Biology subjects

Lane, K.

Publications and source records attributed to Lane, K..

9 recordsLinked to original sources

bioRxiv2026

Regulator-derived growth and fitness costs of Salmonella SPI-2 expression are environment specific and T3SS independent

Bacterial pathogens must evolve regulatory mechanisms that balance the necessity of virulence genes with the costs associated with their expression. Salmonella Pathogenicity Island-2 (SPI-2) is a genomic locus that encodes a Type 3 Secretion System (T3SS) and secreted effectors necessary for intracellular survival and replication. Recent work has shown that SPI-2 is expressed heterogeneously, suggesting uniform or aberrant expression of the system carries fitness costs. Here, we report a negative correlation between microcolony growth and SPI-2 expression, suggesting a growth cost to expression of SPI-2. To quantify and mechanistically evaluate this cost, we employ knockout and synthetic expression of the SPI-2 master regulator ssrB to eliminate or force the expression of SPI-2 genes. We find that ssrB expression causes deficits throughout the growth curve and puts cells at a competitive disadvantage in acidic, nutrient limited media. We further show that these deficits are environment specific, and not observed until stationary phase in neutral, rich media. We also observe unexpected cell morphology effects of ssrB expression, suggesting its effects on the cell extend beyond its regulation of SPI-2 genes. Using known ssrB mutations we find that DNA-binding activity, but not phosphorylation, of SsrB is necessary for growth costs. Lastly, we show that this growth cost is incurred even in the absence of the SPI-2 genetic locus, indicating that it is inherent to expression of ssrB, rather than its primary downstream virulence gene targets. These results demonstrate that SPI-2 expression is coupled to context specific cell growth and fitness deficits induced by its master regulator, offering a potential benefit to heterogeneous expression. SIGNIFICANCEVirulence factors are often heterogeneously expressed in bacteria, offsetting the resource and energetic burdens they impose. Characterizing such burdens reveals the evolutionary pressures that act on virulence genes and uncovers the logic behind regulatory mechanisms that drive heterogeneity in their expression. This study evaluates the growth and fitness costs associated with expression of Salmonella Pathogenicity Island-2 (SPI-2), which encodes a Type 3 Secretion System and secreted effectors required for intracellular survival and replication. The expression of the SPI-2 master regulator ssrB is shown to impose environment-specific growth and fitness deficits. These deficits persist in the absence of T3SS genes, suggesting that costs originate from SsrB acting on other target genes. These results provide critical context to recent observations of heterogeneous SPI-2 expression, elucidating the fitness cost this heterogeneity has evolved to offset.

microbiology↗

bioRxiv2026

Salmonella sensitizes macrophages for context-dependent anti-inflammatory response

Macrophages critical role in the innate immune response depends on their ability to change their activation state in response to environmental signals, generating cells with diverse functions ranging from antimicrobial activity to tissue repair. However, this plasticity in activation state represents a vulnerability that intracellular pathogens have evolved to exploit. Salmonella Typhimurium (STm) harnesses macrophage plasticity to promote anti-inflammatory states conducive to bacterial survival and replication. Although this anti-inflammatory state is well-characterized, how it is established remains unclear. Using smFISH, we find that STm is a weak pro-inflammatory trigger and fails to directly induce anti-inflammatory polarization. Instead, we show that STm creates a sensitized cell state with enhanced responsiveness to the cytokine IL-4. While we found that IL-4R[a] upregulation is a common feature of infected cells, the sensitized state is independent of receptor upregulation. Polarization resulting from this sensitized state is context-dependent, with bacterial load and effector secretion distinctly amplifying expression of the anti-inflammatory genes, Il4ra and Arg1. Together, our results establish a two-signal model in which pathogen-driven sensitization and environmental IL-4 combine to drive macrophage anti-inflammatory polarization.

systems biology↗

bioRxiv2026

Perturbing H-NS function reveals roles in restricting virulence heterogeneity and pathogen adaptation

Bacterial pathogens must balance rapid expression of virulence genes in host niches with tight repression when not needed to avoid fitness costs and ensure survival. Integration of virulence gene regulatory networks with the conserved global repressor H-NS was critical to achieve this balance. H-NS-mediated repression of virulence genes in non-inducing environments is essential for maintaining virulence genes and has shaped pathogen evolution. However, the role of H-NS-mediated repression in virulence gene activation and pathogen evolution in virulence-inducing conditions is less clear. For instance, although virulence gene expression is often heterogeneous, whether relief of H-NS repression contributes to this heterogeneity remains unknown. Furthermore, whether H-NS repression shapes pathogen evolution in virulence-inducing environments is unclear. Here, we use a Salmonella strain with reduced H-NS DNA-binding affinity to investigate the role of H-NS in virulence gene expression in individual bacteria and pathogen adaptation. We find that reduced H-NS repression increases the fraction of virulence gene expressing cells without eliminating bimodality, resulting in enhanced epithelial cell infection in vitro. Using experimental evolution, we demonstrate that the hns genotype constrains adaptive mutations and that disabling virulence gene expression is a common path to improved fitness in intracellular-like environments. Our results expand the role of H-NS-mediated repression from silencing virulence genes in non-inducing conditions to regulating heterogeneity in inducing conditions and demonstrate that evolutionary conservation of H-NS constrains adaptive strategies in intracellular-like environments.

