Nephrotoxicity After the Treatment of Periprosthetic Joint Infection With Antibiotic-Loaded Cement Spacers

Background

Treatment of periprosthetic joint infections commonly involves insertion of an antibiotic-loaded cement spacer (ACS). The risk for acute kidney injury (AKI) related to use of antibiotic spacers has not been well defined. We aimed to identify the incidence of and risk factors for AKI after placement of an ACS.

CFTR rescue with VX-809 and VX-770 favors the repair of primary airway epithelial cell cultures from patients with class II mutations in the presence of Pseudomonas aeruginosa exoproducts

Background

Progressive airway damage due to bacterial infections, especially with Pseudomonas aeruginosa remains the first cause of morbidity and mortality in CF patients. Our previous work revealed a repair delay in CF airway epithelia compared to non-CF. This delay was partially prevented after CFTR correction (with VRT-325) in the absence of infection. Our goals were now to evaluate the effect of the Orkambi combination (CFTR VX-809 corrector?+?VX-770 potentiator) on the repair of CF primary airway epithelia, in infectious conditions.

Reducing the Human Burden of Breast Cancer: Advanced Radiation Therapy Yields Improved Treatment Outcomes

Radiation therapy is an important modality in the treatment of patients with breast cancer. While its efficacy in the treatment of breast cancer was known shortly after the discovery of x‐rays, significant advances in radiation delivery over the past 20 years have resulted in improved patient outcomes. With the development of improved systemic therapy, optimizing local control has become increasingly important and has been shown to improve survival. Better understanding of the magnitude of treatment benefit, as well as patient and biological factors that confer an increased recurrence risk, have allowed radiation oncologists to better tailor treatment decisions to individual patients. Furthermore, significant technological advances have occurred that have reduced the acute and long‐term toxicity of radiation treatment. These advances continue to reduce the human burden of breast cancer. It is important for radiation oncologists and nonradiation oncologists to understand these advances, so that patients are appropriately educated about the risks and benefits of this important treatment modality.     

Measuring anxiety after spinal cord injury: Development and psychometric characteristics of the SCI-QOL Anxiety item bank and linkage with GAD-7

ObjectiveTo develop a calibrated item bank and computer adaptive test to assess anxiety symptoms in individuals with spinal cord injury (SCI), transform scores to the Patient Reported Outcomes Measurement Information System (PROMIS) metric, and create a statistical linkage with the Generalized Anxiety Disorder (GAD)-7, a widely used anxiety measure.DesignGrounded-theory based qualitative item development methods; large-scale item calibration field testing; confirmatory factor analysis; graded response model item response theory analyses; statistical linking techniques to transform scores to a PROMIS metric; and linkage with the GAD-7.SettingFive SCI Model System centers and one Department of Veterans Affairs medical center in the United States.ParticipantsAdults with traumatic SCI.ResultsSeven hundred sixteen individuals with traumatic SCI completed 38 items assessing anxiety, 17 of which were PROMIS items. After 13 items (including 2 PROMIS items) were removed, factor analyses confirmed unidimensionality. Item response theory analyses were used to estimate slopes and thresholds for the final 25 items (15 from PROMIS). The observed Pearson correlation between the SCI-QOL Anxiety and GAD-7 scores was 0.67.ConclusionsThe SCI-QOL Anxiety item bank demonstrates excellent psychometric properties and is available as a computer adaptive test or short form for research and clinical applications. SCI-QOL Anxiety scores have been transformed to the PROMIS metric and we provide a method to link SCI-QOL Anxiety scores with those of the GAD-7.     

