Biology subjects

Fischer, M. S.

Publications and source records attributed to Fischer, M. S..

11 recordsLinked to original sources

bioRxiv2026

An Integrated Experimental and Informatics Strategy Enables Large Proteoform Identification in Top-Down Proteomics

Top-down proteomics enables identification and characterization of intact proteoforms, preserving information on co-occurring sequence variations and post-translational modifications. However, identifying large proteoforms remains challenging due to an exponential decrease in signal-to-noise ratio with increasing molecular weight, interference from coeluting smaller species, unresolved precursor isotopic envelopes, difficulty obtaining informative MS/MS spectra, and limitations of current informatics tools. To address these challenges, here we present an integrated experimental and informatics strategy combining protein separation, data acquisition, precursor mass assignment, and MS/MS interpretation. First, we demonstrate that online two-dimensional liquid chromatography coupling size-exclusion and reversed-phase separations enables facile detection of large proteoforms. We then utilize a precursor mass correction strategy to enable conventional database searching of data-dependent acquisition (DDA) data containing isotopically unresolved precursors. Finally, we use data-independent acquisition (DIA) optimized for large proteins to enhance fragment signal and enable identification through intact-mass-guided candidate discovery and MS/MS validation. Applied to human heart tissue, the integrated strategy identified 703 nonredundant proteoforms from 236 unique proteins, including 65 proteins >30 kDa and 22 proteins >50 kDa, with identifications extending to 223 kDa. This integrated strategy bridges the detection-to-identification gap for large proteins and expands the accessible mass range of discovery top-down proteomics.

biochemistry↗

bioRxiv2025

Structural Heterogeneity of Proteoform-Ligand Complexes in AMP-Activated Protein Kinase Uncovered by Integrated Top-Down Mass Spectrometry

AMP-activated protein kinase (AMPK) is a heterotrimeric complex ({beta}{gamma}) that serves as a master regulator of cellular metabolism, making it a prominent drug target for various diseases. Post-translational modifications (PTMs) and ligand binding significantly affect the activity and function of AMPK. However, the dynamic interplay of PTMs, non-covalent interactions, and higher-order structures of the kinase complex remains poorly understood. Herein, we report the structural heterogeneity of the AMPK complex arising from ligand binding and proteoforms--protein products derived from PTMs, alternative splicing, and genetic variants--using integrated native and denatured top-down mass spectrometry (TDMS). The fully intact AMPK heterotrimeric complex exhibits heterogeneity due to phosphorylation and multiple adenosine monophosphate (AMP) binding states. Native TDMS delineates the subunit composition, AMP binding stoichiometry, and higher-order structure of AMPK complex, whilst denatured TDMS comprehensively characterizes the proteoforms and localizes the phosphorylation site. This is the first study to structurally characterize AMPK proteoform-ligand complexes. Notably, by integrating native TDMS and AlphaFold, we elucidate a flexibly connected regulatory region of AMPK {beta} subunit that has been difficult to visualize with traditional structural biology tools. Our findings uncover previously unresolvable structural features of AMPK, offer new perspectives on protein kinase regulation, and establish a versatile framework for comprehensive characterization of proteoform-ligand complexes.

biochemistry↗

bioRxiv2025

Native Top-Down Proteomics of Endogenous Protein Complexes Enabled by Online Two-Dimensional Liquid Chromatography

Protein complexes are essential for virtually all biological processes, yet their structural characterization remains a major challenge due to their heterogeneous, dynamic nature and the complexity of the proteome. Native top-down mass spectrometry (nTDMS) has emerged as a powerful tool for comprehensive structural characterization of purified protein complexes, but its application to endogenous protein complexes in the proteome is challenging and typically requires labor-intensive and time-consuming prefractionation. Here, for the first time, we develop a nondenaturing online two-dimensional liquid chromatography (2D-LC) method for native top-down proteomics (nTDP), enabling high-throughput structural analysis of endogenous protein complexes. The automated, online interfacing of size-exclusion and mixed-bed ion-exchange chromatography achieves high coverage of endogenous protein complexes. We further develop a multistage nTDMS approach that enables comprehensive structural characterization within the chromatographic timescale, capturing intact non-covalent complexes, released subunits/cofactors, and backbone fragments. Our analysis detected 133 native proteoforms and endogenous protein complexes (up to 350 kDa) from human heart tissue in less than two hours. Such technological leaps in high-throughput structural characterization of endogenous protein complexes will advance large-scale nTDP studies in health and disease.

