C-reactive protein orchestrates acute allograft rejection in vascularized composite allotransplantation via subset-selective monocyte activation.

INTRODUCTION: Despite recent substantial progress in vascularized composite allotransplantation (VCA), such as face transplantations, short- and long-term allograft survival is severely limited by allograft rejection. The acute-phase response, directly after allogeneic transplantation, represents an immune-inflammatory reaction to ischemia/reperfusion and acts as an early initiator of graft rejection. Acute-phase reactants mediate this immune response via crosstalk with the mononuclear phagocyte system. OBJECTIVE: C-reactive protein (CRP), a well-known marker of inflammation, has pro-inflammatory properties and aggravates ischemia/reperfusion injury. Thus, we investigated how CRP impacts acute allograft rejection. METHODS: Based on clinical observations in facial VCAs, we applied a complex hindlimb transplantation model in rats to investigate whether CRP directly affects transplant rejection. We further analyzed subset-specific infiltration and tissue distribution of recipient-derived monocytes in the early phase of acute rejection and assessed their differential regulation by CRP using intravital imaging. RESULTS: We demonstrate that CRP accelerates allograft rejection and reduces allograft survival via selectively activating non-classical monocytes. The therapeutic stabilization of CRP abrogates this activating effect on monocytes, consequently attenuating acute allograft rejection. Intravital imaging of graft-infiltrating, recipient-derived monocytes during the early phase of acute rejection confirmed their differential regulation by CRP and their crucial role in driving the early stage of graft rejection. CONCLUSION: Differential activation of recipient-derived monocytes by CRP aggravates innate immune response and accelerates clinical allograft rejection Thus, therapeutic targeting of CRP represents a novel promising strategy for preventing acute allograft rejection and potentially reducing chronic allograft rejection.

Fractionation and online mass spectrometry based on imaged capillary isoelectric focusing (icIEF) for characterizing charge heterogeneity of therapeutic antibody.

Imaged capillary isoelectric focusing (icIEF) technology has been proved to be robust for the characterization of protein charge heterogeneity due to its high-resolution pI discrimination and high-throughput. Although high performance liquid chromatography (HPLC) tandem mass spectrometry (MS) and offline fraction collection technologies including isoelectric focusing (IEF), ion exchange chromatography (IEX) and free flow electrophoresis (FFE) have been widely utilized for protein charge variant characterization, there are a few applications of MS coupling with icIEF as a front-separation technique and related fractionation technologies for protein charge heterogeneity. However, the application of icIEF-MS has been much less frequent due to difficulties in MS interface, compatible ampholyte and coated capillary cartridge designation, ultimately impeding the breadth of icIEF applications in protein charge heterogeneity. In this study, a therapeutic monoclonal antibody (mAb-M-AT) was used for its charge variant characterization on an integrated icIEF platform with functions including analytical profiling, MS online coupling and fraction collection for charge heterogeneities. The main protein component and its four charge variants were identified using direct icIEF-MS coupling. Additionally, the two major acidic and basic charge variants were collected using preparative fractionation after the protein focused in the separation capillary. The identity of the fractions was confirmed by LC-MS at intact protein level and the results were consistent with those using icIEF-MS online coupling. The multiple operation modes of the icIEF platform described above can be rapidly and flexibly switched just by changing customized capillary separation cartridges without drastically altering instrument configuration. The whole workflow of icIEF-based profiling, fractionation and MS online coupling for protein heterogeneity is straightforward, reliable, and accurate, thus providing comprehensive solutions for in-depth protein heterogeneity characterization.

A fine balance of hydrophobic-electrostatic communication pathways in a pH-switching protein.

Allostery is the phenomenon of coupling between distal binding sites in a protein. Such coupling is at the crux of protein function and regulation in a myriad of scenarios, yet determining the molecular mechanisms of coupling networks in proteins remains a major challenge. Here, we report mechanisms governing pH-dependent myristoyl switching in monomeric hisactophilin, whereby the myristoyl moves between a sequestered state, i.e., buried within the core of the protein, to an accessible state, in which the myristoyl has increased accessibility for membrane binding. Measurements of the pH and temperature dependence of amide chemical shifts reveal protein local structural stability and conformational heterogeneity that accompany switching. An analysis of these measurements using a thermodynamic cycle framework shows that myristoyl-proton coupling at the single-residue level exists in a fine balance and extends throughout the protein. Strikingly, small changes in the stereochemistry or size of core and surface hydrophobic residues by point mutations readily break, restore, or tune myristoyl switch energetics. Synthesizing the experimental results with those of molecular dynamics simulations illuminates atomistic details of coupling throughout the protein, featuring a large network of hydrophobic interactions that work in concert with key electrostatic interactions. The simulations were critical for discerning which of the many ionizable residues in hisactophilin are important for switching and identifying the contributions of nonnative interactions in switching. The strategy of using temperature-dependent NMR presented here offers a powerful, widely applicable way to elucidate the molecular mechanisms of allostery in proteins at high resolution.

Methodological advances and strategies for high resolution structure determination of cellular protein aggregates.

Aggregation of proteins is at the nexus of molecular processes crucial to aging, disease, and employing proteins for biotechnology and medical applications. There has been much recent progress in determining the structural features of protein aggregates that form in cells; yet, owing to prevalent heterogeneity in aggregation, many aspects remain obscure and often experimentally intractable to define. Here, we review recent results of structural studies for cell-derived aggregates of normally globular proteins, with a focus on high-resolution methods for their analysis and prediction. Complementary results obtained by solid-state NMR spectroscopy, FTIR spectroscopy and microspectroscopy, cryo-EM, and amide hydrogen/deuterium exchange measured by NMR and mass spectrometry, applied to bacterial inclusion bodies and disease inclusions, are uncovering novel information on in-cell aggregation patterns as well as great diversity in the structural features of useful and aberrant protein aggregates. Using these advances as a guide, this review aims to advise the reader on which combination of approaches may be the most appropriate to apply to their unique system.

Quenched hydrogen-deuterium amide exchange optimization for high-resolution structural analysis of cellular protein aggregates.

Inclusion bodies (IBs) are large, insoluble aggregates that often form during the overexpression of proteins in bacteria. These aggregates are of broad fundamental and practical significance, for recombinant protein preparation and due to their relevance to aggregation-related medical conditions and their recent emergence as promising functional nanomaterials. Despite their significance, high resolution knowledge of IB structure remains very limited. Such knowledge will advance understanding and control of IB formation and properties in myriad practical applications. Here, we report a detailed quenched hydrogen-deuterium amide exchange (qHDX) method with NMR readout to define the structure of IBs at the level of individual residues throughout the protein. Applying proper control of experimental conditions, such as sample pH, water content, temperature, and intrinsic rate of amide exchange, yields in depth results for these cellular protein aggregates. qHDX results illustrated for Cu, Zn superoxide dismutase 1 (SOD1) and Adnectins show their IBs include native-like structure and some but not all mutations alter IB structure.

High-Resolution NMR H/D Exchange of Human Superoxide Dismutase Inclusion Bodies Reveals Significant Native Features Despite Structural Heterogeneity.

Protein aggregation is central to aging, disease and biotechnology. While there has been recent progress in defining structural features of cellular protein aggregates, many aspects remain unclear due to heterogeneity of aggregates presenting obstacles to characterization. Here we report high-resolution analysis of cellular inclusion bodies (IBs) of immature human superoxide dismutase (SOD1) mutants using NMR quenched amide hydrogen/deuterium exchange (qHDX), FTIR and Congo red binding. The extent of aggregation is correlated with mutant global stability and, notably, the free energy of native dimer dissociation, indicating contributions of native-like monomer associations to IB formation. This is further manifested by a common pattern of extensive protection against H/D exchange throughout nine mutant SOD1s despite their diverse characteristics. These results reveal multiple aggregation-prone regions in SOD1 and illuminate how aggregation may occur via an ensemble of pathways.

Telehealth and in-person training outcomes for novice discrete trial training therapists.

The efficacy and efficiency of telehealth and in-person training were compared while teaching seven undergraduate students to implement components of discrete trial training. A multiple-baseline design across skills with elements of multiple probe and delayed multiple baseline combined with an alternating-treatments design was used to evaluate the effects of behavioral skills training (BST) on (a) implementing a multiple stimulus without replacement preference assessment, (b) setting up an instructional context, (c) delivering antecedent prompts, and (d) delivering consequences for accurate and inaccurate responding. Two skills were trained via telehealth and two skills were trained in-person using BST procedures with a mock student. All participants provided high acceptability ratings for both training procedures. Results also showed that telehealth training was as efficacious and efficient as in-person training for all skills across all participants. Five of six participants showed high levels of maintenance of the newly acquired skills; these five also exhibited the skills during a novel instructional task.

Relationship of causative genetic mutations in maple syrup urine disease with their clinical expression.

Maple syrup urine disease [MSUD] is a rare inborn error of metabolism inherited as an autosomal recessive trait through mutations in any of three different genes that encode components of the branched chain alpha-ketoacid dehydrogenase [BCKD] complex. In this work, the genotype of affected individuals was correlated with their clinical histories. These individuals were diagnosed and followed in a single centralized clinic, and their molecular genetic characterization was done by one laboratory. Three individuals had mutant alleles in the gene for the E1alpha component, five had mutations in the gene for E1beta, and three had mutations in the gene for E2. The results emphasize the diversity of the molecular and clinical presentations for individuals with MSUD and support the complexity of diseases termed "single gene traits." Of primary importance is early identification of at risk infants through newborn screening programs to minimize many of the complications associated with this protein intolerance. Attention to abnormal neurological signs in the neonate or evidence of neurological decompensation in older infants and children by a centralized medical management team minimizes permanent brain damage and improves survival.