Allestimento e test <i>in vivo</i> di un vaccino a DNA contro l’Agalassia Contagiosa

Mycoplasma agalactiae is the aetiological agent of Contagious Agalactia (CA), an economically detrimental disease affecting small ruminants worldwide. The control of M. agalactiae through vaccination is an increasing challenge. Although several inactivated vaccines have proved effective in conferring protection against CA, they carry intrinsic disadvantages and only produce a short-lived humoral immune response. A DNA vaccine against CA has been developed encoding the P48eof Mycoplasma agalactiae inducing the immune responses in BALB/c mice (Chessa et al., 2009). In this study, the vaccinal plasmid pJW/tPA/rP48 expressing the P48 of M. agalactiae in fusion with the signal peptide of tissue plasminogen activator (tPA) was designed and evaluated in vivo. Initially, the production of P48 was evaluated in HEK 293 cells by rtPCR and by Western blotting. A field trial was performed by immunizing sheep by injecting them both with recombinant P48 protein and the plasmid pJW/tPA/rP48. Results demonstrated the production of P48-specific antibodies in immunized sheep, confirming that P48 was expressed and induced significant immune responses. Also we showed that immunization can negatively affect milk-shedding of microorganisms, especially when a prime boost strategy is used. Concluding, it has been demonstrated that even if immunization against rP48 alone induce a significant immune response it does not protect sheep against CA

The liposoluble proteome of Mycoplasma agalactiae: an insight into the minimal protein complement of a bacterial membrane

<p>Abstract</p> <p>Background</p> <p>Mycoplasmas are the simplest bacteria capable of autonomous replication. Their evolution proceeded from gram-positive bacteria, with the loss of many biosynthetic pathways and of the cell wall. In this work, the liposoluble protein complement of <it>Mycoplasma agalactiae</it>, a minimal bacterial pathogen causing mastitis, polyarthritis, keratoconjunctivitis, and abortion in small ruminants, was subjected to systematic characterization in order to gain insights into its membrane proteome composition.</p> <p>Results</p> <p>The selective enrichment for <it>M. agalactiae </it>PG2^Tliposoluble proteins was accomplished by means of Triton X-114 fractionation. Liposoluble proteins were subjected to 2-D PAGE-MS, leading to the identification of 40 unique proteins and to the generation of a reference 2D map of the <it>M. agalactiae </it>liposoluble proteome. Liposoluble proteins from the type strain PG2 and two field isolates were then compared by means of 2D DIGE, revealing reproducible differences in protein expression among isolates. An in-depth analysis was then performed by GeLC-MS/MS in order to achieve a higher coverage of the liposoluble proteome. Using this approach, a total of 194 unique proteins were identified, corresponding to 26% of all <it>M. agalactiae </it>PG2^Tgenes. A gene ontology analysis and classification for localization and function was also carried out on all protein identifications. Interestingly, the 11.5% of expressed membrane proteins derived from putative horizontal gene transfer events.</p> <p>Conclusions</p> <p>This study led to the in-depth systematic characterization of the <it>M. agalactiae </it>liposoluble protein component, providing useful insights into its membrane organization.</p

Renal amyloid‐A amyloidosis in cats: Characterization of proteinuria and biomarker discovery, and associations with kidney histology

