High Number Of Proteins Research Articles

Initial proteomic characterization of IMMODIN, commercially available dialysable leukocytes extract

One of the most significant public health challenges of the twenty-first century is the prevention and treatment of infectious diseases caused by various microorganisms, including resistant bacteria and viruses. With aging, there is a progressive alteration of the immune system and its responses that may result in immune senescence and immunodepression. Nevertheless, immune modulators can speed up recovery. IMMODIN® is a commercially available dialysable leukocyte extract prepared from disintegrated white blood cells of healthy human donors. It is used clinically as a prophylactic or therapeutic agent when cell-mediated immunity plays a key role. This initial proteomic study was aiming to evaluate the polypeptide repertoire present in this immunologic preparation. Using our approach, we identified forty-eight unique proteins associated with blood cells or plasma. From them, twenty-eight were detected in at least two independent analyses of three IMMODIN® preparations. The highest number of proteins was related to innate immunity. There were identified receptors for parasites or microbes that have the ability to recognize the invaders and initiate their inhibition or elimination. We also detected proteins associated with inflammatory response, including those having the potential to inhibit cytokines and treat the cytokine storm. There were found even proteins that can speed up recovery by regulating cell growth and repair.

The metabolic pathways of polyhydroxyalkanoates and exopolysaccharides synthesized by Haloferax mediterranei in response to elevated salinity

How polymer synthesis is mobilized or activated as a biological response of Haloferax mediterranei against hypertonic conditions remains largely unexplored. This study investigated the protein expression of H. mediterranei in response to high salinity by using isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis. The microbes were harvested at end of fermentation at the NaCl salinity of 75 and 250 g L−1. Among the identified 2123 proteins, 170 proteins were differentially expressed. Gene ontology annotation revealed that the highest number of proteins was annotated in biological process category, which was responsible for metabolic process, cellular component and catalytic activity. Differentially expressed proteins were belonged to the class of response to stimulus as well as catalytic activity and binding. Under high salinity conditions, three pathways were established as key responses of PHA and EPS production to hypertonic pressure. Two overexpressed proteins, beta-ketoacyl-ACP reductase and 3-hydroxyacyl-CoA dehydrogenase, enhanced the synthesis of PHAs. The serine-pyruvate transaminase and serine-glyoxylate transaminase were upregulated, thereby increasing the conversion of glucose to PHA. Downregulated levels of sulfate-adenylyl transferase and adenylyl-sulfate kinase could cause diminished EPS synthesis. This study could contribute to better understanding of the proteomic mechanisms of the synthesized polymers in defending against salt stress. SignificanceHaloferax mediterranei, a family member of halophilic archaea, is well known for its fermentative production of poly-β-hydroxyalkanoates (PHAs). PHAs are natural polymers that exhibit great potential in a wide range of applications such as a good alternative to petroleum-based plastics and the biocompatible material. For decades, the functional role of PHAs synthesized by H. mediterranei is deemed to be carbon and energy reservations. The finding proved that differential production of PHA and EPS in H. mediterranei exposed to elevated salinity was caused by differential protein expression. This is the first report on how PHA and EPS synthesized by H. mediterranei is mobilized as the response of increased salinity, contributing to the understanding of halophilic archaea's response to hypertonic stress and the precise control of fermentation production. Despite its advantages as a PHA cell factory, H. mediterranei synthesized EPS simultaneously, thereby lowering the maximum yield of PHA production. Overall, salinity can be used as a vital microbial fermentation parameter to obtain the highest harvest of PHA, as well as the lowest EPS synthesis in industrial fermentation.

