Engineering highly stable variants of Corynactis californica green fluorescent proteins.

Fluorescent proteins (FPs) are versatile biomarkers that facilitate effective detection and tracking of macromolecules of interest in real time. Engineered FPs such as superfolder green fluorescent protein (sfGFP) and superfolder Cherry (sfCherry) have exceptional refolding capability capable of delivering fluorescent readout in harsh environments where most proteins lose their native functions. Our recent work on the development of a split FP from a species of strawberry anemone, Corynactis californica, delivered pairs of fragments with up to threefold faster complementation than split GFP. We present the biophysical, biochemical, and structural characteristics of five full-length variants derived from these split C. californica GFP (ccGFP). These ccGFP variants are more tolerant under chemical denaturation with up to 8 kcal/mol lower unfolding free energy than that of the sfGFP. It is likely that some of these ccGFP variants could be suitable as biomarkers under more adverse environments where sfGFP fails to survive. A structural analysis suggests explanations of the variations in stabilities among the ccGFP variants.

Crystal structures of multidrug efflux transporters from Burkholderia pseudomallei suggest details of transport mechanism.

BpeB and BpeF are multidrug efflux transporters from Burkholderia pseudomallei that enable multidrug resistance. Here, we report the crystal structures of BpeB and BpeF at 2.94 Å and 3.0 Å resolution, respectively. BpeB was found as an asymmetric trimer, consistent with the widely-accepted functional rotation mechanism for this type of transporter. One of the monomers has a distinct structure that we interpret as an intermediate along this functional cycle. Additionally, a detergent molecule bound in a previously undescribed binding site provides insights into substrate translocation through the pathway. BpeF shares structural similarities with the crystal structure of OqxB from Klebsiella pneumoniae, where both are symmetric trimers composed of three "binding"-state monomers. The structures of BpeB and BpeF further our understanding of the functional mechanisms of transporters belonging to the HAE1-RND superfamily.

Effects of lateral instability on ankle coupled motions in vivo using 3D fluoroscopy.

Lateral ankle instability (LAI) compromises the normal kinematics of the ankle, affecting activities of daily living. In vitro kinematics of ankles with LAI during single-plane motions are available, but the active control stability of these motions remains unclear. The current study measured the 3D ankle kinematics during unresisted single-plane motion tests using a bi-plane fluoroscope with a CT model-based 2D/3D registration method in 12 patients with LAI and 14 healthy peers. The coupling of the kinematic components at the talocrural and subtalar joints was quantified by the path difference between the forward and return paths of the coupled motion. Significantly increased path differences were found in the subtalar dorsiflexion/plantarflexion and inversion/eversion components during internal/external rotation tests (p < 0.05). During inversion/eversion, significantly reduced tibiocalcaneal ranges of motion and the path differences in the talocrural and subtalar dorsiflexion/plantarflexion components were noted (p < 0.05). The current results suggest that chronic LAI had compromised control stability at the subtalar joint during internal/external rotation tests and a conservative motion control strategy with significantly reduced ranges of motion to maintain good control of out-of-plane motion components in response to direct challenges of the anterior talofibular ligament during inversion/eversion tests. The current results also suggest that, compared to kinematic patterns of individual components, the path difference of the coupled motion may serve as a better measure of the motion control stability of the ankle in differentiating LAI from healthy controls.

Label-free affinity screening, design and synthesis of inhibitors targeting the Mycobacterium tuberculosis L-alanine dehydrogenase.

The ability of Mycobacterium tuberculosis (Mtb) to persist in its host may enable an evolutionary advantage for drug resistant variants to emerge. A potential strategy to prevent persistence and gain drug efficacy is to directly target the activity of enzymes that are crucial for persistence. We present a method for expedited discovery and structure-based design of lead compounds by targeting the hypoxia-associated enzyme L-alanine dehydrogenase (AlaDH). Biochemical and structural analyses of AlaDH confirmed binding of nucleoside derivatives and showed a site adjacent to the nucleoside binding pocket that can confer specificity to putative inhibitors. Using a combination of dye-ligand affinity chromatography, enzyme kinetics and protein crystallographic studies, we show the development and validation of drug prototypes. Crystal structures of AlaDH-inhibitor complexes with variations at the N6 position of the adenyl-moiety of the inhibitor provide insight into the molecular basis for the specificity of these compounds. We describe a drug-designing pipeline that aims to block Mtb to proliferate upon re-oxygenation by specifically blocking NAD accessibility to AlaDH. The collective approach to drug discovery was further evaluated through in silico analyses providing additional insight into an efficient drug development strategy that can be further assessed with the incorporation of in vivo studies.

