Endothelial Damage Arising From High Salt Hypertension Is Elucidated by Vascular Bed Systematic Profiling.

BACKGROUND: Considerable evidence links dietary salt intake with the development of hypertension, left ventricular hypertrophy, and increased risk of stroke and coronary heart disease. Despite extensive epidemiological and basic science interrogation of the relationship between high salt (HS) intake and blood pressure, it remains unclear how HS impacts endothelial cell (EC) and vascular structure in vivo. This study aims to elucidate HS-induced vascular pathology using a differential systemic decellularization in vivo approach. METHODS: We performed systematic molecular characterization of the endothelial glycocalyx and EC proteomes in mice with HS (8%) diet-induced hypertension versus healthy control animals. Isolation of eGC and EC compartments was achieved using differential systemic decellularization in vivo methodology. Altered protein expression in hypertensive compared to normal mice was characterized by liquid chromatography tandem mass spectrometry. Proteomic results were validated using functional assays, microscopic imaging, and histopathologic evaluation. RESULTS: Proteomic analysis revealed a significant downregulation of eGC and associated proteins in HS diet-induced hypertensive mice (among 1696 proteins identified in this group, 723 were markedly decreased in abundance, while only 168 were increased in abundance. Bioinformatic analysis indicated substantial derangement of the eGC layer, which was subsequently confirmed by fluorescent and electron microscopy assessment of vessel damage ex vivo. In the EC fraction, HS-induced hypertension significantly altered protein mediators of contractility, metabolism, mechanotransduction, renal function, and the coagulation cascade. In particular, we observed dysregulation of integrin subunits α2, α2b, and α5, which was associated with arterial wall inflammation and substantial infiltration of CD68+ monocyte-macrophages. Consequently, HS-induced hypertensive mice also displayed reduced vascular integrity of multiple organs including lungs, kidneys, and heart. CONCLUSIONS: These findings provide novel molecular insight into HS-induced structural changes in eGC and EC composition that may increase cardiovascular risk and potentially guide the development of new diagnostics and therapeutic interventions.

Profiling the 'deamidome' of complex biosamples using mixed-mode chromatography-coupled tandem mass spectrometry.

Deamidation is a spontaneous degenerative protein modification (DPM) that disrupts the structure and function of both endogenous proteins and various therapeutic agents. While deamidation has long been recognized as a critical event in human aging and multiple degenerative diseases, research progress in this field has been restricted by the technical challenges associated with studying this DPM in complex biological samples. Asparagine (Asn) deamidation generates L-aspartic acid (L-Asp), D-aspartic acid (D-Asp), L-isoaspartic acid (L-isoAsp) or D-isoaspartic acid (D-isoAsp) residues at the same position of Asn in the affected protein, but each of these amino acids displays similar hydrophobicity and cannot be effectively separated by reverse phase liquid chromatography. The Asp and isoAsp isoforms are also difficult to resolve using mass spectrometry since they have the same mass and fragmentation pattern in MS/MS. Moreover, the 13C peaks of the amidated peptide are often misassigned as monoisotopic peaks of the corresponding deamidated peptides in protein database searches. Furthermore, typical protein isolation and proteomic sample preparation methods induce artificial deamidation that cannot be distinguished from the physiological forms. To better understand the role of deamidation in biological aging and degenerative pathologies, new technologies are now being developed to address these analytical challenges, including mixed mode electrostatic-interaction modified hydrophilic interaction liquid chromatography (emHILIC). When coupled to high resolution, high accuracy tandem mass spectrometry this technology enables unprecedented, proteome-wide study of the 'deamidome' of complex samples. The current article therefore reviews recent advances in sample preparation methods, emHILIC-MS/MS technology, and MS instrumentation / data processing approaches to achieving accurate and reliable characterization of protein deamidation in complex biological and clinical samples.

Effects of sequential infections of Caenorhabditis elegans with Staphylococcus aureus and Proteus mirabilis.

Caenorhabditis elegans can be used to study the dynamics of polymicrobial infections, specifically those between Gram-positive and Gram-negative bacteria. With C. elegans, Proteus mirabilis acts as an opportunistic pathogen and does not kill this host. Hence, in the present study, C. elegans was immunochallenged by pre-infecting it with the pathogen Staphylococcus aureus in order to study the subsequent effect of P. mirabilis on the host. It was found that 12 hrs of S. aureus and 80 hrs of subsequent P. mirabilis infection significantly reduced the life span of exposed C. elegans by 80%. However, preinfection with S. aureus for 8 and 4 hrs reduced the life span of C. elegans by only 60 and 30%, respectively. Further, there was greater production of reactive oxygen species in the sequentially infected samples than in the S. aureus and P. mirabilis controls. Real time PCR analysis indicated regulation of candidate immune regulatory genes, lysozyme (lys-7), CUB-like proteins (F08G5.6), neuropeptide-like factors (nlp-29), transcription factors of mitogen-activated protein kinase (ATF-7) and daf-2-daf-16 (daf-16), insulin-like signaling pathways and C-type lectin (clec-60 and clec-87) family members during S. aureus and subsequent P. mirabilis-mediated infections, indicating possible roles of, and contributions by, the above factors during host immune responses against these sequential infections. The present findings demonstrate that S. aureus infections increase the vulnerability of the C. elegans host by subverting its immune system, which then permits the opportunistic pathogen P. mirabilis to be pathogenic to this host.