Salivary proteome analysis of crack cocaine dependents

ObjectiveSalivary proteomic analysis may help to understand physiopathological changes in crack cocaine dependents. This study aimed to compare the salivary protein profile between crack cocaine dependents and non-drug users. DesignNine heavy smokers and alcohol consumers men admitted to rehab due to crack cocaine abuse and nine non-drug users age-matched men were evaluated. Unstimulated whole saliva was collected. Proteomic analysis was performed by mass spectrometer. Data were processed using ProteinLynx GlobalServer software. Results were obtained by searching the Homo sapiens database from the UniProt catalog. The search tool IBI-IMIM was used to identify proteins candidates for biomarkers. ResultsThe mean age of crack cocaine and control groups was 36.89 ± 7.78 and 35.78 ± 6.68 years, respectively. 458 salivary proteins were identified in both groups; 305 proteins in the crack cocaine group. Among the 68 proteins presented in both groups, 29 were down-regulated (i.e. “Statherin” and “Transforming growth factor-beta-induced protein ig-h3” were down-regulated at least 10-fold) and 27 up-regulated (i.e. “Negative elongation factor” was up-regulated 19-fold) in the crack cocaine group compared to controls. 90 out of the 458 proteins found in the proteomic analysis were identified as candidates for biomarkers of diseases. Among these, 65 (72.22 %) were detected in the crack cocaine group. ConclusionCrack cocaine dependents with chronic alcohol and tobacco use have a higher number of proteins in saliva compared to non-drug users. 22.3 % of salivary proteins present in crack cocaine dependents were present in controls; 3.9 % of them were expressed in similar quantity.

Study of the Influence of Solvent and Column Temperature on the Separation Effectiveness of LMW Glutenins by RP-HPLC

Gluten contains two fractions, which are represented in equal amounts. These are soluble gliadins and insoluble glutenins. Glutenin fraction is responsible for the dough and gluten viscoelastic properties. The dominant type of protein in glutenins are LMW glutenins. The aim of this paper was to examine the influence of solvent type and column temperature on the separation effectiveness of LMW glutenins. Extraction was performed with 50% (v/v) ethanol, 1-propanol and isopropanol to which Tris-HCl (0.05 mol/l, pH=7.5), urea (2 mol/l) and dithioerythritol (1%) were added. Separation of LMW glutenins was performed on HPLC Agilent Techologies 1260 Infinity apparatus. After the extraction with 50% (v/v) ethanol, 1-propanol and isopropanol to which Tris-HCl (0.05 mol/l, pH=7.5), urea (2 mol/l) and dithioerythritol (1%) were added, the highest number of proteins was observed after extraction with 50% (v/v) 1-propanol and at a column temperature of 40°C (Xsr=20, respectively RC=69.56%), and the lowest number at a column temperature of 45°C (Xsr=14.17, respectively RC=66.42%). The obtained results were read at a wavelength of 210 nm. After the extraction of LMW glutenins with 50% (v/v) ethanol, 1-propanol and isopropanol to which Tris-HCl (0.05 mol/l, pH=7.5), urea (2 mol/l) and dithioerythritol (1%) were added and detected at a wavelength of 280 nm, the highest number of proteins was observed after extraction with 50% (v/v) isopropanol and at a column temperature of 50°C (Xsr=24.17, RC=56.47%) and the lowest number of proteins after extraction with 50% (v/v) ethanol and at a column temperature of 50°C (Xsr=12.17, RC=56.45%).

Proteomic analysis upon peach fruit infection with Monilinia fructicola and M. laxa identify responses contributing to brown rot resistance

Brown rot, caused by Monilinia spp., is a major peach disease worldwide. In this study, the response of peach cultivars Royal Glory (RG) and Rich Lady (RL) to infection by Monilinia fructicola or Monilinia laxa, was characterized. Phenotypic data, after artificial inoculations, revealed that ‘RL’ was relatively susceptible whereas ‘RG’ was moderately resistant to Monilinia spp. Comparative proteomic analysis identified mesocarp proteins of the 2 cultivars whose accumulation were altered by the 2 Monilinia species. Functional analysis indicated that pathogen-affected proteins in ‘RG’ were mainly involved in energy and metabolism, while, differentially accumulated proteins by the pathogen presence in ‘RL’ were involved in disease/defense and metabolism. A higher number of proteins was differentiated in ‘RG’ fruit compared to ‘RL’. Upon Monilinia spp. infection, various proteins were-down accumulated in ‘RL’ fruit. Protein identification by mass spectrometric analysis revealed that several defense-related proteins including thaumatin, formate dehydrogenase, S-formylglutathione hydrolase, CBS domain-containing protein, HSP70, and glutathione S-transferase were up-accumulated in ‘RG’ fruit following inoculation. The expression profile of selected defense-related genes, such as major latex allergen, 1-aminocyclopropane-1-carboxylate deaminase and UDP-glycoltransferase was assessed by RT-PCR. This is the first study deciphering differential regulations of peach fruit proteome upon Monilinia infection elucidating resistance responses.

