Contemporary outcomes of pediatric cardiac transplantation with a positive retrospective crossmatch.

BACKGROUND: A positive crossmatch (+ XM) has traditionally been associated with adverse outcomes following pediatric heart transplantation. However, more recent studies suggest that favorable intermediate-term outcomes may be achieved despite a + XM. This study's hypothesis is that children with a + XM have similar long-term survival, but higher rate of complications such as rejection, coronary allograft vasculopathy (CAV), and infection, compared to patients with a negative (-) XM. METHODS: The Pediatric Heart Transplant Society Registry (PHTS) database was queried from 2010-2021 for all patients <18 years of age with a known XM. Baseline demographics were compared between + XM and - XM groups using appropriate parametric and non-parametric group comparisons. Cox Proportional Hazards Modeling was used to identify risk factors for post-transplant graft loss, rejection, and CAV. RESULTS: Of 4599 pediatric heart transplants during the study period, XM results were available for 3914 (85%), of which 373 (9.5%) had a + XM. Univariate analysis showed lower 10-year survival for patients with + XM (HR = 1.3, p = .04). Multivariate analyses revealed no significant difference in 10-year survival in the 2 groups; however, time to first rejection (p = .0001) remained significantly shorter in the + XM group. CONCLUSIONS: Pediatric patients transplanted across a + XM experience earlier rejection; however, after multivariate adjustment, + XM is not independently associated with intermediate-term graft loss. The risk of heart transplantation against a + XM must be balanced with the ongoing risk of waitlist mortality.

Laminar flow-based microfluidic systems for molecular interaction analysis-Part 2: Data extraction, processing and analysis.

The rate at which fluorescently-labeled biomolecules, that are flowing at a constant speed in a microfluidic channel, diffuse into an adjacent buffer stream can be used to calculate the diffusion coefficient of the molecule, which then gives a measure of its size. Experimentally, determining the rate of diffusion involves capturing concentration gradients in fluorescence microscopy images at different distances along the length of the microfluidic channel, where distance corresponds to residence time, based on the flow velocity. The preceding chapter in this journal covered the development of the experimental setup, including information about the microscope camera detection systems used to acquire fluorescence microscopy data. In order to calculate diffusion coefficients from fluorescence microscopy images, intensity data are extracted from the images and then appropriate methods of processing and analyzing the data, including the mathematical models used for fitting, are applied to the extracted data. This chapter begins with a brief overview of digital imaging and analysis principles, before introducing custom software for extracting the intensity data from the fluorescence microscopy images. Subsequently, methods and explanations for performing the necessary corrections and appropriate scaling of the data are provided. Finally, the mathematics of one-dimensional molecular diffusion is described, and analytical approaches to obtaining the diffusion coefficient from the fluorescence intensity profiles are discussed and compared.

Laminar flow-based microfluidic systems for molecular interaction analysis-Part 1: Chip development, system operation and measurement setup.

The recent advent of laminar flow-based microfluidic systems for molecular interaction analysis has enabled transformative new profiling of proteins in regards to their structure, disordering, complex formation and interactions in general. Based on the diffusive transport of molecules perpendicular to the direction of laminar flow in a microfluidic channel, systems of this type promise continuous-flow, high-throughput screening of complex, multi-molecule interactions, while remaining tolerant to heterogeneous mixtures. Using common microfluidic device processing, the technology provides unique opportunities, as well as device design and experimental challenges, for integrative sample handling approaches that can investigate biomolecular interaction events in complex samples with readily available laboratory equipment. In this first chapter of a two-part series, we introduce system design and experimental setup requirements for a typical laminar flow-based microfluidic system for molecular interaction analysis in the form of what we call the 'LaMInA system' (Laminar flow-based Molecular Interaction Analysis system). We provide microfluidic device development advice on choice of device material, device design, including impact of channel geometry on the signal acquisition, and on design limitations and possible post-fabrication treatments to redress these. Finally. we cover aspects of fluidic actuation, such as selecting, measuring and controlling the flow rate appropriately, and provide a guide to possible fluorescent labels for proteins, as well as options for the fluorescence detection hardware, all in the context of assisting the reader in developing their own laminar flow-based experimental setup for biomolecular interaction analysis.