Aging-induced isoDGR-modified fibronectin activates monocytic and endothelial cells to promote atherosclerosis.

BACKGROUND AND AIMS: Aging is the primary risk factor for cardiovascular disease (CVD), but the mechanisms underlying age-linked atherosclerosis remain unclear. We previously observed that long-lived vascular matrix proteins can acquire 'gain-of-function' isoDGR motifs that might play a role in atherosclerotic pathology. METHODS: IsoDGR-specific mAb were generated and used for ELISA-based measurement of motif levels in plasma samples from patients with coronary artery diseases (CAD) and non-CAD controls. Functional consequences of isoDGR accumulation in age-damaged fibronectin were determined by bioassay for capacity to activate monocytes, macrophages, and endothelial cells (signalling activity, pro-inflammatory cytokine expression, and recruitment/adhesion potential). Mice deficient in the isoDGR repair enzyme PCMT1 were used to assess motif distribution and macrophage localisation in vivo. RESULTS: IsoDGR-modified fibronectin and fibrinogen levels in patient plasma were significantly enhanced in CAD and further associated with smoking status. Functional assays demonstrated that isoDGR-modified fibronectin activated both monocytes and macrophages via integrin receptor 'outside in' signalling, triggering an ERK:AP-1 cascade and expression of pro-inflammatory cytokines MCP-1 and TNFα to drive additional recruitment of circulating leukocytes. IsoDGR-modified fibronectin also induced endothelial cell expression of integrin ß1 to further enhance cellular adhesion and matrix deposition. Analysis of murine aortic tissues confirmed accumulation of isoDGR-modified proteins co-localised with CD68+ macrophages in vivo. CONCLUSIONS: Age-damaged fibronectin features isoDGR motifs that increase binding to integrins on the surface of monocytes, macrophages, and endothelial cells. Subsequent activation of 'outside-in' signalling elicits a range of potent cytokines and chemokines that drive additional leukocyte recruitment to the developing atherosclerotic matrix.

Salmonella enterica Serovar Typhi exposure elicits deliberate physiological alterations and triggers the involvement of ubiquitin mediated proteolysis pathway in Caenorhabditis elegans.

Involvement of several candidate immune regulatory players at transcriptomic levels during microbial interactions were reported by involving C. elegans as a model system for the past few years. In the present study, we have identified a wide range of phenotypical, physiological and biochemical alterations in C. elegans triggered due to S. Typhi infection using standard approaches. We performed several behavioural studies and molecular studies such as liquid-phase IEF, MALDI-MS and bioinformatics analyses. S. Typhi exerted a slow killing against C. elegans and prompted several phenotypical changes such as egg laying defects, pharyngeal arresting, and triggered functional group variations which were disclosed using FT-IR. Proteome analysis using liquid phase IEF and MALDI-ToF-Mass Spectrometry ended up with the identification of 123 proteins which contains human orthologs. Bioinformatics analysis of the MS identified proteins revealed the involvement of ubiquitination pathway which was then validated using immunoblotting. Extensive studies similar to our study with the utility of high-throughput OMICS technologies during host pathogen interactions may pave a way for the identification of biomarkers against bacterial diseases.

Amygdalin-Functionalized Carbon Quantum Dots for Probing ß-Glucosidase Activity for Cancer Diagnosis and Therapeutics.