biochemistry↗

bioRxiv2024

Control of limb loading during active horizontal perturbations at moderate and fast trots in rats

To understand how small animals cope with complex, unstructured, and unpredictable substrates, we analyzed the kinetics of rats (n = 10) moving at a fast and at a moderate trot over an unperturbed substrate and a substrate subjected to active horizontal perturbations. Perturbations were active single forwards or backwards displacements of an instrumented platform by amplitudes of 5mm or 10mm in 0.05s. Single leg ground reaction forces (SLGRF) were collected for unperturbed and perturbed locomotion (hindlimbs: 50/102, forelimbs: 45/130, respectively). When negotiating horizontal perturbations, rats displayed gait resetting (braking, accelerating) and non-resetting behaviors. Feedforward strategies differed between the fore- and hindlimbs. In circa 60% of the perturbed trials, forelimbs started the step in acceleration mode, while hindlimbs began the stance mostly in non-resetting mode ([~]45%). In about 50% of all perturbed steps, the impulse provided by the SLGRF displayed a change in behavior according to the expected response to the perturbation. The remaining 50% retained the feedforward strategy. Still, most perturbed trials displayed changes in SLGRF patterns that indicated passive and active reactions to platform shifts. Our results indicate that rats sensorimotor control system tunes fore- and hindlimbs differently in expectation of a perturbation. In addition, the tendon-muscle systems of the limbs are recruited to prevent leg collapse at the beginning and end of the stance. At lower speeds, spinal and/or higher center commands have enough time to re-adapt limb behavior. At higher speeds of locomotion, rats rely more on their limbs intrinsic stability and on feedforward control.

animal behavior and cognition↗

bioRxiv2023

Soil carbon mineralization and microbial community dynamics in response to PyOM addition

Wildfires can either negatively impact soil carbon (C) stocks through combustion or increase soil carbon stocks through the production of pyrogenic organic matter (PyOM), which is highly persistent and can affect non-pyrogenic soil organic carbon (SOC) mineralization rates. In this study, we used fine-resolution 13CO2 flux tracing to investigate PyOM-C mineralization, soil priming effects, and their impacts on soil microbial communities in a Californian mixed conifer forest Xerumbrept soil burned in the 2014 King Fire. We added PyOM produced from pine biomass at 350 {degrees}C and 550 {degrees}C to the soil and separately traced the mineralization of 13C-labeled water-extractable and non-water-extractable PyOM-C fractions in a short-term incubation. Our results indicate that the water-extractable fraction is 10-50x more mineralizable in both 350 {degrees}C and 550 {degrees}C PyOM treatments than the SOC or non-water-extractable PyOM fraction. 350 {degrees}C PyOM addition led to a short-term positive priming effect, likely due to co-metabolism of easily mineralizable PyOM-C and the SOC, whereas 550 {degrees}C PyOM addition induced negative priming, potentially due to physical protection of SOC. We observed significant shifts in bacterial community composition in response to both 350 {degrees}C and 550 {degrees}C PyOM, with positive PyOM responders belonging to the genera Noviherbaspirillum, Pseudonocardia, and Gemmatimonas. In contrast, fungal communities were less responsive to PyOM additions. Our findings expand our understanding of the post-fire cycling of PyOM and SOC, providing insights into the microbial mineralization of different PyOM-C fractions and their influence on soil C dynamics in fire-affected ecosystems.

ecology↗

bioRxiv2022

Positions of pivot points in quadrupedal locomotion: limbs and trunk global control in four different dog breeds.

Dogs (Canis familiaris) prefer the walk at lower speeds and the more economical trot at speeds ranging from 0.5 Fr up to 3 Fr. Important works were carried out to understand these gaits at the levels of center of mass, joint mechanics, and muscular control. However, less is known about the global control goals for limbs and overall locomotion, and of whether these global control goals are gait or breed specific. For walk and trot, we analyzed dog global dynamics based on motion capture and single leg kinetic data recorded from treadmill locomotion of French Bulldog (N = 4), Whippet (N = 5), Malinois (N = 4) and Beagle (N = 5). Dogs displayed two virtual pivot points (VPP) during walk and trot each. One resembles control of both thoracic (fore) limbs and is roughly located above and caudally to the scapular pivot, while the second is located roughly above and cranially to the hip and mirrors the control of the pelvic (hind-) limbs. The positions of VPPs and the patterns of the legs axial and tangential functions were gait and breed related. However, breed related changes were mainly exposed by the French Bulldog. The position of VPPs relative to the proximal pivots explains the propulsive and breaking forces observed in quadrupedal locomotion and may help to reduce limb work. In combination with former work, from the present study the VPP template emerges as the expression of a simple and general global control rule for both bipeds and quadrupeds.

animal behavior and cognition↗

bioRxiv2022

Prescribed fire selects for a pyrophilous soil subcommunity in a northern California mixed conifer forest.

