New strategies to Reduce Unnecessary Antibiotic Use in the NICU: A Quality Improvement Initiative.

Introduction: Early-onset sepsis (EOS) and late-onset Sepsis (LOS) are common diagnoses entertained in sick newborns treated in neonatal intensive care units (NICUs), and antibiotics are the medications most prescribed in NICUs. Antibiotic stewardship programs have an important impact on limiting unnecessary antibiotic use. Methods: Following the Model for Improvement, between 2/1/16 and 1/31/17, at a level 3 NICU, a multidisciplinary team implemented PDSA cycles to promote antibiotic stewardship practices for newborns at risk of EOS and LOS. The main goal was to decrease the antibiotic usage rate (AUR) safely. Primary strategies included discontinuing antibiotics within 24 hours of life if the newborn was stable, and the blood culture was negative for EOS and implementing an "antibiotic time-out" during rounds. Results: For all newborns admitted to our NICU, the AUR decreased, for EOS from 137 to 32 days per 1000 patient days (77% reduction) and for LOS from 277 to 121 days per 1000 patient days (56% reduction). We demonstrated the sustainability of both EOS-AUR and LOS-AUR during the 2 years postcompletion of the intervention period. There were no adverse effects of reducing the AUR. Conclusion: Interventions that reduce unnecessary antibiotic use in the NICU are safe and prevent excessive antibiotic exposure.

Consolidative mediastinal radiotherapy for advanced-stage classical Hodgkin lymphoma with bulky disease in patients who achieve complete response after chemotherapy in PET-CT era.

Background: The role of consolidation mediastinal radiotherapy (RT) for mediastinal bulky disease in advanced-stage classical Hodgkin lymphoma (cHL) is controversial in the positron emission tomography/computed tomography (PET-CT) era. Materials and methods: We reviewed the medical charts of patients with advanced-stage (clinical stage IIX-IVX) cHL and mediastinal bulky that achieved a complete response after first line chemotherapy treatment between August 2010 and December 2020 and compared the results of those who received with those who did not receive consolidation mediastinal RT. Inclusion criteria required PET-CT imaging for staging and response assessment. Results: We included 115 patients; 91 received mediastinal RT and 24 did not. Patient's characteristics were balanced between the two groups. The median age in patients that received and did not receive mediastinal RT was 28 years and 24.5 years, respectively. Median International Prognostic Score among patients that received and did not receive mediastinal RT was 2 and 2.5, respectively. Disease free survival (DFS) was statistically better in patients that received mediastinal RT (p = 0.013). Two-year DFS for patients that received and did not receive mediastinal RT was 95.2% [95% confidence interval (95% CI): 87.6-98.2%] and 76.4% (95% CI: 52.2-89.4%), respectively. Overall survival (OS) was not different between the two groups (p = 0.617). In multivariate analysis, not receiving mediastinal radiotherapy and only achieving partial response (vs. complete response) after 2 cycles of chemotherapy were factors predictive of lower DFS. Conclusion: DFS, but not OS, was superior in patients that received mediastinal RT.

The impact of immediate adverse donation reactions on the return of volunteers undergoing platelet apheresis.

INTRODUCTION: Immediate adverse reactions experienced during donation decrease return rates among whole-blood donors, but little is known about this effect among platelet apheresis donors. We investigated the impact of immediate adverse reactions on the return rates of volunteer apheresis platelet donors. METHODS: In a sample of 4108 consecutive platelet apheresis donors seen from August 2016 through June 2019, we evaluated whether immediate adverse reactions were associated with returning for a subsequent platelet apheresis donation within a 12-month period. We used propensity score matching to compare donors with and without adverse reactions. RESULTS: An immediate adverse reaction occurred in 312 (7.6%) donors; 98.5% were mild, and 0.3% were severe. Of the original 4108 platelet apheresis donors, only 3211 (72.3%) returned for a subsequent donation within 12 months. Experiencing an immediate adverse reaction during the donation process significantly decreased the return rate for a subsequent donation [HR= 0.74 (0.63-0.87)], especially among female donors [HR= 0.70 (0.53-0.93)], donors aged < 30 years [HR= 0.71 (0.54-0.94)], with a high school educational level [0.63 (0.49-0.81)], donors donating for the first time [HR= 0.73 (0.59-0.90)], and repeat donors with a previous platelet apheresis donation more than 180 days prior [HR= 0.68 (0.50-0.93)]. CONCLUSION: Even mild adverse events reduce the return rates for a subsequent donation among platelet apheresis donors. Female donors, younger donors, and first-time donors are at higher risk of not returning after an immediate adverse reaction. Preventing the incidence of immediate adverse reactions during platelet apheresis donation may increase the rate of donor retention.