Is now the time for a Rubiscuit or Ruburger? Increased interest in Rubisco as a food protein.

Much of the research on Rubisco aims at increasing crop yields, with the ultimate aim of increasing plant production to feed an increasing global population. However, since the identification of Rubisco as the most abundant protein in leaf material, it has also been touted as a direct source of dietary protein. The nutritional and functional properties of Rubisco are on a par with those of many animal proteins, and are superior to those of many other plant proteins. Purified Rubisco isolates are easily digestible, nutritionally complete, and have excellent foaming, gelling, and emulsifying properties. Despite this potential, challenges in efficiently extracting and separating Rubisco have limited its use as a global foodstuff. Leaves are lower in protein than seeds, requiring large amounts of biomass to be processed. This material normally needs to be processed quickly to avoid degradation of the final product. Extraction of Rubisco from the plant material requires breaking down the cell walls and rupturing the chloroplast. In order to obtain high-quality protein, Rubisco needs to be separated from chlorophyll, and then concentrated for final use. However, with increased consumer demand for plant protein, there is increased interest in the potential of leaf protein, and many commercial plants are now being established aimed at producing Rubisco as a food protein, with over US$60 million of funding invested in the past 5 years. Is now the time for increased use of Rubisco in food production as a nitrogen source, rather than just providing a carbon source?

On the utility of microfluidic systems to study protein interactions: advantages, challenges, and applications.

Within the complex milieu of a cell, which comprises a large number of different biomolecules, interactions are critical for function. In this post-reductionist era of biochemical research, the 'holy grail' for studying biomolecular interactions is to be able to characterize them in native environments. While there are a limited number of in situ experimental techniques currently available, there is a continuing need to develop new methods for the analysis of biomolecular complexes that can cope with the additional complexities introduced by native-like solutions. We think approaches that use microfluidics allow researchers to access native-like environments for studying biological problems. This review begins with a brief overview of the importance of studying biomolecular interactions and currently available methods for doing so. Basic principles of diffusion and microfluidics are introduced and this is followed by a review of previous studies that have used microfluidics to measure molecular diffusion and a discussion of the advantages and challenges of this technique.

Platelet-rich plasma as a therapy for androgenic alopecia: a systematic review and meta-analysis.

OBJECTIVE: The past decade has seen platelet-rich plasma (PRP) become a popular therapy around the world as a treatment for androgenetic alopecia (AGA). These systematic review and meta-analyses assess the effectiveness and adverse effects of PRP to determine the role of PRP as a treatment for AGA among the other non-surgical treatment modalities. METHODS: This study follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and is registered under the PROSPERO ID CRD42019136329. Seven databases were searched from inception through May 2019. Meta-analyses of randomized controlled trials (RCTs) were performed to evaluate the effect of PRP treatments on hair density and hair thickness. RESULTS: Thirty studies, including 687 patients, met our inclusion criteria. Twenty-nine studies reported beneficial results, and 24 studies reached statistical significance on a measured outcome. Ten RCTs were included. Our meta-analyses show that PRP treatment increases hair density and hair thickness. CONCLUSIONS: PRP is an autologous treatment that lacks serious adverse effects and effectively improves hair density and hair thickness in men and women with AGA. Future research should include low risk-of-bias RCTs to optimize treatment protocols, investigate variability among studies, and to obtain more data on hair thickness changes.

Late Heart Transplantation in Hypoplastic Left Heart Syndrome With Intact Atrial Septum.

This case report describes 2 patients born with hypoplastic left heart syndrome and an intact atrial septum who underwent a strategy of immediate extracorporeal membrane oxygenation and left atrial decompression followed by hybrid Norwood palliation as a bridge to further palliation. Heart transplantation was ultimately performed in these 2 patients with persisting pulmonary vascular resistance abnormalities.

