Phenotypic Point Of View Research Articles

Genotypic and Phenotypic Characterization of Escherichia coli Isolates Recovered from the Uterus of Mares with Fertility Problems.

Escherichia coli is the bacterial pathogen most frequently associated with mare infertility. Here, we characterized 24 E. coli strains isolated from mares which presented signs of endometritis and infertility from a genotypic and phenotypic point of view. The majority of the isolates belonged to phylogenetic group B1 (9/24, 37.5%). Regarding antibiotic resistance profiles, 10 out of 24 (41.7%) were multidrug-resistant (MDR). Moreover, 17 out of 24 (70.8%) were strong or moderate biofilm producers, and of these eight were MDR strains. Interestingly, 21 out of 24 (87.5%) E. coli strains were phenotypically resistant to ampicillin and 10 of them were also resistant to amoxicillin with clavulanic acid. Regarding the presence of selected virulence factors, 50% of the examined strains carried at least three of them, with fimH detected in all strains, and followed by kpsMTII (11/24, 45.9%). No strain was able to invade HeLa cell monolayers. No relevant differences for all the investigated characteristics were shown by strains that grew directly on plates versus strains requiring the broth-enrichment step before growing on solid media. In conclusion, this work provides new insight into E. coli strains associated with mares' infertility. These results broaden the knowledge of E. coli and, consequently, add useful information to improve prevention strategies and therapeutic treatments contributing to a significant increase in the pregnancy rate in mares.

Plant growth promotion of the forage plant Lupinus albus Var. Orden Dorado using Pseudomonas agronomica sp. nov. and Bacillus pretiosus sp. nov. added over a valorized agricultural biowaste.

The overexploitation of natural ecosystems and the evolution of climate change currently force us to design new strategies for more sustainable agronomic uses. The recovery of plant residues, as an alternative to agrochemicals, can help alleviate these problems, for example, through its use for the synthesis of biofertilizers. In this work, the effect of the organic fertilizer matrix ORGAON® from the valorization of horticultural waste is tested, to which two strains of bacteria (and their consortium) are added (SAICEU11T identified as Bacillus pretiosus and SAICEU22T identified as Pseudomonas agronomica), selected for their demonstrated ability to promote plant growth (PGPB), on the lupine forage plant (Lupinus albus). For the synthesis of the biofertilizer, both strains were added to the ORGAON® organic matrix separately, until reaching a final optical density (OD) of 0.5 McFarland in each case in the irrigation matrix. As a control, sterile ORGAON® (ORGAON®st) was used, also supplemented with the PGPB strains and a chemical fertilizer widely used in agronomy (Chem-F). With these treatments, a 6-week experiment was started under controlled laboratory conditions and on agricultural substrate, to recreate field conditions as accurately as possible. All the tests were carried out with 9 repetitions and 3 replicates of each treatment. After harvest, the improvements on the following biometric variables were studied for each treatment: total weight (Weight_T, g), shoot weight (Weight_S, g), root weight (Weight_R, g), number of leaves (Leaves, No.), shoot length (Length_S), root length (Length_R) and number of secondary roots (Roots, No.). Likewise, the identification of the tested strains and their description as new species was carried out. For this, they were studied from the phenotypic point of view (Transmission electron microscopy (TEM), metabolic profile, PGP activities, fatty acid profile and Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)) and genotypic (sequencing of the main housekeeping genes and sequencing of the whole genome, genomic characteristics (dDDH and ANI) and phylogenetic analysis). After the statistical analysis of the results, it is shown that the individual addition of both strains on the ORGAON® and ORGAON®st organic matrix improve certain biometric variables. In the case of the SAICEU11T (Bacillus pretiosus) strain, the variables root weight (Weight_R, g), total weight (Weight_T, g) and length of the plant, and number of secondary roots (Roots, No.) significantly improve, while in the case of the strain SAICEU22T (Pseudmonas agronomica), a significant improvement of root length (Length_R) and number of secondary roots (Roots, No.) is demonstrated. On the other hand, the genotaxonomic analysis showed that both species have not been described to date. The identification based on the main housekeeping genes, show that for the Bacillus strain (SAICEU11T) the sequence similarity of the 16S rRNA was 100%, gyrB 92.69%, rpoB 97.70% and rpoD 94.67%. For the Pseudomonas strain (SAICEU22T) the results were 100% for 16S rRNA, 98.43% for rpoD and 96.94% for gyrB. However, in both cases, the dDDH and ANI values, as well as the phylogenetic analysis, show that both species are below the species threshold, which would support the hypothesis that both are new species, in line with the chemotaxonomic results obtained by MALDI-TOF spectrometry and fatty acid profile. To verify the biosafety in their handling and release into the natural environment, we have ruled out the presence of genes that encode virulence factors or resistance to antibiotics, concluding that they are suitable for use in the field to improve the yield of crop plants. Type strains are SAICEU11T (= DSM 114702T = CECT30674T) for Bacillus pretiosus and SAICEU22T (= DSM 114959T = CECT30673T) for Pseudomonas agronomicae.

