Biology subjects

Nowak, M.

Publications and source records attributed to Nowak, M..

9 recordsLinked to original sources

bioRxiv2025

Bigger Is Better Than Many: A Strategy to Optimize Multi-Gene Co-Expression

Large plasmids are often avoided in mammalian co-transfection due to the assumption that they transfect poorly, driving the use of multiple smaller plasmids. Here, we pair finite-state-projection modeling with flow cytometry experiments to compare one-, two-, and three-plasmid delivery of GFP/BFP/RFP. Estimated entry rates were size-independent from 4.9 to 16.4 kb, indicating that plasmid length is not the dominant barrier in this range. Our results suggest that using lipofectamine slightly increases co-transfection efficiency due to the ability of lipoplexes to contain multiple plasmids. However, this benefit is limited to only delivering two plasmids. Additionally, we show that contrary to current beliefs, putting all genes onto the same plasmid both increases the probability that a cell will express all genes of interest and results in a tighter correlation of gene expression levels compared to these multi-plasmid systems. Together, these results identify multi-cargo delivery and not plasmid size as the key constraint on co-transfection and show that single-plasmid designs are generally preferable for applications such as viral-vector production. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/675629v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@251928org.highwire.dtl.DTLVardef@196d1cborg.highwire.dtl.DTLVardef@a781e9org.highwire.dtl.DTLVardef@1420928_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Innocent until proven guilty: Tannerella forsythia may attenuate the virulence of Porphyromonas gingivalis

Periodontitis is caused by a dysbiotic microbiome beneath the gum line, primarily driven by the major pathobionts Porphyromonas gingivalis (Pg) and Tannerella forsythia (Tf). Their virulence depends on the excessive, uncontrolled activity of secreted proteases that sustain chronic inflammation, leading to the destruction of tissues supporting the tooth. Paradoxically, Tf also encodes multiple protease inhibitors, including miropin, a serpin with a broad range of targets. Here, we demonstrate that both native and recombinant miropin effectively inhibit lysine-specific gingipain (Kgp) and thiol protease (Tpr), impairing the growth of Pg in peptide-limited media and reducing its virulence in vivo. A rationally designed variant, RVK-miropin, also inhibited both lysine-specific and arginine-specific gingipains, fully suppressing Pg proliferation and virulence in a mouse infection model. Miropin is abundant on the surface of Tf and forms covalent inhibitory complexes with Pg proteases. In an oral gavage model of periodontitis, coinfection with wild-type Tf (but not a miropin-deficient mutant) and Pg significantly reduced alveolar bone loss caused by Pg alone. Miropin thus counteracts Pg virulence factors and host inflammatory responses, revealing an unexpected protective role for Tf. This challenges the traditional view of Tf as a primary periodontal pathogen, suggesting a context-dependent role as a microbial modulator within the dysbiotic biofilm. Beyond periodontal disease, the unique ability of miropin to inhibit structurally diverse proteases makes it a promising candidate for the development of new therapies that restore proteolytic balance in the periodontium.

microbiology↗

bioRxiv2023

Purification of functional mouse skeletal muscle mitochondria using Percoll density gradient centrifugation

ObjectiveOur goal was to isolate purified mitochondria from mouse skeletal muscle using a Percoll density gradient and to assess bioenergetic function and purity via Seahorse Extracellular Flux (XF) Analyses and mass spectrometry. ResultsMitochondria isolated from murine quadriceps femoris skeletal muscle using a Percoll density gradient method allowed for minimally contaminated preparations with time from tissue harvest to mitochondrial isolation and quantification in about 3-4 hours. Percoll purification from 100-200 mg fresh tissue yielded [~]200-400 ug protein. Mitochondrial bioenergetics evaluated using the Seahorse XFe96 analyzer, a high-throughput respirometry platform, showed optimum mitochondrial input at 500 ng with respiratory control ratio ranging from 3.9-7.1 using various substrates demonstrating a high degree of functionality. Furthermore, proteomic analysis of Percoll-enriched mitochondria isolated from skeletal muscle using this method showed significant enrichment of mitochondrial proteins indicating high sample purity. This study established a methodology that ensures sufficient high quality mitochondria for downstream analyses such as mitochondrial bioenergetics and proteomics.

biochemistry↗

bioRxiv2023

Adaptive dynamics of memory-1 strategies in the repeated donation game

Social interactions often take the form of a social dilemma: collectively, individuals fare best if everybody cooperates, yet each single individual is tempted to free ride. Social dilemmas can be resolved when individuals interact repeatedly. Repetition allows individuals to adopt reciprocal strategies which incentivize cooperation. The most basic model to study reciprocity is the repeated donation game, a variant of the repeated prisoners dilemma. Two players interact over many rounds, in which they repeatedly decide whether to cooperate or to defect. To make their decisions, they need a strategy that tells them what to do depending on the history of previous play. Memory-1 strategies depend on the previous round only. Even though memory-1 strategies are among the most elementary strategies of reciprocity, their evolutionary dynamics has been difficult to study analytically. As a result, most previous work relies on simulations. Here, we derive and analyze their adaptive dynamics. We show that the four-dimensional space of memory-1 strategies has an invariant three-dimensional subspace, generated by the memory-1 counting strategies. Counting strategies record how many players cooperated in the previous round, without considering who cooperated. We give a partial characterization of adaptive dynamics for memory-1 strategies and a full characterization for memory-1 counting strategies. Author summaryDirect reciprocity is a mechanism for evolution of cooperation based on the repeated interaction of the same players. In the most basic setting, we consider a game between two players and in each round they choose between cooperation and defection. Hence, there are four possible outcomes: (i) both cooperate; (ii) I cooperate, you defect; (ii) I defect, you cooperate; (iv) both defect. A memory-1 strategy for playing this game is characterized by four quantities which specify the probabilities to cooperate in the next round depending on the outcome of the current round. We study evolutionary dynamics in the space of all memory-1 strategies. We assume that mutant strategies are generated in close proximity to the existing strategies, and therefore we can use the framework of adaptive dynamics, which is deterministic.

evolutionary biology↗

bioRxiv2021

Human motor cortical gamma activity relates to GABAergic signalling and to behaviour

Gamma activity ({gamma}, >30 Hz) is universally demonstrated across brain regions and species. However, the physiological basis and functional role of {gamma} sub-bands (slow-{gamma}, mid-{gamma}, fast-{gamma}) have been predominantly studied in rodent hippocampus; {gamma} activity in the human neocortex is much less well understood. Here we combined neuroimaging and non-invasive brain stimulation to examine the properties of {gamma} activity sub-bands in the primary motor cortex (M1), and their relationship to both local GABAergic activity and to motor learning. In 33 healthy individuals, we quantified movement-related {gamma} activity in M1 using magnetoencephalography, assessed GABAergic signaling using transcranial magnetic stimulation (TMS), and estimated motor learning via a serial reaction time task. We characterised two distinct {gamma} sub-bands (slow-{gamma}, mid-{gamma}) which show movement-related increase in activity during unilateral index finger movements and are characterised by distinct temporal-spectral-spatial profiles. Bayesian correlation analysis revealed strong evidence for a positive relationship between slow-{gamma} ([~]30-60Hz) peak frequency and endogenous GABA signalling during movement preparation (as assessed using the TMS-metric short interval intracortical inhibition). There was also moderate evidence for a relationship between power of the movement-related mid-{gamma} activity (60-90Hz) and motor learning. These relationships were neurochemically- and frequency-specific. These data provide new insights into the neurophysiological basis and functional roles of {gamma} activity in human M1 and allow the development of a new theoretical framework for {gamma} activity in the human neocortex. Significance StatementGamma ({gamma}) activity is ubiquitous in the brain, yet our understanding of the mechanisms and function of {gamma} activity in the human neocortex, and particularly in the human motor cortex, is limited. Using a multimodal approach, we characterised two patterns of movement-related {gamma} activity in the human motor cortex (slow-{gamma} and mid-{gamma}), with different spatial, temporal and spectral properties. Slow-{gamma} peak frequency was correlated to local GABA-A activity, whereas mid-gamma power predicted performance in a subsequent motor learning task. Based on these findings and previous research, we propose a theoretical framework to explain how human motor cortical {gamma} activities may arise and their potential role in plasticity and motor learning, providing new hypotheses to be tested in future studies. Key PointsO_LIWe combined neuroimaging (i.e. MEG) and non-invasive brain stimulation (i.e. TMS) to examine the properties of {gamma} activity sub-bands in the primary motor cortex. C_LIO_LITwo distinct {gamma} sub-bands (slow-{gamma}, mid-{gamma}) show a movement-related increase in activity during finger movements and are characterised by distinct temporal-spectral-spatial profiles. C_LIO_LIWe found strong evidence for a positive relationship between slow-{gamma} ([~]30-60Hz) peak frequency and endogenous GABA signalling during movement preparation (as assessed using the TMS-metric short interval intracortical inhibition). C_LI

neuroscience↗

bioRxiv2020

The scar constituent Collagen I triggers coordinated collective migration and invasion in a 3D spheroid model of early endometriotic lesions

Endometriosis is a painful gynaecological condition characterized by ectopic growth of endometrial cells outside of the uterus. Little is known about the mechanisms by which endometrial fragments invade tissues. This is partially due to a lack of suitable experimental models. In this study, we show that a spheroid 3D model, but not single cells mimic the collective endometrial fragment-like invasion through the extracellular matrix. This model reveals that collagen I, the main constituent of surgical scars, significantly increases the rate of lesion formation by healthy endometrial stromal cells (St-T1b) in vitro compared to the basement membrane-like matrix Matrigel. Stromal cell invasion of collagen I requires MMPs, whereas collective migration of endometriotic epithelial 12Z cells involves Rac-signalling. We show that inhibiting ROCK signalling responsible for actomyosin contraction increases the lesion-size. Moreover, endometriotic epithelial 12Z cells, but not eutopic stromal cells St-T1b migrate on Matrigel. The rate of this migration is decreased by the microRNA miR-200b and increased by miR-145. Our 3D model offers a facile approach to dissect how endometrial fragments invade tissues and is an important step toward developing new personalized therapeutics for endometriosis. Moreover, our model is a suitable tool to screen small molecule drugs and microRNA-based therapeutics.

bioengineering↗

bioRxiv2019

A NanoLuc luciferase-based assay enabling the real-time analysis of protein secretion and injection by bacterial type III secretion systems

The elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a lack of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess secretion and injection through the type III secretion system encoded by Salmonella pathogenicity island 1. The NanoLuc-based secretion assay features a very high signal-to-noise ratio and sensitivity down to the nanoliter scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384-well microplates. We further developed NanoLuc and split-NanoLuc-based assays that enable the monitoring of type III secretion-dependent injection of effector proteins into host cells.\n\nImportanceThe ability to secrete proteins to the bacterial cell surface, to the extracellular environment, or even into target cells is one of the foundations of interbacterial as well as pathogen-host interaction. While great progress has been made in elucidating assembly and structure of secretion systems, our understanding of their secretion mechanism often lags behind, not last because of the challenge to quantitatively assess secretion function. Here, we developed a luciferase-based assay to enable the simple, quick, quantitative, and high throughput-compatible assessment of secretion and injection through virulence-associated type III secretion systems. The assay allows detection of minute amounts of secreted substrate proteins either in the supernatant of the bacterial culture or within eukaryotic host cells. It thus provides an enabling technology to elucidate the mechanisms of secretion and injection of type III secretion systems and is likely adaptable to assay secretion through other bacterial secretion systems.

microbiology↗

bioRxiv2018

A Two-Player Iterated Survival Game

We describe an iterated game between two players, in which the payoff is to survive a number of steps. Expected payoffs are probabilities of survival. A key feature of the game is that individuals have to survive on their own if their partner dies. We consider individuals with hardwired, unconditional behaviors or strategies. When both players are present, each step is a symmetric two-player game. The overall survival of the two individuals forms a Markov chain. As the number of iterations tends to infinity, all probabilities of survival decrease to zero. We obtain general, analytical results for n-step payoffs and use these to describe how the game changes as n increases. In order to predict changes in the frequency of a cooperative strategy over time, we embed the survival game in three different models of a large, well-mixed population. Two of these models are deterministic and one is stochastic. Offspring receive their parents type without modification and fitnesses are determined by the game. Increasing the number of iterations changes the prospects for cooperation. All models become neutral in the limit (n [->] {infty}). Further, if pairs of cooperative individuals survive together with high probability, specifically higher than for any other pair and for either type when it is alone, then cooperation becomes favored if the number of iterations is large enough. This holds regardless of the structure of pairwise interactions in a single step. Even if the single-step interaction is a Prisoners Dilemma, the cooperative type becomes favored. Enhanced survival is crucial in these iterated evolutionary games: if players in pairs start the game with a fitness deficit relative to lone individuals, the prospects for cooperation can become even worse than in the case of a single-step game.

evolutionary biology↗

bioRxiv2017

The distribution of crossovers, and the measure of total recombination

Comparative studies in evolutionary genetics rely critically on evaluation of the total amount of genetic shuffling that occurs during gamete production. However, such studies have been ham-pered by the fact that there has been no direct measure of this quantity. Existing measures consider crossing over by simply counting the average number of crossovers per meiosis. This is qualitatively inadequate because the positions of crossovers along a chromosome are also critical: a crossover towards the middle of a chromosome causes more shuffling than a crossover towards the tip. More-over, traditional measures fail to consider shuffling from independent assortment of homologous chromosomes (Mendels second law). Here, we present a rigorous measure of genome-wide shuffling that does not suffer from these limitations. We define the parameter[r] as the probability that the alleles at two randomly chosen loci will be shuffled in the production of a gamete. This measure can be decomposed into separate contributions from crossover number and position and from independent assortment. Intrinsic implications of this metric include the fact that[r] is larger when crossovers are more evenly spaced, which suggests a novel selective advantage of crossover interference. Utilization of[r] is enabled by powerful emergent methods for determining crossover positions, either cytologically or by DNA sequencing. Application of our analysis to such data from human male and female reveals that: (i)[r] in humans is close to its maximum possible value of 1/2, (ii) this high level of shuffling is due almost entirely to independent assortment, whose contribution is ~30 times greater than that of crossovers.

genetics↗