Evaluating the Impact of Different Sequence Databases on Metaproteome Analysis: Insights from a Lab-Assembled Microbial Mixture

Metaproteomics enables the investigation of the protein repertoire expressed by complex microbial communities. However, to unleash its full potential, refinements in bioinformatic approaches for data analysis are still needed. In this context, sequence databases selection represents a major challenge.This work assessed the impact of different databases in metaproteomic investigations by using a mock microbial mixture including nine diverse bacterial and eukaryotic species, which was subjected to shotgun metaproteomic analysis. Then, both the microbial mixture and the single microorganisms were subjected to next generation sequencing to obtain experimental metagenomic- and genomic-derived databases, which were used along with public databases (namely, NCBI, UniProtKB/SwissProt and UniProtKB/TrEMBL, parsed at different taxonomic levels) to analyze the metaproteomic dataset. First, a quantitative comparison in terms of number and overlap of peptide identifications was carried out among all databases. As a result, only 35% of peptides were common to all database classes; moreover, genus/species-specific databases provided up to 17% more identifications compared to databases with generic taxonomy, while the metagenomic database enabled a slight increment in respect to public databases. Then, database behavior in terms of false discovery rate and peptide degeneracy was critically evaluated. Public databases with generic taxonomy exhibited a markedly different trend compared to the counterparts. Finally, the reliability of taxonomic attribution according to the lowest common ancestor approach (using MEGAN and Unipept software) was assessed. The level of misassignments varied among the different databases, and specific thresholds based on the number of taxon-specific peptides were established to minimize false positives. This study confirms that database selection has a significant impact in metaproteomics, and provides critical indications for improving depth and reliability of metaproteomic results. Specifically, the use of iterative searches and of suitable filters for taxonomic assignments is proposed with the aim of increasing coverage and trustworthiness of metaproteomic data.     

Laminin 332 in junctional epidermolysis bullosa

Laminin 332 is an essential component of the dermal-epidermal junction, a highly specialized basement membrane zone that attaches the epidermis to the dermis and thereby provides skin integrity and resistance to external mechanical forces. Mutations in the LAMA3, LAMB3 and LAMC2 genes that encode the three constituent polypeptide chains, α3, β3 and γ2, abrogate or perturb the functions of laminin 332. The phenotypic consequences are diminished dermal-epidermal adhesion and, as clinical symptoms, skin fragility and mechanically induced blistering. The disorder is designated as junctional epidermolysis bullosa (JEB). This article delineates the signs and symptoms of the different forms of JEB, the mutational spectrum, genotype-phenotype correlations as well as perspectives for future molecular therapies.     

ERP Modulation during Observation of Abstract Paintings by Franz Kline

The aim of this study was to test the involvement of sensorimotor cortical circuits during the beholding of the static consequences of hand gestures devoid of any meaning.In order to verify this hypothesis we performed an EEG experiment presenting to participants images of abstract works of art with marked traces of brushstrokes. The EEG data were analyzed by using Event Related Potentials (ERPs). We aimed to demonstrate a direct involvement of sensorimotor cortical circuits during the beholding of these selected works of abstract art. The stimuli consisted of three different abstract black and white paintings by Franz Kline. Results verified our experimental hypothesis showing the activation of premotor and motor cortical areas during stimuli observation. In addition, abstract works of art observation elicited the activation of reward-related orbitofrontal areas, and cognitive categorization-related prefrontal areas. The cortical sensorimotor activation is a fundamental neurophysiological demonstration of the direct involvement of the cortical motor system in perception of static meaningless images belonging to abstract art. These results support the role of embodied simulation of artist’s gestures in the perception of works of art.     

