Biology subjects

Smith, B. L.

Publications and source records attributed to Smith, B. L..

6 recordsLinked to original sources

bioRxiv2022

Molecular Neurobiology of Loss

Psychological loss is a common experience that erodes well-being and negatively impacts quality of life. The molecular underpinnings of loss are poorly understood. Here, we investigate the mechanisms of loss using an enrichment removal (ER) paradigm in rats. A comprehensive multi-omics investigation of the basolateral amygdala (BLA) revealed alterations in microglia and extracellular matrix (ECM). Follow-up studies indicated that ER decreased microglia size, complexity, and phagocytosis, suggesting reduced immune surveillance. Loss also substantially increased ECM coverage, specifically targeting perineuronal nets surrounding parvalbumin interneurons, suggesting decreased plasticity and increased inhibition in the BLA following loss. Behavioral analyses suggest that these molecular effects are linked to impaired BLA salience evaluation, reflecting emotional blunting observed in human loss. These loss-like behaviors could be rescued by depleting BLA ECM during removal, helping us understand the mechanisms underlying loss and revealing novel molecular targets to ameliorate its impact.

neuroscience↗

bioRxiv2019

Prefrontal Cortex Regulates Chronic Stress-Induced Cardiovascular Susceptibility

The medial prefrontal cortex (mPFC) is necessary for appropriate appraisal of stressful information, as well as coordinating visceral and behavioral processes. However, prolonged stress impairs mPFC function and prefrontal-dependent behaviors. Additionally, chronic stress induces sympathetic predominance, contributing to health detriments associated with autonomic imbalance. Previous studies identified a subregion of rodent prefrontal cortex, infralimbic cortex (IL), as a key regulator of neuroendocrine-autonomic integration after chronic stress, suggesting that IL output may prevent chronic stress-induced autonomic imbalance. In the current study, we tested the hypothesis that the IL regulates hemodynamic, vascular, and cardiac responses to chronic stress. To address this hypothesis, a viral-packaged siRNA construct was used to knockdown vesicular glutamate transporter 1 (vGluT1) and reduce glutamate packaging and release from IL projection neurons. Male rats were injected with a vGluT1 siRNA-expressing construct or GFP control into the IL and then remained as unstressed controls or were exposed to chronic variable stress (CVS). IL vGluT1 knockdown increased heart rate and mean arterial pressure (MAP) reactivity, while CVS increased chronic MAP only in siRNA-treated rats. In a separate cohort, CVS and vGluT1 knockdown interacted to impair both endothelial-dependent and endothelial-independent vasoreactivity ex vivo. Furthermore, vGluT1 knockdown and CVS increased histological markers of fibrosis and hypertrophy. Thus, knockdown of glutamate release from IL projection neurons indicates that these cells are necessary to prevent the enhanced physiological responses to stress that promote susceptibility to cardiovascular pathophysiology. Ultimately, these findings provide evidence for a neurobiological mechanism mediating the relationship between stress and poor cardiovascular health outcomes.\n\nClinical perspectiveWhat is new? O_LIKnockdown of glutamate release from infralimbic cortex increases heart rate and arterial pressure reactivity\nC_LIO_LIDecreased infralimbic glutamate output leads to vascular dysfunction after chronic stress\nC_LIO_LIThese functional changes associate with histological indicators of cardiac hypertrophy, as well as vascular hypertrophy and fibrosis\nC_LI\n\nWhat are the clinical implications? O_LIThese studies provide a neurobiological mechanism that may account for the link between long-term stress and increased cardiovascular disease risk\nC_LI

neuroscience↗

bioRxiv2019

Transcript degradation and codon usage regulate gene expression in a lytic phage

Many viral genomes are small, containing only single- or double-digit numbers of genes and relatively few regulatory elements. Yet viruses successfully execute complex regulatory programs as they take over their host cells. Here, we propose that some viruses regulate gene expression via a carefully balanced interplay between transcription, translation, and transcript degradation. As our model system, we employ bacteriophage T7, whose genome of approximately 60 genes is well annotated and for which there is a long history of computational models of gene regulation. We expand upon prior modeling work by implementing a stochastic gene expression simulator that tracks individual transcripts, polymerases, ribosomes, and RNases participating in the transcription, translation, and transcript-degradation processes occurring during a T7 infection. By combining this detailed mechanistic modeling of a phage infection with high throughput gene expression measurements of several strains of bacteriophage T7, evolved and engineered, we can show that both the dynamic interplay between transcription and transcript degradation, and between these two processes and translation, appear to be critical components of T7 gene regulation. Our results point to a generic gene regulation strategy that may have evolved in many other viruses. Further, our results suggest that detailed mechanistic modeling may uncover the biological mechanisms at work in both evolved and engineered virus variants.

