Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPR.

Organoids, combined with genetic editing strategies, have the potential to offer rapid and efficient investigation of gene function in many models of human disease. However, to date, the editing efficiency of organoids with the use of non-viral electroporation methods has only been up to 30%, with implications for the subsequent need for selection, including turnaround time and exhaustion or adaptation of the organoid population. Here, we describe an efficient method for intestinal organoid editing using a ribonucleoprotein-based CRISPR approach. Editing efficiencies of up to 98% in target genes were robustly achieved across different gut anatomical locations and developmental timepoints from multiple patient samples with no observed off-target editing. The method allowed us to study the effect of loss of the tumour suppressor gene PTEN in normal human intestinal cells. Analysis of PTEN-deficient organoids defined phenotypes that likely relate to its tumour suppressive function in vivo, such as a proliferative advantage and increased organoid budding. Transcriptional profiling revealed differential expression of genes in pathways commonly known to be associated with PTEN loss, including mTORC1 activation.

Diverse types of expertise in facial recognition.

Facial recognition errors can jeopardize national security, criminal justice, public safety and civil rights. Here, we compare the most accurate humans and facial recognition technology in a detailed lab-based evaluation and international proficiency test for forensic scientists involving 27 forensic departments from 14 countries. We find striking cognitive and perceptual diversity between naturally skilled super-recognizers, trained forensic examiners and deep neural networks, despite them achieving equivalent accuracy. Clear differences emerged in super-recognizers' and forensic examiners' perceptual processing, errors, and response patterns: super-recognizers were fast, biased to respond 'same person' and misidentified people with extreme confidence, whereas forensic examiners were slow, unbiased and strategically avoided misidentification errors. Further, these human experts and deep neural networks disagreed on the similarity of faces, pointing to differences in their representations of faces. Our findings therefore reveal multiple types of facial recognition expertise, with each type lending itself to particular facial recognition roles in operational settings. Finally, we show that harnessing the diversity between individual experts provides a robust method of maximizing facial recognition accuracy. This can be achieved either via collaboration between experts in forensic laboratories, or most promisingly, by statistical fusion of match scores provided by different types of expert.

Selecting police super-recognisers.

People vary in their ability to recognise faces. These individual differences are consistent over time, heritable and associated with brain anatomy. This implies that face identity processing can be improved in applied settings by selecting high performers-'super-recognisers' (SRs)-but these selection processes are rarely available for scientific scrutiny. Here we report an 'end-to-end' selection process used to establish an SR 'unit' in a large police force. Australian police officers (n = 1600) completed 3 standardised face identification tests and we recruited 38 SRs from this cohort to complete 10 follow-up tests. As a group, SRs were 20% better than controls in lab-based tests of face memory and matching, and equalled or surpassed accuracy of forensic specialists that currently perform face identification tasks for police. Individually, SR accuracy was variable but this problem was mitigated by adopting strict selection criteria. SRs' superior abilities transferred only partially to body identity decisions where the face was not visible, and they were no better than controls at deciding which visual scene that faces had initially been encountered in. Notwithstanding these important qualifications, we conclude that super-recognisers are an effective solution to improving face identity processing in applied settings.

Single-copy Snail upregulation causes partial epithelial-mesenchymal transition in colon cancer cells.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is an embryonic programme implicated in cancer stem cells, metastasis and therapeutic resistance. Its role in cancer progression remains controversial because the transition can be partial or complete in different models and contexts. METHODS: Using human colon cancer DLD-1 cells, we engineered a cell line with a single-copy of Snail that was doxycycline-inducible and compared it to existing EMT models in DLD-1. The effect of Snail upregulation was characterised functionally, morphologically, and by transcriptional profiling and protein expression. RESULTS: Induction with doxycycline increased Snail expression to a level similar to that observed in cancer cell lines spontaneously expressing Snail and results in partial EMT. In comparison, higher levels of overexpression arising from introduction of episomal-Snail, results in complete EMT. DLD-1 cells with partial EMT show chemoresistance in vitro, increased tumour growth in vivo and decreased apoptosis. CONCLUSIONS: These findings highlight that the amount of bioavailable Snail can dictate phenotypic outcome and that partial EMT may be a preferred outcome of models operating within a natural range of Snail overexpression.

