Engineering a Repeats-in-Toxin Scaffold for Stimulus-Responsive Biotechnology Applications

Protein scaffolds are described as polypeptide frameworks with well-defined tertiary structures that are tolerable to mutagenesis or insertions. These scaffolds have gained significant interest from researchers and clinicians as they have challenged immunoglobulin domains as the preferred protein to address critical problems in biomedical engineering and biotechnology. While engineered antibodies and antibody fragments have been immensely successful, their complex structure, costly production and purification requirements, and large size preclude them from a host of applications. Small, stable proteins devoid of disulfide bond networks that express well recombinantly in prokaryotic systems offer viable alternatives to immunoglobulins. Repeat proteins are characterized structurally by tandem repeats of a consensus motif. These proteins are used in nature to mediate a variety of protein-protein interactions and are appealing scaffolds to bioengineers because of their predictable secondary structures. Several repeat scaffolds have been identified and successfully engineered for in vivo imaging and therapeutic applications. We have identified the repeats-in-toxin (RTX) protein as a potential antibody mimetic and interesting scaffold for protein engineering studies. RTX domains are commonly associated with extracellular proteins secreted through the type 1 secretion system in Gram-negative bacteria. They are composed of tandem repeats of a nonamer calcium binding sequence capped by N and C-termianl flanking regions. These proteins are conformationally dynamic and will fold from an intrinsically disordered state to a compact β-roll secondary structure in response to increasing calcium concentration. We aim to explore the RTX domain as an alternative protein scaffold and exploit the intrinsic conformational response to calcium as a mechanism to mediate molecular interactions. In our first study, we rationally engineer the RTX domain as a calcium-responsive physical cross-linker for hydrogel formation. Protein based materials are favorable for may biomedical applications because of their biocompatibility, tunable mechanical properties, and predictable erosion rates. We have designed a hydrophobic interface on the surface of the RTX domain that is present only in the calcium-bound β-roll conformation. In the absence of calcium, the peptide returns to its disordered state, delocalizing the hydrophobic patch and in turn mitigating the driving force for self-assembly. We show that these mutant RTX domains, with the aid of additional protein cross-linkers, self-assemble into cross-linked macromolecular hydrogel networks, only in the presence of calcium. To expand on this study, we further engineered the RTX domain to contain hydrophobic surfaces on both sides of the folded β-roll simultaneously. By doing this, we doubled the cross-linking capacity of the mutant RTX. This translates to a higher oligomerization state and lower protein concentration required for self-assembly. We also show the double mutant can function as a stand-alone cross-linking domain, eliminating the need for extraneous self-assembling proteins. This designed RTX mutant provides a new platform for stimulus-responsive cross-linking and self-assembly. In our next line of work, we created several synthetic RTX peptides based on a consensus design approach. Such an approach relies on identifying the minimal requirements for a single repeating unit, and concatenating the unit to achieve a desired protein interface. We identified the consensus nonameric unit for the RTX domain and generated several constructs of varying lengths using this sequence. However, it was discovered that these designed RTX peptides undergo a reversible phase change in response to calcium. Rather than abandon these synthetic peptides, we looked to use them as calcium-responsive protein purification tags. By appending a consensus RTX domain to a protein of interest, we were able rapidly and efficiently purify fusions out of cell lysate by precipitation cycling. We were also able to separate the tag from the protein of interest by including a protease recognition site between the two. This system offers an alternative to time consuming and expensive chromatographic techniques for recombinant protein purification. In our final study, we evaluated the RTX domain as a scaffold for evolving molecular recognition. We planned to use the calcium-responsive structural rearrangement as a switch to turn an evolved binding interface "on" and "off". One face of the folded β-roll structure was randomized on the genetic level and the resultant protein constructs were selected against a target protein using ribosome display technology. A consensus binding sequence emerged after several rounds of biopanning and was thoroughly characterized. The evolved β-roll bound the target protein with low micromolar affinity. Although this weak attraction was not suitable for efficiently capturing the target protein in a packed column application, this work provides a platform for evolving the RTX protein for molecular recognition. Several strategies are discussed to achieve higher affinity binders. Overall, this dissertation explores the RTX domain as an alternative stimulus-responsive scaffold for use in a variety of biotechnology applications. We have successfully developed new protein based platforms based on rationally designed or combinatorailly selected RTX proteins for calcium-responsive biomaterials, non-chromatographic protein purification, and calcium-dependent molecular recognition

