Phage-assisted continuous evolution of proteases with altered substrate specificity

Here we perform phage-assisted continuous evolution (PACE) of TEV protease, which canonically cleaves ENLYFQS, to cleave a very different target sequence, HPLVGHM, that is present in human IL-23. A protease emerging from ∼2500 generations of PACE contains 20 non-silent mutations, cleaves human IL-23 at the target peptide bond, and when pre-mixed with IL-23 in primary cultures of murine splenocytes inhibits IL-23-mediated immune signaling. We characterize the substrate specificity of this evolved enzyme, revealing shifted and broadened specificity changes at the six positions in which the target amino acid sequence differed. Mutational dissection and additional protease specificity profiling reveal the molecular basis of some of these changes. This work establishes the capability of changing the substrate specificity of a protease at many positions in a practical time scale and provides a foundation for the development of custom proteases that catalytically alter or destroy target proteins for biotechnological and therapeutic applications

Questionnaire study to gain an insight into the manufacturing and fitting process of artificial eyes in children: an ocularist perspective

Purpose To gain an insight into the manufacturing and fitting of artificial eyes in children and potential improvements to the process. Method An online qualitative survey was distributed to 39 ocularists/prosthetists in Europe and Canada. Participants were recruited through purposive sampling, specifically maximum variation sampling from the researcher’s contacts and an online search. Results The findings highlighted the current impression technique as being the most difficult yet most important part of the current process for both the ocularist and child patient. Negatively affecting obtaining a good impression, the child patients distress can be reduced by their parents by providing encouragement, reassurance, practicing the insertion and removal of the artificial eye and being matter of fact. Whilst improvements to the current process provided mixed views, the incorporation of current technology was perceived as not being able to meet the requirements to produce aesthetically pleasing artificial eyes. Conclusion The current artificial eye process can be seen as an interaction with its success being dependent on the child patient’s acceptance and adjustment which is dependent on the factors associated to the process. Investigation into the needs of the patient and whether technology can improve the process are the next steps in its advancement

Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery

We recently developed base editing, a genome-editing approach that enables the programmable conversion of one base pair into another without double-stranded DNA cleavage, excess stochastic insertions and deletions, or dependence on homology-directed repair. The application of base editing is limited by off-target activity and reliance on intracellular DNA delivery. Here we describe two advances that address these limitations. First, we greatly reduce off-target base editing by installing mutations into our third-generation base editor (BE3) to generate a high-fidelity base editor (HF-BE3). Next, we purify and deliver BE3 and HF-BE3 as ribonucleoprotein (RNP) complexes into mammalian cells, establishing DNA-free base editing. RNP delivery of BE3 confers higher specificity even than plasmid transfection of HF-BE3, while maintaining comparable on-target editing levels. Finally, we apply these advances to deliver BE3 RNPs into both zebrafish embryos and the inner ear of live mice to achieve specific, DNA-free base editing in vivo

Punishment in the Frame: Rethinking the History and Sociology of Art

Images of punishment have featured prominently in Western art and this article explores what might be learnt from studying such pictures of suffering. It seeks to develop an approach to the visual that avoids both the essentialism of art history and the reductionism of sociology by offering a rethinking of the relationships between the two. It begins by setting out the current state of the sociology of art, before discussing ‘new’ art histories that are inspired by social analysis. It then concentrates on how images of punishment have featured in Western art. This substantive material provides a rich resource to understand the force of representation and offers an opportunity to develop an aesthetic sociology that avoids some of the problems identified in the article. The approach developed in the second part is one that seeks to elaborate an aesthetic sociology that combines a historical sensitivity to images with the analytical concerns of social science. It strives to extend the art historian Michael Baxandall’s writings toward more sociological interpretations of visual analysis

Trait impulsivity in Juvenile Myoclonic Epilepsy

Impulsivity is a multidimensional construct that can predispose to psychopathology. Meta‐analysis demonstrates an association between response impulsivity and Juvenile Myoclonic Epilepsy (JME), a common genetic generalized epilepsy. Here, we test the hypotheses that trait impulsivity is (i) elevated in JME compared to controls; (ii) moderated by specific seizure characteristics; and (iii) associated with psychiatric adverse effects of antiepileptic drugs (AEDs)

Lynch syndrome: barriers to and facilitators of screening and disease management

Background Lynch syndrome is a hereditary cancer with confirmed carriers at high risk for colorectal (CRC) and extracolonic cancers. The purpose of the current study was to develop a greater understanding of the factors influencing decisions about disease management post-genetic testing. Methods The study used a grounded theory approach to data collection and analysis as part of a multiphase project examining the psychosocial and behavioral impact of predictive DNA testing for Lynch syndrome. Individual and small group interviews were conducted with individuals from 10 families with the MSH2 intron 5 splice site mutation or exon 8 deletion. The data from confirmed carriers (n = 23) were subjected to re-analysis to identify key barriers to and/or facilitators of screening and disease management. Results Thematic analysis identified personal, health care provider and health care system factors as dominant barriers to and/or facilitators of managing Lynch syndrome. Person-centered factors reflect risk perceptions and decision-making, and enduring screening/disease management. The perceived knowledge and clinical management skills of health care providers also influenced participation in recommended protocols. The health care system barriers/facilitators are defined in terms of continuity of care and coordination of services among providers. Conclusions Individuals with Lynch syndrome often encounter multiple barriers to and facilitators of disease management that go beyond the individual to the provider and health care system levels. The current organization and implementation of health care services are inadequate. A coordinated system of local services capable of providing integrated, efficient health care and follow-up, populated by providers with knowledge of hereditary cancer, is necessary to maintain optimal health

Sex-specific disease modifiers in juvenile myoclonic epilepsy

Juvenile myoclonic epilepsy (JME) is a common idiopathic generalised epilepsy with variable seizure prognosis and sex differences in disease presentation. Here, we investigate the combined epidemiology of sex, seizure types and precipitants, and their influence on prognosis in JME, through cross-sectional data collected by The Biology of Juvenile Myoclonic Epilepsy (BIOJUME) consortium. 765 individuals met strict inclusion criteria for JME (female:male, 1.8:1). 59% of females and 50% of males reported triggered seizures, and in females only, this was associated with experiencing absence seizures (OR = 2.0, p < 0.001). Absence seizures significantly predicted drug resistance in both males (OR = 3.0, p = 0.001) and females (OR = 3.0, p < 0.001) in univariate analysis. In multivariable analysis in females, catamenial seizures (OR = 14.7, p = 0.001), absence seizures (OR = 6.0, p < 0.001) and stress-precipitated seizures (OR = 5.3, p = 0.02) were associated with drug resistance, while a photoparoxysmal response predicted seizure freedom (OR = 0.47, p = 0.03). Females with both absence seizures and stress-related precipitants constitute the prognostic subgroup in JME with the highest prevalence of drug resistance (49%) compared to females with neither (15%) and males (29%), highlighting the unmet need for effective, targeted interventions for this subgroup. We propose a new prognostic stratification for JME and suggest a role for circuit-based risk of seizure control as an avenue for further investigation

HER2-enriched subtype and novel molecular subgroups drive aromatase inhibitor resistance and an increased risk of relapse in early ER+/HER2+ breast cancer

BACKGROUND: Oestrogen receptor positive/ human epidermal growth factor receptor positive (ER+/HER2+) breast cancers (BCs) are less responsive to endocrine therapy than ER+/HER2- tumours. Mechanisms underpinning the differential behaviour of ER+HER2+ tumours are poorly characterised. Our aim was to identify biomarkers of response to 2 weeks’ presurgical AI treatment in ER+/HER2+ BCs. METHODS: All available ER+/HER2+ BC baseline tumours (n=342) in the POETIC trial were gene expression profiled using BC360™ (NanoString) covering intrinsic subtypes and 46 key biological signatures. Early response to AI was assessed by changes in Ki67 expression and residual Ki67 at 2 weeks (Ki672wk). Time-To-Recurrence (TTR) was estimated using Kaplan-Meier methods and Cox models adjusted for standard clinicopathological variables. New molecular subgroups (MS) were identified using consensus clustering. FINDINGS: HER2-enriched (HER2-E) subtype BCs (44.7% of the total) showed poorer Ki67 response and higher Ki672wk (p<0.0001) than non-HER2-E BCs. High expression of ERBB2 expression, homologous recombination deficiency (HRD) and TP53 mutational score were associated with poor response and immune-related signatures with High Ki672wk. Five new MS that were associated with differential response to AI were identified. HER2-E had significantly poorer TTR compared to Luminal BCs (HR 2.55, 95% CI 1.14–5.69; p=0.0222). The new MS were independent predictors of TTR, adding significant value beyond intrinsic subtypes. INTERPRETATION: Our results show HER2-E as a standardised biomarker associated with poor response to AI and worse outcome in ER+/HER2+. HRD, TP53 mutational score and immune-tumour tolerance are predictive biomarkers for poor response to AI. Lastly, novel MS identify additional non-HER2-E tumours not responding to AI with an increased risk of relapse