Biventricular Support Using ProtekDuo Cannula in a Child.

We report a successful pediatric bridge to transplant following application of the ProTekDuo Cannula to provide right ventricular support in a 12-year-old child with biventricular cardiomyopathy and on left ventricular assist device support. We are unaware of any other reports of pediatric use of this device in the medical literature.

Comparative assessment of live cyprinid and salmonid movement networks in England and Wales.

Disease poses a significant threat to aquaculture. While there are a number of factors contributing to pathogen transmission risk, movement of live fish is considered the most important. Understanding live fish movement patterns for different aquaculture sectors is therefore crucial to predicting disease occurrence and necessary for the development of effective, risk-based biosecurity, surveillance and containment policies. However, despite this, our understanding of live movement patterns of key aquaculture species, namely salmonids and cyprinids, within England and Wales remains limited. In this study, networks reflecting live fish movements associated with the cyprinid and salmonid sectors in England and Wales were constructed. The structure, composition and key attributes of each network were examined and compared to provide insight into the nature of trading patterns and connectedness, as well as highlight sites at a high risk of spreading disease. Connectivity at both site and catchment level was considered to facilitate understanding at different resolutions, providing further insight into disease outbreaks, with industry wide implications. The study highlighted that connectivity through live fish movements was extensive for both industries. The salmonid and cyprinid networks comprised 2533 and 3645 nodes, with a network density of 5.81 × 10-4 and 4.2 × 10-4, respectively. The maximum network reach of 2392 in the salmonid network was higher, both in absolute terms and as a proportion of the overall network, compared to maximum network reach of 2085 in the cyprinid network. However, in contrast, the number of sites in the cyprinid network with a network reach greater than one was 513, compared to 171 in the salmonid network. Patterns of connectivity indicated potential for more frequent yet smaller scale disease outbreaks in the cyprinid industry and less frequent but larger scale outbreaks in the salmonid industry. Further, high connectivity between river catchments within both networks was shown, posing challenges for zoning at the catchment level for the purpose of disease management. In addition to providing insight into pathogen transmission and epidemic potential within the salmonid and cyprinid networks, the study highlights the utility of network analysis, and the value of accessible, accurate live fish movement data in this context. The application of outputs from this study, and network analysis methodology, to inform future disease surveillance and control policies, both within England and Wales and more broadly, is discussed.

An unexpected sticking point for carboxysome assembly.

In order to improve photosynthetic efficiency, bacteria often enclose RubisCO and carbonic anhydrase into microcompartments called carboxysomes. Assembly of these complexes requires a protein called CcmM. It had previously been thought that CcmM mediated RubisCO assembly by displacing one of the RubisCO subunits, Ryan et al. show that despite having a three-dimensional structure that closely resembles the RubisCO small subunit, CcmM does not dislodge it, leading to a proposal for an alternative binding location. These results provide a new model for carboxysome assembly with implications for photosynthetic engineering.

Implementation of a Trauma Quality Improvement Program Communications Package Decreases Time-to-Operation for Facial Trauma.

