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We recently developed "autonomous hypermutation yeast surface display" (AHEAD), a technology that enables the rapid generation of potent and specific antibodies in yeast. AHEAD pairs yeast surface display with an error-prone orthogonal DNA replication system (OrthoRep) to continuously and rapidly mutate surface-displayed antibodies, thereby enabling enrichment for stronger binding variants through repeated rounds of cell growth and fluorescence-activated cell sorting. AHEAD currently utilizes a standard galactose induction system to drive the selective display of antibodies on the yeast surface. However, achieving maximal display levels can require up to 48 h of induction. Here we report an updated version of the AHEAD platform that utilizes a synthetic β-estradiol-induced gene expression system to regulate the surface display of antibodies and find that induction is notably faster in achieving surface display for both our AHEAD system and traditional yeast surface display from nuclear plasmids that do not hypermutate. The updated AHEAD platform was fully functional in repeated rounds of evolution to drive the rapid evolution of antibodies.

Orthogonal replication enables rapid continuous biomolecular evolution in Escherichia coli .

Advances in synthetic biology, bioengineering, and computation allow us to rapidly and reliably program cells with increasingly complex and useful functions. However, because the functions we engineer cells to perform are typically burdensome to cell growth, they can be rapidly lost due to the processes of mutation and natural selection. Here, we show that a strategy of terminal differentiation improves the evolutionary stability of burdensome functions in a general manner by realizing a reproductive and metabolic division of labor. To implement this strategy, we develop a genetic differentiation circuit in Escherichia coli using unidirectional integrase-recombination. With terminal differentiation, differentiated cells uniquely express burdensome functions driven by the orthogonal T7 RNA polymerase, but their capacity to proliferate is limited to prevent the propagation of advantageous loss-of-function mutations that inevitably occur. We demonstrate computationally and experimentally that terminal differentiation increases duration and yield of high-burden expression and that its evolutionary stability can be improved with strategic redundancy. Further, we show this strategy can even be applied to toxic functions. Overall, this study provides an effective, generalizable approach for protecting burdensome engineered functions from evolutionary degradation., (© 2022. The Author(s).)

Major changes in the microbiome are associated with health and disease. Some microbiome states persist despite seemingly unfavorable conditions, such as the proliferation of aerobe-anaerobe communities in oxygen-exposed environments in wound infections or small intestinal bacterial overgrowth. Mechanisms underlying transitions into and persistence of these states remain unclear. Using two microbial taxa relevant to the human microbiome, we combine genome-scale mathematical modeling, bioreactor experiments, transcriptomics, and dynamical systems theory to show that multistability and hysteresis (MSH) is a mechanism describing the shift from an aerobe-dominated state to a resilient, paradoxically persistent aerobe-anaerobe state. We examine the impact of changing oxygen and nutrient regimes and identify changes in metabolism and gene expression that lead to MSH and associated multi-stable states. In such systems, conceptual causation-correlation connections break and MSH must be used for analysis. Using MSH to analyze microbiome dynamics will improve our conceptual understanding of stability of microbiome states and transitions between states., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Purpose: In neurofibromatosis type 1 (NF1) and in highly aggressive malignant peripheral nerve sheath tumors (MPNSTs), constitutively active RAS-GTP and increased MAPK signaling are important in tumorigenesis. Dual specificity phosphatases (DUSPs) are negative regulators of MAPK signaling that dephosphorylate p38, JNK, and ERK in different settings. Although often acting as tumor suppressors, DUSPs may also act as oncogenes, helping tumor cells adapt to high levels of MAPK signaling. We hypothesized that inhibiting DUSPs might be selectively toxic to cells from NF1-driven tumors., Experimental Design: We examined DUSP gene and protein expression in neurofibroma and MPNSTs. We used small hairpin RNA (shRNA) to knock down DUSP1 and DUSP6 to evaluate cell growth, downstream MAPK signaling, and mechanisms of action. We evaluated the DUSP inhibitor, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), in MPNST cell lines and in cell-line and patient-derived MPNST xenografts., Results: DUSP1 and DUSP6 are expressed in NF1-deleted tumors. Knockdown of DUSP1 and DUSP6, alone or in combination, reduced MPNST cell growth and led to ERK and JNK hyperactivation increasing downstream TP53 and p-ATM. The DUSP inhibitor, BCI, diminished the survival of NF1-deleted Schwann cells and MPNST cell lines through activation of JNK. In vivo , treatment of an established cell-line xenograft or a novel patient-derived xenograft (PDX) of MPNSTs with BCI increased ERK and JNK activation, caused tumor necrosis and fibrosis, and reduced tumor volume in one model., Conclusions: Targeting DUSP1 and DUSP6 genetically or with BCI effectively inhibits MPNST cell growth and promotes cell death, in vitro and in xenograft models. The data support further investigation of DUSP inhibition in MPNSTs., (©2019 American Association for Cancer Research.)

Medulloblastoma and central nervous system primitive neuroectodermal tumors (CNS-PNET) are aggressive, poorly differentiated brain tumors with limited effective therapies. Using Sleeping Beauty ( SB ) transposon mutagenesis, we identified novel genetic drivers of medulloblastoma and CNS-PNET. Cross-species gene expression analyses classified SB -driven tumors into distinct medulloblastoma and CNS-PNET subgroups, indicating they resemble human Sonic hedgehog and group 3 and 4 medulloblastoma and CNS neuroblastoma with FOXR2 activation. This represents the first genetically induced mouse model of CNS-PNET and a rare model of group 3 and 4 medulloblastoma. We identified several putative proto-oncogenes including Arhgap36, Megf10 , and Foxr2 . Genetic manipulation of these genes demonstrated a robust impact on tumorigenesis in vitro and in vivo . We also determined that FOXR2 interacts with N-MYC, increases C-MYC protein stability, and activates FAK/SRC signaling. Altogether, our study identified several promising therapeutic targets in medulloblastoma and CNS-PNET. SIGNIFICANCE: A transposon-induced mouse model identifies several novel genetic drivers and potential therapeutic targets in medulloblastoma and CNS-PNET., (©2019 American Association for Cancer Research.)