Search for gravitational waves associated with the InterPlanetary Network short gamma ray bursts

We outline the scientific motivation behind a search for gravitational waves associated with short gamma ray bursts detected by the InterPlanetary Network (IPN) during LIGO's fifth science run and Virgo's first science run. The IPN localisation of short gamma ray bursts is limited to extended error boxes of different shapes and sizes and a search on these error boxes poses a series of challenges for data analysis. We will discuss these challenges and outline the methods to optimise the search over these error boxes.Comment: Methods paper; Proceedings for Eduardo Amaldi 9 Conference on Gravitational Waves, July 2011, Cardiff, U

The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis.

Global tuberculosis incidence has declined marginally over the past decade, and tuberculosis remains out of control in several parts of the world including Africa and Asia. Although tuberculosis control has been effective in some regions of the world, these gains are threatened by the increasing burden of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis. XDR tuberculosis has evolved in several tuberculosis-endemic countries to drug-incurable or programmatically incurable tuberculosis (totally drug-resistant tuberculosis). This poses several challenges similar to those encountered in the pre-chemotherapy era, including the inability to cure tuberculosis, high mortality, and the need for alternative methods to prevent disease transmission. This phenomenon mirrors the worldwide increase in antimicrobial resistance and the emergence of other MDR pathogens, such as malaria, HIV, and Gram-negative bacteria. MDR and XDR tuberculosis are associated with high morbidity and substantial mortality, are a threat to health-care workers, prohibitively expensive to treat, and are therefore a serious public health problem. In this Commission, we examine several aspects of drug-resistant tuberculosis. The traditional view that acquired resistance to antituberculous drugs is driven by poor compliance and programmatic failure is now being questioned, and several lines of evidence suggest that alternative mechanisms-including pharmacokinetic variability, induction of efflux pumps that transport the drug out of cells, and suboptimal drug penetration into tuberculosis lesions-are likely crucial to the pathogenesis of drug-resistant tuberculosis. These factors have implications for the design of new interventions, drug delivery and dosing mechanisms, and public health policy. We discuss epidemiology and transmission dynamics, including new insights into the fundamental biology of transmission, and we review the utility of newer diagnostic tools, including molecular tests and next-generation whole-genome sequencing, and their potential for clinical effectiveness. Relevant research priorities are highlighted, including optimal medical and surgical management, the role of newer and repurposed drugs (including bedaquiline, delamanid, and linezolid), pharmacokinetic and pharmacodynamic considerations, preventive strategies (such as prophylaxis in MDR and XDR contacts), palliative and patient-orientated care aspects, and medicolegal and ethical issues

Risk of tuberculosis in patients with diabetes: population based cohort study using the UK Clinical Practice Research Datalink.

BACKGROUND: Previous cohort studies demonstrate diabetes as a risk factor for tuberculosis (TB) disease. Public Health England has identified improved TB control as a priority area and has proposed a primary care-based screening program for latent TB. We investigated the association between diabetes and risk of tuberculosis in a UK General Practice cohort in order to identify potential high-risk groups appropriate for latent TB screening. METHODS: Using data from the UK Clinical Practice Research Datalink we constructed a cohort of patients with incident diabetes. We included 222,731 patients with diabetes diagnosed from 1990-2013 and 1,218,616 controls without diabetes at index date who were matched for age, sex and general practice. The effect of diabetes was explored using a Poisson analysis adjusted for age, ethnicity, body mass index, socioeconomic status, alcohol intake and smoking. We explored the effects of age, diabetes duration and severity. The effects of diabetes on risk of incident TB were explored across strata of chronic disease care defined by cholesterol and blood pressure measurement and influenza vaccination rates. RESULTS: During just under 7 million person-years of follow-up, 969 cases of TB were identified. The incidence of TB was higher amongst patients with diabetes compared with the unexposed group: 16.2 and 13.5 cases per 100,000 person-years, respectively. After adjustment for potential confounders the association between diabetes and TB remained (adjusted RR 1.30, 95 % CI 1.01 to 1.67, P = 0.04). There was no evidence that age, time since diagnosis and severity of diabetes affected the association between diabetes and TB. Diabetes patients with the lowest and highest rates of chronic disease management had a higher risk of TB (P <0.001 for all comparisons). CONCLUSIONS: Diabetes as an independent risk factor is associated with only a modest overall increased risk of TB in our UK General Practice cohort and is unlikely to be sufficient cause to screen for latent TB. Across different consulting patterns, diabetes patients accessing the least amount of chronic disease care are at highest risk for TB.This article presents independent research supported by a National Institute for Health Research (NIHR) In Practice Fellowship to LP (grant number NIHR/IPF/11/05). DAJM received Wellcome Trust funding (grant number 092691/Z/10/Z). LS is supported by a Wellcome Trust Senior Research Fellowship in Clinical Science

