Molecular phylogenetic analysis of Echinococcus granulosus sensu lato infecting sheep in Italy.

Cystic Echinococcosis (CE), caused by the larval form of Echinococcus granulosus sensu lato, is a neglected zoonosis still threatening public health worldwide. In Italy different epidemiological scenarios were reported depending on the geographical area and associated socio-economic activities. Although in northern Italy the occurrence of E. granulosus is considered sporadic, in the southern regions and, particularly in Sardinia, CE prevalence reaches high levels. We analysed CE cysts collected from infected sheep from various areas of mainland Italy and the Sardinia island, with the main objective to investigate intergenotypic and intragenotypic variations at national level. CE cysts were collected from slaughtered sheep following post mortem inspection at local abattoirs. Total genomic DNA was extracted and amplification and sequencing of the partial mitochondrial genes nad5 and cox1 were performed. A Bayesian phylogenetic tree was estimated on a nad5 dataset (n = 260) composed of E. granulosus samples from this study (n = 126) and all the nad5 haplotypes available in GenBank (n = 134). In addition, haplotype network, diversity and neutrality analysis were performed on nad5 and cox1 sequences of Italian origin obtained in this study. E. granulosus sensu stricto (s.s.) was found to be the only Echinococcus species infecting sheep in Italy, mainly represented by G1 genotype (76 %) and, to a lower extent, by G3 genotype (24 %). Phylogenetic analyses revealed 40 nad5 and 33 cox 1 haplotypes, and the presence of two founder haplotypes, belonging to G1 and G3 genotype, showing 100 % similarity with DNA sequences from different geographic regions. The lack of geographical segregation, high haplotype and low nucleotide diversity coupled with significant negative values of Tajima's D and Fu's Fs observed in this study indicated high genetic variation and demographic expansion of E. granulosus s.s. in Italy.

Microtubular Assessment of C6 Rat Glioma Cell Spheroids Developed in Transparent Liquid Marbles or Hanging Drops.

Glioblastoma is a brain tumour frequently used as an experimental model to exploit innovative therapeutic approaches due to its high lethality and refractoriness to therapies. Part of these innovative anticancer therapies address cytoskeletal microtubules (MTs) since specific tubulin post-translational modifications (PTMs) are considered markers of tumour plasticity. In vitro studies, which traditionally employ two-dimensional (2D) culture systems, are now being replaced by three-dimensional (3D) systems that more closely mimic in vivo physiological conditions and allow a better understanding of the signalling between cells. In this work, we compared 2 liquid base 3D methods for the generation of spheroids from C6 rat glioma cells (RGCs) using 30 µL of liquid marble (LM) or the hanging drops (HDs), which contained 2 different cell numbers (5000 or 15,000). After 24 or 48 h of in vitro culture (IVC), the morphology of the spheroids was observed and the behaviour of the two main tubulin PTMs, tyrosinated α-tubulin (Tyr-T) and acetylated α-tubulin (Ac-T), was evaluated by fluorescence and Western blot (WB). RGCs spontaneously formed spherical agglomerates more rapidly in the LM than in the HD system. Cell density influenced the size of the spheroids, which reached a larger size (> of 300 µm Ø), with 15,000 cells compared to 5000 cells (150 µm Ø). Moreover, an increase in Tyr-T and Ac-T was observed in both the HD and LM system from 24 to 48 h, with the highest values shown in the 48 h/LM spheroids of 5000 cells (p < 0.05). In conclusion, by comparing the morphology and microtubular architecture of spheroids from C6 rat glioma cells developed by LM or HD methodology, our findings demonstrate that the use of a fumed silica microbioreactor boosts the induction and maintenance of a high plasticity state in glioma cells. RGCs cultured in LM express levels of tubulin PTMs that can be used to evaluate the efficacy of new anticancer therapies.

Environmental Contamination by Echinococcus spp. Eggs as a Risk for Human Health in Educational Farms of Sardinia, Italy.

Cystic Echinococcosis (CE) is a severe zoonosis caused by the larval stage of the tapeworm Echinococcus granulosus. These parasites are naturally transmitted between canid definitive hosts that harbour the adult stage in the intestine, and mainly ungulate intermediate hosts, with larval cysts developing in their internal organs. The close coexistence between dog and sheep with incorrect hygiene management are the most important factors for the persistence of this parasitic pathology. The Educational Farms (EF) are farms and agritourisms suitably equipped to carry out training activities for people interested in farm practices and agricultural processing, in particular student groups. Public attendance of farms represents a new potential risk factor for the zoonoses transmission. Consumption of contaminated food and water in combination with contact or playing with domestic dogs (Canis familiaris) are possible routes of zoonoses human infection. In fact, Echinococcus spp. eggs may persist in the environment up to several months at low temperatures and moist conditions, having the chance of contaminating different matrices and surfaces. The aim of this investigation was to study environmental contamination by parasitic elements as a risk for zoonoses, such as Echinococcus spp. A total of 116 samples (35 of water, 33 of soil, 23 of vegetables, 25 of dog faeces) were collected in 30 EF in Sardinia. Samples were subjected to biomolecular investigation for the research of specific gene sequences of Echinococcus granulosus, Echinococcus multilocularis and Taenia spp. The study allowed to identify eight positive EF due to the presence of Echinococcusgranulosus in eight dog faeces samples and one positive EF due to the presence of Taenia spp. in a water sample. The work has allowed to develop and harmonise the diagnostic methods and operating protocols essential for controlling the spread of the CE to create "One Health" intervention plans in high endemic areas through the implementation of SOP (standard operating procedures) for monitoring the pathology in animals, humans and environment.

