Highly accurate detection of ovarian cancer using CA125 but limited improvement with serum matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling

Objectives: Our objective was to test the performance of CA125 in classifying serum samples from a cohort of malignant and benign ovarian cancers and age-matched healthy controls and to assess whether combining information from matrix-assisted laser desorption/ionization (MALDI) time-of-flight profiling could improve diagnostic performance. Materials and Methods: Serum samples from women with ovarian neoplasms and healthy volunteers were subjected to CA125 assay and MALDI time-of-flight mass spectrometry (MS) profiling. Models were built from training data sets using discriminatory MALDI MS peaks in combination with CA125 values and tested their ability to classify blinded test samples. These were compared with models using CA125 threshold levels from 193 patients with ovarian cancer, 290 with benign neoplasm, and 2236 postmenopausal healthy controls. Results: Using a CA125 cutoff of 30 U/mL, an overall sensitivity of 94.8% (96.6% specificity) was obtained when comparing malignancies versus healthy postmenopausal controls, whereas a cutoff of 65 U/mL provided a sensitivity of 83.9% (99.6% specificity). High classification accuracies were obtained for early-stage cancers (93.5% sensitivity). Reasons for high accuracies include recruitment bias, restriction to postmenopausal women, and inclusion of only primary invasive epithelial ovarian cancer cases. The combination of MS profiling information with CA125 did not significantly improve the specificity/accuracy compared with classifications on the basis of CA125 alone. Conclusions: We report unexpectedly good performance of serum CA125 using threshold classification in discriminating healthy controls and women with benign masses from those with invasive ovarian cancer. This highlights the dependence of diagnostic tests on the characteristics of the study population and the crucial need for authors to provide sufficient relevant details to allow comparison. Our study also shows that MS profiling information adds little to diagnostic accuracy. This finding is in contrast with other reports and shows the limitations of serum MS profiling for biomarker discovery and as a diagnostic too

OLIgo mass profiling (OLIMP) of extracellular polysaccharides.

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling. An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite. Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)(1) (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself(2), and is amenable to high-throughput analyses(3). While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall. OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes(4). For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase(5, 11, 12, 13), xylan using an endo-beta-(1-4)-xylanase (6,7), or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase (8). Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined (9)

Advancing liquid atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry toward ultra-high-throughput analysis

Label-free high-throughput screening using mass spectrometry has the potential to provide rapid large-scale sample analysis at a speed of more than one sample per second. Such speed is important for compound library, assay and future clinical screening of millions of samples within a reasonable time frame. Herein, we present a liquid atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) setup for high-throughput large-scale sample analysis (>5 samples per second) for three substance classes (peptides, antibiotics and lipids). Liquid support matrices (LSM) were used for the analysis of standard substances as well as complex biological fluids (milk). Throughput and analytical robustness were mainly dependent on the complexity of the sample composition and the current limitations of the commercial hardware. However, the ultimate limits of liquid AP-MALDI in sample throughput can be conservatively estimated to be beyond 10-20 samples per second. This level of analytical speed is highly competitive compared with other label-free MS methods, including electrospray ionization and solid state MALDI, as well as MS methods using multiplexing by labelling, which in principle can also be used in combination with liquid AP-MALDI MS

Evaluation of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the Identification of Group B Streptococcus.

Objective Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and sepsis worldwide. Intrapartum antibiotics given to women carrying GBS are an effective means of reducing disease in the first week of life. Rapid and reliable tests are needed to accurately identify GBS from these women for timely intrapartum antibiotic administration to prevent neonatal disease. Many laboratories now use matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) by direct plating or cell lysis for the identification of GBS isolates. The cell lysis step increases time to results for clinical samples and is more complex to perform. Therefore, we seek to evaluate the sensitivity and specificity of the quicker and more rapid direct plating method in identifying GBS. Results We directly compared swab isolates analysed by both direct plating and cell lysis method and demonstrated that direct plating has a sensitivity and specificity of 0.97 and 1, respectively, compared to an additional cell lysis step. We demonstrated that MALDI-TOF MS can be successfully used for batch processing by the direct plating method which saves time. These results are reassuring for laboratories worldwide who seek to identify GBS from swabs samples as quickly as possible

Nonribosomal peptides, key biocontrol components for Pseudomonas fluorescens In5, isolated from a Greenlandic suppressive soil.

