Antimicrobial activity of prophage endolysins against critical Enterobacteriaceae antibiotic-resistant bacteria.

Enterobacteriaceae species are part of the 2017 World Health Organization antibiotic-resistant priority pathogens list for development of novel medicines. Multidrug-resistant Klebsiella pneumoniae is an increasing threat to public health and has become a relevant human pathogen involved in life-threatening infections. Phage therapy involves the use of phages or their lytic endolysins as bioagents for the treatment of bacterial infectious diseases. Gram-negative bacteria have an outer membrane, making difficult the access of endolysins to the peptidoglycan. Here, three endolysins from prophages infecting three distinct Enterobacterales species, Kp2948-Lys from K. pneumoniae, Ps3418-Lys from Providencia stuartii, and Kaer26608-Lys from Klebsiella aerogenes, were purified and exhibited antibacterial activity against their specific bacterium species verified by zymogram assays. These three endolysins were successfully associated to liposomes composed of dimyristoyl phosphatidyl choline (DMPC), dioleoyl phosphatidyl ethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) at a molar ratio (4:4:2), with an encapsulation efficiency ranging from 24 to 27%. Endolysins encapsulated in liposomes resulted in higher antibacterial activity compared to the respective endolysin in the free form, suggesting that the liposome-mediated delivery system enhances fusion with outer membrane and delivery of endolysins to the target peptidoglycan. Obtained results suggest that Kp2948-Lys appears to be specific for K. pneumoniae, while Ps3418-Lys and Kaer26608-Lys appear to have a broader antibacterial spectrum. Endolysins incorporated in liposomes constitute a promising weapon, applicable in the several dimensions (human, animals and environment) of the One Health approach, against multidrug-resistant Enterobacteriaceae.

Comparison of GFR measurement with a two-blood sample technique using [99mTc]Tc-DTPA vs. creatinine-based equations in potential kidney donors / Comparação da medição da TFG com uma técnica com duas amostras de sangue usando [99mTc]Tc-DTPA vs. equações baseadas em creatinina em potenciais doadores de rim

ABSTRACT Introduction: Accurate determination of glomerular filtration rate (GFR) is crucial for selection of kidney donors. Nuclear medicine methods are considered accurate in measuring GFR but are not always easily available. The four-variable Modification of Diet in Renal Disease (MDRD4), Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI), and Full Age Spectrum (FAS) formulas are common equations for estimating GFR and are recommended for initial assessment of kidney donors. The aim of this study was to evaluate the performance of these GFR estimation equations compared with technetium-99m diethylenetriaminepentaacetic acid ([99mTc]Tc-DTPA) clearance. Methods: We compared GFR estimation by [99mTc]Tc-DTPA clearance using a two-blood sample method with estimation by MDRD4, CKD-EPI, and FAS creatinine-based equations in a population of healthy potential kidney donors. Results: A total of 195 potential kidney donors (68.2% female; mean age 49 years, range 21-75 years) were included in this study. Mean [99mTc]Tc-DTPA measured GFR (mGFR) was 101.5 ± 19.1 mL/min/1.73 m2. All three equations underestimated the GFR value measured by [99mTc]Tc-DTPA (MDRD4: -11.5 ± 18.8 mL/min/1.73 m2; CKD-EPI: -5.0 ± 17.4 mL/min/1.73 m2; FAS: -8.3 ± 17.4 mL/min/1.73 m2). Accuracy within 30% and 10% of the measured GFR value was highest for CKD-EPI. Conclusion: The CKD-EPI equation showed better performance in estimating GFR in healthy potential kidney donors, proving to be a more accurate tool in the initial assessment of kidney donors. However, creatinine-based equations tended to underestimate kidney function. Therefore, GFR should be confirmed by another method in potential kidney donors.

