The Use of Parenteral Nutrition and Disparities in Its Allocation Following Traumatic Injury.

INTRODUCTION: Severe traumatic injury requires rapid and extensive deployment of resources to save the lives of the critically injured. The sequelae of traumatic injuries frequently require extensive intervention obligating patients to a complicated recovery process devoid of meaningful nutrition. In this setting, parenteral nutrition (PN) is key in enabling appropriate wound healing, recovery, and rehabilitation. We sought to examine the use of PN in adult trauma management and to highlight any disparities in the utilization of PN in adult trauma patients. METHODS: We queried the 2017-2019 Trauma Quality Improvement Program (TQIP) for adult patients (aged > 18 y) who sustained blunt or penetrating traumatic injuries and received PN as part of their hospitalization. We compared time to PN administration based on demographics. We then used a multivariable logistic regression model to identify factors associated with the use of PN. We hypothesized that PN would be less commonly employed in the uninsured and minority groups. RESULTS: We identified 2,449,498 patients with sufficient data for analysis. Of these, 1831 patients were treated with PN. On univariate analysis, PN patients were more commonly male (74.7% PN versus 60.2% non-PN; P < 0.001). PN use was more frequent in the Black population (24.3% PN versus 15.5% non-PN; P < 0.001) and less frequent in the White population (72.7% PN versus 81.2% non-PN; P < 0.001). PN use was also much more common among patients covered by Medicaid. Penetrating trauma was over twice as common among PN recipients relative to non-PN patients (% PN versus % non-PN). PN patients had higher injury severity scores (ISSs), more intensive care unit days, longer hospitalizations, and increased mortality compared to non-PN patients. PN patients were half as likely to discharge home and twice as likely to discharge to a long-term care facility. Multivariable analysis including age, race, trauma mechanism, primary payer, and ISS, demonstrated an association of PN use with increasing age (OR 1.01, P < 0.001), cases of penetrating trauma (odds ratio [OR], 2.47; P < 0.001), and patients with high ISS (OR, 0.1.06; P < 0.001). There was decreased use in Uninsured patient (OR, 0.54; P < 0.001). CONCLUSIONS: PN use following traumatic injury is rarely required. Patients treated with PN typically have a resource-intense hospital course. More severe injuries, penetrating trauma, and increased age are more likely to result in PN use. Variations in PN use are apparent based on insurance payer, further examination into allocation of hospital and intensive care resources, as it pertains to patient socioeconomic status, is warranted in light of these findings.

High-throughput functional analysis of autism genes in zebrafish identifies convergence in dopaminergic and neuroimmune pathways.

Advancing from gene discovery in autism spectrum disorders (ASDs) to the identification of biologically relevant mechanisms remains a central challenge. Here, we perform parallel in vivo functional analysis of 10 ASD genes at the behavioral, structural, and circuit levels in zebrafish mutants, revealing both unique and overlapping effects of gene loss of function. Whole-brain mapping identifies the forebrain and cerebellum as the most significant contributors to brain size differences, while regions involved in sensory-motor control, particularly dopaminergic regions, are associated with altered baseline brain activity. Finally, we show a global increase in microglia resulting from ASD gene loss of function in select mutants, implicating neuroimmune dysfunction as a key pathway relevant to ASD biology.

Capturing Fleeting Intermediates in a Claisen Rearrangement Using Nonequilibrium Droplet Imbibition Reaction Conditions.

