Innovations in functional and rehabilitative knee bracing.

New knee brace designs are available that have the potential to improve patient outcomes relative to traditional bracing. For the indications of post-knee injury/surgery recovery, conservative management of knee osteoarthritis (OA), total knee arthroplasty (TKA) pre-habilitation, and the treatment of post-surgical extension deficits/flexion contractures, innovative new bracing designs merit review and discussion. The researchers requested information from industry brace manufacturers, and from the information received, have selected those products considered significant improvements over traditional functional brace designs for review in this article. Clinical research supporting the benefits of the innovative products listed in the article have been cited when available. The authors are both Certified Orthotists with over 50 years of combined knee bracing experience.

Zn2+-binding in the glutamate-rich region of the intrinsically disordered protein prothymosin-alpha.

Prothymosin-α is a small, multifunctional intrinsically disordered protein associated with cell survival and proliferation which binds multiple Zn2+ ions and undergoes partial folding. The interaction between prothymosin-α and at least two of its protein targets is significantly enhanced in the presence of Zn2+ ions, suggesting that Zn2+ binding plays a role in the protein's function. The primary sequence of prothymosin-α is highly acidic, with almost 50% comprised of Asp and Glu, and is unusual for a Zn2+-binding protein as it lacks Cys and His residues. To gain a better understanding of the nature of the Zn2+-prothymosin-α interactions and the protein's ability to discriminate Zn2+ over other divalent cations (e.g., Ca2+, Co2+, Mg2+) we synthesized a set of three model peptides and characterized the effect of metal binding using electrospray ionization mass spectrometry (ESI MS) and circular dichroism (CD) spectroscopy. ESI MS data reveal that the native peptide model of the glutamic acid rich region binds 4 Zn2+ ions with apparent, stepwise Kd values that are, at highest, in the tens of micromolar range. A peptide model with the same amino acid composition as the native sequence, but with the residues arranged randomly, showed no evidence of structural change by CD upon introduction of Zn2+. These results suggest that the high net negative charge of the glutamic acid-rich region of prothymosin-α is not a sufficient criterion for Zn2+ to induce a structural change; rather, Zn2+ binding to prothymosin-α is sequence specific, providing important insight into the behavior of intrinsically disordered proteins.

Effect of 28-mm Cryoballoon Ablation on Major Atrial Ganglionated Plexi.

OBJECTIVES: The authors intended to investigate if 28-mm cryoballoon (CB) ablation also modifies the 4 major atrial ganglionaated plexi (GP). BACKGROUND: The major atrial GP facilitate the initiation and maintenance of atrial fibrillation (AF). The 28-mm CB covers a large surface area of the left atrium and probably the GP areas. METHODS: High-frequency stimulation (20 Hz) was delivered to the area of anterior right GP (ARGP), inferior right GP, superior left (SLGP), and inferior left GP (ILGP). Positive GP sites were defined as a prolongation of R-wave to R-wave (RR) interval during AF by >50%. The area of each GP before and after CB ablation was compared. RESULTS: A total of 18 patients with paroxysmal AF who underwent CB and radiofrequency ablation and had positive GP sites were reviewed. The Wilcoxon signed-rank test was used to assess the effects of CB ablation on each GP. There was a statistically significant difference in the area of all 4 GP after CB ablation: 1) ARGP area: 2.9 cm2 (interquartile range [IQR]: 2.1 to 3.5 cm2) pre-CB, 0.1 cm2 (IQR: 0 to 0.6 cm2) post-CB, p = 0.0002; 2) inferior right GP area: 2.1 cm2 (IQR: 0.9 to 2.9 cm2) pre-CB, 0.5 cm2 (IQR: 0 to 1.7 cm2) post-CB, p = 0.001; 3) SLGP area: 1.4 cm2 (IQR: 0.6 to 2.4 cm2) pre-CB, 0 cm2 (IQR: 0 to 0 cm2) post-CB, p = 0.0002; and 4) ILGP area: 1.3 cm2 (IQR: 0.3 to 2.2 cm2) pre-CB, 0.3 cm2 (IQR: 0 to 1.6 cm2) post-CB, p = 0.008. CONCLUSIONS: The surface area of all 4 of the major atrial GP was substantially reduced by CB ablation. The SLGP and ARGP had the largest, whereas the ILGP had the least percent of reduction following CB ablation. Part of the therapeutic effects of CB ablation may result from modifying the 4 major atrial GP.