RESUMO Introdução: Determinar precisamente a taxa de filtração glomerular (TFG) é crucial para seleção de doadores de rim. Métodos de medicina nuclear são considerados precisos na medição da TFG, mas nem sempre estão facilmente disponíveis. As fórmulas Modification of Diet in Renal Disease de 4 variáveis (MDRD4), Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI), e Full Age Spectrum (FAS) são equações comuns para estimar a TFG, sendo recomendadas para avaliação inicial dos doadores. Este estudo visou avaliar o desempenho destas equações de estimativa da TFG em comparação com o clearance do tecnécio-99m-ácido dietilenotriaminopentacético ([99mTc]Tc-DTPA). Métodos: Comparamos a TFG por clearance de [99mTc]Tc-DTPA usando um método com duas amostras de sangue com estimativa da TFG pelas equações MDRD4, CKD-EPI e FAS baseadas em creatinina em uma população de potenciais doadores saudáveis. Resultados: Incluiu-se 195 potenciais doadores de rim (68,2% mulheres; idade média de 49 anos, intervalo 21-75 anos). A TFG média medida por [99mTc]Tc-DTPA foi 101,5 ± 19,1 mL/min/1,73m2. As três equações subestimaram o valor da TFG medida por [99mTc]Tc-DTPA (MDRD4: -11,5 ± 18,8 mL/min/1,73 m2; CKD-EPI: -5,0 ± 17,4 mL/min/1,73 m2; FAS: -8,3 ± 17,4 mL/min/1,73 m2). A precisão dentro de 30% e 10% do valor da TFG medida foi maior para CKD-EPI. Conclusão: A equação CKD-EPI mostrou melhor desempenho na estimativa da TFG em potenciais doadores de rim saudáveis, revelando-se uma ferramenta mais precisa na avaliação inicial dos doadores. Entretanto, equações baseadas em creatinina tendem a subestimar a função renal. Portanto, a TFG deve ser confirmada por outro método em potenciais doadores.

Comparison of GFR measurement with a two-blood sample technique using [99mTc]Tc-DTPA vs. creatinine-based equations in potential kidney donors.

INTRODUCTION: Accurate determination of glomerular filtration rate (GFR) is crucial for selection of kidney donors. Nuclear medicine methods are considered accurate in measuring GFR but are not always easily available. The four-variable Modification of Diet in Renal Disease (MDRD4), Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI), and Full Age Spectrum (FAS) formulas are common equations for estimating GFR and are recommended for initial assessment of kidney donors. The aim of this study was to evaluate the performance of these GFR estimation equations compared with technetium-99m diethylenetriaminepentaacetic acid ([99mTc]Tc-DTPA) clearance. METHODS: We compared GFR estimation by [99mTc]Tc-DTPA clearance using a two-blood sample method with estimation by MDRD4, CKD-EPI, and FAS creatinine-based equations in a population of healthy potential kidney donors. RESULTS: A total of 195 potential kidney donors (68.2% female; mean age 49 years, range 21-75 years) were included in this study. Mean [99mTc]Tc-DTPA measured GFR (mGFR) was 101.5 ± 19.1 mL/min/1.73 m2. All three equations underestimated the GFR value measured by [99mTc]Tc-DTPA (MDRD4: -11.5 ± 18.8 mL/min/1.73 m2; CKD-EPI: -5.0 ± 17.4 mL/min/1.73 m2; FAS: -8.3 ± 17.4 mL/min/1.73 m2). Accuracy within 30% and 10% of the measured GFR value was highest for CKD-EPI. CONCLUSION: The CKD-EPI equation showed better performance in estimating GFR in healthy potential kidney donors, proving to be a more accurate tool in the initial assessment of kidney donors. However, creatinine-based equations tended to underestimate kidney function. Therefore, GFR should be confirmed by another method in potential kidney donors.

Liposomal Delivery of Newly Identified Prophage Lysins in a Pseudomonas aeruginosa Model.