systems biology↗

bioRxiv2025

Temporal tuning of switch-like virulence expression resolves environmental uncertainty through phenotypic heterogeneity

Gene regulatory networks often evolve in the face of environmental uncertainty, as stimuli are rarely precise and uniform enough to make all or nothing responses advantageous. Virulence gene regulation in intracellular bacterial pathogens is shaped by unique selective pressures to resolve this uncertainty, as host environments are dynamic, hostile, and heterogeneous. Here, we investigate the regulation of Salmonella Pathogenicity Island 2 (SPI-2), a set of genes required for intracellular replication of the enteric pathogen Salmonella Typhimurium (STm), to evaluate whether it has evolved the capacity to tune transcriptional responses to intracellular-like environments. Using live-cell reporters and smFISH in in vitro inducing environments, we identify bimodal expression of SPI-2 in clonal populations. We show cells progressively transition into a SPI-2 expressing state, with multi-generational variation in the timing at which this transition occurs. The rate of these single-cell transitions are modulated by the strength of intracellular-like signals and environmental manipulations of growth rate. We determine that heterogeneity originates at the level of sensor kinase SsrA signaling and is dependent on response regulator SsrB concentration. We identify switch-like dynamics in SPI-2 activation and link single-cell responses to features of cell growth and lineage. Combined, our results show that the fraction of cells expressing SPI-2 in a given timeframe is probabilistically tuned to the likelihood they are experiencing an intracellular environment, a strategy that resolves uncertainty in ambiguous environments.

systems biology↗

bioRxiv2024

Chromosomal Instability in Human Trophoblast Stem Cells and Placentas

The human placenta, a unique tumor-like organ, is typically thought to exhibit rare aneuploidy associated with adverse pregnancy outcomes. Discrepancies in reported aneuploidy prevalence in placenta likely stem from limitations in modeling and the resolution of detection methods. Here, we used isogenic trophoblast stem cells (TSCs) derived from both naive and primed human pluripotent stem cells (hPSCs) to reveal the spontaneous occurrence of aneuploidy, suggesting chromosomal instability (CIN) as an inherent feature of the trophoblast lineage. We identified potential pathways contributing to the occurrence and tolerance of CIN. These findings were further validated using single cell multiome data from human placentas, where we observed a high prevalence of heterogeneous aneuploidy across trophoblast cells. Despite extensive chromosomal abnormalities, TSCs maintained their proliferative and differentiation capacities, suggesting that CIN is a typical aspect of placental development. Our study challenges the traditional view of aneuploidy in the placenta and provides new insights into the role of CIN in normal placental function.

developmental biology↗

bioRxiv2023

A Cortico-Striatal Circuit for Sound-Triggered Prediction of Reward Timing

A crucial aspect of auditory perception is the ability to use sound cues to predict future events and to time actions accordingly. For example, distinct smartphone notification sounds reflect a call that needs to be answered within a few seconds, or a text that can be read later; the sound of an approaching vehicle signals when it is safe to cross the street. Other animals similarly use sounds to plan, time and execute behaviors such as hunting, evading predation and tending to offspring. However, the neural mechanisms that underlie sound-guided prediction of upcoming salient event timing are not well understood. To address this gap, we employed an appetitive sound-triggered reward time prediction behavior in head-fixed mice. We find that mice trained on this task reliably estimate the time from a sound cue to upcoming reward on the scale of a few seconds, as demonstrated by learning-dependent well-timed increases in reward-predictive licking. Moreover, mice showed a dramatic impairment in their ability to use sound to predict delayed reward when the auditory cortex was inactivated, demonstrating its causal involvement. To identify the neurophysiological signatures of auditory cortical reward-timing prediction, we recorded local field potentials during learning and performance of this behavior and found that the magnitude of auditory cortical responses to the sound prospectively encoded the duration of the anticipated sound-reward time interval. Next, we explored how and where these sound-triggered time interval prediction signals propagate from the auditory cortex to time and initiate consequent action. We targeted the monosynaptic projections from the auditory cortex to the posterior striatum and found that chemogenetic inactivation of these projections impairs animals ability to predict sound-triggered delayed reward. Simultaneous neural recordings in the auditory cortex and posterior striatum during task performance revealed coordination of neural activity across these regions during the sound cue predicting the time interval to reward. Collectively, our findings identify an auditory cortical-striatal circuit supporting sound-triggered timing-prediction behaviors.

neuroscience↗

bioRxiv2023

Improved Protocol for Reproducible Human Cortical Organoids Reveals Early Alterations in Metabolism with MAPT Mutations

