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Assessing the stability and degradation of proteins is central to the study of cellular biological processes. Here, we describe a novel pulse-chase method to determine the half-life of cellular proteins that overcomes the limitations of other commonly used approaches. This method takes advantage of pulse-labeling of nascent proteins in living cells with the bioorthogonal amino acid L-azidohomoalanine (AHA) that is compatible with click chemistry-based modifications. We validate this method in both mammalian and yeast cells by assessing both over-expressed and endogenous proteins using various fluorescent and chemiluminescent click chemistry-compatible probes. Importantly, while cellular stress responses are induced to a limited extent following live-cell AHA pulse-labeling, we also show that this response does not result in changes in cell viability and growth. Moreover, this method is not compromised by the cytotoxicity evident in other commonly used protein half-life measurement methods and it does not require the use of radioactive amino acids. This new method thus presents a versatile, customizable, and valuable addition to the toolbox available to cell biologists to determine the stability of cellular proteins.

Reorganization of cellular proteins into subcellular compartments, such as the rearrangement of RNA-binding proteins into cytoplasmic stress granules and P-bodies, is a well-recognized, widely studied physiological process currently under intense investigation. Using the assembly of a novel, inducible, nuclear granule formed from the yeast RNA-binding transcription termination factors Nab3 and Nrd1, we present a freely-accessible, high-throughput and unbiased algorithm written in MATLAB that detects and measures protein distribution, partitioning, and sequestration into subcellular compartments captured by fluorescence microscopy; an invaluable advancement to current image analysis methods which utilize experiment-specific custom scripts or subjective manual counting. Employing our algorithm, we quantified thousands of cells, ensuring rigorous examination of Nab3 granule formation across strains with reproducible statistical analyses. We document strain differences in Nab3 granule formation and an associated growth defect. Additionally, we applied our algorithm to immunofluorescent images of the inducible polymerization into filaments of an enzyme in human cells, demonstrating the algorithm’s versatility and adaptability.SUMMARY STATEMENTWe describe a computational tool that enables the quantification of protein condensation during assembly of a subnuclear compartment. The algorithm scores assembly of fluorescently tagged proteins in yeast or human cells.

Anthraquinones are privileged chemical scaffolds that have been used for centuries in various therapeutic applications. The anthraquinone moiety forms the core of various anticancer agents. However, the emergence of drug-resistant cancers warrants the development of new anticancer agents. The research endeavours towards new anthraquinone-based compounds are increasing rapidly in recent years. They are used as a core chemical template to achieve structural modifications, resulting in the development of new anthraquinone-based compounds as promising anticancer agents. Mechanistically, most of the anthraquinone-based compounds inhibit cancer progression by targeting essential cellular proteins. Herein, we review new anthraquinone analogues that have been developed in recent years as anticancer agents. This includes a systematic review of the recent literature (2005–2021) on anthraquinone-based compounds in cell-based models and key target proteins such as kinases, topoisomerases, telomerases, matrix metalloproteinases and G-quadruplexes involved in the viability of cancer cells. In addition to this, the developments in PEG-based delivery of anthraquinones and the toxicity aspects of anthraquinone derivatives are also discussed. The review dispenses a compact background knowledge to understanding anthraquinones for future research on the expansion of anticancer therapeutics.

Identification of differentially expressed proteins in antiphospholipid syndrome (APS) is a developing area of research for unique profiles of this pathology. Advances in technologies of mass spectrometry brings improvements in proteomics and results in assessment of soluble or cellular proteins which could be candidates for clinical biomarkers of primary APS. The use of blood as a source of proteins ease the acquisition of samples for proteomics analyses and later for disease diagnosis. We performed a systematic review to explore the proteomics studies carried out in circulating released proteins (serum, plasma) or cellular proteins (monocytes and platelets) of APS patients. The study groups differentiate among clinical APS cases with the aim to translate molecular findings to disease stratification and to improve APS diagnosis and prognosis. These studies also include the unravelling of new autoantibodies in non-criteria APS or how post-translational protein modifications provides clues about the pathological mechanisms of antigen-autoantibody recognition. Herein, we identified 82 proteins that were dysregulated in APS across eleven studies. Enrichment analysis revealed its connection to cellular activation and degranulation that eventually leads to thrombosis as the main biological process highlighted by these studies. Validation of APS-relevant proteins by functional and mechanistic studies will be essential for patient stratification and the development of targeted therapies for every clinical subtype of APS.