Two novel platelet biotinylation methods and their impact on stored platelet concentrates in a blood bank environment.

Storage of platelet concentrates (PCs) has an impact on platelet quality and possibly affects their functions after transfusion. The influence of processing and storage conditions of PCs on their in vivo function upon transfusion is unknown. One option for investigating this question is to implement an ex vivo labeling of human platelets, to analyze them after transfusion into heathy volunteers and/or patients. In this study, we developed two labeling methods employing biotin. Two methods of biotinylation were compared to a control (standard PC). The "Bio-Wash" process used washing steps to label all platelets within the PC; for the other method, "Bio-Direct," one fifth of the PC were directly labeled without washing steps. The control and the two biotinylated PCs were analyzed over 7 days of storage. Labeling efficiency, platelet counts, phenotypes, and functions, along with time and costs, were evaluated to select the best process. Both methods achieved a stable labeling through the storage, with similar platelet counts and metabolism in comparison to control PCs. Bio-Wash showed higher activation phenotype and lower aggregation response in comparison to the Bio-Direct method. The Bio-Direct was performed within 1.5 h versus 3 h for the Bio-Wash. However, the Bio-Direct required 12 mg of biotin instead of 8 mg for the other process. We set up two methods of biotinylation that can be easily implemented in a blood bank environment. The Bio-Direct process was preferred to the Bio-Wash because of its similarity, from a functional and phenotypic point of view, with standard PCs.

Network-Based Methods for Approaching Human Pathologies from a Phenotypic Point of View.

Network and systemic approaches to studying human pathologies are helping us to gain insight into the molecular mechanisms of and potential therapeutic interventions for human diseases, especially for complex diseases where large numbers of genes are involved. The complex human pathological landscape is traditionally partitioned into discrete “diseases”; however, that partition is sometimes problematic, as diseases are highly heterogeneous and can differ greatly from one patient to another. Moreover, for many pathological states, the set of symptoms (phenotypes) manifested by the patient is not enough to diagnose a particular disease. On the contrary, phenotypes, by definition, are directly observable and can be closer to the molecular basis of the pathology. These clinical phenotypes are also important for personalised medicine, as they can help stratify patients and design personalised interventions. For these reasons, network and systemic approaches to pathologies are gradually incorporating phenotypic information. This review covers the current landscape of phenotype-centred network approaches to study different aspects of human diseases.

Bronchoalveolar-Lavage-Derived Fibroblast Cell Line (B-LSDM7) as a New Protocol for Investigating the Mechanisms of Idiopathic Pulmonary Fibrosis.

Background: The use of BAL to study ILDs has improved our understanding of IPF pathogenesis. BAL fluid is routinely collected and can be considered a clinical and research tool. The procedure is well tolerated and minimally invasive. No specific cell lines from BAL or immortalized cell lines from IPF patients are available commercially. A method to quickly isolate and characterize fibroblasts from BAL is an unmet research need. Materials and methods: Here we describe a new protocol by which we isolated a cell line from IPF. The cell line was expanded in vitro and characterized phenotypically, morphologically and functionally. Results: This culture showed highly filamentous cells with an evident central nucleus. From the phenotypic point of view, this cell line displays fibroblast/myofibroblast-like features including expression of alpha-SMA, vimentin, collagen type-1 and fibronectin. The results showed high expression of ROS in these cells. Oxidative stress invariably promotes extracellular matrix expression in lung diseases directly or through over-production of pro-fibrotic growth factors. Conclusions: Our protocol makes it possible to obtain fibroblasts BAL that is a routine non-invasive method that offers the possibility of having a large sample of patients. Standardized culture methods are important for a reliable model for testing molecules and eventual novel development therapeutic targets.

