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Introduction: Cognitive decline and mood disorders are two major concerns of people affected by stroke. The extent to which cognition and mood relate to or are independent of stroke severity and post-stroke dependency are unclear. We examined the associations between stroke severity, global measures of cognition, mood and dependency up to 14-weeks post-stroke in a large national study of neurocognitive complications of stroke up to two years, the Rates, Risks and Routes to Reduce Vascular Dementia (R4VaD) study. Methods: R4VaD recruited patients with stroke of all subtypes and severities and collected clinical, cognitive and mood data at baseline (within six-weeks post-stroke), subacutely (6+/-2 weeks later; ie. maximum 14-weeks post-stroke) and 1 and 2 years. We measured baseline stroke severity (National Institute of Health Stroke Scale, NIHSS), pre-stroke and 14-week dependency (Modified Rankin Scale, mRS), cognition (Montreal Cognitive Assessment, MoCA; Modified Telephone Interview for Cognitive Status, TICS-m), and mood (Zung depression scale; Patient Health Questionnaire, PHQ; General Anxiety Disorder scale, GAD-7). We analysed baseline and 14-week cognition and mood using linear models with log-transformed NIHSS, adjusted for mRS, age, sex, education, hypertension, diabetes and smoking. Results: We recruited 2441 participants (mean age=68.2 SD=13.5; 40% female; median NIHSS=2.0, IQR=0-4, range=0-24; median stroke onset to recruitment=6 days, IQR=3-13; median time to follow-up=6.6 weeks, IQR=6.0-7.9). Table 1 shows baseline and subacute cognition and mood scores. At baseline higher NIHSS associated with lower cognition (MoCA: β= -0.22, p

Background Difficulties in top-down and bottom-up emotion generation have been proposed to play a key role in the progression of psychiatric disorders. The aim of the current study was to develop more ecologically valid measures of top-down interpretation biases and bottom-up evoked emotional responses. Methods A total of 124 healthy female participants aged 18–25 took part in the study. We evaluated two sets of 18 brief film clips. The first set of film clips presented ambiguous social situations designed to examine interpretation biases. Participants provided written interpretations of each ambiguous film clip which were subjected to sentiment analysis. We compared the films in terms of the valence of participants interpretations. The second set of film clips presented neutral and emotionally provoking social scenarios designed to elicit subjective and facial emotional responses. While viewing these film clips participants mood ratings and facial affect were recorded and analysed using exploratory factor analyses. Results Most of the 18 ambiguous film clips were interpreted in the expected manner while still retaining some ambiguity. However, participants were more attuned to the negative cues in the ambiguous film clips and three film clips were identified as unambiguous. These films clips were deemed unsuitable for assessing interpretation bias. The exploratory factor analyses of participants’ mood ratings and evoked facial affect showed that the positive and negative emotionally provoking film clips formed their own factors as expected. However, there was substantial cross-loading of the neutral film clips when participants’ facial expression data was analysed. Discussion A subset of the film clips from the two tasks could be used to assess top-down interpretation biases and bottom-up evoked emotional responses. Ambiguous negatively valenced film clips should have more subtle negative cues to avoid ceiling effects and to ensure there is enough room for interpretation.

Surveillance for soil-transmitted helminths, strongyloidiasis, cryptosporidiosis, and giardiasis was conducted in Mississippi, USA. PCR performed on 224 fecal samples for all soil-transmitted helminths and on 370 samples for only Necator americanus and Strongyloides stercoralis identified 1 S. stercoralis infection. Seroprevalences were 8.8% for Toxocara, 27.4% for Cryptosporidium, 5.7% for Giardia, and 0.2% for Strongyloides parasites.