The Claisen rearrangement of aromatic allyl phenyl ether to 2-allyl phenol is known to be induced by heat, acid, and air-water interfacial (on-water) effects. In this work, we show that the combination of acid and interfacial effects in an "on-droplet" experiment accelerates this reaction even further (by a factor >10×). The reaction acceleration was achieved through a droplet imbibition mass spectrometry (MS) experiment that allows reactants to be deposited on rapidly moving (100 m/s), charged microdroplets while avoiding turbulent mixing. In this case, reactants are concentrated mainly at the surface of the short-lived microdroplets (microseconds), enabling enhanced interfacial effects. By doping n-butylamine in the spray solvent and subsequently exposing the resultant electrosprayed microdroplets to formic acid vapor, the ketone intermediate, 6-allylcyclohexa-2,4-dien-1-one, involved in this Claisen rearrangement was captured and characterized by tandem MS, successfully differentiating it from the corresponding isobaric reactant (allyl phenyl ether) and product (2-allyl phenol). Similar results showing rate acceleration and subsequent capture of the ketone intermediate via an instantaneous reaction with n-butylamine were demonstrated for p-methyl and p-nitro substituted allyl phenyl ether. Density functional theory calculations confirmed that the on-droplet reaction condition, with a high abundance of proton sources, is different from the neutral rearrangement. With a calculated free energy of activation of 5.2 kcal mol-1 for the protonated reactant, the on-droplet experimental condition provides a unique mechanism for catalyzing the Claisen rearrangement on the microsecond lifetime of the droplets. This experiment marks the first direct capture and detection of a short-lived ketone intermediate in the Claisen rearrangement, a task that is challenged by a thermodynamically favorable tautomerization step to give a more stabilized product (by 20 kcal/mol).

Anchor-based bisulfite sequencing determines genome-wide DNA methylation.

Whole Genome Bisulfite Sequencing (WGBS) is the current standard for DNA methylation profiling. However, this approach is costly as it requires sequencing coverage over the entire genome. Here we introduce Anchor-Based Bisulfite Sequencing (ABBS). ABBS captures accurate DNA methylation information in Escherichia coli and mammals, while requiring up to 10 times fewer sequencing reads than WGBS. ABBS interrogates the entire genome and is not restricted to the CpG islands assayed by methods like Reduced Representation Bisulfite Sequencing (RRBS). The ABBS protocol is simple and can be performed in a single day.

Molecular Docking as a Tool to Examine Organic Cation Sorption to Organic Matter.

Molecular docking simulations were performed to examine the structural effects of organic cations on their sorption to organic matter. A set of benzylamine compounds was used to assess the sorption trends arising from the systematic structural differences between ring or nitrogen substituents. Binding simulations were performed using AutoDock 4.2 with Schulten's proposed soil organic matter as a representative organic matter structure. The calculated binding energies for the sorbate compounds correlated strongly with the measured sorption energies for Pahokee peat, indicating that the simulated binding energies and their associated sorbate orientations were representative of the experimental conditions. Graphical docking orientations showed primary, secondary, and tertiary aminium compounds to form hydrogen-bond interactions with deprotonated carboxylic acid groups in a pocket of the organic matter structure. Quaternary ammonium compounds formed pi-pi or cation-pi interactions with the aromatic groups elsewhere in the same organic matter pocket. Ring substituents showed no clear trends in sorption energies with the substituent group type for primary aminium compounds. Rather, substituent groups altered the simulated van der Waals, electrostatic, hydrogen-bond, and desolvation energy contributions to the overall sorption energies, in part because of the variations in docking orientations between compounds. Increasing methyl substitution of the aminium nitrogen group was associated with an increase in van der Waals energy contributions and a decrease in electrostatic energy contributions to the overall compound sorption energies because of aminium charge delocalization into methyl substituents and steric hindrance from methyl substituents to form specific interactions. The findings illustrate how molecular docking can be used to explore the effects of organic cation structure on sorption interactions with organic matter.

Defining variant-resistant epitopes targeted by SARS-CoV-2 antibodies: A global consortium study.

Antibody-based therapeutics and vaccines are essential to combat COVID-19 morbidity and mortality after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Multiple mutations in SARS-CoV-2 that could impair antibody defenses propagated in human-to-human transmission and spillover or spillback events between humans and animals. To develop prevention and therapeutic strategies, we formed an international consortium to map the epitope landscape on the SARS-CoV-2 spike protein, defining and structurally illustrating seven receptor binding domain (RBD)­directed antibody communities with distinct footprints and competition profiles. Pseudovirion-based neutralization assays reveal spike mutations, individually and clustered together in variants, that affect antibody function among the communities. Key classes of RBD-targeted antibodies maintain neutralization activity against these emerging SARS-CoV-2 variants. These results provide a framework for selecting antibody treatment cocktails and understanding how viral variants might affect antibody therapeutic efficacy.

