The morbid cutaneous anatomy of the human genome revealed by a bioinformatic approach

Computational approaches have been developed to prioritize candidate genes in disease gene identification. They are based on different pieces of evidences associating each gene with the given disease. In this study, 648 genes underlying genodermatoses have been compared to 1808 genes involved in other genetic diseases using a bioinformatic approach. These genes were studied at the structural, evolutionary and functional levels. Results show that genes underlying genodermatoses present longer CDS and have more exons. Significant differences were observed in nucleotide motif and amino-acid compositions. Evolutionary conservation analysis revealed that genodermatoses genes have less paralogs, more orthologs in Mouse and Dog and are less conserved. Functional analysis revealed that genodermatosis genes seem to be involved in immune system and skin layers. The Bayesian network model returned a rate of good classification of around 80%. This computational approach could help investigators working in the field of dermatology by prioritizing positional candidate genes for mutation screening.     

Improving human embryos selection in IVF: non-invasive metabolomic and chemometric approach

We present here a new metabolomic methodology to predict embryo implantation ability in in vitro fertilization (IVF). In the present study we have included a total of 23 patients scheduled for IVF. Embryos were selected to be transferred by using morphological criteria on day 3 of in vitro culture. The relative amino acid concentrations in the embryo culture media were analyzed by HPLC–MS and HPLC–MS/MS. ¹H NMR metabolomic profiles were also obtained for the embryo culture media. Chemometric models were performed with SIMCA (soft independent modeling of class analogy) for samples from both, non-pregnancy and pregnancy cycles. The metabolic differences between the embryos, with pregnancy and non-pregnancy outcome, can be correlated with the relative amino acid concentrations and with ¹H NMR profiles. We used interval partial least square (iPLS) in order to identify the higher correlation between regions in the ¹H NMR spectra and the embryo implantation capability. The ¹H NMR regions with higher correlation are between 1.2 and 0.5 ppm, that included the signals for cholesterol backbone –C(18)H₃, –CH₃ and CH₂ groups of triglycerides, cholesterol compounds and phospholipids. Our results can allow building a quick, non invasive, useful and feasible chemometric models in order to identify embryos with a high pregnancy rate and embryos unable to achieve successful pregnancies.     

Prolactin isoforms secreted by human prolactinomas.

Prolactin (hPRL) secreted by human prolactinoma cells in culture was purified by gel filtration, lectin affinity chromatography and gel electrophoresis in order to identify the different isoforms of the hormone and to test their respective immunoreactivities and bioactivities. The nonglycosylated hPRL (NG-hPRL), unbound to lectins, was the major form and was a species (NG1-hPRL), of 23,000 (M(r)) apparent molecular weight. The lectin-bound glycosylated hPRL (G-hPRL) consisted of three forms, G1-, G2- and G3-hPRL, of identical molecular weights (25,000 M(r)). Endoglycosidase treatment indicated that these three forms differed by the heterogeneity of their carbohydrate chains. These G-PRLs proved to be 68% less immunoreactive and 50% less bioactive than NG-hPRL. It is concluded from these data that, in prolactinomas, the main variant of the hormone is the nonglycosylated form of PRL.     

Extracellular matrix composition and remodeling in human abdominal aortic aneurysms: a proteomics approach

Abdominal aortic aneurysms (AAA) are characterized by pathological remodeling of the aortic extracellular matrix (ECM). However, besides the well-characterized elastolysis and collagenolysis little is known about changes in other ECM proteins. Previous proteomics studies on AAA focused on cellular changes without emphasis on the ECM. In the present study, ECM proteins and their degradation products were selectively extracted from aneurysmal and control aortas using a solubility-based subfractionation methodology and analyzed by gel-liquid chromatography-tandem MS and label-free quantitation. The proteomics analysis revealed novel changes in the ECM of AAA, including increased expression as well as degradation of collagen XII, thrombospondin 2, aortic carboxypeptidase-like protein, periostin, fibronectin and tenascin. Proteomics also confirmed the accumulation of macrophage metalloelastase (MMP-12). Incubation of control aortic tissue with recombinant MMP-12 resulted in the extensive fragmentation of these glycoproteins, most of which are novel substrates of MMP-12. In conclusion, our proteomics methodology allowed the first detailed analysis of the ECM in AAA and identified markers of pathological ECM remodeling related to MMP-12 activity.     

