In Situ Visualizing the Recognition Between Proteins and Platinum-Damaged DNA in Single-Cells by Correlated Optical and Secondary Ion Mass Spectrometric Imaging

10.26434/chemrxiv.11662335.v1 ◽

2020 ◽

Author(s):

Yu Lin ◽

Kui Wu ◽

Feifei Jia ◽

Ling Chen ◽

Zhaoying Wang ◽

...

Keyword(s):

Single Cells ◽

Mass Spectrometric ◽

Dependent Manner ◽

Mass Spectrometric Imaging ◽

Novel Approach ◽

Molecular Mechanism Of Action ◽

Secondary Ion ◽

Damaged Dna

In situ visualization of the recognition and interactions between proteins and drug damaged DNA at single cell level is highly important for understanding the molecular mechanism of action of DNA targeting drugs, yet a great challenge. We report herein a novel approach, termed as correlated optical and secondary ion mass spectrometric imaging (COSIMSi), to explore the recognition between proteins and cisplatin-damaged DNA in single cells. Genetically encoded EYFP-fused HMGB1, an in vitro well-known specific binder of cisplatin-damaged DNA, dye-stained DNA, and platinum were mapped by LSCM and ToF-SIMS imaging, respectively. The LSCM and SIMS images were aligned with aiding of an addressable silicon wafer to generate fused images, in which the co-localization of the fluorescent and MS signals indicated the formation of HMGB1-Pt-DNA ternary complexes in a dose- and time-dependent manner. In contrast, COSIMSi results showed that little HMGB1<a></a><a>(F37A</a>)-Pt-DNA complex was produced under the same conditions. Moreover, we demonstrated for the first time that cisplatin lesions on DNA prevented DNA-binding proteins Smad3 and Smad7 from interacting with DNA. These results verify that the COSIMSi is an effective and straightforward tool for in situ visualization of recognition and interactions between proteins and specific damaged DNA in single cells.

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In Situ Visualizing the Recognition Between Proteins and Platinum-Damaged DNA in Single-Cells by Correlated Optical and Secondary Ion Mass Spectrometric Imaging

10.26434/chemrxiv.11662335.v3 ◽

2020 ◽

Author(s):

Yu Lin ◽

Kui Wu ◽

Feifei Jia ◽

Ling Chen ◽

Zhaoying Wang ◽

...

Keyword(s):

Single Cells ◽

Mass Spectrometric ◽

Dependent Manner ◽

Mass Spectrometric Imaging ◽

Novel Approach ◽

Molecular Mechanism Of Action ◽

Secondary Ion ◽

Damaged Dna

In situ visualization of the recognition and interaction between proteins and drug damaged DNA at single cell level is highly important for understanding the molecular mechanism of action of DNA targeting drugs, yet a great challenge. We herein report a novel approach, termed as correlated optical and secondary ion mass spectrometric imaging (COSIMSi), for exploring the recognition between proteins and cisplatin-damaged DNA in single cells. Genetically encoded EYFP-fused HMGB1, an in vitro well-known specific binder of cisplatin-damaged DNA, and dye-stained DNA, and cisplatin were mapped by LSCM and ToF-SIMS imaging, respectively. The LSCM and SIMS images were aligned with aiding of an addressable silicon wafer to generate fused images, in which the co-localization of the fluorescence and MS signals indicated the formation of HMGB1-Pt-DNA ternary complexes in a dose- and time-dependent manner. In contrast, COSIMSi showed that little HMGB1(F37A)-Pt-DNA complex was produced under the same conditions. Moreover, we demonstrated for the first time that cisplatin lesion on DNA prevented a DNA-binding protein Smad3 from interacting with DNA. These results verify that the COSIMSi is an effective and straightforward tool for in situ visualization of recognition and interaction between proteins and specific damaged DNA in single cells.

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In Situ Visualizing the Recognition Between Proteins and Platinum-Damaged DNA in Single-Cells by Correlated Optical and Secondary Ion Mass Spectrometric Imaging

10.26434/chemrxiv.11662335.v2 ◽

2020 ◽

Author(s):

Yu Lin ◽

Kui Wu ◽

Feifei Jia ◽

Ling Chen ◽

Zhaoying Wang ◽

...

