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Background: Pancreatic cancer continues to have a 5-year survival of less than 5%. Therefore, more effective therapies are necessary to improve prognosis in this disease. Angiogenesis is required for tumor growth, and subsequently, mediators of angiogenesis are attractive targets for therapy. Vascular endothelial growth factor (VEGF) is a well-characterized mediator of tumor angiogenesis that functions primarily by binding and activating VEGF receptor 2 (VEGFR2). In this study, we evaluate the use of CT-322, a novel biologic (Adnectin). This small protein is based on a human fibronectin domain and has beneficial properties in that it is fully human, stable, and is produced in bacteria. CT-322 binds to and inhibits activation of VEGFR2.Methods: The efficacy of CT-322 was evaluated in vivo using two orthotopic pancreatic tumor models. The first model was a human tumor xenograft where MiaPaCa-2 cells were injected into the tail of the pancreas of nude mice. The second model was a syngeneic tumor using Pan02 cells injected into pancreas of C57BL/6J mice. In both models, therapy was initiated once primary tumors were established. Mice bearing MiaPaCa-2 tumors were treated with vehicle or CT-322 alone. Gemcitabine alone or in combination with CT-322 was added to the treatment regimen of mice bearing Pan02 tumors. Therapy was given twice a week for six weeks, after which the animals were sacrificed and evaluated (grossly and histologically) for primary and metastatic tumor burden. Primary tumors were also evaluated by immunohistochemistry for the level of apoptosis (TUNEL), microvessel density (MECA-32), and VEGF-activated blood vessels (Gv39M).Results: Treatment with CT-322 was effective at preventing pancreatic tumor growth and metastasis in orthotopic xenograft and syngeneic models of pancreatic cancer. Additionally, CT-322 treatment increased apoptosis, reduced microvessel density and reduced the number of VEGF-activated blood vessels in tumors. Finally, CT-322, in combination with gemcitabine was safe and effective at controlling the growth of syngeneic pancreatic tumors in immunocompetent mice.Conclusion: We conclude that CT-322 is an effective anti-VEGFR2 agent and that further investigation of CT-322 for the treatment of pancreatic cancer is warranted. [ABSTRACT FROM AUTHOR]

Objective: We sought to assess whether the presence and extent of lung ultrasound (LUS) findings were associated with asthma exacerbation severity in children., Methods: We enrolled a convenience sample of patients aged 5-18 years presenting with acute asthma exacerbation to a tertiary care pediatric emergency department. Severity of an asthma exacerbation (mild, moderate, severe) was assessed within 1 hour of the LUS using the Hospital Asthma Severity Score, a validated asthma assessment tool. LUS was performed by trained pediatric emergency providers. The presence of LUS findings (B-lines, consolidations, pleural effusion, and pleural line abnormalities) was assessed using a standardized criterion based on consensus guidelines., Results: A total of 111 patients with a median age of 8 years (interquartile range 6-12) were enrolled. LUS was positive in 57% of patients. Pleural line abnormalities were observed in 34%, B-lines in 29%, consolidations <1 cm in 24%, and consolidations ≥1 cm in 7%. Patients with moderate and severe asthma exacerbations were more likely to have any B-lines (31% and 43%, respectively) than patients with mild exacerbations (12%; P = .021); however, the presence of ≥3 B-lines or confluent B-lines did not differ across severity groups. The presence of other findings did not differ based on asthma severity., Conclusions: LUS findings are observed in a substantial portion of children presenting with asthma exacerbations. B-lines were the only LUS finding significantly associated with asthma severity, while lung consolidations <1 cm and >1 cm were not correlated with severity. These findings provide valuable information for the diagnostic use of LUS in pediatric patients with asthma exacerbation., (© 2024 American Institute of Ultrasound in Medicine.)

Supramolecular bioinspired self-assembling peptide hydrogel (SAPH) scaffolds represent a class of fully defined synthetic materials whose chemical and mechanical properties can be finely engineered. In this study, the relationship between SAPHs physicochemical properties and HepG2 cells viability, spheroid formation and function are discussed. We first report that negatively charged SAPHs promote hepatocyte proliferation and spheroids formation in vitro 3D culture while positively charged SAPHs lead to hepatocyte death irrespective of the hydrogel mechanical properties. More specifically HepG2 cultured in 3D in E(FKFE) 2 negatively charged SAPH maintained a differentiated phenotype and assembled into well-defined spheroids with strong cell-cell interactions. Furthermore, HepG2 spheroids responded to acetaminophen exposure with upregulation of key CYP450 enzymes expression clearly showing their potential for drug toxicity testing. These findings demonstrate how fine-tuned functional SAPH scaffolds can be used to identify key scaffolds parameters affecting cells. In this case we demonstrated the potential of negatively charged SAPHs for the 3D culture of HepG2 with potential applications in drug screening.

Electron transfer bifurcation enables biological systems to drive unfavourable (endergonic) electron transfer by coupling it to favourable (exergonic) transfer of a second electron. In electron transfer flavoproteins (ETFs), a domain-scale conformational change is believed to sever the favourable pathway after a single electron has used it, thereby preventing the energy dissipation that would accompany exergonic transfer of the second electron. To understand the conformation change that participates in turnover, we have deployed small-angle neutron scattering (SANS) and computational techniques to characterize the bifurcating ETF from Acidaminococcus fermentans ( Afe ETF). SANS data reveal an overall radius of gyration ( R g ) of 30.1 ± 0.2 Å and a maximum dimension ( D max ) of 100 Å for oxidized Afe ETF. These measurements are 4 Å and 30 Å larger, respectively, than those of any published bifurcating ETF structure. Thus, we find that none of the reported ETF structures can explain the observed scattering, nor can any individual conformation generated by either of our molecular dynamics protocols. To optimize ensembles best able to explain the SANS data, we adapted a genetic algorithm. Successful ensembles contained a compact conformation comparable to one of the crystallographically documented conformations, accompanied by a much more extended one, and these two conformations sufficed to account for the data. The extended conformations identified all have R g s at least 4 Å larger than those of any currently published ETF structures. However, they are strongly populated, constituting 20% of the population of reduced ETF and over 50% of the population of oxidized Afe ETF. Thus, the published (compact) structures provide a seriously incomplete picture of the conformation of Afe ETF in solution. Moreover, because the composition of the conformational ensemble changes upon reduction of Afe ETF's flavins, interconversion of the conformations may contribute to turnover. We propose that the extended conformations can provide energetically accessible paths for rapid interconversion of the open and closed compact conformations that are believed essential at alternating points in turnover., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Self-assembling peptide hydrogels (SAPHs) have been used in the past decade as reliable three-dimensional (3D) synthetic scaffolds for the culture of a variety of mammalian cells in vitro. Thanks to their versatile physicochemical properties, they allow researchers to tailor the hydrogel properties, including stiffness and functionality to the targeted cells and cells' behaviour. One of the advantages of using SAPH scaffolds is the ease of functionalisation. In the present work, we discuss the effect that functionalising the FEFEFKFK (F, phenylalanine; K, lysine; and E, glutamic acid) hydrogel scaffold using the cell-binding RGDS (fibronectin - R, arginine; G, glycine; D, aspartic acid; S, serine) epitope affects the material properties as well as the function of encapsulated human osteoblast cells. RGDS functionalisation resulted in cells adopting an elongated morphology, suggesting attachment and increased proliferation. While this led to higher cell viability, it also resulted in a decrease in extra-cellular matrix (ECM) protein production as well as a decrease in calcium ion deposition, suggesting lower mineralisation capabilities. The work clearly shows that SAPHs are a flexible platform that allow the modification of scaffolds in a controlled manner to investigate cell-material interactions., (© 2024 The Author(s). Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric β-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1., (© 2024. The Author(s).)

