Origin of metazoan cadherin diversity and the antiquity of the classical cadherin/ß-catenin complex.

The evolution of cadherins, which are essential for metazoan multicellularity and restricted to metazoans and their closest relatives, has special relevance for understanding metazoan origins. To reconstruct the ancestry and evolution of cadherin gene families, we analyzed the genomes of the choanoflagellate Salpingoeca rosetta, the unicellular outgroup of choanoflagellates and metazoans Capsaspora owczarzaki, and a draft genome assembly from the homoscleromorph sponge Oscarella carmela. Our finding of a cadherin gene in C. owczarzaki reveals that cadherins predate the divergence of the C. owczarzaki, choanoflagellate, and metazoan lineages. Data from these analyses also suggest that the last common ancestor of metazoans and choanoflagellates contained representatives of at least three cadherin families, lefftyrin, coherin, and hedgling. Additionally, we find that an O. carmela classical cadherin has predicted structural features that, in bilaterian classical cadherins, facilitate binding to the cytoplasmic protein ß-catenin and, thereby, promote cadherin-mediated cell adhesion. In contrast with premetazoan cadherin families (i.e., those conserved between choanoflagellates and metazoans), the later appearance of classical cadherins coincides with metazoan origins.

Insights into the origin of metazoan filopodia and microvilli.

Filopodia are fine actin-based cellular projections used for both environmental sensing and cell motility, and they are essential organelles for metazoan cells. In this study, we reconstruct the origin of metazoan filopodia and microvilli. We first report on the evolutionary assembly of the filopodial molecular toolkit and show that homologs of many metazoan filopodial components, including fascin and myosin X, were already present in the unicellular or colonial progenitors of metazoans. Furthermore, we find that the actin crosslinking protein fascin localizes to filopodia-like structures and microvilli in the choanoflagellate Salpingoeca rosetta. In addition, homologs of filopodial genes in the holozoan Capsaspora owczarzaki are upregulated in filopodia-bearing cells relative to those that lack them. Therefore, our findings suggest that proteins essential for metazoan filopodia and microvilli are functionally conserved in unicellular and colonial holozoans and that the last common ancestor of metazoans bore a complex and specific filopodial machinery.

The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans.

Choanoflagellates are the closest known relatives of metazoans. To discover potential molecular mechanisms underlying the evolution of metazoan multicellularity, we sequenced and analysed the genome of the unicellular choanoflagellate Monosiga brevicollis. The genome contains approximately 9,200 intron-rich genes, including a number that encode cell adhesion and signalling protein domains that are otherwise restricted to metazoans. Here we show that the physical linkages among protein domains often differ between M. brevicollis and metazoans, suggesting that abundant domain shuffling followed the separation of the choanoflagellate and metazoan lineages. The completion of the M. brevicollis genome allows us to reconstruct with increasing resolution the genomic changes that accompanied the origin of metazoans.

Trait impulsivity influences behavioural and physiological responses to threat in a virtual environment.

Trait impulsivity represents a tendency to take action without forethought or consideration of consequences. This trait is multifaceted and can be decomposed into attentional, motor and non-planning subtypes of impulsivity. The purpose of the current study was to investigate how subtypes of trait impulsivity responded to different degrees of threat within room-scale virtual reality (VR) with respect to behaviour and level of physiological activation. Thirty-four participants were required to negotiate a virtual environment (VE) where they walked at height with the continuous threat of a virtual 'fall.' Behavioural measures related to the speed of movement, interaction frequency and risk were collected. Participants also wore ambulatory sensors to collect data from electrocardiogram (ECG) and electrodermal activity (EDA). Our results indicated that participants who scored highly on non-planning impulsivity exhibited riskier behaviour and higher skin conductance level (SCL). Participants with higher motor impulsivity interacted with more objects in the VE when threat was high, they also exhibited contradictory indicators of physiological activation. Attentional impulsivity was associated with a greater number of falls across the VE. The results demonstrate that subtypes of trait impulsivity respond to threats via different patterns of behaviour and levels of physiological activation, reinforcing the multifaceted nature of the trait.

Functional connectivity across dorsal and ventral attention networks in response to task difficulty and experimental pain.