Looking at faces in the wild.

Faces are key to everyday social interactions, but our understanding of social attention is based on experiments that present images of faces on computer screens. Advances in wearable eye-tracking devices now enable studies in unconstrained natural settings but this approach has been limited by manual coding of fixations. Here we introduce an automatic 'dynamic region of interest' approach that registers eye-fixations to bodies and faces seen while a participant moves through the environment. We show that just 14% of fixations are to faces of passersby, contrasting with prior screen-based studies that suggest faces automatically capture visual attention. We also demonstrate the potential for this new tool to help understand differences in individuals' social attention, and the content of their perceptual exposure to other people. Together, this can form the basis of a new paradigm for studying social attention 'in the wild' that opens new avenues for theoretical, applied and clinical research.

Let's talk about faces: Identifying faces from verbal descriptions.

Face descriptions inform real-world identification decisions, for example when eyewitnesses describe criminal perpetrators. However, it is unclear how effective face descriptions are for identification. Here, we examined the accuracy of face identification from verbal descriptions, and how individual differences in face perception relate to producing and using descriptions for identification. In Study 1, participants completed a face communication task in pairs. Each participant saw a single face, and via verbal communication only, the pair decided if they were viewing the same person or different people. Dyads achieved 72% accuracy, compared to 81% when participants completed the task individually by matching face pairs side-by-side. Performance on the face communication and perceptual matching tasks were uncorrelated, perhaps due to low measurement reliability of the face communication task. In subsequent studies, we examined the abilities of face 'describers' (Study 2) and 'identifiers' separately (Study 3). We found that 'super-recognizers' - people with extremely high perceptual face identification abilities - outperformed controls in both studies. Overall, these results show that people can successfully describe faces for identification. Preliminary evidence suggests that this ability - and the ability use facial descriptions for identification - has some association with perceptual face identification skill.

Eye contact and sociability data suggests that Australian dingoes were never domesticated.

Dogs were the first animal to become domesticated by humans, and they represent a classic model system for unraveling the processes of domestication. We compare Australian dingo eye contact and socialization with Basenji and German Shepherd dog (GSD) breeds. Australian dingoes arrived in Australia 5,000-8,000 BP, and there is debate whether they were domesticated before their arrival. The Basenji represents a primitive breed that diverged from the remaining breeds early in the domestication process, while GSDs are a breed dog selected from existing domestic dogs in the late 1800s. We conducted a 4-phase study with unfamiliar and familiar investigators either sitting passively or actively calling each canid. We found 75% of dingoes made eye contact in each phase. In contrast, 86% of Basenjis and 96% of GSDs made eye contact. Dingoes also exhibited shorter eye-gaze duration than breed dogs and did not respond to their name being called actively. Sociability, quantified as a canid coming within 1 m of the experimenter, was lowest for dingoes and highest for GSDs. For sociability duration, dingoes spent less time within 1 m of the experimenter than either breed dog. When compared with previous studies, these data show that the dingo is behaviorally intermediate between wild wolves and Basenji dogs and suggest that it was not domesticated before it arrived in Australia. However, it remains possible that the accumulation of mutations since colonization has obscured historical behaviors, and dingoes now exist in a feralized retamed cycle. Additional morphological and genetic data are required to resolve this conundrum.

The impact of recall timing on the preservation of eyewitness memory.

Two studies investigated the impact of recall timing on eyewitness memory. In Study 1, participants viewed a crime video and then completed the Self-Administered Interview (SAI©) either immediately, after a 24-h delay, after a 1-week delay, or not at all. All participants completed a final recall questionnaire 2 weeks after they had viewed the stimulus video. Study 2 aimed to determine how long the beneficial impact of the SAI© on witnesses' long-term memory lasts. Participants watched a crime video and then either completed the SAI© or did not engage in an immediate recall attempt. Participants then completed a final recall questionnaire after a delay of 24 h, 1 week, 2 weeks, or 1 month. The results indicated that initial recall should be completed within 24 h of an incident and that under these conditions, the beneficial impact of early recall on long-term memory endures for at least 1 month.