Objective: We sought to describe patterns of and indications for surgical specialty consultation for facial laceration repair in pediatric emergency departments (PEDs)., Methods: We performed a multicenter survey of PED leadership throughout the United States and Canada evaluating the practice patterns of surgical specialty consultation for patients presenting for facial lacerations requiring repair. We measured demographics of PEDs, factors influencing the decision to obtain a surgical specialty consultation, and the presence and components of consultation guidelines. Factors related to consultation were ranked on a Likert scale from 1 to 5 (1 = Not at all important, 5 = Extremely important). We evaluated relationships between reported rates of surgical specialty consultation and PED region, annual PED volume, and reported factors associated with PED consultation., Results: Survey responses were received from 67/124 (54%) queried PEDs. The median self-reported rate of surgical specialty consultation for facial lacerations was 10% and ranged from 1% to 70%, with resident physicians performing the repair 71% of the time a subspecialist was consulted. There was regional variability in specialty consultation, with the highest and lowest rate in the Midwest and Canada, respectively ( P = 0.03). The top 4 influential factors prompting consultation with the highest percentage of responses of "Extremely Important" or "Very Important" were: discretion of the physician caring for the patient (95%), parental preference (39%), limited PED resources (32%), and patient requires sedation (32%). Surgical specialty consult guidelines were used in only 6% of PEDs with consensus that depth necessitating more than 2-layer repair or involvement of critical structures should prompt consultation., Conclusions: Surgical specialty usage in the management of patients who present with facial lacerations to PEDs has significant variation related to patient, provider, and department-level factors that influence the decision to consult. Lack of consult guidelines represent a potential opportunity to standardize care delivery to this common presentation., Competing Interests: Disclosure: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Abstract: This review covers common orthopedic injuries seen after acute traumatic injury. A thorough physical examination and radiographic review of these injuries are key to ensuring appropriate management. Although many injuries may require urgent or emergent orthopedic consultation and management, this review focuses on injuries that are low-risk and amenable to splinting with outpatient orthopedic follow-up. This review covers key physical examination features, radiographic findings that help providers assess injuries, as well as reviewing splinting application to help facilitate rapid management of these injuries in the acute care setting., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

With recent advances in the reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs), gene editing technologies, and protocols for the directed differentiation of stem cells into heterogeneous tissues, iPSC-derived kidney organoids have emerged as a useful means to study processes of renal development and disease. Considerable advances guided by knowledge of fundamental renal developmental signaling pathways have been made with the use of exogenous morphogens to generate more robust kidney-like tissues in vitro. However, both biochemical and biophysical microenvironmental cues are major influences on tissue development and self-organization. In the context of engineering the biophysical aspects of the microenvironment, the use of hydrogel extracellular scaffolds for organoid studies has been gaining interest. Two families of hydrogels have recently been the subject of significant attention: self-assembling peptide hydrogels (SAPHs), which are fully synthetic and chemically defined, and gelatin methacryloyl (GelMA) hydrogels, which are semi-synthetic. Both can be used as support matrices for growing kidney organoids. Based on our recently published work, we highlight methods describing the generation of human iPSC (hiPSC)-derived kidney organoids and their maturation within SAPHs and GelMA hydrogels. We also detail protocols required for the characterization of such organoids using immunofluorescence imaging. Together, these protocols should enable the user to grow hiPSC-derived kidney organoids within hydrogels of this kind and evaluate the effects that the biophysical microenvironment provided by the hydrogels has on kidney organoid maturation. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Directed differentiation of human induced pluripotent stem cells (hiPSCs) into kidney organoids and maturation within mechanically tunable self-assembling peptide hydrogels (SAPHs) Alternate Protocol: Encapsulation of day 9 nephron progenitor aggregates in gelatin methacryloyl (GelMA) hydrogels. Support Protocol 1: Human induced pluripotent stem cell (hiPSC) culture. Support Protocol 2: Organoid fixation with paraformaldehyde (PFA) Basic Protocol 2: Whole-mount immunofluorescence imaging of kidney organoids. Basic Protocol 3: Immunofluorescence of organoid cryosections., (© 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.)

Peptide-based supramolecular hydrogels are an attractive class of soft materials for biomedical applications when biocompatibility is a key requirement as they exploit the physical self-assembly of short self-assembling peptides avoiding the need for chemical cross-linking. Based on the knowledge developed through our previous work, we designed two novel peptides, E (FKFE) 2 and K (FEFK) 2 , that form transparent hydrogels at pH 7. We characterized the phase behavior of these peptides and showed the clear link that exists between the charge carried by the peptides and the physical state of the samples. We subsequently demonstrate the cytocompatibility of the hydrogel and its suitability for 3D cell culture using 3T3 fibroblasts and human mesenchymal stem cells. We then loaded the hydrogels with two polymers, poly-l-lysine and dextran. When polymer and peptide fibers carry opposite charges, the size of the elemental fibril formed decreases, while the overall level of fiber aggregation and fiber bundle formation increases. This overall network topology change, and increase in cross-link stability and density, leads to an overall increase in the hydrogel mechanical properties and stability, i.e., resistance to swelling when placed in excess media. Finally, we investigate the diffusion of the polymers out of the hydrogels and show how electrostatic interactions can be used to control the release of large molecules. The work clearly shows how polymers can be used to tailor the properties of peptide hydrogels through guided intermolecular interactions and demonstrates the potential of these new soft hydrogels for use in the biomedical field in particular for delivery or large molecular payloads and cells as well as scaffolds for 3D cell culture.

