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bioRxiv2022

Light-sheet microscopy of network activity in 3D neuronal systems

In vitro systems mimicking brain regions, brain organoids, are revolutionizing the neuroscience field. However, characterization of their electrical activity has remained a challenge as this requires readout at millisecond timescale in 3D at single-neuron resolution. While custom-built microscopes used with genetically encoded sensors are now opening this door, a full 3D characterization of organoid neural activity has not been performed yet, limited by the combined complexity of the optical and the biological system. Here, we introduce an accessible minimalistic light-sheet microscope to the neuroscience community. Designed as an add-on to a standard inverted microscope it can be assembled within one day. In contrast to existing simplistic setups, our platform is suited to record volumetric calcium traces. We successfully extracted 4D calcium traces at high temporal resolution by using a lightweight piezo stage to allow for 5Hz volumetric scanning combined with a processing pipeline for true 3D neuronal trace segmentation. As a proof of principle, we created a 3D connectivity map of a stem cell derived neuron spheroid by imaging its activity. Our fast, low complexity setup empowers researchers to study the formation of neuronal networks in vitro for fundamental and neurodegeneration research.

neuroscience↗

bioRxiv2024

Efficient mRNA Delivery In Vitro and In Vivo Using a Polycharged Biodegradable Nanomaterial

As RNA rises as one of the most significant modalities for clinical applications and life science research, efficient tools for delivering and integrating RNA molecules into biological systems become essential. We hereby report a formulation using a polycharged biodegradable nano-carrier, N1-501, which demonstrates superior efficiency and versatility in mRNA encapsulation and delivery in both cell and animal models. N1-501 is a polymeric material designed to function through a one-step facile formulation process suitable for various research settings. Its capability for mRNA transfection is investigated across a wide range of mRNA doses and in different biological models, including 18 tested cell lines and mouse models. This study also comprehensively analyzes N1-501s application for mRNA transfection by examining factors such as buffer composition and pH, incubation condition, and media type. Additionally, N1-501s superior in vivo mRNA transfection capability ensures its potential as an efficient and consistent tool for advancing mRNA-based therapies and genetic research.

biochemistry↗

bioRxiv2022

Functional characterization of the Co2+ transporter AitP in Sinorhizobium meliloti: a new player in Fe2+ homeostasis

Co2+ induces the increase of the labile-Fe pool (LIP) by Fe-S cluster damage, heme synthesis inhibition and "free" iron import, which affects cell viability. The N2-fixing bacteria, Sinorhizobium meliloti, is a suitable model to determine the roles of Co2+-transporting Cation diffusion facilitator exporters (Co-eCDF) in Fe2+ homeostasis because it has a putative member of this sub-family, AitP, and two specific Fe2+-export systems. An insertional mutant of AitP showed Co2+ sensitivity and accumulation, Fe accumulation and hydrogen peroxide sensitivity, but not Fe2+ sensitivity, despite AitP being a bona fide low affinity Fe2+ exporter as demonstrated by the kinetic analyses of Fe2+ uptake into everted membrane vesicles. Suggesting concomitant Fe2+-dependent induced stress, Co2+ sensitivity was increased in strains carrying mutations in AitP and Fe2+ exporters which did not correlate with the Co2+ accumulation. Growth in the presence of sub-lethal Fe2+ and Co2+ concentrations suggested that free Fe-import might contribute to Co2+ toxicity. Supporting this, Co2+ induced transcription of Fe-import system and genes associated with Fe homeostasis. Analyses of total protoporphyrin content indicates Fe-S cluster attack as the major source for LIP. AitP-mediated Fe2+-export is likely counterbalanced via a non-futile Fe2+-import pathway. Two lines of evidence support this: i) an increased hemin uptake in presence of Co2+ was observed in WT vs. AitP mutant, and ii) hemin reversed the Co2+ sensitivity in the AitP mutant. Thus, the simultaneous detoxification mediated by AitP aids cells to orchestrate an Fe-S cluster salvage response, avoiding the increase in the LIP caused by the disassembly of Fe-S clusters or free iron uptake. ImportanceCross-talk between iron and cobalt has been long recognized in biological systems. This is due to the capacity of cobalt to interfere with proper iron utilization. Cells can detoxify cobalt by exporting mechanisms involving membrane proteins known as exporters. Highlighting the cross-talk, the capacity of several cobalt exporters to also export iron is emerging. Although biologically less important than Fe2+, Co2+ induces toxicity by promoting intracellular Fe release, which ultimately causes additional toxic effects. In this work, we describe how the N2-fixating rhizobial cells solve this perturbation by clearing Fe through a Co2+-exporter, in order to reestablish intracellular Fe-levels by importing non-free Fe, heme. This piggyback-ride type of transport may aid bacterial cells to survive in free-living conditions where high anthropogenic Co2+ content may be encountered.

