Biology subjects

Joshi, K.

Publications and source records attributed to Joshi, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Scaling of stochastic growth and division dynamics: A comparative study of individual rod-shaped cells in the Mother Machine and SChemostat platforms

Microfluidic platforms enable long-term quantification of stochastic behaviors of individual bacterial cells under precisely controlled growth conditions. Yet, quantitative comparisons of physiological parameters and cell behaviors of different microorganisms in different experimental and device modalities is not readily possible owing to experiment-specific details affecting cell physiology in confounding ways. To rigorously assess the effects of mechanical confinement, we designed, engineered, and performed side-by-side experiments under otherwise identical conditions in the Mother Machine (with confinement) and the SChemostat (without confinement), using the latter as the ideal comparator. We established a protocol to cultivate a suitably engineered rod-shaped mutant of Caulobacter crescentus in the Mother Machine, and benchmarked the differences in stochastic growth and division dynamics in the Mother Machine with respect to the SChemostat. While the single-cell growth rate distributions are remarkably similar, the mechanically confined cells in the Mother Machine experience a substantial increase in interdivision times. However, we find that the division ratio distribution precisely compensates for this increase in the interdivision times, which in turn reflects identical emergent simplicities governing stochastic intergenerational homeostasis of cell sizes across device and experimental configurations, provided the cell sizes are appropriately mean-rescaled in each condition. Our results provide insights into the nature of the robustness of the bacterial growth and division machinery.

biophysics↗

bioRxiv2023

Architectural underpinnings of stochastic intergenerational homeostasis

Living systems are naturally complex and adaptive, and offer unique insights into the strategies for achieving and sustaining stochastic homeostasis in different conditions. Here, we focus on homeostasis in the context of stochastic growth and division of individual bacterial cells. We take advantage of high-precision longterm dynamical data that have recently been used to extract emergent simplicities and to articulate empirical intra- and in-tergenerational scaling laws governing these stochastic dynamics. We identify the core motif in the mechanistic coupling between division and growth, which naturally yields these precise rules, thus also bridging the intra- and intergenerational phenomenologies. By developing and utilizing novel techniques for solving a broad class of first passage processes, we derive the exact analytic necessary and sufficient condition for sustaining stochastic intergenerational cell size homeostasis within this framework. Furthermore, we provide predictions for the precision kinematics of cell size homeostasis, and the shape of the interdivision time distribution, which are compellingly borne out by the high-precision data. Taken together, these results provide insights into the functional architecture of control systems that yield robust yet flexible stochastic homeostasis.

biophysics↗

bioRxiv2023

Keras/TensorFlow in Drug Design for Immunity Disorders

Homeostasis of the host immune system is regulated by white blood cells with a variety of cell surface receptors for cytokines. Chemotactic cytokines (chemokines) activate their receptors to evoke the chemotaxis of immune cells in homeostatic migrations or inflammatory conditions towards inflamed tissue or pathogens. Dysregulation of the immune system leading to disorders such as allergies, autoimmune diseases, or cancer requires efficient, fast-acting drugs to minimize the long-term effects of chronic inflammation. Here, we performed structure-based virtual screening (SBVS) assisted by the Keras/TensorFlow neural network (NN) to find novel compound scaffolds acting on three chemokine receptors: CCR2, CCR3 and one CXC receptor CXCR3. Keras/TensorFlow NN was used here not as a typically used binary classifier, but as an efficient multi-class classifier that can discard not only inactive compounds but also low or medium-activity compounds. Several compounds proposed by SBVS and NN were tested in 100 ns all-atom molecular dynamics simulations to confirm their binding affinity. To improve the basic binding affinity of the compounds, new chemical modifications were proposed. The modified compounds were compared with known antagonists of these three chemokine receptors. Known CXCR3 were among the top predicted compounds and thus benefits of using Keras/TensorFlow in drug discovery have been shown in addition to structure-based approaches. Furthermore, we showed that Keras/TensorFlow NN can accurately predict the receptor subtype selectivity of compounds, for which SBVS often fails. We cross-tested chemokine receptor datasets retrieved from ChEMBL and curated datasets for cannabinoid receptors available at: http://db-gpcr-chem.uw.edu.pl. The NN model trained on the cannabinoid receptor datasets retrieved from ChEMBL was the most accurate in the receptor subtype selectivity prediction. Among NN models trained on the chemokine receptor datasets, the CXCR3 model showed the highest accuracy in differentiating the receptor subtype for a given compound dataset.