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium that presents resistance to several antibiotics, thus, representing a major threat to human and animal health. Phage-derived products, namely lysins, or peptidoglycan-hydrolyzing enzymes, can be an effective weapon against antibiotic-resistant bacteria. Whereas in Gram-positive bacteria, lysis from without is facilitated by the exposed peptidoglycan layer, this is not possible in the outer membrane-protected peptidoglycan of Gram-negative bacteria. Here, we suggest the encapsulation of lysins in liposomes as a delivery system against Gram-negative bacteria, using the model of P. aeruginosa. Bioinformatic analysis allowed for the identification of 38 distinct complete prophages within 66 P. aeruginosa genomes (16 of which newly sequenced) and led to the identification of 19 lysins of diverse sequence and function, 5 of which proceeded to wet lab analysis. The four purifiable lysins showed hydrolytic activity against Gram-positive bacterial lawns and, on zymogram assays, constituted of autoclaved P. aeruginosa cells. Additionally, lysins Pa7 and Pa119 combined with an outer membrane permeabilizer showed activity against P. aeruginosa cells. These two lysins were successfully encapsulated in DPPC:DOPE:CHEMS (molar ratio 4:4:2) liposomes with an average encapsulation efficiency of 33.33% and 32.30%, respectively. The application of the encapsulated lysins to the model P. aeruginosa led to a reduction in cell viability and resulted in cell lysis as observed in MTT cell viability assays and electron microscopy. In sum, we report here that prophages may be important sources of new enzybiotics, with prophage lysins showing high diversity and activity. In addition, these enzybiotics following their incorporation in liposomes were able to potentiate their antibacterial effect against the Gram-negative bacteria P. aeruginosa, used as the model.

Cryptic Prophages Contribution for Campylobacter jejuni and Campylobacter coli Introgression.

Campylobacter coli and C. jejuni, the causing agents of campylobacteriosis, are described to be undergoing introgression events, i.e., the transference of genetic material between different species, with some isolates sharing almost a quarter of its genome. The participation of phages in introgression events and consequent impact on host ecology and evolution remain elusive. Three distinct prophages, named C. jejuni integrated elements 1, 2, and 4 (CJIE1, CJIE2, and CJIE4), are described in C. jejuni. Here, we identified two unreported prophages, Campylobacter coli integrated elements 1 and 2 (CCIE1 and CCIE2 prophages), which are C. coli homologues of CJIE1 and CJIE2, respectively. No induction was achieved for both prophages. Conversely, induction assays on CJIE1 and CJIE2 point towards the inducibility of these prophages. CCIE2-, CJIE1-, and CJIE4-like prophages were identified in a Campylobacter spp. population of 840 genomes, and phylogenetic analysis revealed clustering in three major groups: CJIE1-CCIE1, CJIE2-CCIE2, and CJIE4, clearly segregating prophages from C. jejuni and C. coli, but not from human- and nonhuman-derived isolates, corroborating the flowing between animals and humans in the agricultural context. Punctual bacteriophage host-jumps were observed in the context of C. jejuni and C. coli, and although random chance cannot be fully discarded, these observations seem to implicate prophages in evolutionary introgression events that are modulating the hybridization of C. jejuni and C. coli species.

Genomic Analysis of Prophages from Klebsiella pneumoniae Clinical Isolates.

Klebsiella pneumoniae is an increasing threat to public health and represents one of the most concerning pathogens involved in life-threatening infections. The resistant and virulence determinants are coded by mobile genetic elements which can easily spread between bacteria populations and co-evolve with its genomic host. In this study, we present the full genomic sequences, insertion sites and phylogenetic analysis of 150 prophages found in 40 K. pneumoniae clinical isolates obtained from an outbreak in a Portuguese hospital. All strains harbored at least one prophage and we identified 104 intact prophages (69.3%). The prophage size ranges from 29.7 to 50.6 kbp, coding between 32 and 78 putative genes. The prophage GC content is 51.2%, lower than the average GC content of 57.1% in K. pneumoniae. Complete prophages were classified into three families in the order Caudolovirales: Myoviridae (59.6%), Siphoviridae (38.5%) and Podoviridae (1.9%). In addition, an alignment and phylogenetic analysis revealed nine distinct clusters. Evidence of recombination was detected within the genome of some prophages but, in most cases, proteins involved in viral structure, transcription, replication and regulation (lysogenic/lysis) were maintained. These results support the knowledge that prophages are diverse and widely disseminated in K. pneumoniae genomes, contributing to the evolution of this species and conferring additional phenotypes. Moreover, we identified K. pneumoniae prophages in a set of endolysin genes, which were found to code for proteins with lysozyme activity, cleaving the ß-1,4 linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in the peptidoglycan network and thus representing genes with the potential for lysin phage therapy.

