Biology subjects

Sarmah, D.

Publications and source records attributed to Sarmah, D..

5 recordsLinked to original sources

bioRxiv2026

IROP: Ionic Regulation of Permeation for High-Yield AAV Purification

Traditional filtration methods for separating host cell proteins and DNA from adeno-associated viral capsids often result in substantial yield losses. Here, we introduce a new high-yield purification strategy termed Ionic Regulation of Permeation (IROP) for removing major impurities within a single unit operation. Rather than relying on static size exclusion or binding interactions, IROP dynamically modulates AAV transport within a nanoporous membrane. At low ionic strength, AAV permeation was negligible, while high ionic strength markedly increased the sieving coefficient, revealing an electrostatic gating mechanism. AAVs retained under low-salt conditions could not be efficiently recovered by a static high-salt wash, indicating steric-electrostatic trapping within membrane pores. In contrast, high-salt filtration enabled high recovery. Leveraging this mechanism, a two-step IROP process, involving low-salt impurity clearance followed by high-salt AAV recovery, achieved greater than 90% AAV recovery with a greater than 95% reduction in HCPs and host cell DNA.

bioengineering↗

bioRxiv2024

A 96-Well Polyacrylamide Gel for Electrophoresis and Western Blotting

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 (cost and time ~8-fold), and capacity for biological characterization relative to established western blots.

biochemistry↗

bioRxiv2022

Predicting Anti-Cancer Drug Combination Responses with a Temporal Cell State Network Model

Cancer chemotherapy combines multiple drugs, but predicting the effects of drug combinations on cancer cell proliferation remains challenging. 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. 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. This work shows how currently available or attainable information could be combined to predict how cancer cell populations respond to drug combinations.

systems biology↗

bioRxiv2021

Mesowestern Blot: Simultaneous Analysis of Hundreds of Sub-Microliter Lysates

Western blotting is a widely-used technique for molecular-weight-resolved analysis of proteins and their post-translational modifications, but has been refractory to affordable scale-up. Here, we report the Mesowestern blot, which uses a 3D-printable gel-casting mold to enable affordable, high-throughput Western blotting with standard sample preparation and small (<1 uL) sample sizes. The casted polyacrylamide gel contains 336, 0.5 uL micropipette-loadable sample wells arranged within a standard microplate footprint. Polyacrylamide % can be altered to change molecular weight resolution range. Proof-of-concept experiments using both infrared-fluorescent molecular weight protein ladder as well as cell lysate (RIPA buffer) demonstrate protein loaded in Mesowestern gels is amenable to the standard Western blotting steps. The main difference between Mesowestern and traditional Western is that semi-dry horizontal instead of immersed vertical gel electrophoresis is used. The linear range of detection is approximately 2 orders of magnitude, with a limit of detection (for {beta}-actin) of around 30 ng of total protein from mammalian cell lysates (~30-3000 cells). Because the gel mold is 3D-printable, users have significant design freedom for custom layouts, and there are few barriers to adoption by the typical cell and molecular biology laboratory already performing Western blots.

biochemistry↗

bioRxiv2021

Predicting Individual Cell Division Events from Single-Cell ERK and Akt Dynamics

Predictive determinants of stochastic single-cell fates have been elusive, even for the well-studied mammalian cell cycle. What drives proliferation decisions of single cells at any given time? 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 hours). 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. Machine learning algorithms show that simple metrics of central tendency in this time window are most predictive for subsequent cell division; median ERK and Akt activities classify individual division events with an AUC=0.76. Surprisingly, ERK dynamics alone predict division in individual cells with an AUC=0.74, suggesting Akt activity dynamics contribute little 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 are substantially more important than Akt activity dynamics for driving cell division in this non-transformed context. Single cell imaging along with machine learning algorithms provide a better basis to understand cell cycle progression on the single cell level.

systems biology↗