BackgroundAmyloid A (AA) amyloidosis is a protein misfolding disease arising from serum amyloid A (SAA). Systemic AA amyloidosis recently was shown to have a high prevalence in shelter cats in Italy and was associated with azotemia and proteinuria.ObjectivesInvestigate urine protein profiles and diagnostic biomarkers in cats with renal AA amyloidosis.AnimalsTwenty‐nine shelter cats.MethodsCase‐control study. Cats with renal proteinuria that died or were euthanized between 2018 and 2021 with available necropsy kidney, liver and spleen samples, and with surplus urine collected within 30 days before death, were included. Histology was used to characterize renal damage and amyloid amount and distribution; immunohistochemistry was used to confirm AA amyloidosis. Urine protein‐to‐creatinine (UPC) and urine amyloid A‐to‐creatinine (UAAC) ratios were calculated, and sodium dodecyl sulfate‐agarose gel electrophoresis (SDS‐AGE) and liquid chromatography‐mass spectrometry (LC‐MS) of proteins were performed.ResultsTwenty‐nine cats were included. Nineteen had AA amyloidosis with renal involvement. Cats with AA amyloidosis had a higher UPC (median, 3.9; range, 0.6‐12.7 vs 1.5; 0.6‐3.1; P = .03) and UAAC ratios (median, 7.18 × 10$^{−3}$; range, 23 × 10$^{−3}$‐21.29 × 10$^{−3}$ vs 1.26 × 10$^{−3}$; 0.21 × 10$^{−3}$‐6.33 × 10$^{−3}$; P = .04) than unaffected cats. The SDS‐AGE identified mixed‐type proteinuria in 89.4% of cats with AA amyloidosis and in 55.6% without AA amyloidosis (P = .57). The LC‐MS identified 63 potential biomarkers associated with AA amyloidosis (P < .05). Among these, urine apolipoprotein C‐III was higher in cats with AA amyloidosis (median, 1.38 × 10$^{7}$; range, 1.85 × 10$^{5}$‐5.29 × 10$^{7}$ vs 1.76 × 10$^{6}$; 0.0 × 10$^{0}$‐1.38 × 10$^{7}$; P = .01). In the kidney, AA‐amyloidosis was associated with glomerulosclerosis (P = .02) and interstitial fibrosis (P = .05).Conclusions and Clinical ImportanceRenal AA amyloidosis is associated with kidney lesions, increased proteinuria and increased urine excretion of SAA in shelter cats. Additional studies are needed to characterize the role of lipid transport proteins in the urine of affected cats

<i>Mycoplasma agalactiae</i> MAG_5040 is a Mg²⁺-dependent, sugar-nonspecific SNase recognised by the host humoral response during natural infection

In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl2 at temperatures ranging from 37 to 45uC. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants

Oxamate salts as novel agents for the restoration of marble and limestone substrates : case study of ammonium N-phenyloxamate

The ammonium salt of N-phenyloxamic acid (AmPhOxam) was synthesised, characterised by FT-IR, FT-Raman, UV-Vis, 1H-NMR spectroscopic methods and single crystal X-ray diffraction, and evaluated as a protective and consolidating agent for calcareous stone substrates under mild conditions. Hydro-alcoholic solutions of AmPhOxam were tested for the treatment of naturally weathered white marble and biomicritic limestone. Mercury intrusion porosimetry, FT-NIR spectroscopy measurements and SEM microscopy showed the formation of a superficial protective layer of crystals of the corresponding monohydrated calcium salt, CaPhOxam, on both treated stones.Publisher PDFPeer reviewe

The Liposoluble proteome of <i>Mycoplasma agalactiae</i>: an insight into the minimal protein complement of a bacterial membrane

Background Mycoplasmas are the simplest bacteria capable of autonomous replication. Their evolution proceeded from gram-positive bacteria, with the loss of many biosynthetic pathways and of the cell wall. In this work, the liposoluble protein complement of Mycoplasma agalactiae, a minimal bacterial pathogen causing mastitis, polyarthritis, keratoconjunctivitis, and abortion in small ruminants, was subjected to systematic characterization in order to gain insights into its membrane proteome composition. Results The selective enrichment for M. agalactiae PG2T liposoluble proteins was accomplished by means of Triton X-114 fractionation. Liposoluble proteins were subjected to 2-D PAGE-MS, leading to the identification of 40 unique proteins and to the generation of a reference 2D map of the M. agalactiae liposoluble proteome. Liposoluble proteins from the type strain PG2 and two field isolates were then compared by means of 2D DIGE, revealing reproducible differences in protein expression among isolates. An in-depth analysis was then performed by GeLC-MS/MS in order to achieve a higher coverage of the liposoluble proteome. Using this approach, a total of 194 unique proteins were identified, corresponding to 26% of all M. agalactiae PG2T genes. A gene ontology analysis and classification for localization and function was also carried out on all protein identifications. Interestingly, the 11.5% of expressed membrane proteins derived from putative horizontal gene transfer events. Conclusions This study led to the in-depth systematic characterization of the M. agalactiae liposoluble protein component, providing useful insights into its membrane organization