Psychomotor impairments and therapeutic implications revealed by a mutation associated with infantile Parkinsonism-Dystonia.

Parkinson disease (PD) is a progressive, neurodegenerative disorder affecting over 6.1 million people worldwide. Although the cause of PD remains unclear, studies of highly penetrant mutations identified in early-onset familial parkinsonism have contributed to our understanding of the molecular mechanisms underlying disease pathology. Dopamine (DA) transporter (DAT) deficiency syndrome (DTDS) is a distinct type of infantile parkinsonism-dystonia that shares key clinical features with PD, including motor deficits (progressive bradykinesia, tremor, hypomimia) and altered DA neurotransmission. Here, we define structural, functional, and behavioral consequences of a Cys substitution at R445 in human DAT (hDAT R445C), identified in a patient with DTDS. We found that this R445 substitution disrupts a phylogenetically conserved intracellular (IC) network of interactions that compromise the hDAT IC gate. This is demonstrated by both Rosetta molecular modeling and fine-grained simulations using hDAT R445C, as well as EPR analysis and X-ray crystallography of the bacterial homolog leucine transporter. Notably, the disruption of this IC network of interactions supported a channel-like intermediate of hDAT and compromised hDAT function. We demonstrate that Drosophila melanogaster expressing hDAT R445C show impaired hDAT activity, which is associated with DA dysfunction in isolated brains and with abnormal behaviors monitored at high-speed time resolution. We show that hDAT R445C Drosophila exhibit motor deficits, lack of motor coordination (i.e. flight coordination) and phenotypic heterogeneity in these behaviors that is typically associated with DTDS and PD. These behaviors are linked with altered dopaminergic signaling stemming from loss of DA neurons and decreased DA availability. We rescued flight coordination with chloroquine, a lysosomal inhibitor that enhanced DAT expression in a heterologous expression system. Together, these studies shed some light on how a DTDS-linked DAT mutation underlies DA dysfunction and, possibly, clinical phenotypes shared by DTDS and PD.

Upgraded molecular models of the human KCNQ1 potassium channel.

The voltage-gated potassium channel KCNQ1 (KV7.1) assembles with the KCNE1 accessory protein to generate the slow delayed rectifier current, IKS, which is critical for membrane repolarization as part of the cardiac action potential. Loss-of-function (LOF) mutations in KCNQ1 are the most common cause of congenital long QT syndrome (LQTS), type 1 LQTS, an inherited genetic predisposition to cardiac arrhythmia and sudden cardiac death. A detailed structural understanding of KCNQ1 is needed to elucidate the molecular basis for KCNQ1 LOF in disease and to enable structure-guided design of new anti-arrhythmic drugs. In this work, advanced structural models of human KCNQ1 in the resting/closed and activated/open states were developed by Rosetta homology modeling guided by newly available experimentally-based templates: X. leavis KCNQ1 and various resting voltage sensor structures. Using molecular dynamics (MD) simulations, the capacity of the models to describe experimentally established channel properties including state-dependent voltage sensor gating charge interactions and pore conformations, PIP2 binding sites, and voltage sensor-pore domain interactions were validated. Rosetta energy calculations were applied to assess the utility of each model in interpreting mutation-evoked KCNQ1 dysfunction by predicting the change in protein thermodynamic stability for 50 experimentally characterized KCNQ1 variants with mutations located in the voltage-sensing domain. Energetic destabilization was successfully predicted for folding-defective KCNQ1 LOF mutants whereas wild type-like mutants exhibited no significant energetic frustrations, which supports growing evidence that mutation-induced protein destabilization is an especially common cause of KCNQ1 dysfunction. The new KCNQ1 Rosetta models provide helpful tools in the study of the structural basis for KCNQ1 function and can be used to generate hypotheses to explain KCNQ1 dysfunction.