Longitudinal auditory pathophysiology following mild blast-induced trauma.

Blast-induced hearing difficulties affect thousands of veterans and civilians. The long-term impact of even a mild blast exposure on the central auditory system is hypothesized to contribute to lasting behavioral complaints associated with mild blast traumatic brain injury (bTBI). Although recovery from mild blast has been studied separately over brief or long time windows, few, if any, studies have investigated recovery longitudinally over short-term and longer-term (months) time windows. Specifically, many peripheral measures of auditory function either recover or exhibit subclinical deficits, masking deficits in processing complex, real-world stimuli that may recover differently. Thus, examining the acute time course and pattern of neurophysiological impairment using appropriate stimuli is critical to better understanding and intervening in bTBI-induced auditory system impairments. Here, we compared auditory brainstem response, middle-latency auditory-evoked potentials, and envelope following responses. Stimuli were clicks, tone pips, amplitude-modulated tones in quiet and in noise, and speech-like stimuli (iterated rippled noise pitch contours) in adult male rats subjected to mild blast and sham exposure over the course of 2 mo. We found that blast animals demonstrated drastic threshold increases and auditory transmission deficits immediately after blast exposure, followed by substantial recovery during the window of 7-14 days postblast, although with some deficits remaining even after 2 mo. Challenging conditions and speech-like stimuli can better elucidate mild bTBI-induced auditory deficit during this period. Our results suggest multiphasic recovery and therefore potentially different time windows for treatment, and deficits can be best observed using a small battery of sound stimuli.NEW & NOTEWORTHY Few studies on blast-induced hearing deficits go beyond simple sounds and sparsely track postexposure. Therefore, the recovery arc for potential therapies and real-world listening is poorly understood. Evidence suggested multiple recovery phases over 2 mo postexposure. Hearing thresholds largely recovered within 14 days and partially explained recovery. However, midlatency responses, responses to amplitude modulation in noise, and speech-like pitch sweeps exhibited extended changes, implying persistent central auditory deficits and the importance of subclinical threshold shifts.

Alkynyl-Based Covalent Organic Frameworks as High-Performance Anode Materials for Potassium-Ion Batteries.

The development of high-performance organic electrodes for potassium-ion batteries (KIBs) is attracting interest due to their sustainability and low costs. However, the electrolyte systems and moieties that generally proved to be successful in high-performance Li-ion batteries have found relatively little success in KIBs. Herein, two alkynyl-based covalent organic frameworks (COFs) containing 1,3,5-tris(arylethynyl)benzene (TAEB) and dehydrobenzoannulene (DBA) units are utilized as bulk anode materials for KIBs in a localized high-concentration electrolyte. TAEB-COF provides a high capacity value of 254.0 mAh g-1 at ∼100% efficiency after 300 cycles, and DBA-COF 3 provides a capacity of 76.3 mAh g-1 with 98.7% efficiency after 300 cycles. DFT calculations suggest that the alkynyl units of TAEB-COF facilitate the binding of K-ions through both enthalpic and geometric driving forces, leading to high reversible capacities.

A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes.

Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.

Stem cell-derived polarized hepatocytes.

Human stem cell-derived hepatocyte-like cells (HLCs) offer an attractive platform to study liver biology. Despite their numerous advantages, HLCs lack critical in vivo characteristics, including cell polarity. Here, we report a stem cell differentiation protocol that uses transwell filters to generate columnar polarized HLCs with clearly defined basolateral and apical membranes separated by tight junctions. We show that polarized HLCs secrete cargo directionally: Albumin, urea, and lipoproteins are secreted basolaterally, whereas bile acids are secreted apically. Further, we show that enterically transmitted hepatitis E virus (HEV) progeny particles are secreted basolaterally as quasi-enveloped particles and apically as naked virions, recapitulating essential steps of the natural infectious cycle in vivo. We also provide proof-of-concept that polarized HLCs can be used for pharmacokinetic and drug-drug interaction studies. This novel system provides a powerful tool to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism in a more physiologically relevant setting.