Increased Loading Rates During Gait Correlate With Morphology of Unaffected Hip in Juveniles With Treated Developmental Hip Dysplasia.

Long-term follow-up studies on children with surgically treated developmental dysplasia of the hip (DDH) have shown that not only the affected side progresses to premature osteoarthritis, but the unaffected side may also suffer from insidious hip dysplasia or osteonecrosis. The current gait analysis study identified the loading and unloading rates of the ground reaction forces (GRF) and lower limb joint axial forces during gait, and their correlations with the hip morphology in twenty juvenile patients with surgically treated unilateral DDH during toddlerhood. In a gait laboratory, each subject walked at preferred speed on a 10-m walkway while the kinematics and the GRF were measured. Loading and unloading rates of the vertical GRF and the joint axial forces were obtained as the maximum instantaneous slope of these force curves. Radiographic measurements of the hips were taken, and the correlations between the morphological parameters and the loading and unloading rates were obtained. The patients showed greater-than-normal peak loading rates of the joint axial forces, and the loading rates on both the affected and unaffected sides were strongly correlated to the acetabular index on the unaffected side, which was also significantly correlated with the peak unloading rates on the affected side. These results suggest that apart from regular follow-up of the affected hip, routine assessment of the morphological changes and/or increased loading rates on the unaffected hip is also important for early identification of any signs of insidious hip dysplasia and risk of premature degeneration of the cartilage.

Effects of Internal Fixation for Mid-Shaft Clavicle Fractures on Shoulder Kinematics During Humeral Elevations.

BACKGROUND: Mid-shaft clavicle fractures account for 35 to 44% of injuries to the shoulder girdle. There is increasing evidence to support surgical repair, but poor functional outcomes have been reported, and associated factors remain unclear. METHODS: The three-dimensional poses of the shoulder bones during arm elevations were measured in 15 patients treated for mid-shaft clavicle fractures by open reduction and internal fixation, and in 15 healthy controls. RESULTS AND CONCLUSION: No significant between-side differences were found in the clavicle length after surgery (p > 0.05). The patients showed increased scapular protraction at lower elevation angles and reduced scapular retraction at higher elevation angles during frontal-plane elevations, with significantly reduced clavicle retraction (p < 0.05), with unaltered scapular rotation and tilt. The ranges of the observed changes were reduced to arm elevations at 60° and 90° in the scapular and sagittal planes. Similar changes were also found on the unaffected side, suggesting symmetrical bilateral compensation. The results suggest that shoulder kinematics in multi-plane arm elevations should be monitored for any signs of compromised bone motions following surgical treatment, and that rehabilitative training may be needed on both sides to improve the bilateral movement control of the shoulder complex.

Light Guide Layer Thickness Optimization for Enhancement of the Light Extraction Efficiency of Ultraviolet Light-Emitting Diodes.

Consider material machinability and lattice mismatch sapphire as substrates for the ultraviolet-C light-emitting diodes (UV-C LEDs) are commonly used, but their high refractive index can result in the total internal reflection (TIR) of light whereby some light is absorbed, therefore caused reducing light extraction efficiency (LEE). In this study, we propose a method to optimize the thickness of a sapphire substrate light guide layer through first-order optical design which used the optical simulation software Ansys SPEOS to simulate and evaluate the light extraction efficiency. AlGaN UV-C LEDs wafers with a light guide layer thickness of 150-700 µm were used. The simulation proceeded under a center wavelength of 275 nm to determine the optimal thickness design of the light guide layer. Finally, the experimental results demonstrated that the initial light guide layer thickness of 150 µm the reference output power of 13.53 mW, and an increased thickness of 600 um resulted in output power of 20.58 mW. The LEE can be increased by 1.52 times through light guide layer thickness optimization. We propose a method to optimize the thickness of a sapphire substrate light guide layer through first-order optical design. AlGaN UV-C LEDs wafers with a light guide layer thickness of 150-700 µm were used. Finally, the experimental results demonstrated that the LEE can be increased by 1.52 times through light guide layer thickness optimization.

