Biology subjects

Jun, S.

Publications and source records attributed to Jun, S..

50 records · Page 3Linked to original sources

bioRxiv2021

Quantitative examination of five stochastic cell-cycle and cell-size control models for Escherichia coli and Bacillus subtilis

We examine five quantitative models of the cell-cycle and cell-size control in Escherichia coli and Bacillus subtilis that have been proposed over the last decade to explain single-cell experimental data generated with high-throughput methods. After presenting the statistical properties of these models, we test their predictions against experimental data. Based on simple calculations of the defining correlations in each model, we first dismiss the stochastic Helmstetter-Cooper model and the Initiation Adder model, and show that both the Replication Double Adder and the Independent Double Adder model are more consistent with the data than the other models. We then apply a recently proposed statistical analysis method and obtain that the Independent Double Adder model is the most likely model of the cell cycle. By showing that the Replication Double Adder model is fundamentally inconsistent with size convergence by the adder principle, we conclude that the Independent Double Adder model is most consistent with the data and the biology of bacterial cell-cycle and cell-size control. Mechanistically, the Independent Adder Model is equivalent to two biological principles: (i) balanced biosynthesis of the cell-cycle proteins, and (ii) their accumulation to a respective threshold number to trigger initiation and division.

systems biology↗

bioRxiv2021

Temporal characteristics of functional connectome reconfigurations are heritable and related to cognition.

The brains functional connectome is dynamic, constantly reconfiguring in an individual-specific manner. However, which characteristics of such reconfigurations are subject to genetic effects, and to what extent, is largely unknown. Here, we identified heritable dynamic features, quantified their heritability, and determined their association with cognitive phenotypes. In resting-state fMRI, we obtained multivariate features, each describing a temporal or spatial characteristic of connectome dynamics jointly over a set of connectome states. We found strong evidence for heritability of temporal features, particularly fractional occupancy (FO) and transition probability (TP), describing the trajectory of state transitions. Genetic effects explained a substantial proportion of phenotypic variance of these features (h2=.39, 95% CI= [.24.54] for FO; h2=.43, 95% CI=[.29.57] for TP). Moreover, these temporal phenotypes were associated with cognitive performance. Contrarily, we found no robust evidence for heritability of spatial features of the dynamic states (i.e., states Modularity and connectivity pattern). Genetic effects may therefore primarily contribute to how the connectome transitions across states, rather than the precise spatial instantiation of the states in individuals. In sum, genetic effects impact the duration spent in each connectivity configuration and the frequency of shifting between configurations, and such temporal features may act as endophenotypes for cognitive abilities.

neuroscience↗

bioRxiv2021

Cell-type specific requirement for pyruvate dehydrogenase in hematopoiesis and leukemia development.

Cancer cells have different metabolic requirements as compared to their corresponding normal tissues. This is thought to reflect metabolic reprogramming during transformation. An alternative possibility is that some metabolic requirements of cancer cells reflect a maintenance of the metabolism of the specific normal cell type from which cancer cells originate. Here, we investigate this hypothesis by comparing glucose use in normal hematopoiesis and in leukemia. T cell progenitors in the thymus were glucose avid and oxidized more glucose in the tricarboxylic acid (TCA) cycle through pyruvate dehydrogenase (PDH) as compared to hematopoietic stem cells (HSCs) or other hematopoietic cells. PDH deletion reduced the number of double positive (DP) T cell progenitors but had no effect on HSCs, myeloid progenitors and other hematopoietic cells we examined. PDH deletion blocked the development of T cell leukemia from Pten-deficient DP progenitors, but not the development of a myeloid neoplasm from Pten-deficient HSCs or myeloid progenitors. Therefore, the requirement of glucose oxidation for leukemia development is inherited from the normal cell of origin and occurs independently of the driver genetic lesion. PDH was not required in vivo to generate acetyl-CoA or maintain levels of TCA cycle metabolites but to prevent pyruvate accumulation and to maintain glutathione levels and redox homeostasis.

