Isoenzyme A and Urinary N-Acetyl-β-D-Glucosaminidase Activity in Normal Pregnancy

Urinary N-acetyl-β-d-glucosaminidase (NAG) activity has been found to increase during normal uncomplicated pregnancy and such behavior could limit the diagnostic value of this enzyme for detection of subclinical tubular injury. The aim of this study was to evaluate urinary NAG activity and isoenzyme A in normal pregnant women at 30th week of pregnancy and in healthy women, to discriminate between physiological and lesional enzymuria.Enzyme activities in first morning fasting urine samples from 20 nonpregnant control and 20 normal pregnant women at 30th gestational week were evaluated by fluorometric methods.Both total and isoenzyme A activity was significantly higher ( p0.01) in urines of normal pregnant women compared with control urines, whereas ratio between these two parameters was significantly lower ( p0.001).The increase of urinary NAG activity during normal uncomplicated pregnancy appears to be characterized by a prevalent increase in isoenzyme A form, a finding associated with functional (not lesional) enzymuria. The fluorometric assays may represent a simple and rapid method to evaluate whether increase in urinary NAG activity represents a renal physiological adaptation during pregnancy

Optimised production of an anti-fungal antibody in Solanaceae hairy roots to develop new formulations against Candida albicans

Background: Infections caused by fungi are often refractory to conventional therapies and urgently require the development of novel options, such as immunotherapy. To produce therapeutic antibodies, a plant-based expression platform is an attractive biotechnological strategy compared to mammalian cell cultures. In addition to whole plants, hairy roots (HR) cultures can be used, representing an expression system easy to build up, with indefinite growth while handled under containment conditions. Results: In this study the production in HR of a recombinant antibody, proved to be a good candidate for human immunotherapy against fungal infections, is reported. Expression and secretion of this antibody, in an engineered single chain (scFvFc) format, by HR from Nicotiana benthamiana and Solanum lycopersicum have been evaluated with the aim of directly using the deriving extract or culture medium against pathogenic fungi. Although both Solanaceae HR showed good expression levels (up to 68 mg/kg), an optimization of rhizosecretion was only obtained for N. benthamiana HR. A preliminary assessment to explain this result highlighted the fact that not only the presence of proteases, but also the chemical characteristics of the growth medium, can influence antibody yield, with implications on recombinant protein production in HR. Finally, the antifungal activity of scFvFc 2G8 antibody produced in N. benthamiana HR was evaluated in Candida albicans growth inhibition assays, evidencing encouraging results. Conclusions: Production of this anti-fungal antibody in HR of N. benthamiana and S. lycopersicum elucidated factors affecting pharming in this system and allowed to obtain promising ready-to-use immunotherapeutics against C. albicans

Production and Functional Characterization of a Recombinant Predicted Pore-Forming Protein (TVSAPLIP12) of Trichomonas vaginalis in Nicotiana benthamiana Plants

Pore-forming proteins (PFPs) are a group of functionally versatile molecules distributed in all domains of life, and several microbial pathogens notably use members of this class of proteins as cytotoxic effectors. Among pathogenic protists, Entamoeba histolytica, and Naegleria fowleri display a range of pore-forming toxins belonging to the Saposin-Like Proteins (Saplip) family: Amoebapores and Naegleriapores. Following the genome sequencing of Trichomonas vaginalis, we identified a gene family of 12 predicted saposin-like proteins (TvSaplips): this work focuses on investigating the potential role of TvSaplips as cytopathogenetic effectors. We provide evidence that TvSaplip12 gene expression is potently upregulated upon T. vaginalis contact with target cells. We cloned and expressed recombinant TvSaplip12 in planta and we demonstrate haemolytic, cytotoxic, and bactericidal activities of rTvSaplip12 in vitro. Also, evidence for TvSaplip subcellular discrete distribution in cytoplasmic granules is presented. Altogether, our results highlight the importance of TvSaplip in T. vaginalis pathogenesis, depicting its involvement in the cytolytic and bactericidal activities during the infection process, leading to predation on host cells and resident vaginal microbiota for essential nutrients acquisition. This hence suggests a potential key role for TvSaplip12 in T. vaginalis pathogenesis as a candidate Trichopore