INTRODUCTION: Facial trauma is common and carries significant morbidity and cost. Suboptimal interdisciplinary communication is associated with negative health outcomes. This study evaluates the clinical impact of implementation of American College of Surgeons Trauma Quality Improvement Program (TQIP) interdisciplinary communication guidelines between facial surgery and trauma teams. METHODS: Patients with facial trauma presenting to our level 1 trauma center between May and December 2017 were included (N = 812) and split into 3 groups, each anonymously representing a service that treats facial trauma. Services 1 and 2 were controls, and service 3 adopted TQIP communication guidelines. Mean and slope of time-to-operation (TTO) and mean length of stay were assessed 106 days before (n = 95) and 107 days after (n = 77) implementation. RESULTS: For service 3, mean TTO decreased significantly from 6.2 to 2.9 days (P = 0.005) after implementation of the communication intervention. There was no significant difference in mean TTO preimplementation versus postimplementation in either control cohort, including service 1 (4.6 vs 4.9 days; P = 0.59) and service 2 (4.2 vs 4.5 days; P = 0.62). Average length of stay did not differ significantly between the preintervention versus postintervention in any service (service 1: 9.0 vs 8.3 days, P = 0.43; service 2: 4.6 vs 6.6 days, P = 0.85; service 3: 6.7 vs 6.4 days, P = 0.45). CONCLUSION: Our study demonstrates that cost-free TQIP-guided improvement in interdisciplinary communication between the trauma service and a consulting surgical specialist decreases TTO for patients with operative facial trauma. Health care providers should develop strong well-defined communication channels between collaborating teams involved in patient care to optimize patient clinical outcomes.

Functional and solution structure studies of amino sugar deacetylase and deaminase enzymes from Staphylococcus aureus.

N-Acetylglucosamine-6-phosphate deacetylase (NagA) and glucosamine-6-phosphate deaminase (NagB) are branch point enzymes that direct amino sugars into different pathways. For Staphylococcus aureus NagA, analytical ultracentrifugation and small-angle X-ray scattering data demonstrate that it is an asymmetric dimer in solution. Initial rate experiments show hysteresis, which may be related to pathway regulation, and kinetic parameters similar to other bacterial isozymes. The enzyme binds two Zn2+ ions and is not substrate inhibited, unlike the Escherichia coli isozyme. S. aureus NagB adopts a novel dimeric structure in solution and shows kinetic parameters comparable to other Gram-positive isozymes. In summary, these functional data and solution structures are of use for understanding amino sugar metabolism in S. aureus, and will inform the design of inhibitory molecules.

A helical LC3-interacting region mediates the interaction between the retroviral restriction factor Trim5α and mammalian autophagy-related ATG8 proteins.

The retroviral restriction factor tripartite motif-containing 5α (Trim5α) acts during the early postentry stages of the retroviral life cycle to block infection by a broad range of retroviruses, disrupting reverse transcription and integration. The mechanism of this restriction is poorly understood, but it has recently been suggested to involve recruitment of components of the autophagy machinery, including members of the mammalian autophagy-related 8 (ATG8) family involved in targeting proteins to the autophagosome. To better understand the molecular details of this interaction, here we utilized analytical ultracentrifugation to characterize the binding of six ATG8 isoforms and determined the crystal structure of the Trim5α Bbox coiled-coil region in complex with one member of the mammalian ATG8 proteins, autophagy-related protein LC3 B (LC3B). We found that Trim5α binds all mammalian ATG8s and that, unlike the typical LC3-interacting region (LIR) that binds to mammalian ATG8s through a ß-strand motif comprising approximately six residues, LC3B binds to Trim5α via the α-helical coiled-coil region. The orientation of the structure demonstrated that LC3B could be accommodated within a Trim5α assembly that can bind the retroviral capsid. However, mutation of the binding interface does not affect retroviral restriction. Comparison of the typical linear ß-strand LIR with our atypical helical LIR reveals a conservation of the presentation of residues that are required for the interaction with LC3B. This observation expands the range of LC3B-binding proteins to include helical binding motifs and demonstrates a link between Trim5α and components of the autophagosome.

Self-assembly of toroidal proteins explored using native mass spectrometry.

The peroxiredoxins are a well characterised family of toroidal proteins which can self-assemble into a striking array of quaternary structures, including protein nanotubes, making them attractive as building blocks for nanotechnology. Tools to characterise these assemblies are currently scarce. Here, assemblies of peroxiredoxin proteins were examined using native mass spectrometry and complementary solution techniques. We demonstrated unequivocally that tube formation is fully reversible, a useful feature in a molecular switch. Simple assembly of individual toroids was shown to be tunable by pH and the presence of a histidine tag. Collision induced dissociation experiments on peroxiredoxin rings revealed a highly unusual symmetrical disassembly pathway, consistent with the structure disassembling as a hexamer of dimers. This study provides the foundation for the rational design and precise characterisation of peroxiredoxin protein structures where self-assembly can be harnessed as a key feature for applications in nanotechnology.