C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis

The variant rs26232, in the first intron of the chromosome 5 open reading frame 30 (C5orf30) locus, has recently been associated with both risk of developing rheumatoid arthritis (RA) and severity of tissue damage. The biological activities of human C5orf30 are unknown, and neither the gene nor protein show significant homology to any other characterized human sequences. The C5orf30 gene is present only in vertebrate genomes with a high degree of conservation, implying a central function in these organisms. Here, we report that C5orf30 is highly expressed in the synovium of RA patients compared with control synovial tissue, and that it is predominately expressed by synovial fibroblast (RASF) and macrophages in the lining and sublining layer of the tissue. These cells play a central role in the initiation and perpetuation of RA and are implicated in cartilage destruction. RASFs lacking C5orf30 exhibit increased cell migration and invasion in vitro, and gene profiling following C5orf30 inhibition confirmed up-regulation of genes involved in cell migration, adhesion, angiogenesis, and immune and inflammatory pathways. Importantly, loss of C5orf30 contributes to the pathology of inflammatory arthritis in vivo, because inhibition of C5orf30 in the collagen-induced arthritis model markedly accentuated joint inflammation and tissue damage. Our study reveal C5orf30 to be a previously unidentified negative regulator of tissue damage in RA, and this protein may act by modulating the autoaggressive phenotype that is characteristic of RASFs.Rheumatoid arthritis is a chronic systemic autoimmune disease characterized by a symmetrical, inflammatory arthropathy that frequently results in damage to synovial-lined joints with consequent pain, stiffness, and reduced functional capacity. The prevalence of RA is 0.8–1% in Western Europe and North America, and it is believed to arise from an interplay between genetics and the environment. Smoking is known to be a major risk factor particularly for anticitrullinated protein antibody-positive RA (1), whereas consumption of alcohol reduces both the risk and the severity of RA (2). The severity of RA varies from a mild condition with little joint damage to an unremitting condition that leads to extensive bone and cartilage damage. The radiological severity of damage to the hands and feet is widely used to measure outcome of RA and has been shown to have a significant genetic component (3, 4). Loci genetically associated with radiological damage include DRB1 (5), CD40 (6) and TRAF1/C5 (7), IL-4 (8), and IL-15 (9).A genome-wide association study involving 12,277 RA cases and 28,975 controls, all of European descent, reported association of rs26232 in the first intron of chromosome 5 open reading frame 30 (C5orf30) with risk of RA (10). Importantly linkage disequilibrium did not extend to genes in the flanking regions, indicating that the association was arising from C5orf30. This association was subsequently replicated in a British study of 6,108 RA cases and 13,009 controls (11). In a study of three large European RA populations (n = 1,884), we reported an allele dose association of rs26232 with radiological damage (12).The biological activities of human C5orf30 are unknown, and the precise roles it plays in RA have not yet been reported. There is indirect evidence linking human C5orf30 with immune function via its association with intracellular UNC119 (13); the latter increasing both T-cell activation by up-regulating Lck/Fyn activity and Src kinases regulating macrophages activation (14, 15). There are, however, no studies of the biological functions of human C5orf30 and, in view of the genetic association with RA susceptibility and severity, we have undertaken in silico analysis and both in vitro and in vivo experiments to determine its functional activities in RA. Here, we report C5orf30 to be a yet unidentified negative regulator of tissue damage in RA, acting by modulating the autoaggressive phenotype that is characteristic of RA synovial fibroblasts (RASF). It is highly expressed in the synovium of RA patients compared with healthy and osteoarthritis (OA) predominately by RASF in the lining and sublining layer. These cells play an important role in the initiation and perpetuation of RA and are implicated in cartilage destruction (16). Targeting C5orf30 expression by using siRNA technology resulted in increased invasiveness, proliferation and migration of RASFs in vitro, and modulated expression of genes in RA-relevant pathways including migration and adhesion. Importantly, loss of C5orf30 contributes to the pathology of inflammatory arthritis in vivo, because inhibition of C5orf30 in the collagen-induced arthritis (CIA) model mice markedly accentuated joint inflammation and cartilage destruction. These data confirm C5orf30 as a previously unidentified regulator of tissue destruction in RA.     

β-Helical architecture of cytoskeletal bactofilin filaments revealed by solid-state NMR

