Biology subjects

Arnold, J.

Publications and source records attributed to Arnold, J..

31 records · Page 2Linked to original sources

bioRxiv2022

Virtually the same? Evaluating the effectiveness of remote undergraduate research experiences

In-person undergraduate research experiences (UREs) promote students integration into careers in life science research. In 2020, the COVID-19 pandemic prompted institutions hosting summer URE programs to offer them remotely, raising questions about whether undergraduates who participate in remote research can experience scientific integration. To address this, we investigated indicators of scientific integration for students who participated in remote life science URE programs in summer 2020. We found that these students experienced gains in their scientific self-efficacy and scientific identity similar to results reported for in-person UREs. We also found that these students perceived high benefits and low costs of doing research at the outset of their programs, and their perceptions did not change despite the remote circumstances. Yet, their perceptions differed by program, indicating that programs differentially affected students perceptions of the costs of doing research. Finally, we observed that students with prior research experience made greater gains in self-efficacy and identity, as well as in their perceptions of the alignment of their values with those of the scientific community, in comparison to students with no prior research experience. This finding suggests that additional programming may be needed for undergraduates with no prior experience to benefit from remote research.

scientific communication and education↗

bioRxiv2021

Sex-specific transcriptomic and epitranscriptomic signatures of PTSD-like fear acquisition.

Our understanding of the molecular pathology of posttraumatic stress disorder (PTSD) is rapidly evolving and is being driven by advances in sequencing techniques. Conventional short-read RNA sequencing (RNA-seq) is a central tool in transcriptomics research that enables unbiased gene expression profiling. With the recent emergence of Oxford Nanopore direct RNA-seq (dRNA-seq), it is now also possible to interrogate diverse RNA modifications, collectively known as the epitranscriptome. Here, we present our analyses of the male and female mouse amygdala transcriptome and epitranscriptome, obtained using parallel Illumina RNA-seq and Oxford Nanopore dRNA-seq, associated with the acquisition of PTSD-like fear induced by Pavlovian cued-fear conditioning. We report significant sex-specific differences in the amygdala transcriptional response during fear acquisition, and a range of shared and dimorphic epitranscriptomic signatures. Differential RNA modifications are enriched among mRNA transcripts associated with neurotransmitter regulation and mitochondrial function, many of which have been previously implicated in PTSD. Very few differentially modified transcripts are also differentially expressed, suggesting an influential, expression-independent role for epitranscriptional regulation in PTSD-like fear-acquisition. Overall, our application of conventional and newly developed methods provides a platform for future work that will lead to new insights into and therapeutics for PTSD.

neuroscience↗

bioRxiv2021

Behavioral and nociceptor states of inflammatory painacross timescales in 2D and 3D

Ongoing pain is often driven by direct activation of pain-sensing neurons and neuroimmune mediated sensitization. These heightened states of pain alter physiology, reduce motor function, and alter motivation to engage in normal behaviors. The complexity of the pain state has evaded a comprehensive definition, especially in nonverbal animals. Here in mice, we capture the physiological state of sensitized pain neurons at different time points post-inflammation and used computational tools to automatically map behavioral signatures of evoked and spontaneous displays of pain. First, retrograde labeling coupled with electrophysiology of neurons innervating the site of localized inflammation defined critical time points of pain sensitization. Next, we used high-speed videography combined with supervised and unsupervised machine learning tools and uncovered sensory-evoked defensive coping postures to pain. Using 3D pose analytics inspired by natural language processing, we identify movement sequences that correspond to robust representations of ongoing pain states. Surprisingly, with this analytical framework, we find that a commonly used anti-inflammatory painkiller does not return an animals behavior back to a pre-injury state. Together, these findings reveal the previously unidentified signatures of pain and analgesia at timescales when inflammation induces heightened pain states.

neuroscience↗

bioRxiv2021

"How do we do this at a distance?!" A descriptive study of remote undergraduate research programs during COVID-19

The COVID-19 pandemic shut down undergraduate research programs across the U.S. Twenty-three sites offered remote undergraduate research programs in the life sciences during summer 2020. Given the unprecedented offering of remote research experiences, we carried out a study to describe and evaluate these programs. Using structured templates, we documented how programs were designed and implemented, including who participated. Through focus groups and surveys, we identified programmatic strengths and shortcomings as well as recommendations for improvements from the perspectives of participating students. Strengths included the quality of mentorship, opportunities for learning and professional development, and development of a sense of community. Weaknesses included limited cohort building, challenges with insufficient structure, and issues with technology. Although all programs had one or more activities related to diversity, equity, inclusion, and justice, these topics were largely absent from student reports even though programs coincided with a peak in national consciousness about racial inequities and structural racism. Our results provide evidence for designing remote REUs that are experienced favorably by students. Our results also indicate that remote REUs are sufficiently positive to further investigate their affordances and constraints, including the potential to scale up offerings, with minimal concern about disenfranchising students.