Origin, phylogeny, variability and epitope conservation of SARS-CoV-2 worldwide.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses innumerous challenges, like understanding what triggered the emergence of this new human virus, how this RNA virus is evolving or how the variability of viral genome may impact the primary structure of proteins that are targets for vaccine. We analyzed 19471 SARS-CoV-2 genomes available at the GISAID database from all over the world and 3335 genomes of other Coronoviridae family members available at GenBank, collecting SARS-CoV-2 high-quality genomes and distinct Coronoviridae family genomes. Additionally, we analyzed 199,984 spike glycoprotein sequences. Here, we identify a SARS-CoV-2 emerging cluster containing 13 closely related genomes isolated from bat and pangolin that showed evidence of recombination, which may have contributed to the emergence of SARS-CoV-2. The analyzed SARS-CoV-2 genomes presented 9632 single nucleotide variants (SNVs) corresponding to a variant density of 0.3 over the genome, and a clear geographic distribution. SNVs are unevenly distributed throughout the genome and hotspots for mutations were found for the spike gene and ORF 1ab. We describe a set of predicted spike protein epitopes whose variability is negligible. Additionally, all predicted epitopes for the structural E, M and N proteins are highly conserved. The amino acid changes present in the spike glycoprotein of variables of concern (VOCs) comprise between 3.4% and 20.7% of the predicted epitopes of this protein. These results favors the continuous efficacy of the available vaccines targeting the spike protein, and other structural proteins. Multiple epitopes vaccines should sustain vaccine efficacy since at least some of the epitopes present in variability regions of VOCs are conserved and thus recognizable by antibodies.

Warthin Tumor Incidentally Detected on PET/CT Showing Both 68Ga-DOTANOC and 18F-FDG Uptake.

ABSTRACT: A patient with moderately differentiated pancreatic neuroendocrine tumor with synchronous multifocal liver metastases was referred for further staging with PET/CT. The examinations were performed on 2 consecutive days and showed mild 68Ga-DOTANOC and intense 18F-FDG uptake in an incidental right parotid nodule. Differential diagnoses include primary or metastatic neuroendocrine tumor, malignant or benign primary parotid tumor, and intraparotid lymph node. Histology revealed characteristics of a Warthin tumor. While focal FDG uptake in Warthin tumor is frequently described, the somatostatin expression was rarely reported. This clinical case describes 68Ga-DOTANOC and 18F-FDG uptake in a parotid Warthin tumor histologically confirmed.

Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent fluorescence edge-shift.

Among the major challenges in the development of biopharmaceuticals are structural heterogeneity and aggregation. The development of a successful therapeutic monoclonal antibody (mAb) requires both a highly active and also stable molecule. Whilst a range of experimental (biophysical) approaches exist to track changes in stability of proteins, routine prediction of stability remains challenging. The fluorescence red edge excitation shift (REES) phenomenon is sensitive to a range of changes in protein structure. Based on recent work, we have found that quantifying the REES effect is extremely sensitive to changes in protein conformational state and dynamics. Given the extreme sensitivity, potentially this tool could provide a 'fingerprint' of the structure and stability of a protein. Such a tool would be useful in the discovery and development of biopharamceuticals and so we have explored our hypothesis with a panel of therapeutic mAbs. We demonstrate that the quantified REES data show remarkable sensitivity, being able to discern between structurally identical antibodies and showing sensitivity to unfolding and aggregation. The approach works across a broad concentration range (µg-mg/ml) and is highly consistent. We show that the approach can be applied alongside traditional characterisation testing within the context of a forced degradation study (FDS). Most importantly, we demonstrate the approach is able to predict the stability of mAbs both in the short (hours), medium (days) and long-term (months). The quantified REES data will find immediate use in the biopharmaceutical industry in quality assurance, formulation and development. The approach benefits from low technical complexity, is rapid and uses instrumentation which exists in most biochemistry laboratories without modification.