microbiology↗

bioRxiv2017

A Synthetic Microbial Operational Amplifier

Synthetic biology has created oscillators, latches, logic gates, logarithmically linear circuits, and load drivers that have electronic analogs in living cells. The ubiquitous operational amplifier, which allows circuits to operate robustly and precisely has not been built with bio-molecular parts. As in electronics, a biological operational-amplifier could greatly improve the predictability of circuits despite noise and variability, a problem that all cellular circuits face. Here, we show how to create a synthetic 3-stage inducer-input operational amplifier with a differential transcription-factor stage, a CRISPR-based push-pull stage, and an enzymatic output stage with just 5 proteins including dCas9. Our Bio-OpAmp expands the toolkit of fundamental circuits available to bioengineers or biologists, and may shed insight into biological systems that require robust and precise molecular homeostasis and regulation.\n\nOne Sentence SummaryA synthetic bio-molecular operational amplifier that can enable robust, precise, and programmable homeostasis and regulation in living cells with just 5 protein parts is described.

synthetic biology↗

bioRxiv2023

The human developing cerebral cortex is characterized by an increased de novo expression of lncRNAs in excitatory neurons

BackgroundOutstanding human cognitive abilities are computed in the cerebral cortex, a mammalian-specific brain region and the place of massive biological innovation. Long noncoding RNAs (lncRNAs) have emerged as gene regulatory elements with higher evolutionary turnover than mRNAs. The many lncRNAs identified in neural tissues make them candidates for molecular sources of cerebral cortex evolution and disease. Here, we characterized the genomic and cellular shifts that occurred during the evolution of the lncRNA repertoire expressed in the developing cerebral cortex of humans and explored their role in the evolution of this brain region. ResultsUsing systems biology approaches and comparative transcriptomics, we comprehensively annotated the cortical transcriptomes of humans, macaques, mice, and chickens and classified human cortical lncRNAs into evolutionary groups as a function of their predicted minimal ages. LncRNA evolutionary groups showed differences in expression levels, splicing efficiencies, transposable element contents, genomic distributions, and transcription factor binding to their promoters. Furthermore, older lncRNAs showed preferential expression in germinative zones, outer radial glial cells, and cortical inhibitory neurons. In comparison, younger lncRNAs showed preferential expression in cortical excitatory neurons, belonged to human-specific gene coexpression modules, and were dysregulated in autism spectrum disorder. ConclusionsThese results suggest a shift in the roles of cortical lncRNAs over evolution, highlighting the antique lncRNAs as a source of molecular evolution of conserved developmental programs; conversely, the de novo expression of primate and human-specific lncRNAs are sources of molecular evolution and dysfunction of cortical excitatory neurons.

evolutionary biology↗

bioRxiv2024

Elucidating the dynamics of Integrin αIIb3 from native platelet membranes by cryo-EM with build and retrieve method

