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Background: Death with a functioning allograft has become the leading category of graft loss in kidney transplant recipients at all time points. Previous analyses have demonstrated that causes of death in kidney transplant recipients are predominated by comorbidities strongly associated with immunosuppressant medications. Adverse drug events (ADEs) have been strongly associated with nonadherence, health care utilization, and graft loss; clinicians face a difficult decision on whether making immunosuppressant adjustments in the face of ADEs will improve symptomology or simply increase the risk of acute rejection. Clinicians also face a treatment quandary in 50% of kidney transplant recipients with stage 3 or worse chronic kidney disease at 1 year post transplantation, as progressive decline in renal function has been strongly associated with inferior allograft survival., Objective: The primary objective of the CLinical Utility of the omnigrAf biomarkeR Panel In The Care of kidneY Transplant Recipients (CLARITY) trial is to evaluate change in renal function over time in kidney transplant recipients who are undergoing OmniGraf monitoring in conjunction with monitoring of their medication-related symptom burden (MRSB). A secondary objective of this study is to identify the impact of OmniGraf use in conjunction with patient-reported MRSB as part of clinical care on patients' self-efficacy and quality of life., Methods: CLARITY is a 3-year prospective, multisite, observational study of 2000 participants with a matched control, measuring the impact of real-time patients' MRSB and the OmniGraf biomarker panel on change in renal function over time. Secondary outcome measures include the Patient-Reported Outcomes Measurement Information System (PROMIS) Self-Efficacy for Managing Chronic Conditions-Managing Medications and Treatment-Short Form 4a; the PROMIS-29 Profile (version 2.1); the PROMIS Depression Scale, hospitalizations-subcategorized for hospitalizations owing to infections; treated rejections, MRSB, and proportion of participants with overall graft survival at year 3 post transplantation; graft loss or death during the 3-year study follow-up period; and change in provider satisfaction., Results: The primary outcome measure of the study will be a comparison of the slope change in estimated glomerular filtration rate from baseline to the end of follow-up between study participants and a matched control group. Secondary outcome measures include changes over time in PROMIS Self-Efficacy for Managing Chronic Conditions-Managing Medications and Treatment-Short Form 4a, the PROMIS-29 Profile (version 2.1), and PROMIS Depression Scale in the study group, as well as a comparison of hospitalizations and causes, rejections, and graft and patient survival compared between participants and a matched cohort. The anticipated first enrollment in the study is October 2022 with data analysis and publication expected in October 2027., Conclusions: Through this report, we describe the study design, methods, and outcome measures that will be utilized in the ongoing CLARITY trial., Trial Registration: ClinicalTrials.gov NCT05482100; https://clinicaltrials.gov/ct2/show/NCT05482100., International Registered Report Identifier (irrid): PRR1-10.2196/41020., (©James N Fleming, Timothy Cober, Janelle Hickey, Leslie Stach, Allison Kawano, Amanda Szczepanik, Alicia Watson, Yuka Imamura, Juston Weems, Patricia West-Thielke. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 14.12.2022.)

Myasthenia Gravis (MG) is mediated by autoantibodies against acetylcholine receptors that cause loss of the receptors in the neuromuscular junction. Eculizumab, a C5-inhibitor, is the only approved treatment for MG that mechanistically addresses complement-mediated loss of nicotinic acetylcholine receptors. It is an expensive drug and was approved despite missing the primary efficacy endpoint in the Phase 3 REGAIN study. There are two observations to highlight. Firstly, further C5 inhibitors are in clinical development, but other terminal pathway proteins, such as C7, have been relatively understudied as therapeutic targets, despite the potential for lower and less frequent dosing. Secondly, given the known heterogenous mechanisms of action of autoantibodies in MG, effective patient stratification in the REGAIN trial may have provided more favorable efficacy readouts. We investigated C7 as a target and assessed the in vitro function, binding epitopes and mechanism of action of three mAbs against C7. We found the mAbs were human, cynomolgus monkey and/or rat cross-reactive and each had a distinct, novel mechanism of C7 inhibition. TPP1820 was effective in preventing experimental MG in rats in both prophylactic and therapeutic dosing regimens. To enable identification of MG patients that are likely to respond to C7 inhibition, we developed a patient stratification assay and showed in a small cohort of MG patients (n=19) that 63% had significant complement activation and C7-dependent loss of AChRs in this in vitro set up. This study provides validation of C7 as a target for treatment of MG and provides a means of identifying patients likely to respond to anti-C7 therapy based on complement-activating properties of patient autoantibodies., Competing Interests: EL, DGow, CS, IO, LS, DGorm, AS, AB, EW, MB, TW, RP, SK, MF, SD, E-MN are employees and shareholders of GSK. SP-F is an employee of GSK. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lekova, Zelek, Gower, Spitzfaden, Osuch, John-Morris, Stach, Gormley, Sanderson, Bridges, Wear, Petit-Frere, Burden, Priest, Wattam, Kitchen, Feeney, Davis, Morgan and Nichols.)