A fluorescence active nanosystem capable of targeting specific receptors of cancer cells with or without a biorecognition element is advantageous for biosensor studies. Herein, a naturally occurring anticancer drug, amygdalin (synthetic form: Laetrile, a misnomer: vitamin B17), has been modified on the surface of carbon quantum dots, prepared by a hydrothermal method, to probe ß-glucosidase activity. Despite its cyanide toxicity, amygdalin is recently revived to be an anticancer molecule, and the risk factor can be optimized by understanding its binding efficiency with ß-glucosidase in the cancer cells. In this study, an in vitro biorecognition pattern of amygdalin-functionalized carbon quantum dots (Amy@CQDs) toward ß-glucosidase is typically evaluated by an aggregation-induced fluorescence emission mechanism. The optical functionality and structural integrity of CQDs before and after functionalization with amygdalin are comprehensively studied by spectroscopic and microscopic techniques. Our results demonstrate that Amy@CQDs is a stable hydrophilic graphitic carbon nanostructure exhibiting selective fluorescence quenching upon interaction with ß-glucosidase, enabling the lowest detection limit of 134 nM. Hydrolysis products of amygdalin mediated by ß-glucosidase were further confirmed by HPLC and colorimetric methods, indicating the selective binding of the prepared Amy@CQDs, which may find a useful application in cancer diagnosis and therapeutics.

Analysis of Caenorhabditis elegans phosphoproteome reveals the involvement of a molecular chaperone, HSP-90 protein during Salmonella enterica Serovar Typhi infection.

Being a primary and prerequisite Post Translational Modification (PTM), protein phosphorylation mediates the defense mechanisms that presides host defense against a pathogen attack. Hence, the current study was intended to uncover the role of regulatory proteins and their PTMs with special attention to phosphorylation during pathogen attack, using C. elegans as a host and S. Typhi as an interacting pathogen. The study was initiated with the identification of differential regulation of the crucial immune regulatory kinases such as PMK-1, JNK-1 and SGK-1 through immunoblotting analysis, which revealed up-regulation of kinases during 48 h of S. Typhi infection. Subsequent the phosphoproteome profiling of S. Typhi infected C. elegans, using TiO2 Column Chromatography followed by MALDI-ToF-ToF-MS, uncovered the regulated phosphoprotein players resulting in the identification of 166 and 54 proteins from gel-free and gel-based analysis, respectively. HSP-90 was found to be a central player from the interactome analyses and its role during pathogenic defense was validated using immunoblotting. Furthermore, the protein disorders of the identified phosphoproteins have been extensively analysed in silico. This study suggests that S. Typhi interferes with the homeostasis of chaperone molecules by kinetically interfering with the phosphorylation of the downstream pathway players of MAPK and JNK.

Unravelling the wound healing ability and mode of action of pyridine carboxamide oxime using Caenorhabditis elegans as potential prescreen wound model.

AIM: In the current scenario of ethical issues related to animal usage in research, the present study was intended to explore the proficient utility of nematode, Caenorhabditis elegans as wound model in preliminary screening of wound healing therapeutics. MAIN METHODS: In this study, a new wounding protocol and quantitative assessment strategies for various healing parameters [survival, Reactive Oxygen Species (ROS), calcium signals, F-actin dynamics, new collagen synthesis and wound induced anti-microbial peptides] were developed and used for preliminary screening of wound healing actives from natural sources. Wound healing ability of positive lead Tridax procumbens (TP) and its major phytocompounds [Octa decenoic acid (ODA), Pyridine carboxamide oxime, known as Nicotinamide (NA) and Dimethyl Benz[c]acridine (DMB)] were assessed using C. elegans wound model and cell lines scratch wound healing assay. Mode of action of active lead was elucidated using metabolome analysis coupled with MALDI-MS followed by molecular docking. KEY FINDINGS: From the four tested methanolic extracts, TP was chosen as positive lead compared to control, Benzalkonium chloride (BKC) based on survival and new collagen synthesis analyses. Results indicated that the wound healing ability of TP was majorly contributed by NA. Further, it was found that NA acts in chloromethyl nicotinamide derivative form by interacting with the known wound healing biomarker, glycogen synthase kinase 3 (GSK-3) to exert wound healing ability. SIGNIFICANCE: The study evidenced that C. elegans, could be a reliable wound model for high-throughput screening of wound healing actives and to identify their possible mode of action.

Role of DAF-21protein in Caenorhabditis elegans immunity against Proteus mirabilis infection.

UNLABELLED: Caenorhabditis elegans is emerging as one of the handy model for proteome related studies due to its simplest system biology. The present study, deals with changes in protein expression in C. elegans infected with Proteus mirabilis. Proteins were separated using two-dimensional differential gel electrophoresis (2D-DIGE) and identified using MALDI-TOF. Twelve distinctly regulated proteins identified in the infected worms, included heat shock proteins involved stress pathway (HSP-1 and HSP-6), proteins involved in immune response pathway (DAF-21), enzymes involved in normal cellular process (Eukaryotic translation Elongation Factor, actin family member, S-adenosyl homocysteine hydrolase ortholog, glutamate dehydrogenase and Vacuolar H ATPase family member) and few least characterized proteins (H28O16.1 and H08J11.2). The regulation of selected players at the transcriptional level during Proteus mirabilis infection was analyzed using qPCR. Physiological experiments revealed the ability of P. mirabilis to kill daf-21 mutant C. elegans significantly compared with the wild type. This is the first report studying proteome changes in C. elegans and exploring the involvement of MAP Kinase pathway during P. mirabilis infection. BIOLOGICAL SIGNIFICANCE: This is the first report studying proteome changes in C. elegans during P. mirabilis infection. The present study explores the role and contribution of MAP Kinase pathway and its regulator protein DAF-21 involvement in the immunity against opportunistic pathogen P. mirabilis infection. Manipulation of this DAF-21 protein in host, may pave the way for new drug development or disease control strategy during opportunistic pathogen infections.