Changes in night sky brightness after a countywide LED retrofit.

The US National Park Service (NPS) Night Skies Program measured changes in sky brightness resulting from a countywide lighting retrofit project. The retrofit took place in Chelan County, a gateway community to North Cascades National Park and Lake Chelan National Recreation Area in Washington State. The county retrofitted all 3693 county-owned high pressure sodium (HPS) street lamps to full cutoff LEDs. This number is about 60% of the County's total outdoor street and area lights. About 80% of the newly installed lights were 3000 K in color temperature and 20% were 4000 K. The 4000 K LEDs were used to meet Washington State Department of Transportation guidelines. To measure sky brightness, we used the NPS night sky camera system before the retrofit started in 2018 and after its completion in 2019. These images were photometrically calibrated and mosaicked together to provide hemispherical images in V band. For comparison with our ground-based measurement, we obtained the satellite imagery taken by Visible Infrared Imaging Radiometer Suite (VIIRS) onboard the Suomi National Polar-orbiting Partnership satellite. Our measurements show that the post-retrofit skyglow became brighter and extended higher in the sky, but upward radiance, as measured by the day-night band radiometer, decreased. These divergent results are likely explained by a substantial increase in light emitted at wavelengths shorter than 500 nm, and a relative decrease in upward light emission due to better shielded luminaires. These results also demonstrate that earlier models relating VIIRS day-night band data to skyglow will - at a minimum - require substantial revision to account for the different characteristics of solid state luminaires.

Construction, characterization and crystal structure of a fluorescent single-chain Fv chimera.

In vitro display technologies based on phage and yeast have a successful history of selecting single-chain variable fragment (scFv) antibodies against various targets. However, single-chain antibodies are often unstable and poorly expressed in Escherichia coli. Here, we explore the feasibility of converting scFv antibodies to an intrinsically fluorescent format by inserting the monomeric, stable fluorescent protein named thermal green, between the light- and heavy-chain variable regions. Our results show that the scTGP format maintains the affinity and specificity of the antibodies, improves expression levels, allows one-step fluorescent assay for detection of binding and is a suitable reagent for epitope binning. We also report the crystal structure of an scTGP construct that recognizes phosphorylated tyrosine on FcεR1 receptor of the allergy pathway.

Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge.

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.

Teres minor muscle hypertrophy is a negative predictor of outcomes after reverse total shoulder arthroplasty: an evaluation of preoperative magnetic resonance imaging and postoperative implant position.

BACKGROUND: Predictors of outcomes after reverse total shoulder arthroplasty (rTSA) remain unclear. The purpose of this study was to analyze the impact of preoperative muscle quality and postoperative implant positioning on patient-reported outcomes following rTSA. METHODS: We evaluated 88 shoulders treated with rTSA in which preoperative magnetic resonance imaging was available. Preoperative muscle quality was evaluated, including fatty infiltration, rotator cuff muscle volume, and total tear size. Postoperative implant position was determined radiographically. The correlation between imaging parameters and the 2-year postoperative American Shoulder and Elbow Surgeons (ASES) score was examined. Multivariate analyses were performed to adjust for confounding factors including patient demographic characteristics and implant position. RESULTS: Univariate analysis showed that the ASES score was significantly lower in patients with teres minor muscle hypertrophy relative to those with normal muscle (73.3 ± 22.8 vs. 84.2 ± 16.9, P = .02). The functional subscore was significantly lower in patients with grade 2 fatty infiltration of the deltoid muscle relative to those with grade 0 fatty infiltration (26.1 ± 14.6 vs. 34.8 ± 11.6, P = .03). Older age was associated with a higher pain subscore (ρ = 0.32, P = .002). Multivariate analysis demonstrated that teres minor muscle hypertrophy remained a significant independent predictor of the ASES score (ß coefficient = 91.3, P = .03). CONCLUSION: Teres minor muscle hypertrophy is an independent negative predictor of patient-reported outcomes after rTSA.

Tendon release reduced joint stiffness with unaltered leg stiffness during gait in spastic diplegic cerebral palsy.

Biomechanical deviations at individual joints are often identified by gait analysis of patients with cerebral palsy (CP). Analysis of the control of joint and leg stiffness of the locomotor system during gait in children with spastic diplegic CP has been used to reveal their control strategy, but the differences between before and after surgery remain unknown. The current study aimed to bridge the gap by comparing the leg stiffness-both skeletal and muscular components-and associated joint stiffness during gait in 12 healthy controls and 12 children with spastic diplegic CP before and after tendon release surgery (TRS). Each subject walked at a self-selected pace on a 10-meter walkway while their kinematic and forceplate data were measured to calculate the stiffness-related variables during loading response, mid-stance, terminal stance, and pre-swing. The CP group altered the stiffness of the lower limb joints and decreased the demand on the muscular components while maintaining an unaltered leg stiffness during stance phase after the TRS. The TRS surgery improved the joint and leg stiffness control during gait, although residual deficits and associated deviations still remained. It is suggested that the stiffness-related variables be included in future clinical gait analysis for a more complete assessment of gait in children with CP.

Single-Session Video and Electromyography Feedback in Overhead Athletes With Scapular Dyskinesis and Impingement Syndrome.

CONTEXT: Subacromial impingement syndrome (SIS) is associated with scapular dyskinesis, or imbalanced scapular muscle activity. Evidence has shown that feedback can improve scapular control in patients with SIS. However, it is unknown whether real-time video feedback or electromyography (EMG) biofeedback is optimal for improving scapular kinematics and muscle activity during a functional task. OBJECTIVE: To compare the effects of video and EMG feedback sessions on absolute muscle activity (upper trapezius [UT], lower trapezius [LT], serratus anterior), muscle balance ratios (UT/LT, UT/serratus anterior), and scapular kinematics (anterior-posterior tilt, external-internal rotation, upward rotation) in SIS participants during arm elevation and lowering. DESIGN: Randomized controlled clinical trial. SETTING: Research laboratory. PATIENTS OR OTHER PARTICIPANTS: Overhead athletes who were diagnosed with SIS and who also exhibited scapular dyskinesis (N = 41). MAIN OUTCOME MEASURE(S): Three-dimensional kinematics and EMG were recorded before and after feedback training. RESULTS: Lower trapezius muscle activity increased (4.2%-18%, P < .011) and UT/LT decreased (0.56-1.17, P < .013) in the EMG biofeedback training group as compared with those in the video feedback training group. Scapular upward rotation during arm elevation was higher in the video group than in the EMG group after feedback training (2.3°, P = .024). CONCLUSIONS: The EMG biofeedback improved muscle control and video feedback improved the correction of scapular upward rotation in patients with SIS. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov: NCT03252444.

Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix.

Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.

Functional workspace and patient-reported outcomes improve after reverse and total shoulder arthroplasty.

BACKGROUND: Low-cost motion analysis systems (LCMASs) have emerged as easy and practical methods to measure the functional workspace (FWS). Thus, we ventured to apply an LCMAS, the Kinect2 gaming camera, to evaluate the FWS in patients with shoulder osteoarthritis (OA) and patients who underwent total shoulder arthroplasty (TSA) or reverse total shoulder arthroplasty (RTSA). METHODS: A cross-sectional study of participants with OA (n = 53), TSA (n = 70), and RTSA (n = 34) was performed. The FWS as measured by an LCMAS, the American Shoulder and Elbow Surgeons (ASES) Standardized Shoulder Assessment Form score, and the Patient-Reported Outcomes Measurement Information System (PROMIS) score were collected. For participants who underwent TSA or RTSA, the FWS was evaluated at 6, 12, and 24 months postoperatively. The correlation of the FWS with the ASES score and PROMIS score was determined. Significance was set at P < .05. RESULTS: Patients who underwent TSA or RTSA had a significantly higher FWS than patients with shoulder OA at almost all time points. Patients who underwent TSA had a significantly higher FWS than patients who underwent RTSA at 24 months after surgery. PROMIS and ASES scores showed strong correlations with the FWS in patients who underwent TSA (R = 0.75 [P < .001] and R = 0.83 [P < .001], respectively) and RTSA (R = 0.84 [P < .001] and R = 0.73 [P < .001], respectively). CONCLUSION: The FWS measured by an LCMAS is an easy and low-cost method to quantify the reachable space of the hand in patients and shows strong correlations with patient-reported outcome measures. This may be a useful tool to assess upper-extremity range of motion before and after shoulder arthroplasty.

Outcomes of revision arthroplasty for shoulder periprosthetic joint infection: a three-stage revision protocol.

BACKGROUND: This study evaluated outcomes after treatment of shoulder periprosthetic joint infection (PJI) with a 3-stage revision protocol consisting of (1) débridement, explantation, and cement spacer placement, followed by parenteral antibiotics; (2) open biopsy and débridement; and (3) reimplantation if cultures were negative. We hypothesized this protocol would eradicate persistent infection while producing excellent functional and subjective outcomes, and there would be no difference in these parameters for patients with shoulder PJI compared with patients with revision for aseptic indications. METHODS: We retrospectively analyzed a prospectively collected revision shoulder arthroplasty cohort to identify shoulder PJI patients treated with a 3-stage protocol. Demographics, culture data, range of motion, and patient-reported outcomes were collected. Outcomes for patients with shoulder PJI and revision to RTSA were compared with patients revised to RTSA for noninfectious indications. Significance was defined as P < .05. RESULTS: There were 28 cases of shoulder PJI in 27 patients (age, 66.4 ± 11.2 years,); of these, 21 shoulders were revised to RTSA, and 7 shoulders were revised to hemiarthroplasty. There was no recurrent infection at a mean 32-month follow-up. One year after surgery, mean forward flexion was 110° ± 41° and abduction was 106° ± 42°. Mean final American Shoulder and Elbow Surgeons subjective score was 66.5 ± 23.3. The 21 shoulders with PJI revised to RTSA had no differences for functional and subjective outcomes compared with revised patients without shoulder PJI. CONCLUSIONS: A 3-stage revision protocol for shoulder PJI reliably eradicated infection. Patients with PJI revised to RTSA can have similar outcomes as patients with noninfectious revision to RTSA.

BpeB, a major resistance-nodulation-cell division transporter from Burkholderia cenocepacia: construct design, crystallization and preliminary structural analysis.

Burkholderia cenocepacia is an opportunistic pathogen that infects cystic fibrosis patients, causing pneumonia and septicemia. B. cenocepacia has intrinsic antibiotic resistance against monobactams, aminoglycosides, chloramphenicol and fluoroquinolones that is contributed by a homologue of BpeB, which is a member of the resistance-nodulation-cell division (RND)-type multidrug-efflux transporters. Here, the cloning, overexpression, purification, construct design for crystallization and preliminary X-ray diffraction analysis of this BpeB homologue from B. cenocepacia are reported. Two truncation variants were designed to remove possible disordered regions based on comparative sequence and structural analysis to salvage the wild-type protein, which failed to crystallize. The 17-residue carboxyl-terminal truncation yielded crystals that diffracted to 3.6 Šresolution. The efflux function measured using minimal inhibitory concentration assays indicated that the truncation decreased, but did not eliminate, the efflux activity of the transporter.

Chronic kidney disease predicts a lower probability of improvement in patient-reported experience measures among patients with fractures: a prospective multicenter cohort study.

Patient-reported experience measures (PREMs) are integral component of care for fracture patients. Using a multicenter cohort, we showed that the presence of chronic kidney disease (CKD) attenuated the probability of PREM improvement in fracture patients. INTRODUCTION: Assessing PREM can assist physicians in improving patients' experiences. Patients with CKD are at an increased risk of exhibiting poor PREM and developing fractures. We aimed to assess whether CKD influences the probability of PREM improvement during follow-up among patients with fractures. METHODS: We prospectively enrolled patients with hip or vertebral fractures from different institutes into a fracture liaison service program. After registering clinical histories, they received a baseline PREM assessment based on EuroQol group-5 dimension content, including self-care, daily activity, and pain severity using a 5-point Likert scale. A follow-up PREM assessment was arranged 4 months later, and we evaluated whether baseline CKD was predictive of PREM improvement. RESULTS: Among 593 fracture patients (18% with CKD), 37.3% and 62.7% presented with hip and vertebral fractures, respectively. Self-care, daily activity, and pain severity improved after follow-up in 32%, 27%, and 43% participants; those with CKD exhibited worse self-care ability and daily activity than those without. Multivariate logistic regression analyses showed that baseline CKD was significantly associated with lower possibility of improvement in daily activity (odds ratio [OR] 0.58, p = 0.049) and pain severity (OR 0.52, p = 0.01), and an insignificant change in the possibility of improvement in self-care ability (OR 0.61, p = 0.09). CONCLUSIONS: The presence of CKD predicts a significantly lower probability of PREM improvement among fracture patients. An early emphasis on renal function during fracture care should be considered.

The influence of renal dialysis and hip fracture sites on the 10-year mortality of elderly hip fracture patients: A nationwide population-based observational study.

Hip fractures in older people requiring dialysis are associated with high mortality. Our study primarily aimed to evaluate the specific burden of dialysis on the mortality rate following hip fracture. The secondary aim was to clarify the effect of the fracture site on mortality. A retrospective cohort study was conducted using Taiwan's National Health Insurance Research Database to analyze nationwide health data regarding dialysis and non-dialysis patients ≥65 years who sustained a first fragility-related hip fracture during the period from 2001 to 2005. Each dialysis hip fracture patient was age- and sex-matched to 5 non-dialysis hip fracture patients to construct the matched cohort. Survival status of patients was followed-up until death or the end of 2011. Survival analyses using multivariate Cox proportional hazards models and the Kaplan-Meier estimator were performed to compare between-group survival and impact of hip fracture sites on mortality. A total of 61,346 hip fracture patients were included nationwide. Among them, 997 dialysis hip fracture patients were identified and matched to 4985 non-dialysis hip fracture patients. Mortality events were 155, 188, 464, and 103 in the dialysis group, and 314, 382, 1505, and 284 in the non-dialysis group, with adjusted hazard ratios (associated 95% confidence intervals) of 2.58 (2.13-3.13), 2.95 (2.48-3.51), 2.84 (2.55-3.15), and 2.39 (1.94-2.93) at 0 to 3 months, 3 months to 1 year, 1 to 6 years, and 6 to 10 years after the fracture, respectively. In the non-dialysis group, survival was consistently better for patients who sustained femoral neck fractures compared to trochanteric fractures (0-10 years' log-rank test, P < .001). In the dialysis group, survival of patients with femoral neck fractures was better than that of patients with trochanteric fractures only within the first 6 years post-fracture (0-6 years' log-rank, P < .001). Dialysis was a significant risk factor of mortality in geriatric hip fracture patients. Survival outcome was better for non-dialysis patients with femoral neck fractures compared to those with trochanteric fractures throughout 10 years. However, the survival advantage of femoral neck fractures was limited to the first 6 years postinjury among dialysis patients.

Structural basis for DNA recognition by STAT6.

STAT6 participates in classical IL-4/IL-13 signaling and stimulator of interferon genes-mediated antiviral innate immune responses. Aberrations in STAT6-mediated signaling are linked to development of asthma and diseases of the immune system. In addition, STAT6 remains constitutively active in multiple types of cancer. Therefore, targeting STAT6 is an attractive proposition for treating related diseases. Although a lot is known about the role of STAT6 in transcriptional regulation, molecular details on how STAT6 recognizes and binds specific segments of DNA to exert its function are not clearly understood. Here, we report the crystal structures of a homodimer of phosphorylated STAT6 core fragment (STAT6CF) alone and bound with the N3 and N4 DNA binding site. Analysis of the structures reveals that STAT6 undergoes a dramatic conformational change on DNA binding, which was further validated by performing molecular dynamics simulation studies and small angle X-ray scattering analysis. Our data show that a larger angle at the intersection where the two protomers of STAT meet and the presence of a unique residue, H415, in the DNA-binding domain play important roles in discrimination of the N4 site DNA from the N3 site by STAT6. H415N mutation of STAT6CF decreased affinity of the protein for the N4 site DNA, but increased its affinity for N3 site DNA, both in vitro and in vivo. Results of our structure-function studies on STAT6 shed light on mechanism of DNA recognition by STATs in general and explain the reasons underlying STAT6's preference for N4 site DNA over N3.