Probiotic supplementation containing Bacillus velezensis enhances expression of immune regulatory genes against pigeon circovirus in pigeons (Columba livia).

AIMS: In this study, we aimed to isolate and evaluate the efficacy of Bacillus velezensis as a probiotic and to assess its activity towards pigeons infected with pigeon circovirus (PiCV). METHODS AND RESULTS: Bacillus velezensis, isolated from pigeon faeces, was orally administered to pigeons for 60 days. After pigeons were challenged with PiCV, the PiCV viral load and expression of indicator genes for innate immunity were detected in spleen tissue and faeces of pigeons. Bacillus velezensis significantly reduced the PiCV viral load in the faeces and spleen of pigeons 5 days post-challenge (dpc). The mRNA expression levels of treated pigeons showed that interferon-gamma (IFN-γ), myxovirus resistance 1 (Mx1), and signal transducers and activators of transcription 1 (STAT1) genes were upregulated, whereas no expression of interleukin-4 (IL-4) was detected. Moreover, toll-like receptor 2 (TLR2) and 4 (TLR4) were significantly upregulated in probiotic-treated pigeons (P < 0·05). CONCLUSIONS: This is the first report showing that probiotic supplementation can effectively enhance the T-helper type 1 immune response and decrease the PiCV viral loads in pigeons. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proposes that the administration of a probiotic strain, B. velezensis, to pigeons can protect against PiCV infection.

Proteomic changes driven by urban pollution suggest particulate matter as a deregulator of energy metabolism, mitochondrial activity, and oxidative pathways in the rat brain.

The adverse effects of air pollution have been long studied in the lung and respiratory systems, but the molecular changes that this causes at the central nervous system level have yet to be fully investigated and understood. To explore the evolution with time of protein expression levels in the brain of rats exposed to particulate matter of different sizes, we carried out two-dimensional gel electrophoresis followed by determination of dysregulated proteins through Coomassie blue staining-based densities (SameSpots software) and subsequent protein identification using MALDI-based mass spectrometry. Expression differences in dysregulated proteins were found to be statistically significant with p-value <0.05. A systems biology-based approach was utilized to determine critical biochemical pathways involved in the rats' brain response. Our results suggest that rats' brains have a particulate matter size dependent-response, being the mitochondrial activity and the astrocyte function severely affected. Our proteomic study confirms the dysregulation of different biochemical pathways involving energy metabolism, mitochondrial activity, and oxidative pathways as some of the main effects of PM exposure on the rat brain. SIGNIFICANCE: Rat brains exposed to particulate matter with origin in car engines are affected in two main areas: mitochondrial activity, by the dysregulation of many pathways linked to the respiratory chain, and neuronal and astrocytic function, which stimulates brain changes triggering tumorigenesis and neurodegeneration.

Pathway-oriented action of dietary essential oils to prevent muscle protein oxidation and texture deterioration of farmed rainbow trout.

Recently, great attention has been directed towards the use of essential oils from aromatic plants as antimicrobials and antioxidant in food matrix. Fish is well known to be a high perishable food. Indeed, fish muscle is susceptible to suffer protein and lipid oxidation during frozen storage, which can lead to the development of softening and undesirable volatile molecules. However, the possible inclusion of essential oils in fish feed for preserving fish flesh quality during storage is still unclear. For this reason, the potential protective effects of the incorporation of a dietary essential oil constituted by eucalyptol, carvacrol and thymol, to rainbow trout's (Oncorhynchus mykiss) feed were here investigated. Frozen fish fillets resulting from trout fed the essential oil showed a significant protection of specific muscle proteins against the oxidation produced during frozen storage at -10ºC for 6 months. Essential oil-enriched feed decreased carbonylation of specific myofibrillar (α-actinins-1 and -3, myosin heavy chain, myomesin-1, pyruvate kinase, tropomyosin, troponin-T and actin) and sarcoplasmic proteins (glycogen phosphorylase, creatine kinase, fructose-bisphosphate aldolase A and phosphoglycerate mutase 2). Essential oils also increased actin stability and preserved muscle protein solubility and water holding capacity. In addition, essential oils inhibited the onset of lipid oxidation and rancidity, resulting in frozen fish with superior textural quality and sensory scores. As a final conclusion, the inclusion of essential oils in farmed rainbow trout feed is largely efficient for increasing fish quality and shelf life during frozen storage, mainly through a selective-antioxidant effect on muscle proteins.

