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Biology subjects

Alexander, W.

Publications and source records attributed to Alexander, W..

9 recordsLinked to original sources

Combinatorial pathway engineering using multiplex Serine recombinase-Assisted Genome Engineering (mSAGE)

Low efficiency of DNA integration into the chromosome limits multiplexed bacterial genetic engineering. We address this by establishing multiplexed Serine recombinase-Assisted Genome Engineering (mSAGE), enabling simultaneous, site-specific insertion of three DNAs into the chromosome of Pseudomonas putida KT2440. We construct a combinatorial library of isophthalate catabolism and transport genes through a single, pooled transformation of three gene libraries, rapidly identifying the best combination of genes for biodegradation of this plastic comonomer.

microbiology↗

Multilayered regulation by RNA thermometers enables precise control of Cas9 expression

Cas9-based genome editing technologies can rapidly generate mutations to probe a diverse array of mutant genotypes. However, aberrant Cas9 nuclease translation and activity can occur despite the use of inducible promoters to control expression, leading to extensive cell death. This background killing caused by promoter leakiness severely limits the application of Cas9 for generating mutant libraries because of the potential for population skew. We demonstrate the utility of temperature sensitive RNA elements as a layer of post-transcriptional regulation to reduce the impact of promoter leak. We observe significant temperature-dependent increases in cell survival when certain RNA thermometers (RNATs) are placed upstream of the cas9 coding sequence. We show that the most highly repressing RNAT, hsp17rep, significantly reduces population skew with a library of characterized guide RNAs (gRNAs). This information should be applicable to all Cas9-based methods and technologies. NoticeThis manuscript has been authored by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the US Department of Energy (DOE). The US government retains and the publisher, by accepting the article for publication, acknowledges that the US government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for US government purposes. DOE will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (https://www.energy.gov/doe-public-access-plan). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/651296v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1cb97a4org.highwire.dtl.DTLVardef@150bf11org.highwire.dtl.DTLVardef@2ffefforg.highwire.dtl.DTLVardef@10e24c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

Discovering methylated DNA motifs in bacterial nanopore sequencing data with MIJAMP

Bacterial DNA methylation is involved in diverse cellular functions ranging from modulation of gene expression, DNA repair, and restriction-modification systems for defense against viruses and other foreign DNA. Methylome analysis determines sites within a bacterial chromosome that are methylated, revealing motifs that may be targeted by native restriction enzymes. Identification of these motifs is therefore critical to making an organism genetically tractable, where mimicking the methylome patterns in Escherichia coli allows plasmid DNA to be protected from restriction in the target organism and can therefore drastically enhance transformation efficiency. Oxford Nanopore Technologies (ONT) sequencing can detect methylated bases during sequencing, but software is needed to identify the corresponding methylated motifs within the data. Here, we develop MIJAMP (MIJAMP Is Just A MethylBED Parser), a software package that was developed to discover methylated motifs from the output of ONTs Modkit or other data in the methylBED format. MIJAMP employs a human-driven refinement strategy that empirically validates all motifs against genome-wide methylation data, thus eliminating false, under-, or overexplained motifs. MIJAMP can also report methylation data on a specific, user-defined motif. Using MIJAMP, we determined the methylated motifs both in a control strain (wild type E. coli) and in Picosynecococcus sp. strain PCC7002, laying the foundation for improved transformation in this organism. MIJAMP is available at https://code.ornl.gov/alexander-public/mijamp/.

molecular biology↗

Environmentally-mediated selection parallels population divergence across a chimpanzee subspecies contact zone