Phenotypic variability, diversity and genetic-population structure in melon (Cucumis melo L.) Associated with total soluble solids

This study evaluated the phenotypic variation in Total Soluble Solid (TSS) quantified in different environments, together with allelic diversity and genetic-population structure based on 18 microsatellites loci (SSR) in QTLs (Quantitative Trait Loci). The phenotypic characterization in 41 Brazilian accessions of melon and three commercial cultivars revealed a wide variation in TSS, with values higher than 9.0 % for commercial cultivars and lower than 4.0 % in traditional melon accessions. The genetic parameters inferred by the phenotypic analysis revealed low variances but high heritability, indicating that the expression in TSS is highly related to the genotypic potential. In contrast, the significant effect of genotype and environment interaction (G x E) on the studied trait suggests that the selection or recommendation of promising plants for breeding should be based on the behavior of these accessions in more than one environment. High allelic diversity was verified with amplification of SSR loci, ranging from two to ten alleles per locus. High polymorphic content indicates the use of markers such as CMATN22 for mapping and binding analysis. The graphical representation of genetic diversity revealed the formation of two large groups of variation, both in the main components analysis graph and in the weighted neighbor-joining dendrogram. The certification of the distribution was validated by the inclusion of two genotypes of the Spanish access “Piel de Sapo” (PS) in a group different from the exotic Korean access “Songwhan Charmi” (PI161375) and the hybrid PI161375 X A16. The genetic-population structure was inferred using the ad hoc and ΔK method, indicating two well-defined populations and corroborating the graphical information of genetic diversity distribution. However, eight subpopulations were defined, structuring genetic diversity in the analyzed germplasm. The distribution of PS and PI161375 varieties in different subpopulations, despite the structural similarity between the two PS genotypes, indicates an expected pattern in the population structure. The results show the studied germplasm is structured but diversified both from the phenotypic point of view and from the variation in alleles in QTLs for TSS.

The Phenotypic Characterization of the Cammalleri Sisters, an Example of Exceptional Longevity

This article shows demographic, clinical, anamnestic, cognitive, and functional data as well as biochemical, genetic, and epigenetic parameters of two exceptional siblings: Diega (supercentenarian) and Filippa (semisupercentenarian) Cammalleri. The purpose of this study is to provide new insights into the extreme phenotypes represented by semisupercentenarians and supercentenarians. Different studies have been published on supercentenarians, but to the best of our knowledge, this is the only concerning two sisters and the most detailed from a phenotypic point of view. Our findings agree with the suggestion that supercentenarians have an increasing relative resistance to age-related diseases, approximating the limits of the functional human reserve to address successfully the acute causes of death. More interestingly, our data agree with, and extend, the suggestion that inflammation and oxidative stress predict centenarian mortality.

Aciduric Bacterial Communities at Three Levels in Dentin Caries

Dentin caries constitute a complex ecosystem with a diverse microbiota adapted to fluctuations in nutrient concentration and acidity. However, knowledge about the acid tolerant microbiota at different levels in the lesion is rather poor. Hence, the purpose of this study was to investigate the acid tolerant microflora at different levels in established dentin caries lesions using solid pH-selective media. Primary dentin caries lesions were sampled with a bur at three levels (superficial, centre and the clinically caries-free floor of the lesion) in 10 patients. Samples were incubated on pH-neutral and pH-selective (pH 4.0, 4.5, 5.0, 5.5) agars. Numbers of colony-forming units (cfu) were determined and colonies were subsequently characterised morphologically and isolated. The total number of bacteria in the carious lesions, recovered using blood agar (BA), ranged from 5.88 x 103 to 5.85 x 105 (median 2.64 x 105; range 5.80 x 105) and numbers of recovered bacteria decreased with decreasing agar pH. Fewer bacteria were found in the clinically caries-free dentin (P = 0.042), but the mean number of cfu (BA) was still 5.88 x 103 in those samples. Each of the 10 investigated dentin caries lesions harboured a unique microbial flora, indicating that various combinations of aciduric bacteria can colonise, survive in and probably propagate dentin caries. Solid pH-selective agars can be used successfully to select acid-tolerant microorganisms in dentin caries lesions. This could be used to describe this subset of the total microbiome from a phenotypic point of view, an objective that cannot be accomplished using molecular methods.

Influence of Pollen Germination Capacity on a Successful Artificial Hybridization in Cyclamen sp

The artificial hybridization is one of the most important methods to create variability in breeding programs. The success of artificial hybridization very often depends on the fertility of pollen grains which require a minimum 30% germination level. The aim of the present study was to identify the correlation between the level of pollen grain fertility and success of hybridization in several cyclamen cultivars. There were taken under study seven cultivars of Cyclamen sp. very different from phenotypic point of view (maxi, midi, mini). In the first step it was determinated the germination capacity of pollen grains trough viability and germinability percentage and the correlation between these two determinations. The results show a higher ratio of viability then germinability in all cultivars. The best results of artificial hybridization were registered in hybrid combinations in which there were used cultivars with the highest percentage of pollen fertility. Out of seven cyclamen cultivars tested in 18 hybrid combinations, three cultivars showed the highest level of fertility when used as male genitor (Curly White; Curly Magenta Picotee and Allure Salmon Flamed)for a further study on phenol biodegradation especially areas with phenol contamination containing some heavy metals.

