Development and testing of a versatile genome editing application reporter (V-GEAR) system.

CRISPR-Cas9 and novel cas fusion proteins leveraging specific DNA targeting ability combined with deaminases or reverse transcriptases have revolutionized genome editing. However, their efficacy heavily relies upon protein variants, targeting single guide RNAs, and surrounding DNA sequence context within the targeted loci. This necessitates the need for efficient and rapid screening methods to evaluate these editing reagents and designs. Existing plasmid-based reporters lack flexibility, being fixed to specific DNA sequences, hindering direct comparisons between various editing approaches. To address this, we developed the versatile genome editing application reporter (V-GEAR) system. V-GEAR comprises genes detectable after desired editing via base editing, prime editing, or homology-directed repair within relevant genomic contexts. It employs a detectable synthetic cell surface protein (RQR8) followed by a customizable target sequence resembling genomic regions of interest. These genes allow for reliable identification of corrective editing and cell enrichment. We validated the V-GEAR system with base editors, prime editors, and Cas9-mediated homology-directed repair. Furthermore, the V-GEAR system offers versatility by allowing transient screening or stable integration at the AAVS1 safe harbor loci, rapidly achieved through immunomagnetic isolation. This innovative system enables direct comparisons among editing technologies, accelerating the development and testing of genome editing approaches.

Selectivity for local orientation information in visual mirror symmetry perception.

Mirror symmetry is a global percept formed from specific arrangements of matching local information. It has been shown that some features of this local information can interact with the global percept, interfering with symmetry perception. One such feature is orientation; it is well established that the orientation of the symmetry axis has a significant impact on symmetry perception, but the role of local orientation of individual elements is still equivocal. Some studies have argued for no role of local orientation in symmetry perception, while other studies have shown a detrimental effect of certain local orientation combinations. Using dynamic stimuli composed of oriented Gabor elements with increasing temporal delay (SOA) between the onset of the first and second element in a symmetric pair, we systematically map how orientation variation within and between symmetric pairs affected the temporal integration of symmetric patterns in five observers. This method allows consideration of both sensitivity to symmetry (threshold, or T0) as well as the duration of visible persistence of each condition through the visual system (P). Our results show a clear role for local orientation in symmetry perception and highlight the importance of local orientation in symmetry perception. Our findings reinforce the need for more nuanced perceptual models incorporating local element orientation, which is currently unaccounted for.

Post-Concussion Symptoms, Cognition and Brain Connectivity in an Australian Undergraduate Population: A Quantitative Electroencephalography Study.

BACKGROUND: An estimated 99 in 100,000 people experience a traumatic brain injury (TBI), with 85% being mild (mTBI) in nature. The Post-Concussion Symptom Scale (PCSS), is a reliable and valid measure of post-mTBI symptoms; however, diagnostic specificity is challenging due to high symptom rates in the general population. Understanding the neurobiological characteristics that distinguish high and low PCSS raters may provide further clarification on this phenomenon. AIM: To explore the neurobiological characteristics of post-concussion symptoms through the association between PCSS scores, brain network connectivity (using quantitative electroencephalography; qEEG) and cognition in undergraduates. HYPOTHESES: high PCSS scorers will have (1) more network dysregulation and (2) more cognitive dysfunction compared to the low PCSS scorers. METHODS: A sample of 40 undergraduates were divided into high and low PCSS scorers. Brain connectivity was measured using qEEG, and cognition was measured via neuropsychological measures of sustained attention, inhibition, immediate attention, working memory, processing speed and inhibition/switching. RESULTS: Contrary to expectations, greater frontoparietal network dysregulation was seen in the low PCSS score group (p = 0.003). No significant difference in cognitive dysfunction was detected between high and low PCSS scorers. Post-hoc analysis in participants who had experienced mTBI revealed greater network dysregulation in those reporting a more recent mTBI. CONCLUSIONS: Measuring post-concussion symptoms alone is not necessarily informative about changes in underlying neural mechanisms. In an exploratory subset analysis, brain network dysregulation appears to be greater in the early post-injury phase compared to later. Further analysis of underlying PCSS constructs and how to measure these in a non-athlete population and clinical samples is warranted.

BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA.

Mutations in the breast cancer susceptibility gene, BRCA2, greatly increase an individual's lifetime risk of developing breast and ovarian cancers. BRCA2 suppresses tumor formation by potentiating DNA repair via homologous recombination. Central to recombination is the assembly of a RAD51 nucleoprotein filament, which forms on single-stranded DNA (ssDNA) generated at or near the site of chromosomal damage. However, replication protein-A (RPA) rapidly binds to and continuously sequesters this ssDNA, imposing a kinetic barrier to RAD51 filament assembly that suppresses unregulated recombination. Recombination mediator proteins-of which BRCA2 is the defining member in humans-alleviate this kinetic barrier to catalyze RAD51 filament formation. We combined microfluidics, microscopy, and micromanipulation to directly measure both the binding of full-length BRCA2 to-and the assembly of RAD51 filaments on-a region of RPA-coated ssDNA within individual DNA molecules designed to mimic a resected DNA lesion common in replication-coupled recombinational repair. We demonstrate that a dimer of RAD51 is minimally required for spontaneous nucleation; however, growth self-terminates below the diffraction limit. BRCA2 accelerates nucleation of RAD51 to a rate that approaches the rapid association of RAD51 to naked ssDNA, thereby overcoming the kinetic block imposed by RPA. Furthermore, BRCA2 eliminates the need for the rate-limiting nucleation of RAD51 by chaperoning a short preassembled RAD51 filament onto the ssDNA complexed with RPA. Therefore, BRCA2 regulates recombination by initiating RAD51 filament formation.

Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae.

RecA-mediated homologous recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae (SpRecA), in comparison to the Escherichia coli RecA (EcRecA) paradigm. EcRecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block EcRecA nucleofilament growth. We report by direct microscopic analysis of SpRecA filamentation on ssDNA that neither of the two paralogous pneumococcal SSBs could assist the extension of SpRecA nucleopolymers. Instead, we found that the conserved RadA helicase promotes SpRecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native SpRecA nucleopolymer by cryoEM stabilized with ATPγS. It was found to be equivalent to the crystal structure of the EcRecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that SpRecA and EcRecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes.

Contribution of higher-order structure to perception of mirror symmetry: Role of shapes and corners.

Visual mirror symmetry is a global feature that is dependent on specific low-level relationships between component elements. Initially conceptualized as virtual lines between paired elements, it has been suggested that higher-order structure between pairs of symmetric elements forming virtual four cornered shapes may also be important for strengthening the percept of mirror symmetry. We utilize corner elements, formed by joining two Gabor elements along a central midline creating vertices with variable internal angles, in a temporal integration paradigm. This allows us to specifically manipulate the presence and type of higher-order versus lower-order structure in patterns with symmetrically placed elements. We show a significant contribution of higher-order structure to the salience of visual symmetries compared with patterns with only lower-order structures. We also find that although we are more sensitive to patterns with higher-order structure, there is no difference in the temporal processing of higher-order versus lower-order patterns. These findings have important implications for existing spatial filter models of symmetry perception that rely on lower-order structures alone and reinforces the need for elaborated models that can more readily capture the complexities of real-world symmetries.

The Impact of Transparency and Decision Risk on Human-Automation Teaming Outcomes.

OBJECTIVE: Examine the effects of decision risk and automation transparency on the accuracy and timeliness of operator decisions, automation verification rates, and subjective workload. BACKGROUND: Decision aids typically benefit performance, but can provide incorrect advice due to contextual factors, creating the potential for automation disuse or misuse. Decision aids can reduce an operator's manual problem evaluation, and it can also be strategic for operators to minimize verifying automated advice in order to manage workload. METHOD: Participants assigned the optimal unmanned vehicle to complete missions. A decision aid provided advice but was not always reliable. Two levels of decision aid transparency were manipulated between participants. The risk associated with each decision was manipulated using a financial incentive scheme. Participants could use a calculator to verify automated advice; however, this resulted in a financial penalty. RESULTS: For high- compared with low-risk decisions, participants were more likely to reject incorrect automated advice and were more likely to verify automation and reported higher workload. Increased transparency did not lead to more accurate decisions and did not impact workload but decreased automation verification and eliminated the increased decision time associated with high decision risk. CONCLUSION: Increased automation transparency was beneficial in that it decreased automation verification and decreased decision time. The increased workload and automation verification for high-risk missions is not necessarily problematic given the improved automation correct rejection rate. APPLICATION: The findings have potential application to the design of interfaces to improve human-automation teaming, and for anticipating the impact of decision risk on operator behavior.

