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1. Localization of Native Mms13 to the Magnetosome Chain of Magnetospirillum magneticum AMB-1 Using Immunogold Electron Microscopy, Immunofluorescence Microscopy and Biochemical Analysis

   https://doi.org/10.3390/cryst11080874  

   Oestreicher, Zachery ; Valverde-Tercedor, Carmen ; Mumper, Eric ; Pérez-Guzmán, Lumarie ; Casillas-Ituarte, Nadia N. ; Jimenez-Lopez, Concepcion ; Bazylinski, Dennis A. ; Lower, Steven K. ; Lower, Brian H. ( August 2021 , Crystals)

   null (Ed.)

   Magnetotactic bacteria (MTB) biomineralize intracellular magnetite (Fe3O4) crystals surrounded by a magnetosome membrane (MM). The MM contains membrane-specific proteins that control Fe3O4 mineralization in MTB. Previous studies have demonstrated that Mms13 is a critical protein within the MM. Mms13 can be isolated from the MM fraction of Magnetospirillum magneticum AMB-1 and a Mms13 homolog, MamC, has been shown to control the size and shape of magnetite nanocrystals synthesized in-vitro. The objective of this study was to use several independent methods to definitively determine the localization of native Mms13 in M. magneticum AMB-1. Using Mms13-immunogold labeling and transmission electron microscopy (TEM), we found that Mms13 is localized to the magnetosome chain of M. magneticum AMB-1 cells. Mms13 was detected in direct contact with magnetite crystals or within the MM. Immunofluorescence detection of Mms13 in M. magneticum AMB-1 cells by confocal laser scanning microscopy (CLSM) showed Mms13 localization along the length of the magnetosome chain. Proteins contained within the MM were resolved by SDS-PAGE for Western blot analysis and LC-MS/MS (liquid chromatography with tandem mass spectrometry) protein sequencing. Using Anti-Mms13 antibody, a protein band with a molecular mass of ~14 kDa was detected in the MM fraction only. This polypeptide was digested with trypsin, sequenced by LC-MS/MS and identified as magnetosome protein Mms13. Peptides corresponding to the protein’s putative MM domain and catalytic domain were both identified by LC-MS/MS. Our results (Immunogold TEM, Immunofluorescence CLSM, Western blot, LC-MS/MS), combined with results from previous studies, demonstrate that Mms13 and homolog proteins MamC and Mam12, are localized to the magnetosome chain in MTB belonging to the class Alphaproteobacteria. Because of their shared localization in the MM and highly conserved amino acid sequences, it is likely that MamC, Mam12, and Mms13 share similar roles in the biomineralization of Fe3O4 nanocrystals. 

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2. Exosome Isolation by Ultracentrifugation and Precipitation and Techniques for Downstream Analyses

   https://doi.org/10.1002/cpcb.110  

   Coughlan, Christina ; Bruce, Kimberley D. ; Burgy, Olivier ; Boyd, Timothy D. ; Michel, Cole R. ; Garcia‐Perez, Josselyn E. ; Adame, Vanesa ; Anton, Paige ; Bettcher, Brianne M. ; Chial, Heidi J. ; et al ( July 2020 , Current Protocols in Cell Biology)

   Exosomes are 50‐ to 150‐nm‐diameter extracellular vesicles secreted by all mammalian cells except mature red blood cells and contribute to diverse physiological and pathological functions within the body. Many methods have been used to isolate and analyze exosomes, resulting in inconsistencies across experiments and raising questions about how to compare results obtained using different approaches. Questions have also been raised regarding the purity of the various preparations with regard to the sizes and types of vesicles and to the presence of lipoproteins. Thus, investigators often find it challenging to identify the optimal exosome isolation protocol for their experimental needs. Our laboratories have compared ultracentrifugation and commercial precipitation‐ and column‐based exosome isolation kits for exosome preparation. Here, we present protocols for exosome isolation using two of the most commonly used methods, ultracentrifugation and precipitation, followed by downstream analyses. We use NanoSight nanoparticle tracking analysis and flow cytometry (Cytek^®) to determine exosome concentrations and sizes. Imaging flow cytometry can be utilized to both size exosomes and immunophenotype surface markers on exosomes (ImageStream^®). High‐performance liquid chromatography followed by nano‐flow liquid chromatography–mass spectrometry (LCMS) of the exosome fractions can be used to determine the presence of lipoproteins, with LCMS able to provide a proteomic profile of the exosome preparations. We found that the precipitation method was six times faster and resulted in a ∼2.5‐fold higher concentration of exosomes per milliliter compared to ultracentrifugation. Both methods yielded extracellular vesicles in the size range of exosomes, and both preparations included apoproteins. © 2020 Wiley Periodicals LLC.

   Basic Protocol 1: Pre‐analytic fluid collection and processing

   Basic Protocol 2: Exosome isolation by ultracentrifugation

   Alternate Protocol 1: Exosome isolation by precipitation

   Basic Protocol 3: Analysis of exosomes by NanoSight nanoparticle tracking analysis

   Alternate Protocol 2: Analysis of exosomes by flow cytometry and imaging flow cytometry

   Basic Protocol 4: Downstream analysis of exosomes using high‐performance liquid chromatography

   Basic Protocol 5: Downstream analysis of the exosome proteome using nano‐flow liquid chromatography–mass spectrometry

    

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3. Mechanosensitivity Analysis of Breast Cancer Tumor Cells from Needle Biopsy

   Bedoya, Santiago ; Ghosh, Deepraj ; Dawson, Michelle ( April 2018 , The FASEB journal)