Three-dimensional (3D) organoid models have been instrumental in understanding molecular mechanisms responsible for many cellular processes and diseases. However, established organic biomaterial scaffolds used for 3D hydrogel cultures, such as Matrigel, are biochemically complex and display significant batch variability, limiting reproducibility in experiments. Recently, there has been significant progress in the development of synthetic hydrogels for in vitro cell culture that are reproducible, mechanically tuneable, and biocompatible. Self-assembling peptide hydrogels (SAPHs) are synthetic biomaterials that can be engineered to be compatible with 3D cell culture. Here we investigate the ability of PeptiGel® SAPHs to model the mammary epithelial cell (MEC) microenvironment in vitro. The positively charged PeptiGel®Alpha4 supported MEC viability, but did not promote formation of polarised acini. Modifying the stiffness of PeptiGel® Alpha4 stimulated changes in MEC viability and changes in protein expression associated with altered MEC function, but did not fully recapitulate the morphologies of MECs grown in Matrigel. To supply the appropriate biochemical signals for MEC organoids, we supplemented PeptiGels® with laminin. Laminin was found to require negatively charged PeptiGel® Alpha7 for functionality, but was then able to provide appropriate signals for correct MEC polarisation and expression of characteristic proteins. Thus, optimisation of SAPH composition and mechanics allows tuning to support tissue-specific organoids., Competing Interests: Declaration of competing interest Manchester Biogel in part funded this research through a PhD studentship to the lead author (EL). Aline Miller and Alberto Saiani were shareholders, directors, and consultants for Manchester Biogel., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Electron bifurcation produces high-energy products based on less energetic reagents. This feat enables biological systems to exploit abundant mediocre fuel to drive vital but demanding reactions, including nitrogen fixation and CO 2 capture. Thus, there is great interest in understanding principles that can be portable to man-made devices. Bifurcating electron transfer flavoproteins (Bf ETFs) employ two flavins with contrasting reactivities to acquire pairs of electrons from a modest reductant, NADH. The bifurcating flavin then dispatches the electrons individually to a high and a low reduction midpoint potential ( E °) acceptor, the latter of which captures most of the energy. Maximum efficiency requires that only one electron accesses the exergonic path that will 'pay for' the production of the low- E ° product. It is therefore critical that one of the flavins, the 'electron transfer' (ET) flavin, is tuned to execute single-electron (1e - ) chemistry only. To learn how, and extract fundamental principles, we systematically altered interactions with the ET-flavin O2 position. Removal of a single hydrogen bond (H-bond) disfavored the formation of the flavin anionic semiquinone (ASQ) relative to the oxidized (OX) state, lowering by 150 mV and retuning the flavin's tendency for 1e - vs. 2e - reactivity. This was achieved by replacing conserved His 290 with Phe, while also replacing the supporting Tyr 279 with Ile. Although this variant binds oxidized FADs at 90% the WT level, the ASQ state of the ET-flavin is not stable in the absence of H290's H-bond, and dissociates, in contrast to the WT. Removal of this H-bond also altered the ET-flavin's covalent chemistry. While the WT ETF accumulates modified flavins whose formation is believed to rely on an anionic paraquinone methide intermediate, the FADs of the H-bond lacking variant remain unchanged over weeks. Hence the variant that destabilizes the anionic semiquinone also suppresses the anionic intermediate in flavin modification, verifying electronic similarities between these two species. These correlations suggest that the H-bond that stabilizes the crucial flavin ASQ also promotes flavin modification. The two effects may indeed be inseparable, as a Jekyll and Hydrogen bond., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The flavodoxin of Rhodopseudomonas palustris CGA009 (Rp9Fld) supplies highly reducing equivalents to crucial enzymes such as hydrogenase, especially when the organism is iron-restricted. By acquiring those electrons from photodriven electron flow via the bifurcating electron transfer flavoprotein, Rp9Fld provides solar power to vital metabolic processes. To understand Rp9Fld's ability to work with diverse partners, we solved its crystal structure. We observed the canonical flavodoxin (Fld) fold and features common to other long-chain Flds but not all the surface loops thought to recognize partner proteins. Moreover, some of the loops display alternative structures and dynamics. To advance studies of protein-protein associations and conformational consequences, we assigned the 19 F NMR signals of all five tyrosines (Tyrs). Our electrochemical measurements show that incorporation of 3- 19 F-Tyr in place of Tyr has only a modest effect on Rp9Fld's redox properties even though Tyrs flank the flavin on both sides. Meanwhile, the 19 F probes demonstrate the expected paramagnetic effect, with signals from nearby Tyrs becoming broadened beyond detection when the flavin semiquinone is formed. However, the temperature dependencies of chemical shifts and linewidths reveal dynamics affecting loops close to the flavin and regions that bind to partners in a variety of systems. These coincide with patterns of amino acid type conservation but not retention of specific residues, arguing against detailed specificity with respect to partners. We propose that the loops surrounding the flavin adopt altered conformations upon binding to partners and may even participate actively in electron transfer., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: The aim of the study is to assess diagnostic performance of cardiac point-of-care ultrasound (POCUS) performed by pediatric emergency medicine (PEM) physicians in children with preexisting cardiac disease., Methods: We evaluated the use of cardiac POCUS performed by PEM physicians among a convenience sample of children with preexisting cardiac disease presenting to a tertiary care pediatric ED. We assessed patient characteristics and the indication for POCUS. The test characteristics of the sonologist interpretation for the assessment of both pericardial effusion as well as left ventricular systolic dysfunction were compared with expert POCUS review by PEM physicians with POCUS fellowship training., Results: A total of 104 children with preexisting cardiac disease underwent cardiac POCUS examinations between July 2015 and December 2017. Among children with preexisting cardiac disease, structural defects were present in 72%, acquired conditions in 22%, and arrhythmias in 13% of patients. Cardiac POCUS was most frequently obtained because of chest pain (55%), dyspnea (18%), tachycardia (17%), and syncope (10%). Cardiac POCUS interpretation compared with expert review had a sensitivity of 100% (95% confidence interval [CI], 85.7-100) for pericardial effusion and 100% (95% CI, 71.5-100) for left ventricular systolic dysfunction; specificity was 97.5% (95% CI, 91.3.1-99.7) for pericardial effusion and 98.9% (95% CI, 93.8-99.8) for left ventricular systolic dysfunction., Conclusions: Cardiac POCUS demonstrates good sensitivity and specificity in diagnosing pericardial effusion and left ventricular systolic dysfunction in children with preexisting cardiac conditions when technically adequate studies are obtained. These findings support future studies of cardiac POCUS in children with preexisting cardiac conditions presenting to the ED., Competing Interests: Disclosure: The authors declare no conflict of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Objectives: Emergency medicine (EM) physicians and pediatricians who provide acute pediatric care depend on clinical exposure during residency to learn pediatric EM. Increasing volumes of pediatric patients, especially with behavioral health complaints, have stressed pediatric emergency departments (ED) and prompted clinical operations innovations including alternative care sites outside the main ED. We investigated the impact of these recent trends and resulting alternative care sites on the exposure of residents to core pediatric conditions., Methods: This retrospective study reviewed patient encounters between July 1, 2018, and December 31, 2022, at a pediatric ED that hosts one pediatric and three EM residencies. During the study, the hospital employed alternative care sites in response to increased and shifting patient populations. Median patients per resident per academic year were compared before and after the opening of alternative care sites, overall and stratified by patient factors (age, sex, Emergency Severity Index [ESI], and diagnostic category). The study also compared the percentage of residents who saw no patients with a given diagnosis between the two periods., Results: Of 231,101 patient encounters, 199,947 were seen in the main ED and 31,154 in alternative care sites. The median number of patients seen by a single resident in a single academic year ranged from 82 to 136 for pediatric residents and from 128 to 183 for EM residents. The median number of patients per resident per year did not decrease for any age group, sex, ESI level, or diagnosis across the two periods. Residents saw a median of 19 more patients with psychiatric diagnoses (95% CI 15.4-22.7) in the more recent period. Seven diagnoses were not seen by at least 20% of residents during both periods., Conclusions: Current pediatric ED capacity challenges can be addressed with alternative care sites without decreasing volume or variety of patients seen by residents., Competing Interests: The authors declare no conflicts of interest., (© 2023 Society for Academic Emergency Medicine.)

Bifurcating electron transferring flavoproteins (Bf-ETFs) tune chemically identical flavins to two contrasting roles. To understand how, we used hybrid quantum mechanical molecular mechanical calculations to characterize noncovalent interactions applied to each flavin by the protein. Our computations replicated the differences between the reactivities of the flavins: the electron transferring flavin ( ET flavin) was calculated to stabilize anionic semiquinone (ASQ) as needed to execute its single-electron transfers, whereas the Bf flavin ( Bf flavin) was found to disfavor the ASQ state more than does free flavin and to be less susceptible to reduction. The stability of ET flavin ASQ was attributed in part to H-bond donation to the flavin O2 from a nearby His side chain, via comparison of models employing different tautomers of His. This H-bond between O2 and the ET site was uniquely strong in the ASQ state, whereas reduction of ET flavin to the anionic hydroquinone (AHQ) was associated with side chain reorientation, backbone displacement, and reorganization of its H-bond network including a Tyr from the other domain and subunit of the ETF. The Bf site was less responsive overall, but formation of the Bf flavin AHQ allowed a nearby Arg side chain to adopt an alternative rotamer that can H-bond to the Bf flavin O4. This would stabilize the anionic Bf flavin and rationalize effects of mutation at this position. Thus, our computations provide insights on states and conformations that have not been possible to characterize experimentally, offering explanations for observed residue conservation and raising possibilities that can now be tested., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Pediatric cardiac arrest presents an infrequent but high-stakes event for emergency clinicians, who need to maintain expertise in this area. Evidence regarding pediatric resuscitations has been accumulating substantially over the past decade and highlights the unique considerations and challenges when resuscitating children. This issue reviews resuscitation principles of children in cardiac arrest while addressing the newest evidence-based and best-practice recommendations by the American Heart Association.

Introduction: Success in drug discovery remains unpredictable. However, more predictive and relevant disease models are becoming pivotal to demonstrating the clinical benefits of new drugs earlier in the lengthy drug discovery process. Novel hydrogel scaffolds are being developed to transform the relevance of such 3D cell-based in vitro assay systems., Areas Covered: Most traditional hydrogels are still of unknown composition and suffer significant batch-to-batch variations, which lead to technical constraints. This article looks at how a new generation of novel synthetic hydrogels that are based on self-assembling peptides are poised to transform 3D cell-based assay systems by improving their relevance, reproducibility and scalability., Expert Opinion: The emerging advantages of using these novel hydrogels for human 3D screening assays should enable the discovery of more cost-effective drugs, leading to improved patient benefits. Such a disruptive change could also reduce the considerable time lag from obtaining in vitro assay data to initiating clinical trials. There is now a sufficient body of data available in the literature to enable this ambition to become a reality by significantly improving the predictive validity of 3D cell-based assays in early drug discovery. Novel hydrogels are key to unlocking the full potential of these assay systems.