Combined Forward-Backward Asymmetry Measurements in Top-Antitop Quark Production at the Tevatron

The CDF and D0 experiments at the Fermilab Tevatron have measured the asymmetry between yields of forward- and backward-produced top and antitop quarks based on their rapidity difference and the asymmetry between their decay leptons. These measurements use the full data sets collected in proton-antiproton collisions at a center-of-mass energy of $\sqrt s =1.96$ TeV. We report the results of combinations of the inclusive asymmetries and their differential dependencies on relevant kinematic quantities. The combined inclusive asymmetry is $A_{\mathrm{FB}}^{t\bar{t}} = 0.128 \pm 0.025$. The combined inclusive and differential asymmetries are consistent with recent standard model predictions

Global age-sex-specific fertility, mortality, healthy life expectancy (HALE), and population estimates in 204 countries and territories, 1950-2019 : a comprehensive demographic analysis for the Global Burden of Disease Study 2019

Background: Accurate and up-to-date assessment of demographic metrics is crucial for understanding a wide range of social, economic, and public health issues that affect populations worldwide. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019 produced updated and comprehensive demographic assessments of the key indicators of fertility, mortality, migration, and population for 204 countries and territories and selected subnational locations from 1950 to 2019. Methods: 8078 country-years of vital registration and sample registration data, 938 surveys, 349 censuses, and 238 other sources were identified and used to estimate age-specific fertility. Spatiotemporal Gaussian process regression (ST-GPR) was used to generate age-specific fertility rates for 5-year age groups between ages 15 and 49 years. With extensions to age groups 10–14 and 50–54 years, the total fertility rate (TFR) was then aggregated using the estimated age-specific fertility between ages 10 and 54 years. 7417 sources were used for under-5 mortality estimation and 7355 for adult mortality. ST-GPR was used to synthesise data sources after correction for known biases. Adult mortality was measured as the probability of death between ages 15 and 60 years based on vital registration, sample registration, and sibling histories, and was also estimated using ST-GPR. HIV-free life tables were then estimated using estimates of under-5 and adult mortality rates using a relational model life table system created for GBD, which closely tracks observed age-specific mortality rates from complete vital registration when available. Independent estimates of HIV-specific mortality generated by an epidemiological analysis of HIV prevalence surveys and antenatal clinic serosurveillance and other sources were incorporated into the estimates in countries with large epidemics. Annual and single-year age estimates of net migration and population for each country and territory were generated using a Bayesian hierarchical cohort component model that analysed estimated age-specific fertility and mortality rates along with 1250 censuses and 747 population registry years. We classified location-years into seven categories on the basis of the natural rate of increase in population (calculated by subtracting the crude death rate from the crude birth rate) and the net migration rate. We computed healthy life expectancy (HALE) using years lived with disability (YLDs) per capita, life tables, and standard demographic methods. Uncertainty was propagated throughout the demographic estimation process, including fertility, mortality, and population, with 1000 draw-level estimates produced for each metric. Findings: The global TFR decreased from 2·72 (95% uncertainty interval [UI] 2·66–2·79) in 2000 to 2·31 (2·17–2·46) in 2019. Global annual livebirths increased from 134·5 million (131·5–137·8) in 2000 to a peak of 139·6 million (133·0–146·9) in 2016. Global livebirths then declined to 135·3 million (127·2–144·1) in 2019. Of the 204 countries and territories included in this study, in 2019, 102 had a TFR lower than 2·1, which is considered a good approximation of replacement-level fertility. All countries in sub-Saharan Africa had TFRs above replacement level in 2019 and accounted for 27·1% (95% UI 26·4–27·8) of global livebirths. Global life expectancy at birth increased from 67·2 years (95% UI 66·8–67·6) in 2000 to 73·5 years (72·8–74·3) in 2019. The total number of deaths increased from 50·7 million (49·5–51·9) in 2000 to 56·5 million (53·7–59·2) in 2019. Under-5 deaths declined from 9·6 million (9·1–10·3) in 2000 to 5·0 million (4·3–6·0) in 2019. Global population increased by 25·7%, from 6·2 billion (6·0–6·3) in 2000 to 7·7 billion (7·5–8·0) in 2019. In 2019, 34 countries had negative natural rates of increase; in 17 of these, the population declined because immigration was not sufficient to counteract the negative rate of decline. Globally, HALE increased from 58·6 years (56·1–60·8) in 2000 to 63·5 years (60·8–66·1) in 2019. HALE increased in 202 of 204 countries and territories between 2000 and 2019

