global-level-quantification-histone-post-translational-modifications

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid ...

Albert Einstein College of Medicine

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) (MALDI-IMS) provides a technical means for simultaneous analysis of precise anatomic localization and regulation of peptides. We explored the technical capability of matrix-assisted laser desorption ionization mass spectrometry for characterization of peptidomic regulation by an addictive substance along two distinct projection systems in the mouse striatum. The spatial expression patterns of substance P and proenkephalin, marker neuropeptides of two distinct striatal projection neurons, were negatively correlated at baseline. We detected 768 mass/charge (m/z) peaks whose expression levels were mostly negatively and positively correlated with expression levels of substance P and proenkephalin A (amino acids 218-228), respectively, within the dorsal striatum. After nicotine administration, there was a positive shift in correlation of mass/charge peak expression levels with substance P and proenkephalin A (218-228). Our exploratory analyses demonstrate the technical capacity of MALDI-IMS for comprehensive identification of peptidomic regulation patterns along histochemically distinguishable striatal projection pathways. 

Molecular Histochemistry Identifies Peptidomic Organization and Reorganization Along Striatal Projection Units.

The emergence and widespread distribution of multi-drug resistant bacteria are considered as a major public health concern. The inabilities to curb severe infections due to antibiotic resistance have increased healthcare costs as well as patient morbidity and mortality. Bacterial biofilms formed by drug-resistant bacteria add additional challenges to treatment. This study describes a solgel based nanoparticle system loaded with garlic extract (GE-np) that exhibits: i) slow and sustained release of garlic components; ii) stabilization of the active components; and iii) significant enhancement of antimicrobial and antibiofilm activity relative to the free garlic extract. Also, GE-np were efficient in penetrating and disrupting the well-established methicillin-resistant Staphylococcus aureus (MRSA) biofilms. Overall, the study suggests that GE-np might be a promising candidate for the treatment of chronic infections due to biofilm forming drug-resistant bacteria.

Anti-biofilm activity of garlic extract loaded nanoparticles.

Severe aortic stenosis (AS) is prevalent in adults ≥ 65 years, a significant cause of morbidity and mortality, with no medical therapy. Lipid and proteomic alterations of human AS tissue were determined using mass spectrometry imaging (MSI) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to understand histopathology, potential biomarkers of disease, and progression from non-calcified to calcified phenotype. A reproducible MSI method was developed using healthy murine aortic valves (n = 3) and subsequently applied to human AS (n = 2). Relative lipid levels were spatially mapped and associated with different microdomains. Proteomics for non-calcified and calcified microdomains were performed to ascertain differences in expression. Increased pro-osteogenic and inflammatory lysophosphatidylcholine (LPC) 16:0 and 18:0 were co-localized with calcified microdomains. Proteomics analysis identified differential patterns in calcified microdomains with high LPC and low cholesterol as compared to non-calcified microdomains with low LPC and high cholesterol. Calcified microdomains had higher levels of: apolipoproteins (Apo) B-100 (p < 0.001) and Apo A-IV (p < 0.001), complement C3 and C4-B (p < 0.001), C5 (p = 0.007), C8 beta chain (p = 0.013) and C9 (p = 0.010), antithrombotic proteins alpha-2-macroglobulin (p < 0.0001) and antithrombin III (p = 0.002), and higher anti-calcific fetuin-A (p = 0.02), while the osteoblast differentiating factor transgelin (p < 0.0001), extracellular matrix proteins versican, prolargin, and lumican ( p < 0.001) and regulator protein complement factor H (p < 0.001) were higher in non-calcified microdomains. A combined lipidomic and proteomic approach provided insight into factors potentially contributing to progression from non-calcified to calcific disease in severe AS. Additional studies of these candidates and protein networks could yield new targets for slowing progression of AS.

Lipid mass spectrometry imaging and proteomic analysis of severe aortic stenosis.

Proteomics studies generate tables with thousands of entries. A significant component of being a proteomics scientist is the ability to process these tables to identify regulated proteins. Many bioinformatics tools are freely available for the community, some of which within reach for scientists with limited or no background in programming and statistics. However, proteomics has become popular in most other biological and biomedical disciplines, resulting in more and more studies where data processing is delegated to specialists that are not lead authors of the scientific project. This creates a risk or at least a limiting factor, as the biological interpretation of a dataset is contingent of a third-party specialist transforming data without the input of the project leader. We acknowledge in advance that dedicated scripts and software have a higher level of sophistication; but we hereby claim that the approach we describe makes proteomics data processing immediately accessible to every scientist. In this paper, we describe key steps of the typical data transformation, normalization and statistics in proteomics data analysis using a simple spreadsheet. This manuscript aims to demonstrate to those who are not familiar with the math and statistics behind these workflows that a proteomics dataset can be processed, simplified and interpreted in software like Microsoft Excel. With this, we aim to reach the community of non-specialists in proteomics to find a common language and illustrate the basic steps of -omics data processing.

