Microplastics in water resources: Global pollution circle, possible technological solutions, legislations, and future horizon.

Beneath the surface of our ecosystems, microplastics (MPs) silently loom as a significant threat. These minuscule pollutants, invisible to the naked eye, wreak havoc on living organisms and disrupt the delicate balance of our environment. As we delve into a trove of data and reports, a troubling narrative unfolds: MPs pose a grave risk to both health and food chains with their diverse compositions and chemical characteristics. Nevertheless, the peril extends further. MPs infiltrate the environment and intertwine with other pollutants. Worldwide, microplastic levels fluctuate dramatically, ranging from 0.001 to 140 particles.m-3 in water and 0.2 to 8766 particles.g-1 in sediment, painting a stark picture of pervasive pollution. Coastal and marine ecosystems bear the brunt, with each organism laden with thousands of microplastic particles. MPs possess a remarkable ability to absorb a plethora of contaminants, and their environmental behavior is influenced by factors such as molecular weight and pH. Reported adsorption capacities of MPs vary greatly, spanning from 0.001 to 12,700 µg·g-1. These distressing figures serve as a clarion call, demanding immediate action and heightened environmental consciousness. Legislation, innovation, and sustainable practices stand as indispensable defenses against this encroaching menace. Grasping the intricate interplay between microplastics and pollutants is paramount, guiding us toward effective mitigation strategies and preserving our health ecosystems.

Hydrochar as a bio-based adsorbent for heavy metals removal: A review of production processes, adsorption mechanisms, kinetic models, regeneration and reusability.

The spread of heavy metals throughout the ecosystem has extremely endangered human health, animals, plants, and natural resources. Hydrochar has emerged as a promising adsorbent for removal of heavy metals from water and wastewater. Hydrochar, obtained from hydrothermal carbonization of biomass, owns unique physical and chemical properties that are highly potent in capturing heavy metals via surface complexation, electrostatic interactions, and ion exchange mechanisms. This review focuses on removing heavy metals by hydrochar adsorbents from water bodies. The article discusses factors affecting the adsorption capacity of hydrochars, such as contact time, pH, initial metal concentration, temperature, and competing ions. Literature on optimization approaches such as surface modification, composite development, and hybrid systems are reviewed to enlighten mechanisms undertaking the efficiency of hydrochars in heavy metals removal from wastewater. The review also addresses challenges such as hydrochar regeneration and reusability, alongside potential issues related to its disposal and metal leaching. Integration with current water purification methods and the significance of ongoing research and initiatives promoting hydrochar-based technologies were also outlined. The article concludes that combining hydrochar with modern technologies such as nanotechnology and advanced oxidation techniques holds promise for improving heavy metal remediation. Overall, this comprehensive analysis provides valuable insights to guide future studies and foster the development of effective, affordable, and environmentally friendly heavy metal removal technologies to ensure the attainment of safer drinking water for communities worldwide.

Integrating Proteomes for Lung Tissues and Lavage Reveals Pathways That Link Responses in Allergen-Challenged Mice.

To capture interplay between biological pathways, we analyzed the proteome from matched lung tissues and bronchoalveolar lavage fluid (BALF) of individual allergen-naïve and house dust mite (HDM)-challenged BALB/c mice, a model of allergic asthma. Unbiased label-free liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis quantified 2675 proteins from tissues and BALF of allergen-naïve and HDM-exposed mice. In comparing the four datasets, we found significantly greater diversity in proteins between lung tissues and BALF than in the changes induced by HDM challenge. The biological pathways enriched after allergen exposure were compartment-dependent. Lung tissues featured innate immune responses and oxidative stress, while BALF most strongly revealed changes in metabolism. We combined lung tissues and BALF proteomes, which principally highlighted oxidation reduction (redox) pathways, a finding influenced chiefly by the lung tissue dataset. Integrating lung and BALF proteomes also uncovered new proteins and biological pathways that may mediate lung tissue and BALF interactions after allergen challenge, for example, B-cell receptor signaling. We demonstrate that enhanced insight is fostered when different biological compartments from the lung are investigated in parallel. Integration of proteomes from lung tissues and BALF compartments reveals new information about protein networks in response to environmental challenge and interaction between intracellular and extracellular processes.

