Reversible switching between two common protein folds in a designed system using only temperature.

Naturally occurring metamorphic proteins have the ability to interconvert from one folded state to another through either a limited set of mutations or by way of a change in the local environment. Here, we show in a designed system that it is possible to switch reversibly between two of the most common monomeric folds employing only temperature changes. We demonstrate that a latent 3α state can be unmasked from an α/ß-plait topology with a single V90T amino acid substitution, populating both forms simultaneously. The equilibrium between these two states exhibits temperature dependence, such that the 3α state is predominant (>90%) at 5 °C, while the α/ß-plait fold is the major species (>90%) at 30 °C. We describe the structure and dynamics of these topologies, how mutational changes affect the temperature dependence, and the energetics and kinetics of interconversion. Additionally, we demonstrate how ligand-binding function can be tightly regulated by large amplitude changes in protein structure over a relatively narrow temperature range that is relevant to biology. The 3α/αß switch thus represents a potentially useful approach for designing proteins that alter their fold topologies in response to environmental triggers. It may also serve as a model for computational studies of temperature-dependent protein stability and fold switching.

Alfvénic velocity spikes and rotational flows in the near-Sun solar wind.

The prediction of a supersonic solar wind1 was first confirmed by spacecraft near Earth2,3 and later by spacecraft at heliocentric distances as small as 62 solar radii4. These missions showed that plasma accelerates as it emerges from the corona, aided by unidentified processes that transport energy outwards from the Sun before depositing it in the wind. Alfvénic fluctuations are a promising candidate for such a process because they are seen in the corona and solar wind and contain considerable energy5-7. Magnetic tension forces the corona to co-rotate with the Sun, but any residual rotation far from the Sun reported until now has been much smaller than the amplitude of waves and deflections from interacting wind streams8. Here we report observations of solar-wind plasma at heliocentric distances of about 35 solar radii9-11, well within the distance at which stream interactions become important. We find that Alfvén waves organize into structured velocity spikes with duration of up to minutes, which are associated with propagating S-like bends in the magnetic-field lines. We detect an increasing rotational component to the flow velocity of the solar wind around the Sun, peaking at 35 to 50 kilometres per second-considerably above the amplitude of the waves. These flows exceed classical velocity predictions of a few kilometres per second, challenging models of circulation in the corona and calling into question our understanding of how stars lose angular momentum and spin down as they age12-14.

Management of anal intraepithelial neoplasia and anal squamous cell carcinoma at a tertiary referral centre with a dedicated infectious diseases unit: an 18-year review.

INTRODUCTION: Anal squamous cell carcinoma (ASCC) is a rare malignancy with rising incidence rates. Risk factors include human immunodeficiency virus (HIV) infection, high-risk sexual activity and HPV-related genitourinary dysplasia/neoplasia. There is an overlap between high-risk patients and those attending HIV Medicine/Sexual Health (HMSH) services. We hypothesised that HMSH involvement may facilitate earlier referral to colorectal surgeons, with better outcomes. METHODS: Retrospective review of all ASCC and anal intraepithelial neoplasia (AIN) treated at a tertiary-referral hospital with a dedicated HMSH clinic between 2000 and 2018. Comparative analysis was performed of demographics, management and outcomes between HMSH and non-HMSH patients. RESULTS: One hundred and nine patients had anal pathology, eighty-five with ASCC (78%) and twenty-four with AIN (22%). Seventy (64%) were male. Median (range) age at ASCC diagnosis was 51 years (26-88). Thirty-six percent of all patients attended HMSH services, 28% were HIV positive, and 41% of males were men-who-have-sex-with-men (MSM). Eighty-one ASCC patients (97.5%) were treated with curative intent. Sixty-seven (80%) had primary chemoradiation therapy. Fifteen (17.5%) had primary surgical excision. Twelve (14%) developed recurrent disease. Ultimately, seven required salvage APR. Overall 3-year survival (3YS) was 76%. HMSH patients were significantly younger at ASCC diagnosis (p < 0.001), with a higher prevalence of HIV, HPV and MSM. HMSH attenders also tended to be diagnosed at earlier stages, were less likely to develop recurrence and achieved better overall outcomes, with a superior overall 3YS than non-HMSH patients (92% vs 72%, p = 0.037). CONCLUSION: ASCC incidence is increasing worldwide. The HMSH cohort has emerged as a distinct subpopulation of younger, high-risk, male patients. Collaboration between HMSH and colorectal surgeons offers an opportunity for risk reduction strategies and earlier intervention.