Mature teratoma of the pineal region in the paediatric age group: A case report and review of the literature.

INTRODUCTION: Intracranial teratomas account for 0.5% of all intracranial tumours and 2-4% of intracranial tumours in children. However, in terms of tumours of the pineal area, the exact incidence is not ascertained. Although, it is noted that 50-60% of central nervous system (CNS) germ cell tumours are found in the pineal gland. The degree of difficulty in the sampling of lesions in the pineal gland during biopsy emphasizes the importance of correlating the imaging studies, histopathologic findings, and serum and cerebrospinal fluid (CSF) tumour markers. CASE REPORT: This case report is that of a 9-year-old male who presented with frontal headache of eight days, with associated photophobia, nausea and vomiting, and diplopia. Biopsy with intraoperative navigation was done and the specimen was referred for histopathologic evaluation. The biopsy showed findings consistent with a mature teratoma with no histologic findings of an immature component or secondary somatic malignancy. Comparison of the pre-operative and post-operative multiaxial cranial CT scan showed findings that was consistent with a residual lesion. This was correlated with the pre-operative serum tumour markers which showed alpha-fetoprotein of 22.5 ng/mL and beta-HCG of 1.0 mIU/mL(IU/L), and the post-operative tumour markers of the cerebrospinal fluid that showed alpha-fetoprotein of 3.28 ng/mL and beta-HCG of 18.9 mIU/mL (IU/L). CONCLUSION: A review of the literature and comparison with current case in relation to the histopathologic, serum and CSF findings, and imaging studies was done to better understand the mechanism of this lesion.

Variation in levels of non-starch polysaccharides and endogenous endo-1,4-ß-xylanases affects the nutritive value of wheat for poultry.

1. Endo-1,4-ß-xylanase is known to improve the nutritive value of wheat-based diets for poultry by degrading dietary arabinoxylans. However, broilers' response to supplementation of wheat-based diets with exogenous endo-1,4-ß-xylanase is not always observed. 2. In this study, 108 different wheat lots were analysed for levels of extract viscosity as well as for endogenous endo-1,4-ß-xylanase activity, and the impact of these two variables in animal performance was tested. 3. Results revealed that endogenous endo-1,4-ß-xylanase activity and extract viscosity content varied widely among different wheat lots. Thus, a trial was conducted to evaluate the efficacy of exogenous enzyme supplementation in broiler diets using wheats with different levels of extract viscosity and endogenous endo-1,4-ß-xylanase activity. 4. The data revealed that exogenous enzyme supplementation was only effective when the wheat present in the diet had high levels of extract viscosity (14.8 cP) with low endogenous endo-1,4-ß-xylanase activity (347.0 U/kg). Nevertheless, it is apparent that exogenous microbial xylanases reduce digesta extract viscosity and feed conversion ratio independently of the endogenous properties presented by different wheat lots. 5. The data suggest that extract viscosity and/or endogenous endo-1,4-ß-xylanase activity affect the response to enzyme supplementation by poultry fed on wheat-based diets.

Dithiothreitol-based protein equalization technology to unravel biomarkers for bladder cancer.