Trustworthiness perception is mandatory: Task instructions do not modulate fast periodic visual stimulation trustworthiness responses.

Although it is often assumed that humans spontaneously respond to the trustworthiness of others' faces, it is still unclear whether responses to facial trust are mandatory or can be modulated by instructions. Considerable scientific interest lies in understanding whether trust processing is mandatory, given the societal consequences of biased trusting behavior. We tested whether neural responses indexing trustworthiness discrimination depended on whether the task involved focusing on facial trustworthiness or not, using a fast periodic visual stimulation electroencephalography oddball paradigm with a neural marker of trustworthiness discrimination at 1 Hz. Participants judged faces on size without any reference to trust, explicitly formed impressions of facial trust, or were given a financial lending context that primed trust, without explicit trust judgement instructions. Significant trustworthiness discrimination responses at 1 Hz were found in all three conditions, demonstrating the robust nature of trustworthiness discrimination at the neural level. Moreover, no effect of task instruction was observed, with Bayesian analyses providing moderate to decisive evidence that task instruction did not affect trustworthiness discrimination. Our finding that visual trustworthiness discrimination is mandatory points to the remarkable spontaneity of trustworthiness processing, providing clues regarding why these often unreliable impressions are ubiquitous.

A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes.

Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research. Here, we demonstrate that CRISPR-Cas9 RNPs can be used for efficient gene knockout in primary human monocytes. In addition, we demonstrate that intracellular RNases are likely responsible for poor and heterogenous mRNA expression as incorporation of pan-RNase inhibitor allows efficient genome engineering following mRNA-based delivery of Cas9 and base editor enzymes. Moreover, we demonstrate that CRISPR-Cas9 combined with an rAAV vector DNA donor template mediates site-specific insertion and expression of a transgene in primary human monocytes. Finally, we demonstrate that SIRPa knock-out monocyte-derived macrophages have enhanced activity against cancer cells, highlighting the potential for application in cellular immunotherapies.

The influence of three-dimensional cues on body size judgements.

Research has shown that body size judgements are frequently biased, or inaccurate. Critically, judgement biases are further exaggerated for individuals with eating disorders, a finding that has been attributed to difficulties integrating body features into a perceptual whole. However, current understanding of which body features are integrated when judging body size is lacking. In this study, we examine whether individuals integrate three-dimensional (3D) cues to body volume when making body size judgements. Computer-generated body stimuli were presented in a 3D Virtual Reality environment. Participants (N = 412) were randomly assigned to one of the two conditions: in one condition, the to-be-judged body was displayed binocularly (containing 3D cues to body volume); in the other, bodies were presented monocularly (two-dimensional [2D] cues only). Across 150 trials, participants were required to make a body size judgement of a target female body from a third-person point of view using an unmarked visual analogue scale (VAS). It was found that 3D cues significantly influenced body size judgements. Namely, thin 3D bodies were judged smaller, and overweight 3D bodies were judged larger, than their 2D counterpart. Furthermore, to reconcile these effects, we present evidence that the two perceptual biases, regression to the mean and serial dependence, were reduced by the additional 3D feature information. Our findings increase our understanding of how body size is perceptually encoded and creates testable predictions for clinical populations exhibiting integration difficulties.

Serial dependence of facial identity for own- and other-race faces.