Getting Your Sea Legs

Sea travel mandates changes in the control of the body. The process by which we adapt bodily control to life at sea is known as getting one''s sea legs. We conducted the first experimental study of bodily control as maritime novices adapted to motion of a ship at sea. We evaluated postural activity (stance width, stance angle, and the kinematics of body sway) before and during a sea voyage. In addition, we evaluated the role of the visible horizon in the control of body sway. Finally, we related data on postural activity to two subjective experiences that are associated with sea travel; seasickness, and mal de debarquement. Our results revealed rapid changes in postural activity among novices at sea. Before the beginning of the voyage, the temporal dynamics of body sway differed among participants as a function of their (subsequent) severity of seasickness. Body sway measured at sea differed among participants as a function of their (subsequent) experience of mal de debarquement. We discuss implications of these results for general theories of the perception and control of bodily orientation, for the etiology of motion sickness, and for general phenomena of perceptual-motor adaptation and learning.     

Lifelong Expression of Apolipoprotein D in the Human Brainstem: Correlation with Reduced Age-Related Neurodegeneration

The lipocalin apolipoprotein D (Apo D) is upregulated in peripheral nerves following injury and in regions of the central nervous system, such as the cerebral cortex, hippocampus, and cerebellum, during aging and progression of certain neurological diseases. In contrast, few studies have examined Apo D expression in the brainstem, a region necessary for survival and generally less prone to age-related degeneration. We measured Apo D expression in whole human brainstem lysates by slot-blot and at higher spatial resolution by quantitative immunohistochemistry in eleven brainstem nuclei (the 4 nuclei of the vestibular nuclear complex, inferior olive, hypoglossal nucleus, oculomotor nucleus, facial motor nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve, and Roller`s nucleus). In contrast to cortex, hippocampus, and cerebellum, apolipoprotein D was highly expressed in brainstem tissue from subjects (N = 26, 32−96 years of age) with no history of neurological disease, and expression showed little variation with age. Expression was significantly stronger in somatomotor nuclei (hypoglossal, oculomotor, facial) than visceromotor or sensory nuclei. Both neurons and glia expressed Apo D, particularly neurons with larger somata and glia in the periphery of these brainstem centers. Immunostaining was strongest in the neuronal perinuclear region and absent in the nucleus. We propose that strong brainstem expression of Apo D throughout adult life contributes to resistance against neurodegenerative disease and age-related degeneration, possibly by preventing oxidative stress and ensuing lipid peroxidation.     

Biosynthesis of Vitamins and Cofactors in Bacterium-Harbouring Trypanosomatids Depends on the Symbiotic Association as Revealed by Genomic Analyses

Some non-pathogenic trypanosomatids maintain a mutualistic relationship with a betaproteobacterium of the Alcaligenaceae family. Intensive nutritional exchanges have been reported between the two partners, indicating that these protozoa are excellent biological models to study metabolic co-evolution. We previously sequenced and herein investigate the entire genomes of five trypanosomatids which harbor a symbiotic bacterium (SHTs for Symbiont-Haboring Trypanosomatids) and the respective bacteria (TPEs for Trypanosomatid Proteobacterial Endosymbiont), as well as two trypanosomatids without symbionts (RTs for Regular Trypanosomatids), for the presence of genes of the classical pathways for vitamin biosynthesis. Our data show that genes for the biosynthetic pathways of thiamine, biotin, and nicotinic acid are absent from all trypanosomatid genomes. This is in agreement with the absolute growth requirement for these vitamins in all protozoa of the family. Also absent from the genomes of RTs are the genes for the synthesis of pantothenic acid, folic acid, riboflavin, and vitamin B_6. This is also in agreement with the available data showing that RTs are auxotrophic for these essential vitamins. On the other hand, SHTs are autotrophic for such vitamins. Indeed, all the genes of the corresponding biosynthetic pathways were identified, most of them in the symbiont genomes, while a few genes, mostly of eukaryotic origin, were found in the host genomes. The only exceptions to the latter are: the gene coding for the enzyme ketopantoate reductase (EC:1.1.1.169) which is related instead to the Firmicutes bacteria; and two other genes, one involved in the salvage pathway of pantothenic acid and the other in the synthesis of ubiquinone, that are related to Gammaproteobacteria. Their presence in trypanosomatids may result from lateral gene transfer. Taken together, our results reinforce the idea that the low nutritional requirement of SHTs is associated with the presence of the symbiotic bacterium, which contains most genes for vitamin production.     