The attachment of a single O-linked b-N-acetylglucosamine (O-GlcNAc) to serine and threonine residues of numerous proteins in the nucleus, cytoplasm, and mitochondria is a reversible post-translational modification (PTM) and plays an important role as a regulator of various cellular processes in both healthy and disease states. Advances in strategies and tools that allow for the detection of dynamic O-GlcNAcylation on cellular proteins have helped to enhance our initial and ongoing understanding of its dynamic effects on cellular stimuli and given insights into its link to the pathogenesis of several chronic diseases. Furthermore, chemical genetic strategies and related tools have been successfully applied to a myriad of biological systems with a new level of spatiotemporal and molecular precision. These strategies have started to be used in studying and controlling O-GlcNAcylation both in vivo and in vitro. In this mini-review, overviews of recent advances in molecular tools being applied to the detection and identification of O-GlcNAcylation on cellular proteins as well as on individual proteins are provided. In addition, chemical genetic strategies that have already been applied or are potentially usable in O-GlcNAc functional are also discussed.

73 p.-12 fig.-1 tab.-1 graph. abst., Viral infections are a major health problem; therefore, there is an urgent need for novel therapeutic strategies.Antivirals used to target proteins encoded by the viral genome usually enhance drug resistance generated by the virus. A potentialsolution may be drugs acting at host-based targets since viruses are dependent on numerous cellular proteins and phosphorylationevents that are crucial during their life cycle. Repurposing existing kinase inhibitors as antiviral agents would help in the cost andeffectiveness of the process, but this strategy usually does not provide much improvement, and specific medicinal chemistryprograms are needed in thefield. Anyway, extensive use of FDA-approved kinase inhibitors has been quite useful in deciphering therole of host kinases in viral infection. The present perspective aims to review the state of the art of kinase inhibitors that target viralinfections in different development stages., Funding from“la Caixa”Banking Foundation (LCF/PR/HR19/52160012), CSIC (202020E103 and 202080E293),and AEI (Grant PID2019-105600RB-I00) is acknowledged.E.C. holds a JAE Intro fellowship (JAEINT_20_01339) from CSIC., postprint

Author(s): Lam, Yi-Li | Advisor(s): Lew, John | Abstract: One of the hallmarks of Alzheimer’s disease is pathological tau aggregation. We previously reported that cinnamaldehyde, found in cinnamon, could inhibit tau aggregation in vitro, by reacting with one or both of the two cysteines in tau. In my thesis, I use spectrophotometric methods to show that both cysteines display hyperreactivity over model sulfhydryl compounds by as much as four fold. The hyperreactivity is specific to cysteines in tau as opposed to those in other sulfhydryl-containing cellular proteins. Cinnamaldehyde (CA) displays several characteristics that make it favorable as a possible inhibitor of aggregation in vivo: it is a water-soluble, brain-permeable molecule that is non-toxic and inexpensive. I now show in my thesis that the hyperreactivity of CA toward tau specifically, as described in this thesis, is an additional favorable property towards the goal of being a potential therapeutic.

Tailoring stress responses When faced with environmental stress, cells respond by shutting down cellular processes such as translation and nucleocytoplasmic transport. At the same time, cells preserve cytoplasmic messenger RNAs in structures known as stress granules, and many cellular proteins are modified by the covalent addition of ubiquitin, which has long been presumed to reflect degradation of stress-damaged proteins (see the Perspective by Dormann). Maxwell et al. show that cells generate distinct patterns of ubiquitination in response to different stressors. Rather than reflecting the degradation of stress-damaged proteins, this ubiquitination primes cells to dismantle stress granules and reinitiate normal cellular activities once the stress is removed. Gwon et al. show that persistent stress granules are degraded by autophagy, whereas short-lived granules undergo a process of disassembly that is autophagy independent. The mechanism of this disassembly depends on the initiating stress. Science , abc3593 and abf6548, this issue p. eabc3593 and p. eabf6548 ; see also abj2400, p. 1393