A human proteomic dataset from untreated and depleted/enriched serum samples

We present a proteomic dataset generated from a human serum sample and the enriched/depleted fractions obtained by seven commercial products. This report is related to the research article entitled “Comparative evaluation of seven commercial products for human serum enrichment/depletion by shotgun proteomics” [1]. All samples were analyzed by LC-MS/MS, label free quantitation using the spectral counting approach, and Gene Ontology (GO) annotation. Protein relative abundances and functions were reported

Biological colonization on stone monuments: A new low impact cleaning method

In restoration and conservation practices, biocide treatments are considered one of the most practical approaches to remove biological colonization on artworks, including stone. Numerous studies have focused on the short- and long-term effects of these treatments and recently many alternative methods to reduce their potential hazards to human health and the environment have been proposed. In this study, a solvent gel containing dimethyl sulfoxide (DMSO), already used to clean paintings, was applied on colonized marble artifacts at the monumental cemetery of Bonaria (Cagliari - Italy) to remove biological patinas. The protocol efficiency was evaluated by scanning electronic microscopy, rugosimetric and colorimetric measurements and growth tests. A comparative study also was performed to validate the method using biocides currently used in conservation. The results demonstrate that DMSO solvent gel is efficient at removing patinas on stone, of low impact, easy to use, inexpensive and can be considered a more practical alternative to biocide treatments

Ombretta Cocco, Maura Carboni, Laura Solla, Maurizio Serci, Gianfranco Carcangiu, Paola Meloni, Caratterizzazione dei materiali pittorici e delle preparazioni tramite microscopia ottica, spettroscopia infrarossa in trasformata di Fourier e microscopia elettronica a scansione EDS in modalità VP su sezioni microstratigrafiche i

Il volume che documenta l'attività di restauro e delle indagini diagnostiche sul Retablo di Castelsardo, è corredato da un ricco e variegato corredo iconografico che illustra lo stato di conservazione dell'opera sia prima che durante e dopo l'attento restauro al quale l'opera è stata sottoposta. In particolare sono da sottolineare ed evidenziare le fotografie che corredano il saggio dedicato a studiare ed analizzare la caratterizzazione dei materiali pittorici utilizzati. Il Retablo di Castelsardo è uno dei complessi pittorici sardi di maggior rilevanza ed importanza. La pubblicazione del volume intende mettere a completa disposizione del mondo degli studiosi i risultati che sono emersi dal restauro al fine di andare a costituire un ulteriore momento di riflessione per tutti coloro i quali si cimenteranno in futuro con opere policrome appartenenti allo stesso periodo storico e allo stesso ambiente artistico. Il titolo del volume, "Leggere l'invisibile", è stato scelto dai Curatori per indicare chiaramente che quando ci si pone di fronte ad un'opera per analizzarla non bisogna fermarsi ai dati già desumibili con una semplice osservazione di superficie nello spettro del visibile: la fluorescenza a raggi x, la radiografia, la riflettografia all'infrarosso sono oggi divenuti inevitabilmente i nuovi e fondamentali attrezzi del mestiere del restauratore e dello storico dell'arte

Oxalate and oxamate derivatives: novel synthetic strategies in the design of materials for the restoration of marble and biomicrite limestone substrates

Air pollution, and in particular the presence of sulphur and nitrogen oxides, results in acidic rain determining the dissolution of calcite in marble and limestones. This degradation process induces evident roughness of the stone surfaces and the partial loss of carved details in artefacts. The formation of an artificial coating of calcium oxalate, obtained by treatment with ammonium oxalate, was proved to be a very promising technique for the protection of stone items. The functionalization of oxalic acid to give monoesters and monoamides (oxamates) allows tailoring the solubility of the relevant ammonium and calcium salts. In this context, theoretical calculations carried out at Density Functional Theory (DFT) level can help predicting the capability of the investigated compounds in interacting with the calcium carbonate lattice. We summarize here the experimental (X-ray diffraction, Mercury Intrusion Porosimetry, FT-MIR and FT-Raman spectroscopy, SEM) and theoretical (DFT PES and NBO analysis) investigations on the effects induced by the variation of the functionalized anions in oxalate and oxamate salts on the efficiency of the treatment of marble from the Cimitero Monumentale di Bonaria in Cagliari (Italy) and limestone samples from Cava Flore (Santa Caterina di Pittinuri, Oristano, Italy)