This study aimed to assess the benefits of dithiothreitol (DTT)-based sample treatment for protein equalization to assess potential biomarkers for bladder cancer. The proteome of plasma samples of patients with bladder carcinoma, patients with lower urinary tract symptoms (LUTS) and healthy volunteers, was equalized with dithiothreitol (DTT) and compared. The equalized proteomes were interrogated using two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry. Six proteins, namely serum albumin, gelsolin, fibrinogen gamma chain, Ig alpha-1 chain C region, Ig alpha-2 chain C region and haptoglobin, were found dysregulated in at least 70% of bladder cancer patients when compared with a pool of healthy individuals. One protein, serum albumin, was found overexpressed in 70% of the patients when the equalized proteome of the healthy pool was compared with the equalized proteome of the LUTS patients. The pathways modified by the proteins differentially expressed were analyzed using Cytoscape. The method here presented is fast, cheap, of easy application and it matches the analytical minimalism rules as outlined by Halls. Orthogonal validation was done using western-blot. Overall, DTT-based protein equalization is a promising methodology in bladder cancer research.

A cost-effective method to get insight into the peritoneal dialysate effluent proteome.

Protein depletion with acetonitrile and protein equalization with dithiothreitol have been assessed with success as proteomics tools for getting insight into the peritoneal dialysate effluent proteome. The methods proposed are cost-effective, fast and easy of handling, and they match the criteria of analytical minimalism: low sample volume and low reagent consumption. Using two-dimensional gel electrophoresis and peptide mass fingerprinting, a total of 72 unique proteins were identified. Acetonitrile depletes de PDE proteome from high-abundance proteins, such as albumin, and enriches the sample in apolipo-like proteins. Dithiothreitol equalizes the PDE proteome by diminishing the levels of albumin and enriching the extract in immunoglobulin-like proteins. The annotation per gene ontology term reveals the same biological paths being affected for patients undergoing peritoneal dialysis, namely that the largest number of proteins lost through peritoneal dialysate are extracellular proteins involved in regulation processes through binding. SIGNIFICANCE: Renal failure is a growing problem worldwide, and particularly in Europe where the population is getting older. Up-to-date there is a focus of interest in peritoneal dialysis (PD), as it provides a better quality of life and autonomy of the patients than other renal replacement therapies such as haemodialysis. However, PD can only be used during a short period of years, as the peritoneum lost its permeability through time. Therefore to make a breakthrough in PD and consequently contribute to better healthcare system it is urgent to find a group of biomarkers of peritoneum degradation. Here we report on two cost-effective methods for protein depletion in peritoneal dialysate effluent (PDE). The use of ACN and DTT over PDE to deplete high abundant proteins or to equalize the concentration of proteins, respectively, performs well and with similar protein profiles than when the same chemicals are used in human plasma samples. ACN depletes de PDE proteome from large proteins, such as albumin, and enriches the sample in apolipoproteins. DTT equalizes the PDE proteome by diminishing the levels of large proteins such as albumin and enriching the extract in immunoglobulins. Although the number and type of proteins identified are different, the annotation per gene ontology term reveals the same biological paths being affected for patients undergoing peritoneal dialysate. Thus, the largest number of proteins lost through peritoneal dialysate belongs to the group of extracellular proteins involved in regulation processes through binding. As for the searching of biomarkers, DTT seems to be the most promising of the two methods because acts as an equalizer and it allows interrogating more proteins in the same sample.

A methodological approach based on gold-nanoparticles followed by matrix assisted laser desorption ionization time of flight mass spectrometry for the analysis of urine profiling of knee osteoarthritis.