Assessing individual metabolic responsiveness to a lipid challenge using a targeted metabolomic approach

The development of assessment techniques with immediate clinical applicability is a priority for resolving the growing epidemic in metabolic disease. Many imbalances in diet-dependent metabolism are not detectable in the fasted state. Resolving the high inter-individual variability in response to diet requires the development of techniques that can detect metabolic dysfunction at the level of the individual. The intra- and inter-individual variation in lipid metabolism in response to a standardized test meal was determined. Following an overnight fast on three different days, three healthy subjects consumed a test meal containing 40% of their daily calories. Plasma samples were collected at fasting, and 1, 3, 6, and 8 h after the test meal. Plasma fatty acid (FA) concentrations within separated lipid classes and lipoprotein fractions were measured at each time point. The intra-individual variation within each subject across three days was lower than the inter-individual differences among the three subjects for over 50% of metabolites in the triacylglycerol (TG), FA, and phosphatidylcholine (PC) lipid classes at 6 h, and for 25–50% of metabolites across lipid classes at 0, 1, 3, and 8 h. The consistency of response within individuals was visualized by principal component analysis (PCA) and confirmed by ANOVA. Three representative metabolites that discriminated among the three individuals in the apolipoprotein B (ApoB) fraction, TG16:1n7, TG18:2n6, and PC18:3n3, are discussed in detail. The postprandial responses of individuals were unique within metabolites that were individual discriminators (ID) of metabolic phenotype. This study shows that the targeted metabolomic measurement of individual metabolic phenotype in response to a specially formulated lipid challenge is possible even without lead-in periods, dietary and lifestyle control, or intervention over a 3-month period in healthy free-living individuals.     

Unbiased characterization of genotype-dependent metabolic regulations by metabolomic approach in Arabidopsis thaliana

Background  

Metabolites are not only the catalytic products of enzymatic reactions but also the active regulators or the ultimate phenotype of metabolic homeostasis in highly complex cellular processes. The modes of regulation at the metabolome level can be revealed by metabolic networks. We investigated the metabolic network between wild-type and 2 mutant (methionine-over accumulation 1 [mto1] and transparent testa4 [tt4]) plants regarding the alteration of metabolite accumulation in Arabidopsis thaliana.     

Induction of urokinase-type plasminogen activator by UV light in human fetal fibroblasts is mediated through a UV-induced secreted protein.

Plasminogen activator was previously shown to be induced by UV light in human cells with low capacity to repair UV-induced DNA lesions. We now show that in human fetal fibroblasts UV light enhanced the two mRNA species coding for the urokinase-type plasminogen activator (uPA) and the tissue-type plasminogen activator, but immunological analysis revealed exclusively uPA activity. Several independent and complementary experiments indicated that induction of uPA was mediated, apparently entirely, through a UV-induced, secreted protein (UVIS) in the growth medium of irradiated cells. First, elevation of uPA mRNA after irradiation was severely blocked by cycloheximide. Second, replacement of conditioned medium in irradiated cells while the rate of plasminogen activator induction was maximal rapidly and completely stopped any further increase in uPA activity. Third, addition of the same removed conditioned medium to nonirradiated cells mimicked UV light in enhancing the level of uPA activity as well as that of uPA mRNA. Fourth, UVIS activity was completely lost by treating the conditioned medium with trypsin but not with nucleases. Kinetic measurements indicated that the accumulation of UVIS rather than the induction of uPA by UVIS conferred the rate-limiting step in the overall process of uPA induction. Both UV light and UVIS acted synergistically with inhibitors of DNA repair for uPA induction. Based on these results, a model is proposed implicating relaxation of DNA torsional stress of an as yet undefined DNA sequence(s) in the induction of UVIS, which is then responsible for activation of the uPA gene.     

Analysis of biosynthetic fluctuations of cultured Taxus seedlings using a metabolomic approach

Fluctuations in the biosynthesis of taxoids in 1–5 year old cultured seedlings of Taxus chinensis var. mairei were investigated using LC–IT-TOF-MS and a metabolomics approach. In the total ion chromatogram (TIC) of the extracts, 16 prominent peaks were observed. Ten compounds were identified by comparison of retention times and MS/MS spectra with those of reference compounds. An additional 6 taxoids were isolated by preparative HPLC and identified by comparison of their spectroscopic data with those reported in the literature. It was clarified that the relative concentrations of taxoids with 4(20) double bonds are high at early stages of cultivation. On the other hand, relatively higher amounts of 5-acetoxy taxoids oxidized at the 4- and 10- positions and taxoids having 5(20)-oxetane rings were found at later stages of cultivation. This approach provides practical information on the biosynthetic flow of taxoids in cultured yew seedlings.     