Bactofilins are a widespread class of bacterial filament-forming proteins, which serve as cytoskeletal scaffolds in various cellular pathways. They are characterized by a conserved architecture, featuring a central conserved domain (DUF583) that is flanked by variable terminal regions. Here, we present a detailed investigation of bactofilin filaments from Caulobacter crescentus by high-resolution solid-state NMR spectroscopy. De novo sequential resonance assignments were obtained for residues Ala39 to Phe137, spanning the conserved DUF583 domain. Analysis of the secondary chemical shifts shows that this core region adopts predominantly β-sheet secondary structure. Mutational studies of conserved hydrophobic residues located in the identified β-strand segments suggest that bactofilin folding and polymerization is mediated by an extensive and redundant network of hydrophobic interactions, consistent with the high intrinsic stability of bactofilin polymers. Transmission electron microscopy revealed a propensity of bactofilin to form filament bundles as well as sheet-like, 2D crystalline assemblies, which may represent the supramolecular arrangement of bactofilin in the native context. Based on the diffraction pattern of these 2D crystalline assemblies, scanning transmission electron microscopy measurements of the mass per length of BacA filaments, and the distribution of β-strand segments identified by solid-state NMR, we propose that the DUF583 domain adopts a β-helical architecture, in which 18 β-strand segments are arranged in six consecutive windings of a β-helix.Similar to eukaryotes, bacteria use a number of different cytoskeletal elements to ensure the proper temporal and spatial organization of their cellular machinery. Various studies proved the existence of bacterial homologs of eukaryotic cytoskeleton proteins including tubulin homologs such as FtsZ (1), actin homologs such as MreB (2), and intermediate filament (IF)-like proteins (3), which together have important roles in cell division, morphogenesis, polarity determination, and DNA segregation (4–7). In addition, several groups of polymer-forming proteins that are limited to the bacterial domain have been described (6).A recent addition to these bacteria-specific cytoskeletal proteins are the so-called bactofilins (8), a class of proteins that is widespread among most bacterial lineages and involved in a variety of different cellular processes. In the prosthecate α-proteobacterium Caulobacter crescentus, for instance, the two bactofilin paralogues BacA and BacB (Fig. 1A) assemble into membrane-associated polymeric sheets that are specifically localized to the cell pole carrying the stalk (8), a thin protrusion of the cell body involved in cell attachment and nutrient acquisition (9). These assemblies serve as spatial landmarks mediating the polar localization of a cell wall biosynthetic enzyme, PbpC, involved in stalk biogenesis (8) and organization (10). The δ-proteobacterial species Myxococcus xanthus, by contrast, possesses four bactofilin paralogues (BacMNOP) with, at least partly, distinct functions. BacM was shown to form cable- or rod-like structures that are critical for proper cell shape (8, 11). BacP, on the other hand, assembles into short filamentous structures that emanate from the cell poles, recruiting and thus controlling a small GTPase involved in type IV pili-dependent motility (12). As another well-characterized example, the ε-proteobacterium Helicobacter pylori, a human pathogen notorious for causing peptic ulcers, was shown to depend on a bactofilin homolog (CcmA) for maintaining its characteristic helical cell shape, a feature required for cells to efficiently colonize the gastric mucus (13). Moreover, in the γ-proteobacterium Proteus mirabilis, a homolog of the bactofilin CcmA has been implicated in cell shape and swarming motility (14).Open in a separate windowFig. 1.High-resolution ssNMR spectra of BacA filaments. (A) Selected bacterial bactofilins. The total number of amino acids is indicated in parentheses. (B) Amino acid sequence of C. crescentus BacA, together with a synthetic linker peptide (of 17 residues, in red) and a His₆-tag (in blue) attached at the C terminus. The DUF583 domain is highlighted in magenta, and prolines are shown in green. (C) Carbon–carbon 2D correlation spectrum of uniformly [¹³C, ¹⁵N]-labeled BacA. The carbon–carbon magnetization transfer is achieved by PDSD. A short PDSD mixing time of 20 ms was applied, optimal for intraresidue transfer. In the spectrum, a trace through Ile60 is shown to illustrate sensitivity and line width.Bactofilins are usually small proteins (∼20 kDa) that are composed of a central conserved domain of unknown function (DUF583) and flanking N- and C-terminal regions of variable length and sequence. A characteristic of bactofilins is their ability to polymerize spontaneously in the absence of nucleotides or other cofactors (8). Native BacM protofilaments have been isolated from M. xanthus whole-cell lysates by sucrose density centrifugation (11). Moreover, polymers of C. crescentus BacA and BacB were obtained after heterologous expression in Escherichia coli, a species that lacks chromosomally encoded bactofilin homologs (8). Similarly, polymerization was observed for heterologously produced M. xanthus BacN, BacO, and BacP (8, 12). In all cases, the filamentous structures formed were biochemically inert and resistant to nonphysiological salt concentrations and pH values. This behavior is reminiscent of IFs, although there is no evolutionary relationship between these two groups of cytoskeletal elements (6). In particular, bactofilins lack predicted coiled-coil regions, which are a key feature of IF proteins.Up to now, the molecular structure of bactofilins and the mechanism(s) underlying their assembly have remained unknown. This is in large part due to the spontaneous formation, inertness, and insolubility of bactofilin polymers, which makes them difficult substrates for crystallography studies as well as conventional liquid-state NMR methods. We therefore resorted to the use of solid-state NMR (ssNMR) spectroscopy, a technique that has recently been adapted to obtain high-resolution structural information on insoluble and noncrystalline protein assemblies, including functional oligomeric assemblies (15–17), disease-related amyloid fibrils (18–21), and membrane proteins in a lipid bilayer environment (22–26). In this study, we have applied state-of-the-art magic-angle spinning ssNMR spectroscopy in combination with a range of other biophysical methods to filaments of the C. crescentus bactofilin BacA (161 residues). We show that the DUF583 domain serves as a polymerization module that forms the rigid core of BacA filaments, whereas the terminal regions of the protein remain flexible. The core domain folds exclusively into β-sheets, but with an arrangement different from that typically found in amyloids. On the basis of the diffraction pattern of 2D crystalline assemblies observed by transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM) measurements that yield a value for the mass per length (MPL), and the β-strand segments identified by ssNMR, we propose a β-helical arrangement of the BacA subunit. Finally, we performed a mutational analysis of conserved residues in the β-strand segments, which suggests that the folding and/or polymerization of bactofilins are mediated by an extensive and redundant network of hydrophobic interactions. These findings provide for the first time, to our knowledge, insight into the atomic structure of a bacteria-specific cytoskeletal filament and highlight ssNMR as a powerful technique for the analysis of cytoskeletal elements that are unamenable to standard structural biological approaches.     

