Monika Dzieciatkowska

Quantification of Extracellular Matrix Proteins from a Rat Lung Scaffold to Provide a Molecular Readout for Tissue Engineering

The use of extracellular matrix (ECM)1 scaffolds, derived from decellularized tissues for engineered organ generation, holds enormous potential in the field of regenerative medicine. To support organ engineering efforts, we developed a targeted proteomics method to extract and quantify extracellular matrix components from tissues. Our method provides more complete and accurate protein characterization than traditional approaches. This is accomplished through the analysis of both the chaotrope-soluble and -insoluble protein fractions and using recombinantly generated stable isotope labeled peptides for endogenous protein quantification. Using this approach, we have generated 74 peptides, representing 56 proteins to quantify protein in native (nondecellularized) and decellularized lung matrices. We have focused on proteins of the ECM and additional intracellular proteins that are challenging to remove during the decellularization procedure. Results indicate that the acellular lung scaffold is predominantly composed of structural collagens, with the majority of these proteins found in the insoluble ECM, a fraction that is often discarded using widely accepted proteomic methods. The decellularization procedure removes over 98% of intracellular proteins evaluated and retains, to varying degrees, proteoglycans and glycoproteins of the ECM. Accurate characterization of ECM proteins from tissue samples will help advance organ engineering efforts by generating a molecular readout that can be correlated with functional outcome to drive the next generation of engineered organs.

Rejuvenation of chondrogenic potential in a young stem cell microenvironment

Autologous cells suffer from limited cell number and senescence during ex vivo expansion for cartilage repair. Here we found that expansion on extracellular matrix (ECM) deposited by fetal synovium-derived stem cells (SDSCs) (FE) was superior to ECM deposited by adult SDSCs (AE) in promoting cell proliferation and chondrogenic potential. Unique proteins in FE might be responsible for the rejuvenation effect of FE while advantageous proteins in AE might contribute to differentiation more than to proliferation. Compared to AE, the lower elasticity of FE yielded expanded adult SDSCs with lower elasticity which could be responsible for the enhancement of chondrogenic and adipogenic differentiation. MAPK and noncanonical Wnt signals were actively involved in ECM-mediated adult SDSC rejuvenation.

Oxidative modifications of glyceraldehyde 3-phosphate dehydrogenase regulate metabolic reprogramming of stored red blood cells

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) plays a key regulatory function in glucose oxidation by mediating fluxes through glycolysis or the pentose phosphate pathway (PPP) in an oxidative stress-dependent fashion. Previous studies documented metabolic reprogramming in stored red blood cells (RBCs) and oxidation of GAPDH at functional residues upon exposure to pro-oxidants diamide and H2O2 Here we hypothesize that routine storage of erythrocyte concentrates promotes metabolic modulation of stored RBCs by targeting functional thiol residues of GAPDH. Progressive increases in PPP/glycolysis ratios were determined via metabolic flux analysis after spiking (13)C1,2,3-glucose in erythrocyte concentrates stored in Additive Solution-3 under blood bank conditions for up to 42 days. Proteomics analyses revealed a storage-dependent oxidation of GAPDH at functional Cys152, 156, 247, and His179. Activity loss by oxidation occurred with increasing storage duration and was progressively irreversible. Irreversibly oxidized GAPDH accumulated in stored erythrocyte membranes and supernatants through storage day 42. By combining state-of-the-art ultra-high-pressure liquid chromatography-mass spectrometry metabolic flux analysis with redox and switch-tag proteomics, we identify for the first time ex vivo functionally relevant reversible and irreversible (sulfinic acid; Cys to dehydroalanine) oxidations of GAPDH without exogenous supplementation of excess pro-oxidant compounds in clinically relevant blood products. Oxidative and metabolic lesions, exacerbated by storage under hyperoxic conditions, were ameliorated by hypoxic storage. Storage-dependent reversible oxidation of GAPDH represents a mechanistic adaptation in stored erythrocytes to promote PPP activation and generate reducing equivalents. Removal of irreversibly oxidized, functionally compromised GAPDH identifies enhanced vesiculation as a self-protective mechanism in ex vivo aging erythrocytes.

Impact of maternal aging on the molecular signature of human cumulus cells

OBJECTIVE To investigate the impact of maternal aging on the molecular signature of cumulus cells. DESIGN Experimental study. SETTING Research laboratory. PATIENT(S) Patients, young fertile oocyte donors (n = 40) and infertile women of advanced maternal age (40-45 years; n = 48), donated, with Institutional Review Board consent, cumulus cells during routine infertility treatment. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Proteomic and gene expression profiles of cumulus cells. RESULT(S) Proteomic analysis identified a total of 1,423 cumulus cell proteins. Statistical analysis revealed 110 (7.7%) proteins to be differentially expressed in relation to female aging (>1.5-fold change). Pathway annotation revealed significant involvement in metabolism (ACAT2, HSD17B4, ALDH9A1, MVK, CYP11A1, and FDFT1), oxidative phosphorylation (OP; NDUFA1, UQCRC1, MT-ATP6, ATP5I, and MT-ATP8), and post-transcriptional mechanisms (KHSRP, SFPQ, DDX46, SNRPF, ADAR, NHPL1, and U2AF2) relative to advanced maternal age. Gene expression analysis also revealed altered profiles in cumulus cells from women in their early to mid-40s. CONCLUSION(S) This novel study reveals that the cumulus cell molecular signature, at both the gene and protein level, is impacted by advanced maternal aging. A compromised follicular environment is evident with altered energy metabolism and post-transcriptional processes.

Lin28 promotes the proliferative capacity of neural progenitor cells in brain development

Neural progenitor cells (NPCs) have distinct proliferation capacities at different stages of brain development. Lin28 is an RNA-binding protein with two homologs in mice: Lin28a and Lin28b. Here we show that Lin28a/b are enriched in early NPCs and their expression declines during neural differentiation. Lin28a single-knockout mice show reduced NPC proliferation, enhanced cell cycle exit and a smaller brain, whereas mice lacking both Lin28a alleles and one Lin28b allele display similar but more severe phenotypes. Ectopic expression of Lin28a in mice results in increased NPC proliferation, NPC numbers and brain size. Mechanistically, Lin28a physically and functionally interacts with Imp1 (Igf2bp1) and regulates Igf2-mTOR signaling. The function of Lin28a/b in NPCs could be attributed, at least in part, to the regulation of their mRNA targets that encode Igf1r and Hmga2. Thus, Lin28a and Lin28b have overlapping functions in temporally regulating NPC proliferation during early brain development. Summary: The proliferation of neural progenitor cells is regulated by Lin28a/b via their interaction with Imp1 and their regulation of Igf2-mTOR signaling.

Lipooligosaccharide of Campylobacter jejuni SIMILARITY WITH MULTIPLE TYPES OF MAMMALIAN GLYCANS BEYOND GANGLIOSIDES

Campylobacter jejuni is well known for synthesizing ganglioside mimics within the glycan component of its lipooligosaccharide (LOS), which have been implicated in triggering Guillain-Barré syndrome. We now confirm that this pathogen is capable of synthesizing a much broader spectrum of host glycolipid/glycoprotein mimics within its LOS. P blood group and paragloboside (lacto-N-neotetraose) antigen mimicry is exhibited by RM1221, a strain isolated from a poultry source. RM1503, a gastroenteritis-associated strain, expresses lacto-N-biose and sialyl-Lewis c units, the latter known as the pancreatic tumor-associated antigen, DU-PAN-2 (or LSTa). C. jejuni GC149, a Guillain-Barré syndrome-associated strain, expresses an unusual sialic acid-containing hybrid oligosaccharide with similarity to both ganglio and Pk antigens and can, through phase variation of its LOS biosynthesis genes, display GT1a or GD3 ganglioside mimics. We show that the sialyltransferase CstII and the galactosyltransferase CgtD are involved in the synthesis of multiple mimic types, with LOS structural diversity achieved through evolving allelic substrate specificity.

Proteomic Analyses of Human Plasma: Venus versus Mars

BACKGROUND: Plasma is vital for the resuscitation of injured patients and to restore necessary procoagulants, especially Factors (F)II, FV, FVII, FX, and FXIII; however, female plasma has been implicated in the majority of transfusion‐related acute lung injury (TRALI) cases and male‐only plasma transfusion regimens have significantly decreased the incidence of TRALI. Little is known about the human plasma proteome, and no comparisons have been made between male and female plasma; therefore, we hypothesize that there are significant differences between plasma from male and female donors.

Hemolysis exacerbates hyperfibrinolysis, whereas platelolysis shuts down fibrinolysis: evolving concepts of the spectrum of fibrinolysis in response to severe injury.

ABSTRACT Introduction: We have recently identified a spectrum of fibrinolysis in response to injury, in which there is increased mortality in patients who have either excessive fibrinolysis (hyperfibrinolysis [HF]) or impaired fibrinolysis (shutdown). The regulation of the fibrinolytic system after trauma remains poorly understood. Our group’s previous proteomic and metabolomic work identified elevated red blood cell (RBC) degradation products in trauma patients manifesting HF. We therefore hypothesized that hemolysis was contributory to the pathogenesis of HF. Given the central role of platelets in the cell-based model of coagulation, we further investigated the potential role of platelet lysis in mediation of the fibrinolytic system. Methods: Red blood cells from healthy donors were frozen in liquid nitrogen and vortexed to create mechanical membrane disruption. Platelets were prepared in a similar fashion. Assays were performed with citrated whole blood mixed ex vivo with either RBC or platelet lysates. Tissue plasminogen activator (tPA) was then added to promote fibrinolysis, mimicking the tPA release from ischemic endothelium during hemorrhagic shock. The degree of fibrinolysis was evaluated with thromboelastography. To identify the mediators of the fibrinolysis system present in RBC and platelet lysates, these lysates were passed over immobilized tPA and plasminogen affinity columns to capture protein-binding partners from RBC or platelet lysates. Results: The addition of 75 ng/mL of tPA to whole blood increased fibrinolysis from median 30-min lysis of 1.4% (interquartile range [IQR], 0.9%–2.0%) to 8.9% (IQR, 6.5%–11.5%). Red blood cell lysate with tPA increased fibrinolysis to 20.1% (IQR, 12.5%–33.7%), which was nearly three times as much lysis as tPA alone (P < 0.001). Conversely, the addition of platelet lysate decreased tPA-mediated fibrinolysis to 0.35% (IQR, 0.2%–0.8%; P < 0.001). Affinity chromatography coupled with tandem mass spectrometry identified a number of proteins not previously associated with regulation of fibrinolysis and trauma. Conclusion: Red blood cell lysate is a potent enhancer of fibrinolysis, whereas platelet lysate inhibits fibrinolysis. Intracellular proteins from circulating blood cells contain proteins that interact with the two key proteins of tPA-mediated fibrinolysis. Understanding the effect of tissue injury and shock on the lysis of circulating cells may provide insight to comprehending the spectrum of fibrinolysis in response to trauma.

Proteomic analysis of the supernatant of red blood cell units: the effects of storage and leucoreduction

Red blood cell (RBC) transfusion is a life‐saving intervention for critically ill patients; however, it has been linked to increased morbidity and mortality. We hypothesize that a number of important proteins accumulate during routine storage of RBCs, which may explain some of the adverse effects seen in transfused patients.

GeLC-MS/MS Analysis of Complex Protein Mixtures

Discovery-based proteomics has found its place in nearly every facet of biological research. A key objective of this approach is to maximize sequence coverage for proteins across a wide concentration range. Fractionating samples at the protein level is one of the most common ways to circumvent challenges due to sample complexity and improve proteome coverage. Of the available methods, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) is a robust and reproducible method for qualitative and quantitative proteomic analysis. Here we describe a general GeLC-MS/MS protocol and include technical advice and outline caveats to increase the probability of a successful analysis.