Biology subjects

Kuhlman, T. E.

Publications and source records attributed to Kuhlman, T. E..

9 recordsLinked to original sources

bioRxiv2026

Analysis and Design of Frequency-Based Biological Signaling Cascades

Based on our experimental observation of activation state oscillations of different frequencies used to communicate information by the master human stress response regulator protein p38 MAPK 1, we develop a simple graphical approach for understanding and predicting the behavior of complex biological networks acting upon signals carrying information as different frequency waves of chemicals. This approach uses the same techniques used for analyzing and understanding information transmission using waves of electrical currents and fields used in electrical alternating current (AC) circuits. We show how biological components can be organized to behave as standard components found in electronic telecommunications circuits. Finally, we demonstrate how such components can be organized into complex biological signaling cascades whose behavior can be qualitatively and quantitatively understood, and whose output resembles that experimentally observed in p38.

biophysics↗

bioRxiv2026

MosaicLev: Modified Levenshtein distance for mobile element-aware genome comparison

MotivationSomatic genetic mosaicism arises when genomes diverge across cells during development, in part due to the activity of transposons (cut-paste) and retrotransposons (copy-paste). Standard sequence comparison methods are not motif-aware, penalizing mobile element insertions based on length rather than recognizing them as single biological events. ResultsWe introduce a modified Levenshtein distance (mlev) that discounts mobile element insertions via a tunable parameter (m [isin] [0, 1]). Validation on 67 Cluster G1 mycobacteriophage genomes demonstrates bidirectional discrimination between Mycobacteriophage Mobile Element 1 (MPME1) and MPME2 elements: using MPME1 as target yields [~]49% discount for MPME1 carriers but only [~]9% for MPME2 carriers, while using MPME2 reverses this pattern. This approach classifies 35 MPME1 carriers and 11 MPME2 carriers, and identifies 14 phages showing low discount with both motifs, consistent with absence of both elements. Availability and ImplementationPython implementation with Numba JIT compilation freely available at https://doi.org/10.5281/zenodo.18452982.

bioinformatics↗

bioRxiv2025

Clustering of SARS-CoV-2 membrane proteins in lipid bilayer membranes

The accumulation of viral structural proteins along the ER-Golgi intermediate compartment (ERGIC) membrane leads to SARS-CoV-2 self-assembly and budding, driven by the interactions between these proteins, RNA and the ERGIC membrane. The membrane protein (M) is believed to interact with other structural proteins and form clusters needed for the induction of membrane curvature that facilitates virion formation. However, the role played by direct and membrane-mediated interactions between M proteins and their interactions with other proteins in the clustering process remains unclear. Here, we utilize a combination of all-atom molecular dynamics (MD) simulations, continuum modeling and experiments to show that M-M interactions are sufficient to drive clustering in ERGIC-like lipid bilayers in the absence of other proteins or RNA. Using all-atom MD simulations we were able to estimate the membrane thinning induced by M proteins and the resulting membrane-mediated M-M interaction. Combining this with a continuum model that describes the evolution of M protein density in a planar lipid membrane, we identified the existence of a critical, direct M-M interaction energy needed for cluster assembly at a given density. By comparing the model predictions with analysis of atomic force microscopy images of M protein clusters in supported lipid bilayers, we were able to estimate the direct M-M interaction energy and found it to be significantly larger than the membrane mediated interaction energy. Our work therefore establishes that M protein interactions are sufficient to drive clustering and provides a quantitative understanding of the role played by direct and membrane-mediated interactions of M proteins in viral assembly and budding.

biophysics↗

bioRxiv2025

Human Stress Response Specificity through Bioresonance Selectivity

Bold First ParagraphIn eukaryotes, the mitogen activated protein kinase (MAPK) cascade, a multilayered interconnected network of enzymes, connects external stimuli to gene regulation, determining cellular fate 1. Environmental stress sensed by a cell starts a complex chain of reactions between MAPK enzymes that ultimately activates the master stress response regulator protein p38 MAPK 2,3. Thus activated, p38 must then selectively activate targets from a pool of hundreds to initiate appropriate cellular responses 3. Mechanisms for how p38 performs this selection remain unclear 4,5. Here we show that human p38 target selectivity is based on the same principles as modern electronic telecommunications systems, except using waves of chemicals rather than electromagnetic fields or electric currents. p38 encodes information about stimuli as different frequency oscillations of its activation state, and targets are selected through frequency-dependent resonance of oscillating biochemical reactions between p38 and its targets. We demonstrate this mechanism by activating various genetic responses in human cells by applying only sugar at different frequencies. These results unify observations of oscillating signaling components and altered responses 6-19 into a coherent framework to understand and control human gene expression. As failures of this mechanism may contribute to some p38-associated diseases 2,20-28, these findings may have implications for pharmaceutical development and therapeutic strategies.

biophysics↗

bioRxiv2023

Synthesis, Insertion and Characterization of SARS-CoV-2 Membrane Protein Within Lipid Bilayers