Revisiting Old Questions and New Approaches to Investigate the Fungal Cell Wall Construction.

The beginning of our understanding of the cell wall construction came from the work of talented biochemists in the 70-80's. Then came the era of sequencing. Paradoxically, the accumulation of fungal genomes complicated rather than solved the mystery of cell wall construction, by revealing the involvement of a much higher number of proteins than originally thought. The situation has become even more complicated since it is now recognized that the cell wall is an organelle whose composition continuously evolves with the changes in the environment or with the age of the fungal cell. The use of new and sophisticated technologies to observe cell wall construction at an almost atomic scale should improve our knowledge of the cell wall construction. This essay will present some of the major and still unresolved questions to understand the fungal cell wall biosynthesis and some of these exciting futurist approaches.

Shotgun proteomic analysis of protein profile changes in female rabbit meat: the effect of breed and age

To determine the protein profile of rabbit meat, the protein composition in meat of rabbits with different breeds and ages was investigated. In this article, M. Longissimus thoracis et lumborum (LTL) samples were obtained from six groups (ten rabbits in each group) of 60 female rabbits (Ira and Sichuan white; 35, 70, and 105 days old). Their nutritional characteristics, amino acid profile, protein quantity, and protein profile were analysed. The results showed that rabbit meat contained high content of essential amino acids, especially for Lys (2.26%). The analysis of amino acid profile in rabbit meat indicated that it varied significantly with rabbit breeds and ages. The proportion of stromal protein to total protein in rabbit meat was significantly influenced by age (p < .05), and the mean proportions of sarcoplasmic, myofibrillar, and stromal protein to total protein in rabbit meat were 32.57%, 57.20% and 10.23%, respectively. A total of 835 proteins were identified from the samples using shotgun proteomics. These proteins were mainly involved in energy metabolism, material transport and biochemical reactions, and the highest number of proteins was observed in meat of 35-day-old rabbit (p < .05). Although breed had a limited influence on the number of proteins in rabbit meat (p > .05), 69 and 35 unique proteins were identified in Ira and Sichuan white, respectively. In conclusion, both breed and age can affect the protein profile of rabbit meat, and the difference in protein profile may be the reason for the quality variation of different rabbit meat samples.HighlightsComparing the differences between two rabbit breeds from the perspective of proteome.Elucidate proteome in rabbit muscle during its growth. The outcome means that a total of 835 proteins were identified from the samples using shotgun proteomics, and the highest number of unique proteins was observed in meat of 35-day-old rabbit.

Protein and peptide profiles in neonatal meconium.

The proteins accumulated in the meconium reflect the intrauterine environment and are naturally excreted by a neonate. The identification and classification of individual meconium proteins may be a valuable source of information about physiological and pathological processes in utero. Proteomic analysis was used to study the protein composition in pooled 50 serial meconium portions from 10 neonates. The proteins were classified based on the gene ontology database. The amounts and relative number of proteins (%) in the identified categories and their subcategories were assessed. A total of 946 proteins identified in meconium, including 430 represented by two or more peptides were classified into three categories: biological process (n = 401), molecular function (n = 386) and cellular component (n = 422). The highest number of proteins (>25% of the total) was found in the subcategories: developmental processes, signaling, transport, response to stimulus, regulation, metabolic processes, ion binding, extracellular region, membrane and cytoplasm. The composition of meconium proteins identified in this study may be a rich source of new biomarkers for use in neonatology with a potential to predict later development.