To address disparities in COVID-19 outcomes among Latinos with limited English proficiency in Maryland, our team developed a culturally congruent intervention that coupled a statewide social marketing campaign with community-based COVID-19 services. In the first year, we reached 305 122 people through social media advertisements and had 9607 visitors to the Web site. Social marketing campaigns represent an opportunity to promote COVID-19 testing and vaccine uptake among Latino populations, especially when they are paired with community services that simultaneously address structural barriers to care. ( Am J Public Health . 2023;113(3):263-266. https://doi.org/10.2105/10.2105/AJPH.2022.307191).

Study Objective: To explore the association between video-assisted laryngoscopy (use of a videolaryngoscope regardless of where laryngoscopists direct their gaze), first-attempt success, and adverse airway outcomes., Methods: We conducted an observational study using data from 2 airway consortiums that perform prospective surveillance: the National Emergency Airway Registry for Children (NEAR4KIDS) and a pediatric emergency medicine airway education collaborative. Data collected included patient and procedural characteristics and procedural outcomes. We performed multivariable analyses of the association of video-assisted laryngoscopy with individual patient outcomes and evaluated the association between site-level video-assisted laryngoscopy use and tracheal intubation outcomes., Results: The study cohort included 1,412 tracheal intubation encounters performed from January 2017 to March 2021 across 11 participating sites. Overall, the first-attempt success was 70.0%. Video-assisted laryngoscopy was associated with increased odds of first-attempt success (odds ratio [OR] 2.01; 95% confidence interval [CI], 1.48 to 2.73) and decreased odds of severe adverse airway outcomes (OR 0.70; 95% CI, 0.58 to 0.85) including decreased severe hypoxia (OR 0.69; 95% CI, 0.55 to 0.87). Sites varied substantially in the use of video-assisted laryngoscopy (range from 12.9% to 97.8%), and sites with high use of video-assisted laryngoscopy (> 80%) experienced increased first-attempt success even after adjusting for individual patient laryngoscope use (OR 2.30; 95% CI, 1.79 to 2.95)., Conclusion: Video-assisted laryngoscopy is associated with increased first-attempt success and fewer adverse airway outcomes for patients intubated in the pediatric emergency department. There is wide variability in the use of video-assisted laryngoscopy, and the high use is associated with increased odds of first-attempt success., (Copyright © 2022 American College of Emergency Physicians. Published by Elsevier Inc. All rights reserved.)

Background: Decisions about who should perform tracheal intubation in academic settings must balance the needs of trainees to develop competency in pediatric intubation with patient safety. Airway protocols during the COVID-19 pandemic may have reduced opportunities for trainees, representing an opportunity to examine the impact of shifting laryngoscopy responsibilities away from trainees., Methods: This observational study combined data from 11 pediatric emergency departments in North America participating in either the National Emergency Airway Registry for Children (NEAR4KIDS) or a national pediatric emergency medicine airway education collaborative. Sites provided information on airway protocols, patient and procedural characteristics, and clinical outcomes. For the pre-pandemic (January 2017 to March 2020) and pandemic (March 2020 to March 2021) periods, we compared tracheal intubation opportunities by laryngoscopist level of training and specialty. We also compared first-attempt success and adverse airway outcomes between the two periods., Results: There were 1129 intubations performed pre-pandemic and 283 during the pandemic. Ten of 11 sites reported a COVID-19 airway protocol-8 specified which clinician performs tracheal intubation and 10 advocated for videolaryngoscopy. Both pediatric residents and pediatric emergency medicine fellows performed proportionally fewer tracheal intubation attempts during the pandemic: 1.1% of all first attempts versus 6.4% pre-pandemic for residents (p < 0.01) and 38.4% versus 47.2% pre-pandemic for fellows (p = 0.01). Pediatric emergency medicine fellows had greater decrease in monthly intubation opportunities for patients <1 year (incidence rate ratio = 0.35, 95% CI: 0.2, 0.57) than for older patients (incidence rate ratio = 0.79, 95% CI: 0.62, 0.99). Neither the rate of first-attempt success nor adverse airway outcomes differed between pre-pandemic and pandemic periods., Conclusions: The COVID-19 pandemic led to pediatric institutional changes in airway management protocols and resulted in decreased intubation opportunities for pediatric residents and pediatric emergency medicine fellows, without apparent change in clinical outcomes., Competing Interests: All authors report no conflicts of interest., (© 2022 Society for Academic Emergency Medicine.)

Catholic schooling in the United States is suffering from a persistent enrollment crisis that has triggered the need for system-wide organizational reforms. However, most of the changes that the sector has experienced has taken place in individual schools making decisions about how to operationally sustain their individual school community. In this article, we present findings from a qualitative analysis of 26 superintendents of (arch)diocesan Catholic school systems in order to better understand why there has been an absence of system-level change in the Catholic sector in the U.S. at a time when systems thinking has started to spread throughout other sectors domestically and internationally. We show through the findings presented in this paper that many Catholic school systems in the United States do not sustain system-level change because they rely on a decentralized "system of schools" organizational form that superintendents believe limits the possibility for sector-wide organizational reform. We highlight in this paper the ways superintendents are forced to navigate these organizational and political limitations and suggest what the implications of this limited possibility for system-level change are for the Catholic sector and other similarly organized sectors., Competing Interests: Conflict of interestWe have no known conflict of interest to disclose., (© The Author(s), under exclusive licence to Springer Nature B.V. 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

From the outset, canonical electron transferring flavoproteins (ETFs) earned a reputation for containing modified flavin. We now show that modification occurs in the recently recognized bifurcating (Bf) ETFs as well. In Bf ETFs, the 'electron transfer' (ET) flavin mediates single electron transfer via a stable anionic semiquinone state, akin to the FAD of canonical ETFs, whereas a second flavin mediates bifurcation (the Bf FAD). We demonstrate that the ET FAD undergoes transformation to two different modified flavins by a sequence of protein-catalyzed reactions that occurs specifically in the ET site, when the enzyme is maintained at pH 9 in an amine-based buffer. Our optical and mass spectrometric characterizations identify 8-formyl flavin early in the process and 8-amino flavins (8AFs) at later times. The latter have not previously been documented in an ETF to our knowledge. Mass spectrometry of flavin products formed in Tris or bis-tris-aminopropane solutions demonstrates that the source of the amine adduct is the buffer. Stepwise reduction of the 8AF demonstrates that it can explain a charge transfer band observed near 726 nm in Bf ETF, as a complex involving the hydroquinone state of the 8AF in the ET site with the oxidized state of unmodified flavin in the Bf site. This supports the possibility that Bf ETF can populate a conformation enabling direct electron transfer between its two flavins, as has been proposed for cofactors brought together in complexes between ETF and its partner proteins., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Human induced pluripotent stem cell (hiPSC)-derived kidney organoids have prospective applications ranging from basic disease modelling to personalised medicine. However, there remains a necessity to refine the biophysical and biochemical parameters that govern kidney organoid formation. Differentiation within fully-controllable and physiologically relevant 3D growth environments will be critical to improving organoid reproducibility and maturation. Here, we matured hiPSC-derived kidney organoids within fully synthetic self-assembling peptide hydrogels (SAPHs) of variable stiffness (storage modulus, G'). The resulting organoids contained complex structures comparable to those differentiated within the animal-derived matrix, Matrigel. Single-cell RNA sequencing (scRNA-seq) was then used to compare organoids matured within SAPHs to those grown within Matrigel or at the air-liquid interface. A total of 13,179 cells were analysed, revealing 14 distinct clusters. Organoid compositional analysis revealed a larger proportion of nephron cell types within Transwell-derived organoids, while SAPH-derived organoids were enriched for stromal-associated cell populations. Notably, differentiation within a higher G' SAPH generated podocytes with more mature gene expression profiles. Additionally, maturation within a 3D microenvironment significantly reduced the derivation of off-target cell types, which are a known limitation of current kidney organoid protocols. This work demonstrates the utility of synthetic peptide-based hydrogels with a defined stiffness, as a minimally complex microenvironment for the selected differentiation of kidney organoids., Competing Interests: This work was supported, in part, by contribution from Manchester BIOGEL., (© 2022 The Authors.)