cancer biology↗

bioRxiv2020

Single-cell data and correlation analysis support the independent double adder model in both Escherichia coli and Bacillus subtilis

The reference point for cell-size control in the cell cycle is a fundamental biological question. We previously reported that we were unable to reproduce the conclusions of Witz et al.s eLife paper (Witz, van Nimwegen, and Julou 2019) entitled, "Initiation of chromosome replication controls both division and replication cycles in E. coli through a double-adder mechanism", despite extensive efforts. In this replication double adder (RDA) model, both replication and division cycles are determined via replication initiation as the sole implementation point of size control. Witz et al. justified the RDA model using a type of correlation analysis (the "I-value analysis") that they developed. By contrast, we previously showed that, in both Escherichia coli and Bacillus subtilis, replication initiation and cell division are determined by balanced biosynthesis of key cell cycle proteins (e.g., DnaA for initiation and FtsZ for cell division) and their accumulation to their respective threshold numbers, which Witz et al. coined the independent double adder (IDA) model. The adder phenotype is a natural quantitative consequence of these mechanistic principles. In a recent bioRxiv response to our report, Witz and colleagues explicitly confirmed two important limitations of the I-value analysis: (1) it is only applicable to non-overlapping cell cycles, wherein E. coli is known to deviate from the adder principle, and (2) it is only applicable to select biological models and, for example, cannot evaluate the IDA model. These limitations of the I-value analysis were not explained in the original eLife paper and were overlooked during the review process. In this report, we show using data analysis, mathematical modeling, and experiments why the I-value analysis - in its current implementation - cannot compare different biological models. Furthermore, the RDA model is incompatible with the adder principle and is not broadly supported by experimental data. For completeness, we also provide a detailed point-by-point response to Witz et al.s response (Witz, Julou, and van Nimwegen 2020) in the Supplemental Information.

systems biology↗

bioRxiv2020

Comment on 'Initiation of chromosome replication controls both division and replication cycles in E. coli through a double-adder mechanism'

Witz et al. recently performed single-cell mother machine experiments to track growth and the replication cycle in E. coli. They analyzed the correlation structure of selected parameters using both their data and published data, and concluded that E. coli cell-size control is implemented at replication initiation, which challenged the newly emerged division-centric mechanism of cell-size control in bacteria. We repeated Witz et al.s analysis, and performed additional experiments and analytical calculations. These results explain Witz et al.s observation and in fact support the division-centric model.

systems biology↗

bioRxiv2020

Experimental Stroke Induces Chronic Gut Dysbiosis and Neuroinflammation in Male Mice

Recent literature implicates gut epithelia mucosa and intestinal microbiota as important players in post-stroke morbidity and mortality. As most studies have focused on the acute effects of stroke on gut dysbiosis, our study objective was to measure chronic, longitudinal changes in the gut microbiota and intestinal pathology following ischemic stroke. We hypothesized that mice with experimental ischemic stroke would exhibit chronic gut dysbiosis and intestinal pathology up to 36 days post-stroke compared to sham controls. Male C57BL/6J mice were subjected to 60 minutes of transient middle cerebral artery occlusion (tMCAO) or sham surgery. To determine the long-term effects of tMCAO on gut dysbiosis, fecal boli were collected pre- and post-tMCAO on days 0, 3, 14, and 28. Bioinformatics analysis demonstrate significant differences in abundance among Firmicutes and Bacteroidetes taxa at the phylum, family, and species levels in tMCAO compared to sham mice that persisted up to one month post-stroke. The most persistent changes in post-stroke microbial abundance were a decrease in bacteria family S24-7 and significant increases in Ruminococcaceae. Overall, these changes resulted in a persistently increased Firmicutes:Bacteroidetes ratio in stroke animals. Intestinal histopathology showed evidence of chronic intestinal inflammation that included marked increases in immune cell infiltration with mild-moderate epithelial hyperplasia and villous blunting. Increased astrocyte and microglial activity were also detected one-month post-stroke. These results demonstrate that acute, post-stroke disruption of the gut-brain-microbiota axis progresses to chronic gut dysbiosis, intestinal inflammation, and chronic neuroinflammation. Clinical PerspectivesO_LIThe microbiota-gut-brain axis, recently implicated in several neurological disorders, remains largely unexplored at chronic time points post-tMCAO. C_LIO_LIOur results demonstrate chronic gut dysbiosis, prolonged behavioral deficits, and persistent cerebral and intestinal inflammation post-tMCAO in male C57BL/6J mice. C_LIO_LIThese results suggest that manipulation of microbiota may help reduce poor outcomes after stroke and lead to improved post-stroke functional recovery. C_LI

