Controlled levels of protein modification through a chromatography-mediated bioconjugation.

Synthetically modified proteins are increasingly finding applications as well-defined scaffolds for materials. In practice it remains difficult to construct bioconjugates with precise levels of modification because of the limited number of repeated functional groups on proteins. This article describes a method to control the level of protein modification in cases where there exist multiple potential modification sites. A protein is first tagged with a handle using any of a variety of modification chemistries. This handle is used to isolate proteins with a particular number of modifications via affinity chromatography, and then the handle is elaborated with a desired moiety using an oxidative coupling reaction. This method results in a sample of protein with a well-defined number of modifications, and we find it particularly applicable to systems like protein homomultimers in which there is no way to discern between chemically identical subunits. We demonstrate the use of this method in the construction of a protein-templated light-harvesting mimic, a type of system which has historically been difficult to make in a well-defined manner

Decoding Guilty Minds

A central tenet of Anglo-American penal law is that in order for an actor to be found criminally liable, a proscribed act must be accompanied by a guilty mind. While it is easy to understand the importance of this principle in theory, in practice it requires jurors and judges to decide what a person was thinking months or years earlier at the time of the alleged offense, either about the results of his conduct or about some elemental fact (such as whether the briefcase he is carrying contains drugs). Despite the central importance of this task in the administration of criminal justice, there has been very little research investigating how people go about making these decisions, and how these decisions relate to their intuitions about culpability. Understanding the cognitive mechanisms that govern this task is important for the law, not only to explore the possibility of systemic biases and errors in attributions of culpability but also to probe the intuitions that underlie them. In a set of six exploratory studies reported here, we examine the way in which individuals infer others’ legally relevant mental states about elemental facts, using the framework established over fifty years ago by the Model Penal Code (“MPC”). The widely adopted MPC framework delineates and defines the four now-familiar culpable mental states: purpose, knowledge, recklessness, and negligence. Our studies reveal that with little to no training, jury-eligible Americans can apply the MPC framework in a manner that is largely congruent with the basic assumptions of the MPC’s mental state hierarchy. However, our results also indicate that subjects’ intuitions about the level of culpability warranting criminal punishment diverge significantly from prevailing legal practice; subjects tend to regard recklessness as a sufficient basis for punishment under circumstances where the legislatures and courts tend to require knowledge

Validation of a mouse xenograft model system for gene expression analysis of human acute lymphoblastic leukaemia

<p>Abstract</p> <p>Background</p> <p>Pre-clinical models that effectively recapitulate human disease are critical for expanding our knowledge of cancer biology and drug resistance mechanisms. For haematological malignancies, the non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse is one of the most successful models to study paediatric acute lymphoblastic leukaemia (ALL). However, for this model to be effective for studying engraftment and therapy responses at the whole genome level, careful molecular characterisation is essential.</p> <p>Results</p> <p>Here, we sought to validate species-specific gene expression profiling in the high engraftment continuous ALL NOD/SCID xenograft. Using the human Affymetrix whole transcript platform we analysed transcriptional profiles from engrafted tissues without prior cell separation of mouse cells and found it to return highly reproducible profiles in xenografts from individual mice. The model was further tested with experimental mixtures of human and mouse cells, demonstrating that the presence of mouse cells does not significantly skew expression profiles when xenografts contain 90% or more human cells. In addition, we present a novel <it>in silico </it>and experimental masking approach to identify probes and transcript clusters susceptible to cross-species hybridisation.</p> <p>Conclusions</p> <p>We demonstrate species-specific transcriptional profiles can be obtained from xenografts when high levels of engraftment are achieved or with the application of transcript cluster masks. Importantly, this masking approach can be applied and adapted to other xenograft models where human tissue infiltration is lower. This model provides a powerful platform for identifying genes and pathways associated with ALL disease progression and response to therapy <it>in vivo</it>.</p

Parsing the Behavioral and Brain Mechanisms of Third-Party Punishment

The evolved capacity for third-party punishment is considered crucial to the emergence and maintenance of elaborate human social organization and is central to the modern provision of fairness and justice within society. Although it is well established that the mental state of the offender and the severity of the harm he caused are the two primary predictors of punishment decisions, the precise cognitive and brain mechanisms by which these distinct components are evaluated and integrated into a punishment decision are poorly understood. Using a brain-scanning technique known as functional magnetic resonance imaging (fMRI), we implemented a novel experimental design to functionally dissociate the mechanisms underlying evaluation, integration, and decision. This work revealed that multiple parts of the brain – some analytic, some subconscious or emotional – work in a systematic pattern to decide blameworthiness, assess harms, integrate those two decisions, and then ultimately select how a person should be punished. Specifically, harm and mental state evaluations are conducted in two different brain networks and then combined in the medial prefrontal and posterior cingulate areas of the brain, while the amygdala acts as a pivotal hub of the interaction between harm and mental state. This integrated information is then used by the right dorsolateral prefrontal cortex when the brain is making a decision on punishment amount. These findings provide a blueprint of the brain mechanisms by which neutral third parties make punishment decisions

Parsing the Behavioral and Brain Mechanisms of Third-Party Punishment.

UnlabelledThe evolved capacity for third-party punishment is considered crucial to the emergence and maintenance of elaborate human social organization and is central to the modern provision of fairness and justice within society. Although it is well established that the mental state of the offender and the severity of the harm he caused are the two primary predictors of punishment decisions, the precise cognitive and brain mechanisms by which these distinct components are evaluated and integrated into a punishment decision are poorly understood. Using fMRI, here we implement a novel experimental design to functionally dissociate the mechanisms underlying evaluation, integration, and decision that were conflated in previous studies of third-party punishment. Behaviorally, the punishment decision is primarily defined by a superadditive interaction between harm and mental state, with subjects weighing the interaction factor more than the single factors of harm and mental state. On a neural level, evaluation of harms engaged brain areas associated with affective and somatosensory processing, whereas mental state evaluation primarily recruited circuitry involved in mentalization. Harm and mental state evaluations are integrated in medial prefrontal and posterior cingulate structures, with the amygdala acting as a pivotal hub of the interaction between harm and mental state. This integrated information is used by the right dorsolateral prefrontal cortex at the time of the decision to assign an appropriate punishment through a distributed coding system. Together, these findings provide a blueprint of the brain mechanisms by which neutral third parties render punishment decisions.Significance statementPunishment undergirds large-scale cooperation and helps dispense criminal justice. Yet it is currently unknown precisely how people assess the mental states of offenders, evaluate the harms they caused, and integrate those two components into a single punishment decision. Using a new design, we isolated these three processes, identifying the distinct brain systems and activities that enable each. Additional findings suggest that the amygdala plays a crucial role in mediating the interaction of mental state and harm information, whereas the dorsolateral prefrontal cortex plays a crucial, final-stage role, both in integrating mental state and harm information and in selecting a suitable punishment amount. These findings deepen our understanding of how punishment decisions are made, which may someday help to improve them

Overexpression of Map3k7 activates sinoatrial node-like differentiation in mouse ES-derived cardiomyocytes

In vivo, cardiomyocytes comprise a heterogeneous population of contractile cells defined by unique electrophysiologies, molecular markers and morphologies. The mechanisms directing myocardial cells to specific sub-lineages remain poorly understood. Here we report that overexpression of TGFβ-Activated Kinase (TAK1/Map3k7) in mouse embryonic stem (ES) cells faithfully directs myocardial differentiation of embryoid body (EB)-derived cardiac cells toward the sinoatrial node (SAN) lineage. Most cardiac cells in Map3k7-overexpressing EBs adopt markers, cellular morphologies, and electrophysiological behaviors characteristic of the SAN. These data, in addition to the fact that Map3k7 is upregulated in the sinus venous—the source of cells for the SAN—suggest that Map3k7 may be an endogenous regulator of the SAN fate