Molecular Tools for Monitoring the Ecological Sustainability of a Stone Bio-Consolidation Treatment at the Royal Chapel, Granada

Background: Biomineralization processes have recently been applied in situ to protect and consolidate decayed ornamental stone of the Royal Chapel in Granada (Spain). While this promising method has demonstrated its efficacy regarding strengthening of the stone, little is known about its ecological sustainability.Methodology/Principal Findings: Here, we report molecular monitoring of the stone-autochthonous microbiota before and at 5, 12 and 30 months after the bio-consolidation treatment (medium/long-term monitoring), employing the well-known molecular strategy of DGGE analyses. Before the bio-consolidation treatment, the bacterial diversity showed the exclusive dominance of Actinobacteria (100%), which decreased in the community (44.2%) after 5 months, and Gamma-proteobacteria (30.24%) and Chloroflexi (25.56%) appeared. After 12 months, Gamma-proteobacteria vanished from the community and Cyanobacteria (22.1%) appeared and remained dominant after thirty months, when the microbiota consisted of Actinobacteria (42.2%) and Cyanobacteria (57.8%) only. Fungal diversity showed that the Ascomycota phylum was dominant before treatment (100%), while, after five months, Basidiomycota (6.38%) appeared on the stone, and vanished again after twelve months. Thirty months after the treatment, the fungal population started to stabilize and Ascomycota dominated on the stone (83.33%) once again. Members of green algae (Chlorophyta, Viridiplantae) appeared on the stone at 5, 12 and 30 months after the treatment and accounted for 4.25%, 84.77% and 16.77%, respectively.Conclusions: The results clearly show that, although a temporary shift in the bacterial and fungal diversity was observed during the first five months, most probably promoted by the application of the bio-consolidation treatment, the microbiota tends to regain its initial stability in a few months. Thus, the treatment does not seem to have any negative side effects on the stone-autochthonous microbiota over that time. The molecular strategy employed here is suggested as an efficient monitoring tool to assess the impact on the stone-autochthonous microbiota of the application of biomineralization processes as a restoration/conservation procedure.This work was supported by the European Regional Development Fund (ERDF), Junta de Andalucía (Spain) and the “Fortalecimiento de la I+D+i” program from the University of Granada, co-financed by grant RNM-3493 and Research Group BIO-103 from Junta de Andalucía, as well as by the Spanish Government through “José Castillejo” program from the “Ministerio de Educación, Cultura y Deporte” (I+D+i 2008-2011), and by the Austrian Science Fund (FWF) under Grant “Elise-Richter V194-B20”

Contribution of the Microbial Communities Detected on an Oil Painting on Canvas to Its Biodeterioration