immunology↗

bioRxiv2023

The arginine-ornithine transporter, ArgT, plays an essential role in the survival of Brucella in macrophages and mice

Brucella species are facultative intracellular bacterial pathogens that cause the contagious zoonotic disease, brucellosis. Brucella spp. infect a wide range of animals, including livestock, wild animals, and marine mammals. Brucellosis remains endemic to various parts of the world, affecting the economic growth of many countries because of its impact on public health and livestock productivity. There are no human vaccines for brucellosis, and controlling the disease in susceptible animals is crucial for limiting human infections. Although the available live-attenuated vaccines have protective efficacy in animals, they have many disadvantages, including infectivity in humans. Compared with other invasive bacterial pathogens, minimal information is available on the virulence factors of Brucella that enable them to survive in the host. Here, we performed transposon-based random mutagenesis of B. neotomae and identified the arginine/ornithine binding protein, ArgT, as the crucial virulence determinant of Brucella. Deleting ArgT from B. melitensis resulted in its attenuation in macrophages, which was restored upon complementation with an ArgT expression plasmid. We observed that macrophages infected with {Delta}ArgT-Brucella produced elevated levels of NO due to the inability of {Delta}ArgT Brucella to deplete the host intracellular arginine through its importer. Furthermore, defective survival of {Delta}ArgT B. melitensis was observed in the infected mice, which correlated with enhanced NO production in the mice. Our studies revealed that ArgT in Brucella plays a vital role in preventing intracellular killing and contributes to the chronic persistence of Brucella in the host. This study highlights the essential role of arginine in clearing intracellular infections and the subversion of this host defense mechanism by intracellular pathogens for their chronic persistence.

microbiology↗

bioRxiv2023

Non-Markovian memory and emergent simplicities in the stochastic and plastic adaptation of individual cells to dynamic environments

Do individual bacterial cells retain memories of the history of environmental conditions experienced in previous generations? Here we directly address this question through a synthesis of physics theory and high-precision experiments on statistically identical, non-interacting individual bacterial cells, which grow and divide with intrinsic stochasticity in precisely controlled conditions. From these data, we extract "emergent simplicities" in the seemingly complex interplay between history dependence, persistence, and transience in the stochastic memories of the dynamic environments experienced by individuals over multiple generations. First, we find that the instantaneous single-cell growth rate is the key physiologically relevant quantity where intergenerational memory is stored. In contrast, the cell size dynamics are memory free, or Markovian, over intergenerational timescales. Next, we find that the effect of experiencing dynamic environments can be captured quantitatively by recal-ibrating the cellular unit of time by the measured mean instantaneous growth rate; the dynamically rescaled cell age distributions undergo a scaling collapse. Moreover, in a given condition, an individual bacterial cell retains history-dependent, or non-Markovian, memory of its growth rate over tens of generations. We derive from first principles a physically-motivated metric to quantify the degree of non-Markovianity. Furthermore, when conditions change, the instantaneous single-cell growth distribution becomes bimodal, as the bacteriums memory of past environment encountered is reset stochastically and plastically, prior to achieving a new homeostasis.

biophysics↗

bioRxiv2023

Brucella targets host USP8 through the effector protein TcpB to facilitate infection of macrophages

Brucella species are Gram-negative intracellular bacterial pathogens that cause the worldwide zoonotic disease brucellosis. Brucella can infect many mammals, including humans and domestic and wild animals. Brucella manipulates various host cellular processes to invade and multiply in professional and non-professional phagocytic cells. However, the host targets and their modulation by Brucella to facilitate the infection process remain obscure. Here, we report that the host Ubiquitin Specific Protease, USP8 negatively regulates the invasion of Brucella into macrophages through the plasma membrane receptor, CXCR4. Brucella suppressed the expression of USP8 at its early stage of infection in the infected macrophages. Subsequent studies revealed that the Brucella effector protein, TIR-domain containing protein from Brucella, TcpB plays a significant role in downregulating the expression of USP8 by targeting the CREB pathway. Treatment of mice with USP8 inhibitor resulted in enhanced survival of B. melitensis, whereas mice treated with CXCR4 or 14-3-3 antagonist showed a diminished bacterial load. Our experimental data demonstrate a novel role of USP8 in the host defence against microbial intrusion and microbial subversion of host defences.

