Structure and Oxygen Affinity of Crystalline des-His-146β Human Hemoglobin in the T State *

To correlate directly structure with function, the oxygen affinity and the three-dimensional structure of crystals of the T quaternary state of des-His-146beta human hemoglobin have been determined by polarized absorption microspectrophotometry and x-ray diffraction crystallography. In des-His-146beta, the COOH-terminal histidine residues of the beta chains of hemoglobin A have been removed. Oxygen binding to crystalline des-His hemoglobin is non-cooperative and independent of pH. The oxygen affinity is 1.7-fold greater than that of the crystalline state of hemoglobin A. Removal of His-146beta results in a small movement of the truncated COOH-terminal peptide and in a very small change in quaternary structure. Previously, similar studies on T state crystals of des-Arg-141alpha hemoglobin showed that removal of the COOH termini of the alpha chains results in much larger effects on oxygen affinity and on quaternary structure. Kinetic studies in solution reveal that at pH 7.0, the rates of CO combination with deoxygenated des-His-146beta in the absence and presence of inositol hexaphosphate are 2.5- and 1.3-fold, respectively, more rapid than for hemoglobin A. The values for des-Arg are 7.6- and 3.9-fold. The properties of the T state of hemoglobin both in the crystal and in solution are influenced to a greater degree by the interactions associated with Arg-141alpha than those associated with His-146beta

Nuclease Modulates Biofilm Formation in Community-Associated Methicillin-Resistant Staphylococcus aureus

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging contributor to biofilm-related infections. We recently reported that strains lacking sigma factor B (sigB) in the USA300 lineage of CA-MRSA are unable to develop a biofilm. Interestingly, when spent media from a USA300 sigB mutant was incubated with other S. aureus strains, biofilm formation was inhibited. Following fractionation and mass spectrometry analysis, the major anti-biofilm factor identified in the spent media was secreted thermonuclease (Nuc). Considering reports that extracellular DNA (eDNA) is an important component of the biofilm matrix, we investigated the regulation and role of Nuc in USA300. The expression of the nuc gene was increased in a sigB mutant, repressed by glucose supplementation, and was unaffected by the agr quorum-sensing system. A FRET assay for Nuc activity was developed and confirmed the regulatory results. A USA300 nuc mutant was constructed and displayed an enhanced biofilm-forming capacity, and the nuc mutant also accumulated more high molecular weight eDNA than the WT and regulatory mutant strains. Inactivation of nuc in the USA300 sigB mutant background partially repaired the sigB biofilm-negative phenotype, suggesting that nuc expression contributes to the inability of the mutant to form biofilm. To test the generality of the nuc mutant biofilm phenotypes, the mutation was introduced into other S. aureus genetic backgrounds and similar increases in biofilm formation were observed. Finally, using multiple S. aureus strains and regulatory mutants, an inverse correlation between Nuc activity and biofilm formation was demonstrated. Altogether, our findings confirm the important role for eDNA in the S. aureus biofilm matrix and indicates Nuc is a regulator of biofilm formation

Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix.