neuroscience↗

bioRxiv2020

Spike protein binding prediction with neutralizing antibodies of SARS-CoV-2

Coronavirus disease 2019 (COVID-19) is a new emerging human infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, also previously known as 2019-nCoV), originated in Wuhan seafood and animal market, China. Since December 2019, more than 69,000 cases of COVID-19 have been confirmed in China and quickly spreads to other counties. Currently, researchers put their best efforts to identify effective drugs for COVID-19. The neutralizing antibody, which binds to viral capsid in a manner that inhibits cellular entry of virus and uncoating of the genome, is the specific defense against viral invaders. In this study, we investigate to identify neutralizing antibodies that can bind to SARS-CoV-2 Sipke (S) protein and interfere with the interaction between viral S protein and a host receptor by bioinformatic methods. The sequence analysis of S protein showed two major differences in the RBD region of the SARS-CoV-2 S protein compared to SARS-CoV and SARS-CoV related bat viruses (btSARS-CoV). The insertion regions were close to interacting residues with the human ACE2 receptor. Epitope analysis of neutralizing antibodies revealed that SARS-CoV neutralizing antibodies used conformational epitopes, whereas MERS-CoV neutralizing antibodies used a common linear epitope region, which contributes to form the {beta}-sheet structure in MERS-CoV S protein and deleted in SARS-CoV-2 S protein. To identify effective neutralizing antibodies for SARS-CoV-2, the binding affinities of neutralizing antibodies with SARS-CoV-2 S protein were predicted and compared by antibody-antigen docking simulation. The result showed that CR3022 neutralizing antibody from human may have higher binding affinity with SARS-CoV-2 S protein than SARS-CoV S protein. We also found that F26G19 and D12 mouse antibodies could bind to SARS-CoV S protein with high affinity. Our findings provide crucial clues towards the development of antigen diagnosis, therapeutic antibody, and the vaccine against SARS-CoV-2.

bioinformatics↗

bioRxiv2020

Multiplex generation, tracking, and functional screening of substitution mutants using a CRISPR/retron system

We developed a clustered regularly interspaced short palindromic repeats (CRISPR)/retron system for multiplexed generation of substitution mutations by co-utilization of a retron system that continuously expresses donor DNA and a CRISPR/Cas9 cassette that induces cleavage at target genomic loci. Our system efficiently introduces substitution mutation in the Escherichia coli genome in a high-throughput manner. These substitution mutations can be tracked by analysis of retron plasmid sequences without laborious amplification of individual edited loci. We demonstrated that our CRISPR/retron system can introduce thousands of mutations in a single experiment and be used for screening phenotypes related to chemical responses or fitness changes. We expect that our system could facilitate genome-scale substitution screenings.

bioengineering↗

bioRxiv2019

Mother machine image analysis with MM3

The mother machine is a microfluidic device for high-throughput time-lapse imaging of microbes. Here, we present MM3, a complete and modular image analysis pipeline. MM3 turns raw mother machine images, both phase contrast and fluorescence, into a data structure containing cells with their measured features. MM3 employs machine learning and non-learning algorithms, and is implemented in Python. MM3 is easy to run as a command line tool with the occasional graphical user interface on a PC or Mac. A typical mother machine experiment can be analyzed within one day. It has been extensively tested, is well documented and publicly available via Github.