The coronavirus disease 2019 (COVID-19) pandemic has caused more than 2.3 million casualties worldwide and the lack of effective treatments is a major health concern. The development of targeted drugs is held back due to a limited understanding of the molecular mechanisms underlying the perturbation of cell physiology observed after viral infection. Recently, several approaches, aimed at identifying cellular proteins that may contribute to COVID-19 pathology, have been reported. Albeit valuable, this information offers limited mechanistic insight as these efforts have produced long lists of cellular proteins, the majority of which are not annotated to any cellular pathway. We have embarked in a project aimed at bridging this mechanistic gap by developing a new bioinformatic approach to estimate the functional distance between a subset of proteins and a list of pathways. A comprehensive literature search allowed us to annotate, in the SIGNOR 2.0 resource, causal information underlying the main molecular mechanisms through which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and related coronaviruses affect the host–cell physiology. Next, we developed a new strategy that enabled us to link SARS-CoV-2 interacting proteins to cellular phenotypes via paths of causal relationships. Remarkably, the extensive information about inhibitors of signaling proteins annotated in SIGNOR 2.0 makes it possible to formulate new potential therapeutic strategies. The proposed approach, which is generally applicable, generated a literature-based causal network that can be used as a framework to formulate informed mechanistic hypotheses on COVID-19 etiology and pathology.

Mammalian cells process information through coordinated spatiotemporal regulation of proteins. Engineering cellular networks thus relies on efficient tools for regulating protein levels in specific subcellular compartments. To address the need to manipulate the extent and dynamics of protein localization, we developed a platform technology for the target-specific control of protein destination. This platform is based on bifunctional molecules comprising a target-specific nanobody and universal sequences determining target subcellular localization or degradation rate. We demonstrate that nanobody-mediated localization depends on the expression level of the target and the nanobody, and the extent of target subcellular localization can be regulated by combining multiple target-specific nanobodies with distinct localization or degradation sequences. We also show that this platform for nanobody-mediated target localization and degradation can be regulated transcriptionally and integrated within orthogonal genetic circuits to achieve the desired temporal control over spatial regulation of target proteins. The platform reported in this study provides an innovative tool to control protein subcellular localization, which will be useful to investigate protein function and regulate large synthetic gene circuits.

Historically, covalent drugs were avoided in drug development process due to the possible toxicity linked to the covalent binding of such drugs and cellular proteins. However, recent years have witnessed the fast resurgence of the discovery of covalent drugs because of the realization of the advantages of covalent drugs in efficacy, duration of action, therapy-induced resistance, and targeting hard targets. Since December 2019, SARS-CoV-2 has caused nearly 40 million COVID-19 patients and over one million deaths in the whole world as of October 19, 2020. In contrast, effective drugs have not been found at the same time. Therefore, it is of great value to discover and design drugs for the prevention and cure of SARS-CoV-2 infection. Recently, we developed a simple but efficient method named as SCARdock for the discovery of covalent drugs. In this work, we present a detailed protocol of this method in discovering potential anti-SARS-CoV-2 drugs.

The ATG8 family of proteins regulates autophagy in a variety of ways. Recently, ATG8s were demonstrated to conjugate directly to cellular proteins in a process termed “ATG8ylation,” which is amplified by mitochondrial damage and antagonized by ATG4 proteases. ATG8s may have an emerging role as small protein modifiers. Refereed/Peer-reviewed

Extracellular vesicles (EVs) are secreted nano‐sized vesicles that contain cellular proteins, lipids, and nucleic acids. Although EVs are expected to be biologically diverse, current analyses cannot adequately characterize this diversity because most are ensemble methods that inevitably average out information from diverse EVs. Here we describe a single vesicle analysis, which directly visualizes marker expressions of individual EVs using a total internal‐reflection microscopy and analyzes their co‐localization to investigate EV subpopulations. The single‐vesicle imaging and co‐localization analysis successfully illustrated the diversity of EVs and revealed distinct patterns of tetraspanin expressions. Application of the analysis demonstrated similarities and dissimilarities between the EV fractions that had been acquired from different conventional EV isolation methods. The analysis method developed in this study will provide a new and reliable tool for investigating characteristics of single EVs, and the findings of the analysis might increase understanding of the characteristics of EVs.

PROteolysis Targeting Chimeras (PROTACs) are a family of heterobifunctional small molecules that specifically target cellular proteins for degradation. Given that their mode of action is distinct from that of small-molecule inhibitors widely used in clinical practice, PROTACs have the potential to improve current cancer therapies. Multiple studies have suggested that PROTACs exhibit enhanced pharmacodynamics and reduced toxicity both in vitro and in vivo compared to clinically relevant small-molecule kinase inhibitors. In addition, PROTACs have been reported to be less prone to mutation-mediated drug resistance in specific disease settings. Since its development in 2001, the field of targeted protein degradation, in which PROTACs are used, has expanded rapidly. However, earlier studies focused on the advancement of the technology itself, while preclinical and clinical data on the disease-modifying effect of PROTACs have only recently been reported. As preclinical and clinical evidence accumulates, the efficacy of PROTACs as targeted therapeutics-distinct from that of small-molecule kinase inhibitors-suggests potential translational benefit in the clinical setting. In this short review, we aim to describe translational potentials of PROTACs. We offer our perspectives as practicing oncologists on the preclinical and clinical data on PROTACs as novel therapeutics for both solid and hematological malignancies.

Membrane (M) proteins of coronaviruses are the most abundant component of the virus envelope and play crucial roles in virus assembly, virus budding and the regulation of host immunity. To understand more about these functions in the context of PEDV M protein, forty host cell proteins interacting with the M protein were identified in the present study by exploiting the proximity-labeling enzyme APEX2 (a mutant soybean ascorbate peroxidase). Bioinformatic analysis showed that the identified host cell proteins were related to fifty-four signal pathways and a wide diversity of biological processes. Interaction between M and five of the identified proteins (RIG-I, PPID, NHE-RF1, S100A11, CLDN4) was confirmed by co-immunoprecipitation (Co-IP). In addition, knockdown of PPID and S100A11 genes by siRNA significantly improved virus production, indicating that the proteins encoded by the two genes were interfering with or down-regulating virus replication in infected cells. Identification of the host cell proteins accomplished in this study provides new information about the mechanisms underlying PEDV replication and immune evasion. Significance PEDV M protein is an essential structural protein implicated in viral infection, replication and assembly although the precise mechanisms underlying these functions remain enigmatic. In this study, we have identified 40 host cell proteins that interact with PEDV M protein using the proximity-labeling enzyme APEX2. Co-immunoprecipitation subsequently confirmed interactions between PEDV M protein and five host cell proteins, two of which (S100A11 and PPID) were involved in down-regulating virus replication in infected cells. This study is significant in that it formulates a strategy to provide new information about the mechanisms relating to the novel functions of PEDV M protein., Graphical abstract Unlabelled Image

SummarySince the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, investigation of the SARS-CoV-2 infection in the native cellular context is scarce, and there is a lack of comprehensive knowledge on SARS-CoV-2 replicative cycle. Understanding the genome replication, assembly and egress of SARS-CoV-2, a multistage process that involves different cellular compartments and the activity of many viral and cellular proteins, is critically important as it bears the means of medical intervention to stop infection. Here, we investigated SARS-CoV-2 replication in Vero cells under the near-native frozen-hydrated condition using a unique correlative multi-modal, multi-scale cryo-imaging approach combining soft X-ray cryo-tomography and serial cryoFIB/SEM volume imaging of the entire SARS-CoV-2 infected cell with cryo-electron tomography (cryoET) of cellular lamellae and cell periphery, as well as structure determination of viral components by subtomogram averaging. Our results reveal at the whole cell level profound cytopathic effects of SARS-CoV-2 infection, exemplified by a large amount of heterogeneous vesicles in the cytoplasm for RNA synthesis and virus assembly, formation of membrane tunnels through which viruses exit, and drastic cytoplasm invasion into nucleus. Furthermore, cryoET of cell lamellae reveals how viral RNAs are transported from double-membrane vesicles where they are synthesized to viral assembly sites; how viral spikes and RNPs assist in virus assembly and budding; and how fully assembled virus particles exit the cell, thus stablishing a model of SARS-CoV-2 genome replication, virus assembly and egress pathways.

Direct interaction between intrinsically disordered proteins (IDPs) is often difficult to characterize hampering the elucidation of their binding mechanism. Particularly challenging is the study of fuzzy complexes, in which the intrinsically disordered proteins or regions retain conformational freedom within the assembly. To date, nuclear magnetic resonance spectroscopy has proven to be one of the most powerful techniques to characterize at the atomic level intrinsically disordered proteins and their interactions, including those cases where the formed complexes are highly dynamic. Here, we present the characterization of the interaction between a viral protein, the Early region 1A protein from Adenovirus (E1A), and a disordered region of the human CREB-binding protein, namely the fourth intrinsically disordered linker CBP-ID4. E1A was widely studied as a prototypical viral oncogene. Its interaction with two folded domains of CBP was mapped, providing hints for understanding some functional aspects of the interaction with this transcriptional coactivator. However, the role of the flexible linker connecting these two globular domains of CBP in this interaction was never explored before.