Abstract Background Autism spectrum disorder (ASD) has a high cost to affected individuals and society, but treatments for core symptoms are lacking. To expand intervention options, it is crucial to gain a better understanding of potential treatment targets, and their engagement, in the brain. For instance, the striatum (caudate, putamen, and nucleus accumbens) plays a central role during development and its (atypical) functional connectivity (FC) may contribute to multiple ASD symptoms. We have previously shown, in the adult autistic and neurotypical brain, the non-intoxicating cannabinoid cannabidivarin (CBDV) alters the balance of striatal ‘excitatory–inhibitory’ metabolites, which help regulate FC, but the effects of CBDV on (atypical) striatal FC are unknown. Methods To examine this in a small pilot study, we acquired resting state functional magnetic resonance imaging data from 28 men (15 neurotypicals, 13 ASD) on two occasions in a repeated-measures, double-blind, placebo-controlled study. We then used a seed-based approach to (1) compare striatal FC between groups and (2) examine the effect of pharmacological probing (600 mg CBDV/matched placebo) on atypical striatal FC in ASD. Visits were separated by at least 13 days to allow for drug washout. Results Compared to the neurotypicals, ASD individuals had lower FC between the ventral striatum and frontal and pericentral regions (which have been associated with emotion, motor, and vision processing). Further, they had higher intra-striatal FC and higher putamenal FC with temporal regions involved in speech and language. In ASD, CBDV reduced hyperconnectivity to the neurotypical level. Limitations Our findings should be considered in light of several methodological aspects, in particular our participant group (restricted to male adults), which limits the generalizability of our findings to the wider and heterogeneous ASD population. Conclusion In conclusion, here we show atypical striatal FC with regions commonly associated with ASD symptoms. We further provide preliminary proof of concept that, in the adult autistic brain, acute CBDV administration can modulate atypical striatal circuitry towards neurotypical function. Future studies are required to determine whether modulation of striatal FC is associated with a change in ASD symptoms. Trial registration clinicaltrials.gov, Identifier: NCT03537950. Registered May 25th, 2018—Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT03537950?term=NCT03537950&draw=2&rank=1 .

Abstract The glutamatergic modulator ketamine has been shown to rapidly reduce depressive symptoms in patients with treatment-resistant major depressive disorder (TRD). Although its mechanisms of action are not fully understood, changes in cortical excitation/inhibition (E/I) following ketamine administration are well documented in animal models and could represent a potential biomarker of treatment response. Here, we analyse neuromagnetic virtual electrode time series collected from the primary somatosensory cortex in 18 unmedicated patients with TRD and in an equal number of age-matched healthy controls during a somatosensory ‘airpuff’ stimulation task. These two groups were scanned as part of a clinical trial of ketamine efficacy under three conditions: (a) baseline; (b) 6–9 h following subanesthetic ketamine infusion; and (c) 6–9 h following placebo-saline infusion. We obtained estimates of E/I interaction strengths by using dynamic causal modelling (DCM) on the time series, thereby allowing us to pinpoint, under each scanning condition, where each subject’s dynamics lie within the Poincaré diagram—as defined in dynamical systems theory. We demonstrate that the Poincaré diagram offers classification capability for TRD patients, in that the further the patients’ coordinates were shifted (by virtue of ketamine) toward the stable (top-left) quadrant of the Poincaré diagram, the more their depressive symptoms improved. The same relationship was not observed by virtue of a placebo effect—thereby verifying the drug-specific nature of the results. We show that the shift in neural dynamics required for symptom improvement necessitates an increase in both excitatory and inhibitory coupling. We present accompanying MATLAB code made available in a public repository, thereby allowing for future studies to assess individually tailored treatments of TRD.

Abstract Background Although social-emotional difficulties are believed play a key role in anorexia nervosa (AN), there is uncertainty regarding what these difficulties might look like. Previous research has largely focused on a “disease model” of social-emotional processing in AN with little attention paid to positive emotions and experiences. Therefore, the aim of the present study was to obtain a fuller picture of critical life events as identified by those with lived AN experience. Methods Thirty-four participants aged 16–48 with current or past AN completed an online survey describing self-defined positive and difficult critical events. Thematic analysis was used to assess patterns in participants narrative responses. Results Two major themes were identified in the descriptions of positive critical events: Moments of celebration and Unexpected positive outcomes. These major themes revealed increased external focus and some corrective experiences that challenged the participants pre-existing expectations leading to new positive outcomes. Difficult events clustered into life events that were identified as Eating disorder (ED) related and Non-ED related and included the dimensions of relational conflict and feeling unsupported. Discussion The findings suggest that although negative emotionality was identified in the accounts of those with lived experience of AN capacity for “big-picture” thinking with and explicit focus on others was also identified. Moreover, an openness to corrective experiences that worked to challenge negative expectations was evident for some participants. Together these findings have scope as targets for further clinical research and treatment interventions.