systems biology↗

bioRxiv2019

Loss of tissue non-specific alkaline phosphatase (TNAP) enzyme activity in cerebral microvessels is coupled to persistent neuroinflammation and behavioral deficits in late sepsis

Sepsis is characterized as a host response to systemic inflammation and infection that may lead to multi-organ dysfunction and eventual death. While acute brain dysfunction is common among all sepsis patients, chronic neurological impairment is prevalent among sepsis survivors. The brain microvasculature has recently emerged as a major determinant of sepsis-associated brain dysfunction, yet the mechanisms that underlie its associated neuroimmune perturbations and behavioral deficits are not well understood. A growing body of data suggests that the loss of tissue nonspecific alkaline phosphatase (TNAP) enzyme activity in cerebral microvessels may be associated with changes in endothelial cell barrier integrity. The objective of this study was to determine the important mechanisms linking alterations in cerebrovascular TNAP enzyme activity to underlying neurological impairment in late sepsis. We hypothesized that the disruption of TNAP enzymatic activity in cerebral microvessels would be coupled to the sustained loss of brain microvascular integrity, elevated neuroinflammatory responses, and behavioral deficits. Male mice were subjected to cecal ligation and puncture (CLP), a model of experimental sepsis, and assessed up to seven days post-sepsis. All mice were observed daily for sickness behavior and underwent behavioral testing. Our results showed a significant decrease in brain microvascular TNAP enzyme activity in the somatosensory cortex and spinal cord of septic mice but not in the CA1 and CA3 hippocampal regions. Analyses of whole brain myeloid and T-lymphoid cell populations also revealed a persistent elevation of infiltrating leukocytes, which included both neutrophil and monocyte myeloid derived suppressor cells (MDSCs). Regional analyses of the somatosensory cortex, hippocampus, and spinal cord revealed significant astrogliosis and microgliosis in the cortex and spinal cord of septic mice that was accompanied by significant microgliosis in the CA1 and CA3 hippocampal regions. Assessment of behavioral deficits revealed no changes in learning and memory or evoked locomotion. However, the hot plate test uncovered a novel anti-nociceptive phenotype in our septic mice, and we speculate that this phenotype may be a consequence of sustained GFAP astrogliosis and loss of TNAP activity in the somatosensory cortex of septic mice. Taken together, these results demonstrate that the loss of TNAP enzyme activity in cerebral microvessels during late sepsis is coupled to sustained neuroimmune dysfunction which may underlie, in part, the chronic neurological impairments observed in sepsis survivors.\n\nHighlightsO_LIAlkaline phosphatase activity in brain microvessels is diminished in sepsis.\nC_LIO_LILoss of alkaline phosphatase activity is coupled to the loss of barrier integrity.\nC_LIO_LIBrain infiltration of myeloid and T-lymphoid cells persists in late sepsis.\nC_LIO_LISeptic mice exhibit a novel anti-nociceptive phenotype.\nC_LIO_LICortical astrogliosis and microgliosis persist in late sepsis.\nC_LI

neuroscience↗

bioRxiv2019

Gram-positive and Gram-negative Bacteria Share Common Principles to Coordinate Growth and the Cell Cycle at the Single-cell Level

Bacillus subtilis and Escherichia coli are evolutionarily divergent model organisms that have elucidated fundamental differences between Gram-positive and Gram-negative bacteria, respectively. Despite their differences in cell cycle control at the molecular level, both organisms follow the same phenomenological principle for cell size homeostasis known as the adder. We thus asked to what extent B. subtilis and E. coli share common physiological principles in coordinating growth and the cell cycle. To answer this question, we measured physiological parameters of B. subtilis under various steady-state growth conditions with and without translation inhibition at both population and single-cell level. These experiments revealed core shared physiological principles between B. subtilis and E. coli. Specifically, we show that both organisms maintain an invariant cell size per replication origin at initiation, with and without growth inhibition, and even during nutrient shifts at the single-cell level. Furthermore, both organisms also inherit the same \"hierarchy\" of physiological parameters ranked by their coefficient of variation. Based on these findings, we suggest that the basic coordination principles between growth and the cell cycle in bacteria may have been established in the very early stages of evolution.