Comprehensive analysis of the BC200 ribonucleoprotein reveals a reciprocal regulatory function with CSDE1/UNR.

BC200 is a long non-coding RNA primarily expressed in brain but aberrantly expressed in various cancers. To gain a further understanding of the function of BC200, we performed proteomic analyses of the BC200 ribonucleoprotein (RNP) by transfection of 3' DIG-labelled BC200. Protein binding partners of the functionally related murine RNA BC1 as well as a scrambled BC200 RNA were also assessed in both human and mouse cell lines. Stringent validation of proteins identified by mass spectrometry confirmed 14 of 84 protein binding partners and excluded eight proteins that did not appreciably bind BC200 in reverse experiments. Gene ontology analyses revealed general roles in RNA metabolic processes, RNA processing and splicing. Protein/RNA interaction sites were mapped with a series of RNA truncations revealing three distinct modes of interaction involving either the 5' Alu-domain, 3' A-rich or 3' C-rich regions. Due to their high enrichment values in reverse experiments, CSDE1 and STRAP were further analyzed demonstrating a direct interaction between CSDE1 and BC200 and indirect binding of STRAP to BC200 via heterodimerization with CSDE1. Knock-down studies identified a reciprocal regulatory relationship between CSDE1 and BC200 and immunofluorescence analysis of BC200 knock-down cells demonstrated a dramatic reorganization of CSDE1 into distinct nuclear foci.

Activity-Based Protein Profiling of Intraoperative Serine Hydrolase Activities during Cardiac Surgery.

The processes involved in the initiation of acute kidney injury (AKI) following cardiopulmonary bypass (CPB) are thought to occur during the intraoperative period. Such a rapid development might indicate that some of the inductive events are not dependent on de novo protein synthesis, raising the possibility that changes in activities of pre-existing enzymes could contribute to the development of AKI. Activity-based protein profiling (ABPP) was used to compare the serine hydrolase enzyme activities present in the urines of CPB patients who subsequently developed AKI versus those who did not (non-AKI) during the intra- and immediate postoperative periods. Sequential urines collected from a nested case-control cohort of AKI and non-AKI patients were reacted with a serine hydrolase activity probe, fluorophosphonate-TAMRA, and separated by SDS-PAGE. The patterns and levels of probe-labeled proteins in the two groups were initially comparable. However, within 1 h of CPB there were significant pattern changes in the AKI group. Affinity purification and mass spectrometry-based analysis of probe-labeled enzymes in AKI urines at 1 h CPB and arrival to the intensive care unit (ICU) identified 28 enzymes. Quantitative analysis of the activity of one of the identified enzymes, kallikrein-1, revealed some trends suggesting differences in the levels and temporal patterns of enzyme activity between a subset of patients who developed AKI and those who did not. A comparative analysis of affinity-purified probe reacted urinary proteins from these patient groups during the intraoperative period suggested the presence of both shared and unique enzyme patterns. These results indicate that there are intraoperative changes in the levels and types of serine hydrolase activities in patients who subsequently develop AKI. However, the role of these activity differences in the development of AKI remains to be determined.

Inhaled diesel exhaust alters the allergen-induced bronchial secretome in humans.

Diesel exhaust (DE) is a paradigm for traffic-related air pollution. Human adaptation to DE is poorly understood and currently based on oversimplified models. DE promotes allergic responses, but protein expression changes mediated by this interaction have not been systematically investigated. The aim of this study was to define the effect of inhaled DE on allergen-induced proteins in the lung.We performed a randomised and blinded controlled human crossover exposure study. Participants inhaled filtered air or DE; thereafter, contralateral lung segments were challenged with allergen or saline. Using label-free quantitative proteomics, we comprehensively defined DE-mediated alteration of allergen-driven secreted proteins (secretome) in bronchoalveolar lavage. We further examined expression of proteins selected from the secretome data in independent validation experiments using Western blots, ELISA and immunohistochemistry.We identified protein changes unique to co-exposure (DE+allergen), undetected with mono-exposures (DE or allergen alone). Validation studies confirmed that specific proteins (e.g. the antimicrobial peptide cystatin-SA) were significantly enhanced with DE+allergen compared to either mono-exposure.This study demonstrates that common environmental co-exposures can uniquely alter protein responses in the lungs, illuminating biology that mono-exposures cannot. This study highlights the value of complex human in vivo models in detailing airway responses to inhaled pollution.