scientific communication and education↗

bioRxiv2020

Colwellia and Marinobacter metapangenomes reveal species-specific responses to oil and dispersant exposure in deepsea microbial communities

Over 7 million liters of Corexit EC9500A and EC9527A were applied to the Gulf of Mexico in response to the Deepwater Horizon oil spill. The impacts of dispersants remain under debate and negative, positive, and inconclusive impacts have been reported. Here, metatrancriptomics was applied in the context of metapangenomes to microcosms that simulated environmental conditions comparable to the hydrocarbon-rich 1,100 m deep plume. Within this microcosm study, negative effects of dispersants on microbial hydrocarbon degradation were previously reported based on activity measurements and geochemical data. Transcriptional enrichment of Colwellia, a potential dispersant degrader, followed variable time-dependent trajectories due to interactions between oil, dispersants, and nutrients. The Colwellia metapangenome captured a mixture of environmental responses linked to the Colwellia psychrerythraea 34H genome and to the genomes of other members of the Colwellia genus. The activation of genes involved in lipid degradation, nitrogen metabolism, and membrane composition under oil or nutrient availability, suggested an opportunistic growth strategy for Colwellia. In contrast, transcripts of Marinobacter, a natural hydrocarbon degrader, increased only in oil treatments. Marinobacter transcripts largely recruited to the accessory metapangenome of Marinobacter sp. C18, the closest genomic reference. A complex response involving carbon and lipid metabolism, chemotaxis and a type IV secretion system suggested active energy-dependent processes in Marinobacter. These findings highlight chemistry-dependent responses in the metabolism of key hydrocarbon-degrading bacteria and underscore that dispersant-driven selection could temper the ability of the community to respond to hydrocarbon injection.

microbiology↗

bioRxiv2020

Mutation Maker, An Open Source Oligo Design Software for Mutagenesis and De Novo Gene Synthesis Experiments

Protein engineering is the discipline of developing useful proteins for applications in research, therapeutic and industrial processes by modification of naturally occurring proteins or by invention of de novo proteins. Modern protein engineering relies on the ability to rapidly generate and screen diverse libraries of mutant proteins. However, design of mutant libraries is typically hampered by scale and complexity, necessitating development of advanced automation and optimization tools that can improve efficiency and accuracy. At present, automated library design tools are functionally limited or not freely available. To address these issues, we developed Mutation Maker, an open source mutagenic oligo design software for large-scale protein engineering experiments. Mutation Maker is not only specifically tailored to multi-site random and directed mutagenesis protocols, but also pioneers bespoke mutagenic oligo design for de novo gene synthesis workflows. Enabled by a novel bundle of orchestrated heuristics, optimization, constraint-satisfaction and backtracking algorithms, Mutation Maker offers a versatile toolbox for gene diversification design at industrial scale. Supported by in-silico simulations and compelling experimental validation data, Mutation Maker oligos produce diverse gene libraries at high success rates irrespective of genes or vectors used. Finally, Mutation Maker was created as an extensible platform on the notion that directed evolution techniques will continue to evolve and revolutionize current and future-oriented applications.

bioinformatics↗

bioRxiv2020

Hypoxia-Sensing CAR T-Cells Provide Safety and Efficacy in Treating Solid Tumors

There has been significant interest in the prospects of chimeric antigen receptor (CAR) T-cell therapy in the treatment of solid malignancies, and multiple clinical trials are in progress 1. However, the scope of these trials has been restricted by the lack of availability of tumor-specific targets to direct CAR binding. Tumor specificity is crucial as on-target off-tumor activation of CAR T-cells in healthy tissues can result in potentially lethal toxicities due to uncontrolled cytokine release syndrome 2. Here we engineer a stringent hypoxia-sensing CAR T-cell system which achieves selective expression of a pan-ErbB-targeted CAR within a solid tumor, a microenvironment characterized by an inadequate oxygen supply. Using murine xenograft models, we demonstrate that despite widespread expression of ErbB receptors in healthy organs, the approach provides anti-tumor efficacy without off-tumor toxicity. This dynamic on/off oxygen-sensing safety switch has the potential to facilitate the unlimited expansion of the CAR T-cell target repertoire for treating solid malignancies.Competing Interest StatementYes there is potential Competing Interest. J.M. is co-founder and chief scientific officer, T.M. is an employee, and D.M.D., D.L.Y. are consultants to Leucid Bio, which is a spinout company focused on development of cellular therapeutic agents. J.N.A., J.M. and P.K. are named inventors on a patent submitted in relation to this work. All other authors have declared that there are no competing financial interests or conflicts of interest in relation to this study.View Full Text