Trends in Helicobacter pylori resistance to clarithromycin: from phenotypic to genomic approaches.

For a long time Helicobacter pylori infections have been treated using the macrolide antibiotic, clarithromycin. Clarithromycin resistance is increasing worldwide and is the most common cause of H. pylori treatment failure. Here we review the mechanisms of antibiotic resistance to clarithromycin, detailing the individual and combinations of point mutations found in the 23S rRNA gene associated with resistance. Additionally, we consider the methods used to detect clarithromycin resistance, emphasizing the use of high-throughput next-generation sequencing methods, which were applied to 17 newly sequenced pairs of H. pylori strains isolated from the antrum and corpus of a recent colonized paediatric population. This set of isolates was composed of six pairs of resistant strains whose phenotype was associated with two point mutations found in the 23S rRNA gene: A2142C and A2143G. Other point mutations were found simultaneously in the same gene, but, according to our results, it is unlikely that they contribute to resistance. Further, among susceptible isolates, genomic variations compatible with mutations previously associated with clarithromycin resistance were detected. Exposure to clarithromycin may select low-frequency variants, resulting in a progressive increase in the resistance rate due to selection pressure.

Changes in the intestinal mucosal proteome of turkeys (Meleagris gallopavo) infected with haemorrhagic enteritis virus.

Haemorrhagic enteritis (HE) is a viral disease affecting intestinal integrity and barrier function in turkey (Meleagris gallopavo) and resulting in a significant economic loss. Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH-MS) was applied to identify crucial proteins involved in HE infection. A total of 938 proteins were identified and used to generate a reference library for SWATH-MS analysis. In total, 523 proteins were reliably quantified, and 64 proteins were found to be differentially expressed, including 49 up-regulated and 15 down-regulated proteins between healthy and HE-affected intestinal mucosa. Functional analysis suggested that these proteins were involved in the following categories of cellular pathways and metabolisms: 1) energy pathways; 2) intestine lipid and amino acid metabolism; 3) oxidative stress; 4) intestinal immune response. Major findings of this study demonstrated that natural HE infection is related to the changes in abundance of several proteins involved in cell-intrinsic immune defense against viral invasion, systemic inflammation, modulation of excessive inflammation, B and T cell development and function and antigen presentation. mRNA quantitative expression demonstrated that most of the proteins involved in innate immunity that were found to be differentially abundant were produced by intestinal mucosa, suggesting its direct involvement in immune defences against HE infection.

[Medically Assisted Reproduction in Natural Cycle: Outcome Evaluation of a Reproductive Medicine Department]. / Procriação Medicamente Assistida em Ciclo Natural: Avaliação dos Resultados de um Departamento de Medicina da Reprodução.

INTRODUCTION: Medically assisted reproduction in natural cycle has been investigated, especially in women with poor response to conventional ovarian stimulation, with endometrial receptivity improvement, lower cost and possibility of successive cycles. The disadvantages are: lower profitability per treatment cycle and higher cancellation rate. The aim of this study was to determine the rate of clinical pregnancy in infertile women subjected to medically assisted reproduction in natural cycle. MATERIAL AND METHODS: Retrospective study of 149 medically assisted reproduction without ovarian stimulation of 50 infertile women, between January/2011 and October/2014. RESULTS: The mean age of women undergoing medically assisted reproduction in natural cycle was 36.1 years. Approximately half (46.0%) of the cycles were performed in poor responders. On the day of ovulation trigger, the mean diameter of the follicle was 17.5 mm. Twenty-three cycles (15.4%) were canceled prior to ovulation trigger. In 8 cycles (5.3%), ovulation occurred between ovulation trigger and oocyte retrieval. In the majority of cycles (n = 118; 79.2%) oocyte retrieval was executed, a medically assisted reproduction technique was performed in 71 (47.6%), mostly intracytoplasmic injection. The overall fertilization rate was 77.5%. In 40 cycles (26.8%) there was embryo transfer. The implantation rate and the clinical pregnancy rate by embryo transfer was 35.0% and 25.0%, respectively. Most pregnancies occurred in poor responders, according to Bologna criteria. DISCUSSION: Although the pregnancy rate per cycle started was 6.7%, the rate of clinical pregnancy per embryo transfer is quite satisfactory, being a group of women with unfavorable responses in previous treatments. The relatively high rates of cycle cancellation are mitigated by the greater simplicity and lower cost of these cycles. CONCLUSION: The results obtained in this study demonstrate that Medically Assisted Reproduction in natural cycle may be an alternative treatment for ovarian stimulation in patients with poor prognosis, whose only alternative would be oocyte donation.

