"Forcing" new interpretations of molecular tension sensor studies.

Molecular tension sensors are central tools for mechanobiology studies but have limitations in interpretation. Reporting in Cell Reports Methods, Shoyer et al. discover that fluorescent protein photoswitching in concert with sensor extension may expand the use and interpretation of common force-sensing tools.

HUHgle: An Interactive Substrate Design Tool for Covalent Protein-ssDNA Labeling Using HUH-Tags.

HUH-tags have emerged as versatile fusion partners that mediate sequence specific protein-ssDNA bioconjugation through a simple and efficient reaction. Here we present HUHgle, a python-based interactive tool for the visualization, design, and optimization of substrates for HUH-tag mediated covalent labeling of proteins of interest with ssDNA substrates of interest. HUHgle streamlines design processes by integrating an intuitive plotting interface with a search function capable of predicting and displaying protein-ssDNA bioconjugate formation efficiency and specificity in proposed HUH-tag/ssDNA sequence combinations. Validation demonstrates that HUHgle accurately predicts product formation of HUH-tag mediated bioconjugation for single- and orthogonal-labeling reactions. In order to maximize the accessibility and utility of HUHgle, we have implemented it as a user-friendly Google Colab notebook which facilitates broad use of this tool, regardless of coding expertise.

Null regions: a unified conceptual framework for statistical inference.

Ruling out the possibility that there is absolutely no effect or association between variables may be a good first step, but it is rarely the ultimate goal of science. Yet that is the only inference provided by traditional null hypothesis significance testing (NHST), which has been a mainstay of many scientific fields. Reliance on NHST also makes it difficult to define what it means to replicate a finding, and leads to an uncomfortable quandary in which increasing precision in data reduces researchers' ability to perform theory falsification. To solve these problems, in recent years several alternatives to traditional NHST have been proposed. However, each new test is described using its own terminology and practiced in different fields. We describe a simple, unified framework for conceptualizing all these tests so that it is not necessary to learn them separately. Moreover, the framework allows researchers to conduct any of these tests by asking just one question: is the confidence interval entirely outside the null region(s)? This framework may also help researchers choose the test(s) that best answers their research question when simply ruling out 'no effect at all' is not enough.

RAD-TGTs: high-throughput measurement of cellular mechanotype via rupture and delivery of DNA tension probes.

Mechanical forces drive critical cellular processes that are reflected in mechanical phenotypes, or mechanotypes, of cells and their microenvironment. We present here "Rupture And Deliver" Tension Gauge Tethers (RAD-TGTs) in which flow cytometry is used to record the mechanical history of thousands of cells exerting forces on their surroundings via their propensity to rupture immobilized DNA duplex tension probes. We demonstrate that RAD-TGTs recapitulate prior DNA tension probe studies while also yielding a gain of fluorescence in the force-generating cell that is detectable by flow cytometry. Furthermore, the rupture propensity is altered following disruption of the cytoskeleton using drugs or CRISPR-knockout of mechanosensing proteins. Importantly, RAD-TGTs can differentiate distinct mechanotypes among mixed populations of cells. We also establish oligo rupture and delivery can be measured via DNA sequencing. RAD-TGTs provide a facile and powerful assay to enable high-throughput mechanotype profiling, which could find various applications, for example, in combination with CRISPR screens and -omics analysis.

Watson-Crick Base-Pairing Requirements for ssDNA Recognition and Processing in Replication-Initiating HUH Endonucleases.

Replication-initiating HUH endonucleases (Reps) are sequence-specific nucleases that cleave and rejoin single-stranded DNA (ssDNA) during rolling-circle replication. These functions are mediated by covalent linkage of the Rep to its substrate post cleavage. Here, we describe the structures of the endonuclease domain from the Muscovy duck circovirus Rep in complex with its cognate ssDNA 10-mer with and without manganese in the active site. Structural and functional analyses demonstrate that divalent cations play both catalytic and structural roles in Reps by polarizing and positioning their substrate. Further structural comparisons highlight the importance of an intramolecular substrate Watson-Crick (WC) base pairing between the -4 and +1 positions. Subsequent kinetic and functional analyses demonstrate a functional dependency on WC base pairing between these positions regardless of the pair's identity (i.e., A·T, T·A, G·C, or C·G), highlighting a structural specificity for substrate interaction. Finally, considering how well WC swaps were tolerated in vitro, we sought to determine to what extent the canonical -4T·+1A pairing is conserved in circular Rep-encoding single-stranded DNA viruses and found evidence of noncanonical pairings in a minority of these genomes. Altogether, our data suggest that substrate intramolecular WC base pairing is a universal requirement for separation and reunion of ssDNA in Reps. IMPORTANCE Circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses are a ubiquitous group of viruses that infect organisms across all domains of life. These viruses negatively impact both agriculture and human health. All members of this viral family employ a multifunctional nuclease (Rep) to initiate replication. Reps are structurally similar throughout this family, making them targets of interest for viral inhibition strategies. Here, we investigate the functional dependencies of the Rep protein from Muscovy duck circovirus for ssDNA interaction. We demonstrate that this Rep requires an intramolecular Watson-Crick base pairing for origin of replication (Ori) recognition and interaction. We show that noncognate base pair swaps are well tolerated, highlighting a local structural specificity over sequence specificity. Bioinformatic analysis found that the vast majority of CRESS-DNA Oris form base pairs in conserved positions, suggesting this pairing is a universal requirement for replication initiation in the CRESS-DNA virus family.

The Golden Age Is Behind Us: How the Status Quo Impacts the Evaluation of Technology.

New technology invariably provokes concerns over potential societal impacts. Even as risks often fail to materialize, the fear continues. The current research explored the psychological underpinnings of this pattern. Across four studies (N = 2,454 adults recruited via Amazon Mechanical Turk), we found evidence for the role of status quo thinking in evaluating technology. In Study 1, we experimentally manipulated the reported age of unfamiliar technology and found that people evaluate it more favorably when it is described as originating before (vs. after) their birth. In Studies 2 through 4, participants' age at the time of invention strongly predicts attitudes toward a wide range of real-world technologies. Finally, we found that individual differences in status-quo-based decision-making moderated evaluations of technology. These studies provide insight into how people respond to the rapidly changing technological landscape.

The Impact of Training in Transgender Care on Healthcare Providers Competence and Confidence: A Cross-Sectional Survey.

All studies to date demonstrate a lack of access to care for transgender people. A few educational efforts in providing care to transgender people have been successful. However, one challenge in administering training is that there is almost no research on the need of healthcare providers (HCP) to acquire knowledge, as well as on the effect of training on their level of competence and confidence in working with transgender people. Results from an online survey of a convenience sample of HCP across four different European countries (N = 810) showed that 52.7% reported experiences with some form of training on transgender people. The mean confidence level for all HCP (with or without training) in working with transgender people was 2.63, with a significant effect of training on confidence. 92.4% of HCP believed that training would raise their competence, and this belief was significantly higher among HCP with training experience, HCP working in Serbia and Sweden and/or among those HCP who belong to a sexual minority group. General practitioners had the lowest confidence levels of all professions involved. The study provided strong support for the use of training in improving healthcare conditions for transgender people, not only to raise awareness among HCP, but also to increase knowledge, competence and confidence levels of HCP in working with transgender people.

Information without knowledge: the effects of Internet search on learning.

The Internet has radically shifted how people access information. Instead of storing information internally, increasingly, people outsource to the Internet and retrieve it when needed. While this is an efficient strategy in many ways, its downstream consequences remain largely unexplored. This research examines how accessing online information impacts how people remember information in a learning context. Across five experiments, participants studied for a quiz either by searching online to access relevant information or by directly receiving that same information without online search. Those who searched the Internet performed worse in the learning assessment, indicating that they stored less new knowledge in internal memory. However, participants who searched the Internet were as confident, or even more confident, that they had mastered the study material compared to those who did not search online. We argue that, by making information retrievability salient, Internet search reduces the likelihood of information being stored in memory. Further, these results suggest that searching online leads to the misattribution of online information to internal memory, thus masking the Internet-induced learning deficits.

Health disparities between binary and non binary trans people: A community-driven survey.

Background: Genderqueer and nonbinary () people have remained largely invisible in health research. Previous research shows worse outcomes on health indicators for trans people when compared with cisgender controls, but the differences between binary trans and GQNB individuals are inconclusive. Aims: To compare overall health and well-being of GQNB people with controls of trans men and trans women, taking into account the impact of the additive effect of their socio-economic position, as well as their current need for gender affirming medical interventions. Methods: A community-driven survey was conducted in 2016 in five countries (Georgia, Poland, Serbia, Spain, and Sweden). Self-reported health and general well-being were analysed for differences between binary trans and GQNB respondents. The effects of multiple control variables (age, economic situation, educational level, belonging to an ethnic, religious, sexual or ability minority group, sex assigned at birth) as well as the current need for gender affirming medical interventions were controlled for. Results: The sample consisted of 853 respondents aged 16 and older, with 254 trans women (29.8%), 369 trans men (43.2%), and 230 GQNB people (26%). GQNB respondents showed significantly worse self-reported health and worse general well-being in comparison to binary trans respondents. Additional negative impacts of having a lower educational level, having more economic stress, and belonging to a disability minority group were found. Being in need of gender affirming medical interventions contributed significantly to worse self-reported health, whereas being younger contributed to worse general well-being. Discussion: In understanding health disparities between binary trans and GQNB people, it is necessary to take into account the additive effect of multiple socio-economic positions, and the current need for gender affirming medical interventions. The high proportion of GQNB respondents who report worse health outcomes highlights the need for policy makers and health-care providers in creating nonbinary-inclusive environments.

Depathologising gender diversity in childhood in the process of ICD revision and reform.

From 2007 on, the World Health Organisation (WHO) has been revising its diagnostic manual, the International Statistical Classification of Diseases and Related Health Problems (ICD), with approval of ICD-11 due in 2018. The ICD revision has prompted debates on diagnostic classifications related to gender diversity and gender development processes, and specifically on the 'Gender incongruence of childhood' (GIC) code. These debates have taken place at a time an emergent trans depathologisation movement is becoming increasingly international, and regional and international human rights bodies are recognising gender identity as a source of discrimination. With reference to the history of diagnostic classification of gender diversity in childhood, this paper conducts a literature review of academic, activist and institutional documents related to the current discussion on the merits of retaining or abandoning the GIC code. Within this broader discussion, the paper reviews in more detail recent publications arguing for the abandonment of this diagnostic code drawing upon clinical, bioethical and human rights perspectives. The review indicates that gender diverse children engaged in exploring their gender identity and expression do not benefit from diagnosis. Instead they benefit from support from their families, their schools and from society more broadly.