Confocal investigation on colocalization between tubulin posttranslational modifications and associated proteins in rat C6 glioma cells.

Glioblastoma multiforme is the most lethal brain tumor. In the study of mechanisms underlying its development attention has been paid to the microtubular network of its cells, mainly on ßIII tubulin, considered as a marker of malignancy. In the present work, we chose to investigate the tubulin code in glioblastoma cells, analyzing the degree of interaction between tubulin post-translational modifications and different proteins associated with them. The pattern of diverse associated proteins such as EB-1, CLIP-170 and kinesin-1 and their degree of co-distribution with the most abundant post-translational tubulin modifications (tyrosination, acetylation and polyglutamylation) were evaluated. Through immunofluorescence we have shown that EB-1, CLIP-170 and kinesin-1 were well detectable in glioblastoma cells. The double fluorescence and colocalization index between the post-translational modifications of tubulin and associated proteins showed that tyrosinated α-tubulin has significantly high affinity with EB-1, CLIP-170 and kinesin-1, while for acetylated and polyglutamylated tubulin, the degree of interaction with the three associated proteins evaluated was less apparent. Data presented in this paper underline the importance of a thorough analysis of the microtubular mechanics in glioblastoma cells. This may suggest new experimental therapeutic approaches able to act more selectively on the microtubular network of cells in this type of cancer.

Methylcellulose Hydrogel with Melissa officinalis Essential Oil as a Potential Treatment for Oral Candidiasis.

Candida spp. are the most prevalent fungi of the human microbiota and are opportunistic pathogens that can cause oral candidiasis. Management of such infections is limited due to the low number of antifungal drugs available, their relatively high toxicity and the emergence of antifungal resistance. Therefore, much interest in the antimicrobial potential of natural compounds has recently been evident. The use of hydrogels in the delivery of biocides has been explored due to their biocompatibility, ease with drug encapsulation, and due to their potential to confer mechanical and structural properties similar to biological tissue. Methylcellulose hydrogels (10% (w/v)) with 1% (v/v) and 2% (v/v) Melissa officinalis oil were synthesised. The rheological properties and gelation time of the hydrogels were evaluated. Antimicrobial action, the antifungal potential and ability to displace Candida were determined. Rheological tests revealed that the hydrogel jellified in three minutes at 37 °C. Loaded hydrogels successfully inhibited Candida albicans growth as evident by zone of inhibition and time-kill assays. A significant reduction in retained C. albicans was demonstrated with the hydrogel at 2% Melissa officinalis concentration. This work demonstrated that an essential oil-loaded hydrogel had the potential to provide a novel antimicrobial therapy for the treatment of oral candidiasis.

Investigation of the effects of Lycium barbarum polysaccharides against cadmium induced damage in testis.

This study describes the effects of Lycium barbarum polysaccharides (LBP) on testicular damage induced by cadmium (Cd). Adult male rats were i.p. injected with CdCl2 (4mg/Kg, once) with or without LBP pretreatment (300mg/Kg orally, once a day, for 30days). Testis weight, morphological/histological structure and oxidative stress parameters were evaluated. Several adverse effects were observed after CdCl2 injection, with a significant decrease in body/testis weight ratio (P<0.05), gross morphological changes with hyperemia of the parenchyma, increased volume and alteration in the structure of the seminiferous tubules. Furthermore, Cd intoxication caused a significant decrease of glutathione (GSH) and Trolox equivalent antioxidant capacity (TEAC) in testis (P<0.05) together with a significant increase (P<0.01) of 3-nitro-l-tyrosine (3NT) while malondialdehyde (MDA) did not change. LBP pretreatment caused slight signs of improvement in the morphology of the seminiferous tubules. Our results confirm that Cd induces testicular damage and suggest the oxidative stress involvement. LBP could ameliorate Cd testicular damage but further investigations are needed.

Possible antioxidant effect of Lycium barbarum polysaccharides on hepatic cadmium-induced oxidative stress in rats.

The aim of this study was to investigate the potential protective effect of Lycium barbarum polysaccharides (LBP) pretreatment against cadmium (Cd)-induced hepatotoxicity in rats. Wistar rats were divided into control group, LBP group (300 mg/kg orally, once a day, for 30 days), Cd group (CdCl2 4 mg/kg i.p. once), and LBP + Cd group (LBP 300 mg/kg orally, once a day, for 30 days + CdCl2 4 mg/kg i.p. 24 h after the last treatment). Cd liver injury was examined by morphological/histological changes, transaminases, total protein concentration, and oxidative stress evaluated by MDA, 3NT, GSH, SOD, and TEAC activities. Cd intoxication caused gross morphological changes with hyperemia of the parenchyma, increased volume, and disappearance of the anatomical limits of the lobes associated with an increase of ALT, GSH, and TEAC in plasma and a decrease of MDA, GSH, and TEAC in liver, SOD, and total proteins in plasma. LBP pretreatment caused a slight improvement in the histological architecture and in the 3NT amount together with a significant improvement of hematic parameters. On the basis of the obtained results, we can affirm that LBP pretreatment can ameliorate liver conditions, but further studies are needed to better evaluate the protective antioxidant effects of LBP against Cd-induced toxicity.