The dorsal and ventral attention networks (DAN & VAN) provide a framework for studying attentional modulation of pain. It has been argued that cognitive demand distracts attention from painful stimuli via top-down reinforcement of task goals (DAN), whereas pain exerts an interruptive effect on cognitive performance via bottom-up pathways (VAN). The current study explores this explanatory framework by manipulating pain and task demand in combination with functional near-infrared spectroscopy (fNIRS) and Granger Causal Connectivity Analyses (GCCA). Twenty-one participants played a racing game at low and high difficulty levels with or without experimental pain (administered via a cold pressor test). Six channels of fNIRS were collected from bilateral frontal eye fields and intraparietal sulci (DAN), with right-lateralised channels at the inferior frontal gyrus and temporoparietal junction (VAN). Our first analysis revealed increased G-causality from bottom-up pathways (VAN) during the cold pressor test. However, an equivalent experience of experimental pain during gameplay increased G-causality in top-down (DAN) pathways, with the left intraparietal sulcus serving a hub of connectivity. High game difficulty increased G-causality via top-down pathways and implicated the right inferior frontal gyrus as an interhemispheric hub. Our results are discussed with reference to existing models of both networks and attentional modulation of pain.

Cell differentiation and morphogenesis in the colony-forming choanoflagellate Salpingoeca rosetta.

It has been posited that animal development evolved from pre-existing mechanisms for regulating cell differentiation in the single celled and colonial ancestors of animals. Although the progenitors of animals cannot be studied directly, insights into their cell biology may be gleaned from comparisons between animals and their closest living relatives, the choanoflagellates. We report here on the life history, cell differentiation and intercellular interactions in the colony-forming choanoflagellate Salpingoeca rosetta. In response to diverse environmental cues, S. rosetta differentiates into at least five distinct cell types, including three solitary cell types (slow swimmers, fast swimmers, and thecate cells) and two colonial forms (rosettes and chains). Electron microscopy reveals that cells within colonies are held together by a combination of fine intercellular bridges, a shared extracellular matrix, and filopodia. In addition, we have discovered that the carbohydrate-binding protein wheat germ agglutinin specifically stains colonies and the slow swimmers from which they form, showing that molecular differentiation precedes multicellular development. Together, these results help establish S. rosetta as a model system for studying simple multicellularity in choanoflagellates and provide an experimental framework for investigating the origin of animal multicellularity and development.

The psychophysiological mechanisms of real-world time experience.

Our sense of time is fallible, often resulting in the sensation of time flying by quickly or dragging slowly. It has been suggested that changes in sympathetic (SNS) and parasympathetic nervous system (PNS) activity may influence the perceived passage of time, however this proposition has never been tested during real-world temporal experience. The current study directly tested the relationship between the passage of time and SNS-PNS activity in the real-world. Sixty-seven participants completed a normal day's activities whilst wearing sensors to capture electrocardiography (ECG), electrodermal activity (EDA) and movement. They also provided hourly rating of the subjective speed at which time was passing. Results revealed that greater SNS activity (e.g., increased heart rate, frequency of phasic skin conductance response) was associated with time passing more quickly. PNS activity was not related to time experience. Whilst the findings support previous suggestions that changes in physiological arousal are associated with distortions to the passage of time, the effects are small and other factors are likely to contribute to real-world temporal experience.

Complete genome sequence of Algoriphagus sp. PR1, bacterial prey of a colony-forming choanoflagellate.

Bacteria are the primary food source of choanoflagellates, the closest known relatives of animals. Studying signaling interactions between the Gram-negative Bacteroidetes bacterium Algoriphagus sp. PR1 and its predator, the choanoflagellate Salpingoeca rosetta, provides a promising avenue for testing hypotheses regarding the involvement of bacteria in animal evolution. Here we announce the complete genome sequence of Algoriphagus sp. PR1 and initial findings from its annotation.

Classification of Game Demand and the Presence of Experimental Pain Using Functional Near-Infrared Spectroscopy.

Pain tolerance can be increased by the introduction of an active distraction, such as a computer game. This effect has been found to be moderated by game demand, i.e., increased game demand = higher pain tolerance. A study was performed to classify the level of game demand and the presence of pain using implicit measures from functional Near-InfraRed Spectroscopy (fNIRS) and heart rate features from an electrocardiogram (ECG). Twenty participants played a racing game that was configured to induce low (Easy) or high (Hard) levels of demand. Both Easy and Hard levels of game demand were played with or without the presence of experimental pain using the cold pressor test protocol. Eight channels of fNIRS data were recorded from a montage of frontal and central-parietal sites located on the midline. Features were generated from these data, a subset of which were selected for classification using the RELIEFF method. Classifiers for game demand (Easy vs. Hard) and pain (pain vs. no-pain) were developed using five methods: Support Vector Machine (SVM), k-Nearest Neighbour (kNN), Naive Bayes (NB) and Random Forest (RF). These models were validated using a ten fold cross-validation procedure. The SVM approach using features derived from fNIRS was the only method that classified game demand at higher than chance levels (accuracy = 0.66, F1 = 0.68). It was not possible to classify pain vs. no-pain at higher than chance level. The results demonstrate the viability of utilising fNIRS data to classify levels of game demand and the difficulty of classifying pain when another task is present.