microbiology↗

bioRxiv2023

Cellular dynamics under time-varying conditions

Building on the known scaling law that a single timescale, a cellular unit of time, governs stochastic growth and division of individual bacterial cells under constant growth conditions, here we propose that a dynamic rescaling of the cellular unit of time serves to capture the dominant effect of changing conditions on the cell age distribution. This temporal scaling ansatz provides a natural representation for these time-dependent dynamics in whose terms the cell age distribution evolves under time-invariant rules! Finally, we discuss relevance of these results to recent high-precision experiments on individual bacterial cells growing and dividing in dynamic environments.

biophysics↗

bioRxiv2023

Intergenerational scaling law determines the precision kinematics of stochastic individual-cell-size homeostasis

Individual bacterial cells grow and divide stochastically. Yet they maintain their characteristic sizes across generations within a tightly controlled range. What rules ensure intergenerational stochastic homeostasis of individual cell sizes? Valuable clues have emerged from high-precision longterm tracking of individual statistically-identical Caulobacter crescentus cells as reported in [1, 2]: Intergenerational cell size homeostasis is an inherently stochastic phenomenon, follows Markovian or memory-free dynamics, and cells obey an intergenerational scaling law, which governs the stochastic map characterizing generational sequences of cell sizes. These observed emergent simplicities serve as essential building blocks of the data-informed principled theoretical framework we develop here. Our exact analytic fitting-parameter-free results for the predicted intergenerational stochastic map governing the precision kinematics of cell size homeostasis are remarkably well borne out by experimental data, including extant published data on other microorganisms, Escherichia coli and Bacillus subtilis. Furthermore, our framework naturally yields the general exact and analytic condition that is necessary and sufficient to ensure that stochastic homeostasis can be achieved and maintained. Significantly, this condition is more stringent than the known heuristic result from quasi-deterministic frameworks. In turn the fully stochastic treatment we present here extends and updates extant frameworks, and highlights the inherently stochastic behaviors of individual cells in homeostasis.

biophysics↗

bioRxiv2023

Emergent Simplicities in Stochastic Intergenerational Homeostasis

How do complex systems maintain key emergent "state variables" at desired target values to within specified tolerances? This question was first posed in the context of homeostasis in living systems over a century ago, and yet the precise quantitative rules governing this phenomenon have remained fiercely debated. We herein present a direct solution through a synthesis of high-precision experiments and first principles-based physics theory. After introducing a general approach that incorporates the inherently stochastic and dynamic nature of organismal homeostasis, we provide direct experimental evidence that stochastic intergenerational homeostasis is indeed maintained. Next, we identify a series of emergent simplicities hidden in these data. Remarkably, the dynamics of intergenerational homeostasis of organismal sizes are Markovian, or history-independent. The precision data reveal an intergenerational scaling law that fully determines, with no fine-tuning parameters, the exact stochastic map governing homeostasis, as borne out by compelling data- theory matches. These emergent simplicities in turn yield the necessary and sufficient condition for stochastic homeostasis, with surprising implications for the architecture of the underlying control system. Validation across different growth conditions, cell morphologies, experimental modalities, and organisms comprehensively establishes the universality of the results presented here.

biophysics↗

bioRxiv2022

Bacterial Strategies for Damage Management

Organisms are able to partition resources adaptively between growth and repair. The precise nature of optimal partitioning and how this emerges from cellular dynamics including insurmountable trade-offs remains an open question. We construct a mathematical framework to estimate optimal partitioning and the corresponding maximal growth rate constrained by empirical scaling laws. We model a biosynthesis tradeoff governing the partitioning of the ribosome economy between replicating functional proteins and replicating the ribosome pool, and also an energy tradeoff arising from the finite energy budget of the cell. Through exact analytic calculations we predict limits on the range of values partitioning ratios take while sustaining growth. We calculate how partitioning and cellular composition scale with protein and ribosome degradation rates and organism size. These results reveal different classes of optimizing strategies corresponding to phenotypically distinct bacterial lifestyles. We summarize these findings in a quadrant-based taxonomy including: a "greedy" strategy maximally prioritizing growth, a "prudent" strategy maximally prioritizing the management of damaged pools, and "strategically limited" intermediates.