Next Generation Proteomics of Human Neutrophil Granulocytes in Monogenic Disease

Background and objectiveNeutrophil granulocytes (NG) are critical mediators of innate immunity and tissue regeneration. Few monogenic diseases affecting their differentiation and/or function have been described, yet a systems-biology perspective has not yet been opened. We set out to define the proteome and transcriptome of NG from healthy individuals and exome-sequenced patients aiming to develop a multi-dimensional perspective for diagnostic purposes.Patients and methodsWe analyzed highly purified peripheral blood neutrophil granulocytes from 68 healthy individuals, 6 children with severe congenital neutropenia (SCN) associated with ELANE mutations, 5 children with chronic granulomatous disease (CGD) with CYBA (2) or CYBB (3) mutations and 3 children with leukocyte adhesion deficiency (LAD, 2x ITGB2 mutation, 1x SLC35C1 mutation). In addition we analyzed proteomes of 2 children with genetically undetermined neutrophil defects. Cells were isolated from fresh venous blood using magnetic bead-based negative selection (purity >99%). Whole cell proteome analysis was done by data-independent acquisition using a Thermo Fisher QExactive HF mass spectrometer.ResultsWe quantified a total of ~4300 proteins in each sample. This is the first study to analyze highly purified neutrophil granulocytes from a large cohort of healthy individuals as well as from patients with rare diseases affecting differentiation and/or function of NG. Intraindividual reproducibility was shown for one donor sampled at 3 different time points with a correlation coefficient of ~0.89. Principal component analysis demonstrated separation of the proteome of healthy and diseased cells. Interestingly, only 6 proteins constituted for 50% of the neutrophils proteome with the antimicrobial proteins DEF3A and S100A8 being the most abundant.Stringent differential expression analysis (p<0.01) showed specific differences between the neutrophil proteome of healthy individuals and patients with monogenic diseases. In patients with LAD, only few proteins were found to be present in decreased (7 proteins) or increased (5 proteins) abundance. Of note, proteins with lowest expression levels in LAD1 (ITGB2 mutation) were the integrins ITGB2, ITGAM and ITGAX while in LAD2 (SLC35C1 mutation) integrin expression was not affected. In contrast we noticed lack of PRCP, HDHD2 and XRCC4 protein. In patients with CGD, CYBA and CYBB were decreased in expression, independent of the underlying genotype. In contrast, the other members of the NADPH oxidase complex (NCF1/2/4, Rac1/2) were not found to be differentially expressed. This suggests that both membrane-bound proteins CYBB and CYBA stabilize each other. In total, CGD neutrophils showed limited aberrations with 23 overexpressed and 11 underexpressed proteins. In striking contrast to LAD and CGD, SCN patients with ELANE mutations had a much higher number of proteins with increased (93 proteins) or decreased (47 proteins) expression levels. It is currently unknown whether these imbalances are due to the immaturity of peripheral blood neutrophils in SCN or rather secondary to unfolded protein response and cellular stress mechanisms.In ongoing studies, we analyze the transcriptome of healthy neutrophil granulocytes in correlation to the proteome. Preliminary data indicate that there is poor correlation (PCC 0.11) between protein and mRNA levels, indicating that granule proteins are synthesized during earlier steps of differentiation.In two patients where exome sequencing did not reveal any underlying mutation, proteome analysis showed decreased expression of NCF1 and RAB27A, respectively. In both patients we were able to use targeted sequencing of amplified DNA to show the causative mutations in NCF1 and RAB27A. Multi-omic analysis may therefore be superior to unidimensional genomic sequencing to establish a molecular diagnosis.ConclusionIn-depth mass spectrometry-based proteomics provides new insights into disease-specific alterations of neutrophil granulocytes and may help to refine molecular diagnostics. DisclosuresNo relevant conflicts of interest to declare.

Secretomic analysis of Beauveria bassiana related to cattle tick, Rhipicephalus microplus, infection.

