The Reach of the Law: Sin, Crime and Poor Taste

The past decade has been a period of intensive reevaluation of the law. The criminal law, in particular, has been subjected to an especially intensive criticism. These attacks fall largely into two categories: criticisms of the legitimacy of our penal codes, and criticisms of their efficiency. Starting with the Civil Rights Movement of the Kennedy era with its heavy emphasis on civil disobedience as a tool of protest, the legitimacy of many of our laws was called into question. When Rosa Parks sat in the front of the bus in Montgomery, Alabama, she was not simply breaking the law; she was pointing to a law that had no right to be. The mass protests against the Vietnam War similarly were declarations of the demonstrators\u27 belief that the War and the laws which supported it were wrong and illegitimate. Recent minority group protests such as the Gay Liberation Movement and the campaign for equal rights for women again are protests against laws that some groups feel are unfair and unjust

Defining the ecological and physiological traits of phytoplankton across marine ecosystems

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution February 2016Marine phytoplankton are central players in the global carbon cycle, responsible for nearly half of global primary production. The identification of the factors controlling phytoplankton ecology, physiology, and, ultimately, bloom dynamics has been a central problem in the field of biological oceanography for the past century. Molecular approaches enable the direct examination of species-specific metabolic profiles in mixed, natural communities, a task which was previously intractable. In this thesis, I developed and applied novel analytical tools and bioinformatic pipelines to characterize the physiological response of phytoplankton to their environment at various levels of taxonomic grouping. An in silico Bayesian statistical approach was designed to identify stable reference genes from high-throughput sequence data for use in RT-qPCR assays or metatranscriptome studies. Using a metatranscriptomic approach, the role of resource partitioning in the coexistence of two closely related diatom species in an estuarine system was examined. This study demonstrated that co-occurring diatoms in a dynamic coastal system have apparent differences in their capacity to use nitrogen and phosphorus, and that these differences may facilitate the diversity of the phytoplankton. The second field study focused on the diatom, haptophyte, and dinoflagellate functional groups, using simulated blooms to characterize the traits that govern the magnitude and timing of phytoplankton blooms in the oligotrophic ocean. The results indicated that blooms form when phytoplankton are released from limitation by resources and that the mechanistic basis for the success of one functional group over another may be driven by how efficiently the transcriptome is modulated following a nutrient pulse. The final study looked at the sub-species level, examining the balance of phenotypic plasticity and strain diversity in the success of the coccolithophore Emiliania huxleyi. Results indicated strong control of nitrogen on the species complex and showed that nutrient resupply shifted the strain composition as well as transcript abundance of key biogeochemical genes involved in nutrient acquisition and the life stage of the population. Together, these studies demonstrate the breadth of information that can be garnered through the integration of molecular approaches with traditional biological oceanographic surveys, with each illuminating fundamental questions around phytoplankton ecology and bloom formation.have been supported by the MIT Presidential Fellowship, the National Defense Science and Engineering Graduate (NDSEG) Fellowship, the Ocean Life Institute Fellowship, and the WHOI Academic Programs Office. This research was supported by funds from the National Science Foundation (NSF) Environmental Genomics and NSF Biological Oceanography Programs through Grant OCE-0723667 (to S.T.D., B.D.J., and T.A.R.) and Grant OCE-0962208 (to B.D.J.), the Joint Genome Institute/Department of Energy Community Sequencing Program through Grant CSP795793 (to B.D.J., S.T.D., and T.A.R.), the Center for Microbial Oceanography: Research and Education (C-MORE, National Science Foundation Grant EF04-24599), the Gordon and Betty Moore Foundation through Grant 2637 to the National Center for Genome Resources for the MMETSP and Grant 3794 (to D.M.K.). This work was also supported through grants from the Simons Foundation (to D.M.K. and S.T.D.