Species evolve from populations with ancestor-descendant relationships in a bifurcating process shaped by geography, gene flow, genetic drift, and natural selection leading to local adaptation to prevailing environmental and ecological conditions. Building on this foundational understanding, we explored local adaptation in chimpanzees (Pan troglodytes) at a key geographical intersection in Cameroon where the two main chimpanzee phylogenetic lineages converge. The Nigeria-Cameroon chimpanzee (P. t. ellioti) and central chimpanzee (P. t. troglodytes) last shared a common ancestor about 500 thousand years ago, with occasional gene flow between them. The evolutionary processes driving their prolonged separation are not fully understood, but neutral evolutionary mechanisms alone cannot account for the observed divergence pattern. Cameroon is often referred to as Africa in miniature because the Gulf of Guinea Forest, Congo Basin Forest, and savanna converge there, forming an ecotone. Thus, this contact zone between subspecies in Cameroon provides a unique natural laboratory that enabled us to investigate how environmental variation and natural selection shape divergence in chimpanzees. We developed a genome-wide panel of single-nucleotide polymorphisms (SNPs) in 112 wild chimpanzees sampled in multiple habitats across this contact zone. We augmented SNP discovery by sequencing eight new chimpanzee genomes from Cameroon and analyzing them with previously published chimpanzee genomes. We found that P. t. ellioti and P. t. troglodytes diverged from one another around 478,000 years ago and occasionally exchange migrants. We identified 1,690 unique SNPs across 905 genes associated with 31 environmental variables that describe the habitat. These genes are involved in essential biological processes, including immune response, neurological development, behavior, and dietary adaptations. This study highlights the importance of understanding the geographical context of natural selection, paving the way for future studies to interpret evidence for genetic variation with phenotypic traits and deepening our understanding of how populations diverge in response to environmental pressures. Author SummaryWe investigated how local adaptation contributes to shaping the diversification of chimpanzee subspecies at the geographical convergence point for the two major branches of the chimpanzee phylogenetic tree. We analyzed genome-wide SNP genotypes of 112 chimpanzees sampled from natural communities located in this understudied area. We used tiered methods that identified 905 genes subject to selection, each associated with one or more of 31 environmental predictors describing the habitat. We found strong signals of selection in immune response genes that separate P. t. troglodytes from P. t. ellioti, highlighting the important role of different pathogen histories in their evolution. We also found evidence of selection in genes associated with neurological development, behavior, and diet, that separate both the subspecies and populations of P. t. ellioti that occupy different niches. These findings suggest that ecological and cultural factors may also contribute to shaping the diversification of chimpanzees across the contact zone.

genomics↗

Clonal analysis of fetal hematopoietic stem/progenitor cell subsets reveals how post-transplantation capabilities are distributed

It has been proposed that adult haematopoiesis is sustained by multipotent progenitor (MPP) clones that are specified during development. From an immunophenotypic perspective, it is known that hematopoietic stem cell (HSC) and MPPs are present in the fetal liver yet our understanding of how fetal MPPs functionally compare to those in the adult bone marrow is incomplete. Using acute-term transplantations, we found that at a population-level fetal immunophenotypic MPP classes exhibited similar lineage biases as adult cells, albeit with some difference in lymphoid output. Clonal assessment of fetal MPPs engraftment revealed that lineage biases largely resulted from differences in the pattern of single-or bi-lineage differentiation. Immunophenotypic long-term (LT)-and short-term (ST)-HSCs in the fetal liver were distinguished from MPPs according to propensity for clonal multi-lineage differentiation. We also discovered that a large cohort of long-term repopulating units (LT-RU) were within the immunophenotypic ST-HSC population, a significant portion of these were labelled using Flt3-cre. This finding has two implications: (1) use of the CD150+ LT-HSC immunophenotype alone will systematically underestimate the size and diversity of the fetal LT-RU pool; and, (2), given fetal LT-RUs with a ST-HSC immunophenotype have the functional attributes required to persist into adulthood.

developmental biology↗

Degeneracy measures in biologically plausible random Boolean networks

Biological systems show diversity in terms of the underlying network structure and the governing rules of such networks. Yet, different types of biological networks may develop similar adaptation strategies in face of environmental changes. Degeneracy refers to the ability to compensate for compromised function without the need for a redundant component in the system. Highly degenerate systems show resilience to perturbations and damage because the system can compensate for compromised function due to reconfiguration of the underlying network dynamics. Although formal definitions of degeneracy have been proposed, these definitions have only been tested in relatively simple networks involving weighted connections between network nodes. In this study, we test an information theoretic definition of degeneracy on random Boolean networks, frequently used to model gene regulatory networks. Random Boolean networks are discrete dynamical systems with binary connectivity and thus, these networks are well-suited for tracing information flow and the causal effects. By generating networks with random binary wiring diagrams, we test the effects of systematic lesioning of connections and perturbations of the network nodes on the degeneracy measure. Our analysis shows that degeneracy, on average, is the highest in networks in which ~20% of the connections are lesioned while 50% of the nodes are perturbed. Moreover, our results for the networks with no lesions and the fully-lesioned networks are comparable to the degeneracy measures from weighted networks, thus we show that the degeneracy measure is applicable to different networks. Such a generalized applicability implies that degeneracy can be used to make predictions about the variety of systems ability to recover function. Author SummaryDegeneracy - the ability of structurally different elements to perform similar functions - is a property of many biological systems. Systems exhibiting a high degree of degeneracy continue to exhibit the same macroscopic behavior following a lesion even though the underlying network dynamics are significantly different. Degeneracy thus suggests how biological systems can thrive despite changes to internal and external demands. Although degeneracy is a feature of network topologies and seems to be implicated in a wide variety of biological processes, research on degeneracy in biological networks is mostly limited to weighted networks (e.g., neural networks). To date, there has been no extensive investigation of information theoretic measures of degeneracy in other types of biological networks. In this paper, we apply existing approaches for quantifying degeneracy to random Boolean networks used for modeling biological gene regulatory networks. Using random Boolean networks with randomly generated rulesets to generate synthetic gene expression data sets, we systematically investigate the effect of network lesions on measures of degeneracy. Our results are comparable to measures of degeneracy using weighted networks, and this suggests that degeneracy measures may be a useful tool for investigating gene regulatory networks.

bioinformatics↗

Independent and Interacting Value Systems for Reward and Information in the Human Prefrontal Cortex

Theories of Prefrontal Cortex (PFC) as optimizing reward value have been widely deployed to explain its activity in a diverse range of contexts, with substantial empirical support in neuroeconomics and decision neuroscience. Theoretical frameworks of brain function, however, suggest the existence of a second, independent value system for optimizing information during decision-making. To date, however, there has been little direct empirical evidence in favor of such frameworks. Here, by using computational modeling, model-based fMRI analysis, and a novel experimental paradigm, we aim at establishing whether independent value systems exist in human PFC. We identify two regions in the human PFC which independently encode distinct value signals. These value signals are then combined in subcortical regions in order to implement choices. Our results provide empirical evidence for PFC as an optimizer of independent value signals during decision-making under realistic scenarios, with potential implications for the interpretation of PFC activity in both healthy and clinical population. One Sentence SummaryReward and information value are independently optimized in the Human PFC and are then combined in subcortical regions in order to implement choices.

neuroscience↗

Conjunction or co-activation? A multi-level MVPA approach to task set representations

While representing and maintaining rules in order to govern behavior is a critical function of the brain, it remains an open question as to how collections of rules - task sets - are represented in cortex. One possibility is that task sets are represented as the co-activation of representations of the simple rules from which a task set is composed. Alternatively, task sets could be encoded in a conjunctive manner as the unique combination of rules that belong to a task set. Using a novel multi-level MVPA approach in combination with fMRI, we attempted to answer both \"where\" and \"how\" task sets are represented in the brain. Subjects performed a delayed match-to-sample task using task sets composed of multiple, partially overlapping rules that governed which feature dimensions subjects should attend to, and MVPA was used to identify regions that encoded task set information. We identified voxels most relevant for classifying task sets, and, using these voxels as input to a second MVPA analysis, were able to identify regions in prefrontal cortex with activity consistent with co-active representation, while activity in visual cortex was consistent with conjunctive representation. These results highlight the utility of feature selection methods in neuroimaging analyses.

neuroscience↗

Predicting motivation: computational models of PFC can explain neural coding of motivation and effort-based decision-making in health and disease

Human behavior is strongly driven by the pursuit of rewards. In daily life, however, benefits mostly come at a cost, often requiring that effort be exerted in order to obtain potential benefits. Medial prefrontal cortex (MPFC) and dorsolateral prefrontal cortex (DLPFC) are frequently implicated in the expectation of effortful control, showing increased activity as a function of predicted task difficulty. Such activity partially overlaps with expectation of reward, and has been observed both during decision-making and during task preparation. Recently, novel computational frameworks have been developed to explain activity in these regions during cognitive control, based on the principle of prediction and prediction error (PRO model, Alexander and Brown, 2011, HER Model, Alexander and Brown, 2015). Despite the broad explanatory power of these models, it is not clear whether they can also accommodate effects related to the expectation of effort observed in MPFC and DLPFC. Here, we propose a translation of these computational frameworks to the domain of effort-based behavior. First, we discuss how the PRO model, based on prediction error, can explain effort-related activity in MPFC, by reframing effort-based behavior in a predictive context. We propose that MPFC activity reflects monitoring of motivationally relevant variables (such as effort and reward), by coding expectations, and discrepancies from such expectations. Moreover, we derive behavioral and neural model-based predictions for healthy controls and clinical populations with impairments of motivation. Second, we illustrate the possible translation to effort-based behavior of the HER model, an extended version of PRO model based on hierarchical error prediction, developed to explain MPFC-DLPFC interactions. We derive behavioral predictions which describe how effort and reward information is coded in PFC, and how changing the configuration of such environmental information might affect decision-making and task-performance involving motivation.

neuroscience↗