A unified structural model of the mammalian translocator protein (TSPO).

The translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is a membrane protein located on the outer mitochondrial membrane. Experimentally-derived structures of mouse TSPO (mTSPO) and its homologs from bacterial species have been determined by NMR spectroscopy and X-ray crystallography, respectively. These structures and ligand interactions within the TSPO binding pocket display distinct differences. Here, we leverage experimental and computational studies to derive a unified structural model of mTSPO in the presence and absence of the TSPO ligand, PK11195, and study the effects of DPC detergent micelles on the TSPO structure and ligand binding. From this work, we conclude that that the lipid-mimetic system used to solubilize mTSPO for NMR studies thermodynamically destabilizes the protein, introduces structural perturbations, and alters the characteristics of ligand binding. Furthermore, we used Rosetta to construct a unified mTSPO model that reconciles deviating features of the mammalian and bacterial TSPO. These deviating features are likely a consequence of the detergent system used for structure determination of mTSPO by NMR. The unified mTSPO model agrees with available experimental NMR data, appears to be physically realistic (i.e. thermodynamically not frustrated as judged by the Rosetta energy function), and simultaneously shares the structural features observed in sequence-conserved regions of the bacterial proteins. Finally, we identified the binding site for an imaging ligand VUIIS8310 that is currently positioned for clinical translation using NMR spectroscopy and propose a computational model of the VUIIS8310-mTSPO complex.

Structural, functional, and behavioral insights of dopamine dysfunction revealed by a deletion in SLC6A3.

The human dopamine (DA) transporter (hDAT) mediates clearance of DA. Genetic variants in hDAT have been associated with DA dysfunction, a complication associated with several brain disorders, including autism spectrum disorder (ASD). Here, we investigated the structural and behavioral bases of an ASD-associated in-frame deletion in hDAT at N336 (∆N336). We uncovered that the deletion promoted a previously unobserved conformation of the intracellular gate of the transporter, likely representing the rate-limiting step of the transport process. It is defined by a "half-open and inward-facing" state (HOIF) of the intracellular gate that is stabilized by a network of interactions conserved phylogenetically, as we demonstrated in hDAT by Rosetta molecular modeling and fine-grained simulations, as well as in its bacterial homolog leucine transporter by electron paramagnetic resonance analysis and X-ray crystallography. The stabilization of the HOIF state is associated both with DA dysfunctions demonstrated in isolated brains of Drosophila melanogaster expressing hDAT ∆N336 and with abnormal behaviors observed at high-time resolution. These flies display increased fear, impaired social interactions, and locomotion traits we associate with DA dysfunction and the HOIF state. Together, our results describe how a genetic variation causes DA dysfunction and abnormal behaviors by stabilizing a HOIF state of the transporter.

Discovery, Characterization, and Effects on Renal Fluid and Electrolyte Excretion of the Kir4.1 Potassium Channel Pore Blocker, VU0134992.