Interferon γ induced compositional changes in human bone marrow derived mesenchymal stem/stromal cells.

BACKGROUND: Mesenchymal stem/stromal cells (MSC) display a range of immunoregulatory properties which can be enhanced by the exposure to cytokines such interferon γ (IFN-γ). However the compositional changes associated with the 'licensing' of these cells have not been clearly defined. The present study was undertaken to provide a detailed comparative proteomic analysis of the compositional changes that occur in human bone marrow derived MSC following 20 h treatment with IFN-γ. METHODS: 2D LC MSMS analysis of control and IFN-γ treated cells from 5 different healthy donors provided confident identification of more than 8400 proteins. RESULTS: In total 210 proteins were shown to be significantly altered in their expression levels (≥|2SD|) following IFN-γ treatment. The changes for several of these proteins were confirmed by flow cytometry. STRING analysis determined that approximately 30% of the altered proteins physically interacted in described interferon mediated processes. Comparison of the list of proteins that were identified as changed in the proteomic analysis with data for the same proteins in the Interferome DB indicated that ~35% of these proteins have not been reported to be IFN-γ responsive in a range of cell types. CONCLUSIONS: This data provides an in depth analysis of the proteome of basal and IFN-γ treated human mesenchymal stem cells and it identifies a number of novel proteins that may contribute to the immunoregulatory capacity if IFN-γ licensed cells.

Human DDX21 binds and unwinds RNA guanine quadruplexes.

Guanine quadruplexes (G4s) are an important structure of nucleic acids (DNA and RNA) with roles in several cellular processes. RNA G4s require specialized unwinding enzymes, of which only two have been previously identified. We describe the results of a simple and specific mass spectrometry guided method used to screen HEK293T cell lysate for G4 binding proteins. From these results, we validated the RNA helicase protein DDX21. DDX21 is an established RNA helicase, but has not yet been validated as a G4 binding protein. Through biochemical techniques, we confirm that DDX21-quadruplex RNA interactions are direct and mediated via a site of interaction at the C-terminus of the protein. Furthermore, through monitoring changes in nuclease sensitivity we show that DDX21 can unwind RNA G4. Finally, as proof of principle, we demonstrate the ability of DDX21 to suppress the expression of a protein with G4s in the 3΄ UTR of its mRNA.

Peptide Retention Time Prediction in Hydrophilic Interaction Liquid Chromatography: Data Collection Methods and Features of Additive and Sequence-Specific Models.

The development of a peptide retention prediction model for hydrophilic interaction liquid chromatography (XBridge Amide column) is described for a collection of ∼40 000 tryptic peptides. Off-line 2D LC-MS/MS analysis (HILIC-RPLC) of S. cerevisiae whole cell lysate has been used to acquire retention information for a HILIC separation. The large size of the optimization data set (more than 2 orders of magnitude compared to previous reports) permits the accurate assignment of hydrophilic retention coefficients of individual amino acids, establishing both the effects of amino acid position relative to peptide termini and the influence of peptide secondary structure in HILIC. The accuracy of a simple additive model with peptide length correction (R2 value of ∼0.96) was found to be much higher compared to an algorithm of similar complexity applied to RPLC (∼0.91). This indicates significantly smaller influence of peptide secondary structure and interactions with counterions in HILIC. Nevertheless, sequence-specific features were found. Helical peptides that tend to retain stronger than predicted in RPLC exhibit negative prediction errors using an additive HILIC model. N-cap helix stabilizing motifs, which increase retention of amphipathic helical peptides in RP, reduce peptide retention in HILIC independently of peptide amphipathicity. Peptides carrying multiple Pro and Gly (residues with lowest helical propensity) retain stronger than predicted. We conclude that involvement of the peptide backbone's carbonyl and amide groups in hydrogen-bond stabilization of helical structures is a major factor, which determines sequence-dependent behavior of peptides in HILIC. The incorporation of observed sequence dependent features into our Sequence-Specific Retention Calculator HILIC model resulted in 0.98 R2 value correlation, significantly exceeding the predictive performance of all RP and HILIC models developed for complex mixtures of tryptic peptides.

Characterization of Autoantigens Targeted by Anti-Citrullinated Protein Antibodies In Vivo: Prominent Role for Epitopes Derived from Histone 4 Proteins.

Anti-citrullinated protein antibodies (ACPA) have become an integral part of the clinical definition of rheumatoid arthritis, and are hypothesized to be important in the immunopathogenesis of this autoimmune disease. Several citrullinated proteins have been demonstrated to serve as candidate autoantigens for the ACPA, based on in vitro immune reactions between citrullinated peptides/proteins and RA sera. Yet it remains unclear whether the autoantigens identified in vitro are indeed directly and specifically targeted by the ACPA in vivo. Moreover, it is unclear whether ACPA present in RA sera are directed towards the same spectrum of autoantigens as the ACPA present within the synovial compartment. In this study, we isolated ACPA immune complexes from RA synovial fluids (SF) and sera by using immobilized cyclic citrullinated peptides (CCP3) based immune affinity, and characterized the proteins that are directly and specifically associated with them by mass spectrometry. The results demonstrate that four histone proteins are prominent ACPA autoantigens, with the frequency of detection being histone H4 (89%), H2B (63%), H3 (63%), and H2A (58%) in ACPA positive RA SF. We further demonstrate that a histone 4 peptide containing citrulline at position Cit39 was recognized by 100% of ACPA positive RA SF. An adjacent citrulline residue at Cit40 was recognized by 34% of ACPA positive RA SF. An H4 peptide containing Cit39-40 was recognized in the serum of 94% ACPA positive RA, 77% ACPA positive first-degree relatives (FDR) of RA patients, and 2.5% of healthy controls. The Cit39-40 peptide substantially blocked the ACPA reactivity in both SF and serum. Although the spectrum of ACPA we identified was limited to those isolated using immobilized CCP3 peptides, the findings indicate that H4 is a widely recognized RA autoantigen in both the synovial and serum compartments. The identification of this immunodominant ACPA epitope may be valuable in designing approaches to immune tolerance induction in RA.

3D HPLC-MS with Reversed-Phase Separation Functionality in All Three Dimensions for Large-Scale Bottom-Up Proteomics and Peptide Retention Data Collection.

The growing complexity of proteomics samples and the desire for deeper analysis drive the development of both better MS instrument and advanced multidimensional separation schemes. We applied 1D, 2D, and 3D LC-MS/MS separation protocols (all of reversed-phase C18 functionality) to a tryptic digest of whole Jurkat cell lysate to estimate the depth of proteome coverage and to collect high-quality peptide retention information. We varied pH of the eluent and hydrophobicity of ion-pairing modifier to achieve good separation orthogonality (utilization of MS instrument time). All separation modes employed identical LC settings with formic-acid-based eluents in the last dimension. The 2D protocol used a high pH-low pH scheme with 21 concatenated fractions. In the 3D protocol, six concatenated fractions from the first dimension (C18, heptafluorobutyric acid) were analyzed using the identical 2D LC-MS procedure. This approach permitted a detailed evaluation of the analysis output consuming 21× and 126× the analysis time and sample load compared to 1D. Acquisition over 189 h of instrument time in 3D mode resulted in the identification of ∼14 000 proteins and ∼250 000 unique peptides. We estimated the dynamic range via peak intensity at the MS(2) level as approximately 10(4.2), 10(5.6), and 10(6.2) for the 1D, 2D, and 3D protocols, respectively. The uniform distribution of the number of acquired MS/MS, protein, and peptide identifications across all 126 fractions and through the chromatographic time scale in the last LC-MS stage indicates good separation orthogonality. The protocol is scalable and is amenable to the use of peptide retention prediction in all dimensions. All these features make it a very good candidate for large-scale bottom-up proteomic runs, which target both protein identification as well as the collection of peptide retention data sets for targeted quantitative applications.

Identification of broadly conserved cross-species protective Leishmania antigen and its responding CD4+ T cells.