Deubiquitinating enzymes (DUBs) control the removal of ubiquitin and ubiquitin-like proteins from cellular proteins. There are approximately 100 DUBs in the human genome, and they regulate diverse biochemical, cellular and physiological processes. Notably, they are known to control many pathways which are misregulated and affected in human diseases, such as cancer, immunology and neurodegeneration. Due to the broad scope of DUB biology, they are emerging as a target class for inhibitor development. In this chapter, we will describe the promise of targeting DUB in different disease contexts, describe practices for identifying and validating small-molecule inhibitors and physiologically relevant substrates of DUBs and review recent examples of well-characterized DUB inhibitors. These advances underscore the excitement and potential in targeting DUBs for both therapeutic and research purposes.

Covalent attachment of ubiquitin, a small globular polypeptide, to protein substrates is a key post-translational modification that determines the fate, function, and turnover of most cellular proteins. Ubiquitin modification exists as mono- or polyubiquitin chains involving multiple ways how ubiquitin C-termini are connected to lysine, perhaps other amino acid side chains, and N-termini of proteins, often including branching of the ubiquitin chains. Understanding this enormous complexity in protein ubiquitination, the so-called ‘ubiquitin code’, in combination with the ∼1000 enzymes involved in controlling ubiquitin recognition, conjugation, and deconjugation, calls for novel developments in analytical techniques. Here, we review different headways in the field mainly driven by mass spectrometry and chemical biology, referred to as “ubiquitomics”, aiming to understand this system’s biological diversity.

Cavezzi A, Troiani E, Corrao S (2020) COVID-19: hemoglobin, iron, and hypoxia beyond inflammation. A narrative review. Clin Pract 10(2). https://doi.org/10.4081/cp.2020.1271 Garrick M, Ghio A (2020) Iron chelation may harm patients with COVID-19. Eur J Clin Pharmacol Herbert V, Jayatilleke E, Shaw S, Rosman A, Giardina P, Grady R et al (1997) Serum ferritin iron, a new test, measures human body iron stores unconfounded by inflammation. Stem Cells 15(4):291–296 Article Google Scholar Perricone C, Bartoloni E, Bursi R, Cafaro G, Guidelli G, Shoenfeld Y et al (2020) COVID-19 as part of the hyperferritinemic syndromes: the role of iron depletion therapy. Immunol Res 68(4):213–224 Article Google Scholar Edeas M, Saleh J, Peyssonnaux C (2020) Iron: Innocent bystander or vicious culprit in COVID-19 pathogenesis? Int J Infect Dis 97:303–305 Article Google Scholar Liu W, Zhang S, Nekhai S, Liu S (2020) Depriving iron supply to the virus represents a promising adjuvant therapeutic against viral survival. Curr Clin Microbiol Rep 7(2):13–19 Article Google Scholar Shi S, Lai M (2005) Viral and cellular proteins involved in coronavirus replication. Curr Top Microbiol Immunol 287:95–131 PubMed PubMed Central Google Scholar Juang H (2004) Modulation of iron on mitochondrial aconitase expression in human prostatic carcinoma cells. Mol Cell Biochem 265(1/2):185–194 Article Google Scholar Download references Spire Fylde Coast Hospital, St Walburgas Road, Blackpool, FY3 8BP, UK Anis Abobaker You can also search for this author in PubMed Google Scholar Correspondence to Anis Abobaker. The author declares that he has no conflict of interest. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Reprints and Permissions Abobaker, A. Reply: Iron chelation may harm patients with COVID-19. Eur J Clin Pharmacol (2020). https://doi.org/10.1007/s00228-020-02988-9 Download citation Received: 12 August 2020 Accepted: 27 August 2020 Published: 01 September 2020 DOI: https://doi.org/10.1007/s00228-020-02988-9

Targeted Protein Degradation (TPD) has emerged as an exciting new era in chemical biology and drug discovery. PROteolysis TArgeting Chimera (PROTAC) technology targets cellular proteins for degradation by co-opting the Ubiquitin Proteasome System. Over the last five years, numerous studies have expanded our understanding of the unique mode of action and advantages of PROTACs, which has in turn spurred interest in both academia and industry to explore PROTACs as a novel therapeutic strategy. In this review, we first highlight the key advantages of PROTACs and then discuss the spatiotemporal regulation of protein degradation. Next, we explore current chemically tractable E3 ligases focusing on expanding the existing repertoire with novel E3 ligases to uncover the full potential of TPD. Collectively, these studies are guiding the development of the PROTAC technology as it emerges as a new modality in precision medicine.