A Diffusion-like Process Accommodates New Crypts During Clonal Expansion in Human Colonic Epithelium.

BACKGROUND & AIMS: Colorectal cancer (CRC) is thought to arise when the cumulative mutational burden within colonic crypts exceeds a certain threshold that leads to clonal expansion and ultimately neoplastic transformation. Therefore, quantification of the fixation and subsequent expansion of somatic mutations in normal epithelium is key to understanding colorectal cancer initiation. The aim of the present study was to determine how advantaged expansions can be accommodated in the human colon. METHODS: Immunohistochemistry was used to visualize loss of the cancer driver KDM6A in formalin-fixed paraffin-embedded (FFPE) normal human colonic epithelium. Combining microscopy with neural network-based image analysis, we determined the frequencies of KDM6A-mutant crypts and fission/fusion intermediates as well as the spatial distribution of clones. Mathematical modeling then defined the dynamics of their fixation and expansion. RESULTS: Interpretation of the age-related behavior of KDM6A-negative clones revealed significant competitive advantage in intracrypt dynamics as well as a 5-fold increase in crypt fission rate. This was not accompanied by an increase in crypt fusion. Mathematical modeling of crypt spacing identifies evidence for a crypt diffusion process. We define the threshold fission rate at which diffusion fails to accommodate new crypts, which can be exceeded by KRAS activating mutations. CONCLUSIONS: Advantaged gene mutations in KDM6A expand dramatically by crypt fission but not fusion. The crypt diffusion process enables accommodation of the additional crypts up to a threshold value, beyond which polyp growth may occur. The fission rate associated with KRAS mutations offers a potential explanation for KRAS-initiated polyps.

Top-down influences on working memory representations of faces: Evidence from dual-target visual search.

Variability in appearance across different images of the same unfamiliar face often causes participants to perceive different faces. Because perceptual information is not sufficient to link these encounters, top-down guidance may be critical in the initial stages of face learning. Here, we examine the interaction between top-down guidance and perceptual information when forming memory representations of unfamiliar faces. In two experiments, we manipulated the names associated with images of a target face that participants had to find in a search array. In Experiment 1, wrongly labelling two images of the same face with different names resulted in more errors relative to when the faces were labelled correctly. In Experiment 2, we compared this cost of mislabelling with the established "dual-target search cost," where searching for two targets produces more search errors relative to one target. We found search costs when searching for two different faces, but not when searching for mislabelled images of the same face. Together, these results suggest that perceptual and semantic information interact when we form face memory representations. Mislabelling the identity of perceptually similar faces does not cause dual representations to be created, but rather it impedes the process of forming a single robust representation.

Diagnostic feature training improves face matching accuracy.

Identifying unfamiliar faces is surprisingly error-prone, even for experienced professionals who perform this task regularly. Previous attempts to train this ability have been largely unsuccessful, leading many to conclude that face identity processing is hard-wired and not amenable to further perceptual learning. Here, we take a novel expert knowledge elicitation approach to training, based on the feature-based comparison strategy used by high-performing professional facial examiners. We show that instructing novices to focus on the facial features that are most diagnostic of identity for these experts-the ears and facial marks (e.g., scars, freckles and blemishes)-improves accuracy on unfamiliar face matching tasks by 6%. This training takes just 6 min to complete and yet accounts for approximately half of experts' superiority on the task. Benefits of training are strongest when diagnostic features are clearly visible and absent when participants are trained to rely on nondiagnostic features. Our data-driven approach contrasts with theory-driven training that is designed to improve holistic face processing mechanisms associated with familiar face recognition. This suggests that protocols which bypass the core face recognition system-and instead reorient attention to features that are undervalued by novices-offer a more promising route to training for unfamiliar face matching. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Characteristics and re-offending outcomes of 'limiting-term' forensic patients found unfit to stand trial in New South Wales, Australia.

