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  1. Article: A 96-Well Polyacrylamide Gel for Electrophoresis and Western Blotting.

    Birtwistle, Marc R / Huggins, Jonah R / Zadeh, Cameron O / Sarmah, Deepraj / Srikanth, Sujata / Kelly Jones, B / Cascio, Lauren N / Dean, Delphine

    bioRxiv : the preprint server for biology

    2024  

    Abstract: Western blotting is a stalwart technique for analyzing specific proteins and/or their post-translational modifications. However, it remains challenging to accommodate more than ∼10 samples per experiment without substantial departure from trusted, ... ...

    Abstract Western blotting is a stalwart technique for analyzing specific proteins and/or their post-translational modifications. However, it remains challenging to accommodate more than ∼10 samples per experiment without substantial departure from trusted, established protocols involving accessible instrumentation. Here, we describe a 96-sample western blot that conforms to standard 96-well plate dimensional constraints and has little operational deviation from standard western blotting. The main differences are that (i) submerged polyacrylamide gel electrophoresis is operated horizontally (similar to agarose gels) as opposed to vertically, and (ii) a 6 mm thick gel is used, with 2 mm most relevant for membrane transfer (vs ∼1 mm typical). Results demonstrate both wet and semi-dry transfer are compatible with this gel thickness. The major tradeoff is reduced molecular weight resolution, due primarily to less available migration distance per sample. We demonstrate proof-of-principle using gels loaded with molecular weight ladder, recombinant protein, and cell lysates. We expect the 96-well western blot will increase reproducibility, efficiency, and capacity for biological characterization relative to established western blots.
    Language English
    Publishing date 2024-05-11
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2024.05.10.593650
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Predicting anti-cancer drug combination responses with a temporal cell state network model.

    Sarmah, Deepraj / Meredith, Wesley O / Weber, Ian K / Price, Madison R / Birtwistle, Marc R

    PLoS computational biology

    2023  Volume 19, Issue 5, Page(s) e1011082

    Abstract: Cancer chemotherapy combines multiple drugs, but predicting the effects of drug combinations on cancer cell proliferation remains challenging, even for simple in vitro systems. We hypothesized that by combining knowledge of single drug dose responses and ...

    Abstract Cancer chemotherapy combines multiple drugs, but predicting the effects of drug combinations on cancer cell proliferation remains challenging, even for simple in vitro systems. We hypothesized that by combining knowledge of single drug dose responses and cell state transition network dynamics, we could predict how a population of cancer cells will respond to drug combinations. We tested this hypothesis here using three targeted inhibitors of different cell cycle states in two different cell lines in vitro. We formulated a Markov model to capture temporal cell state transitions between different cell cycle phases, with single drug data constraining how drug doses affect transition rates. This model was able to predict the landscape of all three different pairwise drug combinations across all dose ranges for both cell lines with no additional data. While further application to different cell lines, more drugs, additional cell state networks, and more complex co-culture or in vivo systems remain, this work demonstrates how currently available or attainable information could be sufficient for prediction of drug combination response for single cell lines in vitro.
    MeSH term(s) Humans ; Antineoplastic Agents/pharmacology ; Antineoplastic Agents/therapeutic use ; Neoplasms/drug therapy ; Drug Combinations ; Cell Proliferation ; Cell Line, Tumor
    Chemical Substances Antineoplastic Agents ; Drug Combinations
    Language English
    Publishing date 2023-05-01
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
    ZDB-ID 2193340-6
    ISSN 1553-7358 ; 1553-734X
    ISSN (online) 1553-7358
    ISSN 1553-734X
    DOI 10.1371/journal.pcbi.1011082
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Network inference from perturbation time course data.

    Sarmah, Deepraj / Smith, Gregory R / Bouhaddou, Mehdi / Stern, Alan D / Erskine, James / Birtwistle, Marc R

    NPJ systems biology and applications

    2022  Volume 8, Issue 1, Page(s) 42

    Abstract: Networks underlie much of biology from subcellular to ecological scales. Yet, understanding what experimental data are needed and how to use them for unambiguously identifying the structure of even small networks remains a broad challenge. Here, we ... ...

    Abstract Networks underlie much of biology from subcellular to ecological scales. Yet, understanding what experimental data are needed and how to use them for unambiguously identifying the structure of even small networks remains a broad challenge. Here, we integrate a dynamic least squares framework into established modular response analysis (DL-MRA), that specifies sufficient experimental perturbation time course data to robustly infer arbitrary two and three node networks. DL-MRA considers important network properties that current methods often struggle to capture: (i) edge sign and directionality; (ii) cycles with feedback or feedforward loops including self-regulation; (iii) dynamic network behavior; (iv) edges external to the network; and (v) robust performance with experimental noise. We evaluate the performance of and the extent to which the approach applies to cell state transition networks, intracellular signaling networks, and gene regulatory networks. Although signaling networks are often an application of network reconstruction methods, the results suggest that only under quite restricted conditions can they be robustly inferred. For gene regulatory networks, the results suggest that incomplete knockdown is often more informative than full knockout perturbation, which may change experimental strategies for gene regulatory network reconstruction. Overall, the results give a rational basis to experimental data requirements for network reconstruction and can be applied to any such problem where perturbation time course experiments are possible.
    MeSH term(s) Systems Biology/methods ; Algorithms ; Gene Regulatory Networks/genetics ; Signal Transduction/physiology
    Language English
    Publishing date 2022-11-01
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
    ISSN 2056-7189
    ISSN (online) 2056-7189
    DOI 10.1038/s41540-022-00253-6
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Relating individual cell division events to single-cell ERK and Akt activity time courses.