biophysics↗

bioRxiv2022

Compartmentation of putrescine synthesis in plants

Three plant pathways for the synthesis of putrescine have been described to date. These are the synthesis of putrescine from ornithine, by ornithine decarboxylase (ODC); and the synthesis of putrescine from arginine by arginine decarboxylase, agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (NLP1); or arginine decarboxylase and agmatinase. Several enzymes associated with putrescine synthesis have yet to be localized. Here we showed that ODC in soybeans and rice was localized to the ER. In rice, agmatinase is localized to the mitochondria. In A. thaliana there are five isoforms of AIH and three isoforms of NLP1. Stable GFP-tagged transformants of the longest isoforms of AIH and NLP1 showed that both proteins were localized to the ER in leaves and roots of A. thaliana. Four of the isoforms of AIH and all of the isoforms of NLP1 were localized to the ER. However, AIH1.4 was localized to both the ER and the chloroplast. Combining these results with other published data, reveal that putrescine synthesis is excluded from the cytoplasm and is spatially localized to the chloroplast, ER and likely the mitochondria. Synthesis of putrescine in the ER may facilitate cell to cell transport via plasmodesmata, or secretion via vesicles. Differential expression of these pathways may enable putrescine-mediated activation of hormone-responsive genes.

plant biology↗

bioRxiv2022

Single amino acid residues control potential-dependent inactivation of an inner membrane bc-cytochrome

During extracellular electron transfer, Geobacter sulfurreducens constitutively expresses the bc-cytochrome CbcL, yet cells containing only this menaquinone oxidase fail to respire above -0.1 V vs. SHE. By identifying mutations within cbcL that permit growth at higher potentials, we provide evidence that this cytochrome is regulated by redox potential. Strains expressing CbcLV205A, CbcLV205G, and CbcLF525Y were capable of growth with high potential electron acceptors including Fe(III) citrate, Mn(IV) oxides, and electrodes poised at +0.1 V vs. SHE. Electrochemical characterization of wild type CbcL revealed oxidative inactivation of electron transfer above -0.1 V, while CbcLV205A, CbcLV205G, and CbcLF525Y remained active. Growth yields of CbcLV205A, CbcLV205G, and CbcLF525Y were only 50% of WT, consistent with CbcL-dependent electron transfer conserving less energy. These data support the hypothesis that CbcL has evolved to rapidly shut off in response to redox potential to divert electrons to higher yield oxidases that coexist in the Geobacter membrane. TOC image and captionO_ST_ABSTunnel diode behaviorC_ST_ABSElectron flux from cells utilizing the menaquinone oxidase CbcL is attenuated by increased redox potential, preventing use of this low-efficiency pathway when driving forces are high enough to conserve energy via other oxidases. Single amino acid substitutions eliminate this switch-off effect and allow function at all potentials. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/506072v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@11c9b5corg.highwire.dtl.DTLVardef@11be4c6org.highwire.dtl.DTLVardef@4883bborg.highwire.dtl.DTLVardef@c09223_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Heterotypic Endosomal Interactions Drive Emergent Early Endosomal Maturations

Endosomal maturation is critical for robust and timely cargo transport to specific cellular compartments. The most prominent model of early endosomal maturation involves phosphoinositide-driven gain or loss of specific proteins on individual endosomes, emphasising an autonomous and stochastic description. However, limitations in fast, volumetric imaging long hindered direct whole-cell measurements of absolute numbers of maturation events. Here, we use lattice light-sheet imaging and bespoke automated analysis to track individual very early (APPL1-positive) and early (EEA1-positive) endosomes over the entire population, demonstrating that direct interendosomal contact drives maturation. Using fluorescence lifetime, we show that this interaction is underpinned by asymmetric EEA1 binding to very early and early endosomes through its N- and C-termini, respectively. In combination with agent-based simulation that confirms a trigger-and-convert model, our findings indicate that APPL1-to EEA1-positive maturation is driven not by autonomous events but by heterotypic EEA1-mediated interactions, providing a mechanism for temporal and population-level control of maturation.