There are little published data on the characteristics or outcomes of offenders found unfit to stand trial who receive a 'qualified finding of guilt' in a Special Hearing in New South Wales (NSW) and are detained for a 'limiting term' (LT) under the supervision of the NSW Mental Health Review Tribunal (MHRT). We examined NSW MHRT records linked to re-offending data, to report on the characteristics and outcomes of 69 LT patients in a cohort spanning two decades. The most common diagnoses were schizophrenia (54%) and intellectual disability (33%). Patients were detained on average for 4.2 years, which is slightly shorter than the average maximum term imposed. Of the 55 people for whom criminal record data were available, 9.1% were charged with an offence during the first year post-release and 60% overall were charged for at least one post-release offence during a follow-up period ranging from 4.7 to 11.1 years.

Heterogeneity in clone dynamics within and adjacent to intestinal tumours identified by Dre-mediated lineage tracing.

Somatic models of tissue pathology commonly use induction of gene-specific mutations in mice mediated by spatiotemporal regulation of Cre recombinase. Subsequent investigation of the onset and development of disease can be limited by the inability to track changing cellular behaviours over time. Here, a lineage-tracing approach based on ligand-dependent activation of Dre recombinase that can be employed independently of Cre is described. The clonal biology of the intestinal epithelium following Cre-mediated stabilisation of ß-catenin reveals that, within tumours, many new clones rapidly become extinct. Surviving clones show accelerated population of tumour glands compared to normal intestinal crypts but in a non-uniform manner, indicating that intra-tumour glands follow heterogeneous dynamics. In tumour-adjacent epithelia, clone sizes are smaller than in the background epithelia, as a whole. This suggests a zone of ∼seven crypt diameters within which clone expansion is inhibited by tumours and that may facilitate their growth.

Communicating forensic science opinion: An examination of expert reporting practices.

Forensic scientists endeavour to explain complex scientific principles to legal decision-makers with limited scientific training (e.g., police, lawyers, judges, and jurors). Much of the time this communication is limited to written opinions in expert reports. Notwithstanding considerable scientific research and debate about the best way to communicate forensic science opinions, it is unclear how much of the advice has translated into forensic science practice. In conducting this descriptive study, we examined the reporting practices adopted by forensic scientists across a range of forensic science disciplines. Specifically, we used a quantitative content analysis approach to identify the conclusion types and additional information submitted by forensic scientists in proficiency tests during 2016 ("What would be the wording of the Conclusions in your report?"). Our analysis of 500 randomly selected responses in eight disciplines indicated that the conclusion type which has received the most criticism in recent years (categorical statements) remains the preferred means of expression in a clear majority of responses. We also found that the provision of additional information often considered necessary for rational evaluation of the evidence (e.g., information about reliability and validity) was rarely reported. These results suggest limited engagement with recent recommendations and are concerning given the gravity of the legal decisions that hinge on accurate and transparent forensic science communication.

Heavy Metals Make a Chain: A Catenated Bismuth Compound.

Catenation is common for the light main-group elements whereas it is rare for the heavy elements. Herein, we report the first example of a neutral molecule containing a Bi4 chain. It is prepared in a one-step reaction between bismuth trichloride and bis(diisopropylphosphino)amine in methanol suspension. The same reaction carried out in dichloromethane gives quite different products. All products have been characterized spectroscopically and using single-crystal X-ray analysis.

Forensic science evidence: Naive estimates of false positive error rates and reliability.

We do not know how often false positive reports are made in a range of forensic science disciplines. In the absence of this information it is important to understand the naive beliefs held by potential jurors about forensic science evidence reliability. It is these beliefs that will shape evaluations at trial. This descriptive study adds to our knowledge about naive beliefs by: (1) measuring jury-eligible (lay) perceptions of reliability for the largest range of forensic science disciplines to date, over three waves of data collection between 2011 and 2016 (n=674); (2) calibrating reliability ratings with false positive report estimates; and (3) comparing lay reliability estimates with those of an opportunity sample of forensic practitioners (n=53). Overall the data suggest that both jury-eligible participants and practitioners consider forensic evidence highly reliable. When compared to best or plausible estimates of reliability and error in the forensic sciences these views appear to overestimate reliability and underestimate the frequency of false positive errors. This result highlights the importance of collecting and disseminating empirically derived estimates of false positive error rates to ensure that practitioners and potential jurors have a realistic impression of the value of forensic science evidence.