Keeping it light: (re)analyzing community-wide datasets without major infrastructure

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Alexander, H., Johnson, L. K., & Brown, C. T.. Keeping it light: (re)analyzing community-wide datasets without major infrastructure. Gigascience, 8(2),(2019): giy159, doi:10.1093/gigascience/giy159.DNA sequencing technology has revolutionized the field of biology, shifting biology from a data-limited to data-rich state. Central to the interpretation of sequencing data are the computational tools and approaches that convert raw data into biologically meaningful information. Both the tools and the generation of data are actively evolving, yet the practice of re-analysis of previously generated data with new tools is not commonplace. Re-analysis of existing data provides an affordable means of generating new information and will likely become more routine within biology, yet necessitates a new set of considerations for best practices and resource development. Here, we discuss several practices that we believe to be broadly applicable when re-analyzing data, especially when done by small research groups.Funding was provided by the Gordon and Betty Moore Foundation (award GBMF4551 to C.T.B.)

The osmolyte ties that bind: genomic insights into synthesis and breakdown of organic osmolytes in marine microbes

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in McParland, E. L., Alexander, H., & Johnson, W. M. The osmolyte ties that bind: genomic insights into synthesis and breakdown of organic osmolytes in marine microbes. Frontiers in Marine Science, 8, (2021): 689306, https://doi.org/10.3389/fmars.2021.689306.The production and consumption of organic matter by marine organisms plays a central role in the marine carbon cycle. Labile organic compounds (metabolites) are the major currency of energetic demands and organismal interaction, but these compounds remain elusive because of their rapid turnover and concomitant minuscule concentrations in the dissolved organic matter pool. Organic osmolytes are a group of small metabolites synthesized at high intracellular concentrations (mM) to regulate cellular osmolarity and have the potential to be released as abundant dissolved substrates. Osmolytes may represent an essential currency of exchange among heterotrophic prokaryotes and primary and secondary producers in marine food webs. For example, the well-known metabolite dimethylsulfoniopropionate (DMSP) is used as an osmolyte by some phytoplankton and can be subsequently metabolized by 60% of the marine bacterial community, supplying up to 13% of the bacterial carbon demand and 100% of the bacterial sulfur demand. While marine osmolytes have been studied for decades, our understanding of their cycling and significance within microbial communities is still far from comprehensive. Here, we surveyed the genes responsible for synthesis, breakdown, and transport of 14 key osmolytes. We systematically searched for these genes across marine bacterial genomes (n = 897) and protistan transcriptomes (n = 652) using homologous protein profiles to investigate the potential for osmolyte metabolisms. Using the pattern of gene presence and absence, we infer the metabolic potential of surveyed microbes to interact with each osmolyte. Specifically, we identify: (1) complete pathways for osmolyte synthesis in both prokaryotic and eukaryotic marine microbes, (2) microbes capable of transporting osmolytes but lacking complete synthesis and/or breakdown pathways, and (3) osmolytes whose synthesis and/or breakdown appears to be specialized and is limited to a subset of organisms. The analysis clearly demonstrates that the marine microbial loop has the genetic potential to actively recycle osmolytes and that this abundant group of small metabolites may function as a significant source of nutrients through exchange among diverse microbial groups that significantly contribute to the cycling of labile carbon.EM was supported by the Postdoctoral Scholar Program at Woods Hole Oceanographic Institution. WJ was supported by a Research Initiative Award from the College of Arts and Sciences at the University of North Carolina Wilmington. HA was supported by a Independent Research and Development Award from the Woods Hole Oceanographic Institution

Re-assembly, quality evaluation, and annotation of 678 microbial eukaryotic reference transcriptomes

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Johnson, L. K., Alexander, H., & Brown, C. T. Re-assembly, quality evaluation, and annotation of 678 microbial eukaryotic reference transcriptomes. Gigascience, 8(4), (2019): giy158, doi: 10.1093/gigascience/giy158.Background: De novo transcriptome assemblies are required prior to analyzing RNA sequencing data from a species without an existing reference genome or transcriptome. Despite the prevalence of transcriptomic studies, the effects of using different workflows, or “pipelines,” on the resulting assemblies are poorly understood. Here, a pipeline was programmatically automated and used to assemble and annotate raw transcriptomic short-read data collected as part of the Marine Microbial Eukaryotic Transcriptome Sequencing Project. The resulting transcriptome assemblies were evaluated and compared against assemblies that were previously generated with a different pipeline developed by the National Center for Genome Research. Results: New transcriptome assemblies contained the majority of previous contigs as well as new content. On average, 7.8% of the annotated contigs in the new assemblies were novel gene names not found in the previous assemblies. Taxonomic trends were observed in the assembly metrics. Assemblies from the Dinoflagellata showed a higher number of contigs and unique k-mers than transcriptomes from other phyla, while assemblies from Ciliophora had a lower percentage of open reading frames compared to other phyla. Conclusions: Given current bioinformatics approaches, there is no single “best” reference transcriptome for a particular set of raw data. As the optimum transcriptome is a moving target, improving (or not) with new tools and approaches, automated and programmable pipelines are invaluable for managing the computationally intensive tasks required for re-processing large sets of samples with revised pipelines and ensuring a common evaluation workflow is applied to all samples. Thus, re-assembling existing data with new tools using automated and programmable pipelines may yield more accurate identification of taxon-specific trends across samples in addition to novel and useful products for the community.Funding was provided by the Gordon and Betty Moore Foundation under award number GBMF4551 to C.T.B. Jetstream cloud platform was used with XSEDE allocation TG-BIO160028 [66, 67]

Metatranscriptome analyses indicate resource partitioning between diatoms in the field

Diverse communities of marine phytoplankton carry out half of global primary production. The vast diversity of the phytoplankton has long perplexed ecologists because these organisms coexist in an isotropic environment while competing for the same basic resources (e.g., inorganic nutrients). Differential niche partitioning of resources is one hypothesis to explain this “paradox of the plankton,” but it is difficult to quantify and track variation in phytoplankton metabolism in situ. Here, we use quantitative metatranscriptome analyses to examine pathways of nitrogen (N) and phosphorus (P) metabolism in diatoms that cooccur regularly in an estuary on the east coast of the United States (Narragansett Bay). Expression of known N and P metabolic pathways varied between diatoms, indicating apparent differences in resource utilization capacity that may prevent direct competition. Nutrient amendment incubations skewed N/P ratios, elucidating nutrient-responsive patterns of expression and facilitating a quantitative comparison between diatoms. The resource-responsive (RR) gene sets deviated in composition from the metabolic profile of the organism, being enriched in genes associated with N and P metabolism. Expression of the RR gene set varied over time and differed significantly between diatoms, resulting in opposite transcriptional responses to the same environment. Apparent differences in metabolic capacity and the expression of that capacity in the environment suggest that diatom-specific resource partitioning was occurring in Narragansett Bay. This high-resolution approach highlights the molecular underpinnings of diatom resource utilization and how cooccurring diatoms adjust their cellular physiology to partition their niche space.American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowshi

Incidental learning of trust from eye-gaze: Effects of race and facial trustworthiness

Humans rapidly make inferences about individuals’ trustworthiness on the basis of their facial features and perceived group membership. We examine whether incidental learning about trust from shifts in gaze direction is influenced by these facial features. To do so, we examined two types of face category: the race of the face and the initial trustworthiness of the face based on physical appearance. We find that cueing of attention by eye-gaze is unaffected by race or initial levels of trust, whereas incidental learning of trust from gaze behaviour is selectively influenced. That is, learning of trust is reduced for other race faces, as predicted by reduced abilities to identify members of other races (Experiment 1). In contrast, converging findings from an independently gathered set of data showed that the initial trustworthiness of faces did not influence learning of trust (Experiment 2). These results show that learning about the behaviour of other race faces is poorer than for own-race faces, but that this cannot be explained by differences in the perceived trustworthiness of different groups

A phosphate starvation response gene (psr1-like) is present and expressed in Micromonas pusilla and other marine algae

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Fiore, C. L., Alexander, H., Soule, M. C. K., & Kujawinski, E. B. A phosphate starvation response gene (psr1-like) is present and expressed in Micromonas pusilla and other marine algae. Aquatic Microbial Ecology, 86, (2021): 29–46, https://doi.org/10.3354/ame01955.Phosphorus (P) limits primary production in regions of the surface ocean, and many plankton species exhibit specific physiological responses to P deficiency. The metabolic response of Micromonas pusilla, an ecologically relevant marine photoautotroph, to P deficiency was investigated using metabolomics and comparative genomics. The concentrations of some intracellular metabolites were elevated in the P-deficient cells (e.g. xanthine, inosine), and genes involved in the associated metabolic pathways shared a predicted conserved amino acid motif in the non-coding regions of each gene. The presence of the conserved motif suggests that these genes may be co-regulated, and the motif may constitute a regulatory element for binding a transcription factor, specifically that of Psr1 (phosphate starvation response). A putative phosphate starvation response gene ( psr1-like) was identified in M. pusilla with homology to well characterized psr1/ phr1 genes in algae and plants, respectively. This gene appears to be present and expressed in other marine algal taxa (e.g. Emiliania huxleyi) in field sites that are chronically P limited. Results from the present study have implications for understanding phytoplankton taxon-specific roles in mediating P cycling in the ocean.This research was funded by the Gordon and Betty Moore Foundation through Grant GBMF3304 to E.B.K., and it was partially supported by a grant from the Simons Foundation (Award ID 509034 to E.B.K.)

Identifying reference genes with stable expression from high throughput sequence data

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 3 (2012): 385, doi:10.3389/fmicb.2012.00385.Genes that are constitutively expressed across multiple environmental stimuli are crucial to quantifying differentially expressed genes, particularly when employing quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assays. However, the identification of these potential reference genes in non-model organisms is challenging and is often guided by expression patterns in distantly related organisms. Here, transcriptome datasets from the diatom Thalassiosira pseudonana grown under replete, phosphorus-limited, iron-limited, and phosphorus and iron co-limited nutrient regimes were analyzed through literature-based searches for homologous reference genes, k-means clustering, and analysis of sequence counts (ASC) to identify putative reference genes. A total of 9759 genes were identified and screened for stable expression. Literature-based searches surveyed 18 generally accepted reference genes, revealing 101 homologs in T. pseudonana with variable expression and a wide range of mean tags per million. k-means analysis parsed the whole transcriptome into 15 clusters. The two most stable clusters contained 709 genes, but still had distinct patterns in expression. ASC analyses identified 179 genes that were stably expressed (posterior probability < 0.1 for 1.25 fold change). Genes known to have a stable expression pattern across the test treatments, like actin, were identified in this pool of 179 candidate genes. ASC can be employed on data without biological replicates and was more robust than the k-means approach in isolating genes with stable expression. The intersection of the genes identified through ASC with commonly used reference genes from the literature suggests that actin and ubiquitin ligase may be useful reference genes for T. pseudonana and potentially other diatoms. With the wealth of transcriptome sequence data becoming available, ASC can be easily applied to transcriptome datasets from other phytoplankton to identify reference genes.This research was funded by the National Science Foundation grant #OCE-0723667 (to Sonya T. Dyhrman, Mak A. Saito, Bethany D. Jenkins, and Tatiana A. Rynearson). Harriet Alexander is funded under a National Defense Science and Engineering Graduate (NDSEG) Fellowship

Virus-host relationships of marine single-celled eukaryotes resolved from metatranscriptomics

Establishing virus–host relationships has historically relied on culture-dependent approaches. Here we report on the use of marine metatranscriptomics to probe virus–host relationships. Statistical co-occurrence analyses of dsDNA, ssRNA and dsRNA viral markers of polyadenylation-selected RNA sequences from microbial communities dominated by Aureococcus anophagefferens (Quantuck Bay, NY), and diatoms (Narragansett Bay, RI) show active infections by diverse giant viruses (NCLDVs) associated with algal and nonalgal hosts. Ongoing infections of A. anophagefferens by a known Mimiviridae (AaV) occur during bloom peak and decline. Bloom decline is also accompanied by increased activity of viruses other than AaV, including (+) ssRNA viruses. In Narragansett Bay, increased temporal resolution reveals active NCLDVs with both ‘boom-and-bust’ and ‘steady-state infection’-like ecologies that include known as well as novel virus–host interactions. Our approach offers a method for screening active viral infections and develops links between viruses and their potential hosts in situ. Our observations further demonstrate that previously unknown virus–host relationships in marine systems are abundant