Home is where the future is: The BrightFocus Foundation consensus panel on dementia care.

INTRODUCTION: A national consensus panel was convened to develop recommendations on future directions for home-based dementia care (HBDC). METHODS: The panel summarized advantages and challenges of shifting to HBDC as the nexus of care and developed consensus-based recommendations. RESULTS: The panel developed five core recommendations: (1) HBDC should be considered the nexus of new dementia models, from diagnosis to end of life in dementia; (2) new payment models are needed to support HBDC and reward integration of care; (3) a diverse new workforce that spans the care continuum should be prepared urgently; (4) new technologies to promote communication, monitoring/safety, and symptoms management must be tested, integrated, and deployed; and (5) targeted dissemination efforts for HBDC must be employed. DISCUSSION: HBDC represents a promising paradigm shift to improve care for those living with dementia and their family caregivers: these recommendations provide a framework to chart a course forward for HBDC.

The effectiveness of the bone bridge transtibial amputation technique: A systematic review of high-quality evidence.

BACKGROUND: This literature review was undertaken to determine if commonly held views about the benefits of a bone bridge technique are supported by the literature. METHODS: Four databases were searched for articles pertaining to surgical strategies specific to a bone bridge technique of the transtibial amputee. A total of 35 articles were identified as potential articles. Authors included methodology that was applied to separate topics. Following identification, articles were excluded if they were determined to be low quality evidence or not pertinent. RESULTS: Nine articles were identified to be pertinent to one of the topics: Perioperative Care, Acute Care, Subjective Analysis and Function. Two articles sorted into multiple topics. Two articles were sorted into the Perioperative Care topic, 4 articles sorted into the Acute Care topic, 2 articles into the Subjective Analysis topic and 5 articles into the Function topic. DISCUSSION: There are no high quality (level one or two) clinical trials reporting comparisons of the bone bridge technique to traditional methods. There is limited evidence supporting the clinical outcomes of the bone bridge technique. There is no agreement supporting or discouraging the perioperative and acute care aspects of the bone bridge technique. There is no evidence defining an interventional comparison of the bone bridge technique. CONCLUSION: Current level III evidence supports a bone bridge technique as an equivalent option to the non-bone bridge transtibial amputation technique. Formal level I and II clinical trials will need to be considered in the future to guide clinical practice. Clinical relevance Clinical Practice Guidelines are evidence based. This systematic literature review identifies the highest quality evidence to date which reports a consensus of outcomes agreeing bone bridge is as safe and effective as alternatives. The clinical relevance is understanding bone bridge could additionally provide a mechanistic advantage for the transtibial amputee.

A Simple Dewar/Cryostat for Thermally Equilibrating Samples at Known Temperatures for Accurate Cryogenic Luminescence Measurements.

The design and operation of a simple liquid nitrogen Dewar/cryostat apparatus based upon a small fused silica optical Dewar, a thermocouple assembly, and a CCD spectrograph are described. The experiments for which this Dewar/cryostat is designed require fast sample loading, fast sample freezing, fast alignment of the sample, accurate and stable sample temperatures, and small size and portability of the Dewar/cryostat cryogenic unit. When coupled with the fast data acquisition rates of the CCD spectrograph, this Dewar/cryostat is capable of supporting cryogenic luminescence spectroscopic measurements on luminescent samples at a series of known, stable temperatures in the 77-300 K range. A temperature-dependent study of the oxygen quenching of luminescence in a rhodium(III) transition metal complex is presented as an example of the type of investigation possible with this Dewar/cryostat. In the context of this apparatus, a stable temperature for cryogenic spectroscopy means a luminescent sample that is thermally equilibrated with either liquid nitrogen or gaseous nitrogen at a known measureable temperature that does not vary (ΔT < 0.1 K) during the short time scale (~1-10 sec) of the spectroscopic measurement by the CCD. The Dewar/cryostat works by taking advantage of the positive thermal gradient dT/dh that develops above liquid nitrogen level in the Dewar where h is the height of the sample above the liquid nitrogen level. The slow evaporation of the liquid nitrogen results in a slow increase in h over several hours and a consequent slow increase in the sample temperature T over this time period. A quickly acquired luminescence spectrum effectively catches the sample at a constant, thermally equilibrated temperature.

