk of any type of migraine, though the statistical significance was greatest in migraine with aura. The homocysteine serum levels were significantly higher in migraine with aura compared to migraine without aura. In a pediatric population MTHFR T/T homozygosity influences susceptibility to migraine.

Catheter ablation in children and young adults: is there an additional benefit from remote magnetic navigation?

PURPOSE: Although rare, children and young adults can suffer from significant cardiac arrhythmia, especially in the context of congenital malformations and after cardiac surgery. METHODS: A total of 62 patients (32 female, median age 20 years) underwent an invasive electrophysiology study between 2008-2011: half had normal cardiac anatomy, whereas the remaining patients had various types of congenital heart disease. All patients were treated using either conventional techniques (CVN) or remote magnetic navigation (RMN). RESULTS: Patients treated with the RMN system differed substantially from patients in the CVN group with respect to presence of congenital heart disease (67 % vs. 37 %), previous cardiac surgery (59 % vs. 20 %) or failed previous conventional ablation (22 % vs. 9 %), respectively. Although these more complex arrhythmias resulted in longer median procedure duration (180 vs. 130 min, p = 0.034), the median overall fluoroscopy exposure in the RMN group was significantly lower (4.1 vs. 5.2 min, p = 0.020). Clinical outcome was comparable in both groups without complications caused by the ablation. CONCLUSIONS: Catheter ablation using remote magnetic navigation is safe and feasible in children and young adults and is especially valuable in patients with abnormal cardiac morphologies. RMN resulted in significantly lower radiation exposure compared with the conventional technique.

[Tuberculosis risk assessment in Italian healthcare centres]. / La valutazione del rischio tubercolosi nelle strutture sanitarie.

BACKGROUND: Tuberculosis transmission is a significant hazard in healthcare settings. METHODS: Risk factors suggested by CDC guidelines in 1994, which were adopted by the Italian Ministry of Health, were assessed in 29 centres via questionnaires in 2005. RESULTS: Few centers were equipped with negative pressure, respiratory isolation rooms. Half of the centres had high or ongoing risk. CONCLUSIONS: The hazard is underestimated mostly because of a high number of initially undiagnosed TB patients.

Distinct antithrombotic consequences of platelet glycoprotein Ibalpha and VI deficiency in a mouse model of arterial thrombosis.

BACKGROUND: Collagen and von Willebrand factor (VWF) are considered essential to initiate platelet deposition at sites of vascular injury, but their respective roles remain to be elucidated. METHODS: We used a model of carotid artery thrombosis induced by a ferric chloride injury to compare the time to first occlusion and occlusion rate at 25 min postinjury in mice lacking the collagen receptor, glycoprotein (GP) VI, or the ligand-binding domain of the VWF receptor, GP Ibalpha. RESULTS: In normal mice used as controls (n = 12), a complete obstruction of blood flow developed within 8.05 +/- 0.47 min (mean +/- SEM), and the occlusion rate was 100%. The results were variable in 26 GP VI(-/-) mice. The artery never occluded in eight mice, but the time to first occlusion in the remaining 18 (8.36 +/- 0.27 min) was not different from normal (P = 0.556). Nonetheless, the occlusion rate was 42%, because in seven mice the occluded artery reopened and stayed patent at 25 min. In contrast, the artery never occluded in 12 mice lacking GP Ibalpha. In ex vivo perfusion experiments, GP VI(-/-) platelets failed to form thrombi onto collagen type I fibrils, but formed thrombi of normal size when exposed to endothelial or fibroblast extracellular matrix. CONCLUSIONS: Absence of GP Ibalpha function has a more profound antithrombotic effect in vivo than absence of the GP VI-dependent pathway of collagen-induced adhesion/activation. Components of the extracellular matrix may elicit a thrombogenic response in the absence of GP VI but not GP Ibalpha.

[Phenomenon of occupational accidents in hospitals: a retrospective study with reference to introduction of preventive measures and safety specified by D.Lgs 626/94]. / Il fenomeno infortunistico in ospedale: studio retrospettivo con riferimento all'introduzione delle misure preventive e di sicurezza previste dal D.Lgs 626/94.

The work analyzes the phenomenon of the professional accidents in the Policlinico S. Matteo in Pavia, in the quadrenium 1995-1998 to appraise the effects of the application of the D. Lgs 626/94. The obtained data highlight an aggregative diminution of the number of accidents in the biennium 1997-1998 in comparison with the previous biennium 1995-1996. The reduction of the phenomenon of professional accidents is linked substantially with a diminution of the number of accidents with biologic risk. In the quadrennium considered no case of serum conversion has been recorded. It doesn't result significantly altered the number of events of professional accidents linked with the manual handling of loads and with different traumatisms without biological risk. It is highlighted the necessity of more and more close collaboration between the operators of the prevention and the workers.

Circulating monocyte-platelet aggregates are an early marker of acute myocardial infarction.

OBJECTIVES: We investigated whether elevated levels of circulating monocyte-platelet aggregates (MPA) can be used to identify patients with acute myocardial infarction (AMI). BACKGROUND: Commonly used blood markers of AMI reflect myocardial cell death, but do not reflect the earlier pathophysiologic processes of plaque rupture, platelet activation and resultant thrombus formation. Circulating MPA form after platelet activation. METHODS: In a single center between October 1998 and November 1999, we measured circulating MPA in a blinded fashion by whole blood flow cytometry in 211 consecutive patients who presented to the emergency department (ED) with chest pain and were admitted to rule out AMI. Acute myocardial infarction was diagnosed by a CK-MB fraction greater than three times control. RESULTS: Patients with AMI (n = 61), as compared with those without AMI (n = 150), had significantly higher numbers of circulating MPA (11.6 +/- 11.4 vs. 6.4 +/- 3.6, mean +/- SD, p < 0.0001). After controlling for age, the adjusted odds of developing AMI for patients in the 2nd, 3rd and 4th quartiles of MPA, in comparison with patients in the lowest quartile (odds ratio = 1.0), were 2.1 (95% confidence interval [CI]: 0.7, 6.8), 4.4 (95% CI: 1.5, 13.1) and 10.8 (95% CI: 3.6, 32.0), respectively. The number of circulating MPA in patients with AMI presenting within 4 h of symptom onset (14.4) was significantly greater than those presenting after 4 h (9.4) and after 8 h (7.0), (p < 0.001). Of the 61 patients with AMI, 35 (57%) had a normal creatine kinase isoenzyme ratio at the time of presentation to the ED, but had high levels of circulating MPA (13.3). CONCLUSIONS: Circulating MPA are an early marker of AMI.

Unique pathway of thrombin-induced platelet aggregation mediated by glycoprotein Ib.

Thrombin plays a central role in normal and abnormal hemostatic processes. It is assumed that alpha-thrombin activates platelets by hydrolyzing the protease-activated receptor (PAR)-1, thereby exposing a new N-terminal sequence, a tethered ligand, which initiates a cascade of molecular reactions leading to thrombus formation. This process involves cross-linking of adjacent platelets mediated by the interaction of activated glycoprotein (GP) IIb/IIIa with distinct amino acid sequences, LGGAKQAGDV and/or RGD, at each end of dimeric fibrinogen molecules. We demonstrate here the existence of a second alpha-thrombin-induced platelet-activating pathway, dependent on GP Ib, which does not require hydrolysis of a substrate receptor, utilizes polymerizing fibrin instead of fibrinogen, and can be inhibited by the Fab fragment of the monoclonal antibody LJIb-10 bound to the GP Ib thrombin-binding site or by the cobra venom metalloproteinase, mocarhagin, that hydrolyzes the extracellular portion of GP Ib. This alternative alpha-thrombin pathway is observed when PAR-1 or GP IIb/IIIa is inhibited. The recognition sites involved in the cross-linking of polymerizing fibrin and surface integrins via the GP Ib pathway are different from those associated with fibrinogen. This pathway is insensitive to RGDS and anti-GP IIb/IIIa antibodies but reactive with a mutant fibrinogen, gamma407, with a deletion of the gamma-chain sequence, AGDV. The reaction is not due to simple trapping of platelets by the fibrin clot, since ligand binding, signal transduction, and second messenger formation are required. The GP Ib pathway is accompanied by mobilization of internal calcium and the platelet release reaction. This latter aspect is not observed with ristocetin-induced GP Ib-von Willebrand factor agglutination nor with GP Ib-von Willebrand factor-polymerizing fibrin trapping of platelets. Human platelets also respond to gamma-thrombin, an autoproteolytic product of alpha-thrombin, through PAR-4. Co-activation of the GP Ib, PAR-1, and PAR-4 pathways elicit synergistic responses. The presence of the GP Ib pathway may explain why anti-alpha-thrombin/anti-platelet regimens fail to completely abrogate thrombosis/restenosis in the cardiac patient.

Adhesive properties of the isolated amino-terminal domain of platelet glycoprotein Ibalpha in a flow field.

We have examined the interaction between the amino-terminal domain of platelet glycoprotein (GP) Ibalpha and immobilized von Willebrand Factor (vWF) under flow conditions in the absence of other components of the GP Ib-IX-V complex. Latex beads were coated with a recombinant fragment containing GP Ibalpha residues 1-302, either with normal sequence or with the single G233V substitution that causes enhanced affinity for plasma vWF in platelet-type pseudo-von-Willebrand disease. Beads coated with native fragment adhered to vWF in a manner comparable to platelets, showing surface translocation that reflected the transient nature of the bonds formed. Thus, the GP Ibalpha extracellular domain is necessary and sufficient for interacting with vWF under high shear stress. Beads coated with the mutated fragment became tethered to vWF in greater number and had lower velocity of translocation than beads coated with the normal counterpart, suggesting that the G233V mutation lowers the rate of bond dissociation. Our findings define an approach for studying the biomechanical properties of the GP Ibalpha-vWF bond and suggest that this interaction is tightly regulated to allow rapid binding at sites of vascular injury, while permitting the concurrent presence of receptor and ligand in the circulation.

Identification of three tyrosine residues of glycoprotein Ib alpha with distinct roles in von Willebrand factor and alpha-thrombin binding.

The interaction between von Willebrand factor (vWF) and the platelet membrane glycoprotein (GP) Ib-IX-V complex is essential for platelet adhesion at sites of vascular injury under high shear stress flow conditions. Moreover, GP Ib-IX-V may contribute to the mechanisms of platelet activation through its high affinity binding of alpha-thrombin. There are two distinct but partially overlapping regions of GP Ib alpha thought to be involved in interacting with vWF (residues 251-279) and alpha-thrombin (residues 271-284); they share three tyrosine residues (positions 276, 278, and 279) that have recently been shown to be sulfated (Dong, J., Li, C. Q., and Lopez, J.A. (1994) Biochemistry 33, 13946-13953). To define the functional role of these three residues, we have introduced selected mutations in a soluble recombinant GP Ib alpha fragment (corresponding to the sequence 1-302 of the mature protein) that binds vWF and alpha-thrombin with the same attributes as intact GP Ib-IX-V complex. Fragments containing a single Tyr-->Phe substitution either at position 276 or 278 or 279 exhibited normal interaction with vWF but markedly reduced or absent binding of alpha-thrombin. GP Ib alpha fragment with normal sequence but synthesized under sulfate-free conditions also failed to bind alpha-thrombin and, in addition, had markedly reduced interaction with vWF. The simultaneous substitution of three neighboring Asp residues with Asn at positions 272, 274, and 277, a multiple mutation that may impair Tyr sulfation, also resulted in loss of binding of both ligands. These results define distinct structural features of GP Ib alpha selectively involved in supporting the interaction with vWF or alpha-thrombin.

Point mutation in a leucine-rich repeat of platelet glycoprotein Ib alpha resulting in the Bernard-Soulier syndrome.

Leucine-rich repeats are a conserved structural motif, of yet undefined significance, found in a group of proteins from different species. Among these are the four components of the human platelet glycoprotein Ib-IX-V complex, a membrane receptor that performs an essential role in the thrombogenic function of platelets by interacting with the adhesive protein, von Willebrand factor. We have found that a single amino acid substitution (Ala156-->Val) within one of the six leucine-rich repeats in the alpha-subunit of glycoprotein Ib results in a variant form of the congenital bleeding disorder, Bernard-Soulier syndrome, characterized by giant dysfunctional platelets. Genetic studies of the propositus and his family members were complemented by immunological and functional analysis of expressed recombinant GP Ib alpha fragments to demonstrate that the observed mutation is the cause of defective von Willebrand factor binding. These studies define the molecular basis of the Bernard-Soulier syndrome within this family and demonstrate that structural integrity of a leucine-rich repeat is necessary for normal function of the glycoprotein Ib-IX-V receptor complex and, possibly, for normal platelet morphology.

Expression of human platelet glycoprotein Ib alpha in transgenic mice.

Platelets are cytoplasmic fragments of megakaryocytes and, therefore, their membrane proteins cannot be manipulated by expression methods in culture. To overcome this limitation, we have expressed human glycoprotein (GP) Ib alpha in transgenic mouse megakaryocytes and found that it was present on the surface of platelets associated with the mouse GP Ib beta subunit. This finding demonstrates that assembly of the heterooligomeric GP Ib complex occurs through mechanisms conserved across species. In contrast, the receptor function of GP Ib exhibited restricted species specificity, since only the chimeric complex containing human GP Ib alpha bound human von Willebrand factor and supported platelet aggregation mediated by ristocetin. These studies demonstrate the transgenic engineering of a platelet adhesion receptor under control of the human GP Ib alpha promoter and illustrate a new approach to manipulate platelet receptors and study structure-function relationships in hemostasis and thrombosis.

[The glycoprotein IIb/IIIa complex of the platelets. An activation-dependent integrin]. / Il complesso glicoproteico IIb/IIIa delle piastrine. Un'integrina attivazione-dipendente.

The glycoprotein (GP) IIb/IIIa complex is the most abundant platelet membrane receptor (approximately 80,000 copies/platelet). The GP IIb/IIIa complex is an adhesion receptor belonging to the integrin superfamily; it can bind five adhesive proteins containing the arginine-glycine-aspartic acid (RGD) sequence in their structure: fibrinogen (Fg), von Willebrand factor (vWf), thrombospondin (Tsp), fibronectin (Fn) and vitronectin (Vn). Fg mediates platelet aggregation; vWf, Tsp and Fn are large molecules that support platelet adhesion to vessel wall; Vn is a molecular connection among hemostasis and others physiological processes. The complex is presents at any time on the platelet surface, but macromolecular ligands cannot access to their receptor because of steric hindrances intrinsic to the complex itself or its microenvironment. Adhesive proteins can bind to the complex only after platelets become activated; following platelet stimulation and ligand binding a conformational change takes place, accompanied by expression of new epitopes termed LIBS (ligand-induced binding sites). The complex-bound fibrinogen undergoes to a progressive rearrangement which increases the adhesive function of the molecule. The RGD sequence present in adhesive proteins, in addition to its receptor role, may serve as a trigger sequence that induces a high affinity ligand-binding state in the GP IIb/IIIa complex. The different domains of adhesive proteins can bind to platelet surface receptors, other than GP IIb/IIIa, so realizing multiple ligand-receptor interactions.

Diagnosis of Glanzmann's thrombasthenia and carrier detection using monoclonal antibodies to platelet glycoprotein IIb and IIIa in immunoblotting.

Glanzmann's thrombasthenia is a rare hemorrhagic syndrome, characterized by a quantitative or functional defect of the platelet glycoprotein GPIIb-IIIa complex. The authors describe a method to diagnose thrombasthenic patients and identify carrier subjects by using monoclonal antibodies specific for GPIIb and GPIIIa in an immunoblotting technique. The immunoreaction patterns of two thrombasthenic patients lacking GPIIb or GPIIIa, respectively, are shown. The described method produces further evidence concerning the biochemical heterogeneity of Glanzmann's thrombasthenia.

Platelet glycoprotein Ib polymorphism in the Italian population.

The glycoprotein Ib (GP Ib) is the major sialoglycoprotein on the platelet membrane and plays an important role in primary hemostasis. It was demonstrated that GP Ib is polymorphic: four different species of GP Ib (designated A, B, C and D) were observed, having molecular weights of 168, 162, 159 and 153 kiloDaltons, respectively. The polymorphism was first studied in the Japanese population and, subsequently, in Americans; a significant difference between the phenotype frequencies in the two populations was found. The authors report about GP Ib polymorphism patterns in the Italian population. GP Ib from normal subjects was analyzed by means of platelet protein SDS-PAGE followed by Western blotting; GP Ib species immobilized onto nitrocellulose filters were coupled with wheat germ agglutinin and stained using immunoperoxidase. Despite some differences in phenotype distribution, statistical analysis did not show differences between Italian and U.S. Caucasian phenotype frequencies: this is attributable to rather similar gene frequencies in the two populations. In our platelet samples we only observed three GP Ib types, precisely the B, C and D types; therefore we can confirm the rarity of the A type in Caucasians, while it was well represented in the Japanese population.

A monoclonal antibody to platelet glycoprotein IIb induces aggregation of platelet rich plasma.

The monoclonal antibody (MAb) GAP 5.9 directed against platelet glycoprotein IIb is described. This MAb is able to induce aggregation of platelet rich plasma in the absence of further stimuli and likely recognizes a glycoprotein epitope not closely related to calcium binding sites on the molecule.