biophysics↗

bioRxiv2022

Single cells tell their own story: An updated framework for understanding stochastic variations in cell cycle progression in bacteria

Our current understanding of the bacterial cell cycle is framed largely by population-based experiments that focus on the behavior of idealized average cells. Most famously, the contributions of Cooper and Helmstetter help to contextualize the phenomenon of overlapping replication cycles observed in rapidly growing bacteria. Despite the undeniable value of these approaches, their necessary reliance on the behavior of idealized average cells washes out the stochasticity inherent in single cell growth and physiology limiting their mechanistic value. To bridge this gap, we propose an updated and agnostic framework, informed by extant single-cell data, that quantitatively accounts for stochastic variations in single-cell dynamics and the impact of medium composition on cell growth and cell cycle progression. In this framework, stochastic timers sensitive to medium composition impact the relationship between cell cycle events, accounting for observed differences in the relationship between cell cycle events in slow and fast growing cells. We conclude with a roadmap for potential application of this framework to longstanding open questions in the bacterial cell cycle field.

biophysics↗

bioRxiv2022

Emergent periodicity in the collective synchronous flashing of fireflies

In isolation from their peers, Photinus carolinus fireflies flash with no intrinsic period between successive bursts. Yet, when congregating into large mating swarms, these fireflies transition into predictability, synchronizing with their neighbors with a rhythmic periodicity. Here we propose a mechanism for emergence of synchrony and periodicity, and formulate the principle in a mathematical framework. Remarkably, with no fitting parameters, analytic predictions from this simple principle and framework agree strikingly well with data. Next, we add further sophistication to the framework using a computational approach featuring groups of random oscillators via integrate-and-fire interactions controlled by a tunable parameter. This agent-based framework of P. carolinus fireflies interacting in swarms of increasing density also shows quantitatively similar phenomenology and reduces to the analytic framework in the appropriate limit of the tunable coupling strength. We discuss our findings and note that the resulting dynamics follow the style of a decentralized follow-the-leader synchronization, where any of the randomly flashing individuals may take the role of the leader of any subsequent synchronized flash burst.

animal behavior and cognition↗

bioRxiv2021

Geobacter sulfurreducens inner membrane cytochrome CbcBA controls electron transfer and growth yield near the energetic limit of respiration.

Geobacter sulfurreducens utilizes extracellular electron acceptors such as Mn(IV), Fe(III), syntrophic partners, and electrodes that vary from +0.4 to -0.3 V vs. Standard Hydrogen Electrode (SHE), representing a potential energy span that should require a highly branched electron transfer chain. Here we describe CbcBA, a bc-type cytochrome essential near the thermodynamic limit of respiration when acetate is the electron donor. Mutants lacking cbcBA ceased Fe(III) reduction at -0.21 V vs. SHE, could not transfer electrons to electrodes between -0.21 and -0.28 V, and could not reduce the final 10% - 35% of Fe(III) minerals. As redox potential decreased during Fe(III) reduction, cbcBA was induced with the aid of the regulator BccR to become one of the most highly expressed genes in G. sulfurreducens. Growth yield (CFU/mM Fe(II)) was 112% of WT in {Delta}cbcBA, and deletion of cbcL (a different bc-cytochrome essential near -0.15 V) in {Delta}cbcBA increased yield to 220%. Together with ImcH, which is required at high redox potentials, CbcBA represents a third cytoplasmic membrane oxidoreductase in G. sulfurreducens. This expanding list shows how these important metal-reducing bacteria may constantly sense redox potential to adjust growth efficiency in changing environments.

microbiology↗

bioRxiv2021

Host F-box protein 22 enhances the uptake of Brucella by macrophages and drives a sustained release of pro-inflammatory cytokines through degradation of the anti-inflammatory effector proteins of Brucella.