A Potent d-Protein Antagonist of VEGF-A is Nonimmunogenic, Metabolically Stable, and Longer-Circulating in Vivo.

Polypeptides composed entirely of d-amino acids and the achiral amino acid glycine (d-proteins) inherently have in vivo properties that are proposed to be near-optimal for a large molecule therapeutic agent. Specifically, d-proteins are resistant to degradation by proteases and are anticipated to be nonimmunogenic. Furthermore, d-proteins are manufactured chemically and can be engineered to have other desirable properties, such as improved stability, affinity, and pharmacokinetics. Thus, a well-designed d-protein therapeutic would likely have significant advantages over l-protein drugs. Toward the goal of developing d-protein therapeutics, we previously generated RFX001.D, a d-protein antagonist of natural vascular endothelial growth factor A (VEGF-A) that inhibited binding to its receptor. However, RFX001.D is unstable at physiological temperatures (Tm = 33 °C). Here, we describe RFX037.D, a variant of RFX001.D with extreme thermal stability (Tm > 95 °C), high affinity for VEGF-A (Kd = 6 nM), and improved receptor blocking. Comparison of the two enantiomeric forms of RFX037 revealed that the d-protein is more stable in mouse, monkey, and human plasma and has a longer half-life in vivo in mice. Significantly, RFX037.D was nonimmunogenic in mice, whereas the l-enantiomer generated a strong immune response. These results confirm the potential utility of synthetic d-proteins as alternatives to therapeutic antibodies.

How much gene flow is needed to avoid inbreeding depression in wild tiger populations?

The number and size of tiger populations continue to decline owing to habitat loss, habitat fragmentation and poaching of tigers and their prey. As a result, tiger populations have become small and highly structured. Current populations have been isolated since the early 1970s or for approximately seven generations. The objective of this study is to explore how inbreeding may be affecting the persistence of remaining tiger populations and how dispersal, either natural or artificial, may reduce the potentially detrimental effect of inbreeding depression. We developed a tiger simulation model and used published levels of genetic load in mammals to simulate inbreeding depression. Following a 50 year period of population isolation, we introduced one to four dispersing male tigers per generation to explore how gene flow from nearby populations may reduce the negative impact of inbreeding depression. For the smallest populations, even four dispersing male tigers per generation did not increase population viability, and the likelihood of extinction is more than 90% within 30 years. Unless habitat connectivity is restored or animals are artificially introduced in the next 70 years, medium size wild populations are also likely to go extinct, with only four to five of the largest wild tiger populations likely to remain extant in this same period without intervention. To reduce the risk of local extinction, habitat connectivity must be pursued concurrently with efforts to increase population size (e.g. enhance habitat quality, increase habitat availability). It is critical that infrastructure development, dam construction and other similar projects are planned appropriately so that they do not erode the extent or quality of habitat for these populations so that they can truly serve as future source populations.

Modeling, characterizing, and accommodating static birefringence in circular and linear dichroism spectroscopy.

Nearly all circular dichroism (CD) and linear dichroism (LD) spectrometers use a photoelastic modulator (PEM) in which an optical element is stressed using a high-tension voltage (HT) signal to induce birefringence. The birefringence consequently produces a phase difference between perpendicular polarization states of light passing through the PEM that is appropriate to CD or LD measurements. However, even without external stress (i.e., at zero HT) the PEM exhibits an inherent static birefringence. This article discusses the characterization of the static birefringence inherent to a PEM and its effect on the measurement of CD and LD, as well as the development and implementation of a novel model that accommodates for the presence of static birefringence. The model is validated with CD and LD experiments using purely chiral or linearly structured molecules (camphorsulfonic acid and chrysazin).