Platelets fulfill their essential physiological roles sensing the extracellular environment through their membrane proteins. The native membrane environment provides essential regulatory cues that impact the protein structure and mechanism of action. Single-particle cryogenic electron microscopy (cryo-EM) has transformed structural biology by allowing high-resolution structures of membrane proteins to be solved from homogeneous samples. Our recent breakthroughs in data processing now make it feasible to obtain atomic-level-resolution protein structures from crude preparations in their native environments by integrating cryo-EM with the "Build-and-Retrieve" (BaR) data processing methodology. We applied this iterative bottom-up methodology on resting human platelet membranes for an in-depth systems biology approach to uncover how lipids, metal binding, post-translational modifications, and co-factor associations in the native environment regulate platelet function at the molecular level. Here, we report using cryo-EM followed by the BaR method to solve the unmodified integrin IIb{beta}3 structure directly from resting human platelet membranes in its inactivated and intermediate states at 2.75[A] and 2.67[A], respectively. Further, we also solved a novel dimer conformation of IIb{beta}3 at 2.85[A] formed by two intermediate-states of IIb{beta}3. This may indicate a previously unknown self-regulatory mechanism of IIb{beta}3 in its native environment. In conclusion, our data show the power of using cryo-EM with the BaR method to determine three distinct structures including a novel dimer directly from natural sources. This approach allows us to identify unrecognized regulation mechanisms for proteins without artifacts due to purification processes. These data have the potential to enrich our understanding of platelet signaling circuitry. Key pointsO_LIWe report the first structural analysis of platelet membrane proteins extracted directly from human platelet membranes. C_LIO_LIOur novel structural-omics approach allowed us to solve integrin IIb{beta}3 structures in two distinct states from resting human platelets. C_LIO_LIRegulatory cues of integrin IIb{beta}3 were preserved from the native resource and revealed on the models with atomic resolutions. C_LIO_LIThis study opens the potential to build the platelet membrane protein atlas to understand platelet physiology. C_LI

biochemistry↗

bioRxiv2025

Kmo restricts Salmonella in a whole organism infection model by promoting macrophage lysosomal acidification through kainate receptor antagonism.

The kynurenine pathway of tryptophan degradation has been implicated in various diseases, including cancer, neurodegenerative disorders, and infectious diseases. A key branchpoint in this pathway is production of the metabolite 3-hydroxy-kynurenine (3-HK) by the enzyme kynurenine 3-monooxygenase (Kmo). We have previously reported that administration of exogenous 3-HK promotes survival of zebrafish larvae to Salmonella Typhimurium infection by restricting bacterial expansion via a systemic mechanism that targets kainate sensitive glutamate receptor (KAR) ion channels and that the endogenous production of 3-HK by Kmo is required for defense against systemic Salmonella infection. Here we show that endogenous 3-HK promotes lysosomal acidification to contribute to macrophage microbicidal activity, with its absence leading to increased host susceptibility to infection. Further, 3-HK promotes lysosomal acidification in a KAR-dependent manner. We thus reveal a novel link between KARs and macrophage lysosomal acidification, and a novel mechanism by which 3-HK promotes control of bacterial infection. Author SummaryStandard therapy for bacterial infections involves antibiotics to clear pathogens. However, the host immune system can also efficiently eliminate bacteria. We have recently shown that a metabolite of the kynurenine metabolic pathway, 3-hydroxy-kynurenine (3-HK), plays a role in the innate immune response to bacterial infection. Here, we show that the kynurenine pathway promotes macrophage clearance of intracellular bacteria by increasing lysosomal acidification of engulfed bacteria and that 3-HK does so by antagonizing kainate receptors. Together, this adds to our understanding of how multiple biological systems, including metabolic and immune pathways, interact to boost defense against bacteria.

immunology↗

bioRxiv2019

piNET: a versatile web platform for downstream analysis and visualization of proteomics data

Large proteomics data, including those generated by mass spectrometry, are being generated to characterize biological systems at the protein level. Computational methods and tools to identify and quantify peptides, proteins and post-translational modifications (PTMs) that are captured in modern mass spectrometers have matured over the years. On the other hand, tools for downstream analysis, interpretation and visualization of proteomics data sets, in particular those involving PTMs, require further improvement and integration to accelerate scientific discovery and maximize the impact of proteomics studies by connecting them better with biological knowledge across not only proteomics, but also other Omics domains. With the goal of addressing these challenges, the piNET server has been developed as a versatile web platform to facilitate mapping, annotation, analysis and visualization of peptide, PTM, and protein level quantitative data generated by either targeted, shotgun or other proteomics approaches. Building on our experience with large scale analysis of gene and protein expression profiles as part of the Library of Integrated Network Cellular Signatures (LINCS) project, piNET has been designed as a fast, versatile and easy to use web-based tool with three modules that provide mapping from peptides (with PTMs) to proteins, from PTM sites to modifying enzymes that target those sites, and finally from proteins (with PTMs) to pathways, and for further mechanistic insights to LINCS signatures of chemical and genetic perturbations. piNET is freely available at http://www.pinet-server.org.