Annexin 1 is secreted by the human prostate

The human prostate expresses very high concentrations of annexins 1, 4, and 5 and secretes high concentrations of annexins 1 and 5. Although the biological roles of these proteins in prostate secretions are not known, these studies emphasize the need to consider extracellular sites for physiological functions of annexins. The clear demonstration of secretion of proteins that have blocked N-termini and lack hydrophobic signal sequences raises the possibility that novel cellular secretory pathways exist. Preliminary immunohistochemical experiments in collaboration with Dr. James Fallon indicate that both annexins 1 and 4 are expressed in prostate ductal secretory epithelium. Since annexin 1, but not annexin 4, is secreted, a comparison of the cellular fate of these two related proteins in the prostate may provide a useful model system for determining the structural elements that direct the secretion of proteins which lack conventional signal sequences.     

LsrR quorum sensing "switch" is revealed by a bottom-up approach

Quorum sensing (QS) enables bacterial multicellularity and selective advantage for communicating populations. While genetic "switching" phenomena are a common feature, their mechanistic underpinnings have remained elusive. The interplay between circuit components and their regulation are intertwined and embedded. Observable phenotypes are complex and context dependent. We employed a combination of experimental work and mathematical models to decipher network connectivity and signal transduction in the autoinducer-2 (AI-2) quorum sensing system of E. coli. Negative and positive feedback mechanisms were examined by separating the network architecture into sub-networks. A new unreported negative feedback interaction was hypothesized and tested via a simple mathematical model. Also, the importance of the LsrR regulator and its determinant role in the E. coli QS "switch", normally masked by interfering regulatory loops, were revealed. Our simple model allowed mechanistic understanding of the interplay among regulatory sub-structures and their contributions to the overall native functioning network. This "bottom up" approach in understanding gene regulation will serve to unravel complex QS network architectures and lead to the directed coordination of emergent behaviors.     

Protein expression profiles in an endosymbiotic cyanobacterium revealed by a proteomic approach

Molecular mechanisms behind adaptations in the cyanobacterium (Nostoc sp.) to a life in endosymbiosis with plants are still not clarified, nor are the interactions between the partners. To get further insights, the proteome of a Nostoc strain, freshly isolated from the symbiotic gland tissue of the angiosperm Gunnera manicata Linden, was analyzed and compared with the proteome of the same strain when free-living. Extracted proteins were separated by two-dimensional gel electrophoresis and were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry combined with tandem mass spectrometry. Even when the higher percentage of differentiated cells (heterocysts) in symbiosis was compensated for, the majority of the proteins detected in the symbiotic cyanobacteria were present in the free-living counterpart, indicating that most cellular processes were common for both stages. However, differential expression profiling revealed a significant number of proteins to be down-regulated or missing in the symbiotic stage, while others were more abundant or only expressed in symbiosis. The differential protein expression was primarily connected to i) cell envelope-associated processes, including proteins involved in exopolysaccharide synthesis and surface and membrane associated proteins, ii) to changes in growth and metabolic activities (C and N), including upregulation of nitrogenase and proteins involved in the oxidative pentose phosphate pathway and downregulation of Calvin cycle enzymes, and iii) to the dark, microaerobic conditions offered inside the Gunnera gland cells, including changes in relative phycobiliprotein concentrations. This is the first comprehensive analysis of proteins in the symbiotic state.     

Lysis of human immunodeficiency virus type 1 by a specific secreted human phospholipase A2

Phospholipase A2 (PLA2) proteins affect cellular activation, signal transduction, and possibly innate immunity. A specific secretory PLA2, sPLA2-X, is shown here to neutralize human immunodeficiency virus type 1 (HIV-1) through degradation of the viral membrane. Catalytic function was required for antiviral activity, and the target cells of infection were unaffected. sPLA2-X potently reduced gene transfer of HIV-1 Env-pseudotyped lentivirus vectors and inhibited the replication of both CCR5- and CXCR4-tropic HIV-1 in human CD4+ T cells. Virions resistant to damage by antibody and complement were sensitive to lysis by sPLA2-X, suggesting a novel mechanism of antiviral surveillance independent of the acquired immune system.     