Human pluripotent stem cell (hPSC)-derived cortical organoids are powerful models but are often limited by low efficiency, variability, and stress-related artifacts. To address these challenges, we developed a scalable organoid platform with end-to-end quality control (QC) metrics spanning manufacturing and single-cell RNA-sequencing (scRNA-seq), developed using eight MAPT mutation isogenic line sets relevant to frontotemporal dementia (FTD-tau). Using a 96 slit-well format, we achieved [~]100% production efficiency across 64 lines. Controlled-release FGF2 enhanced iPSC pluripotency and reduced mesendodermal contaminants, while optimized SB431542 dosing enhanced cortical patterning across lines with variable TGFBR1/ALK5 expression. The resulting organoids displayed transcriptomic profiles and low-stress signatures closely aligned with the developing human cortex. Applying a cortical organoid scRNA-seq index (CortiCOSI), we identified early dysregulation of phosphatase regulators (PPP2CA, ANP32A) and the prefoldin subunit PFDN6 in MAPT V337M excitatory neurons before tau hyperphosphorylation and oligomerization. This platform improves scalability, reproducibility, and mechanistic insight in cortical organoid studies.

neuroscience↗

bioRxiv2021

Glutamatergic dysfunction precedes neuron loss in cerebral organoids with MAPT mutation.

Frontotemporal dementia (FTD) due to MAPT mutation causes pathological accumulation of tau and glutamatergic cortical neuronal death by unknown mechanisms. We used human induced pluripotent stem cell (iPSC)-derived cerebral organoids expressing tau-V337M and isogenic corrected controls to discover early alterations due to the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, and glutamatergic signaling pathways and regulators including the RNA-binding protein ELAVL4. Over the following 4 months, mutant organoids accumulate splicing changes, disruption of autophagy function and build-up of tau and P-tau S396. By 6 months, tau-V337M organoids show specific loss of glutamatergic neurons of layers affected in patients. Mutant neurons are susceptible to glutamate toxicity which was rescued pharmacologically by treatment with the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede cell death, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD.

neuroscience↗

bioRxiv2019

Identifying Women at Risk for Polycystic Ovary Syndrome Using a Mobile Health Application

BackgroundPolycystic ovary syndrome (PCOS) is an endocrine disrupting disorder affecting at least 10 percent of reproductive-aged women. Women with PCOS are at increased risk for diabetes and cardiovascular disease. In North America and Europe, the diagnosis of PCOS may be delayed several years and may require multiple doctors resulting in lost time for risk-reducing interventions. Menstrual tracking applications are one potential tool to alert women of their risk for PCOS while also prompting them to seek evaluation from a medical professional.\n\nObjectiveThe objective of this study was to develop the Irregular Cycles Feature (ICF), an adaptive questionnaire, on the mobile phone application (app) Clue(R) to generate a probability of a virtual test subjects risk for PCOS. The secondary objective was to assess the accuracy of the ICF by comparing the probability of risk generated by the app to a probability generated by a physician.\n\nMethodsFirst, a literature review was conducted to generate a list of signs and symptoms of PCOS, termed variables. These include, but are not limited to, hirsutism, acne, and alopecia. Probabilities were assigned to each variable and built into a Bayesian network. The network served as the backbone of the ICF, which identified potential subjects through self-reported menstrual cycles and answers to medical history questions. Upon completion of the questionnaire, a Result Screen summarizing the virtual test subjects probability of having PCOS is displayed. For each eligible virtual test subject, a Doctors Report containing information regarding tracked menstrual cycles and self-reported medical history is generated. Both of these documents share information about PCOS and detailed explanations for facilitating a diagnosis by a medical provider. Virtual test subjects were assigned probabilities by a) the ICF and b) a board-certified reproductive endocrinology/infertility physician-scientist, which served as the gold standard. The ICF was set to recommend individuals with a score greater than or equal to 25% to follow-up with their physician. Differences between the network and physician probability scores were assessed using a t-test and a Pearson correlation coefficient. An additional iteration was performed to improve the ICFs prediction capability.\n\nResultsThe first iteration of the ICF produced only one false positive compared to the physician screening score and had an absolute mean difference of 15.5% (SD= 15.1%) amongst virtual test subjects. Upon modification of the ICF, the second iteration had two false positives as compared to the physician screening score and had an absolute mean difference of 18.8% (SD = 13.6%). The majority of virtual test subjects had an ICF score that over predicted PCOS when compared to the physician. However, there was strong positive significant correlation between the ICF and the physician score (Pearson correlation coefficient= 0.69; p < 0.01). The second iteration performed worse with a Pearson correlation coefficient of 0.54; p > 0.01).\n\nConclusionThe first iteration ICF, as compared to the second, was better able to predict the probability of PCOS and can potentially be used as a screening tool to prompt a high-risk subject to seek evaluation by a medical professional.

bioinformatics↗