Background: Large-scale longitudinal and multi-centre studies are used to explore neuroimaging markers of normal ageing, and neurodegenerative and mental health disorders. Longitudinal changes in brain structure are typically small, therefore the reliability of automated techniques is crucial. Determining the effects of different factors on reliability allows investigators to control those adversely affecting reliability, calculate statistical power, or even avoid particular brain measures with low reliability. This study examined the impact of several image acquisition and processing factors and documented the test-retest reliability of structural MRI measurements. Methods: In Phase I, 20 healthy adults (11 females; aged 20–30 years) were scanned on two occasions three weeks apart on the same scanner using the ADNI-3 protocol. On each occasion, individuals were scanned twice (repetition), after re-entering the scanner (reposition) and after tilting their head forward. At one year follow-up, nine returning individuals and 11 new volunteers were recruited for Phase II (11 females; aged 22–31 years). Scans were acquired on two different scanners using the ADNI-2 and ADNI-3 protocols. Structural images were processed using FreeSurfer (v5.3.0, 6.0.0 and 7.1.0) to provide subcortical and cortical volume, cortical surface area and thickness measurements. Intra-class correlation coefficients (ICC) were calculated to estimate test-retest reliability. We examined the effect of repetition, reposition, head tilt, time between scans, MRI sequence and scanner on reliability of structural brain measurements. Mean percentage differences were also calculated in supplementary analyses. Results: Using the FreeSurfer v7.1.0 longitudinal pipeline, we observed high reliability for subcortical and cortical volumes, and cortical surface areas at repetition, reposition, three weeks and one year (mean ICCs>0.97). Cortical thickness reliability was lower (mean ICCs>0.82). Head tilt had the greatest adverse impact on ICC estimates, for example reducing mean right cortical thickness to ICC=0.74. In contrast, changes in ADNI sequence or MRI scanner had a minimal effect. We observed an increase in reliability for updated FreeSurfer versions, with the longitudinal pipeline consistently having a higher reliability than the cross-sectional pipeline. Discussion: Longitudinal studies should monitor or control head tilt to maximise reliability. We provided the ICC estimates and mean percentage differences for all FreeSurfer brain regions, which may inform power analyses for clinical studies and have implications for the design of future longitudinal studies.

BackgroundPrevious theoretical models and reviews have documented a strong connection between emotion dysregulation eating disorder (ED) psychopathology among the general and clinical populations. The aim of this review was to build on this previous work by conducting a network meta-analysis to explore associations between adaptive and maladaptive emotion regulation strategies and ED psychopathology trans-diagnostically across the ED spectrum to identify areas of emotion dysregulation that have the strongest association with symptomatology.MethodologyA total of 104 studies were included in the meta-analysis and correlation coefficient representing the associations between specific emotion regulation strategies and ED symptomatology were extracted. We ran a Bayesian random effects network meta-analysis and the initial network was well-connected with each emotion regulation strategy being linked to at least one other strategy. We also conducted a network meta-regression to explore whether between-study differences in body mass index (BMI), age, and whether the sample consisted of solely female participants explained any possible network inconsistency.ResultsThe network meta-analysis revealed that ruminations and non-acceptance of emotions were most closely associated with ED psychopathology. There was no significant network inconsistency but two comparisons approached significance and thus meta-regressions were conducted. The meta-regressions revealed a significant effect of BMI such that the associations between different emotion regulation strategies and ED symptomatology were weaker among those with low BMI.DiscussionThe present findings build on previous work and highlight the role of rumination and difficulties with accepting emotions as key emotion regulation difficulties in EDs. Additionally, the finding that the associations were weaker among ED patients with low BMI may point toward a complex relationship between ED behaviors and emotion regulation. Taken together, our findings call for interventions that target emotion regulation, specifically rumination and difficulties accepting emotions, in the treatment of EDs.Systematic Review Registrationhttps://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021249996, PROSPERO, identifier: CRD42021249996.