Keyword(s):

Single Cells ◽

Mass Spectrometric ◽

Dependent Manner ◽

Mass Spectrometric Imaging ◽

Novel Approach ◽

Molecular Mechanism Of Action ◽

Secondary Ion ◽

Damaged Dna

In situ visualization of the recognition and interactions between proteins and drug damaged DNA at single cell level is highly important for understanding the molecular mechanism of action of DNA targeting drugs, yet a great challenge. We report herein a novel approach, termed as correlated optical and secondary ion mass spectrometric imaging (COSIMSi), to explore the recognition between proteins and cisplatin-damaged DNA in single cells. Genetically encoded EYFP-fused HMGB1, an in vitro well-known specific binder of cisplatin-damaged DNA, dye-stained DNA, and platinum were mapped by LSCM and ToF-SIMS imaging, respectively. The LSCM and SIMS images were aligned with aiding of an addressable silicon wafer to generate fused images, in which the co-localization of the fluorescent and MS signals indicated the formation of HMGB1-Pt-DNA ternary complexes in a dose- and time-dependent manner. In contrast, COSIMSi results showed that little HMGB1<a></a><a>(F37A</a>)-Pt-DNA complex was produced under the same conditions. Moreover, we demonstrated for the first time that cisplatin lesions on DNA prevented DNA-binding proteins Smad3 and Smad7 from interacting with DNA. These results verify that the COSIMSi is an effective and straightforward tool for in situ visualization of recognition and interactions between proteins and specific damaged DNA in single cells.

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Optics-Free Visualization of Proteins in Single Cells by Time of Flight-Secondary Ion Mass Spectrometry Coupled with Genetically Encoded Chemical Tags

10.26434/chemrxiv.12046125 ◽

2020 ◽

Author(s):

Feifei Jia ◽

Jie Wang ◽

Yanyan Zhang ◽

Qun Luo ◽

Luyu Qi ◽

...

Keyword(s):

Mass Spectrometry ◽

Secondary Ion Mass Spectrometry ◽

Single Cells ◽

Time Of Flight ◽

E Coli ◽

Tof Sims ◽

Ion Mass Spectrometry ◽

Chemical Tags ◽

Secondary Ion

In situ visualization of proteins of interest at single cell level is attractive in cell biology, molecular biology and biomedicine, which usually involves photon, electron or X-ray based imaging methods. Herein, we report an optics-free strategy that images a specific protein in single cells by time of flight-secondary ion mass spectrometry (ToF-SIMS) following genetic incorporation of fluorine-containing unnatural amino acids as a chemical tag into the protein via genetic code expansion technique. The method was developed and validated by imaging GFP in E. coli and human HeLa cancer cells, and then utilized to visualize the distribution of chemotaxis protein CheA in E. coli cells and the interaction between high mobility group box 1 protein and cisplatin damaged DNA in HeLa cells. The present work highlights the power of ToF-SIMS imaging combined with genetically encoded chemical tags for in situ visualization of proteins of interest as well as the interactions between proteins and drugs or drug damaged DNA in single cells.

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INTEGRINS MEDIATE PLACENTAL EXTRACELLULAR VESICLE TRAFFICKING TO LUNG AND LIVER IN VIVO

10.1101/2020.09.22.309047 ◽

2020 ◽

Author(s):

Sean L. Nguyen ◽

Soo Hyun Ahn ◽

Jacob W. Greenberg ◽

Benjamin W. Collaer ◽

Dalen W. Agnew ◽

...

Keyword(s):

Immune Cells ◽

Extracellular Vesicle ◽

Dependent Manner ◽

Cellular Phenotype ◽

Reporter Mouse ◽

Membrane Bound ◽

First Time

ABSTRACTMembrane-bound extracellular vesicles (EVs) mediate intercellular communication in all organisms, and those produced by placental mammals have become increasingly recognized as significant mediators of fetal-maternal communication. Here, we aimed to identify maternal cells targeted by placental EVs and elucidate the mechanisms by which they traffic to these cells. Exogenously administered pregnancy-associated EVs traffic specifically to the lung; further, placental EVs associate with lung interstitial macrophages and liver Kupffer cells in an integrin-dependent manner. Localization of EV to maternal lungs was confirmed in unmanipulated pregnancy using a transgenic reporter mouse model, which also provided in situ and in vitro evidence that fetally-derived EVs, rarely, may cause genetic alteration of maternal cells. These results provide for the first time direct in vivo evidence for targeting of placental EVs to maternal immune cells, and further, evidence that EVs can alter cellular phenotype.

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Identification of prolyl carboxypeptidase as an alternative enzyme for processing of renal angiotensin II using mass spectrometry

AJP Cell Physiology ◽

10.1152/ajpcell.00346.2012 ◽

2013 ◽

Vol 304 (10) ◽

pp. C945-C953 ◽

Cited By ~ 28

Author(s):

Nadja Grobe ◽

Nathan M. Weir ◽

Orly Leiva ◽

Frank S. Ong ◽

Kenneth E. Bernstein ◽

...