Guide for protein fold change and p-value calculation for non-experts in proteomics.

The intracellular parasite Toxoplasma gondii infects a large proportion of humans worldwide and can cause adverse complications in the settings of immune-compromise and pregnancy. T. gondii thrives within many different cell types due in part to its residence within a specialized and heavily modified compartment in which the parasite divides, termed the parasitophorous vacuole. Within this vacuole, numerous proteins optimize intracellular survival following their secretion by the parasite. We investigated the contribution of one of these proteins, TgPPM3C, predicted to contain a PP2C-class serine/threonine phosphatase domain and previously shown to interact with the protein MYR1, an essential component of a putative vacuolar translocon that mediates effector export into the host cell. Parasites lacking the TgPPM3C gene exhibit a minor growth defect in vitro, are avirulent during acute infection in mice, and form fewer cysts in mouse brain during chronic infection. Phosphoproteomic assessment of TgPPM3C deleted parasite cultures demonstrated alterations in the phosphorylation status of many secreted vacuolar proteins including two exported effector proteins, GRA16 and GRA28, as well as MYR1. Parasites lacking TgPPM3C are defective in GRA16 and GRA28 export, but not in the export of other MYR1-dependant effectors. Phosphomimetic mutation of two GRA16 serine residues results in export defects, suggesting that de-phosphorylation is a critical step in the process of GRA16 export. These findings provide another example of the emerging role of phosphatases in regulating the complex environment of the T. gondii parasitophorous vacuole and influencing the export of specific effector proteins from the vacuolar lumen into the host cell.

Toxoplasma gondii PPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a World Health Organization priority pathogen. CCHFV infections cause a highly lethal hemorrhagic fever for which specific treatments and vaccines are urgently needed. Here, we characterize the human immune response to natural CCHFV infection to identify potent neutralizing monoclonal antibodies (nAbs) targeting the viral glycoprotein. Competition experiments showed that these nAbs bind six distinct antigenic sites in the Gc subunit. These sites were further delineated through mutagenesis and mapped onto a prefusion model of Gc. Pairwise screening identified combinations of non-competing nAbs that afford synergistic neutralization. Further enhancements in neutralization breadth and potency were attained by physically linking variable domains of synergistic nAb pairs through bispecific antibody (bsAb) engineering. Although multiple nAbs protected mice from lethal CCHFV challenge in pre- or post-exposure prophylactic settings, only a single bsAb, DVD-121-801, afforded therapeutic protection. DVD-121-801 is a promising candidate suitable for clinical development as a CCHFV therapeutic.

Protective neutralizing antibodies from human survivors of Crimean-Congo hemorrhagic fever.

The immune system has been described to play a role in the development of Alzheimer's disease (AD), but the distribution of immunoglobulins and their subclasses in brain tissue has not been explored. In this study, examination of pathologically diagnosed frontal cortex gray matter revealed significantly higher levels of IgM and IgG in late-stage AD (Braak and Braak stages V and VI) compared to age-matched controls. While levels of IgG2 and IgG4 constant region fragments were higher in late-stage AD, concentration of native-state IgG4 with free Fc regions was increased in AD III and VI. RNA analysis did not support parenchymal B-cell production of IgG4 in AD III and V, indicating possible peripheral or meningeal B-cell involvement. Changes in the profile of IgM, IgG and IgG subclasses in AD frontal cortex may provide insight into understanding disease pathogenesis and progression.

Article	Year
Molecular Histochemistry Identifies Peptidomic Organization and Reorganization Along Striatal Projection Units. Biological psychiatry\| PubMed ID: 26520239	2016
Anti-biofilm activity of garlic extract loaded nanoparticles. Nanomedicine : nanotechnology, biology, and medicine\| PubMed ID: 31085344	2019
Lipid mass spectrometry imaging and proteomic analysis of severe aortic stenosis. Journal of molecular histology\| PubMed ID: 32794037	2020
Guide for protein fold change and p-value calculation for non-experts in proteomics. Molecular omics\| PubMed ID: 32968743	2020
Toxoplasma gondii PPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export. PLoS pathogens\| PubMed ID: 33370417	2020
Protective neutralizing antibodies from human survivors of Crimean-Congo hemorrhagic fever. Cell\| PubMed ID: 34077751	2021
Altered abundances of human immunoglobulin M and immunoglobulin G subclasses in Alzheimer's disease frontal cortex. Scientific reports\| PubMed ID: 35484384	2022

Jennifer T. Aguilan

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