The inward rectifier potassium (Kir) channel Kir4.1 (KCNJ10) carries out important physiologic roles in epithelial cells of the kidney, astrocytes in the central nervous system, and stria vascularis of the inner ear. Loss-of-function mutations in KCNJ10 lead to EAST/SeSAME syndrome, which is characterized by epilepsy, ataxia, renal salt wasting, and sensorineural deafness. Although genetic approaches have been indispensable for establishing the importance of Kir4.1 in the normal function of these tissues, the availability of pharmacological tools for acutely manipulating the activity of Kir4.1 in genetically normal animals has been lacking. We therefore carried out a high-throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library for small-molecule modulators of Kir4.1. The most potent inhibitor identified was 2-(2-bromo-4-isopropylphenoxy)-N-(2,2,6,6-tetramethylpiperidin-4-yl)acetamide (VU0134992). In whole-cell patch-clamp electrophysiology experiments, VU0134992 inhibits Kir4.1 with an IC50 value of 0.97 µM and is 9-fold selective for homomeric Kir4.1 over Kir4.1/5.1 concatemeric channels (IC50 = 9 µM) at -120 mV. In thallium (Tl+) flux assays, VU0134992 is greater than 30-fold selective for Kir4.1 over Kir1.1, Kir2.1, and Kir2.2; is weakly active toward Kir2.3, Kir6.2/SUR1, and Kir7.1; and is equally active toward Kir3.1/3.2, Kir3.1/3.4, and Kir4.2. This potency and selectivity profile is superior to Kir4.1 inhibitors amitriptyline, nortriptyline, and fluoxetine. Medicinal chemistry identified components of VU0134992 that are critical for inhibiting Kir4.1. Patch-clamp electrophysiology, molecular modeling, and site-directed mutagenesis identified pore-lining glutamate 158 and isoleucine 159 as critical residues for block of the channel. VU0134992 displayed a large free unbound fraction (fu) in rat plasma (fu = 0.213). Consistent with the known role of Kir4.1 in renal function, oral dosing of VU0134992 led to a dose-dependent diuresis, natriuresis, and kaliuresis in rats. Thus, VU0134992 represents the first in vivo active tool compound for probing the therapeutic potential of Kir4.1 as a novel diuretic target for the treatment of hypertension.

Mechanisms of KCNQ1 channel dysfunction in long QT syndrome involving voltage sensor domain mutations.

Mutations that induce loss of function (LOF) or dysfunction of the human KCNQ1 channel are responsible for susceptibility to a life-threatening heart rhythm disorder, the congenital long QT syndrome (LQTS). Hundreds of KCNQ1 mutations have been identified, but the molecular mechanisms responsible for impaired function are poorly understood. We investigated the impact of 51 KCNQ1 variants with mutations located within the voltage sensor domain (VSD), with an emphasis on elucidating effects on cell surface expression, protein folding, and structure. For each variant, the efficiency of trafficking to the plasma membrane, the impact of proteasome inhibition, and protein stability were assayed. The results of these experiments combined with channel functional data provided the basis for classifying each mutation into one of six mechanistic categories, highlighting heterogeneity in the mechanisms resulting in channel dysfunction or LOF. More than half of the KCNQ1 LOF mutations examined were seen to destabilize the structure of the VSD, generally accompanied by mistrafficking and degradation by the proteasome, an observation that underscores the growing appreciation that mutation-induced destabilization of membrane proteins may be a common human disease mechanism. Finally, we observed that five of the folding-defective LQTS mutant sites are located in the VSD S0 helix, where they interact with a number of other LOF mutation sites in other segments of the VSD. These observations reveal a critical role for the S0 helix as a central scaffold to help organize and stabilize the KCNQ1 VSD and, most likely, the corresponding domain of many other ion channels.

Computational design of membrane proteins using RosettaMembrane.

Computational membrane protein design is challenging due to the small number of high-resolution structures available to elucidate the physical basis of membrane protein structure, multiple functionally important conformational states, and a limited number of high-throughput biophysical assays to monitor function. However, structural determination of membrane proteins has made tremendous progress in the past years. Concurrently the field of soluble computational design has made impressive inroads. These developments allow us to tackle the formidable challenge of designing functional membrane proteins. Herein, Rosetta is benchmarked for membrane protein design. We evaluate strategies to cope with the often reduced quality of experimental membrane protein structures. Further, we test the usage of symmetry in design protocols, which is particularly important as many membrane proteins exist as homo-oligomers. We compare a soluble scoring function with a scoring function optimized for membrane proteins, RosettaMembrane. Both scoring functions recovered around half of the native sequence when completely redesigning membrane proteins. However, RosettaMembrane recovered the most native-like amino acid property composition. While leucine was overrepresented in the inner and outer-hydrophobic regions of RosettaMembrane designs, it resulted in a native-like surface hydrophobicity indicating that it is currently the best option for designing membrane proteins with Rosetta.

Protocols for Molecular Modeling with Rosetta3 and RosettaScripts.