Familiarity does not inhibit image-specific encoding of faces.

When matching and recognizing familiar faces, performance is unaffected by changes to image-specific details such as lighting, head angle, and expression. In contrast, these changes have a substantial impact on performance when faces are unfamiliar. What process can account for this difference? Recent evidence shows a memory disadvantage for remembering specific images of familiar people compared to unfamiliar people, suggesting that image invariance in familiar face processing may be supported by loss of image-specific details in memory. Here, we examine whether this cost results from loss of image specific details during encoding of familiar faces. Participants completed four tasks that required participants to retain image-specific information in working memory: duplicate detection (Experiment 1), change detection (Experiment 2), short-term recognition memory (Experiment 3 and 5), and visual search (Experiment 4). Across all experiments (combined n = 270), our results consistently show equivalent memory performance for specific images of familiar and unfamiliar faces. We conclude that familiarity does not influence encoding of pictorial details, suggesting that loss of image-specificity reported in previous work is a result of longer-term storage mechanisms. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Hydrogenolysis of Dinuclear PCNR Ligated PdII µ-Hydroxides and Their Mononuclear PdII Hydroxide Analogues.

The hydrogenolysis of mono- and dinuclear PdII hydroxides was investigated both experimentally and computationally. It was found that the dinuclear µ-hydroxide complexes {[(PCNR )Pd]2 (µ-OH)}(OTf) (PCNH =1-[3-[(di-tert-butylphosphino)methyl]phenyl]-1H-pyrazole; PCNMe =1-[3-[(di-tert-butylphosphino)methyl]phenyl]-5-methyl-1H-pyrazole) react with H2 to form the analogous dinuclear hydride species {[(PCNR )Pd]2 (µ-H)}(OTf). The dinuclear µ-hydride complexes were fully characterized, and are rare examples of structurally characterized unsupported singly bridged µ-H PdII dimers. The {[(PCNMe )Pd]2 (µ-OH)}(OTf) hydrogenolysis mechanism was investigated through experiments and computations. The hydrogenolysis of the mononuclear complex (PCNH )Pd-OH resulted in a mixed ligand dinuclear species [(PCNH )Pd](µ-H)[(PCC)Pd] (PCC=a dianionic version of PCNH bound through phosphorus P, aryl C, and pyrazole C atoms) generated from initial ligand "rollover" C-H activation. Further exposure to H2 yields the bisphosphine Pd0 complex Pd[(H)PCNH ]2 . When the ligand was protected at the pyrazole 5-position in the (PCNMe )Pd-OH complex, no hydride formed under the same conditions; the reaction proceeded directly to the bisphosphine Pd0 complex Pd[(H)PCNMe ]2 . Reaction mechanisms for the hydrogenolysis of the monomeric and dimeric hydroxides are proposed.

Quantifying Microsatellite Mutation Rates from Intestinal Stem Cell Dynamics in Msh2-Deficient Murine Epithelium.

Microsatellite sequences have an enhanced susceptibility to mutation, and can act as sentinels indicating elevated mutation rates and increased risk of cancer. The probability of mutant fixation within the intestinal epithelium is dictated by a combination of stem cell dynamics and mutation rate. Here, we exploit this relationship to infer microsatellite mutation rates. First a sensitive, multiplexed, and quantitative method for detecting somatic changes in microsatellite length was developed that allowed the parallel detection of mutant [CA]n sequences from hundreds of low-input tissue samples at up to 14 loci. The method was applied to colonic crypts in Mus musculus, and enabled detection of mutant subclones down to 20% of the cellularity of the crypt (∼50 of 250 cells). By quantifying age-related increases in clone frequencies for multiple loci, microsatellite mutation rates in wild-type and Msh2-deficient epithelium were established. An average 388-fold increase in mutation per mitosis rate was observed in Msh2-deficient epithelium (2.4 × 10-2) compared to wild-type epithelium (6.2 × 10-5).