Phenotypic and genetic relationships between indicators of the mammary gland health status and milk composition, coagulation, and curd firming in dairy sheep

The present study investigated the effect of somatic cell count, lactose, and pH on sheep milk composition, coagulation properties (MCP), and curd firming (CF) parameters. Individual milk samples were collected from 1,114 Sarda ewes reared in 23 farms. Milk composition, somatic cell count, single point MCP (rennet coagulation time, RCT; curd firming time, k20; and curd firmness, a30, a45, and a60), and CF model parameters were achieved. Phenotypic traits were statistically analyzed using a mixed model to estimate the effects of the different levels of milk somatic cell score (SCS), lactose, and pH, respectively. Additive genetic, herd, and residual correlations among these 3 traits, and with milk composition, MCP and CF parameters, were inferred using a Bayesian approach. From a phenotypic point of view, higher SCS levels caused a delayed gelification of milk. Lactose concentration and pH were significant for many milk quality traits, with a very intense effect on both coagulation times and curd firming. These traits (RCT, RCT estimated using the curd firming over time equation, and k20) showed an unfavorable increase of about 20% from the highest to the lowest level of lactose. Milk samples with pH values lower than 6.56 versus higher than 6.78 were characterized by an increase of RCT (from 6.00 to 14.3 min) and k20 (from 1.65 to 2.65 min) and a decrease of all the 3 curd firmness traits. From a genetic point of view, the marginal posterior distribution of heritability estimates evidenced a large and exploitable variability for all 3 phenotypes. The mean intra-farm heritability estimates were 0.173 for SCS, 0.418 for lactose content, and 0.206 for pH. Lactose (favorably), and SCS and pH (unfavorably), at phenotypic and genetic levels, were correlated mainly with RCT and RCT estimated using the curd firming over time equation and scarcely with the other curd firming traits. The SCS, lactose, and pH were significantly correlated with each other's. In conclusion, results reported in the present study suggest that SCS, pH, and lactose affect, contemporarily and independently, milk quality and MCP. These phenotypes, easily available during milk recording schemes measured by infrared spectra prediction, could be used as potential indicators traits for improving cheese-making ability of ovine milk.

Mechanisms of Speciation

Mechanisms of Speciation

Abstract 3013: Study of three-dimensional cell culturing influence on DNA methylation profile

Abstract In this study, we purpose the analysis of the DNA methylation profile of four different human breast cell lines, two non transformed and two tumoral, taking into account the influence of the environment in cell culturing. The extracellular matrix was initially thought to serve as nothing more than a physical support for the cells. However, nowadays is very well known that the extracellular matrix is a key component in cell biology, regulating cell biology and tissue homeostasis. This suggests that there is a bidirectional crosstalk between the cells and the surrounding extracellular matrix. Cell culturing is a commonly used technique in all areas of biology research. Usually, adherent cells are spreading in two-dimensional plates where they growth attached to the plastic surface in two directions. Mammary epithelial cells that are cultured on this conventional way fail to form acinar-like structures and loss tissue specific protein expression. Many of the microenvironment influences can be restored in cell culturing models by using three-dimensional cultures in extracellular matrix gels. 3D culturing of epithelial cells can help to reconstruct the acinar morphology seen in vivo and restore some mammary-specific function, like milk proteins production. DNA methylation plays a significant role in the epigenetic regulation of chromatin structure and gene expression. In this work, we tried to analyze the DNA methylation profile of four different human breast cell lines growth in 2 and 3 dimensions to assess the influence of DNA methylation in the two models. HMEC (finite lifespan human mammary epithelial cell line), MCF10A (spontaneously immortalized, but not transform, human breast epithelial cell line), MCF7 (human breast adenocarcinoma cell line, metastasic) and MDA-MB-231 (human breast adenocarcinoma cell line, metastasic) were growth in plates with and without MatrigelTM basement Membrane Matrix Growth Factor Reduced (BD). 3D culturing was done by spreading the cells in the matrix surface and allowing them to form the characteristic acini of the mammary gland. Morphological differences are evident between the 2 and 3D culture models, being much more accurate the 3D model from a phenotypic point of view. DNA mehtylation status was done by illumina's Infinium Microarrays, comparing the methylation profile of each cell line growth in 2 and 3D. We determined in this way which genes are differentially methylated in 2 and 3 dimensions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3013. doi:10.1158/1538-7445.AM2011-3013