We developed a new approach that couples Southwestern blotting and mass spectrometry to discover proteins that bind extracellular DNA (eDNA) in bacterial biofilms. Using Staphylococcus aureus as a model pathogen, we identified proteins with known DNA-binding activity and uncovered a series of lipoproteins with previously unrecognized DNA-binding activity. We demonstrated that expression of these lipoproteins results in an eDNA-dependent biofilm enhancement. Additionally, we found that while deletion of lipoproteins had a minimal impact on biofilm accumulation, these lipoprotein mutations increased biofilm porosity, suggesting that lipoproteins and their associated interactions contribute to biofilm structure. For one of the lipoproteins, SaeP, we showed that the biofilm phenotype requires the lipoprotein to be anchored to the outside of the cellular membrane, and we further showed that increased SaeP expression correlates with more retention of high-molecular-weight DNA on the bacterial cell surface. SaeP is a known auxiliary protein of the SaeRS system, and we also demonstrated that the levels of SaeP correlate with nuclease production, which can further impact biofilm development. It has been reported that S. aureus biofilms are stabilized by positively charged cytoplasmic proteins that are released into the extracellular environment, where they make favorable electrostatic interactions with the negatively charged cell surface and eDNA. In this work we extend this electrostatic net model to include secreted eDNA-binding proteins and membrane-attached lipoproteins that can function as anchor points between eDNA in the biofilm matrix and the bacterial cell surface.IMPORTANCE Many bacteria are capable of forming biofilms encased in a matrix of self-produced extracellular polymeric substances (EPS) that protects them from chemotherapies and the host defenses. As a result of these inherent resistance mechanisms, bacterial biofilms are extremely difficult to eradicate and are associated with chronic wounds, orthopedic and surgical wound infections, and invasive infections, such as infective endocarditis and osteomyelitis. It is therefore important to understand the nature of the interactions between the bacterial cell surface and EPS that stabilize biofilms. Extracellular DNA (eDNA) has been recognized as an EPS constituent for many bacterial species and has been shown to be important in promoting biofilm formation. Using Staphylococcus aureus biofilms, we show that membrane-attached lipoproteins can interact with the eDNA in the biofilm matrix and promote biofilm formation, which suggests that lipoproteins are potential targets for novel therapies aimed at disrupting bacterial biofilms

Evryscope Science: Exploring the Potential of All-Sky Gigapixel-Scale Telescopes

Low-cost mass-produced sensors and optics have recently made it feasible to build telescope arrays which observe the entire accessible sky simultaneously. In this article, we discuss the scientific motivation for these telescopes, including exoplanets, stellar variability, and extragalactic transients. To provide a concrete example we detail the goals and expectations for the Evryscope, an under-construction 780 MPix telescope which covers 8660 sq. deg. in each 2-minute exposure; each night, 18,400 sq. deg. will be continuously observed for an average of ≈6 hr. Despite its small 61 mm aperture, the system's large field of view provides an étendue which is ∼10% of LSST. The Evryscope, which places 27 separate individual telescopes into a common mount which tracks the entire accessible sky with only one moving part, will return 1%-precision, many-year-length, high-cadence light curves for every accessible star brighter than ∼16th magnitude. The camera readout times are short enough to provide near-continuous observing, with a 97% survey time efficiency. The array telescope will be capable of detecting transiting exoplanets around every solar-type star brighter than mV = 12, providing at least few-millimagnitude photometric precision in long-term light curves. It will be capable of searching for transiting giant planets around the brightest and most nearby stars, where the planets are much easier to characterize; it will also search for small planets nearby M-dwarfs, for planetary occultations of white dwarfs, and will perform comprehensive nearby microlensing and eclipse-timing searches for exoplanets inaccessible to other planet-finding methods. The Evryscope will also provide comprehensive monitoring of outbursting young stars, white dwarf activity, and stellar activity of all types, along with finding a large sample of very-long-period M-dwarf eclipsing binaries. When relatively rare transients events occur, such as gamma-ray bursts (GRBs), nearby supernovae, or even gravitational wave detections from the Advanced LIGO/Virgo network, the array will return minute-by-minute light curves without needing pointing toward the event as it occurs. By coadding images, the system will reach V ∼ 19 in 1-hr integrations, enabling the monitoring of faint objects. Finally, by recording all data, the Evryscope will be able to provide pre-event imaging at 2-minute cadence for bright transients and variable objects, enabling the first high-cadence searches for optical variability before, during and after all-sky events

MABEL photon-counting laser altimetry data in Alaska for ICESat-2 simulations and development

Ice, Cloud, and land Elevation Satellite-2 (ICESat-2) is scheduled to launch in late 2017 and will carry the Advanced Topographic Laser Altimeter System (ATLAS) which is a photon-counting laser altimeter and represents a new approach to satellite determination of surface elevation. Given the new technology of ATLAS, an airborne instrument, the Multiple Altimeter Beam Experimental Lidar (MABEL), was developed to provide data needed for satellite-algorithm development and ICESat-2 error analysis. MABEL was deployed out of Fairbanks, Alaska, in July 2014 to provide a test dataset for algorithm development in summer conditions with water-saturated snow and ice surfaces. Here we compare MABEL lidar data to in situ observations in Southeast Alaska to assess instrument performance in summer conditions and in the presence of glacier surface melt ponds and a wet snowpack. Results indicate the following: (1) based on MABEL and in situ data comparisons, the ATLAS 90m beam-spacing strategy will provide a valid assessment of across-track slope that is consistent with shallow slopes (< 1) of an ice-sheet interior over 50 to 150m length scales; (2) the dense along-track sampling strategy of photon counting systems can provide crevasse detail; and (3) MABEL 532 nm wavelength light may sample both the surface and subsurface of shallow (approximately 2m deep) supraglacial melt ponds. The data associated with crevasses and melt ponds indicate the potential ICESat-2 will have for the study of mountain and other small glaciers.Acknowledgements. Funding for this project was through the NASA ICESat-2 Project Science Office. Funding for J. M. Amundson was provided by NSF-PLR 1303895. We acknowledge the considerable efforts of the Project, Science, and Instrument teams of NASA’s ICESat-2 and MABEL missions. We thank the following people: Eugenia De Marco (ASRC Aerospace Corp., NASA/GSFC) and Dan Reed (Sigma Space Corp., NASA/GSFC) for MABEL instrument support; Scott Luthcke (NASA/GSFC), David Hancock (NASA/WFF), and Jeff Lee (NASA/WFF) for MABEL data calibration; Scott McGee and Ya’ Shonti Bridgers (JIRP) for GPS field data collection and data processing support; and NASA/AFRC (specifically ER-2 pilots Tim Williams and Denis Steele) for Alaska airborne support. WorldView imagery was provided by the Polar Geospatial Center at the University of Minnesota, which is supported by NSF-PLR 1043681. GPS receivers for the survey of the terminus of the Lower Taku Glacier were provided by UNAVCO. GPS receivers for the JIRP survey were provided by Werner Stempfhuber of the Beuth University of Applied Sciences. And, finally, we thank two anonymous reviewers for their highly constructive suggestions.Ye

Human monoclonal antibodies against Staphylococcus aureus surface antigens recognize in vitro and in vivo biofilm

Implant-associated Staphylococcus aureus infections are difficult to treat because of biofilm formation. Bacteria in a biofilm are often insensitive to antibiotics and host immunity. Monoclonal antibodies (mAbs) could provide an alternative approach to improve the diagnosis and potential treatment of biofilm-related infections. Here, we show that mAbs targeting common surface components of S. aureus can recognize clinically relevant biofilm types. The mAbs were also shown to bind a collection of clinical isolates derived from different biofilm-associated infections (endocarditis, prosthetic joint, catheter). We identify two groups of antibodies: one group that uniquely binds S. aureus in biofilm state and one that recognizes S. aureus in both biofilm and planktonic state. Furthermore, we show that a mAb recognizing wall teichoic acid (clone 4497) specifically localizes to a subcutaneously implanted pre-colonized catheter in mice. In conclusion, we demonstrate the capacity of several human mAbs to detect S. aureus biofilms in vitro and in vivo

Rheumatoid arthritis - treatment: 180. Utility of Body Weight Classified Low-Dose Leflunomide in Japanese Rheumatoid Arthritis

Background: In Japan, more than 20 rheumatoid arthritis (RA) patients died of interstitial pneumonia (IP) caused by leflunomide (LEF) were reported, but many of them were considered as the victims of opportunistic infection currently. In this paper, efficacy and safety of low-dose LEF classified by body weight (BW) were studied. Methods: Fifty-nine RA patients were started to administrate LEF from July 2007 to July 2009. Among them, 25 patients were excluded because of the combination with tacrolimus, and medication modification within 3 months before LEF. Remaining 34 RA patients administered 20 to 50 mg/week of LEF were followed up for 1 year and enrolled in this study. Dose of LEF was classified by BW (50 mg/week for over 50 kg, 40 mg/week for 40 to 50 kg and 20 to 30 mg/week for under 40 kg). The average age and RA duration of enrolled patients were 55.5 years old and 10.2 years. Prednisolone (PSL), methotrexate (MTX) and etanercept were used in 23, 28 and 2 patients, respectively. In case of insufficient response or adverse effect, dosage change or discontinuance of LEF were considered. Failure was defined as dosages up of PSL and MTX, or dosages down or discontinuance of LEF. Last observation carried forward method was used for the evaluation of failed patients at 1 year. Results: At 1 year after LEF start, good/ moderate/ no response assessed by the European League Against Rheumatism (EULAR) response criteria using Disease Activity Score, including a 28-joint count (DAS28)-C reactive protein (CRP) were showed in 14/ 10/ 10 patients, respectively. The dosage changes of LEF at 1 year were dosage up: 10, same dosage: 5, dosage down: 8 and discontinuance: 11 patients. The survival rate of patients in this study was 23.5% (24 patients failed) but actual LEF continuous rate was 67.6% (11 patients discontinued) at 1 year. The major reason of failure was liver dysfunction, and pneumocystis pneumonia was occurred in 1 patient resulted in full recovery. One patient died of sepsis caused by decubitus ulcer infection. DAS28-CRP score was decreased from 3.9 to 2.7 significantly. Although CRP was decreased from 1.50 to 0.93 mg/dl, it wasn't significant. Matrix metalloproteinase (MMP)-3 was decreased from 220.0 to 174.2 ng/ml significantly. Glutamate pyruvate transaminase (GPT) was increased from 19 to 35 U/l and number of leukocyte was decreased from 7832 to 6271 significantly. DAS28-CRP, CRP, and MMP-3 were improved significantly with MTX, although they weren't without MTX. Increase of GPT and leukopenia were seen significantly with MTX, although they weren't without MTX. Conclusions: It was reported that the risks of IP caused by LEF in Japanese RA patients were past IP history, loading dose administration and low BW. Addition of low-dose LEF is a potent safe alternative for the patients showing unsatisfactory response to current medicines, but need to pay attention for liver function and infection caused by leukopenia, especially with MTX. Disclosure statement: The authors have declared no conflicts of interes

Oral abstracts 1: SpondyloarthropathiesO1. Detecting axial spondyloarthritis amongst primary care back pain referrals

Background: Inflammatory back pain (IBP) is an early feature of ankylosing spondylitis (AS) and its detection offers the prospect of early diagnosis of AS. However, since back pain is very common but only a very small minority of back pain sufferers have ASpA or AS, screening of back pain sufferers for AS is problematic. In early disease radiographs are often normal so that fulfilment of diagnostic criteria for AS is impossible though a diagnosis of axial SpA can be made if MRI evidence of sacroiliitis is present. This pilot study was designed to indicate whether a cost-effective pick up rate for ASpA/early AS could be achieved by identifying adults with IBP stratified on the basis of age. Methods: Patients aged between 18 and 45 years who were referred to a hospital physiotherapy service with back pain of more than 3 months duration were assessed for IBP. All were asked to complete a questionnaire based on the Berlin IBP criteria. Those who fulfilled IBP criteria were also asked to complete a second short questionnaire enquiring about SpA comorbidities, to have a blood test for HLA-B27 and CRP level and to undergo an MRI scan of the sacroiliac joints. This was a limited scan, using STIR, diffusion-weighted, T1 and T2 sequences of the sacroiliac joints to minimize time in the scanner and cost. The study was funded by a research grant from Abbott Laboratories Ltd. Results: 50 sequential patients agreed to participate in the study and completed the IBP questionnaire. Of these 27 (54%) fulfilled criteria for IBP. Of these, 2 patients reported a history of an SpA comorbidity - 1 psoriasis; 1 ulcerative colitis - and 3 reported a family history of an SpA comorbidity - 2 psoriasis; 1 Crohn's disease. 4 were HLA-B27 positive, though results were not available for 7. Two patients had marginally raised CRP levels (6, 10 -NR ≤ 5). 19 agreed to undergo MRI scanning of the sacroiliac joints and lumbar spine; 4 scans were abnormal, showing evidence of bilateral sacroiliitis on STIR sequences. In all cases the changes met ASAS criteria but were limited. Of these 4 patients 3 were HLA-B27 positive but none gave a personal or family history of an SpA-associated comorbidity and all had normal CRP levels. Conclusions: This was a pilot study yielding only limited conclusions. However, it is clear that: Screening of patients referred for physiotherapy for IBP is straightforward, inexpensive and quick. It appears that IBP is more prevalent in young adults than overall population data suggest so that targeting this population may be efficient. IBP questionnaires could be administered routinely during a physiotherapy assessment. HLA-B27 testing in this group of patients with IBP is a suitable screening tool. The sacroiliac joint changes identified were mild and their prognostic significance is not yet clear so that the value of early screening needs further evaluation. Disclosure statement: C.H. received research funding for this study from Abbott. A.K. received research funding for this study, and speaker and consultancy fees, from Abbott. All other authors have declared no conflicts of interes

BHPR research: qualitative1. Complex reasoning determines patients' perception of outcome following foot surgery in rheumatoid arhtritis

Background: Foot surgery is common in patients with RA but research into surgical outcomes is limited and conceptually flawed as current outcome measures lack face validity: to date no one has asked patients what is important to them. This study aimed to determine which factors are important to patients when evaluating the success of foot surgery in RA Methods: Semi structured interviews of RA patients who had undergone foot surgery were conducted and transcribed verbatim. Thematic analysis of interviews was conducted to explore issues that were important to patients. Results: 11 RA patients (9 ♂, mean age 59, dis dur = 22yrs, mean of 3 yrs post op) with mixed experiences of foot surgery were interviewed. Patients interpreted outcome in respect to a multitude of factors, frequently positive change in one aspect contrasted with negative opinions about another. Overall, four major themes emerged. Function: Functional ability & participation in valued activities were very important to patients. Walking ability was a key concern but patients interpreted levels of activity in light of other aspects of their disease, reflecting on change in functional ability more than overall level. Positive feelings of improved mobility were often moderated by negative self perception ("I mean, I still walk like a waddling duck”). Appearance: Appearance was important to almost all patients but perhaps the most complex theme of all. Physical appearance, foot shape, and footwear were closely interlinked, yet patients saw these as distinct separate concepts. Patients need to legitimize these feelings was clear and they frequently entered into a defensive repertoire ("it's not cosmetic surgery; it's something that's more important than that, you know?”). Clinician opinion: Surgeons' post operative evaluation of the procedure was very influential. The impact of this appraisal continued to affect patients' lasting impression irrespective of how the outcome compared to their initial goals ("when he'd done it ... he said that hasn't worked as good as he'd wanted to ... but the pain has gone”). Pain: Whilst pain was important to almost all patients, it appeared to be less important than the other themes. Pain was predominately raised when it influenced other themes, such as function; many still felt the need to legitimize their foot pain in order for health professionals to take it seriously ("in the end I went to my GP because it had happened a few times and I went to an orthopaedic surgeon who was quite dismissive of it, it was like what are you complaining about”). Conclusions: Patients interpret the outcome of foot surgery using a multitude of interrelated factors, particularly functional ability, appearance and surgeons' appraisal of the procedure. While pain was often noted, this appeared less important than other factors in the overall outcome of the surgery. Future research into foot surgery should incorporate the complexity of how patients determine their outcome Disclosure statement: All authors have declared no conflicts of interes