The ophthalmic diagnosis and management of four siblings with Werner syndrome.

INTRODUCTION: Werner syndrome is a rare autosomal recessive disorder caused by mutations in the Werner syndrome WRN gene, on chromosome 8. Those affected manifest early the features of ageing. DISCUSSION: Cataract surgery is prone to post-operative complications in those with Werner syndrome. The development of cystoid macular oedema (CMO) is likely multifactorial. Patients with WS have diabetes mellitus type 2 which can contribute to macular oedema. There is a deposition of abnormal WRN proteins in the macula which also predisposes to macular oedema. The trauma of cataract surgery appears to be the main stimulus for the development of CMO. CMO may, as a result, be difficult to manage in Werner syndrome patients. CONCLUSION: Further study is needed to elucidate the precise role of retinal WRN protein expression in the development of CMO in those with Werner syndrome. A tailored and more successful approach to the treatment of CMO in such patients may result.

Characterization of the NISTmAb Reference Material using small-angle scattering and molecular simulation : Part I: Dilute protein solutions.

Both conformational and colloidal stability of therapeutic proteins must be closely monitored and thoroughly characterized to assess the long-term viability of drug products. We characterized the IgG1 NISTmAb reference material in its histidine formulation buffer and report our findings on the higher order structure and interactions of NISTmAb under a range of conditions. In this paper we present the analysis of experimental small-angle scattering data with atomistic molecular simulations to characterize the monodisperse dilute solution of NISTmAb. In part II we describe the characterization of the NISTmAb at high protein concentration (Castellanos et al. 2018). The NISTmAb was found to be a flexible protein with a radius of gyration of 49.0 ± 1.2 Å in histidine formulation buffer using a variety of neutron and X-ray scattering measurements. Scattering data were then modeled using molecular simulation. After building and validating a starting NISTmAb structure from the Fc and Fab crystallographic coordinates, molecular dynamics and torsion-angle Monte Carlo simulations were performed to explore the configuration space sampled in the NISTmAb and obtain ensembles of structures with atomistic detail that are consistent with the experimental data. Our results indicate that the small-angle scattering profiles of the NISTmAb can be modeled using ensembles of flexible structures that explore a wide configuration space. The NISTmAb is flexible in solution with no single preferred orientation of Fc and Fab domains, but with some regions of configuration space that are more consistent with measured scattering profiles. Analysis of inter-domain atomistic contacts indicated that all ensembles contained configurations where residues between domains are ≤ 4 Å, although few contacts were observed for variable and C H 3 regions. Graphical Abstract Heavy atom self contact maps of the NISTmAb indicate a highly-flexible structure.

Establishing the feasibility of a community and primary health care intervention to raise awareness of symptoms of Type 1 Diabetes-The Early Detection of Type 1 Diabetes in Youth (EDDY) study.

AIMS: To design, develop, and evaluate the feasibility of delivering a multi-component community based intervention to parents and primary health care professionals to raise awareness of the symptoms of Type 1 diabetes (T1D) in childhood in 3 adjoining borough counties of South Wales. MATERIALS AND METHODS: Parent and primary health care advisory groups were established to design the intervention. Qualitative interviews with stakeholders and parents assessed the acceptability, feasibility and any potential impact of the intervention. RESULTS: The parent component of the intervention developed was a re-useable shopping bag with the 4 main symptoms of T1D illustrated on the side, based on the road traffic system of red warning triangles and an octagon "stop" sign stating "Seek Medical Help". Accompanying the bag was an A5 leaflet giving further information. Both were overwrapped with clear plastic and delivered to 98% (323/329) schools, equating to 101 371 children. The primary health care professional component was a dual glucose/ketone meter, single use lancets, stickers, the A5 parent leaflet displayed as a poster and an educational visit from a Community Diabetes Liaison Nurse. 87% (73/84) of GP practices received the intervention, 100% received the materials. The intervention was delivered within Cardiff, the Vale of Glamorgan and Bridgend. Qualitative analyses suggest that the intervention raised awareness and had some impact. CONCLUSION: This study showed that it is feasible and acceptable to design, develop and deliver a community based intervention to raise awareness of T1D. There is some suggestion of impact but a definitive evaluation of effectiveness is still required.