Preserving biodiversity and its many components is a priority of conservation science and how to efficiently allocate resources to preserve healthy populations of as many species, habitats, and ecosystems as possible. We used the U.S. Geological Survey (USGS) Gap Analysis Project (GAP) species models released in 2018, which identify predicted habitats for terrestrial vertebrates in the conterminous United States, to illustrate hotspots of biodiversity for the major taxonomic groups. This collection represents the first complete compilation of terrestrial vertebrate species models for the conterminous United States (U.S. Geological Survey (USGS), 2018a). We used the species models but not the available subspecies models; this resulted in the inclusion of 282 amphibian models, 621 bird models, 365 mammal models, and 322 reptiles in our analysis. We also used population trend information and made spatial queries to characterize species in three dimensions: geographic range (small or large), habitat breadth (narrow or wide), and population trend (decreasing vs stable or increasing). This characterization allowed us to divide the species into eight groups (A-H) with similar characteristics. Group A species (large geographic range, wide habitat breadth, and stable or increasing population trend) are species that are common now with no indication of becoming rare. Species B-H have theoretical or known characteristics that could lead them to become rare with the H species exhibiting small geographic range, narrow habitat breadth, and decreasing population trend. Finally, we evaluated the prevalence of mapped habitat on protected lands for each species, exploring the patterns of representation in the rare species groups by ecoregion. The species we identified with population and habitat use characteristics that potentially predispose them to being or becoming rare represented a large percentage of each taxon. Potentially rare species were widely distributed among ecoregions. Of the 20 ecoregions in the country, 14 have a greater number of rare species than the national average for at least one taxon. Protection of the habitat for the majority of these rare species is below that recommended (17% of available habitat) by the Convention on Biological Diversity (CBD). The Everglades ecoregion was the only ecoregion that protected more than half of its rare or potentially rare species.

School counselors support the academic, career, and social/emotional development of all students in their care, and one important method of delivery of a comprehensive school counseling program is the use of classroom instruction. In this exploratory, sequential mixed-methods study, we examined participant responses from nine focus groups regarding the school counselors' experiences with delivery of classroom lessons, in combination with a national survey of 247 practicing school counselors. Results underscored participants' desire to spend more time in classrooms and ongoing barriers to classroom access. We discuss implications for school counseling practice and school counselor education.

Aims: Left atrial (LA) remodelling is a common feature of many cardiovascular pathologies and is a sensitive marker of adverse cardiovascular outcomes. The aim of this study was to establish normal ranges for LA parameters derived from coronary computed tomographic angiography (CCTA) imaging using a standardised image processing pipeline to establish normal ranges in a previously described cohort. Methods: CCTA imaging from 193 subjects recruited to the Budapest GLOBAL twin study was analysed. Indexed LA cavity volume (LACVi), LA surface area (LASAi), wall thickness and LA tissue volume (LATVi) were calculated. Wall thickness maps were combined into an atlas. Indexed LA parameters were compared with clinical variables to identify early markers of pathological remodelling. Results: LACVi is similar between sexes (31 ml/m2 v 30 ml/m2) and increased in hypertension (33 ml/m2 v 29 ml/m2, p = 0.009). LASAi is greater in females than males (47.8 ml/m2 v 45.8 ml/m2 male, p = 0.031). Median LAWT was 1.45 mm. LAWT was lowest at the inferior portion of the posterior LA wall (1.14 mm) and greatest in the septum (median = 2.0 mm) (p