Tetrazolium salts discussed here are of the 2-H type, and contain one or more five-membered heterocyclic rings, each of which carries a delocalized positive charge. Such a tetrazolium compound may be represented by a pair of Kekule forms. Tetrazolium salts can be reduced to formazans by a variety of mild reductants, including those of enzyme-catalyzed biological systems. Formazans with suitable chelating groups can give rise to metal coordination complexes under histochemical conditions. To facilitate histochemical application, and to localize enzyme activities, tetrazolium salts should be water soluble, and derived formazans should be insoluble in water and routine processing solvents. The fact that reduction of tetrazolium salts to formazans involves loss of a positive charge, and hence a diminution of hydrophilicity, is therefore advantageous. Localization of staining to the vicinity of the enzymic sites is however largely controlled by generation of formazans with large conjugated systems, because this property enhances binding to cellular proteins.

Interactions between the pancreatic extracellular matrix (ECM) and islet cells are known to regulate multiple aspects of islet physiology, including survival, proliferation, and glucose-stimulated insulin secretion. Recognizing the essential role of ECM in islet survival and function, various engineering approaches have been developed that aim to utilize ECM-based materials to recreate a native-like microenvironment. However, a major impediment to the success of these approaches has been the lack of a robust and comprehensive characterization of the human pancreatic proteome. Herein, by combining mass spectrometry (MS) and multiplex ELISA, we have provided an improved workflow for the in-depth profiling of the proteome, including minor constituents that are generally underrepresented. Moreover, we have further validated the effectiveness of our detergent-free decellularization protocol in the removal of cellular proteins and retention of the matrisome. It has also been established that the decellularized ECM and its derivatives can provide more tissue-specific cues than traditionally used biological scaffolds and are therefore more physiologically relevant for the development of hydrogels, bioinks and medium additives, in order to create a pancreatic niche. The data generated in this study would contribute significantly to the efforts of comprehensively defining the ECM atlas and also serve as a standard for the human pancreatic proteome to provide further guidance for design and engineering strategies for improved tissue engineering scaffolds.

Background: Leptospirosis, caused mainly by Leptospira interrogans, is a global zoonotic infectious disease. Macrophages and vascular endothelial cells are the main host cells for L. interrogans during infection, but the proteins released from the pathogen and the two host cells during infection remain mostly unknown.Results: Cellular supernatant proteins (CSPs) from human THP-1 macrophages or umbilical vein endothelial cells (HUVECs) infected with L. interrogans strain Lai were extracted by TCA/FASP methods. The exoproteins in the CSPs were identified by LC-MS/MS. Viability of the leptospires and host cells during infection was confirmed by confocal microscopy and MTT. The results showed that higher co-culture temperature (from 28°C to 37°C) and different biochemical environments cause a large change in the exoproteome of the spirochete. L. interrogans increased levels of leptospiral exoproteins related to stress, signal transduction and virulence factors, while the lipoprotein antigens LipL41, LipL21 and/or Loa22 were not detected. During infection of macrophages and endothelial cells, there was a large increase in host-cell exoproteins involved in stress response, complement pathways (C4/5/7/8), inflammatory cytokines (IL-6, TNF-α, MIF, MCP-1 and GM-CSF), extracellular matrix proteins (FN, LN and COLs), and blood coagulation factors. One-third of the leptospires and infected THP-1 macrophages died during macrophage infection, but nearly all the leptospires and endothelial cells remained viable during endothelial cell infection.Conclusions: Infection causes stress reponses for both leptospires and human macrophages and vascular endothelial cells and release of virulence factors, alteration of surface leptospiral lipoprotein antigens and secretion of complement components and inflammatory cytokines from host cells.

Isolevuglandins (IsoLGs) are a family of highly reactive 4-ketoaldehydes formed by lipid peroxidation that modify the lysyl residues of cellular proteins. Modification of proteins by IsoLGs have been shown to contribute to disease processes such as the development of hypertension. Accurate quantitation of the extent of protein modification by IsoLGs is essential for understanding the mechanisms whereby these modifications contribute to disease and the efficacy of interventions designed to prevent this modification. The previously described LC/MS assay to quantitate IsoLG protein adducts was extremely labor-intensive and time consuming, and while it offered reasonably low intra-day variation for replicate samples, variation when replicate samples were processed on separate days was significant. These limitations significantly restricted utilization of this approach. We therefore performed a series of studies to optimize the assay. We now report a significantly simplified LC/MS assay for measurement of IsoLG protein adducts with increased sensitivity and lower intra-day and inter-day variability.