Novel technique for improvement in calibration of the photoelastic modulator in circular and linear dichroism spectroscopy.

Both the voltage-induced and the inherent (static) birefringence of a photoelastic modulator (PEM) affect the phase difference between orthogonal components of light passing through the PEM. This phase difference determines the polarization state of the light and is essential for determining true circular and linear dichroism (CD and LD, respectively) spectroscopy. Presented here are a more complete theoretical model of CD and LD and a new technique to determine the phase difference and static birefringence of a PEM in CD and LD. The intensity of the light for various configurations of the analyzer is interpreted (by using Mueller matrices and Stokes parameters) to calibrate the voltage-induced phase difference and to characterize the static birefringence in the photoelastic modulator. The effects of the static birefringence as well as intermediate polarization states (between left- and right-circularly polarized light) on LD and CD are characterized. Appropriate adjustments to the voltage applied to the PEM in order to mitigate these effects are discussed. It is expected that the techniques presented here will have a broad impact on the calibration of CD and LD spectrometers.

Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography.

Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF(165) to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {D-protein antagonist + L-protein form of VEGF-A}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 Å. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 Å(2) in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.

CsSnI3: Semiconductor or metal? High electrical conductivity and strong near-infrared photoluminescence from a single material. High hole mobility and phase-transitions.

CsSnI(3) is an unusual perovskite that undergoes complex displacive and reconstructive phase transitions and exhibits near-infrared emission at room temperature. Experimental and theoretical studies of CsSnI(3) have been limited by the lack of detailed crystal structure characterization and chemical instability. Here we describe the synthesis of pure polymorphic crystals, the preparation of large crack-/bubble-free ingots, the refined single-crystal structures, and temperature-dependent charge transport and optical properties of CsSnI(3), coupled with ab initio first-principles density functional theory (DFT) calculations. In situ temperature-dependent single-crystal and synchrotron powder X-ray diffraction studies reveal the origin of polymorphous phase transitions of CsSnI(3). The black orthorhombic form of CsSnI(3) demonstrates one of the largest volumetric thermal expansion coefficients for inorganic solids. Electrical conductivity, Hall effect, and thermopower measurements on it show p-type metallic behavior with low carrier density, despite the optical band gap of 1.3 eV. Hall effect measurements of the black orthorhombic perovskite phase of CsSnI(3) indicate that it is a p-type direct band gap semiconductor with carrier concentration at room temperature of ∼ 10(17) cm(-3) and a hole mobility of ∼585 cm(2) V(-1) s(-1). The hole mobility is one of the highest observed among p-type semiconductors with comparable band gaps. Its powders exhibit a strong room-temperature near-IR emission spectrum at 950 nm. Remarkably, the values of the electrical conductivity and photoluminescence intensity increase with heat treatment. The DFT calculations show that the screened-exchange local density approximation-derived band gap agrees well with the experimentally measured band gap. Calculations of the formation energy of defects strongly suggest that the electrical and light emission properties possibly result from Sn defects in the crystal structure, which arise intrinsically. Thus, although stoichiometric CsSnI(3) is a semiconductor, the material is prone to intrinsic defects associated with Sn vacancies. This creates highly mobile holes which cause the materials to appear metallic.

Noise characterization in circular dichroism spectroscopy.