synthetic biology↗

bioRxiv2020

Unveiling Crucivirus Diversity by Mining Metagenomic Data

The discovery of cruciviruses revealed the most explicit example of a common protein homologue between DNA and RNA viruses to date. Cruciviruses are a novel group of circular Rep-encoding ssDNA (CRESS-DNA) viruses that encode capsid proteins (CPs) that are most closely related to those encoded by RNA viruses in the family Tombusviridae. The apparent chimeric nature of the two core proteins encoded by crucivirus genomes suggests horizontal gene transfer of CP genes between DNA and RNA viruses. Here, we identified and characterized 451 new crucivirus genomes and ten CP-encoding circular genetic elements through de novo assembly and mining of metagenomic data. These genomes are highly diverse, as demonstrated by sequence comparisons and phylogenetic analysis of subsets of the protein sequences they encode. Most of the variation is reflected in the replication associated protein (Rep) sequences, and much of the sequence diversity appears to be due to recombination. Our results suggest that recombination tends to occur more frequently among groups of cruciviruses with relatively similar capsid proteins, and that the exchange of Rep protein domains between cruciviruses is rarer than gene exchange. Altogether, we provide a comprehensive and descriptive characterization of cruciviruses. IMPORTANCEViruses are the most abundant biological entities on Earth. In addition to their impact on animal and plant health, viruses have important roles in ecosystem dynamics as well as in the evolution of the biosphere. Circular Rep-encoding single-stranded (CRESS) DNA viruses are ubiquitous in nature, many are agriculturally important, and are viruses that appear to have multiple origins from prokaryotic plasmids. CRESS-DNA viruses such as the cruciviruses, have homologues of capsid proteins (CPs) encoded by RNA viruses. The genetic structure of cruciviruses attests to the transfer of capsid genes between disparate groups of viruses. However, the evolutionary history of cruciviruses is still unclear. By collecting and analyzing cruciviral sequence data, we provide a deeper insight into the evolutionary intricacies of cruciviruses. Our results reveal an unexpected diversity of this virus group, with frequent recombination as an important determinant of variability.

evolutionary biology↗

bioRxiv2019

RNA-dependent RNA polymerase speed and fidelity are not the only determinants of the mechanism or efficiency of recombination.

Using the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV) as our model system, we have shown that Lys-359 in motif-D functions as a general acid in the mechanism of nucleotidyl transfer. A K359H (KH) RdRp derivative is slow and faithful relative to wild-type enzyme. In the context of the virus, RdRp-coding sequence evolves, selecting for the following substitutions: I331F (IF, motif-C) and P356S (PS, motif-D). We have evaluated IF-KH, PS-KH, and IF-PS-KH viruses and enzymes. The speed and fidelity of each double mutant are equivalent. Each exhibits a unique recombination phenotype, with IF-KH being competent for copy-choice recombination and PS-KH being competent for forced-copy-choice recombination. Although the IF-PS-KH RdRp exhibits biochemical properties within twofold of wild type, the virus is impaired substantially for recombination in cells. We conclude that there are biochemical properties of the RdRp in addition to speed and fidelity that determine the mechanism and efficiency of recombination. The interwoven nature of speed, fidelity, the undefined property suggested here, and recombination makes it impossible to attribute a single property of the RdRp to fitness. However, the derivatives described here may permit elucidation of the importance of recombination on the fitness of the viral population in a background of constant polymerase speed and fidelity.\n\nSignificanceThe availability of a \"universal\" method to create attenuated viruses for use as vaccine strains would permit a rapid response to outbreaks of newly emerging viruses. Targeting RdRp fidelity has emerged as such a universal approach. However, because polymerase fidelity and speed are inextricably linked, the effort to attribute the attenuated phenotype to a single biochemical property of the RdRp may be futile. Here, we show that this circumstance is even more complex. We provide evidence for the existence of a biochemical parameter that combines with fidelity and speed to govern the mechanism and/or efficiency of recombination. We conclude that the field will be served best by continued emphasis on discovery of manipulatable functions of the RdRp instead of debating the importance of individual properties.

microbiology↗

bioRxiv2019

Medial prefrontal cortex guides goal-directed decision-making about hunger and thirst

Decision-making guided by self-evaluation of bodily need states (interoception) is thought to be important for cognitive control over eating or drinking1. However, interoception of body states is notoriously unreliable2-4 because hunger and thirst have similar motivational characteristics5. Consequently, individuals may inaccurately assess their need state and consume food when dehydrated, leading some healthcare professionals to advise overweight patients to drink water before eating6-8. Neuroimaging in humans9-11 and recordings in rodents12-15 indicate medial prefrontal frontal cortex (mPFC) involvement in need state-dependent decisions about hunger and thirst, but mPFC surgical lesions16,17 and electrical activity perturbations have little influence on eating or drinking18,19. To investigate need-state dependent decision-making as well as the function of mPFC in hunger and thirst, we developed an instrumental foraging task for mice that mimics key elements of human decisions. When homeostatic need state was variable, mice did not show intrinsic knowledge of hunger or thirst state but, instead, rapidly identified their need after consumption of small portions of food and water (outcome evaluation). We observed a food-seeking bias, even in thirsty mice, that required outcome evaluation for mice to correctly seek water. mPFC was required for need state-dependent decisions about hunger and thirst, specifically under variable need state conditions. Food-seeking or water-seeking choices were controlled by multiple decision-making processes, and mPFC was involved in goal-directed decisions about the identity of need states. Thus, we have discovered a role for mPFC in decision-making about hunger and thirst, which is relevant for human behaviors that contribute to obesity.

neuroscience↗

bioRxiv2019

More than efficacy revealed by single-cell analysis of antiviral therapeutics

Development of antiviral therapeutics emphasizes minimization of the effective dose and maximization of the toxic dose, first in cell culture and later in animal models. Long-term success of an antiviral therapeutic is determined not only by its efficacy but also by the duration of time required for drug-resistance to evolve. We have developed a microfluidic device comprised of ~6000 wells, with each well containing a microstructure to capture single cells. We have used this device to characterize enterovirus inhibitors with distinct mechanisms of action. In contrast to population methods, single-cell analysis reveals that each class of inhibitor interferes with the viral infection cycle in a manner that can be distinguished by principal component analysis. Single-cell analysis of antiviral candidates reveals not only efficacy but also properties of the members of the viral population most sensitive to the drug, the stage of the lifecycle most affected by the drug, and perhaps even if the drug targets an interaction of the virus with its host.

microbiology↗

bioRxiv2018

Continuous in vivo metabolism by NMR

Dense time-series metabolomics data are essential for unraveling the underlying dynamic properties of metabolism. Here we extend high-resolution-magic angle spinning (HR-MAS) to enable continuous in vivo monitoring of metabolism by NMR (CIVM-NMR) and provide analysis tools for these data. First, we reproduced a result in human chronic lymphoid leukemia cells by using isotope-edited CIVM-NMR to rapidly and unambiguously demonstrate unidirectional flux in branched-chain amino acid metabolism. We then collected untargeted CIVM-NMR datasets for Neurospora crassa, a classic multicellular model organism, and uncovered dynamics between central carbon metabolism, amino acid metabolism, energy storage molecules, and lipid and cell wall precursors. Virtually no sample preparation was required to yield a dynamic metabolic fingerprint over hours to days at ~4-min temporal resolution with little noise. CIVM-NMR is simple and readily adapted to different types of cells and microorganisms, offering an experimental complement to kinetic models of metabolism for diverse biological systems.

systems biology↗

bioRxiv2017

Unexpected sequences and structures of mtDNA required for efficient transcription from the first heavy-strand promoter

Human mtDNA contains three promoters, suggesting a need for differential expression of the mitochondrial genome. Studies of mitochondrial transcription have used a reductionist approach, perhaps masking differential regulation. Here we evaluate transcription from light-strand (LSP) and heavy-strand (HSP1) promoters using templates that mimic their natural context. These studies reveal sequences upstream, hypervariable in the human population (HVR3), and downstream of the HSP1 transcription start site required for maximal yield. The carboxy-terminal tail of TFAM is essential for activation of HSP1 but not LSP. Images of the template obtained by atomic force microscopy show that TFAM creates loops in a discrete region, the formation of which correlates with activation of HSP1; looping is lost in tail-deleted TFAM. Identification of HVR3 as a transcriptional regulatory element may contribute to between-individual variability in mitochondrial gene expression. The unique requirement of HSP1 for the TFAM tail may enable its regulation by post-translational modifications.

biochemistry↗