Beauveria bassiana is widely studied as an alternative to chemical acaricides in controlling the cattle tick Rhipicephalus microplus. Although its biocontrol efficiency has been proved in laboratory and field scales, there is a need to a better understanding of host interaction process at molecular level related to biocontrol activity. In this work, applying a proteomic technique multidimensional protein identification technology (MudPIT), the differential secretome of B. bassiana induced by the host R. microplus cuticle was evaluated. The use of the host cuticle in a culture medium, mimicking an infection condition, is an established experimental model that triggers the secretion of inducible enzymes. From a total of 236 proteins, 50 proteins were identified exclusively in infection condition, assigned to different aspects of infection like host adhesion, cuticle penetration and fungal defense, and stress. Other 32 proteins were considered up- or down-regulated. In order to get a meaningful global view of the secretome, several bioinformatic analyses were performed. Regarding molecular function classification, the highest number of proteins in the differential secretome was assigned in to hydrolase activity, enzyme class of all cuticle-degrading enzymes like lipases and proteases. These activities were also further validated through enzymatic assays. The results presented here reveal dozens of specific proteins and different processes potentially implicated in cattle tick infection improving the understanding of molecular basis of biocontrol of B. bassiana against R. microplus.

Q-switched 1064nm Nd-Yag nanosecond laser effects on skin barrier function and on molecular rejuvenation markers in keratinocyte-fibroblasts interaction.

Skin represents an interface between internal and external environment; it protects human body by regulating the water loss and the maintenance of body temperature, defending against irritant and pathogen agents, and against physical, chemical, and UV damage. It provides to essential physiological functions, such as the important antioxidant defense capacity; its protective/defensive function is performed by a high number of proteins, and shows important functions in maintenance of skin barrier homeostasis. Keratinocytes and fibroblasts play a pivotal role to determine or prevent skin aging in response to intrinsic or extrinsic stimuli, modulating cytokines and several biochemical factors. Non-ablative technologies are playing an increasing role in the management of skin aging, inducing a dermal remodeling without a visible epidermal damage. The objective of this study was to evaluate the effect of Q-switched 1064 Nd-YAG laser (Medlite Conbio C6 Nd-YAG laser, Cynosure USA) in skin barrier function, analyzing the constituents which are strongly altered in aging skin. Particularly, we evaluated the expression of filaggrin, TGase, HSP70, and aquaporins, on HaCaT cells. The expression of proinflammatory cytokines has been investigated too.As a second step of the study, we analyzed the modulation of the rejuvenation molecular markers on human skin fibroblasts (HDFs) stimulated with keratinocytes conditioned medium (KCM).Our results demonstrated that Q-switched 1064nm Nd:YAG laser acts on the skin barrier function, increasing the expression of aquaporins, filaggrin, TGase, and HSP70, modulating the proinflammatory cytokines. In fibroblasts stimulated with keratinocytes conditioned medium (KCM) and irradiated with Q-switched 1064nm Nd:YAG laser, we can observe a reduction of MMP-1 and an increase in procollagen, collagen type I, and elastin. Our results highlight that Q-switched 1064nm Nd:YAG laser treatment could represent an effective weapon to fight skin aging.

Expressed protein profile of a Tectomicrobium and other microbial symbionts in the marine sponge Aplysina aerophoba as evidenced by metaproteomics

Aplysina aerophoba is an emerging model marine sponge, with a well-characterized microbial community in terms of diversity and structure. However, little is known about the expressed functional capabilities of its associated microbes. Here, we present the first metaproteomics-based study of the microbiome of A. aerophoba. We found that transport and degradation of halogenated and chloroaromatic compounds are common active processes in the sponge microbiomes. Our data further reveal that the highest number of proteins were affiliated to a sponge-associated Tectomicrobium, presumably from the family Entotheonellaceae, as well as to the well-known symbiont “Candidatus Synechococcus spongiarium”, suggesting a high metabolic activity of these two microorganisms in situ. Evidence for nitric oxide (NO) conversion to nitrous oxide was consistently observed for Tectomicrobia across replicates, by production of the NorQ protein. Moreover, we found a potential energy-yielding pathway through CO oxidation by putative Chloroflexi bacteria. Finally, we observed expression of enzymes that may be involved in the transformation of chitin, glycoproteins, glycolipids and glucans into smaller molecules, consistent with glycosyl hydrolases predicted from analyses of the genomes of Poribacteria sponge symbionts. Thus, this study provides crucial links between expressed proteins and specific members of the A. aerophoba microbiome.

Spiroplasma affects host aphid proteomics feeding on two nutritional resources

Bacterial symbionts are broadly distributed among insects, influencing their bioecology to different degrees. Aphids carry a number of secondary symbionts that can influence aphid physiology and fitness attributes. Spiroplasma is seldom reported as an aphid symbiont, but a high level of infection has been observed in one population of the tropical aphid Aphis citricidus. We used sister isolines of Spiroplasma-infected (Ac-BS) and Spiroplasma-free (Ac-B) aphids reared on sweet orange (optimum host) and orange jasmine (suboptimum host) to demonstrate the effects of Spiroplasma infection in the aphid proteome profile. A higher number of proteins were differently abundant in aphids feeding on orange jasmine, indicating an impact of host plant quality. In both host plants, the majority of proteins affected by Spiroplasma infection were heat shock proteins, proteins linked to cell function and structure, and energy metabolism. Spiroplasma also induced changes in proteins involved in antimicrobial activity, carbohydrate processing and metabolism, amino acid synthesis and metabolism in aphids feeding on orange jasmine. We discuss on how the aphid host proteome is differentially affected by Spiroplasma infection when the host is exploiting host plants with different nutritional values.

Saturated or unsaturated fat supplemented maternal diets influence omental adipose tissue proteome of suckling goat-kids

The aim of the present study was to investigate how maternal diet can influence the adipose tissue of goat kids. Omental adipose tissue proteomes of goat-kids from mothers fed with diet enriched with stearic acid (ST-kids), fish oil (FO-kids) and standard diets (CTRL) were determined by quantitative iTRAQ 2D-LC-MS/MS analysis. Twenty proteins were found to be differentially expressed in suckling kids' omental adipose tissue. Stearic acid induces changes in a higher number of proteins when compared to fish oil. Eleven proteins, namely AARS, ECl1, PMSC2, CP, HSPA8, GPD1, RPL7, OGDH, RPL24, FGA and RPL5 were decreased in ST-kids only. Four proteins, namely DLST, EEF1G, BCAP31 and RALA were decreased in FO-kids only, and one, NUCKS1, was increased. Four proteins, namely PMSC1, PPIB, TUB5×2 and EIF5A1, were be less abundant in both ST- and FO- kids. Most of the protein whose abundance was decreased in ST kids (10 out of 15) are involved in protein metabolism and catabolism pathways. Qualitative gene expression analysis confirmed that all the proteins identified by mass spectrometry, with the exception of FGA, were produced by adipose tissue. Quantitative gene expression analysis demonstrated that two proteins, namely CP, a minor acute phase protein, and ECl1, involved in fatty acid beta oxidation, were downregulated at mRNA level as well. ECl1 gene expression was downregulated in ST-kids AT as compared to Ctrl-kids and CP was downregulated in both ST- and FO-kids. The present results demonstrate that it is possible to influence adipose goat-kid proteome by modifying the maternal diet.

S-nitrosylation/denitrosylation as a regulatory mechanism of salt stress sensing in sunflower seedlings.

Nitric oxide (NO) and various reactive nitrogen species produced in cells in normal growth conditions, and their enhanced production under stress conditions are responsible for a variety of biochemical aberrations. The present findings demonstrate that sunflower seedling roots exhibit high sensitivity to salt stress in terms of nitrite accumulation. A significant reduction in S-nitrosoglutathione reductase (GSNOR) activity is evident in response to salt stress. Restoration of GSNOR activity with dithioerythritol shows that the enzyme is reversibly inhibited under conditions of 120 mM NaCl. Salt stress-mediated S-nitrosylation of cytosolic proteins was analyzed in roots and cotyledons using biotin-switch assay. LC-MS/MS analysis revealed opposite patterns of S-nitrosylation in seedling cotyledons and roots. Salt stress enhances S-nitrosylation of proteins in cotyledons, whereas roots exhibit denitrosylation of proteins. Highest number of proteins having undergone S-nitrosylation belonged to the category of carbohydrate metabolism followed by other metabolic proteins. Of the total 61 proteins observed to be regulated by S-nitrosylation, 17 are unique to cotyledons, 4 are unique to roots whereas 40 are common to both. Eighteen S-nitrosylated proteins are being reported for the first time in plant systems, including pectinesterase, phospholipase d-alpha and calmodulin. Further physiological analysis of glyceraldehyde-3-phosphate dehydrogenase and monodehydroascorbate reductase showed that salt stress leads to a reversible inhibition of both these enzymes in cotyledons. However, seedling roots exhibit enhanced enzyme activity under salinity stress. These observations implicate the role of S-nitrosylation and denitrosylation in NO signaling thereby regulating various enzyme activities under salinity stress in sunflower seedlings.

Protein expression during early stages of bone regeneration under hydrophobic and hydrophilic titanium domes. A pilot study.

There is significant evidence that, during the early stages of osseointegration, moderately rough hydrophilic (SLActive) surfaces can accelerate osteogenesis and increase bone-to-implant contact in comparison to hydrophobic (SLA) surfaces. However, very little is known regarding the molecular mechanisms behind the influence that surface chemistry modifications to increase hydrophilicity determine on bone healing. The aim of this study was to describe for the first time the proteins and related signalling pathways expressed during early osseous healing stages under SLA and SLActive titanium domes for guided bone regeneration. One SLA and 1 SLActive dome with an internal diameter of 5.0mm and a height of 3.0mm were secured to the parietal bones of nine 6-month-old male New Zealand rabbits. Three animals were randomly euthanized at 4, 7 and 14days and the newly formed tissues retrieved under the domes were analysed with liquid chromatography-mass spectrometry/mass spectrometry. STRING and KEGG databases were applied for Gene Ontology and pathway analyses. A different modulation of several pathways was detected between the 2 groups at all healing times. The main differences in the osseous healing response associated to the 2 surfaces were related to pathways involved in regulating the inflammatory response, differentiation of osteoblast precursors and skeletogenesis. At day 7, the highest number of proteins and the highest cellular activity were observed in both groups, although a more complex and articulated proteome in terms of cellular metabolism and signal transduction was observed in SLActive samples. This is the first study describing the proteome expressed during early healing stages of guided bone regeneration and osseointegration. A combination of enhanced early osteogenic response and reduced inflammatory response were suggested for the hydrophilic group. Future studies are needed to corroborate these findings and explore the molecular effects of different titanium surfaces on the cascade of events taking place during bone formation.

Influence of NanoLC Column and Gradient Length as well as MS/MS Frequency and Sample Complexity on Shotgun Protein Identification of Marine Bacteria

Protein identification by shotgun proteomics, i.e., nano-liquid chromatography (nanoLC) peptide separation online coupled to electrospray ionization (ESI) mass spectrometry (MS)/MS, is the most widely used gel-free approach in proteome research. While the mass spectrometer accounts for mass accuracy and MS/MS frequency, the nanoLC setup and gradient time influence the number of peptides available for MS analysis, which ultimately determine the number of proteins identifiable. Here, we report on the influence of (i) analytical column length (15, 25, or 50 cm) coupled to (ii) the applied gradient length (120, 240, 360, 480, or 600 min), as well as (iii) MS/MS frequency on peptide/protein identification by shotgun proteomics of (iv) 2 marine bacteria. Longer gradients increased the number of peptides/proteins identified as well as the reproducibility of identification. Furthermore, longer analytical columns strictly enlarge the covered proteome complement. Notably, the proteome complement identified with a short column and applying a long gradient is also covered when using longer columns with shorter gradients. Coverage of the proteome complement further increases with higher MS/MS frequency. Compilation of peptide lists of replicate analyses (same gradient length) improves protein identification, while compilation of analyses with different gradient lengths yields a similar or even higher number of proteins using comparable or even less total analysis time.

Differential proteomics analysis of Frankliniella occidentalis immune response after infection with Tomato spotted wilt virus (Tospovirus)

Tomato spotted wilt virus (TSWV) is mainly vectored by Frankliniella occidentalis Pergande, and it potentially activates the vector's immune response. However, molecular background of the altered immune response is not clearly understood. Therefore, using a proteomic approach, we investigated the immune pathways that are activated in F. occidentalis larvae after 24 h exposure to TSWV. Two-dimensional isoelectric focusing/sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D-IEF/SDS/PAGE) combined with mass spectrometry (MS), were used to identify proteins that were differentially expressed upon viral infection. High numbers of proteins were abundantly expressed in F. occidentalis exposed to TSWV (73%) compared to the non-exposed (27%), with the majority functionally linked to the innate immune system such as: signaling, stress response, defense response, translation, cellular lipids and nucleotide metabolism. Key proteins included: 70 kDa heat shock proteins, Ubiquitin and Dermcidin, among others, indicative of a responsive pattern of the vector's innate immune system to viral infection.

Identification of the major proteins present in the seminal plasma of European eel, and how hormonal treatment affects their evolution. Correlation with sperm quality

By first time, 2DE protein profile of European eel seminal plasma has been determined. 14 different proteins corresponding to 9 major families were identified in seminal plasma, through hormonal treatment. Some of them play a part in sperm maturation, including carbonic anhydrase which is responsible for modulating the pH of seminal plasma, and warm temperature acclimation protein, which may play an important role in the final maturation of this species, due to the warm temperature of their spawning ground (in the Sargasso Sea).Sperm samples were classified into three motility categories depending on the percentage of motile cells, I: 0–25%, II: 25–50% and III: >50%. Different protein profiles were observed depending on the sperm motility categories, specifically, with the apolipoproteins and complement C3. Higher numbers of proteins from the apolipoprotein family were registered at lower motilities; whereas the complement C3-like family was higher in the samples with the highest percentage of motile cells. These results suggest that the proteins linked to the transportation of lipids (apolipoprotein) and to the immune system (complement C3) may carry out their functions at different stages of spermatogenesis. Using SDS-PAGE analysis, 13 bands were identified, most of which migrated between 20 to 60kDa. In the last weeks of treatment significant increases were observed in the percentage of motile spermatozoa, curvilinear velocity and beat cross frequency. This improvement in sperm quality coincided with a higher amount of proteins located at 19KDa, therefore, this protein could be involved in sperm motility of the European eel.

Temporal regulation of proteome profile in the fruit fly, Drosophila melanogaster.

Background. Diurnal rhythms of protein synthesis controlled by the biological clock underlie the rhythmic physiology in the fruit fly, Drosophila melanogaster. In this study, we conducted a proteome-wide investigation of rhythmic protein accumulation in D. melanogaster.Materials and Methods. Total protein collected from fly samples harvested at 4 h intervals over the 24 h period were subjected to two-dimensional gel electrophoresis, trypsin digestion and MS/MS analysis. Protein spots/clusters were identified with MASCOT search engine and Swiss-Prot database. Expression of proteins was documented as percentage of volume contribution using the Image Master 2D Platinum software.Results. A total of 124 protein spots/clusters were identified using MS/MS analysis. Significant variation in the expression of 88 proteins over the 24-h period was observed. A relatively higher number of proteins was upregulated during the night compared to the daytime. The complexity of temporal regulation of the D. melanogaster proteome was further reflected from functional annotations of the differently expressed proteins, with those that were upregulated at night being restricted to the heat shock proteins and proteins involved in metabolism, muscle activity, protein synthesis/folding/degradation and apoptosis, whilst those that were overexpressed in the daytime were apparently involved in metabolism, muscle activity, ion-channel/cellular transport, protein synthesis/folding/degradation, redox homeostasis, development and transcription.Conclusion. Our data suggests that a wide range of proteins synthesized by the fruit fly, D. melanogaster, is under the regulation of the biological clock.