Exploring Leptin Antagonism in Ophthalmic Cell Models

Background

Emerging evidence suggests that angiogenic and pro-inflammatory cytokine leptin might be implicated in ocular neovascularization. However, the potential of inhibiting leptin function in ophthalmic cells has never been explored. Here we assessed mitogenic, angiogenic, and signaling leptin activities in retinal and corneal endothelial cells and examined the capability of a specific leptin receptor (ObR) antagonist, Allo-aca, to inhibit these functions.

Methods and Results

The experiments were carried out in monkey retinal (RF/6A) and bovine corneal (BCE) endothelial cells. Leptin at 50-250 ng/mL stimulated the growth of both cell lines in a dose-dependent manner. The maximal mitogenic response (35±7 and 27±3% in RF6A and BCE cells, respectively) was noted at 24 h of 250 ng/mL leptin treatments. Leptin-dependent proliferation was reduced to base levels with 10 and 100 nM Allo-aca in BCE and RF6A cells, respectively. In both cell lines, leptin promoted angiogenic responses, with the maximal increase in tube formation (163±10 and 133±8% in RF6A and BCE cultures, respectively) observed under a 250 ng/mL leptin treatment for 3 h. Furthermore, in both cell lines 250 ng/mL leptin modulated the activity or expression of several signaling molecules involved in proliferation, inflammatory activity and angiogenesis, such as STAT3, Akt, and ERK1/2, COX2, and NFκB. In both cell lines, leptin-induced angiogenic and signaling responses were significantly inhibited with 100 nM Allo-aca. We also found that leptin increased its own mRNA and protein expression in both cell lines, and this autocrine effect was abolished by 100-250 nM Allo-aca.

Conclusions

Our data provide new insights into the role of leptin in ocular endothelial cells and represent the first original report on targeting ObR in ophthalmic cell models.     

Effects of Highly Conserved Major Histocompatibility Complex (MHC) Extended Haplotypes on Iron and Low CD8+ T Lymphocyte Phenotypes in HFE C282Y Homozygous Hemochromatosis Patients from Three Geographically Distant Areas

Hereditary Hemochromatosis (HH) is a recessively inherited disorder of iron overload occurring commonly in subjects homozygous for the C282Y mutation in HFE gene localized on chromosome 6p21.3 in linkage disequilibrium with the human leukocyte antigen (HLA)-A locus. Although its genetic homogeneity, the phenotypic expression is variable suggesting the presence of modifying factors. One such genetic factor, a SNP microhaplotype named A-A-T, was recently found to be associated with a more severe phenotype and also with low CD8⁺T-lymphocyte numbers. The present study aimed to test whether the predictive value of the A-A-T microhaplotype remained in other population settings. In this study of 304 HH patients from 3 geographically distant populations (Porto, Portugal 65; Alabama, USA 57; Nord-Trøndelag, Norway 182), the extended haplotypes involving A-A-T were studied in 608 chromosomes and the CD8⁺ T-lymphocyte numbers were determined in all subjects. Patients from Porto had a more severe phenotype than those from other settings. Patients with A-A-T seemed on average to have greater iron stores (p = 0.021), but significant differences were not confirmed in the 3 separate populations. Low CD8⁺ T-lymphocytes were associated with HLA-A*03-A-A-T in Porto and Alabama patients but not in the greater series from Nord-Trøndelag. Although A-A-T may signal a more severe iron phenotype, this study was unable to prove such an association in all population settings, precluding its use as a universal predictive marker of iron overload in HH. Interestingly, the association between A-A-T and CD8⁺ T-lymphocytes, which was confirmed in Porto and Alabama patients, was not observed in Nord-Trøndelag patients, showing that common HLA haplotypes like A*01–B*08 or A*03–B*07 segregating with HFE/C282Y in the three populations may carry different messages. These findings further strengthen the relevance of HH as a good disease model to search for novel candidate loci associated with the genetic transmission of CD8⁺ T-lymphocyte numbers.