systems biology↗

bioRxiv2019

Single-cell analysis of a mutant library generated using CRISPR-guided deaminase

CRISPR-based screening methods using single-cell RNA sequencing (scRNA-seq) technology enable comprehensive profiling of gene perturbations from knock-out mutations. However, evaluating substitution mutations using scRNA-seq is currently limited. We combined CRISPR RNA-guided deaminase and scRNA-seq technology to develop a platform for introducing mutations in multiple genes and assessing the mutation-associated signatures. Using this platform, we generated a library consisting of 420 sgRNAs, performed sgRNA tracking analysis, and assessed the effect size of the response to vemurafenib in the melanoma cell line, which has been well-studied via knockout-based drop-out screens. However, a substitution mutation library screen has not been applied and transcriptional information for mechanisms of action was not assessed. Our platform permits discrimination of several candidate mutations that function differently from other mutations by integrating sgRNA candidates and gene expression readout. We anticipate that our platform will enable high-throughput analyses of the mechanisms related to a variety of biological events.

bioengineering↗

bioRxiv2018

Mechanistic origin of cell-size control and homeostasis in bacteria

Evolutionarily divergent bacteria share a common phenomenological strategy for cell-size homeostasis under steady-state conditions. In the presence of inherent physiological stochasticity, cells following this \"adder\" principle gradually return to their steady-state size by adding a constant volume between birth and division regardless of their size at birth. However, the mechanism of the adder has been unknown despite intense efforts. In this work, we show that the adder is a direct consequence of two general processes in biology: (1) threshold -- accumulation of initiators and precursors required for cell division to a respective fixed number, and (2) balanced biosynthesis -- maintenance of their production proportional to volume growth. This mechanism is naturally robust to static growth inhibition, but also allows us to \"reprogram\" cell-size homeostasis in a quantitatively predictive manner in both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. By generating dynamic oscillations in the concentration of the division protein FtsZ, we were able to oscillate cell size at division and systematically break the adder. In contrast, periodic induction of replication initiator protein DnaA caused oscillations in cell size at initiation, but did not alter division size or the adder. Finally, we were able to restore the adder phenotype in slow growing E. coli, the only known steady-state growth condition wherein E. coli significantly deviates from the adder, by repressing active degradation of division proteins. Together these results show that division and replication are independently controlled, and that division processes exclusively drive cell-size homeostasis in bacteria.

systems biology↗

bioRxiv2016

Deconstructing cell size control into physiological modules in Escherichia coli

It is generally assumed that the allocation and synthesis of total cellular resources in microorganisms are uniquely determined by the growth conditions. Adaptation to a new physiological state leads to a change in cell size via reallocation of cellular resources. However, it has not been understood how cell size is coordinated with biosynthesis and robustly adapts to physiological states. We show that cell size in Escherichia coli can be predicted for any steady-state condition by projecting all biosynthesis into three measurable variables representing replication initiation, replication-division cycle, and the global biosynthesis rate. These variables can be decoupled by selectively controlling their respective core biosynthesis using CRISPR interference and antibiotics, verifying our predictions that different physiological states can result in the same cell size. We performed extensive growth inhibition experiments, and discovered that cell size at replication initiation per origin, namely the initiation mass or \"unit cell,\" is remarkably invariant under perturbations targeting transcription, translation, ribosome content, replication kinetics, fatty acid and cell-wall synthesis, cell division, and cell shape. Based on this invariance and balanced resource allocation, we explain why the total cell size is the sum of all unit cells. These results provide an overarching framework with quantitative predictive power over cell size in bacteria.

microbiology↗