    Stern, Alan D / Smith, Gregory R / Santos, Luis C / Sarmah, Deepraj / Zhang, Xiang / Lu, Xiaoming / Iuricich, Federico / Pandey, Gaurav / Iyengar, Ravi / Birtwistle, Marc R

    Scientific reports

    2022  Volume 12, Issue 1, Page(s) 18077

    Abstract: Biochemical correlates of stochastic single-cell fates have been elusive, even for the well-studied mammalian cell cycle. We monitored single-cell dynamics of the ERK and Akt pathways, critical cell cycle progression hubs and anti-cancer drug targets, ... ...

    Abstract Biochemical correlates of stochastic single-cell fates have been elusive, even for the well-studied mammalian cell cycle. We monitored single-cell dynamics of the ERK and Akt pathways, critical cell cycle progression hubs and anti-cancer drug targets, and paired them to division events in the same single cells using the non-transformed MCF10A epithelial line. Following growth factor treatment, in cells that divide both ERK and Akt activities are significantly higher within the S-G2 time window (~ 8.5-40 h). Such differences were much smaller in the pre-S-phase, restriction point window which is traditionally associated with ERK and Akt activity dependence, suggesting unappreciated roles for ERK and Akt in S through G2. Simple metrics of central tendency in this time window are associated with subsequent cell division fates. ERK activity was more strongly associated with division fates than Akt activity, suggesting Akt activity dynamics may contribute less to the decision driving cell division in this context. We also find that ERK and Akt activities are less correlated with each other in cells that divide. Network reconstruction experiments demonstrated that this correlation behavior was likely not due to crosstalk, as ERK and Akt do not interact in this context, in contrast to other transformed cell types. Overall, our findings support roles for ERK and Akt activity throughout the cell cycle as opposed to just before the restriction point, and suggest ERK activity dynamics may be more important than Akt activity dynamics for driving cell division in this non-transformed context.
    MeSH term(s) Animals ; Proto-Oncogene Proteins c-akt/metabolism ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Signal Transduction ; Cell Division ; Cell Cycle ; Mammals/metabolism
    Chemical Substances Proto-Oncogene Proteins c-akt (EC 2.7.11.1) ; Extracellular Signal-Regulated MAP Kinases (EC 2.7.11.24)
    Language English
    Publishing date 2022-10-27
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2615211-3
    ISSN 2045-2322 ; 2045-2322
    ISSN (online) 2045-2322
    ISSN 2045-2322
    DOI 10.1038/s41598-022-23071-6
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Mesowestern Blot: Simultaneous Analysis of Hundreds of Submicroliter Lysates.

    Zadeh, Cameron O / Huggins, Jonah R / Sarmah, Deepraj / Westbury, Baylee C / Interiano, William R / Jordan, Micah C / Phillips, S Ashley / Dodd, William B / Meredith, Wesley O / Harold, Nicholas J / Erdem, Cemal / Birtwistle, Marc R

    ACS omega

    2022  Volume 7, Issue 33, Page(s) 28912–28923

    Abstract: Western blotting is a widely used technique for molecular-weight-resolved analysis of proteins and their posttranslational modifications, but high-throughput implementations of the standard slab gel arrangement are scarce. The previously developed ... ...

    Abstract Western blotting is a widely used technique for molecular-weight-resolved analysis of proteins and their posttranslational modifications, but high-throughput implementations of the standard slab gel arrangement are scarce. The previously developed Microwestern requires a piezoelectric pipetting instrument, which is not available for many labs. Here, we report the Mesowestern blot, which uses a 3D-printable gel casting mold to enable high-throughput Western blotting without piezoelectric pipetting and is compatible with the standard sample preparation and small (∼1 μL) sample sizes. The main tradeoffs are reduced molecular weight resolution and higher sample-to-sample CV, making it suitable for qualitative screening applications. The casted polyacrylamide gel contains 336, ∼0.5 μL micropipette-loadable sample wells arranged within a standard microplate footprint. Polyacrylamide % can be altered to change molecular weight resolution profiles. Proof-of-concept experiments using both infrared-fluorescent molecular weight protein ladder and cell lysate (RIPA buffer) demonstrate that the protein loaded in Mesowestern gels is amenable to the standard Western blotting steps. The main difference between Mesowestern and traditional Western is that semidry horizontal instead of immersed vertical gel electrophoresis is used. The linear range of detection is at least 32-fold, and at least ∼500 attomols of β-actin can be detected (∼29 ng of total protein from mammalian cell lysates: ∼100-300 cells). Because the gel mold is 3D-printable, users with access to additive manufacturing cores have significant design freedom for custom layouts. We expect that the technique could be easily adopted by any typical cell and molecular biology laboratory already performing Western blots.
    Language English
    Publishing date 2022-08-11
    Publishing country United States
    Document type Journal Article
    ISSN 2470-1343
    ISSN (online) 2470-1343
    DOI 10.1021/acsomega.2c02201
    Database MEDical Literature Analysis and Retrieval System OnLINE

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