In this study, we investigated the microbial community (bacteria and fungi) colonising an oil painting on canvas, which showed visible signs of biodeterioration. A combined strategy, comprising culture-dependent and -independent techniques, was selected. The results derived from the two techniques were disparate. Most of the isolated bacterial strains belonged to related species of the phylum Firmicutes, as Bacillus sp. and Paenisporosarcina sp., whereas the majority of the non-cultivable members of the bacterial community were shown to be related to species of the phylum Proteobacteria, as Stenotrophomonas sp. Fungal communities also showed discrepancies: the isolated fungal strains belonged to different genera of the order Eurotiales, as Penicillium and Eurotium, and the non-cultivable belonged to species of the order Pleosporales and Saccharomycetales. The cultivable microorganisms, which exhibited enzymatic activities related to the deterioration processes, were selected to evaluate their biodeteriorative potential on canvas paintings; namely Arthrobacter sp. as the representative bacterium and Penicillium sp. as the representative fungus. With this aim, a sample taken from the painting studied in this work was examined to determine the stratigraphic sequence of its cross-section. From this information, “mock paintings,” simulating the structure of the original painting, were prepared, inoculated with the selected bacterial and fungal strains, and subsequently examined by micro-Fourier Transform Infrared spectroscopy, in order to determine their potential susceptibility to microbial degradation. The FTIR-spectra revealed that neither Arthrobacter sp. nor Penicillium sp. alone, were able to induce chemical changes on the various materials used to prepare “mock paintings.” Only when inoculated together, could a synergistic effect on the FTIR-spectra be observed, in the form of a variation in band position on the spectrum.The FTIR analyses performed in this study were financed by the Junta de Andalucía (RNM-325 group). The molecular analyses performed in this study were financed by the Austrian Science Fund (FWF) project ‘Hertha-Firnberg T137’ and the Spanish Ministry of Science and Innovation (Project CTQ2008-06727-C03-03). G. Piñar also thanks the “Elise-Richter V194-B20” projects

Long-term effectiveness, safety, and liver stiffness dynamics of PBC treatment with obeticholic acid in real-world

Background & Aims: Several studies have assessed the short-term effectiveness and safety of obeticholic acid (OCA) in the real-world setting. We aimed to extend knowledge on the real-world effectiveness and safety of OCA treatment by expanding sample size and follow-up, and by exploring changes in liver stiffness measurement (LSM) over time. Methods: The RECAPITULATE project involves centres belonging to the “Italian PBC registry” and/or the “Club Epatologi Ospedalieri” PBC working group. Effectiveness was evaluated as biochemical response according to POISE and normal range (NR) criteria (normal alkaline phosphatase/alanine aminotransferase/bilirubin). Safety was assessed as the incidence of de novo/worsening pruritus and discontinuation rate/causes. Available LSMs were also captured. Results: We included 747 patients from 66 Italian centres: mean age 58 years; female/male 88%/14%; median follow-up 24 months [IQR 12-42]; 28% with cirrhosis, and 14% with autoimmune hepatitis (AIH)/PBC overlap syndrome. Probabilities of POISE and NR response increased from baseline to 57% and 20%, respectively, by the 42nd month. The probabilities of response were lower in patients with cirrhosis (p = 0.02 and p = 0.004 for POISE and NR), but not different between patients with AIH/PBC and pure PBC (p = 0.8). Overall, 130 patients (17%) discontinued treatment, mainly due to pruritus (36.9%), while 28.5% did so after developing hepatic events. The discontinuation rate was higher in patients with cirrhosis (p <0.001). LSM was available in 573 patients (∼77%), of whom 255 had multiple measurements. LSM variation over time differed based on the attainment of POISE biochemical response (expected mean annual variation -0.48 [-0.78, -0.19] in responders vs. +0.33 [-0.07, 0.73] in non-responders, respectively, p <0.001). Conclusions: Our findings confirm the effectiveness and safety profiles of OCA in the medium/long term and demonstrate that biochemical response is associated with the change in LSM over time. Impact and Implications: After the conditional approval of OCA for the treatment of PBC, the main confirmatory study failed to demonstrate OCA's ability to reduce liver-related events, leading the EMA to revoke the drug's marketing authorization. The ensuing scientific debate highlights an urgent need for further evidence from real-world practice. In the largest real-world series of patients treated with OCA to date, we confirm that the drug's effectiveness and safety profiles are maintained over a medium-to-long follow-up period. Valuable data for the management of the drug in relevant subgroups of patients, such as those with cirrhosis and autoimmune hepatitis/PBC overlap syndrome, are also provided. Our original results on liver stiffness measurement variation over time suggest a favourable impact of OCA on fibrosis progression, particularly in patients achieving a biochemical response to the drug. Overall, these data provide important insights for clinicians managing patients with PBC and contribute to the ongoing scientific debate about the effectiveness/safety profile of this drug