Introdução: As técnicas de procriação medicamente assistida em ciclo natural têm sido investigadas, sobretudo em mulheres com má resposta à estimulação ovárica convencional, observando-se melhor recetividade endometrial, custo inferior e possibilidade de realização de ciclos sucessivos. Como desvantagens salientam-se: menor eficácia por ciclo de tratamento e maior taxa de cancelamento. O objetivo definido para este trabalho foi determinar a taxa de gravidez evolutiva em mulheres inférteis, submetidas a procriação medicamente assistida em ciclo natural. Material e Métodos: Estudo retrospetivo de 149 ciclos de procriação medicamente assistida sem estimulação ovárica de 50 mulheres inférteis, entre janeiro de 2011 e outubro de 2014. Resultados: As mulheres submetidas a procriação medicamente assistida em ciclo natural tinham, em média, 36,1 anos. Aproximadamente metade (46,0%) dos ciclos realizaram-se em más respondedoras. No dia do desencadeamento da ovulação o diâmetro médio do folículo foi 17,5 mm. Cancelaram-se 23 ciclos (15,4%) previamente ao desencadeamento. Em 8 ciclos (5,3%) ocorreu ovulação entre o desencadeamento e a punção folicular. Na maioria dos ciclos (n = 118; 79,2%) efetuou-se punção folicular, realizando-se técnica de procriação medicamente assistida em 71 (47,6%), maioritariamente injeção intracitoplasmática. A taxa de fecundação global foi 63,8%. Em 40 ciclos (26,8%) houve transferência embrionária. A taxa de implantação e de gravidez evolutiva por transferência embrionária foram de 35,0% e 25,0%, respetivamente. A maioria das gestações ocorreu em más respondedoras, conforme critérios de Bolonha. Discussão: Apesar de a taxa de gravidez por ciclo iniciado ser de 6,7%, a taxa de gravidez evolutiva por transferência embrionária é bastante satisfatória, sendo mulheres com respostas desfavoráveis em tratamentos prévios. As taxas relativamente elevadas de cancelamento do ciclo são atenuadas pela simplicidade e menor custo destes ciclos. Conclusão: Os resultados obtidos neste trabalho demonstram que as técnicas de procriação medicamente assistida em ciclo natural podem ser uma alternativa de tratamento à estimulação ovárica em doentes com mau prognóstico, cuja alternativa seria o recurso à doação de ovócitos.

Saturated or unsaturated fat supplemented maternal diets influence omental adipose tissue proteome of suckling goat-kids.

The aim of the present study was to investigate how maternal diet can influence the adipose tissue of goat kids. Omental adipose tissue proteomes of goat-kids from mothers fed with diet enriched with stearic acid (ST-kids), fish oil (FO-kids) and standard diets (CTRL) were determined by quantitative iTRAQ 2D-LC-MS/MS analysis. Twenty proteins were found to be differentially expressed in suckling kids' omental adipose tissue. Stearic acid induces changes in a higher number of proteins when compared to fish oil. Eleven proteins, namely AARS, ECl1, PMSC2, CP, HSPA8, GPD1, RPL7, OGDH, RPL24, FGA and RPL5 were decreased in ST-kids only. Four proteins, namely DLST, EEF1G, BCAP31 and RALA were decreased in FO-kids only, and one, NUCKS1, was increased. Four proteins, namely PMSC1, PPIB, TUB5×2 and EIF5A1, were be less abundant in both ST- and FO- kids. Most of the protein whose abundance was decreased in ST kids (10 out of 15) are involved in protein metabolism and catabolism pathways. Qualitative gene expression analysis confirmed that all the proteins identified by mass spectrometry, with the exception of FGA, were produced by adipose tissue. Quantitative gene expression analysis demonstrated that two proteins, namely CP, a minor acute phase protein, and ECl1, involved in fatty acid beta oxidation, were downregulated at mRNA level as well. ECl1 gene expression was downregulated in ST-kids AT as compared to Ctrl-kids and CP was downregulated in both ST- and FO-kids. The present results demonstrate that it is possible to influence adipose goat-kid proteome by modifying the maternal diet.

Immunohistochemical Expression of FXR1 in Canine Normal Tissues and Melanomas.

Fragile X mental retardation-related protein 1 (FXR1) is a cytoplasmic RNA-binding protein highly conserved among vertebrates. It has been studied for its role in muscle development, inflammation, and tumorigenesis, being related, for example, to metastasizing behavior in human and canine uveal melanoma. Anti-FXR1 antibodies have never been validated in the canine species. To investigate FXR1 expression in canine melanocytic tumors, the present study tested two commercially available polyclonal anti-human FXR1 antibodies, raised in goat and rabbit, respectively. The cross-reactivity of the anti-FXR1 antibodies was assessed by Western blot analysis, and the protein was localized by IHC in a set of normal canine tissues and in canine melanocytic tumors (10 uveal and 10 oral). Western blot results demonstrated that the antibody raised in rabbit specifically recognized the canine FXR1, while the antibody raised in goat did not cross-react with this canine protein. FXR1 protein was immunodetected using rabbit anti-FXR1 antibody, in canine normal tissues with different levels of intensity and distribution. It was also detected in 10/10 uveal and 9/10 oral melanocytic tumors. The present study validated for the first time the use of anti-FXR1 antibody in dogs and highlighted different FXR1 protein expression in canine melanocytic tumors, the significance of which is undergoing further investigations.

Widespread extrahepatic expression of acute-phase proteins in healthy chicken (Gallus gallus) tissues.

Acute phase proteins (APP) are plasma proteins that can modify their expression in response to inflammation caused by tissue injury, infections, immunological disorders or stress. Although APP are produced mainly in liver, extrahepatic production has also been described. As a prerequisite to get insight the expression of APP in chicken during diseases, this study investigated the presence of five APP, including alpha1-acid glycoprotein (AGP), Serum Amyloid A (SAA), PIT54, C-Reactive protein (CRP) and Ovotransferrin (OVT) in twenty tissues collected from healthy chicken (Gallus gallus) by quantitative Real Time PCR and immunohistochemistry. As expected, APP gene abundance was higher in liver compared with other tissues. The mRNA coding for CRP, OVT and SAA was detected in all analyzed tissues with a higher expression in gastrointestinal tract, respiratory and lymphatic samples. SAA expression was particularly high in cecal tonsil, lung, spleen and Meckel's diverticulum, whereas OVT in lung, bursa of Fabricius and pancreas. AGP and PIT54 mRNA expression were detected in all tissues but at negligible levels. Immunohistochemical expression of AGP and OVT was variably detected in different organs, being identified in endothelium of every tissue. Positive cells were present in the epithelium of the mucosal layer of gastrointestinal tract and kidney. Lung and central nervous system stained for both proteins. No positive staining was detected in lymphoid tissues and muscle. These results suggest that most tissues can express different amount of APP even in healthy conditions and are therefore capable to mount a local acute phase reaction.

Allergy to grass pollen: mapping of Dactylis glomerata and Phleum pratense allergens for dogs by two-dimensional immunoblotting.