bioinformatics↗

bioRxiv2023

Thermodynamically consistent determination of free energies and rates in kinetic cycle models

Kinetic and thermodynamic models of biological systems are commonly used to connect microscopic features to system function in a bottom-up multiscale approach. The parameters of such models--free energy differences for equilibrium properties and in general rates for equilibrium and out-of-equilibrium observables--have to be measured by different experiments or calculated from multiple computer simulations. All such parameters necessarily come with uncertainties so that when they are naively combined in a full model of the process of interest, they will generally violate fundamental statistical mechanical equalities, namely detailed balance and an equality of forward/backward rate products in cycles due to T. Hill. If left uncorrected, such models can produce arbitrary outputs that are physically inconsistent. Here we develop a maximum likelihood approach (named multibind) based on the so-called potential graph to combine kinetic or thermodynamic measurements to yield state resolved models that are thermodynamically consistent while being most consistent with the provided data and their uncertainties. We demonstrate the approach with two theoretical models, a generic two-proton binding site and a simplified model of a sodium/proton antiporter. We also describe an algorithm to use the multibind approach to solve the inverse problem of determining microscopic quantities from macroscopic measurements and as an example we predict the microscopic pKas and protonation states of a small organic molecule from 1D NMR data. The multibind approach is applicable to any thermodynamic or kinetic model that describes a system as transitions between well-defined states with associated free energy differences or rates between these states. A Python package multibind, which implements the approach described here, is made publicly available under the MIT Open Source license. WHY IT MATTERSThe increase in computational efficiency and rapid advances in methodology for quantitative free energy and rate calculations has allowed for the construction of increasingly complex thermodynamic or kinetic "bottom-up" models of chemical and biological processes. These multi-scale models serve as a framework for analyzing aspects of cellular function in terms of microscopic, molecular properties and provide an opportunity to connect molecular mechanisms to cellular function. The underlying model parameters--free energy differences or rates--are constrained by thermodynamic identities over cycles of states but these identities are not necessarily obeyed during model construction, thus potentially leading to inconsistent models. We address these inconsistencies through the use of a maximum likelihood approach for free energies and rates to adjust the model parameters in such a way that they are maximally consistent with the input parameters and exactly fulfill the thermodynamic cycle constraints. This approach enables formulation of thermodynamically consistent multi-scale models from simulated or experimental measurements.

biophysics↗

bioRxiv2025

Overcoming preservation challenges to enable single-cell proteomics of fixed cell and tissue samples with retained proteome integrity

The ability to assay the molecular composition of biological systems with single-cell resolution has revolutionised our understanding of tissue heterogeneity and function. Recent advances in single-cell proteomics (SCP) now enable the unbiased quantification of the proteome to a depth of several thousand proteins across hundreds of cells. Yet the broader adoption beyond specialised groups remains limited due to the need for specific equipment and expertise. A major challenge in making these analyses more broadly available is sample preservation for transporting biological material to SCP-capable facilities. To address this issue and provide practical solutions; we first evaluated various cell preservation methods from monolayer culture samples, then tested our optimised methodology on both cultured cells and, for the first time, preserved animal tissue from an in vivo mouse model. Our findings highlight the feasibility of SCP analyses in preserved tissues, significantly expanding its current applicability. By optimising upstream processing, our approach enables robust single-cell proteome analysis of both cells and tissues, making SCP more accessible to the wider scientific community. Ultimately, this advancement expands the potential applications of SCP, particularly in disciplines where analysing rare or heterogeneous populations is beneficial.

biochemistry↗

bioRxiv2025

Oxidant-mediated amino acid conversion of angiotensin II: Effects on gene expression in vascular smooth muscle cells.