Acrolein-mediated alpha-synuclein pathology involvement in the early post-injury pathogenesis of mild blast-induced Parkinsonian neurodegeneration.

Survivors of blast-induced traumatic brain injury (bTBI) have increased susceptibility to Parkinson's disease (PD), characterized by α-synuclein aggregation and the progressive degeneration of nigrostriatal dopaminergic neurons. Using an established bTBI rat model, we evaluated the changes of α-synuclein and tyrosine hydroxylase (TH), known hallmarks of PD, and acrolein, a reactive aldehyde and marker of oxidative stress, with the aim of revealing key pathways leading to PD post-bTBI. Indicated in both animal models of PD and TBI, acrolein is likely a point of pathogenic convergence. Here we show that after a single mild bTBI, acrolein is elevated up to a week, systemically in urine, and in whole brain tissue, specifically the substantia nigra and striatum. Acrolein elevation is accompanied by heightened α-synuclein oligomerization, dopaminergic dysregulation, and acrolein/α-synuclein interaction in the same brain regions. We further show that acrolein can directly modify and oligomerize α-synuclein in vitro. Taken together, our data suggests acrolein likely plays an important role in inducing PD pathology following bTBI by encouraging α-synuclein aggregation. These results are expected to advance our understanding of the long-term post-bTBI pathological changes leading to the development of PD, and suggest intervention targets to curtail such pathology.

Stereo- and Regioselective Synthesis of Molecular Baskets.

We describe a stereoselective method for obtaining multigram quantities of molecular basket 1 syn in overall 11% yield, using inexpensive cyclopentadiene and diethyl fumarate as starting materials. First, an asymmetric synthesis of enantioenriched bromo(trimethylstannyl)alkene (-)-8 was accomplished by the stereoselective bromination of dibromonorbornene (+)-4 guided by anchimeric assistance and subsequent syn- exo-elimination of tetrabromonorbornane (-)-5a as the key steps. Subsequent Cu(I)-catalyzed cyclotrimerization of (-)-8 was optimized to give 1 syn/ anti in 85% yield and 1:1 ratio of diastereomers. Importantly, the results of our mechanistic experiments were in line with the cyclotrimerization occurring in a chain-type fashion with racemization of a Cu(I) homochiral dimeric intermediate, reducing the stereoselectivity of the transformation. Enabled by more facile access to molecular baskets of type 1 syn, a range of recognition studies can now be completed for producing novel supramolecular catalysts, organophosphorus scavengers, and nanostructured materials.

Functional Peptide Nanofibers with Unique Tumor Targeting and Enzyme-Induced Local Retention Properties.

An effective tumoral delivery system should show minimal removal by the reticuloendothelial system (RES), promote tumor uptake and penetration, and minimize on-site clearance. This study reports the design and synthesis of advanced self-assembling peptide nanofiber precursor (NFP) analogues. The peptidic nature of NFP offers the design flexibility for on-demand customization with imaging agents and surface charges while maintaining a set size, allowing for real-time monitoring of kinetic and dynamic tumoral delivery by multimodal fluorescence/positron emission tomography/computed tomography (fluo/PET/CT) imaging, for formulation optimization. The optimized glutathione (GSH)-NFP displays a reduced capture by the RES as well as excellent tumor targeting and tissue invasion properties compared to naive NFP. Inside a tumor, GSH-NFP can structurally transform into ten times larger interfibril networks, serving as in situ depot that promotes weeks-long local retention. This nanofiber, which can further be designed to release the active pharmacophores within a tumor microenvironment, displays a superior therapeutic efficacy for inhibiting disease progression and improving the survival of animals bearing triple-negative breast cancer tumors compared to free drug and liposome formulation of the drug, in addition to a favorable toxicity profile.

Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites.