Circular dichroism (CD), defined as the difference in absorption between left and right circularly polarized light, is used to spectroscopically study the structures of chiral materials. In this article, various methodologies are presented for characterizing the performance of CD spectrometers to determine (1) experimental conditions for optimal data collection, (2) noise characteristics dependent on machine parameters, (3) the relative significance of spectral data as a function of detector gain, and (4) stray light and dark current as a function of wavelength. The results of case studies of two commercial CD spectrometers (specifically, Jasco J810 and J815) are described. The analyses show that the variation of CD signal is Poisson distributed and hence can be considered shot noise. Also, optimum scan parameters are established and a weighting function of CD data significance is produced so that wavelength-dependent gain (as determined by the high tension, HT, voltage applied to the photomultiplier tube, PMT, detector) can be accommodated. Lastly, the amount of stray light and dark current for the photomultiplier tube is determined. Though specific to the Jasco CD spectrometers characterized in this study, it is expected that all CD spectrometers exhibit similar behavior and the methodology described here can be usefully applied to characterize CD spectrometers independent of manufacturer.

Ligand binding assays in the 21st century laboratory: recommendations for characterization and supply of critical reagents.

Critical reagents are essential components of ligand binding assays (LBAs) and are utilized throughout the process of drug discovery, development, and post-marketing monitoring. Successful lifecycle management of LBA critical reagents minimizes assay performance problems caused by declining reagent activity and can mitigate the risk of delays during preclinical and clinical studies. Proactive reagent management assures adequate supply. It also assures that the quality of critical reagents is appropriate and consistent for the intended LBA use throughout all stages of the drug development process. This manuscript summarizes the key considerations for the generation, production, characterization, qualification, documentation, and management of critical reagents in LBAs, with recommendations for antibodies (monoclonal and polyclonal), engineered proteins, peptides, and their conjugates. Recommendations are given for each reagent type on basic and optional characterization profiles, expiration dates and storage temperatures, and investment in a knowledge database system. These recommendations represent a consensus among the authors and should be used to assist bioanalytical laboratories in the implementation of a best practices program for critical reagent life cycle management.

Calorimetric investigation of copper(II) binding to Aß peptides: thermodynamics of coordination plasticity.

Metal ions have been shown to play a critical role in ß-amyloid (Aß) neurotoxicity, thus prompting an intense investigation into the formation of metal-Aß complexes. Isothermal titration calorimetry (ITC) has been widely used to determine binding constants (K) for a variety of metal-protein interactions, including those in metal-Aß complexes. In this study, ITC was used to more fully quantify the thermodynamics (K, ΔG, ΔH, and TΔS) of Cu(2+) binding to Aß16, N-acetyl-Aß16, Aß28, N-acetyl-Aß28, and Aß28 variants (H6A, H13A, H14A) at pH 7.4 and 37 °C. After deconvolution of competing reactions, K for Aß16 was found to be 1.1 (±0.13) × 10(9) and is in strong agreement with literature values measured under similar conditions. Further, a similar K value was obtained at two additional concentrations of competing ligand, suggesting that ternary complex formation is not significant. The acetylated peptide analogs reveal a marked decrease in the overall free energy upon binding, which is the result of less favorable enthalpic and entropic contributions. Circular dichroism spectroscopy shows conformational changes that are consistent with these results. Most importantly, data for Aß28 variants lacking a potential Cu(2+)-binding histidine residue reveal that the overall free energy of binding remains constant, which is the result of entropy/enthalpy compensation. These data provide fundamental thermodynamic evidence for coordination plasticity in Cu(2+) binding to Aß and other intrinsically disordered peptides.

Characterization of amyloidogenic intermediate states through a combined use of CD and NMR spectroscopy.

Characterization of amyloidogenic intermediate states is of central importance in understanding the molecular mechanism of amyloid formation. In this study, we utilized CD and NMR spectroscopy to investigate secondary structure of the monomeric amyloidogenic intermediate of a beta-structured SH3 domain, which was induced by trifluoroethanol (TFE). The combined biophysical studies showed that the native state SH3 domain is gradually converted to the amyloidogenic intermediate state at TFE concentrations of 20-26% (v/v) and the aggregation-prone state contains substantial amount of the beta-sheet conformation ( approximately 30%) with disordered (54%) and some helical characters (16%). Under weaker amyloidogenic conditions of higher TFE concentrations (>40%), the beta-sheet structures were gradually changed to helical conformations and the relative content of the helical and beta-sheet conformations was highly correlated with the aggregation propensity of the SH3 domain. This indicates that the beta-sheet characters of the amyloidogenic states may be critical to the effective amyloid formation.