Brucella species are intracellular bacterial pathogens, causing the world-wide zoonotic disease, brucellosis. Brucella invade professional and non-professional phagocytic cells, followed by resisting intracellular killing and establishing a replication permissive niche. Brucella also modulate the innate and adaptive immune responses of the host for their chronic persistence. The complex intracellular cycle of Brucella majorly depends on multiple host factors but limited information is available on host and bacterial proteins that play essential role in the invasion, intracellular replication and modulation of host immune responses. By employing an siRNA screening, we identified a role for the host protein, FBXO22 in Brucella-macrophage interaction. FBXO22 is the key element in the SCF E3 ubiquitination complex where it determines the substrate specificity for ubiquitination and degradation of various host proteins. Downregulation of FBXO22 by siRNA or CRISPR-Cas9 system, resulted diminished uptake of Brucella into macrophages, which was dependent on NF-{kappa}B-mediated regulation of phagocytic receptors. FBXO22 expression was upregulated in Brucella-infected macrophages that resulted induction of phagocytic receptors and enhanced production of pro-inflammatory cytokines through NF-{kappa}B. Furthermore, we found that FBXO22 recruits the effector proteins of Brucella, including the anti-inflammatory proteins, TcpB and OMP25 for degradation through the SCF complex. We did not observe any role for another F-box containing protein of SCF complex, {beta}-TrCP in Brucella-macrophage interaction. Our findings unravel novel functions of FBXO22 in host-pathogen interaction and its contribution to pathogenesis of infectious diseases. Author SummaryBrucellosis is a major zoonotic disease world-wide that poses a serious veterinary and public health problem in various countries, impacting their economic development. Brucellosis is caused by the species of intracellular bacterial pathogen, Brucella that replicates in professional and non-professional phagocytic cells. Brucella is considered as a stealthy pathogen as it invades/suppresses host defense responses using various virulence strategies. Brucella hijacks many cellular processes for gaining entry into the target cells, followed by establishing a replication permissive niche. However, host proteins that are involved in Brucella-macrophage interaction remains obscure. Here, we identified the host protein, FBXO22 that recruits target proteins to SCF E3 ubiquitination complex for their ubiquitination and degradation. We found that down-regulation and upregulation of FBXO22 decreased and enhanced the uptake of Brucella by macrophages, respectively. Our subsequent studies revealed that Brucella induces the expression of FBXO22 that resulted activation of NF-{kappa}B and the concomitant upregulation of phagocytic receptors that might have contributed to the enhanced uptake of Brucella. The Brucella-induced expression of FBXO22 resulted enhanced production of pro-inflammatory cytokines. We have also found that FBXO22 targets Brucella effectors, including the anti-inflammatory effector proteins for degradation through the SCF complex. Our experimental data reveals that FBXO22 plays an important role in the uptake of microbial pathogens by macrophages and pathogenesis of infectious diseases that is resulting from overt inflammatory responses.

microbiology↗

bioRxiv2021

Epitranscriptomic Dysregulation in Stress-induced Psychopathologies

To date, over 100 different chemical modifications to RNA have been identified. Collectively known as the epitranscriptome, these modifications function to regulate RNA stability and as such, represent another mechanistic layer of post-transcriptional gene regulation. N6-methyladenosine (m6A) is the most common RNA modification in the mammalian brain and has been implicated in a number of processes relevant to neurodevelopment, brain function and behaviour. Here, following brief descriptions on epitranscriptomic mechanisms, we will review the literature on the potential functions of the m6A-methylome in fine-tuning gene expression which include prescribing localisation of transcripts in distal compartments as well as interactions with microRNAs and long non-coding RNAs. We will then discuss findings from rodent and human studies for stress-induced disorders - major depression and post-traumatic stress disorder - which support a hypothesis for a dysregulation of the m6A-methylome and the m6A-machinery in the pathophysiology. To support this, we have included a bioinformatic analysis of publicly available single-cell RNA-sequencing and bulk transcriptomics datasets which suggests an altered m6A-methylome as a consequence of dysregulated cell- and regionally-specific expression of key enzymes involved in the writing, reading and erasing of m6A. We hope this review will generate further interest in the field of epitranscriptomics, opening up new lines of research into its involvement in psychiatric disorders.

neuroscience↗