There is currently no clinically effective vaccine against leishmaniasis because of poor understanding of the antigens that elicit dominant T cell immunity. Using proteomics and cellular immunology, we identified a dominant naturally processed peptide (PEPCK335-351) derived from Leishmania glycosomal phosphoenolpyruvate carboxykinase (PEPCK). PEPCK was conserved in all pathogenic Leishmania, expressed in glycosomes of promastigotes and amastigotes, and elicited strong CD4(+) T cell responses in infected mice and humans. I-A(b)-PEPCK335-351 tetramer identified protective Leishmania-specific CD4(+) T cells at a clonal level, which comprised ~20% of all Leishmania-reactive CD4(+) T cells at the peak of infection. PEPCK335-351-specific CD4(+) T cells were oligoclonal in their T cell receptor usage, produced polyfunctional cytokines (interleukin-2, interferon-γ, and tumor necrosis factor), and underwent expansion, effector activities, contraction, and stable maintenance after lesion resolution. Vaccination with PEPCK peptide, DNA expressing full-length PEPCK, or rPEPCK induced strong durable cross-species protection in both resistant and susceptible mice. The effectiveness and durability of protection in vaccinated mice support the development of a broadly cross-species protective vaccine against different forms of leishmaniasis by targeting PEPCK.

Comparative proteomic analyses demonstrate enhanced interferon and STAT-1 activation in reovirus T3D-infected HeLa cells.

As obligate intracellular parasites, viruses are exclusively and intimately dependent upon their host cells for replication. During replication viruses induce profound changes within cells, including: induction of signaling pathways, morphological changes, and cell death. Many such cellular perturbations have been analyzed at the transcriptomic level by gene arrays and recent efforts have begun to analyze cellular proteomic responses. We recently described comparative stable isotopic (SILAC) analyses of reovirus, strain type 3 Dearing (T3D)-infected HeLa cells. For the present study we employed the complementary labeling strategy of iTRAQ (isobaric tags for relative and absolute quantitation) to examine HeLa cell changes induced by T3D, another reovirus strain, type 1 Lang, and UV-inactivated T3D (UV-T3D). Triplicate replicates of cytosolic and nuclear fractions identified a total of 2375 proteins, of which 50, 57, and 46 were significantly up-regulated, and 37, 26, and 44 were significantly down-regulated by T1L, T3D, and UV-T3D, respectively. Several pathways, most notably the Interferon signaling pathway and the EIF2 and ILK signaling pathways, were induced by virus infection. Western blots confirmed that cells were more strongly activated by live T3D as demonstrated by elevated levels of key proteins like STAT-1, ISG-15, IFIT-1, IFIT-3, and Mx1. This study expands our understanding of reovirus-induced host responses.

Towards further defining the proteome of mouse saliva.

BACKGROUND: Knowledge of the mouse salivary proteome is not well documented and as a result, very limited. Currently, several salivary proteins remain unidentified and for some others, their function yet to be determined. The goal of the present study is to utilize mass spectrometry analysis to widen our knowledge of mouse salivary proteins, and through extensive database searches, provide further insight into the array of proteins that can be found in saliva. A comprehensive mouse salivary proteome will also facilitate the development of mouse models to study specific biomarkers of many human diseases. RESULTS: Individual saliva samples were collected from male and female mice, and later pooled according to sex. Two pools of saliva from female mice (2 samples/pool) and 2 pools of saliva from male mice were used for analysis utilizing high performance liquid chromatograph mass spectrometry (nano-RPLC-MS/MS). The resulting datasets identified 345 proteins: 174 proteins were represented in saliva obtained from both sexes, as well as 82 others that were more female specific and 89 that were more male specific. Of these sex linked proteins, twelve were identified as exclusively sex-limited; 10 unique to males and 2 unique to females. Functional analysis of the 345 proteins identified 128 proteins with catalytic activity characteristics; indicative of proteins involved in digestion, and 35 proteins associated with stress response, host defense, and wound healing functions. Submission of the list of 345 proteins to the BioMart data mining tool in the Ensembl database further allowed us to identify a total of 283 orthologous human genes, of which, 131 proteins were recently reported to be present in the human salivary proteome. CONCLUSIONS: The present study is the most comprehensive list to date of the proteins that constitute the mouse salivary proteome. The data presented can serve as a useful resource for identifying potentially useful biomarkers of human health and disease.