systems biology↗

bioRxiv2018

Combinatorial Approaches to Viral Attenuation

Attenuated viruses have numerous applications, in particular in the context of live viral vaccines. However, purposefully designing attenuated viruses remains challenging, in particular if the attenuation is meant to be resistant to rapid evolutionary recovery. Here we develop and analyze a new attenuation method, promoter ablation, using an established viral model, bacteriophage T7. Ablating promoters of the two most highly expressed T7 proteins (scaffold and capsid) led to major reductions in transcript abundance of the affected genes, with the effect of the double knockout approximately additive of the effects of single knockouts. Fitness reduction was moderate and also approximately additive; fitness recovery on extended adaptation was partial and did not restore the promoters. The fitness effect of promoter knockouts combined with a previously tested codon deoptimization of the capsid gene was less than additive, as anticipated from their competing mechanisms of action. In one design, the engineering created an unintended consequence that led to further attenuation, the effect of which was studied and understood in hindsight. Overall, the mechanisms and effects of genome engineering on attenuation behaved in a predictable manner. Therefore, this work suggests that the rational design of viral attenuation methods is becoming feasible.\n\nImportanceLive viral vaccines rely on attenuated viruses that can successfully infect their host but have reduced fitness or virulence. Such attenuated viruses were originally developed through trial- and-error, typically by adaptation of the wild-type virus to novel conditions. That method was haphazard, with no way of controlling the degree of attenuation, the number of attenuating mutations, or preventing evolutionary reversion. Synthetic biology now enables rational design and engineering of viral attenuation, but rational design must be informed by biological principles to achieve stable, quantitative attenuation. This work shows that in a model system for viral attenuation, bacteriophage T7, attenuation can be obtained from rational design principles, and multiple different attenuation approaches can be combined for enhanced overall effect.

microbiology↗

bioRxiv2017

Reduced protein expression in a virus attenuated by codon deoptimization

The engineering of hundreds of synonymous codon changes into a viral genome appears to provide a general means of achieving attenuation. The mechanistic underpinnings of this approach remain enignmatic, however. Using quantitative proteomics and RNA sequencing, we explore the molecular basis of attenuation in a strain of bacteriophage T7 whose major capsid gene was engineered to carry 182 suboptimal codons. As expected, there was no evident effect of the recoding on transcription. Proteomic observations revealed that translation is halved for the recoded major capsid gene, and a smaller reduction applies to a few genes downstream, potentially caused by translational coupling. Viral burst size is also approximately halved, and the fitness drop accompanying attenuation is compatible with the reduced burst size. Overall, the fitness effect and molecular basis of attenuation by codon deoptimization are compatible with a relatively simple model of reduced translation of a few genes and a consequent diminished virion assembly. This mechanism is simpler than that operating in eukaryotic viruses.

systems biology↗

bioRxiv2016

The E. coli molecular phenotype under different growth conditions

Modern systems biology requires extensive, carefully curated measurements of cellular components in response to different environmental conditions. While high-throughput methods have made transcriptomics and proteomics datasets widely accessible and relatively economical to generate, systematic measurements of both mRNA and protein abundances under a wide range of different conditions are still relatively rare. Here we present a detailed, genome-wide transcriptomics and proteomics dataset of E. coli grown under 34 different conditions. We manipulate concentrations of sodium and magnesium in the growth media, and we consider four different carbon sources glucose, gluconate, lactate, and glycerol. Moreover, samples are taken both in exponential and stationary phase, and we include two extensive time-courses, with multiple samples taken between 3 hours and 2 weeks. We find that exponential-phase samples systematically differ from stationary-phase samples, in particular at the level of mRNA. Regulatory responses to different carbon sources or salt stresses are more moderate, but we find numerous differentially expressed genes for growth on gluconate and under salt and magnesium stress. Our data set provides a rich resource for future computational modeling of E. coli gene regulation, transcription, and translation.

bioinformatics↗