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Cryptococcus neoformans is an opportunistic fungal pathogen with a polysaccharide capsule that becomes greatly enlarged in the mammalian host and during in vitro growth under host-like conditions. To understand how individual environmental signals affect capsule size and gene expression, we grew cells in all combinations of 5 signals implicated in capsule size and systematically measured cell and capsule sizes. We also sampled these cultures over time and performed RNA-seq in quadruplicate, yielding 881 RNA-seq samples. Analysis of the resulting data sets showed that capsule induction in tissue culture medium, typically used to represent host-like conditions, requires the presence of either CO2 or exogenous cyclic AMP. Surprisingly, adding either of these pushes overall gene expression in the opposite direction from tissue culture media alone, even though both are required for capsule development. Another unexpected finding was that rich medium blocks capsule growth completely. Statistical analysis further revealed many genes whose expression is associated with capsule thickness; deletion of one of these significantly reduced capsule size. Beyond illuminating capsule induction, our massive, uniformly collected data set will be a significant resource for the research community., Competing Interests: Conflicts of interest: The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Cryptococcus neoformans is an opportunistic fungal pathogen with a polysaccharide capsule that becomes greatly enlarged in the mammalian host and during in vitro growth under host-like conditions. To understand how individual environmental signals affect capsule size and gene expression, we grew cells in all combinations of five signals implicated in capsule size and systematically measured cell and capsule sizes. We also sampled these cultures over time and performed RNA-Seq in quadruplicate, yielding 881 RNA-Seq samples. Analysis of the resulting data sets showed that capsule induction in tissue culture medium, typically used to represent host-like conditions, requires the presence of either CO 2 or exogenous cyclic AMP (cAMP). Surprisingly, adding either of these pushes overall gene expression in the opposite direction from tissue culture media alone, even though both are required for capsule development. Another unexpected finding was that rich medium blocks capsule growth completely. Statistical analysis further revealed many genes whose expression is associated with capsule thickness; deletion of one of these significantly reduced capsule size. Beyond illuminating capsule induction, our massive, uniformly collected dataset will be a significant resource for the research community., Competing Interests: COMPETING INTERESTS The authors declare that they have no competing interests.

Genetic and pharmacological perturbation of the cytoskeleton enhances the regenerative potential of neurons. This response requires Dual-leucine Zipper Kinase (DLK), a neuronal stress sensor that is a central regulator of axon regeneration and degeneration. The damage and repair aspects of this response are reminiscent of other cellular homeostatic systems, suggesting that a cytoskeletal homeostatic response exists. In this study, we propose a framework for understanding DLK mediated neuronal cytoskeletal homeostasis. We demonstrate that low dose nocodazole treatment activates DLK signaling. Activation of DLK signaling results in a DLK-dependent transcriptional signature, which we identify through RNA-seq. This signature includes genes likely to attenuate DLK signaling while simultaneously inducing actin regulating genes. We identify alterations to the cytoskeleton including actin-based morphological changes to the axon. These results are consistent with the model that cytoskeletal disruption in the neuron induces a DLK-dependent homeostatic mechanism, which we term the Cytoskeletal Stress Response (CSR) pathway., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 DeVault et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Genetic and pharmacological perturbation of the cytoskeleton enhances the regenerative potential of neurons. This response requires Dual-leucine Zipper Kinase (DLK), a neuronal stress sensor that is a central regulator of axon regeneration and degeneration. The damage and repair aspects of this response are reminiscent of other cellular homeostatic systems, suggesting that a cytoskeletal homeostatic response exists. In this study, we propose a framework for understanding DLK mediated neuronal cytoskeletal homeostasis. We demonstrate that a) low dose nocodazole treatment activates DLK signaling and b) DLK signaling mitigates the microtubule damage caused by the cytoskeletal perturbation. We also perform RNA-seq to discover a DLK-dependent transcriptional signature. This signature includes genes likely to attenuate DLK signaling while simultaneously inducing actin regulating genes and promoting actin-based morphological changes to the axon. These results are consistent with the model that cytoskeletal disruption in the neuron induces a DLK-dependent homeostatic mechanism, which we term the Cytoskeletal Stress Response (CSR) pathway.

Calling Cards is a platform technology to record a cumulative history of transient protein-DNA interactions in the genome of genetically targeted cell types. The record of these interactions is recovered by next-generation sequencing. Compared with other genomic assays, readouts of which provide a snapshot at the time of harvest, Calling Cards enables correlation of historical molecular states to eventual outcomes or phenotypes. To achieve this, Calling Cards uses the piggyBac transposase to insert self-reporting transposon "Calling Cards" into the genome, leaving permanent marks at interaction sites. Calling Cards can be deployed in a variety of in vitro and in vivo biological systems to study gene regulatory networks involved in development, aging, and disease. Out of the box, it assesses enhancer usage but can be adapted to profile-specific transcription factor (TF) binding with custom TF-piggyBac fusion proteins. The Calling Cards workflow has five main stages: delivery of Calling Cards reagents, sample preparation, library preparation, sequencing, and data analysis. Here, we first present a comprehensive guide for experimental design, reagent selection, and optional customization of the platform to study additional TFs. Then, we provide an updated protocol for the five steps, using reagents that improve throughput and decrease costs, including an overview of a newly deployed computational pipeline. This protocol is designed for users with basic molecular biology experience to process samples into sequencing libraries in 2 days. Familiarity with bioinformatic analysis and command line tools is required to set up the pipeline in a high-performance computing environment and to conduct downstream analyses. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparation and delivery of Calling Cards reagents Support Protocol 1: Next-generation sequencing quantification of barcode distribution within self-reporting transposon plasmid pool and adeno-associated virus genome Basic Protocol 2: Sample collection and RNA purification Support Protocol 2: Library density quantitative PCR Basic Protocol 3: Sequencing library preparation Basic Protocol 4: Library pooling and sequencing Basic Protocol 5: Data analysis., (© 2023 Wiley Periodicals LLC.)

Calling Cards is a platform technology to record a cumulative history of transient protein-DNA interactions in the genome of genetically targeted cell types. The record of these interactions is recovered by next generation sequencing. Compared to other genomic assays, whose readout provides a snapshot at the time of harvest, Calling Cards enables correlation of historical molecular states to eventual outcomes or phenotypes. To achieve this, Calling Cards uses the piggyBac transposase to insert self-reporting transposon (SRT) "Calling Cards" into the genome, leaving permanent marks at interaction sites. Calling Cards can be deployed in a variety of in vitro and in vivo biological systems to study gene regulatory networks involved in development, aging, and disease. Out of the box, it assesses enhancer usage but can be adapted to profile specific transcription factor binding with custom transcription factor (TF)-piggyBac fusion proteins. The Calling Cards workflow has five main stages: delivery of Calling Card reagents, sample preparation, library preparation, sequencing, and data analysis. Here, we first present a comprehensive guide for experimental design, reagent selection, and optional customization of the platform to study additional TFs. Then, we provide an updated protocol for the five steps, using reagents that improve throughput and decrease costs, including an overview of a newly deployed computational pipeline. This protocol is designed for users with basic molecular biology experience to process samples into sequencing libraries in 1-2 days. Familiarity with bioinformatic analysis and command line tools is required to set up the pipeline in a high-performance computing environment and to conduct downstream analyses. Basic Protocol 1: Preparation and delivery of Calling Cards reagentsBasic Protocol 2: Sample preparationBasic Protocol 3: Sequencing library preparationBasic Protocol 4: Library pooling and sequencingBasic Protocol 5: Data analysis.

Members of the bacterial T 6SS a midase e ffector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions., Competing Interests: AR, AS, SF, CH, HS, RK, SM, ST, CM, JB, ZZ, LS, WH, WV, AR, JS, SA, PB, BH No competing interests declared, SC currently the President and CEO of Arcadia Science, a for-profit research company focused on non-model organisms. While work presented in this manuscript is not related and will not be continued at Arcadia, I thought it would be prudent to declare this position which may be perceived by some as a competing interest, (© 2022, Radkov et al.)

A vector's susceptibility and ability to transmit a pathogen-termed vector competency-determines disease outcomes, yet the ecological factors influencing tick vector competency remain largely unknown. Ixodes pacificus, the tick vector of Borrelia burgdorferi (Bb) in the western U.S., feeds on rodents, birds, and lizards. Rodents and birds are reservoirs for Bb and infect juvenile ticks, while lizards are refractory to Bb and cannot infect feeding ticks. Additionally, the lizard bloodmeal contains borreliacidal properties, clearing previously infected feeding ticks of their Bb infection. Despite I. pacificus feeding on a range of hosts, it is undetermined how the host identity of the larval bloodmeal affects future nymphal vector competency. We experimentally evaluate the influence of larval host bloodmeal on Bb acquisition by nymphal I. pacificus. Larval I. pacificus were fed on either lizards or mice and after molting, nymphs were fed on Bb-infected mice. We found that lizard-fed larvae were significantly more likely to become infected with Bb during their next bloodmeal than mouse-fed larvae. We also conducted the first RNA-seq analysis on whole-bodied I. pacificus and found significant upregulation of tick antioxidants and antimicrobial peptides in the lizard-fed group. Our results indicate that the lizard bloodmeal significantly alters vector competency and gene regulation in ticks, highlighting the importance of host bloodmeal identity in vector-borne disease transmission and upends prior notions about the role of lizards in Lyme disease community ecology., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)