N-capping motifs promote interaction of amphipathic helical peptides with hydrophobic surfaces and drastically alter hydrophobicity values of individual amino acids.

Capping rules, which govern interactions of helical peptides with hydrophobic surfaces, were never established before due to lack of methods for the direct measurement of polypeptide structure on the interphase boundary. We employed proteomic techniques and peptide retention modeling in reversed-phase chromatography to generate a data set sufficient for amino acid population analysis at helix ends. We found that interactions of amphipathic helical peptides with a hydrophobic C18 phase are induced by a unique motif featuring hydrophobic residues in the N1 and N2 positions adjacent to the N-cap (Asn, Asp, Ser, Thr, Gly), followed by Glu, Gln, or Asp in position N3 to complete a capping box. A favorable N-capping arrangement prior to amphipathic helix may result in the highest hydrophobicity (retention on C18 columns) of Asp/Asn (or Glu/Gln) peptide analogues among all naturally occurring amino acids when placed in N-cap or N3 position, respectively. These results contradict all previously reported hydrophobicity scales and provide new insights into our understanding of the phenomenon of hydrophobic interactions.

Insights into electron flux through manipulation of fermentation conditions and assessment of protein expression profiles in Clostridium thermocellum.

While annotation of the genome sequence of Clostridium thermocellum has allowed predictions of pathways catabolizing cellobiose to end products, ambiguities have persisted with respect to the role of various proteins involved in electron transfer reactions. A combination of growth studies modulating carbon and electron flow and multiple reaction monitoring (MRM) mass spectrometry measurements of proteins involved in central metabolism and electron transfer was used to determine the key enzymes involved in channeling electrons toward fermentation end products. Specifically, peptides belonging to subunits of ferredoxin-dependent hydrogenase and NADH:ferredoxin oxidoreductase (NFOR) were low or below MRM detection limits when compared to most central metabolic proteins measured. The significant increase in H2 versus ethanol synthesis in response to either co-metabolism of pyruvate and cellobiose or hypophosphite mediated pyruvate:formate lyase inhibition, in conjunction with low levels of ferredoxin-dependent hydrogenase and NFOR, suggest that highly expressed putative bifurcating hydrogenases play a substantial role in reoxidizing both reduced ferredoxin and NADH simultaneously. However, product balances also suggest that some of the additional reduced ferredoxin generated through increased flux through pyruvate:ferredoxin oxidoreductase must be ultimately converted into NAD(P)H either directly via NADH-dependent reduced ferredoxin:NADP(+) oxidoreductase (NfnAB) or indirectly via NADPH-dependent hydrogenase. While inhibition of hydrogenases with carbon monoxide decreased H2 production 6-fold and redirected flux from pyruvate:ferredoxin oxidoreductase to pyruvate:formate lyase, the decrease in CO2 was only 20 % of that of the decrease in H2, further suggesting that an alternative redox system coupling ferredoxin and NAD(P)H is active in C. thermocellum in lieu of poorly expressed ferredoxin-dependent hydrogenase and NFOR.

Proteomic characterization of serine hydrolase activity and composition in normal urine.

BACKGROUND: Serine hydrolases constitute a large enzyme family involved in a diversity of proteolytic and metabolic processes which are essential for many aspects of normal physiology. The roles of serine hydrolases in renal function are largely unknown and monitoring their activity may provide important insights into renal physiology. The goal of this study was to profile urinary serine hydrolases with activity-based protein profiling (ABPP) and to perform an in-depth compositional analysis. METHODS: Eighteen healthy individuals provided random, mid-stream urine samples. ABPP was performed by reacting urines (n = 18) with a rhodamine-tagged fluorophosphonate probe and visualizing on SDS-PAGE. Active serine hydrolases were isolated with affinity purification and identified on MS-MS. Enzyme activity was confirmed with substrate specific assays. A complementary 2D LC/MS-MS analysis was performed to evaluate the composition of serine hydrolases in urine. RESULTS: Enzyme activity was closely, but not exclusively, correlated with protein quantity. Affinity purification and MS/MS identified 13 active serine hydrolases. The epithelial sodium channel (ENaC) and calcium channel (TRPV5) regulators, tissue kallikrein and plasmin were identified in active forms, suggesting a potential role in regulating sodium and calcium reabsorption in a healthy human model. Complement C1r subcomponent-like protein, mannan binding lectin serine protease 2 and myeloblastin (proteinase 3) were also identified in active forms. The in-depth compositional analysis identified 62 serine hydrolases in urine independent of activity state. CONCLUSIONS: This study identified luminal regulators of electrolyte homeostasis in an active state in the urine, which suggests tissue kallikrein and plasmin may be functionally relevant in healthy individuals. Additional serine hydrolases were identified in an active form that may contribute to regulating innate immunity of the urinary tract. Finally, the optimized ABPP technique in urine demonstrates its feasibility, reproducibility and potential applicability to profiling urinary enzyme activity in different renal physiological and pathophysiological conditions.

Influenza A infection of primary human airway epithelial cells up-regulates proteins related to purine metabolism and ubiquitin-related signaling.

Virus-host interactions are important determinants of virus replication and immune responses, but they are not well-defined. In this study we analyzed quantitative host protein alterations in nuclei-enriched fractions from multiple primary human bronchial airway epithelial (HBAE) cells infected by an H1N1 influenza A virus (A/PR/8/34). We first developed an effective detergent-free nuclear lysis method that was coupled with in-solution digestion and LC-MS/MS. Using SILAC, we identified 837 HBAE nuclear proteins in three different donors and compared their responses to infection at 24 h. Some proteins were altered in all three donors, of which 94 were up-regulated and 13 were down-regulated by at least 1.5-fold. Many of these up-regulated proteins clustered into purine biosynthesis, carbohydrate metabolism, and protein modification. Down-regulated proteins were not associated with any specific pathways or processes. These findings further our understanding of cellular processes that are altered in response to influenza in primary epithelial cells and may be beneficial in the search for host proteins that may be targeted for antiviral therapy.

Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression.

BACKGROUND: Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase. RESULTS: Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase, demonstrated differential expression during transition from exponential to stationary phase. CONCLUSIONS: Relative expression profiles demonstrate which proteins are likely utilized in carbohydrate utilization and end-product synthesis and suggest that H2 synthesis occurs via bifurcating hydrogenases while ethanol synthesis is predominantly catalyzed by a bifunctional aldehyde/alcohol dehydrogenase. Differences in expression profiles of core metabolic proteins in response to growth phase may dictate carbon and electron flux towards energy storage compounds and end-products. Combined knowledge of relative protein expression levels and their changes in response to physiological conditions may aid in targeted metabolic engineering strategies and optimization of fermentation conditions for improvement of biofuels production.

Response of primary human airway epithelial cells to influenza infection: a quantitative proteomic study.

Influenza A virus exerts a large health burden during both yearly epidemics and global pandemics. However, designing effective vaccine and treatment options has proven difficult since the virus evolves rapidly. Therefore, it may be beneficial to identify host proteins associated with viral infection and replication to establish potential new antiviral targets. We have previously measured host protein responses in continuously cultured A549 cells infected with mouse-adapted virus strain A/PR/8/34(H1N1; PR8). We here identify and measure host proteins differentially regulated in more relevant primary human bronchial airway epithelial (HBAE) cells. A total of 3740 cytosolic HBAE proteins were identified by 2D LC-MS/MS, of which 52 were up-regulated ≥2-fold and 41 were down-regulated ≥2-fold after PR8 infection. Up-regulated HBAE proteins clustered primarily into interferon signaling, other host defense processes, and molecular transport, whereas down-regulated proteins were associated with cell death signaling pathways, cell adhesion and motility, and lipid metabolism. Comparison to influenza-infected A549 cells indicated some common influenza-induced host cell alterations, including defense response, molecular transport proteins, and cell adhesion. However, HBAE-specific alterations consisted of interferon and cell death signaling. These data point to important differences between influenza replication in continuous and primary cell lines and/or alveolar and bronchial epithelial cells.