S-Fatty-acylation is the covalent attachment of long chain fatty acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine (Cys) residues via a thioester linkage on proteins. This post-translational and reversible lipid modification regulates protein function and localization in eukaryotes and is important in mammalian physiology and human diseases. While chemical labeling methods have improved the detection and enrichment of S-fatty-acylated proteins, mapping sites of modification and characterizing the endogenously attached fatty acids are still challenging. Here, we describe the integration and optimization of fatty acid chemical reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated proteins and direct tagging of modified Cys residues to selectively map lipid modification sites. This afforded improved enrichment and direct identification of many protein S-fatty-acylation sites compared to previously described methods. Notably, we directly identified the S-fatty-acylation sites of IFITM3, an important interferon-stimulated inhibitor of virus entry, and we further demonstrated that the highly conserved Cys residues are primarily modified by palmitic acid. The methods described here should facilitate the direct analysis of protein S-fatty-acylation sites and their endogenously attached fatty acids in diverse cell types and activation states important for mammalian physiology and diseases.

IKKß is a ß-catenin kinase that regulates mesenchymal stem cell differentiation.

Mesenchymal stem cells (MSCs) can give rise to both adipocytes and osteoblasts, but the molecular mechanisms underlying MSC fate determination remain poorly understood. IκB kinase ß (IKKß), a central coordinator of inflammation and immune responses through activation of NF-κB, has been implicated as a critical molecular link between obesity and metabolic disorders. Here, we show that IKKß can reciprocally regulate adipocyte and osteoblast differentiation of murine and human MSCs through an NF-κB-independent mechanism. IKKß is a ß-catenin kinase that phosphorylates the conserved degron motif of ß-catenin to prime it for ß-TrCP-mediated ubiquitination and degradation, thereby increasing adipogenesis and inhibiting osteogenesis in MSCs. Animal studies demonstrated that deficiency of IKKß in BM mesenchymal stromal cells increased bone mass and decreased BM adipocyte formation in adult mice. In humans, IKKß expression in adipose tissue was also positively associated with increased adiposity and elevated ß-catenin phosphorylation. These findings suggest IKKß as a key molecular switch that regulates MSC fate, and they provide potentially novel mechanistic insights into the understanding of the cross-regulation between the evolutionarily conserved IKKß and Wnt/ß-catenin signaling pathways. The IKKß-Wnt axis we uncovered may also have important implications for development, homeostasis, and disease pathogenesis.

Pathological correlations between traumatic brain injury and chronic neurodegenerative diseases.

Traumatic brain injury is among the most common causes of death and disability in youth and young adults. In addition to the acute risk of morbidity with moderate to severe injuries, traumatic brain injury is associated with a number of chronic neurological and neuropsychiatric sequelae including neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. However, despite the high incidence of traumatic brain injuries and the established clinical correlation with neurodegeneration, the causative factors linking these processes have not yet been fully elucidated. Apart from removal from activity, few, if any prophylactic treatments against post-traumatic brain injury neurodegeneration exist. Therefore, it is imperative to understand the pathophysiological mechanisms of traumatic brain injury and neurodegeneration in order to identify potential factors that initiate neurodegenerative processes. Oxidative stress, neuroinflammation, and glutamatergic excitotoxicity have previously been implicated in both secondary brain injury and neurodegeneration. In particular, reactive oxygen species appear to be key in mediating molecular insult in neuroinflammation and excitotoxicity. As such, it is likely that post injury oxidative stress is a key mechanism which links traumatic brain injury to increased risk of neurodegeneration. Consequently, reactive oxygen species and their subsequent byproducts may serve as novel fluid markers for identification and monitoring of cellular damage. Furthermore, these reactive species may further serve as a suitable therapeutic target to reduce the risk of post-injury neurodegeneration and provide long term quality of life improvements for those suffering from traumatic brain injury.

Chemical proteomics reveals ADP-ribosylation of small GTPases during oxidative stress.