The propagation of epileptic seizure activity in the brain is a widespread pathophysiology that, in principle, should yield to intervention techniques guided by mathematical models of neuronal ensemble dynamics. During a seizure, neural activity will deviate from its current dynamical regime to one in which there are significant signal fluctuations. In silico treatments of neural activity are an important tool for the understanding of how the healthy brain can maintain stability, as well as of how pathology can lead to seizures. The hope is that, contained within the mathematical foundations of such treatments, there lie potential strategies for mitigating instabilities, e.g. via external stimulation. Here, we demonstrate that the dynamic causal modelling neuronal state equation generalises to a Fokker-Planck formalism if one extends the framework to model the ways in which activity propagates along the structural connections of neural systems. Using the Jacobian of this generalised state equation, we show that an initially unstable system can be rendered stable via a reduction in diffusivity-i.e., by lowering the rate at which neuronal fluctuations disperse to neighbouring regions. We show, for neural systems prone to epileptic seizures, that such a reduction in diffusivity can be achieved via external stimulation. Specifically, we show that this stimulation should be applied in such a way as to temporarily mirror the activity profile of a pathological region in its functionally connected areas. This counter-intuitive method is intended to be used pre-emptively-i.e., in order to mitigate the effects of the seizure, or ideally even prevent it from occurring in the first place. We offer proof of principle using simulations based on functional neuroimaging data collected from patients with idiopathic generalised epilepsy, in which we successfully suppress pathological activity in a distinct sub-network prior to seizure onset. Our hope is that this technique can form the basis for future real-time monitoring and intervention devices that are capable of treating epilepsy in a non-invasive manner.

People with anorexia nervosa (AN) commonly exhibit social difficulties, which may be related to problems with understanding the perspectives of others, commonly known as Theory of Mind (ToM) processing. However, there is a dearth of literature investigating the neural basis of these differences in ToM and at what age they emerge. This study aimed to test for differences in the neural correlates of ToM processes in young women with AN, and young women weight-restored (WR) from AN, as compared to healthy control participants (HC). Based on previous findings in AN, we hypothesized that young women with current or prior AN, as compared to HCs, would exhibit a reduced neural response in the medial prefrontal cortex (mPFC), the inferior frontal gyrus, and the temporo-parietal junction (TPJ) whilst completing a ToM task. We recruited 73 young women with AN, 45 WR young women, and 70 young women without a history of AN to take part in the current study. Whilst undergoing a functional magnetic resonance imaging (fMRI) scan, participants completed the Frith-Happé task, which is a commonly used measure of ToM with demonstrated reliability and validity in adult populations. In this task, participants viewed the movements of triangles, which depicted either action movements, simple interactions, or complex social interactions. Viewing trials with more complex social interactions in the Frith-Happé task was associated with increased brain activation in regions including the right TPJ, the bilateral mPFC, the cerebellum, and the dorsolateral prefrontal cortex. There were no group differences in neural activation in response to the ToM contrast. Overall, these results suggest that the neural basis of spontaneous mentalizing is preserved in most young women with AN.

In many clinical and scientific situations the optimal neuroimaging sequence may not be known prior to scanning and may differ for each individual being scanned, depending on the exact nature and location of abnormalities. Despite this, the standard approach to data acquisition, in such situations, is to specify the sequence of neuroimaging scans prior to data acquisition and to apply the same scans to all individuals. In this paper, we propose and illustrate an alternative approach, in which data would be analysed as it is acquired and used to choose the future scanning sequence: Active Acquisition. We propose three Active Acquisition scenarios based around multiple MRI modalities. In Scenario 1, we propose a simple use of near-real time analysis to decide whether to acquire more or higher resolution data, or acquire data with a different field-of-view. In Scenario 2, we simulate how multimodal MR data could be actively acquired and combined with a decision tree to classify a known outcome variable (in the simple example here, age). In Scenario 3, we simulate using Bayesian optimisation to actively search across multiple MRI modalities to find those which are most abnormal. These simulations suggest that by actively acquiring data, the scanning sequence can be adapted to each individual. We also consider the many outstanding practical and technical challenges involving normative data acquisition, MR physics, statistical modelling and clinical relevance. Despite these, we argue that Active Acquisition allows for potentially far more powerful, sensitive or rapid data acquisition, and may open up different perspectives on individual differences, clinical conditions, and biomarker discovery.