Aims: To establish levels of use of tobacco, alcohol and illegal drugs among 18-20 year old men and women of Asian (Punjabi) and non-Asian origin compared with levels four years earlier and consider the role of religion and culture in abstinent behaviour., Design: Structured self-complete questionnaire used with 94% of pupils with South Asian names recorded by the Greater Glasgow education department in 1991 and a proportionate random sample of pupils in the same years who did not have South Asian names. Followed up in 1996 in an interviewer-led structured questionnaire in their own homes., Setting: Greater Glasgow, largest city in the west of Scotland., Participants: Eight hundred and twenty-four overwhelmingly British-born 14-15 year olds in 1992, 492 followed up aged 18-20 years in 1996., Measurements: Self-report measures of ever having tried alcohol, tobacco and drugs and the quantities consumed at age 14-15 and 18-20. Indication of reasons for abstinence from substance use at age 18-20., Findings: Asians were much more abstinent from all these substances at both ages (p < 0.001), except for smoking at 18-20. However, religiously specific patterns of abstinence were particularly strong for alcohol (Muslim odds ratio 7- to 9-fold lower at 14-15, 16- to 25-fold lower at 18-20) and smoking (Sikh/Hindu odds ratio 10-fold lower than Muslims, 20-fold than Christians at 18-20), though there is a shared Asian tendency for women to observe these patterns more than men at 18-20., Conclusions: At age 14-15 abstinence was high in the largely British-born generation of Asians mainly for cultural reasons common to religious groups. Four years later culturally determined abstinence has atrophied, and abstinence reflects the specific influence of ascetic religious traditions, though some cultural influence remains in that women are more affected. Intergenerational changes are similar. The erosion of constraints on smoking presents a threat to health.

Objectives: To identify and explore explanatory models of cancer among Irish and white British people living in Britain., Methods: Ethnographic in-depth interviews and focus groups were conducted in London, Manchester and Glasgow, with a total of 58 (n = 58) Irish and 57 (n = 57) white British participants. The study samples were broadly similar in socio-demographic characteristics., Results: We explored explanatory models (lay beliefs) used by the Irish and white British to understand their cancer-related beliefs and behaviours. Among both groups there was confusion about causation, poor knowledge of signs and symptoms, and a general pessimism about cancer prevention and treatments. The narratives of the Irish were, however, qualitatively different from those of the white British. Historical, cultural, social and economic circumstances, both in the UK and in the past in Ireland, appeared to influence views of cancer and health-seeking behaviours. Recollections of negative family experiences of cancers linked to 'stigma' and 'secrecy', poor outcomes and medical practices in rural Ireland, particularly among the older Irish, influenced Irish understanding of cancers and help-seeking behaviours. The second generation also appeared to retain some beliefs that were common amongst the first generation migrants. The context of migration was also felt by the Irish group to have exposed them to living and working environments that made them susceptible to cancers., Conclusion: The Irish frame of reference was firmly embedded in a specific historical, social and economic context which may contribute to cultural constraints on discussions about cancers and to the lack of engagement with preventative behaviours and health care services.

This paper considers the ways in which accounts from Glasgow Catholics diverge from those of Protestants and explores the reasons why people leave jobs, including health grounds. Accounts reveal experiences distinctive to Catholics, of health-threatening stress, obstacles to career progression within (mainly) private-sector organisations, and interactional difficulties which create particular problems for (mainly) middle class men. This narrows the employment options for upwardly mobile Catholics, who may then resort to self-employment or other similarly stressful options. The paper considers whether the competence of Catholics or Catholic cultural factors are implicated in thwarting social mobility among Catholics or, alternatively, whether institutional sectarianism is involved. We conclude that, of these options, theories of institutional sectarianism provide the hypothesis which currently best fits these data. In Glasgow, people of indigenous Irish descent are recognisable from their names and Catholic background and are identified as Catholic by others. Overt historical exclusion of Catholics from middle class employment options now seems to take unrecognised forms in routine assumptions and practices which restrict Catholic employment opportunities. It is argued that younger Catholics use education to overcome the obstacles to mobility faced by older people and circumvent exclusions by recourse to middle class public-sector employment. This paper aims to link historical, structural and sectarian patterns of employment experience to accounts of health and work, and in so doing to contribute to an explanation for the relatively poor health of Catholic Glaswegians with Irish roots.

Religiosity is often associated with mental health in adult populations, but not in a consistent direction. Conflicting results reflect the multidimensional nature of both concepts. Few studies have addressed the relationship between religiosity and mental health among children. In this paper, we examine the relation of weekly church attendance to measures of mental health for 11 year olds from the two main Christian denominations in West Scotland. Levels of church-attendance were low among those affiliated with the Church of Scotland and relatively high among Catholics. The only mental health measure to show a similar relationship with church attendance in both denominations was aggression, which was less prevalent among weekly attenders. Self-esteem, anxiety and depression all demonstrated an interaction, such that weekly church attendance was associated either with advantage for Catholics, disadvantage for children with a Church of Scotland affiliation, or both. Teasing/bullying acted in a small way as a mediating factor in these relationships. In an education system with separate Catholic and 'non-denominational' schools, we hypothesise that the relationship between church attendance and mental health may be contingent on whether church attendance is normative within the peer group.