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

BACKGROUND: Amyloid 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. OBJECTIVES: Investigate urine protein profiles and diagnostic biomarkers in cats with renal AA amyloidosis. ANIMALS: Twenty-nine shelter cats. METHODS: Case-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. RESULTS: Twenty-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 × 107 ; range, 1.85 × 105 -5.29 × 107 vs 1.76 × 106 ; 0.0 × 100 -1.38 × 107 ; P = .01). In the kidney, AA-amyloidosis was associated with glomerulosclerosis (P = .02) and interstitial fibrosis (P = .05). CONCLUSIONS AND CLINICAL IMPORTANCE: Renal 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.

All Cats are Gray in the Dark: Enrichment/Depletion Approaches for Biomarker Discovery on Felis catus Plasma.

In veterinary medicine, assay performance is often affected by the lack of species-specific diagnostic tools. Reliable biomarkers might be identified by investigating biological fluids of the species of interest, but protein sequence databases are often incomplete and human-specific devices for reducing sample complexity might fail when applied to animal plasma. Here, seven commercial methods based on different capturing agents (anti-human antibodies, affinity ligands, mixture of antibodies and ligands, and combinatorial peptide ligand libraries) are applied to cat plasma and evaluated in terms of yield, identified proteins/ peptides, and relative abundance by high-resolution shotgun proteomics and label-free quantitation. As a result, anti-human antibody-based methods are unsatisfactory. Most fail in reducing albumin and immunoglobulins, and some lead to a substantial removal of other highly abundant proteins, probably because of nonspecific interactions. A protein A/dye ligand-based method is efficient in reducing immunoglobulins, fibrinogen, and apolipoprotein A1 and A2, but not albumin, and protein identifications do not increase. Only peptide ligand libraries flatten the dynamic range, and increased protein identification (59.0%). Albumin and immunoglobulins are successfully depleted (60.7% and 35.9%, respectively). Although further studies will be required for reinforcing our observations, this work can provide a useful guide for cat plasma pretreatment in biomarker discovery studies.

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.

Comparative evaluation of seven commercial products for human serum enrichment/depletion by shotgun proteomics.

Seven commercial products for human serum depletion/enrichment were tested and compared by shotgun proteomics. Methods were based on four different capturing agents: antibodies (Qproteome Albumin/IgG Depletion kit, ProteoPrep Immunoaffinity Albumin and IgG Depletion Kit, Top 2 Abundant Protein Depletion Spin Columns, and Top 12 Abundant Protein Depletion Spin Columns), specific ligands (Albumin/IgG Removal), mixture of antibodies and ligands (Albumin and IgG Depletion SpinTrap), and combinatorial peptide ligand libraries (ProteoMiner beads), respectively. All procedures, to a greater or lesser extent, allowed an increase of identified proteins. ProteoMiner beads provided the highest number of proteins; Albumin and IgG Depletion SpinTrap and ProteoPrep Immunoaffinity Albumin and IgG Depletion Kit resulted the most efficient in albumin removal; Top 2 and Top 12 Abundant Protein Depletion Spin Columns decreased the overall immunoglobulin levels more than other procedures, whereas specifically gamma immunoglobulins were mostly removed by Albumin and IgG Depletion SpinTrap, ProteoPrep Immunoaffinity Albumin and IgG Depletion Kit, and Top 2 Abundant Protein Depletion Spin Columns. Albumin/IgG Removal, a resin bound to a mixture of protein A and Cibacron Blue, behaved less efficiently than the other products.

Zika Virus spreading in South America: Evolutionary analysis of emerging neutralizing resistant Phe279Ser strains.

OBJECTIVE: To investigate the genetic diversity of Zika Virus (ZIKV) and the relationships existing among these circulating viruses worldwide. To evaluate the genetic polymorphisms harbored from ZIKV that can have an influence on the virus circulation. METHODS: Three different ZIKV dataset were built. The first dataset included 63 E gene sequences, the second one 22 NS3 sequences and the third dataset was composed of 108 NS5 gene sequences. Phylogenetic and selective pressure analysis was performed. The edited nucleic acid alignment from the Envelope dataset was used to generate a conceptual translation to the corresponding peptide sequences through UGene software. RESULTS: The phylogeographic reconstruction was able to discriminate unambiguously that the Brazilian strains are belonged to the Asian lineage. The structural analysis reveals instead the presence of the Ser residue in the Brazilian sequences (however already observed in other previously reported ZIKV infections) that could suggest the presence of a neutralization-resistant population of viruses. CONCLUSIONS: Phylogenetic, evolutionary and selective pressure analysis contributed to improve the knowledge on the circulation of ZIKV.

Global microRNA profiling of peripheral blood mononuclear cells in patients with Behçet's disease.

OBJECTIVES: To explore the post-transcriptional regulation of the peripheral blood mononuclear cells (PBMCs) transcriptome by microRNAs in Behçet's disease (BD). METHODS: Using TaqMan Low Density Array-based microRNAs expression profiling, the expression of 750 mature human microRNAs in PBMCs from 5 BD patients and 3 healthy controls (HC) was compared. The expression of deregulated microRNAs was then validated by quantitative real time-polymerase chain reaction (qRT-PCR), in 42 BD patients and 8 HC. RESULTS: In the initial screening, 13 microRNAs appeared deregulated in BD vs HC. Among them, the differential expression of miR-720 and miR-139-3p was confirmed by qRT-PCR, (p<0.05 and FDR<5%). Areas under the receiver operating characteristic curve for miR-139-3p, miR-720 and miR-139-3p+miR-720 in the validation cohort were 0.84, 0.87 and 0.92 respectively, indicating good discrimination between BD patients and HC. Post-hoc analysis showed that 9 out of 13 microRNAs from the discovery phase were significantly upregulated in active vs. quiescent BD, suggesting inflammation as a key regulator of microRNAs machinery in BD. In silico analysis revealed that several BD candidate susceptibility genes are predicted target of significantly deregulated microRNAs in active BD. A significant enrichment in microRNAs targeting elements of the Toll-like receptor (TLR) and T-cell receptor signalling pathways was also assumed. CONCLUSIONS: miR199-3p and miR720 deserve further confirmation as biomarkers of BD in larger studies. PBMCs from active BD displayed a unique signature of microRNAs which may be implicated in regulation of innate immunity activation and T-cell function.

Mycoplasma agalactiae 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 45°C. 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.

Ovis aries Papillomavirus 3: a prototype of a novel genus in the family Papillomaviridae associated with ovine squamous cell carcinoma.

Papillomaviruses play an important role in human cancer development, and have been isolated from a number of animal malignancies. However, the association of papillomaviruses with tumors has been poorly investigated in sheep. In this study, a novel ovine Papillomavirus, OaPV3, was cloned from sheep squamous cell carcinoma. Unlike the already known ovine papillomaviruses, belonging to the Delta genus, OaPV3 lacks the E5 open reading frame and maintains the conserved retinoblastoma motif in the E7 gene. OaPV3 infects exclusively epithelial cells, and was found in skin of healthy sheep of geographically separated flocks located in Sardinia (Italy). This new virus is transcriptionally active in tumors and shares low homology with all the other papillomaviruses, establishing a new genus. Taken together, the co-occurrence of OaPV3 and tumors, its cell and tissue tropism, and its gene repertoire, suggests a role for this virus in development of sheep squamous cell carcinoma.

The liposoluble proteome of Mycoplasma agalactiae: 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.