   Tumor stiffness has been associated with malignancy and increased risk for metastasis. Extensive research has been done investigating breast cancer cell lines’ responsiveness to surfaces of varying rigidities as well as examining the biophysical properties of breast cancer tumor samples. However, there is a critical gap regarding the relationship between cells’ mechanosensitivity in conjunction to biophysical properties of their extracellular matrix environment. To explore this relationship, we will analyze single-cell mechanosensitivity in comparison to tumor rigidity via shearwave ultrasound elastogrophy (SWE). Given the putative affiliation, we hypothesize that cells expressing invasive mechanosensitivity profiles will correlate with stiffer tumor regions. Using collagen gels containing different cell types, we derived biopsy-sized samples allowing us to optimize single-cell mechanosensitivity analysis. Cells were stained using different dyes corresponding to invasiveness. Subsequently, we analyzed their morphology. Morphological identification within organoid environments would allow for single-cell analysis without the aggression of tissue digestion, though preliminary results suggest high heterogeneity may not allow for confident cell identification solely on morphology. Thus, inquisition into cell viability and integrity was explored by analyzing the effects of tissue digestion with HyQtase on single-cells. Cell count and live-dead stain via flow cytometry allowed for analysis of single-cell viability. Lastly, cell integrity was evaluated by a 2D adhesion assay of isolated cells. The live/dead stain revealed that digestion resulted in isolation of approximately 10% of the original 500,000 cell population with 90–97% of the isolated population being live-cells (invasive and non-invasive respectively). Furthermore, the adhesion assay showed that these isolated single cells retained the ability to adhere to new surfaces, with no difference between the invasive and non-invasive cell types. These results show that cells are able to retain mechanosensitive properties following enzymatic digestion. However, they also suggest our digestion procedure is not aggressive enough to isolate invasive subpopulations that are more strongly imbedded in the original tissues. Development of these novel techniques will allow for accurate and confident analysis of precious human biopsy samples. Insight into the relationship between single-cell mechanosensitivity and tumor biophysical properties could elucidate pathways for metastasis inhibition and prevention. 

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4. Electroformed Inverse‐Opal Nanostructures for Surface‐Marker‐Specific Isolation of Extracellular Vesicles Directly from Complex Media

   https://doi.org/10.1002/admt.202201622  

   Lin, Andrew A. ; Jiang, Zhimin ; Yee, Stephanie S. ; Carpenter, Erica L. ; Pikul, James H. ; Issadore, David ( January 2023 , Advanced Materials Technologies)

   Extracellular vesicles (EVs) – nanoscale membranous particles that carry multiple proteins and nucleic acid cargoes from their mother cells of origin into circulation – have enormous potential as biomarkers. However, devices appropriately scaled to the nanoscale to match the size of EVs (30–200 nm) have orders of magnitude too low throughput to process clinical samples (10¹²EVs mL⁻¹in serum). To address this challenge, we develop a novel approach that incorporates billions of nanomagnetic sorters that act in parallel to precisely isolate sparse EVs based on immunomagnetic labeling directly from clinical samples at flow rates billions of times greater than that of a single nanofluidic device. To fabricate these chips, the ferromagnetic metals are electro‐deposited into a self‐assembled microlattice, achieving >10⁹nanoscale magnetophoretic sorting devices in a 3D postage stamp‐sized lattice with >70x magnetic traps and >20x enrichment of magnetic nanoparticles versus our previous work. The immunomagnetically labeled EVs are isolated and achieve a ≈100% increase in yield as well as increased purity compared to conventional methods. Building on the proof‐of‐concept demonstrations in this manuscript, this new approach has the potential to enhance the future clinical translation of EV biomarkers by enabling rapid, sensitive, and specific isolation of EV subpopulations from clinical samples.

    

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5. Cytochrome C with peroxidase-like activity encapsulated inside the small DPS protein nanocage

   https://doi.org/10.1039/D1TB00234A  

   Waghwani, Hitesh Kumar ; Douglas, Trevor ( April 2021 , Journal of Materials Chemistry B)

   null (Ed.)

   Nature utilizes self-assembled protein-based structures as subcellular compartments in prokaryotes to sequester catalysts for specialized biochemical reactions. These protein cage structures provide unique isolated environments for the encapsulated enzymes. Understanding these systems is useful in the bioinspired design of synthetic catalytic organelle-like nanomaterials. The DNA binding protein from starved cells (Dps), isolated from Sulfolobus solfataricus , is a 9 nm dodecameric protein cage making it the smallest known naturally occurring protein cage. It is naturally over-expressed in response to oxidative stress. The small size, natural biodistribution to the kidney, and ability to cross the glomerular filtration barrier in in vivo experiments highlight its potential as a synthetic antioxidant. Cytochrome C (CytC) is a small heme protein with peroxidase-like activity involved in the electron transport chain and also plays a critical role in cellular apoptosis. Here we report the encapsulation of CytC inside the 5 nm interior cavity of Dps and demonstrate the catalytic activity of the resultant Dps nanocage with enhanced antioxidant behavior. The small cavity can accommodate a single CytC and this was achieved through self-assembly of chimeric cages comprising Dps subunits and a Dps subunit to which the CytC was fused. For selective isolation of CytC containing Dps cages, we utilized engineered polyhistidine tag present only on the enzyme fused Dps subunits (6His-Dps-CytC). The catalytic activity of encapsulated CytC was studied using guaiacol and 3,3′,5,5′-tetramethylbenzidine (TMB) as two different peroxidase substrates and compared to the free (unencapsulated) CytC activity. The encapsulated CytC showed better pH dependent catalytic activity compared to free enzyme and provides a proof-of-concept model to engineer these small protein cages for their potential as catalytic nanoreactors. 

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