Keyword(s):

Ph Control ◽

Mass Spectrometric ◽

Acidic Ph ◽

Ang Ii ◽

Converting Enzyme ◽

Wild Type ◽

Angiotensin Converting Enzyme 2 ◽

Genetic Mouse Models

Angiotensin-converting enzyme 2 (ACE2) catalyzes conversion of ANG II to ANG-(1–7). The present study uses newly established proteomic approaches and genetic mouse models to examine the contribution of alternative renal peptidases to ACE2-independent formation of ANG-(1–7). In situ and in vitro mass spectrometric characterization showed that substrate concentration and pH control renal ANG II processing. At pH ≥6, ANG-(1–7) formation was significantly reduced in ACE2 knockout (KO) mice. However, at pH <6, formation of ANG-(1–7) in ACE2 KO mice was similar to that in wild-type (WT) mice, suggesting alternative peptidases for renal ANG II processing. Furthermore, the dual prolyl carboxypeptidase (PCP)-prolyl endopeptidase (PEP) inhibitor Z-prolyl-prolinal reduced ANG-(1–7) formation in ACE2 KO mice, while the ACE2 inhibitor MLN-4760 had no effect. Unlike the ACE2 KO mice, ANG-(1–7) formation from ANG II in PEP KO mice was not different from that in WT mice at any tested pH. However, at pH 5, this reaction was significantly reduced in kidneys and urine of PCP-depleted mice. In conclusion, results suggest that ACE2 metabolizes ANG II in the kidney at neutral and basic pH, while PCP catalyzes the same reaction at acidic pH. This is the first report demonstrating that renal ANG-(1–7) formation from ANG II is independent of ACE2. Elucidation of ACE2-independent ANG-(1–7) production pathways may have clinically important implications in patients with metabolic and renal disease.

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Determination of mosquito bloodmeal pH in situ by ion-selective microelectrode measurement: implications for the regulation of malarial gametogenesis

Parasitology ◽

10.1017/s0031182099005946 ◽

2000 ◽

Vol 120 (6) ◽

pp. 547-551 ◽

Cited By ~ 38

Author(s):

O. BILLKER ◽

A. J. MILLER ◽

R. E. SINDEN

Keyword(s):

Xanthurenic Acid ◽

Mosquito Vector ◽

Dependent Manner ◽

Ph Increase ◽

Ph Range ◽

Dose Dependent

Malarial gametocytes circulate in the peripheral blood of the vertebrate host as developmentally arrested intra-erythrocytic cells, which only resume development into gametes when ingested into the bloodmeal of the female mosquito vector. The ensuing development encompasses sexual reproduction and mediates parasite transmission to the insect. In vitro the induction of gametogenesis requires a drop in temperature and either a pH increase from physiological blood pH (ca pH 7·4) to about pH 8·0, or the presence of a gametocyte-activating factor recently identified as xanthurenic acid (XA). However, it is unclear whether either the pH increase or XA act as natural triggers in the mosquito bloodmeal. We here use pH-sensitive microelectrodes to determine bloodmeal pH in intact mosquitoes. Measurements taken in the first 30 min after ingestion, when malarial gametogenesis is induced in vivo, revealed small pH increases from 7·40 (mouse blood) to 7·52 in Aedes aegypti and to 7·58 in Anophěles stephensi. However, bloodmeal pH was clearly suboptimal if compared to values required to induce gametogenesis in vitro. Xanthurenic acid is shown to extend the pH-range of exflagellation in vitro in a dose-dependent manner to values that we have observed in the bloodmeal, suggesting that in vivo malarial gametogenesis could be further regulated by both these factors.

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In-Vivo Toxicity Studies and In-Vitro Inactivation of SARS-CoV-2 by Povidone-iodine In-situ Gel Forming Formulations

10.1101/2020.05.18.103184 ◽

2020 ◽

Cited By ~ 2

Author(s):

Bo Liang ◽

Xudong Yuan ◽

Gang Wei ◽

Wei Wang ◽

Ming Zhang ◽

...