From the Cover: A simple nutrient-dependence mechanism for predicting the stoichiometry of marine ecosystems

It is widely recognized that the stoichiometry of nutrient elements in phytoplankton varies within the ocean. However, there are many conflicting mechanistic explanations for this variability, and it is often ignored in global biogeochemical models and carbon cycle simulations. Here we show that globally distributed particulate P:C varies as a linear function of ambient phosphate concentrations, whereas the N:C varies with ambient nitrate concentrations, but only when nitrate is most scarce. This observation is consistent with the adjustment of the phytoplankton community to local nutrient availability, with greater flexibility of phytoplankton P:C because P is a less abundant cellular component than N. This simple relationship is shown to predict the large-scale, long-term average composition of surface particles throughout large parts of the ocean remarkably well. The relationship implies that most of the observed variation in N:P actually arises from a greater plasticity in the cellular P:C content, relative to N:C, such that as overall macronutrient concentrations decrease, N:P rises. Although other mechanisms are certainly also relevant, this simple relationship can be applied as a first-order basis for predicting organic matter stoichiometry in large-scale biogeochemical models, as illustrated using a simple box model. The results show that including variable P:C makes atmospheric CO₂ more sensitive to changes in low latitude export and ocean circulation than a fixed-stoichiometry model. In addition, variable P:C weakens the relationship between preformed phosphate and atmospheric CO₂ while implying a more important role for the nitrogen cycle.Nutrient elements are used by phytoplankton to synthesize molecules, in order to accomplish biochemical functions. Some of these molecules are absolutely necessary, and the nutrient elements have no substitutes. Examples are P in nucleic acids, N in amino acids, and Fe in the photosynthetic apparatus (1). However, there is a degree of plasticity in the molecular assemblage required per phytoplankton cell, which varies between species and between clades (2, 3). Furthermore, there is a capacity for plasticity in molecular composition of even a given species, as shown in culture experiments (4, 5). Such plasticity leads to variability in the elemental ratios of nutrients in marine phytoplankton, widely documented in laboratory and field measurements (2, 6, 7). Recent analyses of global nutrient and particulate observations have shown that N:P, the most commonly discussed ratio, varies regionally, including low N:P in the high-latitude Southern Ocean and high N:P in the oligotrophic regions (7–9). Explanations of high N:P in oligotrophic waters have often invoked an enhanced reliance on N-rich proteins for gathering scarce resources (1, 10), whereas low N:P in the Southern Ocean has been variously attributed to the abundance of P-rich molecules in cold, fast-growing plankton (11), or to the availability of Si, which supports P-rich diatom communities (8, 12).Despite an abundant literature on stoichiometric variability and its potential causes, no simple predictive relationship has been widely adopted in global biogeochemical models. Instead, the vast majority of global biogeochemical models assumes fixed C:N:P in organic matter, including most participants in the recent Coupled Model Intercomparison Project, CMIP5 (13). Thus, the potential impact of changes in organic matter stoichiometry on ocean carbon storage and oxygen consumption remain largely unexplored. The neglect of stoichiometric variability is due, at least in part, to the lack of a clear predictive framework.Here, it is argued that the concentration of a nutrient element in seawater can provide a suitable predictive framework, because it is a critical determinant of the rate at which that element will tend to be taken up by the organisms in the local community. This hypothesis builds on classic resource competition theory (14), which argues that if the concentration of an element is low, such that uptake is difficult, the community will be dominated by organisms that are well adapted to a low cellular quota of that nutrient (10). If, on the other hand, the concentration is high, facilitating high uptake rates, the community will be dominated by organisms that are capable of taking advantage of that nutrient to grow faster. This suggestion leads to clear predictions with significant biogeochemical consequences, as outlined below.