INTRODUCTION: Much less is known about grass-pollen allergens to dogs, when compared with humans. Genetic-based patterns might play an important role in sensitization profiles, conditioning the success of allergen-specific immunotherapy. AIM: Mapping of Dactylis glomerata (D. glomerata) and Phleum pratense (P. pratense) allergens for grass pollen-sensitized atopic dogs, for better understanding how individual allergograms may influence the response to grass-pollen immunotherapy. MATERIAL AND METHODS: To identify D. glomerata and P. pratense allergoms for dogs, 15 individuals allergic to grass pollen and sensitized to D. glomerata and P. pratense were selected. D. glomerata and P. pratense proteomes were separated by isoelectric focusing (IEF), one-dimensional (1-D) and two-dimensional (2-D) sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were blotted onto Polyvinylidene difluoride (PVDF) membranes and allergens were identified by patient sera IgE in Western Blotting (WB). RESULTS: In D. glomerata, 17 allergens were identified from IEF and 11 from 1-D SDS-PAGE, while from P. pratense, 18 and 6 allergens were identified, respectively. From 2-D SDS-PAGE 13 spots were identified from D. glomerata and 27 from P. pratense. CONCLUSIONS: Several similarities were found between dog and human D. glomerata and P. pratense sensitization profiles but no relationship between clinical signs and a specific pattern of allergen recognition was observed. Similarities were found in each patient pattern of sensitization between D. glomerata and P. pratense, also suggesting cross-reactive phenomena. Further molecular epidemiology approach is needed to understand the role of the sensitization pattern in allergen-specific immunotherapy effectiveness in grass-pollen allergic dogs.

Identification of a Bitter-Taste Receptor Gene Repertoire in Different Lagomorphs Species.

The repertoires of bitter-taste receptor (T2R) gene have been described for several animal species, but these data are still scarce for Lagomorphs. The aim of the present work is to identify potential repertoires of T2R in several Lagomorph species, covering a wide geographical distribution. We studied these genes in Lepus timidus, L. europaeus, Oryctolagus cuniculus algirus, Romerolagus diazi, and Sylvilagus floridanus, using O. cuniculus cuniculus as control species for PCR and DNA sequencing. We studied the identities of the DNA sequences and built the corresponding phylogenetic tree. Sequencing was successful for both subspecies of O. cuniculus for all T2R genes studied, for five genes in Lepus, and for three genes in R. diazi and S. floridanus. We describe for the first time the partial repertoires of T2R genes for Lagomorphs species, other than the common rabbit. Our phylogenetic analyses indicate that sequence proximity levels follow the established taxonomic classification.

The effect of transport stress on turkey (Meleagris gallopavo) liver acute phase proteins gene expression.

The aim of this study was to investigate the effects of transport-related stress on the liver gene expression of four acute phase proteins (APP), namely α1-acid glycoprotein (AGP), C-Reactive Protein (CRP), Serum Amyloid A (SAA) and PIT54, in turkeys (Meleagris gallopavo). A group of seven BUT BIG 6 commercial hens was subjected to a two-hour long road transportation and the quantitative gene expression of APP in the liver was compared to that of a non transported control group. The expression of AGP and CRP mRNA was found to be increased in animals slaughtered after road transport. The presence of AGP protein was also confirmed by immunohistochemistry and Western blotting. The results of this study showed that road-transport may induce the mRNA expression of immune related proteins. The finding that AGP and CRP can be upregulated during transport could suggest their use as for the assessment of turkey welfare during transport.

The localization and differential expression of Serum Amyloid A in bovine liver and adipose tissue depots.

In this article the localization of the acute phase protein Serum Amyloid A (SAA) in different depots of bovine adipose tissue (AT) and liver is reported. Quantitative (Real Time) PCR was paired to immunohistochemistry after the production of a specific polyclonal antibody. SAA's mRNA was found in all analyzed AT depots included in the present study, the AT located in the withers being the major source of SAA mRNA. A polyclonal antibody was raised against bovine SAA and was used to validate gene expression analyses. Western Blotting confirmed that SAA is present in all the seven adipose tissue depots include in the present experiment. Anti-SAA polyclonal antibody also stained diffusely adipocytes. In liver, intracytoplasmic immunolabeling was observed in hepatocytes. Staining was generally mild and not diffuse: negative hepatocytes were intermixed with positive ones. A positive intracytoplasmic immunostaining was occasionally observed in endothelial cells lining small blood vessels within AT septa and liver parenchyma. Our data confirm that bovine AT may provide an important source of SAA in healthy subjects. It remains to be determined which is the contribution of AT in the serum concentration of SAA.