Peripheral Blood Dendritic Cell Subsets from Patients with Monoclonal Gammopathies Show an Abnormal Distribution and Are Functionally Impaired

Objectives. The information currently available about dendritic cells (DCs) in patients with different types of monoclonal gammopathy (MG) is limited and frequently controversial. In the present study, we analyzed the ex vivo distribution as well as the phenotypic and functional characteristics of peripheral blood (PB) DCs from different types of MG. Methods. For this purpose, 61 untreated patients in total with MG were analyzed-MG of undetermined significance (MGUS), 29 cases; multiple myeloma (MM), 28 cases; and plasma cell leukemia (PCL), 4 cases-in comparison with a group of 10 healthy controls. Results. Our results show an absolute overall higher number of all subsets of PB DCs in PCL, together with lower numbers of myeloid DCs in MM patients. From a phenotypic point of view, PB DC subsets from all types of MG expressed significantly higher levels of HLA molecules and altered patterns of expression of the CD2, CD11c, CD16, CD22, CD62L, and CD86 molecules, in association with altered patterns of secretion of inflammatory cytokines. Conclusion. In summary, we show the existence of significant abnormalities in the distribution, phenotype, and pattern of secretion of inflammatory cytokines by different subsets of PB DCs from patients with MGs, which could reflect a potentially altered homing of DCs, together with a greater in vivo activation and lower responsiveness of PB DCs, which are already detectable in MGUS patients.

Immunophenotyping of acute leukemias and myelodysplastic syndromes.

Alberto Orfao,* Francisco Ortuno, Maria de Santiago, Antonio Lopez, and Jesus San Miguel Servicio General de Citometria, Universidad de Salamanca, Salamanca, Spain Centro de Investigacion del Cancer y Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain Servicio de Hematologia y Oncologia Medica, Hospital General Universitario J.M. Morales Meseguer, Murcia, Spain Servicio de Hematologia, Hospital Universitario de Salamanca, Salamanca, Spain

Impact of genotypic studies on mycobacterial taxonomy: the new mycobacteria of the 1990s.

The advancement of genetic techniques has greatly boosted taxonomic studies in recent years. Within the genus Mycobacterium, 42 new species have been detected since 1990, most of which were grown from clinical samples. Along with species for which relatively large numbers of strains have been reported, some of the new species of mycobacteria have been detected rarely or even only once. From the phenotypic point of view, among the new taxa, chromogens exceed nonchromogens while the numbers of slowly and rapidly growing species are equivalent. Whereas conventional identification tests were usually inconclusive, an important role was played by lipid analyses and in particular by high-performance liquid chromatography. Genotypic investigations based on sequencing of 16S rRNA gene have certainly made the most important contribution. The investigation of genetic relatedness led to the redistribution of the species previously included in the classically known categories of slow and rapid growers into new groupings. Within slow growers, the intermediate branch related to Mycobacterium simiae and the cluster of organisms related to Mycobacterium terrae have been differentiated; among rapid growers, the group of thermotolerant mycobacteria has emerged. The majority of species are resistant to isoniazid and, to a lesser extent, to rifampin. Many of the new species of mycobacteria are potentially pathogenic, and there are numerous reports of their involvement in diseases. Apart from disseminated and localized diseases in immunocompromised patients, the most frequent infections in immunocompetent people involve the lungs, skin, and, in children, cervical lymph nodes. The awareness of such new mycobacteria, far from being a merely speculative exercise, is therefore important for clinicians and microbiologists.

Poor prognosis of mediastinal non-Hodgkin's lymphoma with an immature phenotype of CD2+, CD7 (or CD5)+, CD3-, CD4-, and CD8-.

Nine children with mediastinal non-Hodgkin's lymphoma (NHL) were treated according to our new regimen which is characterized by intensified therapy with high-dose cytosine arabinoside (HDCA). After induction therapy with a combination of five drugs, such as vincristine, doxorubicin, cyclophosphamide, 1-asparaginase, and prednisolone, intermediate dosages of methotrexate (MTX) (1 g/m2) and HDCA (1.5 g/m2 x 12 doses) were administered. All but one patient (88.9%) achieved complete remission and then received this intensified therapy. With a median follow-up period of 25.5 months, five patients are still in complete remission, but three patients have relapsed. From the phenotypic point of view, these relapsed patients showed only very immature T-cell differentiation antigens such as CD2 and CD7 (or CD5). These results suggest that HDCA as intensified therapy for children with mediastinal NHL seems to be effective. However, for patients with an immature phenotype of T-lineage cells, more sophisticated regimens should be prepared.