Immobilization of tobacco etch virus (TEV) protease on a high surface area protein nanofibril scaffold.

Tobacco etch virus (TEV) protease is widely used for the removal of poly-histidine affinity tags from proteins. In solution, it is a one-time use enzyme for tag cleavage that has low stability, and is therefore a good candidate for immobilization. Amyloid fibrils can act as a versatile nanoscaffold by providing a large surface area for biomolecule immobilization. Immobilization of TEV protease to amyloid fibrils grown from the surface of a small glass bead, using physisorption, successfully immobilized active TEV protease. The bead retained activity over several uses and successfully cleaved a poly-histidine tag from several his-tagged proteins. This is first time that TEV protease has been immobilized to insulin amyloid fibrils, or any protein based support. Such functionalized surface assembled amyloid fibrils show promise as a novel nanosupport for the creation of functional bionanomaterials, for example, active surface coatings for the production of fine chemicals, chemical detoxification, or biosensing. Insulin amyloid fibrils provide a new nanosupport for the immobilization of TEV protease, which could allow for the reuse of the enzyme, saving on production costs for recombinantly expressed poly-histidine tagged proteins. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1506-1512, 2018.

Structural and functional analyses of Rubisco from arctic diatom species reveal unusual posttranslational modifications.

The catalytic performance of the major CO2-assimilating enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), restricts photosynthetic productivity. Natural diversity in the catalytic properties of Rubisco indicates possibilities for improvement. Oceanic phytoplankton contain some of the most efficient Rubisco enzymes, and diatoms in particular are responsible for a significant proportion of total marine primary production as well as being a major source of CO2 sequestration in polar cold waters. Until now, the biochemical properties and three-dimensional structures of Rubisco from diatoms were unknown. Here, diatoms from arctic waters were collected, cultivated, and analyzed for their CO2-fixing capability. We characterized the kinetic properties of five and determined the crystal structures of four Rubiscos selected for their high CO2-fixing efficiency. The DNA sequences of the rbcL and rbcS genes of the selected diatoms were similar, reflecting their close phylogenetic relationship. The Vmax and Km for the oxygenase and carboxylase activities at 25 °C and the specificity factors (Sc/o) at 15, 25, and 35 °C were determined. The Sc/o values were high, approaching those of mono- and dicot plants, thus exhibiting good selectivity for CO2 relative to O2 Structurally, diatom Rubiscos belong to form I C/D, containing small subunits characterized by a short ßA-ßB loop and a C-terminal extension that forms a ß-hairpin structure (ßE-ßF loop). Of note, the diatom Rubiscos featured a number of posttranslational modifications of the large subunit, including 4-hydroxyproline, ß-hydroxyleucine, hydroxylated and nitrosylated cysteine, mono- and dihydroxylated lysine, and trimethylated lysine. Our studies suggest adaptation toward achieving efficient CO2 fixation in arctic diatom Rubiscos.

Quaternary structure influences the peroxidase activity of peroxiredoxin 3.

Peroxiredoxins are abundant peroxidase enzymes that are key regulators of the cellular redox environment. A major subgroup of these proteins, the typical 2-Cys peroxiredoxins, can switch between dimers and decameric or dodecameric rings, during the catalytic cycle. The necessity of this change in quaternary structure for function as a peroxidase is not fully understood. In order to explore this, human peroxiredoxin 3 (Prx3) protein was engineered to form both obligate dimers (S75E Prx3) and stabilised dodecameric rings (S78C Prx3), uncoupling structural transformations from the catalytic cycle. The obligate dimer, S75E Prx3, retained catalytic activity towards hydrogen peroxide, albeit significantly lower than the wildtype and S78C proteins, suggesting an evolutionary advantage of having higher order self-assemblies.

Can granulomatosis with polyangiitis be diagnosed earlier in primary care? A case-control study.

BACKGROUND: People with granulomatosis with polyangiitis (GPA) commonly described long delays before diagnosis. AIM: To study the natural history of GPA prior to diagnosis using primary care data, and determine whether clinical features could be identified to help earlier diagnosis. DESIGN: Case-control study using the Clinical Practice Research Datalink. METHODS: We compared primary care activity and clinical features between cases and 10 matched controls. RESULTS: We identified 757 cases and matched 7546 controls. Compared to controls, cases had more GP consultations and overall healthcare activity in the 5 years prior to their diagnosis, with a marked increase in the year before diagnosis, and particularly in the last 3 months. However, consultations were mostly for symptoms that were not specifically related to GPA. In the year prior to diagnosis, the most frequent and strongly predictive clinical features of GPA were Ear Nose and Throat (ENT) symptoms [34.5% of cases, odds ratio (OR) 10.5, 95% confidence intervals (CI) 8.6-12.7], and general (constitutional) symptoms (21.5% of cases, OR 9.0, 95% CI 7.1-11.3). In the year before diagnosis a larger number of cases attended secondary care (382, 50.5%) than had records of clinical features of GPA. CONCLUSIONS: After discussing our findings, we conclude that it would be difficult to identify cases of GPA earlier in primary care. Our results support a need for heightened awareness of this condition among secondary care clinicians, especially those assessing emergency admissions, and in the clinics which were most frequently attended by cases 3-12 months prior to diagnosis.

Plant DHDPR forms a dimer with unique secondary structure features that preclude higher-order assembly.

Dihydrodipicolinate reductase (DHDPR) catalyses the second reaction in the diaminopimelate pathway of lysine biosynthesis in bacteria and plants. In contrast with the tetrameric bacterial DHDPR enzymes, we show that DHDPR from Vitis vinifera (grape) and Selaginella moellendorffii are dimeric in solution. In the present study, we have also determined the crystal structures of DHDPR enzymes from the plants Arabidopsis thaliana and S. moellendorffii, which are the first dimeric DHDPR structures. The analysis of these models demonstrates that the dimer forms through the intra-strand interface, and that unique secondary features in the plant enzymes block tetramer assembly. In addition, we have also solved the structure of tetrameric DHDPR from the pathogenic bacteria Neisseria meningitidis Measuring the activity of plant DHDPR enzymes showed that they are much more prone to substrate inhibition than the bacterial enzymes, which appears to be a consequence of increased flexibility of the substrate-binding loop and higher affinity for the nucleotide substrate. This higher propensity to substrate inhibition may have consequences for ongoing efforts to increase lysine biosynthesis in plants.

Formation of supramolecular protein structures on gold surfaces.

Recent research has highlighted the exciting possibilities enabled by the use of protein structures as nanocomponents to form functional nanodevices. To this end, control over protein-protein and protein-surface interactions is essential. In this study, the authors probe the interaction of human peroxiredoxin 3 with gold surfaces, a protein that has been previously identified as having potential use in nanotechnology. Analytical ultracentrifugation and transmission electron microscopy revealed the pH mediated assembly of protein toroids into tubular structures across a small pH range. Quartz crystal microbalance with dissipation measurements showed differences in absorbed protein mass when pH is switched from pH 8.0 to 7.2, in line with the formation of supramolecular structures observed in solution studies. Scanning tunneling microscopy under ambient conditions showed that these protein tubes form on surfaces in a concentration dependent manner, with a tendency for protein adsorption and supramolecular assembly at the edges of Au(111) terraces. Finally, self-assembled monolayer modification of Au surfaces was explored as a means to control the adsorption and orientation of pH triggered protein structures.