Previously, we published an article providing an overview of the Rosetta suite of biomacromolecular modeling software and a series of step-by-step tutorials [Kaufmann, K. W., et al. (2010) Biochemistry 49, 2987-2998]. The overwhelming positive response to this publication we received motivates us to here share the next iteration of these tutorials that feature de novo folding, comparative modeling, loop construction, protein docking, small molecule docking, and protein design. This updated and expanded set of tutorials is needed, as since 2010 Rosetta has been fully redesigned into an object-oriented protein modeling program Rosetta3. Notable improvements include a substantially improved energy function, an XML-like language termed "RosettaScripts" for flexibly specifying modeling task, new analysis tools, the addition of the TopologyBroker to control conformational sampling, and support for multiple templates in comparative modeling. Rosetta's ability to model systems with symmetric proteins, membrane proteins, noncanonical amino acids, and RNA has also been greatly expanded and improved.

Documentation of an Imperative To Improve Methods for Predicting Membrane Protein Stability.

There is a compelling and growing need to accurately predict the impact of amino acid mutations on protein stability for problems in personalized medicine and other applications. Here the ability of 10 computational tools to accurately predict mutation-induced perturbation of folding stability (ΔΔG) for membrane proteins of known structure was assessed. All methods for predicting ΔΔG values performed significantly worse when applied to membrane proteins than when applied to soluble proteins, yielding estimated concordance, Pearson, and Spearman correlation coefficients of <0.4 for membrane proteins. Rosetta and PROVEAN showed a modest ability to classify mutations as destabilizing (ΔΔG < -0.5 kcal/mol), with a 7 in 10 chance of correctly discriminating a randomly chosen destabilizing variant from a randomly chosen stabilizing variant. However, even this performance is significantly worse than for soluble proteins. This study highlights the need for further development of reliable and reproducible methods for predicting thermodynamic folding stability in membrane proteins.

An Integrated Framework Advancing Membrane Protein Modeling and Design.

Membrane proteins are critical functional molecules in the human body, constituting more than 30% of open reading frames in the human genome. Unfortunately, a myriad of difficulties in overexpression and reconstitution into membrane mimetics severely limit our ability to determine their structures. Computational tools are therefore instrumental to membrane protein structure prediction, consequently increasing our understanding of membrane protein function and their role in disease. Here, we describe a general framework facilitating membrane protein modeling and design that combines the scientific principles for membrane protein modeling with the flexible software architecture of Rosetta3. This new framework, called RosettaMP, provides a general membrane representation that interfaces with scoring, conformational sampling, and mutation routines that can be easily combined to create new protocols. To demonstrate the capabilities of this implementation, we developed four proof-of-concept applications for (1) prediction of free energy changes upon mutation; (2) high-resolution structural refinement; (3) protein-protein docking; and (4) assembly of symmetric protein complexes, all in the membrane environment. Preliminary data show that these algorithms can produce meaningful scores and structures. The data also suggest needed improvements to both sampling routines and score functions. Importantly, the applications collectively demonstrate the potential of combining the flexible nature of RosettaMP with the power of Rosetta algorithms to facilitate membrane protein modeling and design.

Inverted topologies in membrane proteins: a mini-review.

Helical membrane proteins such as transporters, receptors, or channels often exhibit structural symmetry. Symmetry is perfect in homo-oligomers consisting of two or more copies of the same protein chain. Intriguingly, in single chain membrane proteins, often internal pseudo-symmetry is observed, in particular in transporters and channels. In several cases single chain proteins with pseudo-symmetry exist, that share the fold with homo-oligomers suggesting evolutionary pathways that involve gene duplication and fusion. It has been hypothesized that such evolutionary pathways allow for the rapid development of large proteins with novel functionality. At the same time symmetry can be leveraged to recognize highly symmetric substrates such as ions. Here we review helical transporter proteins with an inverted two-fold pseudo-symmetry. In this special scenario the symmetry axis lies in the membrane plane. As a result, the putative ancestral monomeric protein would insert in both directions into the membrane and its open-to-the-inside and open-to-the-outside conformations would be structurally identical and iso-energetic, giving a possible evolutionary pathway to create a transporter protein that needs to flip between the two states.