Why high-error-rate random mutagenesis libraries are enriched in functional and improved proteins

Recently, several groups have used error-prone polymerase chain reactions to construct mutant libraries containing up to 27 nucleotide mutations per gene on average, and reported a striking observation: although retention of protein function initially declines exponentially with mutations as has previously been observed, orders of magnitude more proteins remain viable at the highest mutation rates than this trend would predict. Mutant proteins having improved or novel activity were isolated disproportionately from these heavily mutated libraries, leading to the suggestion that distant regions of sequence space are enriched in useful cooperative mutations and that optimal mutagenesis should target these regions. If true, these claims have profound implications for laboratory evolution and for evolutionary theory. Here, we demonstrate that properties of the polymerase chain reaction can explain these results and, consequently, that average protein viability indeed decreases exponentially with mutational distance at all error rates. We show that high-error-rate mutagenesis may be useful in certain cases, though for very different reasons than originally proposed, and that optimal mutation rates are inherently protocol-dependent. Our results allow optimal mutation rates to be found given mutagenesis conditions and a protein of known mutational robustness.Comment: Optimality results improved. 26 pages, 4 figures, 3 table

Supersymmetric Monojets at the Large Hadron Collider

Supersymmetric monojets may be produced at the Large Hadron Collider by the process qg -> squark neutralino_1 -> q neutralino_1 neutralino_1, leading to a jet recoiling against missing transverse momentum. We discuss the feasibility and utility of the supersymmetric monojet signal. In particular, we examine the possible precision with which one can ascertain the neutralino_1-squark-quark coupling via the rate for monojet events. Such a coupling contains information on the composition of the neutralino_1 and helps bound dark matter direct detection cross-sections and the dark matter relic density of the neutralino_1. It also provides a check of the supersymmetric relation between gauge couplings and gaugino-quark-squark couplings.Comment: 46 pages, 10 figures. The appendix has been rewritten to correct an error that appears in all previous versions of the appendix. This error has no effect on the results in the main body of the pape

Nucleotide flipping by restriction enzymes analyzed by 2-aminopurine steady-state fluorescence

Many DNA modification and repair enzymes require access to DNA bases and therefore flip nucleotides. Restriction endonucleases (REases) hydrolyze the phosphodiester backbone within or in the vicinity of the target recognition site and do not require base extrusion for the sequence readout and catalysis. Therefore, the observation of extrahelical nucleotides in a co-crystal of REase Ecl18kI with the cognate sequence, CCNGG, was unexpected. It turned out that Ecl18kI reads directly only the CCGG sequence and skips the unspecified N nucleotides, flipping them out from the helix. Sequence and structure conservation predict nucleotide flipping also for the complexes of PspGI and EcoRII with their target DNAs (/CCWGG), but data in solution are limited and indirect. Here, we demonstrate that Ecl18kI, the C-terminal domain of EcoRII (EcoRII-C) and PspGI enhance the fluorescence of 2-aminopurines (2-AP) placed at the centers of their recognition sequences. The fluorescence increase is largest for PspGI, intermediate for EcoRII-C and smallest for Ecl18kI, probably reflecting the differences in the hydrophobicity of the binding pockets within the protein. Omitting divalent metal cations and mutation of the binding pocket tryptophan to alanine strongly increase the 2-AP signal in the Ecl18kI–DNA complex. Together, our data provide the first direct evidence that Ecl18kI, EcoRII-C and PspGI flip nucleotides in solution

ABCB1 (MDR1) polymorphisms and ovarian cancer progression and survival: A comprehensive analysis from the Ovarian Cancer Association Consortium and The Cancer Genome Atlas

<b>Objective</b> <i>ABCB1</i> encodes the multi-drug efflux pump P-glycoprotein (P-gp) and has been implicated in multi-drug resistance. We comprehensively evaluated this gene and flanking regions for an association with clinical outcome in epithelial ovarian cancer (EOC).<p></p> <b>Methods</b> The best candidates from fine-mapping analysis of 21 <i>ABCB1</i> SNPs tagging C1236T (rs1128503), G2677T/A (rs2032582), and C3435T (rs1045642) were analysed in 4616 European invasive EOC patients from thirteen Ovarian Cancer Association Consortium (OCAC) studies and The Cancer Genome Atlas (TCGA). Additionally we analysed 1,562 imputed SNPs around ABCB1 in patients receiving cytoreductive surgery and either ‘standard’ first-line paclitaxel–carboplatin chemotherapy (n = 1158) or any first-line chemotherapy regimen (n = 2867). We also evaluated ABCB1 expression in primary tumours from 143 EOC patients.<p></p> <b>Result</b> Fine-mapping revealed that rs1128503, rs2032582, and rs1045642 were the best candidates in optimally debulked patients. However, we observed no significant association between any SNP and either progression-free survival or overall survival in analysis of data from 14 studies. There was a marginal association between rs1128503 and overall survival in patients with nil residual disease (HR 0.88, 95% CI 0.77–1.01; p = 0.07). In contrast, <i>ABCB1</i> expression in the primary tumour may confer worse prognosis in patients with sub-optimally debulked tumours.<p></p> <b>Conclusion</b> Our study represents the largest analysis of <i>ABCB1</i> SNPs and EOC progression and survival to date, but has not identified additional signals, or validated reported associations with progression-free survival for rs1128503, rs2032582, and rs1045642. However, we cannot rule out the possibility of a subtle effect of rs1128503, or other SNPs linked to it, on overall survival.<p></p&gt

Python as a Federation Tool for GENESIS 3.0

The GENESIS simulation platform was one of the first broad-scale modeling systems in computational biology to encourage modelers to develop and share model features and components. Supported by a large developer community, it participated in innovative simulator technologies such as benchmarking, parallelization, and declarative model specification and was the first neural simulator to define bindings for the Python scripting language. An important feature of the latest version of GENESIS is that it decomposes into self-contained software components complying with the Computational Biology Initiative federated software architecture. This architecture allows separate scripting bindings to be defined for different necessary components of the simulator, e.g., the mathematical solvers and graphical user interface. Python is a scripting language that provides rich sets of freely available open source libraries. With clean dynamic object-oriented designs, they produce highly readable code and are widely employed in specialized areas of software component integration. We employ a simplified wrapper and interface generator to examine an application programming interface and make it available to a given scripting language. This allows independent software components to be ‘glued’ together and connected to external libraries and applications from user-defined Python or Perl scripts. We illustrate our approach with three examples of Python scripting. (1) Generate and run a simple single-compartment model neuron connected to a stand-alone mathematical solver. (2) Interface a mathematical solver with GENESIS 3.0 to explore a neuron morphology from either an interactive command-line or graphical user interface. (3) Apply scripting bindings to connect the GENESIS 3.0 simulator to external graphical libraries and an open source three dimensional content creation suite that supports visualization of models based on electron microscopy and their conversion to computational models. Employed in this way, the stand-alone software components of the GENESIS 3.0 simulator provide a framework for progressive federated software development in computational neuroscience

Fertility, Living Arrangements, Care and Mobility

There are four main interconnecting themes around which the contributions in this book are based. This introductory chapter aims to establish the broad context for the chapters that follow by discussing each of the themes. It does so by setting these themes within the overarching demographic challenge of the twenty-first century – demographic ageing. Each chapter is introduced in the context of the specific theme to which it primarily relates and there is a summary of the data sets used by the contributors to illustrate the wide range of cross-sectional and longitudinal data analysed

Reduced dielectric screening and enhanced energy transfer in single and few-layer MoS2

We report highly efficient non-radiative energy transfer from cadmium selenide (CdSe) quantum dots to monolayer and few-layer molybdenum disulfide (MoS2). The quenching of the donor quantum dot photoluminescence increases as the MoS2 flake thickness decreases, with the highest efficiency (>95%) observed for monolayer MoS2. This counterintuitive result arises from reduced dielectric screening in thin layer semiconductors having unusually large permittivity and a strong in-plane transition dipole moment, as found in MoS2. Excitonic energy transfer between a 0D emitter and a 2D absorber is fundamentally interesting and enables a wide range of applications including broadband optical down-conversion, optical detection, photovoltaic sensitization, and color shifting in light-emitting devices.Comment: 14 pages, 4 figure

A randomized phase III trial of stereotactic ablative radiotherapy for patients with up to 10 oligometastases and a synchronous primary tumor (SABR-SYNC): study protocol

BACKGROUND Emerging randomized data, mostly from phase II trials, have suggested that patients with oligometastatic cancers may benefit from ablative treatments such as stereotactic ablative radiotherapy (SABR). However, phase III data testing this paradigm are lacking, and many studies have examined SABR in the setting of metachronous oligometastatic disease. The goal of the SABR-SYNC trial is to assess the effect of SABR in patients with oligometastatic cancers and a synchronous primary tumor. METHODS One hundred and eighty patients will be randomized in a 1:2 ratio between standard of care (SOC) palliative-intent treatments vs. SOC + ablative therapy (SABR preferred) to all sites of known disease. Randomization will be stratified based on histology and number of metastases at enrollment. SABR may be delivered in 1-, 3- and 5-fraction regimens, with recommended doses of 20 Gy, 30 Gy, and 35 Gy, respectively. Non-SABR local modalities (e.g. surgery, thermal ablation, conventional radiation) may be used for treatment of the primary or metastases at the discretion of the treating physicians, if those modalities are clinically preferred. The primary endpoint is overall survival, and secondary endpoints include progression-free survival, time to development of new metastatic lesions, time to initiation of next systemic therapy, quality of life, and toxicity. Translational endpoints include assessment of circulating tumor DNA and immunological predictors of outcomes. DISCUSSION SABR-SYNC will provide phase III data to assess the impact of SABR on overall survival in a population of patients with synchronous oligometastases. The translational component will attempt to identify novel prognostic and predictive biomarkers to aid in clinical decision making. TRIAL REGISTRATION Clinicaltrials.gov NCT05717166 (registration date: Feb. 8, 2023)

Assessment of data quality in a multi-centre cross-sectional study of participation and quality of life of children with cerebral palsy

BACKGROUND: SPARCLE is a cross-sectional survey in nine European regions, examining the relationship of the environment of children with cerebral palsy to their participation and quality of life. The objective of this report is to assess data quality, in particular heterogeneity between regions, family and item non-response and potential for bias. METHODS: 1,174 children aged 8–12 years were selected from eight population-based registers of children with cerebral palsy; one further centre recruited 75 children from multiple sources. Families were visited by trained researchers who administered psychometric questionnaires. Logistic regression was used to assess factors related to family non-response and self-completion of questionnaires by children. RESULTS: 431/1,174 (37%) families identified from registers did not respond: 146 (12%) were not traced; of the 1,028 traced families, 250 (24%) declined to participate and 35 (3%) were not approached. Families whose disabled children could walk unaided were more likely to decline to participate. 818 children entered the study of which 500 (61%) self-reported their quality of life; children with low IQ, seizures or inability to walk were less likely to self-report. There was substantial heterogeneity between regions in response rates and socio-demographic characteristics of families but not in age or gender of children. Item non-response was 2% for children and ranged from 0.4% to 5% for questionnaires completed by parents. CONCLUSION: While the proportion of untraced families was higher than in similar surveys, the refusal rate was comparable. To reduce bias, all analyses should allow for region, walking ability, age and socio-demographic characteristics. The 75 children in the region without a population based register are unlikely to introduce bias