Genetic organization, length conservation, and evolution of RNA polymerase II carboxyl-terminal domain.

With a simple tandem iterated sequence, the carboxyl terminal domain (CTD) of eukaryotic RNA polymerase II (RNAP II) serves as the central coordinator of mRNA synthesis by harmonizing a diversity of sequential interactions with transcription and processing factors. Despite intense research interest, many key questions regarding functional and evolutionary constraints on the CTD remain unanswered; for example, what selects for the canonical heptad sequence, its tandem array across organismal diversity, and constant CTD length within given species and finally and how a sequence-identical, repetitive structure can orchestrate a diversity of simultaneous and sequential, stage-dependent interactions with both modifying enzymes and binding partners? Here we examine comparative sequence evolution of 58 RNAP II CTDs from diverse taxa representing all six major eukaryotic supergroups and employ integrated evolutionary genetic, biochemical, and biophysical analyses of the yeast CTD to further clarify how this repetitive sequence must be organized for optimal RNAP II function. We find that the CTD is composed of indivisible and independent functional units that span diheptapeptides and not only a flexible conformation around each unit but also an elastic overall structure is required. More remarkably, optimal CTD function always is achieved at approximately wild-type CTD length rather than number of functional units, regardless of the characteristics of the sequence present. Our combined observations lead us to advance an updated CTD working model, in which functional, and therefore, evolutionary constraints require a flexible CTD conformation determined by the CTD sequence and tandem register to accommodate the diversity of CTD-protein interactions and a specific CTD length rather than number of functional units to correctly order and organize global CTD-protein interactions. Patterns of conservation of these features across evolutionary diversity have important implications for comparative RNAP II function in eukaryotes and can more clearly direct specific research on CTD function in currently understudied organisms.

Structural disorder in silk proteins reveals the emergence of elastomericity.

Spider silks combine basic amino acids into strong and versatile fibers where the quality of the elastomer is attributed to the interaction of highly adapted protein motifs with a complex spinning process. The evaluation, however, of the interaction has remained elusive. Here, we present a novel analysis to study silk formation by examining the secondary structures of silk proteins in solution. Using the seven different silks of Nephila edulis as a benchmark system, we define a structural disorder parameter (the folding index, gamma). We found that gamma is highly correlated with the ratio of glycine present. Testing the correlation between glycine content and the folding index (gamma) against a selected range of silks, we find quantitatively that, in order to achieve specialization with changes in mechanical performance, the spider's silks require higher structural flexibility at the expense of reduced stability and consequently an increased conversion-energy cost. Taken together, our biophysical and evolutionary findings reveal that silk elastomericity evolved in tandem with specializations in the process of silk spinning.

Fesselin is a natively unfolded protein.

Fesselin is a heat stable proline-rich actin binding protein. The stability, amino acid composition, and ability to bind to several proteins suggested that fesselin may be unfolded under native conditions. While the complete sequence of fesselin is unknown an analysis of a closely related protein, synaptopodin 2 from Gallus gallus, indicates that fesselin consists of a series of unstructured regions interspersed between short folded regions. To determine if fesselin is natively unfolded, we compared fesselin to a known globular protein (myosin S1) and a known unfolded protein Cad22 (the COOH terminal 22 kDa fragment of caldesmon). Fesselin, and Cad22, had larger Stokes radii than globular proteins of equivalent mass. The environments of tryptophan residues of fesselin and Cad22 were the same in the presence and absence of 6 M guanidine hydrochloride. Fesselin had a circular dichroism spectrum that was primarily random coil. Changes in pH over the range of 1.5-11.5 did not alter that spectrum. Increasing the temperature to 85 degrees C caused an increase in the degree of secondary structure. Calmodulin binding to fesselin altered the environment of the tryptophan residues so that they became less sensitive to the quencher acrylamide. These results show that fesselin is a natively unfolded protein.