UnlabelledPotatoes are cultivated in southwest Greenland without the use of pesticides and with limited crop rotation. Despite the fact that plant-pathogenic fungi are present, no severe-disease outbreaks have yet been observed. In this report, we document that a potato soil at Inneruulalik in southern Greenland is suppressive against Rhizoctonia solani Ag3 and uncover the suppressive antifungal mechanism of a highly potent biocontrol bacterium, Pseudomonas fluorescens In5, isolated from the suppressive potato soil. A combination of molecular genetics, genomics, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) imaging mass spectrometry (IMS) revealed an antifungal genomic island in P. fluorescens In5 encoding two nonribosomal peptides, nunamycin and nunapeptin, which are key components for the biocontrol activity by strain In5 in vitro and in soil microcosm experiments. Furthermore, complex microbial behaviors were highlighted. Whereas nunamycin was demonstrated to inhibit the mycelial growth of R. solani Ag3, but not that of Pythium aphanidermatum, nunapeptin instead inhibited P. aphanidermatum but not R. solani Ag3. Moreover, the synthesis of nunamycin by P. fluorescens In5 was inhibited in the presence of P. aphanidermatum. Further characterization of the two peptides revealed nunamycin to be a monochlorinated 9-amino-acid cyclic lipopeptide with similarity to members of the syringomycin group, whereas nunapeptin was a 22-amino-acid cyclic lipopeptide with similarity to corpeptin and syringopeptin.ImportanceCrop rotation and systematic pest management are used to only a limited extent in Greenlandic potato farming. Nonetheless, although plant-pathogenic fungi are present in the soil, the farmers do not experience major plant disease outbreaks. Here, we show that a Greenlandic potato soil is suppressive against Rhizoctonia solani, and we unravel the key biocontrol components for Pseudomonas fluorescens In5, one of the potent biocontrol bacteria isolated from this Greenlandic suppressive soil. Using a combination of molecular genetics, genomics, and microbial imaging mass spectrometry, we show that two cyclic lipopeptides, nunamycin and nunapeptin, are important for the biocontrol activity of P. fluorescens In5 both in vitro and in microcosm assays. Furthermore, we demonstrate that the synthesis of nunamycin is repressed by the oomycete Pythium aphanidermatum. Overall, our report provides important insight into interkingdom interference between bacteria and fungi/oomycetes

A comprehensive evaluation of colonic mucosal isolates of Sutterella wadsworthensis from inflammatory bowel disease

Peer reviewedPublisher PD

Investigation of the effects of weight loss on Ovis aries muscle: a proteomic study on three breeds with different levels of adaptation to nutritional stress

Tese de mestrado. Biologia (Biologia Celular e Biotecnologia). Universidade de Lisboa, Faculdade de Ciências, 2011Nos trópicos, a produção animal em regime extensivo enfrenta grandes obstáculos, talvez o maior dos quais é a perda de peso sazonal, devida à época seca existente nos países tropicais com uma pluviosidade muito baixa ou até mesmo inexistente. Em muitos países tropicais, a produção animal foca-se sobretudo na ovelha e, principalmente nos sistemas tradicionais, recorre ao uso de espécies nativas pois estas, duma forma geral, apresentam maior resistência às condições ambientais comparativamente com as espécies seleccionadas. Factores importantes para a escolha das espécies nativas sobre as seleccionadas são a tolerância a longos períodos sem alimento nem água, às altas temperaturas e a insectos causadores de doenças, como por exemplo a miíase – designação geral para doença parasitária causada pela larva de moscas ou outros dípteros. A escassez de alimento leva a que o animal perca peso, podendo atingir uma diminuição de peso na ordem de 40% em ovelhas. É evidente a necessidade para desenvolvimentos nas áreas da produção animal para evitar as quebras na produção e rendimento. Na Austrália, uma medida para reduzir essa diminuição de produção passa pelo progressivo abandono das raças e sistemas de produção tradicionais e pela adopção de raças ‘alternativas’. Um típico exemplo é a passagem de sistemas de produção de lã para sistemas de produção de carne. Os sistemas de produção de lã, normalmente com apenas a raça Merino, estão numa tendência de abandono, devido principalmente aos custos elevados de manutenção e mão-de-obra bem como à diminuição do valor da lã nos mercados internacionais. Sistemas de produção de lã baseados em Merino passam a ser sistemas de produção de carne com raças de pêlo, como por exemplo as raças Dorper e Damara, as raças ‘alternativas’ mais importantes na Austrália. Estas três raças apresentam diferenças ao nível da tolerância à escassez de alimento: a Merino, sendo uma raça oriunda de selecção, apresenta o nível mais baixo de tolerância; a Dorper apresenta o nível de tolerância intermédio; a Damara é a raça mais tolerante, das três, à escassez de alimento. Dado que a escassez de alimento conduzirá eventualmente à perda de peso, a perda de peso deve-se essencialmente pela utilização das reservas energéticas do corpo do animal: primeiro há consumo do tecido adiposo com reduzido consumo proteico, seguindo-se um aumento da degradação das proteínas, é de esperar que haja diferenças na expressão das proteínas entre um animal bem alimentado e outro em perda de peso (subalimentado). Assim, este trabalho pretende identificar essas proteínas cuja expressão varia entre condições de favoráveis e desfavoráveis ao ganho de peso. Para tal recorreu-se à técnica de Electroforese Bidimensional usando 6 grupos experimentais, grupo de controlo e grupo com subnutrição para cada uma das três raças, com quatro réplicas biológicas, amostras de quatro animais diferentes por grupo experimental. Obtiveram-se assim 24 géis, com um total de 1228 spots, dos quais apenas 22 eram estatisticamente significativos (p<0.05) e tinham um poder de pelo menos 0,8. Todos os 22 spots foram seleccionados para identificação, em que 16 foram identificados com sucesso. Estas 16 proteínas distribuem-se, de acordo com a sua função, por proteínas do metabolismo (23%), proteínas estruturais (18%) e proteínas do aparelho contráctil (32%).In tropical countries, extensive ruminant production faces several constraints, of which the most important is seasonal weight loss, which is caused by a dry season with very low rain levels or no rain at all. In the majority of these countries, the animal most used for production is sheep, namely indigenous breeds, as these breeds can, generally speaking, endure harsher environment conditions than selected breeds. Being able to better tolerate lack of food and water is a very important feature an animal should have regarding extensive production systems. In Australia, currently, the sheep industry is mainly focused on the Merino breed. This breed, introduced in Australia by the Europeans and a derivative from the European breed, i.e. a selected breed, is wool breed and as such is mainly used for wool production, with very few animals being used for meat. This, however, creates a problem for these industries: with the decrease in price of wool and the increase of labour costs, it is becoming less and less economically viable to produce Merino for wool. Because of this, increasing number of producers are looking for alternative breeds that can better withstand the lack of food and tolerate some diseases that affect greatly Merino sheep. Two breeds are standing out due to their ability to withstand prolonged undernutrition: the Damara and the Dorper. These two breeds have high and intermediate tolerance to undernutrition, respectively, while the Merino have very low tolerance to undernutrition. If the undernutrition, and the associated weight loss, is long enough, there will be protein break down in response to the need for energy to maintain the animal’s metabolism. In regard to the interested in selecting traits like weight loss resistance for increased animal production rates, this work aims at providing information about protein markers which may be used to access tolerance to undernutrition and be used as a tool to better select the desired traits in the animals. A proteomic approach of Two-Dimensional Gel Electrophoresis was used to compare gastrocnemius muscle protein expression patterns between six experimental groups: three sheep breeds (Damara, Dorper and Australian Merino) grouped in a control (not feed restricted) and a feed restricted group; samples from four animals for each group were used. A total of 24 gels was obtained. After staining and analysis, 22 spots were detected as significantly (p<0.05) different and with a power of at least 0.8. All spots were selected for identification, 16 were successfully identified, with the identified proteins ranging from structural, metabolic and contractile apparatus roles

Advances in Microfluidics and Lab-on-a-Chip Technologies

Advances in molecular biology are enabling rapid and efficient analyses for effective intervention in domains such as biology research, infectious disease management, food safety, and biodefense. The emergence of microfluidics and nanotechnologies has enabled both new capabilities and instrument sizes practical for point-of-care. It has also introduced new functionality, enhanced sensitivity, and reduced the time and cost involved in conventional molecular diagnostic techniques. This chapter reviews the application of microfluidics for molecular diagnostics methods such as nucleic acid amplification, next-generation sequencing, high resolution melting analysis, cytogenetics, protein detection and analysis, and cell sorting. We also review microfluidic sample preparation platforms applied to molecular diagnostics and targeted to sample-in, answer-out capabilities