Malignant mesothelioma (MM) is poorly responsive to systemic cytotoxic chemotherapy and invariably fatal. Here we describe a screen of 94 drugs in 15 exome-sequenced MM lines and the discovery of a subset defined by loss of function of the nuclear deubiquitinase BRCA associated protein-1 (BAP1) that demonstrate heightened sensitivity to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand). This association is observed across human early passage MM cultures, mouse xenografts and human tumour explants. We demonstrate that BAP1 deubiquitinase activity and its association with ASXL1 to form the Polycomb repressive deubiquitinase complex (PR-DUB) impacts TRAIL sensitivity implicating transcriptional modulation as an underlying mechanism. Death receptor agonists are well-tolerated anti-cancer agents demonstrating limited therapeutic benefit in trials without a targeting biomarker. We identify BAP1 loss-of-function mutations, which are frequent in MM, as a potential genomic stratification tool for TRAIL sensitivity with immediate and actionable therapeutic implications.

Health Canada, the national department responsible for regulating food, health and consumer products to keep Canadians safe, published the Guidance Document: Information and Submission Requirements for Biosimilar Biologic Drugs on 14 November 2016 to update its framework for the market authorization of biosimilars in Canada. Biosimilars are the off-patent copies of biologics, a class of therapeutic agents derived from complex organic processing. Biosimilars are only “similar and not identical” to their reference biologic because biologics cannot be perfectly duplicated. As a result, biosimilars may not have the same safety profile as their reference biologic. As a number of biologics will be coming off patent in the coming years, Health Canada’s goal was to prepare by updating and clarifying the process surrounding market authorization. Santé Canada, le ministère en charge de la réglementation des produits de consommation et des produits de santé au Canada, a publié le 14 novembre 2016 la Ligne directrice : exigences en matière de renseignements et de présentation relatives aux médicaments biologiques biosimilaires, pour actualiser le cadre législatif des autorisations de mise sur le marché de biosimilaires au Canada. Les biosimilaires sont des copies de médicaments biologiques (une classe d’agents thérapeutiques issus de procédés organiques complexes) dont le brevet a expiré. Les biosimilaires sont “similaires mais non identiques” à leur médicament biologique de référence, parce que des médicaments biologiques ne peuvent être parfaitement dupliqués. Il en résulte que les biosimilaires ne peuvent offrir tout à fait les mêmes garanties d’emploi que leur médicament de référence. Compte tenu du fait qu’un grand nombre de brevets de médicaments biologiques vont expirer dans les années à venir, le but de Santé Canada était de se préparer en actualisant et clarifiant son processus de mise sur le marché.

Background: The behavioural literature in anorexia nervosa (AN) has suggested impairments in psychosocial functioning and studies using facial expression processing tasks (FEPT) have reported poorer recognition and slower identification of emotions. Methods: Functional magnetic resonance imaging (fMRI) was used alongside a FEPT, depicting neutral, mildly happy and happy faces, to examine the neural correlates of implicit emotion processing in AN. Participants were instructed to specify the gender of the faces. Levels of depression, anxiety, obsessive–compulsive symptoms and eating disorder behaviour were obtained and principal component analysis (PCA) was performed to acquire uncorrelated variables. Results: fMRI analysis revealed a greater blood-oxygenation level dependent (BOLD) response in AN in the right fusiform gyrus to all facial expressions. This response showed a linear increase with the happiness of the facial expression and was found to be stronger in those not taking medication. PCA analysis revealed a single component indicating a greater level of general clinical symptoms. Conclusion: Neuroimaging findings would suggest that alterations in implicit emotion processing in AN occur during early perceptual processing of social signals and illustrate greater engagement on the FEPT. The lack of separate components using PCA suggests that the questionnaires used might not be suited as predictive measures.

What is the optimal level of chaos in a computational system? If a system is too chaotic, it cannot reliably store information. If it is too ordered, it cannot transmit information. A variety of computational systems exhibit dynamics at the “edge of chaos”, the transition between the ordered and chaotic regimes. In this work, we examine the evolved neural networks of Polyworld, an artificial life model consisting of a simulated ecology populated with biologically inspired agents. As these agents adapt to their environment, their initially simple neural networks become increasingly capable of exhibiting rich dynamics. Dynamical systems analysis reveals that natural selection drives these networks toward the edge of chaos until the agent population is able to sustain itself. After this point, the evolutionary trend stabilizes, with neural dynamics remaining on average significantly far from the transition to chaos.

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification.