Four small puzzles that Rosetta doesn't solve

A complete macromolecule modeling package must be able to solve the simplest structure prediction problems. Despite recent successes in high resolution structure modeling and design, the Rosetta software suite fares poorly on deceptively small protein and RNA puzzles, some as small as four residues. To illustrate these problems, this manuscript presents extensive Rosetta results for four well-defined test cases: the 20-residue mini-protein Trp cage, an even smaller disulfide-stabilized conotoxin, the reactive loop of a serine protease inhibitor, and a UUCG RNA tetraloop. In contrast to previous Rosetta studies, several lines of evidence indicate that conformational sampling is not the major bottleneck in modeling these small systems. Instead, approximations and omissions in the Rosetta all-atom energy function currently preclude discriminating experimentally observed conformations from de novo models at atomic resolution. These molecular "puzzles" should serve as useful model systems for developers wishing to make foundational improvements to this powerful modeling suite.Comment: Published in PLoS One as a manuscript for the RosettaCon 2010 Special Collectio

Heavy Meson Electromagnetic Mass Differences from QCD

We compute the electromagnetic mass differences of mesons containing a single heavy quark in terms of measurable data using QCD-based arguments in heavy-quark effective theory. We derive an unsubtracted dispersion relation that shows that the mass differences are calculable in terms of the properties of the lowest-lying physical intermediate states. We then consider the problem in the large-$N$ limit, where $N$ is the number of QCD colors. In this limit, we can write a kind of double-dispersion relation for the amplitude required to determine the electromagnetic mass difference. We use this to derive analogs of the Weinberg sum rules for heavy meson matrix elements valid to leading order in $1/N$ and to $O(1/m_Q)$ in the heavy quark expansion. In order to obtain our final result, we assume that the electromagnetic mass differences and sum rules are dominated by the lowest-lying states in analogy with the situation for the $\pi^+$--$\pi^0$ mass difference. Despite the fact that some of the matrix elements appearing in our final result have not yet been accurately measured, we can obtain useful numerical estimates: for example, we obtain (M_{B^+} - M_{B^0})^{EM} \simeq +1.8 \MeV. We argue that our results are accurate to about $30\%$.Comment: 20 pages, plain TeX, 1 uuencoded postscript figur

Hemagglutinin Receptor Binding Avidity Drives Influenza A Virus Antigenic Drift

Refer to Web version on PubMed Central for supplementary material.Rapid antigenic evolution in the influenza A virus hemagglutinin precludes effective vaccination with existing vaccines. To understand this phenomenon, we passaged virus in mice immunized with influenza vaccine. Neutralizing antibodies selected mutants with single–amino acid hemagglutinin substitutions that increased virus binding to cell surface glycan receptors. Passaging these high-avidity binding mutants in naïve mice, but not immune mice, selected for additional hemagglutinin substitutions that decreased cellular receptor binding avidity. Analyzing a panel of monoclonal antibody hemagglutinin escape mutants revealed a positive correlation between receptor binding avidity and escape from polyclonal antibodies. We propose that in response to variation in neutralizing antibody pressure between individuals, influenza A virus evolves by adjusting receptor binding avidity via amino acid substitutions throughout the hemagglutinin globular domain, many of which simultaneously alter antigenicity.National Institute of Mental Health (U.S.). Division of Intramural ResearchNational Institute of Allergy and Infectious Diseases (U.S.)Singapore-MIT Alliance for Research and TechnologyNational Institute of General Medical Sciences (U.S.) (GM 57073)National Institute of General Medical Sciences (U.S.) (U54GM62116

Protocol on a multicentre statistical and economic modelling study of risk-based stratified and personalised screening for diabetes and its complications in India (SMART India)