Effortful listening: Sympathetic activity varies as a function of listening demand but parasympathetic activity does not.

Research on listening effort has used various physiological measures to examine the biological correlates of listening effort but a systematic examination of the impact of listening demand on cardiac autonomic nervous system activity is still lacking. The presented study aimed to close this gap by assessing cardiac sympathetic and parasympathetic responses to variations in listening demand. For this purpose, 45 participants performed four speech-in-noise tasks differing in listening demand-manipulated as signal-to-noise ratio varying between +23 dB and -16 dB-while their pre-ejection period and respiratory sinus arrythmia responses were assessed. Cardiac responses showed the expected effect of listening demand on sympathetic activity, but failed to provide evidence for the expected listening demand impact on parasympathetic activity: Pre-ejection period reactivity increased with increasing listening demand across the three possible listening conditions and was low in the very high (impossible) demand condition, whereas respiratory sinus arrythmia did not show this pattern. These findings have two main implications. First, cardiac sympathetic responses seem to be the more sensitive correlate of the impact of task demand on listening effort compared to cardiac parasympathetic responses. Second, very high listening demand may lead to disengagement and correspondingly low effort and reduced cardiac sympathetic response.

The Role of the Prefrontal Cortex and Functional Connectivity during Maritime Operations: An fNIRS study.

INTRODUCTION: Watchkeeping is a significant activity during maritime operations, and failures of sustained attention and decision-making can increase the likelihood of a collision. METHODS: A study was conducted in a ship bridge simulator where 40 participants (20 experienced/20 inexperienced) performed: (1) a 20-min period of sustained attention to locate a target vessel and (2) a 10-min period of decision-making/action selection to perform an evasive maneuver. Half of the participants also performed an additional task of verbally reporting the position of their vessel. Activation of the prefrontal cortex (PFC) was captured via a 15-channel functional near-infrared spectroscopy (fNIRS) montage, and measures of functional connectivity were calculated frontal using graph-theoretic measures. RESULTS: Neurovascular activation of right lateral area of the PFC decreased during sustained attention and increased during decision-making. The graph-theoretic analysis revealed that density declined during decision-making in comparison with the previous period of sustained attention, while local clustering declined during sustained attention and increased when participants prepared their evasive maneuver. A regression analysis revealed an association between network measures and behavioral outcomes, with respect to spotting the target vessel and making an evasive maneuver. CONCLUSIONS: The right lateral area of the PFC is sensitive to watchkeeping and decision-making during operational performance. Graph-theoretic measures allow us to quantify patterns of functional connectivity and were predictive of safety-critical performance.

Assessment of threat and negativity bias in virtual reality.

Negativity bias, i.e., tendency to respond strongly to negative stimuli, can be captured via behavioural and psychophysiological responses to potential threat. A virtual environment (VE) was created at room-scale wherein participants traversed a grid of ice blocks placed 200 m above the ground. Threat was manipulated by increasing the probability of encountering ice blocks that disintegrated and led to a virtual fall. Participants interacted with the ice blocks via sensors placed on their feet. Thirty-four people were recruited for the study, who were divided into High (HN) and Low (LN) Neuroticism groups. Movement data were recorded alongside skin conductance level and facial electromyography from the corrugator supercilii and zygomaticus major. Risk-averse behaviours, such as standing on 'safe' blocks and testing blocks prior to movement, increased when threat was highest. HN individuals exhibited more risk-averse behaviour than the LN group, especially in the presence of high threat. In addition, activation of the corrugator muscle was higher for HN individuals in the period following a movement to an ice block. These findings are discussed with respect to the use of room-scale VE as a protocol for emotion induction and measuring trait differences in negativity bias within VR.

A Neuroergonomics Approach to Mental Workload, Engagement and Human Performance.

The assessment and prediction of cognitive performance is a key issue for any discipline concerned with human operators in the context of safety-critical behavior. Most of the research has focused on the measurement of mental workload but this construct remains difficult to operationalize despite decades of research on the topic. Recent advances in Neuroergonomics have expanded our understanding of neurocognitive processes across different operational domains. We provide a framework to disentangle those neural mechanisms that underpin the relationship between task demand, arousal, mental workload and human performance. This approach advocates targeting those specific mental states that precede a reduction of performance efficacy. A number of undesirable neurocognitive states (mind wandering, effort withdrawal, perseveration, inattentional phenomena) are identified and mapped within a two-dimensional conceptual space encompassing task engagement and arousal. We argue that monitoring the prefrontal cortex and its deactivation can index a generic shift from a nominal operational state to an impaired one where performance is likely to degrade. Neurophysiological, physiological and behavioral markers that specifically account for these states are identified. We then propose a typology of neuroadaptive countermeasures to mitigate these undesirable mental states.

Routine Plasma-Based Genotyping to Comprehensively Detect Germline, Somatic, and Reversion BRCA Mutations among Patients with Advanced Solid Tumors.

PURPOSE: PARP inhibitors (PARPi) are efficacious in multiple cancers harboring germline (and possibly somatic) BRCA1/2 mutations. Acquired reversions can restore BRCA1/2 function, causing resistance to PARPi and/or platinum-based chemotherapy. The optimal method of identifying patients with germline, somatic, and/or reversion mutations in BRCA1/2 has not been established. Next-generation sequencing (NGS) of cell-free DNA (cfDNA) provides a platform to identify these three types of BRCA1/2 mutations. EXPERIMENTAL DESIGN: Patients with advanced breast, ovarian, prostate, or pancreatic cancer were tested using a clinically validated 73-gene cfDNA assay that evaluates single-nucleotide variants and insertion-deletion mutations (indels) in BRCA1/2, and distinguishes somatic/reversion from germline mutations with high accuracy. RESULTS: Among 828 patients, one or more deleterious BRCA1/2 mutations were detected in 60 (7.2%) patients, including germline (n = 42) and somatic (n = 18) mutations. Common coexisting mutations included TP53 (61.6%), MYC (30%), PIK3CA (26.6%), BRAF (15%), and ESR1 (11.5%). Polyclonal reversion mutations (median, 5) were detected in 9 of 42 (21.4%) germline BRCA1/2-mutant patients, the majority (77.7%) of whom had prior PARPi exposure (median duration, 10 months). Serial cfDNA demonstrated emergence of reversion BRCA mutations under therapeutic pressure from initial PARPi exposure, which contributed to subsequent resistance to PARPi and platinum therapy. CONCLUSIONS: cfDNA NGS identified high rates of therapeutically relevant mutations without foreknowledge of germline or tissue-based testing results, including deleterious somatic BRCA1/2 mutations missed by germline testing and reversion mutations that can have important treatment implications. Further research is needed to confirm clinical utility of these findings to guide precision medicine approaches for patients with advanced malignancies.

Molecular Mechanisms of Acquired Resistance to MET Tyrosine Kinase Inhibitors in Patients with MET Exon 14-Mutant NSCLC.

PURPOSE: Molecular mechanisms of acquired resistance to MET tyrosine kinase inhibitors (TKI) are poorly understood. We aimed to characterize the genomic mechanisms of resistance to type I and type II MET TKIs and their impact on sequential MET TKI therapy outcomes in patients with metastatic MET exon 14-mutant NSCLC. EXPERIMENTAL DESIGN: Genomic alterations occurring at the time of progression on MET TKIs were studied using plasma and tissue next-generation sequencing (NGS). RESULTS: A total of 20 patients had tissue or plasma available for analysis at the time of acquired resistance to a MET TKI. Genomic alterations known or suspected to be mechanisms of resistance were detected in 15 patients (75%). On-target acquired mechanisms of resistance, including single and polyclonal MET kinase domain mutations in codons H1094, G1163, L1195, D1228, Y1230, and high levels of amplification of the MET exon 14-mutant allele, were observed in 7 patients (35%). A number of off-target mechanisms of resistance were detected in 9 patients (45%), including KRAS mutations and amplifications in KRAS, EGFR, HER3, and BRAF; one case displayed both on- and off-target mechanisms of resistance. In 2 patients with on-target resistant mutations, switching between type I and type II MET TKIs resulted in second partial responses. CONCLUSIONS: On-target secondary mutations and activation of bypass signaling drive resistance to MET TKIs. A deeper understanding of these molecular mechanisms can support the development of sequential or combinatorial therapeutic strategies to overcome resistance.

Identification of osimertinib-resistant EGFR L792 mutations by cfDNA sequencing: oncogenic activity assessment and prevalence in large cfDNA cohort.