The aim of this work is to develop a nanoparticle-based methodology to find out biomarkers of diagnostic for knee osteoarthritis, KOA, through the use of matrix assisted laser desorption ionization time-of-flight-based mass spectrometry profiling. Urine samples used for this study were obtained from KOA patients (42 patients), patients with prosthesis (58 patients), and controls (36 individuals) with no history of joint disease. Gold-nano particle MALDI-based urine profiling was optimized and then applied over the 136 individuals. Jaccard index and 10 different classifiers over MALDI MS datasets were used to find out potential biomarkers. Then, the specificity and sensitivity of the method were evaluated. The presence of ten m/z signals as potential biomarkers in the healthy versus non-healthy approach suggests that patients (KOA and prosthesis) are differentiable from the healthy volunteers through profiling. The automatic diagnostic study confirmed these preliminary conclusions. The sensitivity and the specificity for the urine profiling criteria here reported, achieved by the C4.5 classifier, is 97% and 69% respectively. Thus, it is confirmed the utility of the method proposed in this work as an additional fast, non-expensive and robust test for KOA diagnostic. When the proposed method is compared with those used in common practice it is found that sensitivity is the highest, thus with a low false negative rate for diagnostic KOA patients in the population studied. Specificity is lower but in the range accepted for diagnostic objectives.

Mass-Up: an all-in-one open software application for MALDI-TOF mass spectrometry knowledge discovery.

BACKGROUND: Mass spectrometry is one of the most important techniques in the field of proteomics. MALDI-TOF mass spectrometry has become popular during the last decade due to its high speed and sensitivity for detecting proteins and peptides. MALDI-TOF-MS can be also used in combination with Machine Learning techniques and statistical methods for knowledge discovery. Although there are many software libraries and tools that can be combined for these kind of analysis, there is still a need for all-in-one solutions with graphical user-friendly interfaces and avoiding the need of programming skills. RESULTS: Mass-Up, an open software multiplatform application for MALDI-TOF-MS knowledge discovery is herein presented. Mass-Up software allows data preprocessing, as well as subsequent analysis including (i) biomarker discovery, (ii) clustering, (iii) biclustering, (iv) three-dimensional PCA visualization and (v) classification of large sets of spectra data. CONCLUSIONS: Mass-Up brings knowledge discovery within reach of MALDI-TOF-MS researchers. Mass-Up is distributed under license GPLv3 and it is open and free to all users at http://sing.ei.uvigo.es/mass-up.

A journey through PROTEOSONICS.

Ultrasonic energy is gaining momentum in Proteomics. It helps to shorten many proteomics workflows in an easy and efficient manner. Ultrasonic energy is nowadays used for protein extraction, solubilisation and cell disruption, to speed protein identification, protein quantification, peptide profiling, metal-protein complexes characterisation and imaging mass spectrometry. The present review gives a perspective of the latest achievements in ultrasonic-based sample treatment for proteomics as well as provides the basic concepts and the tools of the trade to efficiently implement this tool in proteomics labs.

A comprehensive factorial design study of variables affecting protein extraction from formalin-fixed kidney tissue samples.

Formalin-fixed tissues are an important source of biological samples for biomedical research. However, proteomics analysis of formalin-fixed tissues has been set aside by formalin-induced protein modifications, which reduce protein extraction efficiency. In this study, a two level full factorial experimental design (2(4)) was used to determine the effects of the extracting conditions in the efficiency of protein recovery from formalin-fixed kidney samples. The following variables were assessed: temperature of extraction, pH of extraction, composition of the extracting buffer and the use ultrasonic energy applied with probe. It is clearly demonstrated that when hating and ultrasonic energy are used in conjunction, a 7-fold increase (p < 0.05) in protein extraction is obtained if compared to extracting conditions for which neither heating nor ultrasonic energy are used. The optimization study was done following the amount of protein extracted by UV (Nanodrop(®) technology, protein ABS at 280 nm) and by 1D SDS-PAGE. Extracts obtained with the optimized conditions were subjected to LC-MALDI MS/MS. A total of 112 proteins were identified.

An assessment of the indirect high intensity ultrasonic assisted cleavage of complex proteomes with immobilized trypsin.

The indirect high intensity ultrasonic assisted cleavage of complex proteomes using immobilized trypsin has been assessed. It has been found that the formation of aggregates between the beds supporting the immobilized trypsin is promoted. This affects the efficiency of the digestion process, which can be 100 times lower than the digestion efficiency obtained with in-solution trypsin. Through the use of isotopic peptide labelling with 18-O, it has been quantified that the digestion efficiency over serum samples can be 1.2-100 times higher for the 70% of the peptides when indirect ultrasonication is replaced by direct ultrasonication. Therefore, direct high intensity ultrasonic assisted cleavage of proteins is proposed as an alternative to be combined with immobilized trypsin.

Probe-based chemical modulations of tissues for IMS.

Chemical modulation imaging over a tissue is gaining momentum in the field of mass spectrometry. Some endogenous or exogenous compounds present in a tissue can be visualized by imaging mass spectrometry after chemical derivatization. This approach gives researchers the possibility to elude chemical interferences in components of the tissues, such as lipids or salts, as well as interferences caused by the matrix. The use of primary and secondary antibodies, the chemical derivatization of peptides and small molecules, and the use of (18)O labeling are various examples reviewed in this article to demonstrate the importance and potential of this emerging aspect of imaging mass spectrometry.

Ultrasonic energy as a tool to overcome some drawbacks in the determination of lead in brain tissue and urine of rats.

An ultrasonic assisted solid-liquid extraction method was developed to determine the level of lead in the brain and urine of rats. Lead was determined by electrothermal atomic absorption spectrometry with longitudinal-Zeeman background correction. Several analytical drawbacks were addressed and overcome, namely small brain sample mass and the formation of precipitate in the urine samples. Utrasonication provided by an ultrasonic probe succeeded in extracting lead from brain samples. Furthermore, it was demonstrated that the formation of a precipitate lowered the lead content in the liquid phase of the urine. Lead was back extracted from the precipitate to the liquid phase with the aid of ultrasonic energy and acidifying the urine with 10% v/v nitric acid. A microwave-assisted acid digestion protocol was used to check the completeness of the lead extraction. The within bath and between bath precision was 5% (n=9) and 7% (n=3) respectively. The limit of quantification was 1.05 µg g(-1) for brain samples and 2.1 µg L(-1) for urine samples. A total of 6 samples of urine and 12 samples of brain from control rats and another 6 samples of urine and 12 samples of brain from rats fed with tap water rich in lead acetate were used in this research. Lead levels in brain and urine from exposed rats ranged from 1.9 ± 0.2 µg g(-1) to 3.5 ± 0.2 µg g(-1) and from 752 ± 56 µg L(-1) to 60.9 ± 1.2 mg L(-1) respectively. Statistically significant differences of levels of lead in brain and urine were found between exposed and non exposed rats.

Plant extracts as an alternative to control Leucoptera coffeella (Guérin-Mèneville) (Lepidoptera: Lyonetiidae).

We evaluated the effects of crude extracts from the plantain Plantago lanceolata and the bitter gourd Momordica charantia on the oviposition preference and development of the coffee leaf miner Leucoptera coffeella Guérin-Mèneville & Perrottet under laboratory and/or greenhouse conditions. The ovicidal effects of these extracts were also studied in a greenhouse. Plantago lanceolata and M. charantia extracts also underwent fractionation directed by oviposition tests with the coffee leaf miner. The extracts of both plants reduced L. coffeella oviposition and egg hatching, apparently as a result of action of plant metabolites on the embryo. Adults originating from eggs treated with the extracts exhibited similar survival rates, but a higher female/male ratio. Fecundity was reduced for females obtained from eggs treated with the M. charantia extract. Partial chemical analysis indicated that both extracts produced polar fractions that reduced the oviposition of L. coffeella on coffee leaves under laboratory conditions. The extracts of P. lanceolata and M. charantia have potential for use in the development of new products to control the coffee leaf miner.

Does usnic acid affect microtubules in human cancer cells? / O ácido úsnico pode afetar microtúbulos em células cancerosas humanas?

Usnic acid, a lichen metabolite, is known to exert antimitotic and antiproliferative activities against normal and malignant human cells. Many chemotherapy agents exert their activities by blocking cell cycle progression, inducing cell death through apoptosis. Microtubules, protein structure involved in the segregation of chromosomes during mitosis, serve as chemotherapeutical targets due to their key role in cellular division as well as apoptosis. The aim of this work was to investigate whether usnic acid affects the formation and/or stabilisation of microtubules by visualising microtubules and determining mitotic indices after treatment. The breast cancer cell line MCF7 and the lung cancer cell line H1299 were treated with usnic acid 29 µM for 24 hours and two positive controls: vincristine (which prevents the formation of microtubules) or taxol (which stabilizes microtubules). Treatment of MCF7 and H1299 cells with usnic acid did not result in any morphological changes in microtubules or increase in the mitotic index. These results suggest that the antineoplastic activity of usnic acid is not related to alterations in the formation and/or stabilisation of microtubules.

O ácido úsnico, um metabólito de liquens, é conhecido por sua atividade antimitótica e antiproliferativa em células humanas normais e malignas. Muitos quimioterápicos exercem suas atividades bloqueando a progressão do ciclo celular e induzindo morte celular por apoptose. Os microtúbulos, estruturas protéicas envolvidas na segregação dos cromossomos durante a mitose, servem como alvo quimioterapêutico devido ao seu importante papel tanto na divisão celular quanto nos mecanismos de morte celular por apoptose. O objetivo deste trabalho foi investigar se o ácido úsnico afeta a formação e/ou estabilização dos microtúbulos, a partir da visualização de microtúbulos e determinação de índices mitóticos após o tratamento. Células de câncer de mama MCF7 e de câncer de pulmão H1299 foram tratadas por 24 horas com 29 µM de ácido úsnico e dois controles positivos: vincristina (que impede a formação de microtúbulos) e taxol (que estabiliza microtúbulos). O tratamento das células MCF7 e H1299 com o ácido úsnico não resultou em aumento do índice mitótico. Os resultados sugerem que a atividade antineoplásica do ácido úsnico não está relacionada a alterações na formação e/ou estabilização de microtúbulos.

Does usnic acid affect microtubules in human cancer cells?

Usnic acid, a lichen metabolite, is known to exert antimitotic and antiproliferative activities against normal and malignant human cells. Many chemotherapy agents exert their activities by blocking cell cycle progression, inducing cell death through apoptosis. Microtubules, protein structure involved in the segregation of chromosomes during mitosis, serve as chemotherapeutical targets due to their key role in cellular division as well as apoptosis. The aim of this work was to investigate whether usnic acid affects the formation and/or stabilisation of microtubules by visualising microtubules and determining mitotic indices after treatment. The breast cancer cell line MCF7 and the lung cancer cell line H1299 were treated with usnic acid 29 microM for 24 hours and two positive controls: vincristine (which prevents the formation of microtubules) or taxol (which stabilizes microtubules). Treatment of MCF7 and H1299 cells with usnic acid did not result in any morphological changes in microtubules or increase in the mitotic index. These results suggest that the antineoplastic activity of usnic acid is not related to alterations in the formation and/or stabilisation of microtubules.

Latest developments in sample treatment for 18O-isotopic labeling for proteomics mass spectrometry-based approaches: a critical review.

Nowadays isotopic (18)O-labeling of peptides has recalled the attention of researchers due to its simplicity of application and high versatility for proteomics studies. Protein quantification, differential peptide mass mapping, studies regarding proteins overexpressed or underexpressed, or the searching of biomarkers can be accomplished by using (18)O-labeling. In this critical review we comment on the different ways in which (18)O-labeling can be done, highlighting the key parameters of the different sample treatments to obtain a reliable and reproducible labeling. In addition we describe and compare the latest improvement in terms of sample treatment that allows to reduce the handling and to increase the throughput for this sample treatment. Finally, we hypothesize on the future trends of these methods under the light of the new technological advances to speed protein cleavage.