Both natural influenza infection and current seasonal influenza vaccines primarily induce neutralizing antibody responses against highly diverse epitopes within the "head" of the viral hemagglutinin ...(HA) protein. There is increasing interest in redirecting immunity toward the more conserved HA stem or stalk as a means of broadening protective antibody responses. Here we examined HA stem-specific B cell and T follicular helper (Tfh) cell responses in the context of influenza infection and immunization in mouse and monkey models. We found that during infection, the stem domain was immunologically subdominant to the head in terms of serum antibody production and antigen-specific B and Tfh cell responses. Similarly, we found that HA stem immunogens were poorly immunogenic compared with the full-length HA with abolished sialic acid binding activity, with limiting Tfh cell elicitation a potential constraint to the induction or boosting of anti-stem immunity by vaccination. Finally, we confirm that currently licensed seasonal influenza vaccines can boost preexisting memory responses against the HA stem in humans. An increased understanding of the immune dynamics surrounding the HA stem is essential to inform the design of next-generation influenza vaccines for broad and durable protection.
Recent advances in cell-free gene expression (CFE) systems have enabled their use for a host of synthetic biology applications, particularly for rapid prototyping of genetic circuits and biosensors. ...Despite the proliferation of cell-free protein synthesis platforms, the large number of currently existing protocols for making CFE extracts muddles the collective understanding of how the extract preparation method affects its functionality. A key aspect of extract performance relevant to many applications is the activity of the native host transcriptional machinery that can mediate protein synthesis. However, protein yields from genes transcribed in vitro by the native Escherichia coli RNA polymerase are variable for different extract preparation techniques, and specifically low in some conventional crude extracts originally optimized for expression by the bacteriophage transcriptional machinery. Here, we show that cell-free expression of genes under bacterial σ70 promoters is constrained by the rate of transcription in crude extracts, and that processing the extract with a ribosomal runoff reaction and subsequent dialysis alleviates this constraint. Surprisingly, these processing steps only enhance protein synthesis in genes under native regulation, indicating that the translation rate is unaffected. We further investigate the role of other common extract preparation process variants on extract performance and demonstrate that bacterial transcription is inhibited by including glucose in the growth culture but is unaffected by flash-freezing the cell pellet prior to lysis. Our final streamlined and detailed protocol for preparing extract by sonication generates extract that facilitates expression from a diverse set of sensing modalities including protein and RNA regulators. We anticipate that this work will clarify the methodology for generating CFE extracts that are active for biosensing using native transcriptional machinery and will encourage the further proliferation of cell-free gene expression technology for new applications.
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IJS, KILJ, NUK, PNG, UL, UM
Recent advances in cell-free synthetic biology have spurred the development of in vitro molecular diagnostics that serve as effective alternatives to whole-cell biosensors. However, cell-free sensors ...for detecting manmade organic water contaminants such as pesticides are sparse, partially because few characterized natural biological sensors can directly detect such pollutants. Here, we present a platform for the cell-free detection of one critical water contaminant, atrazine, by combining a previously characterized cyanuric acid biosensor with a reconstituted atrazine-to-cyanuric acid metabolic pathway composed of several protein-enriched bacterial extracts mixed in a one pot reaction. Our cell-free sensor detects atrazine within an hour of incubation at an activation ratio superior to previously reported whole-cell atrazine sensors. We also show that the response characteristics of the atrazine sensor can be tuned by manipulating the ratios of enriched extracts in the cell-free reaction mixture. Our approach of utilizing multiple metabolic steps, encoded in protein-enriched cell-free extracts, to convert a target of interest into a molecule that can be sensed by a transcription factor is modular. Our work thus serves as an effective proof-of-concept for a scheme of “metabolic biosensing”, which should enable rapid, field-deployable detection of complex organic water contaminants.
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IJS, KILJ, NUK, PNG, UL, UM
Our ability to routinely engineer genetic networks for applications is limited by the scarcity of highly specific and non-cross-reacting (orthogonal) gene regulators with predictable behavior. Though ...antisense RNAs are attractive contenders for this purpose, quantitative understanding of their specificity and sequence-function relationship sufficient for their design has been limited. Here, we use rationally designed variants of the RNA-IN-RNA-OUT antisense RNA-mediated translation system from the insertion sequence IS10 to quantify >500 RNA-RNA interactions in Escherichia coli and integrate the data set with sequence-activity modeling to identify the thermodynamic stability of the duplex and the seed region as the key determinants of specificity. Applying this model, we predict the performance of an additional ~2,600 antisense-regulator pairs, forecast the possibility of large families of orthogonal mutants, and forward engineer and experimentally validate two RNA pairs orthogonal to an existing group of five from the training data set. We discuss the potential use of these regulators in next-generation synthetic biology applications.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
•In this series of 55 patients with a periprosthetic distal femur fracture treated with lateral plate fixation, the overall complication rate was 24%.•The overall rate of nonunion was 18%, no ...surgical technique factors were identified as a risk factor for nonunion.•Further work is needed to identify strategies to reduce the complication rate in this patient population
Periprosthetic fractures of the distal femur can be challenging injuries to treat; nonunion rates of up to 22% have been reported. The purpose of this study was to determine the rate of complications and nonunion in a multicenter series, and to identify patient or surgical factors that were associated with nonunion.
Retrospective comparative study
Three Level 1 trauma centers
Fifty-five patients with a periprosthetic distal femur fracture proximal to a total knee arthroplasty. Minimum follow up for inclusion was six months or until union or failure.