Biophysical characterization and structure of the Fab fragment from the NIST reference antibody, RM 8671.

Monoclonal antibody pharmaceuticals are the fastest-growing class of therapeutics, with a wide range of clinical applications. To assure their safety, these protein drugs must demonstrate highly consistent purity and stability. Key to these objectives is higher order structure measurements validated by calibration to reference materials. We describe preparation, characterization, and crystal structure of the Fab fragment prepared from the NIST Reference Antibody RM 8671 (NISTmAb). NISTmAb is a humanized IgG1κ antibody, produced in murine cell culture and purified by standard biopharmaceutical production methods, developed at the National Institute of Standards and Technology (NIST) to serve as a reference material. The Fab fragment was derived from NISTmAb through papain cleavage followed by protein A based purification. The purified Fab fragment was characterized by SDS-PAGE, capillary gel electrophoresis, multi-angle light scattering, size exclusion chromatography, mass spectrometry, and x-ray crystallography. The crystal structure at 0.2 nm resolution includes four independent Fab molecules with complete light chains and heavy chains through Cys 223, enabling assessment of conformational variability and providing a well-characterized reference structure for research and engineering applications. This nonproprietary, publically available reference material of known higher-order structure can support metrology in biopharmaceutical applications, and it is a suitable platform for validation of molecular modeling studies.

MUTYH-Associated Polyposis: The Irish Experience>.

MUTYH is involved in DNA damage repair. Bi-allelic MUTYH mutations predispose to polyposis and gastrointestinal malignancies, distinct genetically from autosomal dominant familial adenomatous polyposis coli. Two common European MUTYH mutations account for 90% of MUTYH-associated polyposis (MAP). We aimed to examine the incidence of MAP in Ireland. A retrospective cohort study was undertaken. Patients undergoing MUTYH testing from 2003-2016 were identified by searching electronic databases using terms "MUTYH" and "MYH". Phenotypic and genotypic details were obtained by chart review. Bi-allelic mutations were confirmed in 26 individuals (17 families), of whom 16 (62%) developed colorectal malignancies, and 22(85%) polyposis. Eleven families had bi-allelic status for one/both common European mutations. Regional variation was noted, with over-representation of bi-allelic mutation carriers in the South-west of Ireland. MAP is under-diagnosed in Ireland. Increased awareness is required to facilitate appropriate identification and surveillance of bi-allelic mutation carriers for colorectal pathology.

An analysis of the association between prostate cancer risk loci, PSA levels, disease aggressiveness and disease-specific mortality.

BACKGROUND: Genome-wide association studies have identified multiple single-nucleotide polymorphsims (SNPs) associated with prostate cancer (PCa). Although these SNPs have been clearly associated with disease risk, their relationship with clinical outcomes is less clear. Our aim was to assess the frequency of known PCa susceptibility alleles within a single institution ascertainment and to correlate risk alleles with disease-specific outcomes. METHODS: We genotyped 1354 individuals treated for localised PCa between June 1988 and December 2007. Blood samples were prospectively collected and de-identified before being genotyped and matched to phenotypic data. We investigated associations between 61 SNPs and disease-specific end points using multivariable analysis and also determined if SNPs were associated with PSA at diagnosis. RESULTS: Seven SNPs showed associations on multivariable analysis (P<0.05), rs13385191 with both biochemical recurrence (BR) and castrate metastasis (CM), rs339331 (BR), rs1894292, rs17178655 and rs11067228 (CM), and rs11902236 and rs4857841 PCa-specific mortality. After applying a Bonferroni correction for number of SNPs (P<0.0008), the only persistent significant association was between rs17632542 (KLK3) and PSA levels at diagnosis (P=1.4 × 10(-5)). CONCLUSIONS: We confirmed that rs17632542 in KLK3 is associated with PSA at diagnosis. No significant association was seen between loci and disease-specific end points when accounting for multiple testing. This provides further evidence that known PCa risk SNPs do not predict likelihood of disease progression.

Editor's choice--Angulation of the C-arm during complex endovascular aortic procedures increases radiation exposure to the head.

OBJECTIVES/BACKGROUND: The increased complexity of endovascular aortic repair necessitates longer procedural time and higher radiation exposure to the operator, particularly to exposed body parts. The aims were to measure directly exposure to radiation of the bodies and heads of the operating team during endovascular repair of thoracoabdominal aortic aneurysms (TAAA), and to identify factors that may increase exposure. METHODS: This was a single-centre prospective study. Between October 2013 and July 2014, consecutive elective branched and fenestrated TAAA repairs performed in a hybrid operating room were studied. Electronic dosimeters were used to measure directly radiation exposure to the primary (PO) and assistant (AO) operator in three different areas (under-lead, over-lead, and head). Fluoroscopy and digital subtraction angiography (DSA) acquisition times, C-arm angulation, and PO/AO height were recorded. RESULTS: Seventeen cases were analysed (Crawford II-IV), with a median operating time of 280 minutes (interquartile range 200-330 minutes). Median age was 76 years (range 71-81 years); median body mass index was 28 kg/m(2) (25-32 kg/m(2)). Stent-grafts incorporated branches only, fenestrations only, or a mixture of branches and fenestrations. A total of 21 branches and 38 fenestrations were cannulated and stented. Head dose was significantly higher in the PO compared with the AO (median 54 µSv [range 24-130 µSv] vs. 15 µSv [range 7-43 µSv], respectively; p = .022), as was over-lead body dose (median 80 µSv [range 37-163 µSv] vs. 32 µSv [range 6-48 µSv], respectively; p = .003). Corresponding under-lead doses were similar between operators (median 4 µSv [range 1-17 µSv] vs. 1 µSv [range 1-3 µSv], respectively; p = .222). Primary operator height, DSA acquisition time in left anterior oblique (LAO) position, and degrees of LAO angulation were independent predictors of PO head dose (p < .05). CONCLUSIONS: The head is an unprotected area receiving a significant radiation dose during complex endovascular aortic repair. The deleterious effects of exposure to this area are not fully understood. Vascular interventionalists should be cognisant of head exposure increasing with C-arm angulation, and limit this manoeuvre.

X-ray crystal structure of the streptococcal specific phage lysin PlyC.

Bacteriophages deploy lysins that degrade the bacterial cell wall and facilitate virus egress from the host. When applied exogenously, these enzymes destroy susceptible microbes and, accordingly, have potential as therapeutic agents. The most potent lysin identified to date is PlyC, an enzyme assembled from two components (PlyCA and PlyCB) that is specific for streptococcal species. Here the structure of the PlyC holoenzyme reveals that a single PlyCA moiety is tethered to a ring-shaped assembly of eight PlyCB molecules. Structure-guided mutagenesis reveals that the bacterial cell wall binding is achieved through a cleft on PlyCB. Unexpectedly, our structural data reveal that PlyCA contains a glycoside hydrolase domain in addition to the previously recognized cysteine, histidine-dependent amidohydrolases/peptidases catalytic domain. The presence of eight cell wall-binding domains together with two catalytic domains may explain the extraordinary potency of the PlyC holoenyzme toward target bacteria.

Distance as a risk factor for amputation in patients with diabetes: a case-control study.

We studied the association between amputation and distance of patients' residences to a diabetes care centre. We performed a case-control study matching each case (amputation) with 5 controls (no amputation) by age and sex. We compared the distance of residence to the diabetes centre, duration and type of diabetes, haemoglobin-A1c levels and foot examination findings for cases and controls. We analysed the association between distance and the strongest predictors of amputation. Sixty-six cases of amputation and 313 controls were identified. Distance of residence was 12.1km greater for cases (p = 0.028). In multivariate analysis, only diabetes duration (OR/year 1.07, 1.03 to 1.11) and neuropathy (OR 10.73, 4.55 to 25.74) were significantly associated with amputation. Patients with neuropathy resided 97 km further than those without neuropathy (p = 0.01). Patients requiring amputation reside at greater distances from the diabetes centre, possibly due to higher rates of neuropathy.

Germline single nucleotide polymorphisms associated with response of urothelial carcinoma to platinum-based therapy: the role of the host.

BACKGROUND: Variations in urothelial carcinoma (UC) response to platinum chemotherapy are common and frequently attributed to genetic and epigenetic variations of somatic DNA. We hypothesized that variations in germline DNA may contribute to UC chemosensitivity. PATIENTS AND METHODS: DNA from 210 UC patients treated with platinum-based chemotherapy was genotyped for 80 single nucleotide polymorphisms (SNPs). Logistic regression was used to examine the association between SNPs and response, and a multivariable predictive model was created. Significant SNPs were combined to form a SNP score predicting response. Eleven UC cell lines were genotyped as validation. RESULTS: Six SNPs were significantly associated with 101 complete or partial responses (48%). Four SNPs retained independence association and were incorporated into a response prediction model. Each additional risk allele was associated with a nearly 50% decrease in odds of response [odds ratio (OR) = 0.51, 95% confidence interval 0.39-0.65, P = 1.05 × 10(-7)). The bootstrap-adjusted area under the curves of this model was greater than clinical prognostic factors alone (0.78 versus 0.64). The SNP score showed a positive trend with chemosensitivity in cell lines (P = 0.115). CONCLUSIONS: Genetic variants associated with response of UC to platinum-based therapy were identified in germline DNA. A model using these genetic variants may predict response to chemotherapy better than clinical factors alone.

Optimal scaling of weight and waist circumference to height for maximal association with DXA-measured total body fat mass by sex, age and race/ethnicity.

BACKGROUND: Body mass index (BMI; weight (Wt)/height (Ht) (in kg m(-2)) and waist circumference (WC) are widely used as proxy anthropometric measures for total adiposity. Little is known about what scaling power of 'x' in both Wt(kg)/Ht(m)(x) and WC(m)/Ht(m)(x) is maximally associated with measured total body fat mass (TBFM). Establishing values for x would provide the information needed to create optimum anthropometric surrogate measures of adiposity. OBJECTIVE: To estimate the value of 'x' that renders Wt/Ht(x) and WC/Ht(x) maximally associated with DXA-measured TBFM. SUBJECTS: Participants of the NHANES 1999-2004 surveys, stratified by sex (men, women), race/ethnicity (non-Hispanic whites, non-Hispanic blacks, Mexican-Americans), and age(18-29, 30-49, 50-84 years). METHODS: We apply a grid search by increasing x from 0.0-3.0 by increments of 0.1 to the simple regression models, TBFM=b0+b1*(Wt/Ht(x)) and TBFM=b0+b1*(WC/Ht(x)) to obtain an estimate of x that results in the greatest R(2), taking into account complex survey design features and multiply imputed data. RESULTS: R(2)'s for BMI are 0.86 for men (N=6544) and 0.92 for women (N=6362). The optimal powers x for weight are 1.0 (R(2)=0.90) for men and 0.8 (R(2)=0.96) for women. The optimal power x for WC is 0, that is, no scaling of WC to height, for men (R(2)=0.90) or women (R(2)=0.82). The optimal powers for weight across nine combinations of race/ethnicity and age groups for each sex vary slightly (x=0.8-1.3) whereas the optimal scaling powers for WC are all 0 for both sexes except for non-Hispanic black men aged 18-29y (x=0.1). Although the weight-for-height indices with optimal powers are not independent of height, they yield more accurate TBFM estimates than BMI. CONCLUSION: In reference to TBFM, Wt/Ht and Wt/Ht(0.8) are the optimal weight-for-height indices for men and women, respectively, whereas WC alone, without Ht adjustment, is the optimal WC-for-height index for both sexes. Thus, BMI, an index independent of height, may be less useful when predicting TBFM.

Outpatient parenteral antimicrobial therapy (OPAT) in the Republic of Ireland: results of a national survey.

Outpatient parenteral antimicrobial therapy (OPAT) services are not well developed in the Republic of Ireland. A national programme is being instituted to standardise care. This survey aims to assess the current use of outpatient intravenous antibiotics and to quantify the needs that physicians identify in the development of a national programme. General medical consultant physicians and clinical microbiology consultants were contacted through the Royal College of Physicians of Ireland (RCPI) from April to June 2012. Data were analysed using SPSS version 20. A total of 512 physicians were contacted, of which 55 (10.7 %) responded. The majority, 38/55 (69 %), practice general internal medicine in combination with a medical specialty, 2 (4 %) general internal medicine alone, 8 (15 %) clinical microbiology and 7 (13 %) a medical specialty alone. Of those practising a medical specialty, 12 (27 %) practice infectious diseases. Seventy-four percent reported having discharged patients with intravenous antibiotics; however, 47 % did not have a designated service available. Of those with no service, 100 % identified a need for these resources. Of those responsible for an OPAT service, 56 % had not audited their service. The most common indications were skin and soft tissue infections, osteomyelitis and respiratory tract infection. Flucloxacillin was the most commonly reported antibiotic. Eleven percent responded that they never monitor laboratory studies for patients discharged with intravenous antibiotics. While OPAT services in Ireland are not well developed, patients are being discharged with intravenous antibiotics. This survey underscores the need to develop the national programme to standardise care and ensure patients receive safe and efficient therapy.

The effects of dependence and function on costs of care for Alzheimer's disease and mild cognitive impairment in Ireland.

OBJECTIVE: To explore the incremental effects of patient dependence and function on costs of care for patients with Alzheimer's disease (AD) and amnestic mild cognitive impairment (MCI) in Ireland. METHODS: Cost analysis based on reported resource use for a cross-section of 100 community-based people with AD and MCI. Formal care included general practice visits, hospitalizations, outpatient clinic consultations, accident and emergency visits, respite care, meals on wheels services and other health and social care professional consultations. Informal care included time input provided by caregivers. Resource unit costs were applied to value formal care and the opportunity cost method was used to value informal care. Patient dependence on others was measured using the Dependence Scale and patient functional capacity using the Disability Assessment for Dementia scale. Multivariate regression analysis was used to model the cost of care. RESULTS: Both dependence and function were independently and significantly associated with total formal and informal care cost: a one point increase in dependence was associated with a €796 increase in total cost and a one point improvement in function with a €417 reduction in total cost over 6 months. Patient function was significantly associated with formal care costs, whereas patient function and dependence were both significantly associated with informal care costs. CONCLUSION: The costs of care for patients with AD and MCI in Ireland are substantial. Interventions that reduce patient dependence on others and functional decline may be associated with important economic benefits.

Monomeric crystal structure of the vaccine carrier protein CRM197 and implications for vaccine development.

CRM197 is a genetically detoxified mutant of diphtheria toxin (DT) that is widely used as a carrier protein in conjugate vaccines. Protective immune responses to several bacterial diseases are obtained by coupling CRM197 to glycans from these pathogens. Wild-type DT has been described in two oligomeric forms: a monomer and a domain-swapped dimer. Their proportions depend on the chemical conditions and especially the pH, with a large kinetic barrier to interconversion. A similar situation occurs in CRM197, where the monomer is preferred for vaccine synthesis. Despite 30 years of research and the increasing application of CRM197 in conjugate vaccines, until now all of its available crystal structures have been dimeric. Here, CRM197 was expressed as a soluble, intracellular protein in an Escherichia coli strain engineered to have an oxidative cytoplasm. The purified product, called EcoCRM, remained monomeric throughout crystallization. The structure of monomeric EcoCRM is reported at 2.0 Šresolution with the domain-swapping hinge loop (residues 379-387) in an extended, exposed conformation, similar to monomeric wild-type DT. The structure enables comparisons across expression systems and across oligomeric states, with implications for monomer-dimer interconversion and for the optimization of conjugation.