INTRODUCTION: The aim of this study is to develop practical and affordable models to (a) diagnose people with diabetes and prediabetes and (b) identify those at risk of diabetes complications so that these models can be applied to the population in low-income and middle-income countries (LMIC) where laboratory tests are unaffordable. METHODS AND ANALYSIS: This statistical and economic modelling study will be done on at least 48 000 prospectively recruited participants aged 40 years or above through community screening across 20 predefined regions in India. Each participant will be tested for capillary random blood glucose (RBG) and complete a detailed health-related questionnaire. People with known diabetes and all participants with predefined levels of RBG will undergo further tests, including point-of-care (POC) glycated haemoglobin (HbA1c), POC lipid profile and POC urine test for microalbuminuria, retinal photography using non-mydriatic hand-held retinal camera, visual acuity assessment in both eyes and complete quality of life questionnaires. The primary aim of the study is to develop a model and assess its diagnostic performance to predict HbA1c diagnosed diabetes from simple tests that can be applied in resource-limited settings; secondary outcomes include RBG cut-off for definition of prediabetes, diagnostic accuracy of cost-effective risk stratification models for diabetic retinopathy and models for identifying those at risk of complications of diabetes. Diagnostic accuracy inter-tests agreement, statistical and economic modelling will be performed, accounting for clustering effects. ETHICS AND DISSEMINATION: The Indian Council of Medical Research/Health Ministry Screening Committee (HMSC/2018–0494 dated 17 December 2018 and institutional ethics committees of all the participating institutions approved the study. Results will be published in peer-reviewed journals and will be presented at national and international conferences. TRIAL REGISTRATION: ISRCTN57962668 V1.0 24/09/2018

A robust system for RNA interference in the chicken using a modified microRNA operon

AbstractRNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo

Protocol on a multicentre statistical and economic modelling study of risk-based stratified and personalised screening for diabetes and its complications in India (SMART India)

Introduction The aim of this study is to develop practical and affordable models to (a) diagnose people with diabetes and prediabetes and (b) identify those at risk of diabetes complications so that these models can be applied to the population in low-income and middle-income countries (LMIC) where laboratory tests are unaffordable. Methods and analysis This statistical and economic modelling study will be done on at least 48 000 prospectively recruited participants aged 40 years or above through community screening across 20 predefined regions in India. Each participant will be tested for capillary random blood glucose (RBG) and complete a detailed health-related questionnaire. People with known diabetes and all participants with predefined levels of RBG will undergo further tests, including point-of-care (POC) glycated haemoglobin (HbA1c), POC lipid profile and POC urine test for microalbuminuria, retinal photography using non-mydriatic hand-held retinal camera, visual acuity assessment in both eyes and complete quality of life questionnaires. The primary aim of the study is to develop a model and assess its diagnostic performance to predict HbA1c diagnosed diabetes from simple tests that can be applied in resource-limited settings; secondary outcomes include RBG cut-off for definition of prediabetes, diagnostic accuracy of cost-effective risk stratification models for diabetic retinopathy and models for identifying those at risk of complications of diabetes. Diagnostic accuracy inter-tests agreement, statistical and economic modelling will be performed, accounting for clustering effects. Ethics and dissemination The Indian Council of Medical Research/Health Ministry Screening Committee (HMSC/2018–0494 dated 17 December 2018 and institutional ethics committees of all the participating institutions approved the study. Results will be published in peer-reviewed journals and will be presented at national and international conferences. Trial registration number ISRCTN57962668 V1.0 24/09/2018

Atomic-accuracy prediction of protein loop structures through an RNA-inspired ansatz

Consistently predicting biopolymer structure at atomic resolution from sequence alone remains a difficult problem, even for small sub-segments of large proteins. Such loop prediction challenges, which arise frequently in comparative modeling and protein design, can become intractable as loop lengths exceed 10 residues and if surrounding side-chain conformations are erased. This article introduces a modeling strategy based on a 'stepwise ansatz', recently developed for RNA modeling, which posits that any realistic all-atom molecular conformation can be built up by residue-by-residue stepwise enumeration. When harnessed to a dynamic-programming-like recursion in the Rosetta framework, the resulting stepwise assembly (SWA) protocol enables enumerative sampling of a 12 residue loop at a significant but achievable cost of thousands of CPU-hours. In a previously established benchmark, SWA recovers crystallographic conformations with sub-Angstrom accuracy for 19 of 20 loops, compared to 14 of 20 by KIC modeling with a comparable expenditure of computational power. Furthermore, SWA gives high accuracy results on an additional set of 15 loops highlighted in the biological literature for their irregularity or unusual length. Successes include cis-Pro touch turns, loops that pass through tunnels of other side-chains, and loops of lengths up to 24 residues. Remaining problem cases are traced to inaccuracies in the Rosetta all-atom energy function. In five additional blind tests, SWA achieves sub-Angstrom accuracy models, including the first such success in a protein/RNA binding interface, the YbxF/kink-turn interaction in the fourth RNA-puzzle competition. These results establish all-atom enumeration as a systematic approach to protein structure that can leverage high performance computing and physically realistic energy functions to more consistently achieve atomic resolution.Comment: Identity of four-loop blind test protein and parts of figures 5 have been omitted in this preprint to ensure confidentiality of the protein structure prior to its public releas