Changes in Caenorhabditis elegans immunity and Staphylococcal virulence factors during their interactions.

The nematode Caenorhabditis elegans is used as a model system for the study of host-pathogen interactions. Lipoteichoic acid (LTA) is one of the major virulent and immunostimulatory components found in gram positive bacteria. The current study used LTA isolated from Staphylococcus aureus and pathogenic and non-pathogenic Staphylococcus epidermidis. The overall physiological assays revealed that LTA exposed C. elegans show a significant reduction in the life span, production of eggs and progenies. To understand the involvement of innate immune specific players at the mRNA level, the regulation of few candidate antimicrobial genes was studied during Staphylococcal LTA exposures. qPCR analysis indicated an upregulation of antimicrobial peptides during LTA exposures. To understand the involvement of LTA and other virulent genes during infection, the regulation of LTA synthase and a few virulence genes was monitored during host exposure. The qPCR analyses indicated the upregulation of ltaS and other virulence genes (atoxin, sak, ssaA and fbe) during infection. Ability of the pathogens to modify their internal machinery during host presence was monitored by Fourier transform infrared spectroscopy, electrochemical impedance spectroscopy and cyclic voltametric analyses. The FTIR results indicated distinct alterations of peaks from Staphylococcal LTA composition between control and the host exposed. Further, EIS and CV data displayed clear differences between the host exposed Staphylococcal samples compared to their respective unexposed controls. The pathogenic and non-pathogenic strains showed different types of regulations and interactions during host exposures. The observed modifications clearly suggest that the Gram positive pathogen changes its LTA production and possibly the structure to cause a severe pathogenic effect on an interacting host.

A MAP Kinase pathway in Caenorhabditis elegans is required for defense against infection by opportunistic Proteus species.

Caenorhabditis elegans innate immunity requires a conserved mitogen activated protein kinase (MAPK) pathway that regulates the basal and pathogen-induced expression of immune effectors. Being in the group of opportunistic pathogens, Proteus spp. cause large number of nosocomial infections. Since, Proteus spp. do not cause death in wild type C. elegans, to understand the role and contribution of MAP Kinase pathway, the mutants (sek-1 and pmk-1) of this pathway were employed. Physiological experiments revealed that the Proteus spp. were able to kill MAP Kinase pathway mutant's C. elegans significantly. To understand the involvement of innate immune pathways specific players at the mRNA level, the regulation of few candidate antimicrobial genes were kinetically investigated during Proteus spp. infections. Real-time PCR analysis indicated a regulation of few candidate immune regulatory genes (F08G5.6, lys-7, nlp-29, ATF-7 and daf-16) during the course of Proteus spp. infections. In addition, the lipopolysaccharides (LPS) isolated from Proteus mirabilis upon exposure to mutant C. elegans showed modifications at their functional regions suggesting that the pathogen modifies its internal machinery according to the specific host for effective pathogenesis.

Changes in Caenorhabditis elegans life span and selective innate immune genes during Staphylococcus aureus infection.

Caenorhabditis elegans has been increasingly used to study the innate immunity and for the screening of microbe/host-specific pathogenic factors. Staphylococcus aureus-mediated infections with live C. elegans were performed on solid (full-lawn) and liquid assays. S. aureus required 90 ± 10 h for the complete killing of C. elegans, but the infection was started only after 32 h of exposure with 20% inoculum of S. aureus. The short time exposure studies revealed that, in 20% of inoculum, continuous exposure to the pathogen was required for the killing of nematode. In 100% of inoculum, only 8 h of exposure was sufficient to kill the C. elegans. To evaluate kinetically at the innate immune level, the regulation of representative candidate antimicrobial genes was investigated. Both semi-quantitative reverse transcriptase polymerase chain reaction (PCR) and real-time PCR analyses indicated the regulation of candidate immune regulatory genes of lysozyme (lys-7), cysteine protease (cpr-2), and C-type lectin (clec-60 and clec-87) family members during the course of S. aureus infections, indicating the possible contribution of the above players during the host immune response against S. aureus exposures.