Angiotensin II (Ang II) is a major mediator of pathophysiological processes. Angiotensin-converting enzyme 2 (ACE2) regulates Ang II levels by cleaving it into Ang 1-7. Our previous study showed that arginine at the second position (Arg2) of Ang II plays an important role in regulating ACE2 peptidase activity. The present study examined the mechanisms and functions of Arg2 in Ang II. Cleavage of Arg2 from Ang II, or single amino acid replacement of a positively charged Arg with other amino acids, particularly negatively charged amino acids such as glutamic acid (Glu), eliminated Ang IIs ability to compete with the fluorogenic substrate used in the ACE2 activity assay. Arg2-to-Glu substitution also affected the ability of angiotensin I and angiotensin III to interact with ACE2. These results confirmed the important roles of Arg2 in the ACE2 enzymatic mechanism. Since Arg and proline (Pro) residues are susceptible to protein carbonylation, and both residues produce glutamic semialdehyde, which can be further oxidized to Glu, we previously proposed that Arg, Pro, and Glu could be interchangeable, and that ROS promote amino acid conversions within protein structures post-translationally in biological systems. Thus, Arg2 of Ang II could undergo oxidant-mediated amino acid residue conversion to become Glu or Pro. The presence of both circulating Ang II (Arg2Glu) and Ang II (Arg2Pro) in rats were confirmed by mass spectrometry. Global gene expression analyses revealed that Arg2Glu- and Arg2Pro-converted Ang II peptides elicit different cellular signaling responses compared with wild-type Ang II, suggesting that Arg2-modifed Ang II peptides exhibit distinct biological functions.

biochemistry↗

bioRxiv2025

Exploring Proteomic Differences in PBMCs for Sex-Specific Insights into Alzheimer's Disease

Peripheral blood mononuclear cells (PBMCs) offer a minimally invasive window into systemic biology and immune dysregulation in Alzheimers disease (AD). We performed quantitative proteomic profiling of PBMCs from male and female AD patients and controls to assess sex differences. AD was associated with proteomic remodeling, with complement activation, coagulation, and neuronal signaling enriched in males, whereas females showed increased steroid hormone secretion, lipid metabolism, and acute-phase response with reduced translation and DNA maintenance. Despite distinct patterns, both sexes exhibited immune and hemostatic activation, underscoring shared systemic mechanisms and the need for sex-specific biomarkers and therapeutic strategies in AD.

neuroscience↗

bioRxiv2018

An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar

How viruses evolve within hosts can dictate infection outcomes; however, reconstructing this process is challenging. We evaluated our multiplexed amplicon approach - PrimalSeq - to demonstrate how virus concentration, sequencing coverage, primer mismatches, and replicates influence the accuracy of measuring intrahost virus diversity. We developed an experimental protocol and computational tool (iVar) for using PrimalSeq to measure virus diversity using Illumina and compared the results to Oxford Nanopore sequencing. We demonstrate the utility of PrimalSeq by measuring Zika and West Nile virus diversity from varied sample types and show that the accumulation of genetic diversity is influenced by experimental and biological systems.

evolutionary biology↗

bioRxiv2021

BioNERO: an all-in-one R/Bioconductor package for comprehensive and easy biological network reconstruction

SummaryCurrently, standard network analysis workflows rely on many different packages, often requiring users to have a solid statistics and programming background. Here, we present BioNERO, an R package that aims to integrate all aspects of network analysis workflows, including expression data preprocessing, gene coexpression and regulatory network inference, functional analyses, and intra and interspecies network comparisons. The state-of-the-art methods implemented in BioNERO ensure that users can perform all analyses with a single package in a simple pipeline, without needing to learn a myriad of package-specific syntaxes. BioNERO offers a user-friendly framework that can be easily incorporated in systems biology pipelines. Availability and implementationThe package is available at Bioconductor (http://bioconductor.org/packages/BioNERO).

bioinformatics↗

bioRxiv2022

Symbolic Kinetic Models in Python (SKiMpy): Intuitive modeling of large-scale biological kinetic models

MotivationLarge-scale kinetic models are an invaluable tool to understand the dynamic and adaptive responses of biological systems. The development and application of these models have been limited by the availability of computational tools to build and analyze large-scale models efficiently. The toolbox presented here provides the means to implement, parametrize and analyze large-scale kinetic models intuitively and efficiently. ResultsWe present a Python package (SKiMpy) bridging this gap by implementing an efficient kinetic modeling toolbox for the semiautomatic generation and analysis of large-scale kinetic models for various biological domains such as signaling, gene expression, and metabolism. Furthermore, we demonstrate how this toolbox is used to parameterize kinetic models around a steady-state reference efficiently. Finally, we show how SKiMpy can imple-ment multispecies bioreactor simulations to assess biotechnological processes. AvailabilityThe software is available as a Python 3 package on GitHub: https://github.com/EPFL-LCSB/SKiMpy, along with adequate documentation. Contactvassily.hatzimanikatis@epfl.ch

bioinformatics↗

bioRxiv2022

Laser Ablation Tomography (LATscan) as a new tool for anatomical studies of woody plants