Hydrogels are versatile materials that have emerged in the last few decades as promising candidates for a range of applications in the biomedical field, from tissue engineering and regenerative medicine to controlled drug delivery. In the drug delivery field, in particular, they have been the subject of significant interest for the spatially and temporally controlled delivery of anticancer drugs and therapeutics. Self-assembling peptide-based hydrogels, in particular, have recently come to the fore as potential candidate vehicles for the delivery of a range of drugs. In order to explore how drug-peptide interactions influence doxorubicin (Dox) release, five β-sheet-forming self-assembling peptides with different physicochemical properties were used for the purpose of this study, namely: FEFKFEFK (F8), FKFEFKFK (FK), FEFEFKFE (FE), FEFKFEFKK (F8K), and KFEFKFEFKK (KF8K) (F: phenylalanine; E: glutamic acid; K: lysine). First, Dox-loaded hydrogels were characterized to ensure that the incorporation of the drug did not significantly affect the hydrogel properties. Subsequently, Dox diffusion out of the hydrogels was investigated using UV absorbance. The amount of drug retained in F8/FE composite hydrogels was found to be directly proportional to the amount of charge carried by the peptide fibers. When cation-π interactions were used, the position and number of end-lysine were found to play a key role in the retention of Dox. In this case, the amount of Dox retained in F8/KF8K composite hydrogels was linked to the amount of end-lysine introduced, and an end-lysine/Dox interaction stoichiometry of 3/1 was obtained. For pure FE and KF8K hydrogels, the maximum amount of Dox retained was also found to be related to the overall concentration of the hydrogels and, therefore, to the overall fiber surface area available for interaction with the drug. For 14 mM hydrogel, ∼170-200 μM Dox could be retained after 24 h. This set of peptides also showed a broad range of susceptibilities to enzymatic degradation opening the prospect of being able to control also the rate of degradation of these hydrogels. Finally, the Dox released from the hydrogel was shown to be active and affect 3T3 mouse fibroblasts viability in vitro. Our study clearly shows the potential of this peptide design as a platform for the formulation of injectable or sprayable hydrogels for controlled drug delivery.

Objectives: To investigate the rates of radiographic pneumonia and clinical outcomes of children with suspected pneumonia and subcentimeter, subpleural consolidations on point-of-care lung ultrasound., Study Design: We enrolled a prospective convenience sample of children aged 6 months to 18 years undergoing chest radiography (CXR) for pneumonia evaluation in a single tertiary-care pediatric emergency department. Point-of-care lung ultrasound was performed by an emergency medicine physician with subsequent expert review. We determined rates of radiographic pneumonia and clinical outcomes in the children with subcentimeter, subpleural consolidations, stratified by the presence of larger (>1 cm) sonographic consolidations. The children were followed prospectively for 2 weeks to identify a delayed diagnosis of pneumonia., Results: A total of 188 patients, with a median age of 5.8 years (IQR, 3.5-11.0 years), were evaluated. Of these patients, 62 (33%) had subcentimeter, subpleural consolidations on lung ultrasound, and 23 (37%) also had larger (>1 cm) consolidations. Patients with subcentimeter, subpleural consolidations and larger consolidations had the highest rates of definite radiographic pneumonia (61%), compared with 21% among children with isolated subcentimeter, subpleural consolidations. Overall, 23 children with isolated subcentimeter, subpleural consolidations (59%) had no evidence of pneumonia on CXR. Among 16 children with isolated subcentimeter, subpleural consolidations and not treated with antibiotics, none had a subsequent pneumonia diagnosis within the 2-week follow-up period., Conclusions: Children with subcentimeter, subpleural consolidations often had radiographic pneumonia; however, this occurred most frequently when subcentimeter, subpleural consolidations were identified in combination with larger consolidations. Isolated subcentimeter, subpleural consolidations in the absence of larger consolidations should not be viewed as synonymous with pneumonia; CXR may provide adjunctive information in these cases., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bifurcating electron transfer flavoproteins (Bf ETFs) are important redox enzymes that contain two flavin adenine dinucleotide (FAD) cofactors, with contrasting reactivities and complementary roles in electron bifurcation. However, for both the "electron transfer" (ET) and the "bifurcating" (Bf) FADs, the only charged amino acid within 5 Å of the flavin is a conserved arginine (Arg) residue. To understand how the two sites produce different reactivities utilizing the same residue, we investigated the consequences of replacing each of the Arg residues with lysine, glutamine, histidine, or alanine. We show that absence of a positive charge in the ET site diminishes accumulation of the anionic semiquinone (ASQ) that enables the ET flavin to act as a single electron carrier, due to depression of the oxidized versus. ASQ reduction midpoint potential, E° OX/ASQ . Perturbation of the ET site also affected the remote Bf site, whereas abrogation of Bf FAD binding accelerated chemical modification of the ET flavin. In the Bf site, removal of the positive charge impaired binding of FAD or AMP, resulting in unstable protein. Based on pH dependence, we propose that the Bf site Arg interacts with the phosphate(s) of Bf FAD or AMP, bridging the domain interface via a conserved peptide loop ("zipper") and favoring nucleotide binding. We further propose a model that rationalizes conservation of the Bf site Arg even in non-Bf ETFs, as well as AMP's stabilizing role in the latter, and provides a mechanism for coupling Bf flavin redox changes to domain-scale motion., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Liver fibrosis, a condition characterized by extensive deposition and cross-linking of extracellular matrix (ECM) proteins, is idiosyncratic in cases of chronic liver injury. The dysregulation of ECM remodeling by hepatic stellate cells (HSCs), the main mediators of fibrosis, results in an elevated ECM stiffness that drives the development of chronic liver disease such as cirrhosis and hepatocellular carcinoma. Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is a key element in the regulation of ECM remodeling, which modulates the degradation and turnover of ECM components. We have previously reported that a rigid, fibrotic-like substrate can impact TIMP-1 expression at the protein level in HSCs without altering its mRNA expression. While HSCs are known to be highly susceptible to mechanical stimuli, the mechanisms through which mechanical cues regulate TIMP-1 at the post-translational level remain unclear. Here, we show a mechanism of regulation of plasma membrane tension by matrix stiffness. We found that this effect is orchestrated by the β1 integrin/RhoA axis and results in elevated exocytosis and secretion of TIMP-1 in a caveolin-1- and dynamin-2-dependent manner. We then show that TIMP-1 and caveolin-1 expression increases in cirrhosis and hepatocellular carcinoma. These conditions are associated with fibrosis, and this effect can be recapitulated in 3D fibrosis models consisting of hepatic stellate cells encapsulated in a self-assembling polypeptide hydrogel. This work positions stiffness-dependent membrane tension as a key regulator of enzyme secretion and function and a potential target for therapeutic strategies that aim at modulating ECM remodeling in chronic liver disease.