Prediction of peptide reactivity with human IVIg through a knowledge-based approach

The prediction of antibody-protein (antigen) interactions is very difficult due to the huge variability that characterizes the structure of the antibodies. The region of the antigen bound to the antibodies is called epitope. Experimental data indicate that many antibodies react with a panel of distinct epitopes (positive reaction). The Challenge 1 of DREAM5 aims at understanding whether there exists rules for predicting the reactivity of a peptide/epitope, i.e., its capability to bind to human antibodies. DREAM 5 provided a training set of peptides with experimentally identified high and low reactivities to human antibodies. On the basis of this training set, the participants to the challenge were asked to develop a predictive model of reactivity. A test set was then provided to evaluate the performance of the model implemented so far.We developed a logistic regression model to predict the peptide reactivity, by facing the challenge as a machine learning problem. The initial features have been generated on the basis of the available knowledge and the information reported in the dataset. Our predictive model had the second best performance of the challenge. We also developed a method, based on a clustering approach, able to "in-silico" generate a list of positive and negative new peptide sequences, as requested by the DREAM5 "bonus round" additional challenge.The paper describes the developed model and its results in terms of reactivity prediction, and highlights some open issues concerning the propensity of a peptide to react with human antibodies.     

Trafficking and postsecretory events responsible for the formation of secreted human salivary peptides: a proteomics approach

To elucidate the localization of post-translational modifications of different classes of human salivary proteins and peptides (acidic and basic proline-rich proteins (PRPs), Histatins, Statherin, P-B peptide, and "S type" Cystatins) a comparative reversed phase HPLC-ESI-MS analysis on intact proteins of enriched granule preparations from parotid and submandibular glands as well as parotid, submandibular/sublingual (Sm/Sl), and whole saliva was performed. The main results of this study indicate the following. (i) Phosphorylation of all salivary peptides, sulfation of Histatin 1, proteolytic cleavages of acidic and precursor basic PRPs occur before granule storage. (ii) In agreement with previous studies, basic PRPs are secreted by the parotid gland only, whereas all isoforms of acidic PRPs (aPRPs) are secreted by both parotid and Sm/Sl glands. (iii) Phosphorylation levels of aPRPs, Histatin 1, and Statherin are higher in the parotid gland, whereas the extent of cleavage of aPRP is higher in Sm/Sl glands. (iv) O-Sulfation of tyrosines of Histatin 1 is a post-translational modification specific for the submandibular gland. (v) The concentration of Histatin 3, Histatin 5, and Histatin 6, but not Histatin 1, is higher in parotid saliva. (vi) Histatin 3 is submitted to the first proteolytic cleavage (generating Histatins 6 and 5) during granule maturation, and it occurs to the same relative extent in both glands. (vii) The proteolytic cleavages of Histatin 5 and 6, generating a cascade of Histatin 3 fragments, take place after granule secretion and are more extensive in parotid secretion. (viii) Basic PRPs are cleaved in the oral cavity by unknown peptidases, generating various small proline-rich peptides. (ix) C-terminal removal from Statherin is more extensive in parotid saliva. (x) P-B peptide is secreted by both glands, and its relative quantity is higher in submandibular/sublingual secretion. (xi) In agreement with previous studies, S type Cystatins are mainly the product of Sm/Sl glands.     

Identification of human plasma metabolites exhibiting time-of-day variation using an untargeted liquid chromatography-mass spectrometry metabolomic approach