Surgical fixation using a precontoured lateral locking plate
Fracture union was the primary outcome. Patient demographic and injury variables (age, comorbidities, fracture classification and characteristics) and surgical technique factors (mode of plate fixation, plate material, working length, screw density, and proximal screw type) were identified and compared between patients who developed a nonunion and those who did not. Regression analysis was performed to identify independent risk factors for nonunion.
The overall rate of nonunion was 18% and the total complication rate was 24%. After additional surgery, 49 of 55 patients went on to heal (89%). There were no statistical differences in patient demographic or injury variables between the union and nonunion groups, and none of the variables studied were independent risk factors for nonunion in the regression analysis.
In this series of 55 patients with periprosthetic distal femur fractures treated with precontoured lateral locking plates, 18% developed nonunion and the overall complication rate was 24%. No patient or surgical variables were identified as risk factors. Future research should seek to identify patients at high risk for complication and nonunion who could benefit from alternative fixation strategies or distal femoral replacement.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Transthoracic echocardiography (TTE) and cine cardiac magnetic resonance imaging (CMR) are established imaging methods of the aortic root. We aimed to evaluate the comparability of ...measurements in TTE and standard cine CMR sequences of the aortic root. Our study included 741 subjects (mean age 63.5 ± 8 years, 43.7% female) from the Hamburg City Health Study (HCHS). Subjects underwent CMR and TTE. Aortic root measurements were performed at the level of the aortic annulus (AoAn), sinus of Valsalva (SoV), and sinotubular junction (STJ) by standard cine CMR in left ventricular long axis and left ventricular outflow tract view. Measurements were performed applying the leading-edge to leading-edge (LL) convention and inner-edge to inner-edge (II) convention in TTE and the II convention in CMR. Inter correlation coefficients (ICCs) demonstrated high inter- and intraobserver reproducibility for CMR and TTE measurements of SoV and STJ (ICCs 0.9–0.98) and moderate reproducibility for AoAn (ICCs 0.68–0.91). CMR measurements of SoV and STJ showed strong agreement with TTE: while correlations were comparable (r = 0.75–0.85) bias was lower with TTE II (bias − 0.1 to − 0.74) versus TTE LL measurements (mean bias − 1.49 to − 2.58 mm). The agreement for AoAn was fair (r = 0.51–0.57) with variable bias (mean bias 0.39–3.9). Standard cine CMR and TTE derived aortic root measurements are reproducible and comparable with higher agreement for TTE II instead of LL measurements. These results support an interchangeable application of TTE and standard CMR for screening of aortic root diseases thereby possibly reducing redundant multimodality imaging.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
CO, ethylene, and H2 demonstrate divergent adsorption enthalpies upon interaction with a series of anion-exchanged Ni2X2BTDD materials (X = OH, F, Cl, Br; H2BTDD = ...bis(1H-1,2,3-triazolo4,5-b4′,5′-i)dibenzo1,4dioxin)). The dissimilar responses of these conventional π-acceptor gaseous ligands are in contrast with the typical behavior that may be expected for gas sorption in metal–organic frameworks (MOFs), which generally follows similar periodic trends for a given set of systematic changes to the host MOF structure. A combination of computational and spectroscopic data reveals that the divergent behavior, especially between CO and ethylene, stems from a predominantly σ-donor interaction between the former and Ni2+ and a π-acceptor interaction for the latter. These findings will facilitate further deliberate postsynthetic modifications of MOFs with open metal sites to control the equilibrium selectivity of gas sorption.
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IJS, KILJ, NUK, PNG, UL, UM
Sequence census methods reduce molecular measurements such as transcript abundance and protein-nucleic acid interactions to counting problems via DNA sequencing. We focus on a novel assay utilizing ...this approach, called selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), that can be used to characterize RNA secondary and tertiary structure. We describe a fully automated data analysis pipeline for SHAPE-Seq analysis that includes read processing, mapping, and structural inference based on a model of the experiment. Our methods rely on the solution of a series of convex optimization problems for which we develop efficient and effective numerical algorithms. Our results can be easily extended to other chemical probes of RNA structure, and also generalized to modeling polymerase drop-off in other sequence census-based experiments.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Genomic variation is widespread, and both neutral and selective processes can generate similar patterns in the genome. These processes are not mutually exclusive, so it is difficult to infer the ...evolutionary mechanisms that govern population and species divergence. Boechera stricta is a perennial relative of Arabidopsis thaliana native to largely undisturbed habitats with two geographic and ecologically divergent subspecies. Here, we delineate the evolutionary processes driving the genetic diversity and population differentiation in this species.
Using whole-genome re-sequencing data from 517 B. stricta accessions, we identify four genetic groups that diverged around 30-180 thousand years ago, with long-term small effective population sizes and recent population expansion after the Last Glacial Maximum. We find three genomic regions with elevated nucleotide diversity, totaling about 10% of the genome. These three regions of elevated nucleotide diversity show excess of intermediate-frequency alleles, higher absolute divergence (d
), and lower relative divergence (F
) than genomic background, and significant enrichment in immune-related genes, reflecting long-term balancing selection. Scattered across the genome, we also find regions with both high F
and d
among the groups, termed F
-islands. Population genetic signatures indicate that F
-islands with elevated divergence, which have experienced directional selection, are derived from divergent sorting of ancient polymorphisms.
Our results suggest that long-term balancing selection on disease resistance genes may have maintained ancestral haplotypes across different geographical lineages, and unequal sorting of balanced polymorphisms may have generated genomic regions with elevated divergence. This study highlights the importance of ancestral balanced polymorphisms as crucial components of genome-wide variation.
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