Background/objectives: Antibiotic indication documentation at the time of order entry is mandated by the Joint Commission. Inclusion of indication at order entry may have an impact on the time to administration. Our primary objective was to evaluate agreement between indication selected during order entry and clinical notes. Our secondary objective was to observe if there was a change in time to administration after indications were required during order entry., Methods: Patients ≤18 years old who received ≥1 dose of vancomycin or ceftriaxone during a preintervention period and 3 postintervention periods were included. Indication for use, agreement between order and clinical note, and timing of antibiotic administration were collected., Results: Most common indication for vancomycin (total: 789) was sepsis (26%, n = 204). Common indications for ceftriaxone (total: 1071) were sepsis (12%, n = 127), perforated appendicitis (12%, n = 125), and urinary tract infection (10%, n = 107). Postintervention, agreement between the indication selected during order entry and indication documented in clinical note for ceftriaxone and vancomycin orders were 41% and 46%, respectively. Median time to administration decreased among patients who received ceftriaxone ( P < .01) but had no significant impact on time to administration of vancomycin ( P = .49)., Conclusions: Indication for ceftriaxone and vancomycin selected during order entry and reported in clinical notes inconsistently matched. Inclusion of antibiotic indication may impact time to administration.

The terminal pathway of complement is implicated in the pathology of multiple diseases and its inhibition is, therefore, an attractive therapeutic proposition. The practicalities of inhibiting this pathway, however, are challenging, as highlighted by the very few molecules in the clinic. The proteins are highly abundant, and assembly is mediated by high-affinity protein-protein interactions. One strategy is to target neoepitopes that are present transiently and only exist on active or intermediate complexes but not on the abundant native proteins. Here, we describe an antibody discovery campaign that generated neoepitope-specific mAbs against the C5b6 complex, a stable intermediate complex in terminal complement complex assembly. We used a highly diverse yeast-based antibody library of fully human IgGs to screen against soluble C5b6 antigen and successfully identified C5b6 neoepitope-specific antibodies. These antibodies were diverse, showed good binding to C5b6, and inhibited membrane attack complex (MAC) formation in a solution-based assay. However, when tested in a more physiologically relevant membrane-based assay these antibodies failed to inhibit MAC formation. Our data highlight the feasibility of identifying neoepitope binding mAbs, but also the technical challenges associated with the identification of functionally relevant, neoepitope-specific inhibitors of the terminal pathway.

Several pathologies have been associated with the AAA+ ATPase p97, an enzyme essential to protein homeostasis. Heterozygous polymorphisms in p97 have been shown to cause neurological disease, while elevated proteotoxic stress in tumours has made p97 an attractive cancer chemotherapy target. The cellular processes reliant on p97 are well described. High-resolution structural models of its catalytic D2 domain, however, have proved elusive, as has the mechanism by which p97 converts the energy from ATP hydrolysis into mechanical force to unfold protein substrates. Here, we describe the high-resolution structure of the p97 D2 ATPase domain. This crystal system constitutes a valuable tool for p97 inhibitor development and identifies a potentially druggable pocket in the D2 domain. In addition, its P6 1 symmetry suggests a mechanism for substrate unfolding by p97. DATABASE: The atomic coordinates and structure factors have been deposited in the PDB database under the accession numbers 6G2V, 6G2W, 6G2X, 6G2Y, 6G2Z and 6G30., (© 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Pseudomonas aeruginosa has three type VI secretion systems (T6SSs), H1-, H2-, and H3-T6SS, each belonging to a distinct group. The two T6SS components, TssB/VipA and TssC/VipB, assemble to form tubules that conserve structural/functional homology with tail sheaths of contractile bacteriophages and pyocins. Here, we used cryoelectron microscopy to solve the structure of the H1-T6SS P. aeruginosa TssB1C1 sheath at 3.3 Å resolution. Our structure allowed us to resolve some features of the T6SS sheath that were not resolved in the Vibrio cholerae VipAB and Francisella tularensis IglAB structures. Comparison with sheath structures from other contractile machines, including T4 phage and R-type pyocins, provides a better understanding of how these systems have conserved similar functions/mechanisms despite evolution. We used the P. aeruginosa R2 pyocin as a structural template to build an atomic model of the TssB1C1 sheath in its extended conformation, allowing us to propose a coiled-spring-like mechanism for T6SS sheath contraction., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The AAA+ (ATPases associated with diverse cellular activities) ATPase p97 is essential to a wide range of cellular functions, including endoplasmic reticulum-associated degradation, membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes, which are regulated by ubiquitination. p97 acts downstream from ubiquitin signaling events and utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes. A multitude of p97 cofactors have evolved which are essential to p97 function. Ubiquitin-interacting domains and p97-binding domains combine to form bi-functional cofactors, whose complexes with p97 enable the enzyme to interact with a wide range of ubiquitinated substrates. A set of mutations in p97 have been shown to cause the multisystem proteinopathy inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia. In addition, p97 inhibition has been identified as a promising approach to provoke proteotoxic stress in tumors. In this review, we will describe the cellular processes governed by p97, how the cofactors interact with both p97 and its ubiquitinated substrates, p97 enzymology and the current status in developing p97 inhibitors for cancer therapy., (© 2017 The Author(s).)

A rapid test to detect methicillin resistance in Staphylococcus aureus through the penicillin-binding protein 2a antigen was introduced for pediatric patients hospitalized for purulent skin and soft tissue infections. Integration of this test with physician education and stewardship guidance improved targeted antibiotic use for infections caused by methicillin-susceptible S. aureus (44%-80%, P < 0.001), including when final culture results were not available.

Background and Objectives: Hospitalists increasingly serve as the primary physicians for children hospitalized with infections. Consequently, hospitalists frequently interact with institutional antimicrobial stewardship programs (ASPs). Understanding how these services interact can inform ongoing practice improvement efforts. The objectives of this study were to identify factors associated with ASP recommendations among hospitalist-managed children, and to determine the association of ASP interventions with clinical outcomes for hospitalist-managed patients., Methods: We retrospectively analyzed ASP reviews of hospitalist patients from a children's hospital from March 2008 to June 2013. Clinical factors associated with an ASP recommendation were determined. Length of stay and 30-day readmission were compared between cases of agreement and disagreement with ASP recommendations., Results: The ASP reviewed 2163 hospitalist patients, resulting in 350 recommendations (16.2% of reviews). Hospitalists agreed with ASP recommendations in 86.9% of cases. The odds of an ASP recommendation decreased during the study period. Ceftriaxone was the most common antibiotic associated with a recommendation (154/350, 44.0%); community-acquired pneumonia was the most common diagnosis (105/350, 30.0%). Antibiotic discontinuation was the most often recommendation; hospitalists most often disagreed with consulting infectious diseases. Disagreement with ASP recommendations was associated with a decreased length of stay of 15.4 (95% confidence interval -33.2 to 1.1) hours but not 30-day readmission prevalence., Conclusions: Pediatric hospitalists and ASPs can form an effective collaboration that improves antibiotic use while providing safe care. Better characterization of the areas of disagreement between hospitalists and ASPs is needed. Future studies are needed to identify ASP strategies that will be beneficial in other hospitalist settings., (Copyright © 2015 by the American Academy of Pediatrics.)

The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs), RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N) in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Scaffold-assisted signaling cascades guide cellular decision-making. In budding yeast, one such signal transduction pathway called the mitotic exit network (MEN) governs the transition from mitosis to the G1 phase of the cell cycle. The MEN is conserved and in metazoans is known as the Hippo tumor-suppressor pathway. We found that signaling through the MEN kinase cascade was mediated by an unusual two-step process. The MEN kinase Cdc15 first phosphorylated the scaffold Nud1. This created a phospho-docking site on Nud1, to which the effector kinase complex Dbf2-Mob1 bound through a phosphoserine-threonine binding domain, in order to be activated by Cdc15. This mechanism of pathway activation has implications for signal transmission through other kinase cascades and might represent a general principle in scaffold-assisted signaling.

One major signaling method employed by Mycobacterium tuberculosis, the causative agent of tuberculosis, is through reversible phosphorylation of proteins mediated by protein kinases and phosphatases. This study concerns one of these enzymes, the serine/threonine protein kinase PknF, that is encoded in an operon with Rv1747, an ABC transporter that is necessary for growth of M. tuberculosis in vivo and contains two forkhead-associated (FHA) domains. FHA domains are phosphopeptide recognition motifs that specifically recognize phosphothreonine-containing epitopes. Experiments to determine how PknF regulates the function of Rv1747 demonstrated that phosphorylation occurs on two specific threonine residues, Thr-150 and Thr-208. To determine the in vivo consequences of phosphorylation, infection experiments were performed in bone marrow-derived macrophages and in mice using threonine-to-alanine mutants of Rv1747 that prevent specific phosphorylation and revealed that phosphorylation positively modulates Rv1747 function in vivo. The role of the FHA domains in this regulation was further demonstrated by isothermal titration calorimetry, using peptides containing both phosphothreonine residues. FHA-1 domain mutation resulted in attenuation in macrophages highlighting the critical role of this domain in Rv1747 function. A mutant deleted for pknF did not, however, have a growth phenotype in an infection, suggesting that other kinases can fulfill its role when it is absent. This study provides the first information on the molecular mechanism(s) regulating Rv1747 through PknF-dependent phosphorylation but also indicates that phosphorylation activates Rv1747, which may have important consequences in regulating growth of M. tuberculosis.

C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca(2+)-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mM Ca(2+) exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s(20,w)(0) of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca(2+), CRP partially dissociates into its protomers and the NaCl concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration R(G) values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged K(D) value of 21 microM in solution (140 mM NaCl, 2 mM Ca(2+)) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar K(D) value of 23 microM (140 mM NaCl, 2 mM Ca(2+)), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mM Ca(2+) and 140 mM NaCl are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response.

Forkhead-associated (FHA) domains have gained considerable prominence as ubiquitous phosphothreonine-dependent binding modules; however, their precise roles in serine and threonine kinase (STK) pathways and mechanisms of regulation remain unclear. From experiments with Rv1827, an FHA domain-containing protein from Mycobacterium tuberculosis, we derived a complete molecular description of an FHA-mediated STK signaling process. First, binding of the FHA domain to each of three metabolic enzyme complexes regulated their catalytic activities but did not require priming phosphorylation. However, phosphorylation of a threonine residue within a conserved amino-terminal motif of Rv1827 triggered its intramolecular association with the FHA domain of Rv1827, thus blocking its interactions with each of the three enzymes. The solution structure of this inactivated form and further mutagenic studies showed how a previously unidentified intramolecular phosphoswitch blocked the access of the target enzymes to a common FHA interaction surface and how this shared surface accommodated three functionally related, but structurally diverse, binding partners. Thus, our data reveal an unsuspected versatility in the FHA domain that allows for the transformation of multiple kinase inputs into various downstream regulatory signals.