O_LITraditionally, botanists study the anatomy of plants by carefully sectioning samples, histological staining to highlight tissues of interests, then imaging slides under light microscopy. This approach generates significant details; however, this traditional workflow is laborious and time consuming, and ultimately yields two-dimensional (2D) images. Laser Ablation Tomography (LATscan) is a high-throughput imaging system that yields hundreds of images per minute. This method has proven useful for studying the structure of delicate plant tissues, however its utility in understanding the structure of tougher woody tissues is underexplored. C_LIO_LIWe report LATscan-derived anatomical data from several woody stems (ca. 20 mm) of eight species and compare these results to those obtained through traditional anatomical techniques. C_LIO_LILATscan successfully allows the description of tissue composition by differentiating cell type, size, and shape, but also permits the recognition of distinct cell wall composition (e.g., lignin, suberin, cellulose) based on differential fluorescent signals on unstained samples. C_LIO_LILATscan generate high-resolution 2D images and 3D reconstructions of woody plant samples, therefore this new technology is useful for both qualitative and quantitative analyses. This high-throughput imaging technology has the potential to bolster phenotyping of vegetative and reproductive anatomy, wood anatomy, and other biological systems such as plant-pathogen and parasitic plant associations. C_LI

plant biology↗

bioRxiv2018

PathMe: Merging and exploring mechanistic pathway knowledge

BackgroundThe complexity of representing biological systems is compounded by an ever-expanding body of knowledge emerging from multi-omics experiments. A number of pathway databases have facilitated pathway-centric approaches that assist in the interpretation of molecular signatures yielded by these experiments. However, the lack of interoperability between pathway databases has hindered the ability to harmonize these resources and to exploit their consolidated knowledge. Such a unification of pathway knowledge is imperative in enhancing the comprehension and modeling of biological abstractions.\n\nResultsHere, we present PathMe, a Python package that transforms pathway knowledge from three major pathway databases into a unified abstraction using Biological Expression Language as the pivotal, integrative schema. PathMe is complemented by a novel web application (freely available at https://pathme.scai.fraunhofer.de/) which allows users to comprehensively explore pathway crosstalks and compare areas of consensus and discrepancies.\n\nConclusionsThis work has harmonized three major pathway databases and transformed them into a unified schema in order to gain a holistic picture of pathway knowledge. We demonstrate the utility of the PathMe framework in: i) integrating pathway landscapes at the database level, ii) comparing the degree of consensus at the pathway level, and iii) exploring pathway crosstalk and investigating consensus at the molecular level.

bioinformatics↗

bioRxiv2026

Effects of different concentrations and combinations of antibiotics on the dynamics of intracellular transposition in Escherichia coli

The use of antimicrobial compounds in humans, animals and in agriculture leads to environmental antimicrobial contamination through domestic, industrial and agricultural wastewater. Efforts have been made to perform environmental risk assessments based on the potential of these compounds to select for antimicrobial resistance (AMR) at certain concentrations in bacteria. This has resulted in predicted no effect concentrations (PNEC) which determine the minimum thresholds required to select for resistance. However, the effects of these compounds on intracellular transposition within bacterial isolates, a major driver of AMR, have not been previously assessed. Here, we assess the effect of differing sub-inhibitory concentrations of the third-generation cephalosporin, ceftriaxone, on the rate of intracellular transposition in combination with colistin and kanamycin. Two triple replicons systems (RS1 and RS2) were developed to assess this, each containing a chromosome, plasmid and entrapment vector. We show that sub-inhibitory concentrations of ceftriaxone exert hormetic effects on the intracellular transposition rate in RS1 and a steady linear increase in RS2. This defines a predicted no effect concentration for transposition (PNECT) for ceftriaxone as 320 ng/L in RS1 and 3200 ng/L in RS2. This provides a minimum threshold for the environmental impact of ceftriaxone on biological systems at the sub-cellular scale, which is applicable to industrial standards of waste management, where consideration of ecological impact is central.

microbiology↗