It is well established that individuals are better at recognising faces of their own-race compared with other-races; however, there is ongoing debate regarding the perceptual mechanisms that may be involved and therefore sensitive to face-race. Here, we ask whether serial dependence of facial identity, a bias where the perception of a face's identity is biased towards a previously presented face, shows an other-race effect. Serial dependence is associated with face recognition ability and appears to operate on high-level, face-selective representations, like other candidate mechanisms (e.g., holistic processing). We therefore expected to find an other-race effect for serial dependence for our Caucasian and Asian participants. While participants showed robust effects of serial dependence for all faces, only Caucasian participants showed stronger serial dependence for own-race faces. Intriguingly, we found that individual variation in own-race, but not other-race, serial dependence was significantly associated with face recognition abilities. Preliminary evidence also suggested that other-race contact is associated with other-race serial dependence. In conclusion, though we did not find an overall difference in serial dependence for own- versus other-race faces in both participant groups, our results highlight that this bias may be functionally different for own- versus other-race faces and sensitive to racial experience.

Autistic Traits are Associated with Less Precise Perceptual Integration of Face Identity.

Face recognition difficulties are common in autism and could be a consequence of perceptual atypicalities that disrupt the ability to integrate current and prior information. We tested this theory by measuring the strength of serial dependence for faces (i.e. how likely is it that current perception of a face is biased towards a previously seen face) across the broader autism phenotype. Though serial dependence was not weaker in individuals with more autistic traits, more autistic traits were associated with greater integration of less similar faces. These results suggest that serial dependence is less specialised, and may not operate optimally, in individuals with more autistic traits and could therefore be a contributing factor to autism-linked face recognition difficulties.

Sizing up the crowd: Assessing spatial integration difficulties in body size judgements across eating disorder symptomatology.

Introduction: Body size judgements are frequently biased, or inaccurate, and these errors are further exaggerated for individuals with eating disorders. Within the eating disorder literature, it has been suggested that exaggerated errors in body size judgements are due to difficulties with integration. Across two experiments, we developed a novel integration task, named the Ebbinghaus Illusion for Bodies in Virtual Reality (VR), to assess whether nearby bodies influence the perceived size of a single body. VR was used to simulate the appearance of a small crowd around a central target body. Method and Results: In Experiment 1 (N = 412), participants were required to judge the size of a central female target within a crowd. Experiment 1 revealed an Ebbinghaus Illusion, in which a central female appeared larger when surrounded by small distractors, but comparatively smaller when surrounded by large distractors. In other words, the findings of Experiment 1 demonstrate that surrounding crowd information is integrated when judging an individual's body size; a novel measure of spatial integration (i.e., an Ebbinghaus Illusion for Bodies in VR). In Experiment 2 (N = 96), female participants were selected based on high (n = 43) and low (n = 53) eating disorder symptomatology. We examined whether the magnitude of this illusion would differ amongst those with elevated versus low eating disorder symptomatology, in accordance with weak central coherence theory, with the high symptomatology group displaying less spatial integration relative to the low group. The results of Experiment 2 similarly found an Ebbinghaus Illusion for Bodies in VR. However, illusion magnitude did not vary across high and low symptomatology groups. Discussion: Overall, these findings demonstrate that surrounding crowd information is integrated when judging individual body size; however, those with elevated eating disorder symptomatology did not show any integration deficit on this broader measure of spatial integration.

Blinded by bodies: Elevated eating disorder symptomatology is associated with increased attentional priority for thin bodies.

Selective processing of female thin-ideal body imagery is associated with greater body dissatisfaction, and eating disorder-specific rumination has been shown to mediate this relationship. Across two studies, we employed a modified rapid serial visual presentation task (similar to that used within the emotion-induced blindness literature), such that participants searched for a task-relevant target that was sometimes preceded by a thin body, non-thin body, or neutral task-irrelevant distractor. Our first experiment (N = 372) revealed a "body-induced blindness" in an unselected female sample, such that bodies in general distracted attention more than neutral images, and non-thin bodies distracted more than thin-ideal bodies. In our second experiment, female participants were selected based on eating disorder symptomatology (N = 114). Females that exhibited elevated eating disorder symptoms were distracted more by thin bodies compared to those low in symptomatology, greater distraction from thin bodies was associated with greater body dissatisfaction, and this relationship was mediated by eating disorder-specific rumination. Altogether, our findings illustrate the persistent nature of attentional distortions that occur early in cognitive processing and across time for those high in eating disorder symptomatology.

Using ThinPrep Papanicolaou test samples to evaluate sexually transmitted infection screening practices.

INTRODUCTION: We sought to evaluate the use of Papanicolaou samples as a screening tool for sexually transmitted infections (STIs). METHODS: Retrospective chart review analyzing Papanicolaou samples for STI. Samples were processed and results compared to clinical data to assess this technique's viability. Cases and controls were matched by sample date. Characteristics of women with STI testing were compared in bivariate analyses. RESULTS: We analyzed 50 STI-positive and 50 date-matched samples. Thirteen (26.0%) of the STI-positive patients were not screened at their visit. Women without STI screening were older (39.5 vs. 30.0 years, P = 0.001); non-Hispanic White (65.9% vs. 46.4%, P = 0.05); and married (60.0% vs. 26.9%, P = 0.005) than women with STI screening. Fifty-eight were offered and accepted STI testing at their visit; 37 samples were STI-positive: 17 (29.3%) Mycoplasma genitalium (Mgen), 10 (17.2%) chlamydia, 6 (10.3%) trichomoniasis, 1 (1.7%) gonorrhea, and 3 (5.2%) had two STIs. Among the 42 patients without STI testing, 12 (28.6%) had positive samples: 6 (14.3%) chlamydia, 5 (12.0%) Mgen, and 1 (2.4%) trichomoniasis. CONCLUSIONS: Over one-quarter of STI-positive patients were not screened; though low-risk by current screening criteria, a significant number may harbor untreated STIs; using Papanicolaou samples may allow for increased screening in this population.

The temporal integration windows for visual mirror symmetry.

Symmetry perception in dot patterns is tolerant to temporal delays of up to 60 ms within and between element pairs. However, it is not known how factors effecting symmetry discrimination in static patterns might affect temporal integration in dynamic patterns. One such feature is luminance polarity. Using dynamic stimuli with increasing temporal delay (SOA) between the onset of the first and second element in a symmetric pair, we investigated how four different luminance-polarity conditions affected the temporal integration of symmetric patterns. All four luminance polarity conditions showed similar upper temporal limits of approximately 60 ms. However psychophysical performance over all delay durations showed significantly higher symmetry thresholds for unmatched-polarity patterns at short delays, but also significantly less sensitivity to increasing temporal delay relative to matched-polarity patterns. These varying temporal windows are consistent with the involvement of a fast, sensitive first-order mechanism for matched-polarity patterns, and a slower, more robust second-order mechanism for unmatched-polarity patterns. Temporal integration windows for unmatched-polarity patterns were not consistent with performance expected from attentional mechanisms alone, and instead supports the involvement of second-order mechanisms that combines information from ON and OFF channels.

Comparison of surgical versus medical termination of pregnancy between 13-20 weeks of gestation in Ethiopia: A quasi-experimental study.

BACKGROUND: Dilation and evacuation is a method of second trimester pregnancy termination introduced recently in Ethiopia. However, little is known about the safety and effectiveness of this method in an Ethiopian setting. Therefore, the study is intended to determine the safety and effectiveness of dilation and evacuation for surgical abortion as compared to medical abortion between 13-20 weeks' gestational age. METHODS: This is a quasi-experimental study of women receiving second trimester termination of pregnancy between 13-20 weeks. Patients were allocated to either medical or surgical abortion based on their preference. A structured questionnaire was used to collect demographic information and clinical data upon admission. Procedure related information was collected after the procedure was completed and before the patient was discharged. Additionally, women were contacted 2 weeks after the procedure to evaluate for post-procedural complications. The primary outcome of the study was a composite complication rate. Data were collected using Open Data Kit and then analyzed using Stata version 14.2. Univariate analyses were performed using means (standard deviation), or medians (interquartile range) when the distribution was not normal. Multiple logistic regression was also performed to control for confounders. RESULTS: Two hundred nineteen women chose medical abortion and 60 chose surgical abortion. The composite complication rate is not significantly different among medical and surgical abortion patients (15% versus 10%; p = 0.52). Nine patients (4.1%) in the medical arm required additional intervention to complete the abortion, while none of the surgical abortion patients required additional intervention. Median (IQR) hospital stay was significantly longer in the medical group at 24 (12-24) hours versus 6(4-6) hours in the surgical group p<0.001. CONCLUSION: From the current study findings, we concluded that there is no difference in safety between surgical and medical methods of abortion. This study demonstrates that surgical abortion can be used as a safe and effective alternative to medical abortion and should be offered equivalently with medical abortion, per the patient's preference.

ZBTB20 regulates WNT/CTNNB1 signalling pathway by suppressing PPARG during hepatocellular carcinoma tumourigenesis.

BACKGROUND & AIMS: Zinc finger and BTB domain containing 20 (ZBTB20) has been implicated as a potential oncogene in liver cancer. However, knockout studies have shown it to be a transcriptional repressor of the alpha-foetoprotein (Afp) gene in adult liver, and reduced levels of ZBTB20 allow for upregulation of AFP with increased tumour severity in certain cases of hepatocellular carcinoma (HCC). As there are many discrepancies in the literature regarding its role in liver tumourigenesis, the aim of this study was to elucidate the role of ZBTB20 in HCC tumourigenesis. METHODS: A reverse genetic study using the Sleeping Beauty (SB) transposon system in mice was performed to elucidate the role of ZBTB20 in HCC tumourigenesis. In vitro ZBTB20 gain- and loss-of-function experiments were used to assess the relationship amongst ZBTB20, peroxisome proliferator activated receptor gamma (PPARG) and catenin beta 1 (CTNNB1). RESULTS: Transgenic overexpression of ZBTB20 in hepatocytes and in the context of transformation related protein (T r p53) inactivation induced hepatic hypertrophy, activation of WNT/CTNNB1 signalling, and development of liver tumours. In vitro overexpression and knockout experiments using CRISPR/Cas9 demonstrated the important role for ZBTB20 in downregulating PPARG, resulting in activation of the WNT/CTNNB1 signalling pathway and its downstream effectors in HCC tumourigenesis. CONCLUSIONS: These findings demonstrate a novel interaction between ZBTB20 and PPARG, which leads to activation of the WNT/CTNNB1 signalling pathway in HCC tumourigenesis. LAY SUMMARY: ZBTB20 has been implicated as a potential oncogene in liver cancer. Herein, we uncover its important role in liver cancer development. We show that it interacts with PPARG to upregulate the WNT/CTNNB1 signalling pathway, leading to tumourigenesis.

Serial dependence of facial identity reflects high-level face coding.

Serial dependence of facial identity is a type of bias where the perceived identity of a face is biased towards a previously presented face. There are individual differences in serial dependence strength and tuning (how the strength varies depending on stimuli similarity), and previous research has shown that both stronger and more narrowly tuned serial dependence of facial identity is associated with better face recognition abilities. These results are consistent with the idea that this bias plays a functional role in face perception. It is important, therefore, to determine whether serial dependence of facial identity reflects a high-level face-coding mechanism acting on the identity of a face or instead predominantly reflects a bias in low-level features, which are also subject to serial dependence. We first sought evidence that serial dependence of facial identity survived changes in low-level visual features, by varying face viewpoint between successive stimuli. We found that serial dependence persisted across changes in viewpoint, arguing against an entirely low-level locus for this bias. We next tested whether the bias was affected by inversion, as sensitivity to inversion is argued to be a characteristic of high-level face-selective processing. Serial dependence was stronger and more narrowly tuned for upright than inverted faces. Taken together, our results are consistent with the view that serial dependence of facial identity affects high-level visual representations and may reflect a face-coding mechanism that is operating at the level of facial identity.