Cell-free DNA (cfDNA) next-generation sequencing has the potential to capture tumor heterogeneity and genomic evolution under treatment pressure in a non-invasive manner. Here, we report the detection of EGFR L792 mutations, a non-covalent mechanism of osimertinib resistance, using Guardant360 cfDNA testing in a patient with metastatic EGFR-mutant non-small cell lung cancer (NSCLC) whose disease progressed on osimertinib. We subsequently analyzed a large cohort of over 1800 additional patient samples harboring an EGFR T790M mutation and identified a concomitant L792 mutation in a total of 22 (1.2%) cases. In vitro functional assays demonstrated that the EGFR L858R/T790M/L792F/H mutations conferred intermediate-level resistance to osimertinib. Further understanding of potential acquired resistance mechanisms to targeted therapy may help inform treatment strategy in EGFR-mutant NSCLC.

Analysis of Cell-Free DNA from 32,989 Advanced Cancers Reveals Novel Co-occurring Activating RET Alterations and Oncogenic Signaling Pathway Aberrations.

PURPOSE: RET is an emerging oncogenic target showing promise in phase I/II clinical trials. An understudied aspect of RET-driven cancers is the extent to which co-occurring genomic alterations exist and how they may impact prognosis or therapeutic response. EXPERIMENTAL DESIGN: Somatic activating RET alterations were identified among 32,989 consecutive patients with metastatic solid tumors tested with a clinical cell-free circulating tumor DNA (cfDNA) assay. This comprehensive next-generation sequencing (NGS) assay evaluates single-nucleotide variants, and select indels, fusions, and copy number gains in 68-73 clinically relevant cancer genes. RESULTS: A total of 176 somatic activating RET alterations were detected in 170 patients (143 fusions and 33 missense mutations). Patients had non-small cell lung (NSCLC, n = 125), colorectal (n = 15), breast (n = 8), thyroid (n = 8), or other (n = 14) cancers. Alterations in other oncogenic signaling pathway genes were frequently identified in RET-positive samples and varied by specific RET fusion gene partner. RET fusions involving partners other than KIF5B were enriched for alterations in MAPK pathway genes and other bona fide oncogenic drivers of NSCLC, particularly EGFR. Molecular and clinical data revealed that these variants emerged later in the genomic evolution of the tumor as mechanisms of resistance to EGFR tyrosine kinase inhibitors. CONCLUSIONS: In the largest cancer cohort with somatic activating RET alterations, we describe novel co-occurrences of oncogenic signaling pathway aberrations. We find that KIF5B-RET fusions are highly specific for NSCLC. In our study, only non-KIF5B-RET fusions contributed to anti-EGFR therapy resistance. Knowledge of specific RET fusion gene partner may have clinical significance.

Validation of Microsatellite Instability Detection Using a Comprehensive Plasma-Based Genotyping Panel.

PURPOSE: To analytically and clinically validate microsatellite instability (MSI) detection using cell-free DNA (cfDNA) sequencing. EXPERIMENTAL DESIGN: Pan-cancer MSI detection using Guardant360 was analytically validated according to established guidelines and clinically validated using 1,145 cfDNA samples for which tissue MSI status based on standard-of-care tissue testing was available. The landscape of cfDNA-based MSI across solid tumor types was investigated in a cohort of 28,459 clinical plasma samples. Clinical outcomes for 16 patients with cfDNA MSI-H gastric cancer treated with immunotherapy were evaluated. RESULTS: cfDNA MSI evaluation was shown to have high specificity, precision, and sensitivity, with a limit of detection of 0.1% tumor content. In evaluable patients, cfDNA testing accurately detected 87% (71/82) of tissue MSI-H and 99.5% of tissue microsatellite stable (863/867) for an overall accuracy of 98.4% (934/949) and a positive predictive value of 95% (71/75). Concordance of cfDNA MSI with tissue PCR and next-generation sequencing was significantly higher than IHC. Prevalence of cfDNA MSI for major cancer types was consistent with those reported for tissue. Finally, robust clinical activity of immunotherapy treatment was seen in patients with advanced gastric cancer positive for MSI by cfDNA, with 63% (10/16) of patients achieving complete or partial remission with sustained clinical benefit. CONCLUSIONS: cfDNA-based MSI detection using Guardant360 is highly concordant with tissue-based testing, enabling highly accurate detection of MSI status concurrent with comprehensive genomic profiling and expanding access to immunotherapy for patients with advanced cancer for whom current testing practices are inadequate.See related commentary by Wang and Ajani, p. 6887.