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Background: In an era of shifting global agendas and expanded emphasis on non-communicable diseases and injuries along with communicable diseases, sound evidence on trends by cause at the national level is essential. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) provides a systematic scientific assessment of published, publicly available, and contributed data on incidence, prevalence, and mortality for a mutually exclusive and collectively exhaustive list of diseases and injuries. Methods: GBD estimates incidence, prevalence, mortality, years of life lost (YLLs), years lived with disability (YLDs), and disability-adjusted life-years (DALYs) due to 369 diseases and injuries, for two sexes, and for 204 countries and territories. Input data were extracted from censuses, household surveys, civil registration and vital statistics, disease registries, health service use, air pollution monitors, satellite imaging, disease notifications, and other sources. Cause-specific death rates and cause fractions were calculated using the Cause of Death Ensemble model and spatiotemporal Gaussian process regression. Cause-specific deaths were adjusted to match the total all-cause deaths calculated as part of the GBD population, fertility, and mortality estimates. Deaths were multiplied by standard life expectancy at each age to calculate YLLs. A Bayesian meta-regression modelling tool, DisMod-MR 2.1, was used to ensure consistency between incidence, prevalence, remission, excess mortality, and cause-specific mortality for most causes. Prevalence estimates were multiplied by disability weights for mutually exclusive sequelae of diseases and injuries to calculate YLDs. We considered results in the context of the Socio-demographic Index (SDI), a composite indicator of income per capita, years of schooling, and fertility rate in females younger than 25 years. Uncertainty intervals (UIs) were generated for every metric using the 25th and 975th ordered 1000 draw values of the posterior distribution. Findings: Global health has steadily improved over the past 30 years as measured by age-standardised DALY rates. After taking into account population growth and ageing, the absolute number of DALYs has remained stable. Since 2010, the pace of decline in global age-standardised DALY rates has accelerated in age groups younger than 50 years compared with the 1990–2010 time period, with the greatest annualised rate of decline occurring in the 0–9-year age group. Six infectious diseases were among the top ten causes of DALYs in children younger than 10 years in 2019: lower respiratory infections (ranked second), diarrhoeal diseases (third), malaria (fifth), meningitis (sixth), whooping cough (ninth), and sexually transmitted infections (which, in this age group, is fully accounted for by congenital syphilis; ranked tenth). In adolescents aged 10–24 years, three injury causes were among the top causes of DALYs: road injuries (ranked first), self-harm (third), and interpersonal violence (fifth). Five of the causes that were in the top ten for ages 10–24 years were also in the top ten in the 25–49-year age group: road injuries (ranked first), HIV/AIDS (second), low back pain (fourth), headache disorders (fifth), and depressive disorders (sixth). In 2019, ischaemic heart disease and stroke were the top-ranked causes of DALYs in both the 50–74-year and 75-years-and-older age groups. Since 1990, there has been a marked shift towards a greater proportion of burden due to YLDs from non-communicable diseases and injuries. In 2019, there were 11 countries where non-communicable disease and injury YLDs constituted more than half of all disease burden. Decreases in age-standardised DALY rates have accelerated over the past decade in countries at the lower end of the SDI range, while improvements have started to stagnate or even reverse in countries with higher SDI. Interpretation: As disability becomes an increasingly large component of disease burden and a larger component of health expenditure, greater research and developm nt investment is needed to identify new, more effective intervention strategies. With a rapidly ageing global population, the demands on health services to deal with disabling outcomes, which increase with age, will require policy makers to anticipate these changes. The mix of universal and more geographically specific influences on health reinforces the need for regular reporting on population health in detail and by underlying cause to help decision makers to identify success stories of disease control to emulate, as well as opportunities to improve. Funding: Bill & Melinda Gates Foundation. © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licens

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder