Co-Functionalization of Gold Nanoparticles with C7H2 and HuAL1 Peptides: Enhanced Antimicrobial and Antitumoral Activities.

The functionalization of nanoparticles with therapeutic peptides has been pointed out as a promising strategy to improve the applications of these molecules in the field of health sciences. Peptides are highly bioactive but face several limitations such as low bioavailability due to the difficulty of overcoming the physiological barriers in the body and their degradation by enzymes. In this work, gold nanoparticles (AuNPs) were co-functionalized with two therapeutic peptides simultaneously. The peptides from the complementary determining region of monoclonal antibodies, composed of the amino acid sequences YISCYNGATSYNQKFK (C7H2) and RASQSVSSYLA (HuAL1) were chosen for having exhibited antitumor and antimicrobial activity before. The peptides-conjugated AuNPs were characterized regarding size, morphology, and metal concentration by using TEM, dynamic light scattering, and ICP-OES techniques. Then, peptides-conjugated AuNPs were evaluated regarding the antimicrobial activity against E. coli, P. aeruginosa, and C. albicans. The antitumoral activity was evaluated in vitro by cell viability assays with metastatic melanoma cell line (B16F10-Nex2) and the cytotoxicity was evaluated against human foreskin fibroblast (Hs68) cell line. Finally, in vivo assays were performed by using a syngeneic animal model of metastatic melanoma. Our findings have highlighted the potential application of the dual-peptide AuNPs in order to enhance the antitumor and antimicrobial activity of peptides.

Molecular detection of Leishmania (Sauroleishmania) tarentolae in human blood and Leishmania (Leishmania) infantum in Sergentomyia minuta: unexpected host-parasite contacts.

The detection of atypical Kinetoplastida in vertebrate hosts and vectors might suggest unexpected host-parasite contacts. Aside to major vectors of Leishmania (Leishmania) infantum in Italy (e.g. Phlebotomus perniciosus and Phlebotomus perfiliewi), the sand fly fauna also includes Sergentomyia minuta, herpetophilic and proven vector of Leishmania (Sauroleishmania) tarentolae, in which records of blood meal on mammals and detection of L. infantum DNA are increasing. This study was conducted in Central Italy aiming to molecularly detect potential atypical Leishmania host-vector contacts. Detection of Leishmania spp. DNA was performed by polymerase chain reaction (SSU rRNA, ITS1 targets) on field-collected sand fly females (N = 344), blood samples from humans (N = 185) and dogs (N = 125). Blood meal identification was also performed on engorged sand flies. Leishmania spp. DNA was found in 13.1% sand flies, 3.7% humans and 14.4% dogs. Sequence analysis identified L. infantum in S. minuta (4.4%), P. perniciosus (9.1%), humans (2.2%) and dogs (14.4%). Leishmania tarentolae was detected in S. minuta (12.6%), P. perfiliewi (6.6%) and human (1.6%) samples. Of 28 S. minuta examined for blood meal, 3.6 and 21.4% scored positive for human and lizard DNA, respectively. These results indicate the importance of one-health approach to explore new potential routes of transmission of leishmaniasis involving S. minuta.

Dirofilaria repens Infection Mimicking Lung Melanoma Metastasis.

We describe a rare case of Dirofilaria repens infection presenting as peripheral lung nodules and mimicking a metastatic focus from a previously diagnosed cutaneous melanoma. To avoid invasive investigations before arriving at the correct diagnosis, dirofilariasis should be included as a part of the diagnostic process in subjects with lung nodules who live in (or have traveled to) endemic regions.

Receptor homodimerization plays a critical role in a novel dominant negative P2RY12 variant identified in a family with severe bleeding.

Essentials Three dominant variants for the autosomal recessive bleeding disorder type-8 have been described. To date, there has been no phenotype/genotype correlation explaining their dominant transmission. Proline plays an important role in P2Y12R ligand binding and signaling defects. P2Y12R homodimer formation is critical for the receptor function and signaling. SUMMARY: Background Although inherited platelet disorders are still underdiagnosed worldwide, advances in molecular techniques are improving disease diagnosis and patient management. Objective To identify and characterize the mechanism underlying the bleeding phenotype in a Caucasian family with an autosomal dominant P2RY12 variant. Methods Full blood counts, platelet aggregometry, flow cytometry and western blotting were performed before next-generation sequencing (NGS). Detailed molecular analysis of the identified variant of the P2Y12 receptor (P2Y12R) was subsequently performed in mammalian cells overexpressing receptor constructs. Results All three referred individuals had markedly impaired ADP-induced platelet aggregation with primary wave only, despite normal total and surface P2Y12R expression. By NGS, a single P2RY12:c.G794C substitution (p.R265P) was identified in all affected individuals, and this was confirmed by Sanger sequencing. Mammalian cell experiments with the R265P-P2Y12R variant showed normal receptor surface expression versus wild-type (WT) P2Y12R. Agonist-stimulated R265P-P2Y12R function (both signaling and surface receptor loss) was reduced versus WT P2Y12R. Critically, R265P-P2Y12R acted in a dominant negative manner, with agonist-stimulated WT P2Y12R activity being reduced by variant coexpression, suggesting dramatic loss of WT homodimers. Importantly, platelet P2RY12 cDNA cloning and sequencing in two affected individuals also revealed three-fold mutant mRNA overexpression, decreasing even further the likelihood of WT homodimer formation. R265 located within extracellular loop 3 (EL3) is one of four residues that are important for receptor functional integrity, maintaining the binding pocket conformation and allowing rotation following ligand binding. Conclusion This novel dominant negative variant confirms the important role of R265 in EL3 in the functional integrity of P2Y12R, and suggests that pathologic heterodimer formation may underlie this family bleeding phenotype.

Mean platelet diameter measurements to classify inherited thrombocytopenias.

INTRODUCTION: Mean platelet volume (MPV) assists the differential diagnosis of inherited thrombocytopenia (IT) but lacks standardisation and varies between automated analysers. Classification of IT based on mean platelet diameter (MPD) has been proposed by an international collaborative study but has not been validated. METHODS: To assess the applicability of MPD to classify forms of IT, digital images of blood films from patients with established genetic causes for IT were generated, and the MPD measured (ZEISS Axio-scanner and Image J software) by a blinded reviewer. Comparison was made to the proposed classification system. RESULTS: Mean platelet volume was measured in thrombocytopenia with different genetic aetiologies, bilallelic BSS (bBSS) (n = 1), monoallelic BSS (mBSS) (n = 2), MYH9-related disorders (MYH9-RD) (n = 11), GFI1B-related thrombocytopenia (RT) (n = 15), FLI1-RT (n = 2), TUBB1-RT (n = 3), ITGA2B/ITGB3-RT (n = 1), RUNX1-RT (n = 2) and controls (n = 54). bBSS and 82% of MYH9-RD samples had MPD >4 µm which correlated with "IT with giant platelets." Only 55% of samples expected in the "large platelet group" had MPD meeting the classification cut-off (MPD >3.2 µm). FLI1-RT MPD were significantly larger than expected whilst ITGA2B/ITGB3-RT MPD were smaller than proposed. MPD in FPD/AML were "normal." CONCLUSION: Platelet MPD measurements are a useful guide to classify IT, but the time taken to record measurements may limit clinical applicability.

Thrombocytopenia and CD34 expression is decoupled from α-granule deficiency with mutation of the first growth factor-independent 1B zinc finger.

Essentials The phenotypes of different growth factor-independent 1B (GFI1B) variants are not established. GFI1B variants produce heterogeneous clinical phenotypes dependent on the site of mutation. Mutation of the first non-DNA-binding zinc-finger causes a mild platelet and clinical phenotype. GFI1B regulates the CD34 promoter; platelet CD34 expression is an indicator of GFI1B mutation. SUMMARY: Background Mutation of the growth factor-independent 1B (GFI1B) fifth DNA-binding zinc-finger domain causes macrothrombocytopenia and α-granule deficiency leading to clinical bleeding. The phenotypes associated with GFI1B variants disrupting non-DNA-binding zinc-fingers remain uncharacterized. Objectives To determine the functional and phenotypic consequences of GFI1B variants disrupting non-DNA-binding zinc-finger domains. Methods The GFI1B C168F variant and a novel GFI1B c.2520 + 1_2520 + 8delGTGGGCAC splice variant were identified in four unrelated families. Phenotypic features, DNA-binding properties and transcriptional effects were determined and compared with those in individuals with a GFI1B H294 fs mutation of the fifth DNA-binding zinc-finger. Patient-specific induced pluripotent stem cell (iPSC)-derived megakaryocytes were generated to facilitate disease modeling. Results The DNA-binding GFI1B variant C168F, which is predicted to disrupt the first non-DNA-binding zinc-finger domain, is associated with macrothrombocytopenia without α-granule deficiency or bleeding symptoms. A GFI1B splice variant, c.2520 + 1_2520 + 8delGTGGGCAC, which generates a short GFI1B isoform that lacks non-DNA-binding zinc-fingers 1 and 2, is associated with increased platelet CD34 expression only, without quantitative or morphologic platelet abnormalities. GFI1B represses the CD34 promoter, and this repression is attenuated by different GFI1B zinc-finger mutations, suggesting that deregulation of CD34 expression occurs at a direct transcriptional level. Patient-specific iPSC-derived megakaryocytes phenocopy these observations. Conclusions Disruption of GFI1B non-DNA-binding zinc-finger 1 is associated with mild to moderate thrombocytopenia without α-granule deficiency or bleeding symptomatology, indicating that the site of GFI1B mutation has important phenotypic implications. Platelet CD34 expression appears to be a common feature of perturbed GFI1B function, and may have diagnostic utility.

Molecular and phylogenetic analysis of the filarial nematode Micipsella numidica from the hare Lepus europaeus in Italy.

The genus Micipsella comprises three species of filariae to date identified in lagomorphs only, whereas the other genera belonging to the subfamily Splendidofilariinae are described as parasites of birds, reptiles and mammals. In the present study seven specimens of Micipsella numidica (Seurat, 1917), collected from the hare Lepus europaeus in Italy, were characterized genetically by molecular amplification of the mitochondrial genes (12S rDNA; cox1) and the 5S rDNA gene spacer region. Phylogenetic trees inferred using available sequences from filariae and those identified in this study evidenced a close relationship between M. numidica and Splendidofilariinae of other mammals and reptiles (Rumenfilaria andersoni and Madathamugadia hiepei). The present findings, apart from adding new data about the hosts in Italy, support the taxonomic position of M. numidica and highlight the substantial biological and molecular differences existing between Splendidofilariinae and other Onchocercidae. The study also contributes to our knowledge of the molecular/genetic diagnosis of filarial parasites of veterinary and medical concern in any vertebrate or invertebrate host.

Molecular epidemiology and genetic diversity of Blastocystis infection in humans in Italy.

In order to describe the molecular epidemiology of Blastocystis infection in Italy, 189 isolates, which had been collected during the years 2012-2014 from mildly symptomatic patients, or those affected by inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) or chronic diarrhoea, or otherwise immunosuppressed, were subtyped by sequence analysis of the SSU rRNA gene (536 bp). Six subtypes (STs) were detected: ST1 (15·3%), ST2 (13·8%), ST3 (46·0%), ST4 (21·7%), ST6 (3·2%) and ST8 (0·5%). They clustered in distinct clades, as inferred from Bayesian inference phylogenetic and median joining network analyses. A high genetic differentiation was found at the inter-subtype level; it ranged from Jukes-Cantor (JC) distance = 0·02 (between ST1 and ST4) to JC = 0·11 (between ST6 and ST2). At the intra-ST level, a high genetic homogeneity was registered in ST4, whereas higher genetic variation was found in isolates corresponding to ST1 and ST2. Accordingly, high values of haplotype and nucleotide diversity were observed in ST1, ST2 and ST3. No association was found between patient gender and ST, whereas ST3 and ST1 were significantly more prevalent in patients aged 15-50 years. A significant occurrence of Blastocystis ST4 in patients suffering from IBS, IBD or chronic diarrhoea was observed; in addition, a slight significant association between ST1 and ST3 and IBS patients was found. Multiple correspondence analysis showed some significant contribution of different variables (subtypes, haplotypes, age) in the observed pattern of ordination of the 189 patients in the symptom categories.

Monitoring game-based motor rehabilitation of patients at home for better plans of care and quality of life.

This paper describes the biomedical, remote monitoring infrastructure developed and currently tested in the EU REHAB@HOME project to support home rehabilitation of the upper extremity of persons post-stroke and in persons with other neurological disorders, such as Multiple Sclerosis patients, in order to track their progress over therapy and improve their Quality of Life. The paper will specifically focus on describing the initial testing of the tele-rehabilitation system's components for patients' biomedical monitoring over therapy, which support the delivery and monitoring of more personalized, engaging plans of care by rehabilitation centers and services.

Strongyloides stercoralis infection in allogeneic stem cell transplant: a case report and review of the literature.

Strongyloides stercoralis infections may be documented in low-endemicity areas, particularly in immigrants from endemic areas. The case of a patient from Bangladesh, an immigrant to Italy who developed a S. stercoralis infection after allogeneic stem cell transplant, is described, and 7 further cases are reviewed. Because of the atypical clinical presentation, the low predictive role of the eosinophil count, and the low sensitivity of the microbiological tests, diagnosis of strongyloidiasis is a challenging problem. When a case of S. stercoralis infection is suspected, previous exposure may be the only clue to guide the diagnostic approach.

Sonographic evaluation of the lower uterine segment thickness in women with a single previous Cesarean section.

AIM: The aim of this paper was to evaluate the lower uterine segment (LUS) thickness through transvaginal sonography in late preterm and full term pregnancies with a single previous Cesarean section, to correlate the obtained LUS measurements with intraoperative observations, and to identify a predictive cut-off value in order to select the best candidates for a vaginal birth after Cesarean delivery (VBAC). METHODS: Two hundred and fourteen women with a single previous Cesarean section who had an ultrasound measurement of the LUS thickness (stratified in S1, S2 and S3) in pregnancy were enrolled. The outcome of interest was the visual finding of a thin uterine scar at the time of the iterative Cesarean section. Linear regression was used to correlate the LUS thickness with gestational age (GA). A ROC curve has been used to determine the detection rate (DR) and the risk of each actual value of LUS thickness versus a thin uterine scar (outcome of interest). RESULTS: The LUS thickness was correlated with the gestational age (R2=0.034, P-value =0.005). The DR as estimated by ROC curves to detect a translucent lower uterine segment (S3) was 94.1% at a false positive rate (FPR) of 20%. The correspondent cut-off value was 1.8 mm. Finally a likelihood ratio (LR) of observing S3 was estimated. At the quoted cut-off of 1.8 mm the LR was 3. As demonstrated, for a segment of 1 mm the LR was instead about 13. CONCLUSION: The obtained values lead us to the conclusion that a thickness less than 1.8 mm can be reasonably considered a valid cut-off value to identify patients with a higher risk of thin uterine scar.

New insights into the epidemiology of bovine piroplasmoses in Italy.

Few studies have been published on bovine piroplasmoses in Italy, and therefore a clear picture of the epidemiology of these infections is difficult to obtain. Vertebrate and invertebrate hosts in Central and Northern Regions of Italy were investigated in 2005 and 2006, when microscopy, molecular tools and serological tests were applied to 468 blood samples drawn from cattle in order to evaluate the presence of these protozoa and identify possible risk factors. Ticks were also collected, identified and analyzed by molecular techniques. Microscopy identified 6.5% of the animals as positive, whereas PCR detected piroplasm DNA in 21.6%. BLAST analysis showed 67 amplicons (17.0%) referable to the Theileria sergenti/buffeli/orientalis group, 17 (4.3%) to Theileria annae, and 1 to Babesia divergens. Serology evidenced a prevalence of 45.4% for Babesia bovis, 17.4% for Babesia bigemina, and 34.9% for B. divergens. The 127 collected ticks were identified as belonging to 5 species, mostly represented by Rhipicephalus bursa, Hyalomma marginatum and Ixodes ricinus. Molecular analyses evidenced the presence of B. bovis and B. bigemina, in 3 and 5 ticks, respectively. Our findings suggest that different species of piroplasms are circulating in bovine populations in Central and Northern Italy, and provide new insights into the complex epidemiology of bovine piroplasmoses in Italy.

Subcutaneous dirofilariosis in South-East Serbia--case report.

Human dirofilariosis is a relatively rare infection caused by filarial worms of the genus Dirofilaria. We herein report the first case of human subcutaneous dirofilariosis in the southeastern part of Serbia. A complete alive nematode was removed from a nodule in the periorbital region of a 50-year-old woman. The nematode was morphologically identified as a D. repens-like immature female. The diagnosis was confirmed with molecular methods. The patient was probably infected in the South-East Serbia as she had not travelled abroad, nor in other parts of Serbia such as Vojvodina, recently identified as a hyperendemic area for D. repens infection of dogs.

Bilateral cleft lip and palate without premaxillary protrusion is associated with lethal aneuploidies.

OBJECTIVE: To investigate the clinical implications of two categories of fetal bilateral cleft lip and palate (BCLP): with premaxillary protrusion and with a flattened profile. METHODS: This was a retrospective study of fetuses with a prenatal diagnosis of BCLP at the Department of Obstetrics and Gynecology of the University of Bologna in the period 1991-2005. RESULTS: BCLP was diagnosed prenatally in 14 cases (mean gestational age at diagnosis, 21 (range, 12-36) weeks). In nine of these, there was a premaxillary pseudomass; in the remaining five, the profile was flat. Associated structural and/or chromosomal anomalies were found in two of the nine with a premaxillary pseudomass and in all five of those with a flat profile (P = 0.02). All fetuses with a flat profile had aneuploidies (three trisomy 18, one trisomy 13, one trisomy 8 mosaic), as did one of the nine with a premaxillary pseudomass. Eight of the pregnancies were terminated, including three of those with a premaxillary pseudomass and all five of those without. All continuing pregnancies resulted in live births, although one neonate affected by Krabbe's disease died shortly after birth. CONCLUSIONS: Our findings suggest that a third of cases of BCLP diagnosed in utero have a flat profile and these are at high risk of lethal aneuploidies.

Endoparasites of red fox (Vulpes vulpes) in central Italy.

A parasitologic study on 129 red foxes (Vulpes vulpes) from Tuscany (central Italy) was carried out in 2004-2006. Five intestinal species were found at necropsy: Dipylidium caninum (prevalence 57.3%), Mesocestoides lineatus (45.4%), Uncinaria stenocephala (39.1%), Toxocara canis (9.1%), and Toxascaris leonina (5.4%). Other parasites not associated with the intestine included Crenosoma vulpis (14.7%), Capillaria aerophila (7.0%), Angiostrongylus vasorum (7.0%), and filarial parasites (17.8%). Coprologic tests were less sensitive and less specific in identifying parasites than direct examinations at necropsy. Trichinella larvae were not found in muscles submitted to artificial digestion. By immunologic assay, antigens of Echinococcus spp. were detected in fecal samples of 20 foxes, but results could not be confirmed by fecal examination or molecular tests.

Canine piroplasmosis in Italy: epidemiological aspects in vertebrate and invertebrate hosts.

In Italy, canine piroplasmosis is believed to be widespread, but few data are available on its presence in most areas. In 2005 and 2006, vertebrate and invertebrate hosts were investigated in Central and Northern Regions of the Country. Microscopy on blood smears, molecular tools and serological tests were applied to 420 blood samples collected from dogs, in order to evaluate the presence of these protozoa and to identify possible risk factors. Moreover, ticks were analyzed by molecular techniques. Microscopy identified as positive 2.8% of the animals, all from Central Italy, and PCR detected 'piroplasm' DNA in 6.0%. Serology evidenced a mean prevalence of 34.0% with a decreasing trend from Central to Northern areas. The 507 collected ticks were identified as belonging to 8 species, mostly represented by Rhipicephalus sanguineus (n=376) and Ixodes ricinus (n=58). Molecular analyses evidenced the presence of babesial parasites (Babesia canis canis, B. canis vogeli, B. microti-like) in 25 (4.9%) of them; in Rh. sanguineus there was also demonstration of the vertical transmission of B. canis canis. Statistical analysis identified 'kennel' as risk factor for Babesia infection. Our findings evidenced that different species of piroplasms potentially infectious for dogs are circulating in Italy, and that epidemiological aspects of these infections are more complex than expected. Vector importance of both Rh. sanguineus and I. ricinus is hypothesized, but further investigation is needed.

Morphology and genetics of a Babesia isolate from Capreolus capreolus.

A Babesia isolate that was morphologically distinct from Babesia capreoli and very similar to B. divergens was found in the blood of a roe deer (Capreolus capreolus) found dead in central Italy. Sequences corresponding to the full coding region of the 18S ribosomal RNA (rRNA) gene were identical to a sequence reported for Babesia divergens from a reindeer (Rangifer tarandus) and 99.9% and 99.8% similar to those reported for B. capreoli and bovine origin B. divergens, respectively.

Aedes albopictus and Culex pipiens implicated as natural vectors of Dirofilaria repens in central Italy.

To identify the natural vectors of Dirofilaria repens Railliet et Henry, entomological samplings were carried out in four sites within the Lazio region, foci of canine subcutaneous dirofilariasis. Collections were made in 2002-2003 by means of dog-baited and miniature Centers for Disease Control and Prevention traps as well as on humans. Microscopy identified 1576 attracted mosquito females as belonging to six species, but molecular diagnostics detected filarial DNA only in Culex pipiens L. and Aedes albopictus (Skuse, 1894). Dirofilaria immitis Leidy DNA, D. repens DNA, or both were found in the head and thorax of both mosquitoes. The simultaneous presence of vectors showing diurnal and nocturnal activity patterns is of concern for animal and human health. The finding of D. immitis DNA in mosquitoes in areas where only D. repens was been recovered in dogs also demonstrates that this filarial parasite circulates among carnivores (wild or domesticated pets).

Prospective evaluation of the risk of pre-eclampsia using logistic regression analysis.

OBJECTIVES: To calculate the risk of developing pre-eclampsia (PET) in a consecutive series of low-risk women at 18-24 weeks' gestation, using recently published logistic regression models. METHODS: This was a prospective study, with complete follow-up, in a consecutive series of unselected low-risk singleton pregnancies. Uterine artery pulsatility index as well as a combination of maternal factors were recorded at 18-24 weeks' gestation. The distribution of the estimated risks for the 16 PET patients was compared with that obtained for 136 women who had a normal pregnancy, as assessed by routine testing. A receiver-operating characteristics (ROC) curve was plotted to evaluate the detection rate at fixed false-positive rates (FPRs) of 5%, 10% and 20% and the corresponding odds cut-offs. RESULTS: Just 1/16 (6.2%) women with PET developed the disease before the 34(th) week of gestation. Using the 'All PET' logistic regression model, for 16 PET cases the overall median odds was 1 : 1454, higher compared with that of 1 : 41635 estimated for controls. Using the 'PET >or= 34 weeks' model, the median odds of the 15 women who developed PET late was 1 : 3405, compared with 1 : 40785 for controls. In the case of PET before 34 weeks, the risk was 1 : 426373 vs. 1 : 4159823126 estimated for controls ('PET < 34 weeks' model). Detection rates for the All PET model were 18%, 50% and 62% at a FPR of 5%, 10% and 20%, respectively. For the PET >or= 34 weeks model these detection rates were 6%, 46% and 60%, respectively. CONCLUSION: Even though the individual odds estimation is too low to represent the real risk of PET, the recently published logistic regression models detected more than 60% of PET at a FPR of 20% for both All PET and PET >or= 34 weeks models. Using these models in clinical practice does not seem to give any significant improvement over Doppler alone in the prediction of PET, but the use of a PET-specific odds instead of an actual Doppler value alone seems to be useful for clinical management.

Occurrence of the G3 Indian buffalo strain of Echinococcus granulosus in cattle.

During a survey carried out to define the occurrence of Echinococcus granulosus in cattle bred in the province of Rieti (Central Italy), molecular diagnostics (PCR amplification and sequencing of a partial region of the mitochondrial CO1 gene) showed that 6/10 positive bovines harboured hydatid cysts (No.=16) genetically identical (95.8-100%) to the Indian buffalo genotype G3. As far the location of the 16 cysts, 11 of them were found in the lungs of three animals, whereas 5 cysts were in the liver of three parasitized hosts. The occurrence of genotype G3 in 60% of parasitized bovines living in an area never studied before provides more definite evidence about the existence of the strain in this region, and proves that cattle have to be considered a non-accidental host.