Keyword(s):

Povidone Iodine ◽

Early Stage ◽

Etiologic Agent ◽

Dependent Manner ◽

Forming Technology ◽

Long Acting ◽

Dose Dependent

AbstractTo curb the spread of SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, we characterize the virucidal activity of long-acting Povidone Iodine (PVP-I) compositions developed using an in-situ gel forming technology. The PVP-I gel forming nasal spray (IVIEW-1503) and PVP-I gel forming ophthalmic eye drop (IVIEW-1201) rapidly inactivated SARS-CoV-2, inhibiting the viral infection of VERO76 cells. No toxicity was observed for the PVP-I formulations. Significant inactivation was noted with preincubation of the virus with these PVP-I formulations at the lowest concentrations tested. It has been demonstrated that both PVP-I formulations can inactivate SARS-CoV-2 virus efficiently in both a dose-dependent and a time-dependent manner. These results suggest IVIEW-1503 and IVIEW-1201 could be potential agents to reduce or prevent the transmission of the virus through the nasal cavity and the eye, respectively. Further studies are needed to clinically evaluate these formulations in early-stage COVID-19 patients.

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High Proliferative Placenta-Derived Multipotent Cells Express Cytokeratin 7 at Low Level

BioMed Research International ◽

10.1155/2019/2098749 ◽

2019 ◽

Vol 2019 ◽

pp. 1-13

Author(s):

V. Shablii ◽

M. Kuchma ◽

H. Svitina ◽

I. Skrypkina ◽

P. Areshkov ◽

...

Keyword(s):

Gene Expression ◽

Single Cells ◽

Placental Tissue ◽

First Trimester ◽

Mesenchymal Cells ◽

Culture Conditions ◽

Multipotent Cells ◽

Genes Expression

The purpose of this study was to investigate the immunophenotypes and gene expression profile of high proliferative placenta-derived multipotent cells (PDMCs) population at different stages of culture. We demonstrated that the colonies resulting from single cells were either positive or negative for CK7, whereas only PDMC clones with weak CK7 expression (CK7low-clones) were highly proliferative. Interestingly, vimentin positive (Vim+) placental stromal mesenchymal cells did not express CK7 in situ, but double CK7+Vim+ cells detection in tissue explants and explants outgrowth indicated CK7 inducible expression in vitro. PCNA presence in CK7+Vim+ cells during placental explants culturing confirmed belonging of these cells to proliferative subpopulation. Transcription factors CDX2 and EOMES were expressed in both CK7low-clones and subset of stromal mesenchymal cells of first-trimester placental tissue in situ. Meanwhile, CK7low -clones and stromal mesenchymal cells of full-term placental tissue in situ expressed ERG heterogeneously. SPP1, COL2A1, and PPARG2 mesodermal-related genes expression by CK7low-clones additionally confirms their mesenchymal origin. Inherent stem cell-related gene expression (IFTM3, POU5F1, and VASA) in CK7low-clones might indicate their enrichment for progenitors. Finally, in CK7low-clones we observed expression of such trophoblast-associated genes as CGB types I and II, fusogenic ERVW-1, GCM1, and GATA3. Thus, our results indicate that PDMCs acquired the representative immunophenotype signature under culture conditions.

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Peptide decoys selected by phage display block in vitro and in vivo activity of a human anti-FVIII inhibitor

Blood ◽

10.1182/blood-2002-06-1886 ◽

2003 ◽

Vol 102 (3) ◽

pp. 949-952 ◽

Cited By ~ 43

Author(s):

Sylvie Villard ◽

Sébastien Lacroix-Desmazes ◽

Thomas Kieber-Emmons ◽

Dominique Piquer ◽

Sabrina Grailly ◽

...

Keyword(s):

Dependent Manner ◽

Peptide Epitope ◽

Fviii Inhibitor ◽

Novel Approach ◽

Fviii Activity ◽

Life Threatening ◽

Normal Hemostasis ◽

Phage Displayed Peptide Library

Abstract Hemophilia A is a life-threatening, hemorrhagic, X-linked recessive disorder resulting in deficient factor VIII (FVIII) activity. After the infusion of therapeutic FVIII, 25% of patients develop anti-FVIII antibodies that inhibit FVIII procoagulant activity, thus precluding further administration of FVIII. Here we report a novel approach aimed at neutralizing the activity of FVIII inhibitors by peptide epitope surrogates. To illustrate our concept, we chose the human anti-FVIII monoclonal antibody, Bo2C11, as a representative of anti-FVIII antibodies and a phage-displayed peptide library approach to obtain surrogate peptides. We selected a series of constrained dodecapeptides with the core sequence W-NR, which specifically interacts with the combining site of Bo2C11. The peptides mimic the epitope recognized by Bo2C11 and are able to inhibit specifically and in a dose-dependent manner the binding of Bo2C11 to FVIII. Peptide 107, in particular, neutralized the activity of Bo2C11 in vitro and restored normal hemostasis in hemophilic mice. Thus, the use of peptide decoys may be a promising new approach for the neutralization of pathologic antibodies.

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