Low intensity prescribed fire is a critical strategy for mitigating the effects of catastrophic wildfires. The above-ground response to fire has been well-documented, including many ecosystem benefits associated with prescribed burning, but fewer studies have directly addressed the effect of prescribed fire on soil organisms. We aimed to understand how soil microbial communities respond to prescribed fire and to determine the ecological processes driving their dynamics. We extensively sampled four plots for 17 months in a mixed conifer forest in northern California, USA; immediately following a low-intensity prescribed fire, a higher-intensity prescribed fire, and two no-burn control plots. We found that prescribed fire significantly altered the community structure for both fungi (ITS) and bacteria (16S), which was sustained throughout the time-series. By comparing our community profiling results with a model of neutral community assembly, we found that the presence of most taxa across all experimental conditions could be explained by neutral processes. However, combining threshold indicator taxa analysis and correlation network analysis with the neutral model identified a cohort of taxa that responded deterministically to prescribed fire. The subcommunity identified through this series of analyses includes both known and new pyrophilous taxa. Beyond this, our analyses revealed network modules within postfire communities which were responsive to fire-intensity. Taken together, these results lay the foundation for building a process-driven understanding of microbial community assembly in the context of the classical disturbance regime of fire.

ecology↗

bioRxiv2022

Limb, joint and pelvic kinematic control in the quail coping with step perturbations

Small cursorial birds display remarkable walking skills and can negotiate complex and unstructured terrains with ease. The neuromechanical control strategies necessary to adapt to these challenging terrains are still not well understood. Here, we analyzed the 2D- and 3D pelvic and leg kinematic strategies employed by the common quail to negotiate visible step-up and step-down perturbations of 1 cm, 2.5 cm, and 5 cm. We used biplanar fluoroscopy to accurately describe joint positions in three dimensions and performed semi-automatic landmark localization using deep learning. Quails negotiated vertical perturbations without major problems and rapidly regained steady-state locomotion. When coping with step-up perturbations, the quail mostly adapted the trailing limb to permit the leading leg to step on the elevated substrate in a similar way as it did during level locomotion. When the quail negotiated step-down perturbations, both legs showed significant adaptations. For small and moderate perturbations (not inducing aerial running) the quail kept the function of the distal joints (i.e., their kinematic pattern) largely unchanged during uneven locomotion, and most changes occurred in proximal joints. The hip regulated leg length, while the distal joints maintained the spring-damped limb patterns. However, to negotiate the largest visible step perturbations, more dramatic kinematic alterations were observed. For these large perturbations, all joints contributed to leg lengthening/ shortening in the trailing leg and both the trailing and leading legs stepped more vertically and less abducted. This indicates a shift from a dynamic walking program to strategies that are focused on maximizing safety.

animal behavior and cognition↗

bioRxiv2021

Pyrolyzed substrates induce aromatic compound metabolism in the post-fire fungus, Pyronema domesticum

Wildfires represent a fundamental and profound disturbance in many ecosystems, and their frequency and severity are increasing in many regions of the world. Fire affects soil by removing carbon in the form of CO2 and transforming remaining surface carbon into pyrolyzed organic material (PyOM). Fires also generate substantial necromass at depths where the heat kills soil organisms but does not catalyze the formation of PyOM. Pyronema species strongly dominate soil fungal communities within weeks to months after fire. However, the carbon pool (i.e. necromass or PyOM) that fuels their rise in abundance is unknown. We used a Pyronema domesticum isolate from the catastrophic 2013 Rim Fire (CA, USA) to ask if P. domesticum is capable of metabolizing PyOM. P. domesticum grew readily on agar media where the sole carbon source was PyOM (specifically, pine wood PyOM produced at 750 {degrees}C). Using RNAseq, we investigated the response of P. domesticum to PyOM and observed a comprehensive induction of genes involved in the metabolism and mineralization of aromatic compounds, typical of those found in PyOM. Lastly, we used 13C-labeled 750 {degrees}C PyOM to demonstrate that P. domesticum is capable of mineralizing PyOM to CO2. Collectively, our results indicate a robust potential for P. domesticum to liberate carbon from PyOM in post-fire ecosystems and return it to the bioavailable carbon pool. IMPORTANCEFires are increasing in frequency and severity in many regions across the world. Thus, its critically important to understand how our ecosystems respond to inform restoration and recovery efforts. Fire transforms the soil, removing many nutrients while leaving behind both nutritious necromass and complex pyrolyzed organic matter, which is often recalcitrant. Filamentous fungi of the genus Pyronema strongly dominate soil fungal communities soon after fire. While Pyronema are key pioneer species in post-fire environments, the nutrient source that fuels their rise in abundance is unknown. In this manuscript, we used a P. domesticum isolate from the catastrophic 2013 Rim Fire (CA, USA) to demonstrate that P. domesticum metabolizes pyrolyzed organic material, effectively liberating this complex pyrolyzed carbon and returning it to the bioavailable carbon pool. The success of Pyronema in post-fire ecosystems has the potential to kick-start growth of other organisms and influence the entire trajectory of post-fire recovery.

microbiology↗

bioRxiv2020

A three-dimensional musculoskeletal model of the dog

Dogs are an interesting object of investigation because of the wide range of body size, body mass, and physique. In the last several years, the number of clinical and biomechanical studies on dog locomotion has increased. However, the relationship between body structure and joint load during locomotion, as well as between joint load and degenerative diseases of the locomotor system (e.g. dysplasia), are not sufficiently understood. In vivo measurements/records of joint forces and loads or deep/small muscles are complex, invasive, and sometimes ethically questionable. The use of detailed musculoskeletal models may help in filling that knowledge gap. We describe here the methods we used to create a detailed musculoskeletal model with 84 degrees of freedom and 134 muscles. Our model has three key-features: Three-dimensionality, scalability, and modularity. We tested the validity of the model by identifying forelimb muscle synergies of a beagle at walk. We used inverse dynamics and static optimization to estimate muscle activations based on experimental data. We identified three muscle synergy groups by using hierarchical clustering. Predicted activation patterns exhibited good agreement with experimental data for most of the forelimb muscles. We expect that our model will speed up the analysis of how body size, physique, agility, and disease influence joint neuronal control and loading in dog locomotion.

bioengineering↗

bioRxiv2018

A network of MAP-Kinase pathways and transcription factors regulates cell-to-cell communication and cell wall integrity in Neurospora crassa

Maintenance of cell integrity and cell-to-cell communication are fundamental biological processes. Filamentous fungi, such as Neurospora crassa, depend on communication to locate compatible cells, coordinate cell fusion, and establish a robust hyphal network. Two MAP-Kinase pathways are essential for communication and cell fusion in N. crassa; the Cell Wall Integrity/MAK-1 pathway and the MAK-2 (signal response) pathway. Previous studies have demonstrated several points of cross talk between the MAK-1 and MAK-2 pathways, which is likely necessary for oordinating chemotropic growth toward an extracellular signal, and then mediating cell fusion. Canonical MAP-Kinase pathways begin with signal reception and end with a transcriptional response. Two transcription factors, ADV-1 and PP-1, are essential for communication and cell fusion. PP-1 is the conserved target of MAK-2, while it is unclear what targets ADV-1. We did RNAseq on{Delta} adv-1, {Delta}pp-1, and wild-type cells and found that ADV-1 and PP-1 have a shared regulon including many genes required for communication, cell fusion, growth, development, and stress response. We identified ADV-1 and PP-1 binding sites across the genome by adapting the in vitro method of DNA-Affinity Purification sequencing (DAP-seq) for N. crassa. To elucidate the regulatory network, we misexpressed each transcription factor in each upstream MAPK deletion mutant. Misexpression of adv-1 was sufficient to fully suppress the phenotype of the{Delta} pp-1 mutant and partially suppress the phenotype of the{Delta} mak-1 mutant. Collectively, our data demonstrate that the MAK-1-ADV-1 and MAK-2- PP-1 pathways form a tight regulatory network that maintains cell integrity and mediates communication and cell fusion.

genetics↗