Objectives: We sought to describe the test characteristics of cardiac point-of-care ultrasound (POCUS) performed by pediatric emergency medicine (PEM) physicians after structured cardiac POCUS training., Methods: We evaluated the use of clinically indicated cardiac POCUS by PEM physicians in a single tertiary care pediatric emergency department after implementation of a focused cardiac POCUS training curriculum. The test characteristics of the sonologist interpretation were compared with expert POCUS review, by PEM physicians who have completed PEM POCUS fellowship training, for the assessment of both pericardial effusion and left ventricular systolic dysfunction., Results: A total of 1241 cardiac POCUS examinations were performed between July 2015 and December 2017, of which 456 were clinically indicated and underwent expert POCUS review and comprised the study sample. These examinations were performed by 33 different PEM attending sonologists. Chest pain (52%), dyspnea (20%), and tachycardia (18%) were the most common indications for cardiac POCUS. Prevalence of pericardial effusion and global systolic dysfunction based on expert POCUS review were 11% (48/443) and 4% (16/435), respectively. Real-time cardiac POCUS interpretation had a sensitivity and specificity of 100% and 99.5%, respectively, for both pericardial effusion and left ventricular systolic dysfunction when compared with expert POCUS review., Conclusions: Cardiac POCUS is both sensitive and specific for identifying pericardial effusion and left ventricular systolic dysfunction when performed by PEM attendings with focused training., Competing Interests: Disclosure: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: Traditionally, patient-reported fasting time has been the primary objective presedation measure of aspiration risk. Recently, gastric ultrasound has been used to assess gastric volume for the determination of aspiration risk in patients undergoing anesthesia in the operative setting. We sought to determine the correlation of gastric volume estimated by point-of-care ultrasound (POCUS) to reported fasting time., Methods: We included children 4 to 18 years of age who presented with an acute traumatic injury. Enrolled children underwent POCUS to calculate gastric volume, which was calculated using a validated formula: Volume (mL) = -7.8 + (3.5 × Cross-sectional Area [CSA]) + (0.127 × Age in months). The CSA was measured (CSA = (anterior-posterior diameter × craniocaudal diameter × π)/4). We analyzed the relationship between time since last reported oral intake and measured gastric volume using Spearman rank correlation (ρ)., Results: A total of 103 patients with a median age of 10.5 years (interquartile range, 7.3-13.7 years) were enrolled. The gastric antrum was identified and measured in 88 (85%) patients; air obstructing the posterior surface of the gastric antrum prevented measurement in 14 of the 15 remaining patients. We observed a weak inverse correlation between fasting time (either liquid or solid) and estimated gastric volume (ρ = -0.33), with no significant difference based on type of intake (solids, ρ = 0.28; liquids, ρ = 0.22)., Conclusion: Gastric volume can be estimated by POCUS and is not strongly correlated with fasting time in children in the emergency department setting., Competing Interests: Disclosure: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Flavins are central to countless enzymes but display different reactivities depending on their environments. This is understood to reflect modulation of the flavin electronic structure. To understand changes in orbital natures, energies, and correlation over the ring system, we begin by comparing seven flavin variants differing at C8, exploiting their different electronic spectra to validate quantum chemical calculations. Ground state calculations replicate a Hammett trend and reveal the significance of the flavin π-system. Comparison of higher-level theories establishes CC2 and ACD(2) as methods of choice for characterization of electronic transitions. Charge transfer character and electron correlation prove responsive to the identity of the substituent at C8. Indeed, bond length alternation analysis demonstrates extensive conjugation and delocalization from the C8 position throughout the ring system. Moreover, we succeed in replicating a particularly challenging UV/Vis spectrum by implementing hybrid QM/MM in explicit solvents. Our calculations reveal that the presence of nonbonding lone pairs correlates with the change in the UV/Vis spectrum observed when the 8-methyl is replaced by NH 2 , OH, or SH. Thus, our computations offer routes to understanding the spectra of flavins with different modifications. This is a first step toward understanding how the same is accomplished by different binding environments.

Background: Children with limp or hip pain often undergo radiographs and ultrasound as part of their initial evaluation. Previous research suggests that hip radiography may have limited utility, and early use of ultrasound may safely reduce the use of radiographs., Objectives: We sought to assess the utility of radiography in addition to ultrasound by evaluating the rate of bony abnormalities present on hip radiographs among children with and without effusion on ultrasound. We also assessed the agreement of point-of-care and Radiology-performed ultrasounds for the detection of effusion., Methods: This is a retrospective cohort study of children presenting to a pediatric emergency department with acute atraumatic limp or hip pain. Data from patients who received both hip ultrasound and hip radiography as part of their evaluation were analyzed. We included both point-of-care and Radiology-performed hip ultrasounds., Results: We identified 134 patients who received both hip ultrasound and hip radiographs. Sixty-eight patients (51%) had a hip effusion present on ultrasound and none of these had bony abnormalities on radiography (0%, 95% confidence interval 0-5.3%). Of the 66 patients (49%) who had no effusion on hip ultrasound, 2 patients were found to have a bony abnormality (3%, 95% confidence interval 0.4-10.5%). For patients who received both point-of-care and Radiology-performed ultrasound, the overall agreement for diagnosis of effusion was 92.6% (kappa = 0.82)., Conclusions: We observed that no children with an effusion on ultrasound had bony pathology on plain radiography, suggesting that the routine performance of hip radiography may not be indicated in all children. Future studies are needed to evaluate the negative predictive value of effusion in larger numbers of patients with known bony abnormalities., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Self-assembling peptide hydrogels (SAPH) are a popular biomaterial due to their biocompatibility with a wide range of cell types, synthetic design, structural properties that provide a more accurate 3D microenvironment, and potential for cell- and/or drug-delivery system. Mimicking solid tumors in vitro using hydrogels is one method of testing anti-cancer drug efficacy and observing cancerous cell-ECM interactions within a 3D system. In this study, a SAPH, PeptiGel®Alpha1, was used to model in vitro the 3D breast tumor microenvironment. PeptiGel®Alpha1 is composed of entangled nanofibers with consistent diameter and mechanical properties similar to breast cancer that more accurately mimic the stiffness of breast tumor tissue than Matrigel® or collagen type I. PeptiGel®Alpha1 supported the viability and growth of the breast cancer cell lines MCF-7 and MDA-MB-231 and recapitulated key features of solid tumors such as hypoxia and invasion. MCF-7 cells in the hydrogels formed large spheroids resembling acini, while MDA-MB-231 remained dispersed. When treated with tamoxifen, PeptiGel®Alpha1 acted as a barrier, providing drug penetration geometry similar to that in vivo, providing better prediction of the drug effect. Finally, it was observed that MCF-7 cells engulfed the peptide matrix after 14 days, highlighting a potential use in drug delivery. PeptiGel®Alpha1 is a suitable platform for in vitro modeling of breast cancer., (Copyright © 2021. Published by Elsevier B.V.)

Electron transfer bifurcation allows production of a strongly reducing carrier at the expense of a weaker one, by redistributing energy among a pair of electrons. Thus, two weakly-reducing electrons from NADH are consumed to produce a strongly reducing ferredoxin or flavodoxin, paid for by reduction of an oxidizing acceptor. The prevailing mechanism calls for participation of a strongly reducing flavin semiquinone which has been difficult to observe with site-certainly in multi-flavin systems. Using blue light (450 nm) to photoexcite the flavins of bifurcating electron transfer flavoprotein (ETF), we demonstrate accumulation of anionic flavin semiquinone in excess of what is observed in equilibrium titrations, and establish its ability to reduce the low-potential electron acceptor benzyl viologen. This must occur at the bifurcating flavin because the midpoint potentials of the electron transfer (ET) flavin are not sufficiently negative. We show that bis-tris propane buffer is an effective electron donor to the flavin photoreduction, but that if the system is prepared with the ET flavin chemically reduced, so that only the bifurcating flavin is oxidized and photochemically active, flavin anionic semiquinone is formed more rapidly. Thus, excited bifurcating flavin is able to draw on an electron stored at the ET flavin. Flavin semiquinone photogenerated at the bifurcation site must therefore be accompanied by additional semiquinone formation by oxidation of the ET flavin. Consistent with the expected instability of bifurcating flavin semiquinone, it subsides immediately upon cessation of illumination. However comparison with yields of semiquinone in equilibrium titrations suggest that during continuous illumination at pH 9 a steady state population of 0.3 equivalents of bifurcating flavin semiquinone accumulates, and then undergoes further photoreduction to the hydroquinone. Although transient, the population of bifurcating flavin semiquinone explains the system's ability to conduct light-driven electron transfer from bis-tris propane to benzyl viologen, in effect trapping energy from light., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The tumor microenvironment plays a critical role in modulating cancer cell migration, metabolism, and malignancy, thus, highlighting the need to develop in vitro culture systems that can recapitulate its abnormal properties. While a variety of stiffness-tunable biomaterials, reviewed here, have been developed to mimic the rigidity of the tumor extracellular matrix, culture systems that can recapitulate the broader extracellular context of the tumor microenvironment (including pH and temperature) remain comparably unexplored, partially due to the difficulty in independently tuning these parameters. Here, we investigate a self-assembled polypeptide network hydrogel as a cell culture platform and demonstrate that the culture parameters, including the substrate stiffness, extracellular pH and temperature, can be independently controlled. We then use this biomaterial as a cell culture substrate to assess the effect of stiffness, pH and temperature on Suit2 cells, a pancreatic cancer cell line, and demonstrate that these microenvironmental factors can regulate two critical transcription factors in cancer: yes-associated protein 1 (YAP) and hypoxia inducible factor (HIF-1A).