Although daily rhythms regulate multiple aspects of human physiology, rhythmic control of the metabolome remains poorly understood. The primary objective of this proof-of-concept study was identification of metabolites in human plasma that exhibit significant 24-h variation. This was assessed via an untargeted metabolomic approach using liquid chromatography-mass spectrometry (LC-MS). Eight lean, healthy, and unmedicated men, mean age 53.6 (SD ± 6.0) yrs, maintained a fixed sleep/wake schedule and dietary regime for 1 wk at home prior to an adaptation night and followed by a 25-h experimental session in the laboratory where the light/dark cycle, sleep/wake, posture, and calorific intake were strictly controlled. Plasma samples from each individual at selected time points were prepared using liquid-phase extraction followed by reverse-phase LC coupled to quadrupole time-of-flight MS analysis in positive ionization mode. Time-of-day variation in the metabolites was screened for using orthogonal partial least square discrimination between selected time points of 10:00 vs. 22:00 h, 16:00 vs. 04:00 h, and 07:00 (d 1) vs. 16:00 h, as well as repeated-measures analysis of variance with time as an independent variable. Subsequently, cosinor analysis was performed on all the sampled time points across the 24-h day to assess for significant daily variation. In this study, analytical variability, assessed using known internal standards, was low with coefficients of variation <10%. A total of 1069 metabolite features were detected and 203 (19%) showed significant time-of-day variation. Of these, 34 metabolites were identified using a combination of accurate mass, tandem MS, and online database searches. These metabolites include corticosteroids, bilirubin, amino acids, acylcarnitines, and phospholipids; of note, the magnitude of the 24-h variation of these identified metabolites was large, with the mean ratio of oscillation range over MESOR (24-h time series mean) of 65% (95% confidence interval [CI]: 49-81%). Importantly, several of these human plasma metabolites, including specific acylcarnitines and phospholipids, were hitherto not known to be 24-h variant. These findings represent an important baseline and will be useful in guiding the design and interpretation of future metabolite-based studies.     

Evidence of a robust resident bacteriophage population revealed through analysis of the human salivary virome

Viruses are the most abundant known infectious agents on the planet and are significant drivers of diversity in a variety of ecosystems. Although there have been numerous studies of viral communities, few have focused on viruses within the indigenous human microbiota. We analyzed 2 267 695 virome reads from viral particles and compared them with 263 516 bacterial 16S rRNA gene sequences from the saliva of five healthy human subjects over a 2- to 3-month period, in order to improve our understanding of the role viruses have in the complex oral ecosystem. Our data reveal viral communities in human saliva dominated by bacteriophages whose constituents are temporally distinct. The preponderance of shared homologs between the salivary viral communities in two unrelated subjects in the same household suggests that environmental factors are determinants of community membership. When comparing salivary viromes to those from human stool and the respiratory tract, each group was distinct, further indicating that habitat is of substantial importance in shaping human viromes. Compared with coexisting bacteria, there was concordance among certain predicted host–virus pairings such as Veillonella and Streptococcus, whereas there was discordance among others such as Actinomyces. We identified 122 728 virulence factor homologs, suggesting that salivary viruses may serve as reservoirs for pathogenic gene function in the oral environment. That the vast majority of human oral viruses are bacteriophages whose putative gene function signifies some have a prominent role in lysogeny, suggests these viruses may have an important role in helping shape the microbial diversity in the human oral cavity.     

Responses of sympatric canids to human development revealed through citizen science

Measuring wildlife responses to anthropogenic activities often requires long‐term, large‐scale datasets that are difficult to collect. This is particularly true for rare or cryptic species, which includes many mammalian carnivores. Citizen science, in which members of the public participate in scientific work, can facilitate collection of large datasets while increasing public awareness of wildlife research and conservation. Hunters provide unique benefits for citizen science given their knowledge and interest in outdoor activities. We examined how anthropogenic changes to land cover impacted relative abundance of two sympatric canids, coyote (Canis latrans), and red fox (Vulpes vulpes) at a large spatial scale. In order to assess how land cover affected canids at this scale, we used citizen science data from bow hunter sighting logs collected throughout New York State, USA, during 2004–2017. We found that the two species had contrasting responses to development, with red foxes positively correlated and coyotes negatively correlated with the percentage of low‐density development. Red foxes also responded positively to agriculture, but less so when agricultural habitat was fragmented. Agriculture provides food and denning resources for red foxes, whereas coyotes may select forested areas for denning. Though coyotes and red foxes compete in areas of sympatry, we did not find a relationship between species abundance, likely a consequence of the coarse spatial resolution used. Red foxes may be able to coexist with coyotes by altering their diets and habitat use, or by maintaining territories in small areas between coyote territories. Our study shows the value of citizen science, and particularly hunters, in collection of long‐term data across large areas (i.e., the entire state of New York) that otherwise would unlikely be obtained.