SUMMARY/ABSTRACTThe membrane protein (M) is the most abundant structural protein in the SARS-CoV-2 virus and functions exclusively as a membrane-embedded homodimer. M protein is required for the formation of the SARS-CoV-2 virus particle and has been shown to interact with the Spike and Envelope proteins, as well as the RNA-packaging Nucleocapsid protein. Our knowledge of M protein is very limited due to its small size and challenges in expressing enough protein for use in structural and biophysical experiments. We report the successful development of a SUMO tag-based expression system to produce and purify significant quantities of M protein, and a method to insert the synthesized dimers into a suspended lipid membrane in a homogeneous orientation. We used AFM and Cryo-EM to image individual membrane-bound M protein dimers and characterize the configurations that they can assume. Our experimental results are in agreement with our molecular dynamics simulations which predict thinning of the membrane around the M protein and a propensity to induce local membrane curvature. Taken together, our results shed new light on M protein properties within the lipid bilayer and suggest mechanisms that could contribute to viral assembly and budding.

biophysics↗

bioRxiv2023

Phylogenetic Distribution of Prokaryotic Non-Homologous End Joining DNA Repair Systems in Bacteria and Archaea

Nonhomologous end-joining (NHEJ) is a repair mechanism for double strand breaks (DSBs) of DNA. This mechanism is ubiquitously observed within the eukaryotic domain; however, its presence is not as pervasive among prokaryotes and archaea. Notably, in prokaryotes, it has been discerned that multiple distinct NHEJ pathways have evolved in contrast to the singular NHEJ pathway prevalent in eukaryotes. We performed phylogenetic analysis to gain deeper insights into the distribution of these prokaryotic NHEJ pathways. Concurrently, components of the prokaryotic NHEJ pathways were used to find if any archaea carry the genes required and may be able to carry out NHEJ. The results show that few prokaryotes carry the components required for NHEJ, but multiple pathways may be active in a single species. In the context of Archaea, the analysis revealed that a substantial number of species contain fragments or segments of prokaryotic NHEJ elements. Nevertheless, the presence of all the necessary components for the complete execution of the NHEJ pathway remains relatively rare within the archaeal domain.

microbiology↗

bioRxiv2023

A Low-Cost Stage-Top Incubation Device For Human Cell Imaging Using Rapid Prototyping Methods

Live imaging of human or other mammalian cells at multi-hour time scales with minimal perturbation to their growth state requires that the specimens optimal growth conditions are met while fixed to a microscope stage. In general, ideal conditions include culturing in complete growth media, an ambient temperature of 36-37 C, and a humidity-controlled atmosphere comprising typically 5-7% CO2. Commercially available devices that achieve these conditions are not a financially viable option for many labs, with the price ranging anywhere from $12000 to $40000. The advent of 3D printing technology has allowed for low-cost rapid prototyping with precision comparable to traditional fabrication methods, opening the possibility for in-lab design and production of otherwise prohibitively expensive equipment such as stage-top incubation devices. The continued usefulness and widespread availability of single-board computers (SBC) such as Arduino and Raspberry Pi also simplify the process by which these devices can be controlled. Here we report the production of a do-it-yourself (DIY) device for stage-top incubation with temperature and atmospheric control with a cost reduction of approximately 100x.

cell biology↗

bioRxiv2022

IS200/IS605 Family-Associated TnpB Increases Transposon Activity and Retention

The IS200/IS605 family of insertion sequences are abundant mobile elements associated with one of the most numerous genes found in nature, tnpB1-3. Previous studies suggest that TnpB protein may be an evolutionary precursor to CRISPR Cas enzymes, and TnpB has received renewed interest having itself been shown to function as a Cas-like RNA-guided DNA endonuclease3,4. However, interpretation of the fundamental role of TnpB in transposition and how it contributes to genome dynamics5 remains controversial without direct, real-time measurement in live cells. Here, using a suite of fluorescent reporters coupled to transposition in live Escherichia coli, we show that IS608-TnpB causes increased transposon activity, and assists in preventing transposon loss from host genomes. Analyzing our results through a mathematical model of transposon dynamics, we discuss the multifaceted roles it may play in transposon regulation. The mutually beneficial transposon-TnpB interaction may explain the prevalence of tnpB, creating conditions for the appropriation of TnpBs RNA-guided endonuclease activity for adaptive immunity. SIGNIFICANCE STATEMENTPhylogenetic evidence suggests that tnpB, one of the most numerous genes found in nature, is the ancestral form of CRISPR-Cas enzymes and played a critical role in the evolution of adaptive immunity. However, the role TnpB plays in transposition that has contributed to its wide distribution remains unclear. Here, we use a unique approach that couples fluorescent reporters to transposition to non-perturbatively quantify transpositional dynamics in live cells. In contrast to previous indirect methods suggesting that TnpB suppresses transposition, our results instead clearly demonstrate that TnpB significantly increases transposition rates and enhances transposon retention within the host genome, resulting in a mutually beneficial interaction between transposons and TnpB that can account for its wide distribution.

synthetic biology↗

bioRxiv2016

Environment determines evolutionary trajectory in a constrained phenotypic space

Constraints on phenotypic variation limit the capacity of organisms to adapt to the multiple selection pressures encountered in natural environments. To better understand evolutionary dynamics in this context, we select Escherichia coli for faster migration through a porous environment, a process which depends on both motility and growth. We find that a trade-off between swimming speed and growth rate constrains the evolution of faster migration. Evolving faster migration in rich medium results in slow growth and fast swimming, while evolution in minimal medium results in fast growth and slow swimming. In each condition parallel genomic evolution drives adaptation through different mutations. We show that the trade-off is mediated by antagonistic pleiotropy through mutations that affect negative regulation. A model of the evolutionary process shows that the genetic capacity of an organism to vary traits can qualitatively alter the evolutionary trajectory when selection pressures are complex.

evolutionary biology↗