Intervertebral disc (IVD) degeneration is a process that starts in the central nucleus pulposus (NP) and leads to inflammation, extracellular matrix (ECM) degradation, and progressive loss of disc height. Early treatment of IVD degeneration is critical to the reduction of low back pain and related disability. As such, minimally invasive therapeutic approaches that can halt and reverse NP degeneration at the early stages of the disease are needed. Recently, we developed an injectable graphene oxide (GO) - self-assembling peptide FEFKFEFK (F: phenylalanine; K: lysine; E: glutamic acid) hybrid hydrogels as potential delivery platform for cells and/or drugs in the NP. In this current study, we explored the possibility of using the GO present in these hybrid hydrogels as a vehicle for the sequestration and controlled delivery of transforming growth factor beta-3 (TGF-β3), an anabolic growth factor (GF) known to direct NP cell fate and function. For this purpose, we first investigated the potential of GO to bind and sequestrate TGF-β3. We then cultured bovine NP cells in the new functional scaffolds and investigated their response to the presence of GO and TGF-β3. Our results clearly showed that GO flakes can sequestrate TGF-β3 through strong binding interactions resulting in a slow and prolonged release, with the GF remaining active even when bound to the GO flakes. The adsorption of the GF on the GO flakes to create TGF-β3-loaded GO flakes and their subsequent incorporation in the hydrogels through mixing, [(GO/TGF-β3 Ads )-F8] hydrogel, led to the upregulation of NP-specific genes, accompanied by the production and deposition of an NP-like ECM, rich in aggrecan and collagen II. NP cells actively interacted with TGF-β3-loaded GO flakes and remodeled the scaffolds through endocytosis. This work highlights the potential of using GO as a nanocarrier for the design of functional hybrid peptide-based hydrogels. STATEMENT OF SIGNIFICANCE: Intervertebral disc (IVD) degeneration is a process that starts in the central nucleus pulposus (NP) and leads to inflammation, extracellular matrix (ECM) degradation, and progressive loss of disc height. As such, minimally invasive therapeutic approaches that can halt and reverse NP degeneration at the early stages of the disease are needed. In this current study, we explored the possibility of using peptide - GO hybrid hydrogels as a vehicle for the sequestration and controlled delivery of transforming growth factor beta-3 (TGF-β3), an anabolic growth factor (GF) known to direct NP cell fate and function., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Flavodoxin is a small protein that employs a non-covalently bound flavin to mediate single-electron transfer at low potentials. The long-chain flavodoxins possess a long surface loop that is proposed to interact with partner proteins. We have incorporated 19 F-labeled tyrosine in long-chain flavodoxin from Rhodopseudomonas palustris to gain a probe of possible loop dynamics, exploiting the presence of a Tyr in the long loop in addition to Tyr residues near the flavin. We report 19 F resonance assignments for all four Tyrs, and demonstration of a pair of resonances in slow exchange, both corresponding to a Tyr adjacent to the flavin. We also provide evidence for dynamics affecting the Tyr in the long loop. Thus, we show that 19 F NMR of 19 F-Tyr labeled flavodoxin holds promise for monitoring possible changes in conformation upon binding to partner proteins., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Heart failure (HF) remains one of the leading causes of death worldwide; most commonly developing after myocardial infarction (MI). Since adult cardiomyocytes characteristically do not proliferate, cells lost during MI are not replaced. As a result, the heart has a limited regenerative capacity. There is, therefore, a need to develop novel cell-based therapies to promote the regeneration of the heart after MI. The delivery and retention of cells at the injury site remains a significant challenge. In this context, we explored the potential of using an injectable, RGDSP-functionalised self-assembling peptide - FEFEFKFK - hydrogel as scaffold for the delivery and retention of rat cardiac progenitor cells (CPCs) into the heart. Our results show that culturing CPCs in vitro within the hydrogel for one-week promoted their spontaneous differentiation towards adult cardiac phenotypes. Injection of the hydrogel on its own, or loaded with CPCs, into the rat after injury resulted in a significant reduction in myocardial damage and left ventricular dilation., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

A remarkable charge transfer (CT) band is described in the bifurcating electron transfer flavoprotein (Bf-ETF) from Rhodopseudomonas palustris ( Rpa ETF). Rpa ETF contains two FADs that play contrasting roles in electron bifurcation. The Bf-FAD accepts electrons pairwise from NADH, directs one to a lower-reduction midpoint potential ( E °) carrier, and the other to the higher- E ° electron transfer FAD (ET-FAD). Previous work noted that a CT band at 726 nm formed when ET-FAD was reduced and Bf-FAD was oxidized, suggesting that both flavins participate. However, existing crystal structures place them too far apart to interact directly. We present biochemical experiments addressing this conundrum and elucidating the nature of this CT species. We observed that Rpa ETF missing either FAD lacked the 726 nm band. Site-directed mutagenesis near either FAD produced altered yields of the CT species, supporting involvement of both flavins. The residue substitutions did not alter the absorption maximum of the signal, ruling out contributions from residue orbitals. Instead, we propose that the residue identities modulate the population of a protein conformation that brings the ET-flavin and Bf-flavin into direct contact, explaining the 726 nm band based on a CT complex of reduced ET-FAD and oxidized Bf-FAD. This is corroborated by persistence of the 726 nm species during gentle protein denaturation and simple density functional theory calculations of flavin dimers. Although such a CT complex has been demonstrated for free flavins, this is the first observation of such, to our knowledge, in an enzyme. Thus, Bf-ETFs may optimize electron transfer efficiency by enabling direct flavin-flavin contact., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Duan et al.)

Hydrogels' hydrated fibrillar nature makes them the material of choice for the design and engineering of 3D scaffolds for cell culture, tissue engineering, and drug-delivery applications. One particular class of hydrogels which has been the focus of significant research is self-assembling peptide hydrogels. In the present work, we were interested in exploring how fiber-fiber edge interactions affect the self-assembly and gelation properties of amphipathic peptides. For this purpose, we investigated two β-sheet-forming peptides, FEFKFEFK (F8) and KFEFKFEFKK (KF8K), the latter one having the fiber edges covered by lysine residues. Our results showed that the addition of the two lysine residues did not affect the ability of the peptides to form β-sheet-rich fibers, provided that the overall charge carried by the two peptides was kept constant. However, it did significantly reduce edge-driven hydrophobic fiber-fiber associative interactions, resulting in reduced tendency for KF8K fibers to associate/aggregate laterally and form large fiber bundles and consequently network cross-links. This effect resulted in the formation of hydrogels with lower moduli but faster dynamics. As a result, KF8K fibers could be aligned only under high shear and at high concentration while F8 hydrogel fibers were found to align readily at low shear and low concentration. In addition, F8 hydrogels were found to fragment at high concentration because of the high aggregation state stabilizing the fiber bundles, resulting in fiber breakage rather than disentanglement and alignment.

Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms. The reaction is thought to proceed via electrophilic aromatic substitution, catalyzed by enzymatic deprotonation of substrate, in single-component hydroxylases that use flavin as a cofactor (group A). However, two-component hydroxylases (group D), which use reduced flavin as a co-substrate, are less amenable to spectroscopic investigation. Herein, we employed 19 F NMR in conjunction with fluorinated substrate analogs to directly measure p K a values and to monitor protein events in hydroxylase active sites. We found that the single-component monooxygenase 3-hydroxybenzoate 6-hydroxylase (3HB6H) depresses the p K a of the bound substrate analog 4-fluoro-3-hydroxybenzoate (4F3HB) by 1.6 pH units, consistent with previously proposed mechanisms. 19 F NMR was applied anaerobically to the two-component monooxygenase 4-hydroxyphenylacetate 3-hydroxylase (HPAH), revealing depression of the p K a of 3-fluoro-4-hydroxyphenylacetate by 2.5 pH units upon binding to the C 2 component of HPAH. 19 F NMR also revealed a p K a of 8.7 ± 0.05 that we attributed to an active-site residue involved in deprotonating bound substrate, and assigned to His-120 based on studies of protein variants. Thus, in both types of hydroxylases, we confirmed that binding favors the phenolate form of substrate. The 9 and 14 kJ/mol magnitudes of the effects for 3HB6H and HPAH-C 2 , respectively, are consistent with p K a tuning by one or more H-bonding interactions. Our implementation of 19 F NMR in anaerobic samples is applicable to other two-component flavin-dependent hydroxylases and promises to expand our understanding of their catalytic mechanisms., (© 2020 Pitsawong et al.)

Objective: We sought to assess interrater reliability (IRR) of lung point-of-care ultrasound (POCUS) findings among pediatric patients with suspected pneumonia., Methods: A convenience sample of patients between the ages of 6 months and 18 years with a clinical suspicion of pneumonia had a lung ultrasound performed by a POCUS-credentialed emergency medicine physician with subsequent expert review. Each lung zone was assessed as either normal or abnormal, and specific ultrasound findings were recorded. IRR was assessed by intraclass correlation coefficient (ICC) and kappa statistics., Results: Seventy-one patients, with a total of 852 lung zones imaged, were included. The sonographer assessment of normal versus abnormal, across each of the zones, demonstrated moderate agreement with ICC 0.46 (95% CI: 0.41, 0.52) and kappa 0.56. Right-sided zones demonstrated moderate agreement [0.43 (CI 0.35, 0.51)] while left-sided zones, specifically left-sided anterior zones, showed only fair agreement [0.36 (0.28, 0.44)]. IRR varied between specific findings: ICC for B-lines 0.52 (95% CI: 0.46, 0.57), pleural effusion 0.40 (0.34, 0.45), consolidation 0.39 (0.33, 0.44), subpleural consolidation 0.31 (0.25, 0.37), and pleural line irregularity 0.16 (0.10, 0.23). A composite indicator of typical pneumonia findings (consolidation, B-lines, and pleural effusion) demonstrated moderate [ICC 0.52 (0.46, 0.57)] reliability., Conclusions: We found moderate interrater reliability of lung POCUS findings for the assessment of pediatric patients with suspected pneumonia. B-lines had the highest reliability. Further assessment of lung POCUS is necessary to guide proper training and optimal scanning techniques to ensure adequate reliability of ultrasound findings in the assessment of pediatric pneumonia., (Copyright © 2019 Elsevier Inc. All rights reserved.)

DNA and RNA biomarkers have not progressed beyond the automated specialized clinic due to failure in the reproducibility necessary to standardize robust and rapid nucleic acid detection at the point of care, where health outcomes can be most improved by early-stage diagnosis and precise monitoring of therapy and disease prognosis. We demonstrate here a new analytical platform to meet this challenge using functional 3D hydrogels engineered from peptide and oligonucleotide building blocks to provide sequence-specific, PCR-free fluorescent detection of unlabeled nucleic acid sequences. We discriminated at picomolar detection limits (<7 pM) "perfect-match" from mismatched sequences, down to a single nucleotide mutation, buried within longer lengths of the target. Detailed characterization by NMR, TEM, mass spectrometry, and rheology provided the structural understanding to design these hybrid peptide-oligonucleotide biomaterials with the desired sequence sensitivity and detection limit. We discuss the generic design, which is based on a highly predictable secondary structure of the oligonucleotide components, as a platform to detect genetic abnormalities and to screen for pathogenic conditions at the level of both DNA (e.g., SNPs) and RNA (messenger, micro, and viral genomic RNA).

Background: Prior studies have shown racial differences in concussion awareness and outcome. Objective: To assess if racial or ethnic differences exist in Emergency Department (ED) utilization and diagnosis for children with sports-related head injuries. Methods: We performed a retrospective, cross-sectional analysis of ED visits from 2008 to 2017 using National Electronic Injury Surveillance System (NEISS) data. Population-weighted ED visits for children age 7-18 years with a sport-related injury were included. We compared the probability of an ED visit being for an injury to the head or diagnosed as a concussion between children of different races/ethnicities. Analyses were adjusted for age, gender, sport, year, and location where the injury occurred. Results: We identified 11,529,994 population-weighted ED visits for pediatric sports-related injuries, of which 1,497,717 (13.0%) were injuries to the head and 619,714 (5.4%) received a diagnosis of concussion. Black children were significantly less likely than non-Hispanic white children to have their ED visit be for an injury to the head [Odds Ratio (OR) 0.72, 95%CI 0.65-0.79] or concussion (OR 0.58, 95%CI 0.50-0.68). Black children presenting to the ED with an injury to their head were less likely than non-Hispanic white children to be diagnosed with a concussion (OR = 0.71, 95%CI 0.59-0.85). Conclusions: Racial differences exist in both ED utilization for pediatric sports-related head injuries and in the diagnosis of concussion. Further work is needed to understand these differences to ensure all brain injured athletes receive optimal care, regardless of race.

Cell-based therapies have shown significant promise in tissue engineering with one key challenge being the delivery and retention of cells. As a result, significant efforts have been made in the past decade to design injectable biomaterials to host and deliver cells at injury sites. Intervertebral disc (IVD) degeneration, a major cause of back pain, is a particularly relevant example where a minimally-invasive cellular therapy could bring significant benefits specifically at the early stages of the disease, when a cell-driven process starts in the gelatinous core of the IVD, the nucleus pulposus (NP). In this present study we explore the use of graphene oxide (GO) as nano-filler for the reinforcement of FEFKFEFK (β-sheet forming self-assembling peptide) hydrogels. Our results confirm the presence of strong interactions between FEFKFEFK and GO flakes with the peptide coating and forming short thin fibrils on the surface of the flakes. These strong interactions were found to affect the bulk properties of hybrid hydrogels. At pH 4 electrostatic interactions between the peptide fibres and the peptide-coated GO flakes are thought to govern the final bulk properties of the hydrogels while at pH 7, after conditioning with cell culture media, electrostatic interactions are removed leaving the hydrophobic interactions to govern hydrogel final properties. The GO-F820 hybrid hydrogel, with mechanical properties similar to the NP, was shown to promote high cell viability and retained cell metabolic activity in 3D over the 7 days of culture and therefore shown to harbour significant potential as an injectable hydrogel scaffold for the in-vivo delivery of NP cells. STATEMENT OF SIGNIFICANCE: Short self-assembling peptide hydrogels (SAPHs) have attracted significant interest in recent years as they can mimic the natural extra-cellular matrix, holding significant promise for the ab initio design of cells' microenvironments. Recently the design of hybrid hydrogels for biomedical applications has been explored through the incorporation of specific nanofillers. In this study we exploited graphene oxide (GO) as nanofiller to design hybrid injectable 3Dscaffolds for the delivery of nucleus pulposus cells (NPCs) for intervertebral disc regeneration. Our work clearly shows the presence of strong interactions between peptide and GO, mimicking the mechanical properties of the NP tissue and promoting high cell viability and metabolic activity. These hybrid hydrogels therefore harbour significant potential as injectable scaffolds for the in vivo delivery of NPCs., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)