ADP-ribosylation is a post-translational modification that is known to be involved in cellular homeostasis and stress but has been challenging to analyze biochemically. To facilitate the detection of ADP-ribosylated proteins, we show that an alkyne-adenosine analog, N6-propargyl adenosine (N6pA), is metabolically incorporated in mammalian cells and enables fluorescence detection and proteomic analysis of ADP-ribosylated proteins. Notably, our analysis of N6pA-labeled proteins that are upregulated by oxidative stress revealed differential ADP-ribosylation of small GTPases. We discovered that oxidative stress induced ADP-ribosylation of Hras on Cys181 and Cys184 in the C-terminal hypervariable region, which are normally S-fatty-acylated. Downstream Hras signaling is impaired by ADP-ribosylation during oxidative stress, but is rescued by ADP-ribosyltransferase inhibitors. Our study demonstrates that ADP-ribosylation of small GTPases not only is mediated by bacterial toxins but is endogenously regulated in mammalian cells. N6pA provides a useful tool to characterize ADP-ribosylated proteins and their regulatory mechanisms in cells.

αIIbß3 binding to a fibrinogen fragment lacking the γ-chain dodecapeptide is activation dependent and EDTA inducible.

Platelet integrin receptor αIIbß3 supports platelet aggregation by binding fibrinogen. The interaction between the fibrinogen C-terminal γ-chain peptide composed of residues γ-404-411 (GAKQAGDV) and the Arg-Gly-Asp (RGD) binding pocket on αIIbß3 is required for fibrinogen-mediated platelet aggregation, but data suggest that other ancillary binding sites on both fibrinogen and αIIbß3 may lead to higher-affinity fibrinogen binding and clot retraction. To identify additional sites, we analyzed the ability of platelets and cells expressing normal and mutant αIIbß3 to adhere to an immobilized fibrinogen plasmin fragment that lacks intact γ-404-411 ('D98'). We found the following: (1) Activated, but not unactivated, platelets adhere well to immobilized 'D98.' (2) Cells expressing constitutively active αIIbß3 mutants, but not cells expressing normal αIIbß3 or αVß3, adhere well to 'D98.' (3) Monoclonal antibodies 10E5 and 7E3 inhibit the adhesion to 'D98' of activated platelets and cells expressing constitutively active αIIbß3, as do small-molecule inhibitors that bind to the RGD pocket. (4) EDTA paradoxically induces normal αIIbß3 to interact with 'D98.' Because molecular modeling and molecular dynamics simulations suggested that the αIIb L151-D159 helix may contribute to the interaction with 'D98,' we studied an αIIbß3 mutant in which the αIIb 148-166 loop was swapped with the corresponding αV loop; it failed to bind to fibrinogen or 'D98.' Our data support a model in which conformational changes in αIIbß3 and/or fibrinogen after platelet activation and the interaction between γ-404-411 and the RGD binding pocket make new ancillary sites available that support higher-affinity fibrinogen binding and clot retraction.

Estrogens Suppress a Behavioral Phenotype in Zebrafish Mutants of the Autism Risk Gene, CNTNAP2.

Autism spectrum disorders (ASDs) are a group of devastating neurodevelopmental syndromes that affect up to 1 in 68 children. Despite advances in the identification of ASD risk genes, the mechanisms underlying ASDs remain unknown. Homozygous loss-of-function mutations in Contactin Associated Protein-like 2 (CNTNAP2) are strongly linked to ASDs. Here we investigate the function of Cntnap2 and undertake pharmacological screens to identify phenotypic suppressors. We find that zebrafish cntnap2 mutants display GABAergic deficits, particularly in the forebrain, and sensitivity to drug-induced seizures. High-throughput behavioral profiling identifies nighttime hyperactivity in cntnap2 mutants, while pharmacological testing reveals dysregulation of GABAergic and glutamatergic systems. Finally, we find that estrogen receptor agonists elicit a behavioral fingerprint anti-correlative to that of cntnap2 mutants and show that the phytoestrogen biochanin A specifically reverses the mutant behavioral phenotype. These results identify estrogenic compounds as phenotypic suppressors and illuminate novel pharmacological pathways with relevance to autism.