Solving the conundrum of intra-specific variation in metabolic rate: A multidisciplinary conceptual and methodological toolkit: New technical developments are opening the door to an understanding of why metabolic rate varies among individual animals of a species: New technical developments are opening the door to an understanding of why metabolic rate varies among individual animals of a species.

Researchers from diverse disciplines, including organismal and cellular physiology, sports science, human nutrition, evolution and ecology, have sought to understand the causes and consequences of the surprising variation in metabolic rate found among and within individual animals of the same species. Research in this area has been hampered by differences in approach, terminology and methodology, and the context in which measurements are made. Recent advances provide important opportunities to identify and address the key questions in the field. By bringing together researchers from different areas of biology and biomedicine, we describe and evaluate these developments and the insights they could yield, highlighting the need for more standardisation across disciplines. We conclude with a list of important questions that can now be addressed by developing a common conceptual and methodological toolkit for studies on metabolic variation in animals.

Acyl Capping Group Identity Effects on α-Helicity: On the Importance of Amide·Water Hydrogen Bonds to α-Helix Stability.

Acyl capping groups stabilize α-helices relative to free N-termini by providing one additional C═Oi···Hi+4-N hydrogen bond. The electronic properties of acyl capping groups might also directly modulate α-helix stability: electron-rich N-terminal acyl groups could stabilize the α-helix by strengthening both i/i + 4 hydrogen bonds and i/i + 1 n → π* interactions. This hypothesis was tested in peptides X-AKAAAAKAAAAKAAGY-NH2, where X = different acyl groups. Surprisingly, the most electron-rich acyl groups (pivaloyl and iso-butyryl) strongly destabilized the α-helix. Moreover, the formyl group induced nearly identical α-helicity to that of the acetyl group, despite being a weaker electron donor for hydrogen bonds and for n → π* interactions. Other acyl groups exhibited intermediate α-helicity. These results indicate that the electronic properties of the acyl carbonyl do not directly determine the α-helicity in peptides in water. In order to understand these effects, DFT calculations were conducted on α-helical peptides. Using implicit solvation, α-helix stability correlated with acyl group electronics, with the pivaloyl group exhibiting closer hydrogen bonds and n → π* interactions, in contrast to the experimental results. However, DFT and MD calculations with explicit water solvation revealed that hydrogen bonding to water was impacted by the sterics of the acyl capping group. Formyl capping groups exhibited the closest water-amide hydrogen bonds, while pivaloyl groups exhibited the longest. In α-helices in the PDB, the highest frequency of close amide-water hydrogen bonds is observed when the N-cap residue is Gly. The combination of experimental and computational results indicates that solvation (hydrogen bonding of water) to the N-terminal amide groups is a central determinant of α-helix stability.

4,4-Difluoroproline as a Unique 19F NMR Probe of Proline Conformation.

Despite the importance of proline conformational equilibria (trans versus cis amide and exo versus endo ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive 19F NMR-based probe of proline conformational biases and cis-trans isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (ΔδFF = 0-3 ppm) when a trans X-Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (ΔδFF = 5-12 ppm) difference in chemical shift in a cis X-Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that ΔδFF directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudo-axial (i.e., the pro-4R-F in an exo ring pucker, or the pro-4S-F in an endo ring pucker) is downfield, while a fluorine which is pseudo-equatorial (i.e., pro-4S-F when exo, or pro-4R-F when endo) is upfield. Thus, when a proline is disordered (a mixture of exo and endo ring puckers, as at trans-Pro in peptides in water), it exhibits a small Δδ. In contrast, when the Pro is ordered (i.e., when one ring pucker is strongly preferred, as in cis-Pro amide bonds, where the endo ring pucker is strongly favored), a large Δδ is observed. Dfp can be used to identify inherent induced order in peptides and to quantify proline cis-trans isomerism. Using Dfp, we discovered that the stable polyproline II helix (PPII) formed in the denatured state (8 M urea) exhibits essentially equal populations of the exo and endo proline ring puckers. In addition, the data with Dfp suggested the specific stabilization of PPII by water over other polar solvents. These data strongly support the importance of carbonyl solvation and n → π* interactions for the stabilization of PPII. Dfp was also employed to quantify proline cis-trans isomerism as a function of phosphorylation and the R406W mutation in peptides derived from the intrinsically disordered protein tau. Dfp is minimally sterically disruptive and can be incorporated in expressed proteins, suggesting its broad application in understanding proline cis-trans isomerization, protein folding, and local order in intrinsically disordered proteins.

Helical twists and ß-turns in structures at serine-proline sequences: Stabilization of cis-proline and type VI ß-turns via C-H/O interactions.

Structures at serine-proline sites in proteins were analyzed using a combination of peptide synthesis with structural methods and bioinformatics analysis of the PDB. Dipeptides were synthesized with the proline derivative (2S,4S)-(4-iodophenyl)hydroxyproline [hyp(4-I-Ph)]. The crystal structure of Boc-Ser-hyp(4-I-Ph)-OMe had two molecules in the unit cell. One molecule exhibited cis-proline and a type VIa2 ß-turn (BcisD). The cis-proline conformation was stabilized by a C-H/O interaction between Pro C-Hα and the Ser side-chain oxygen. NMR data were consistent with stabilization of cis-proline by a C-H/O interaction in solution. The other crystallographically observed molecule had trans-Pro and both residues in the PPII conformation. Two conformations were observed in the crystal structure of Ac-Ser-hyp(4-I-Ph)-OMe, with Ser adopting PPII in one and the ß conformation in the other, each with Pro in the δ conformation and trans-Pro. Structures at Ser-Pro sequences were further examined via bioinformatics analysis of the PDB and via DFT calculations. Ser-Pro versus Ala-Pro sequences were compared to identify bases for Ser stabilization of local structures. C-H/O interactions between the Ser side-chain Oγ and Pro C-Hα were observed in 45% of structures with Ser-cis-Pro in the PDB, with nearly all Ser-cis-Pro structures adopting a type VI ß-turn. 53% of Ser-trans-Pro sequences exhibited main-chain COi•••HNi+3 or COi•••HNi+4 hydrogen bonds, with Ser as the i residue and Pro as the i + 1 residue. These structures were overwhelmingly either type I ß-turns or N-terminal capping motifs on α-helices or 310-helices. These results indicate that Ser-Pro sequences are particularly potent in favoring these structures. In each, Ser is in either the PPII or ß conformation, with the Ser Oγ capable of engaging in a hydrogen bond with the amide N-H of the i + 2 (type I ß-turn or 310-helix; Ser χ1 t) or i + 3 (α-helix; Ser χ1 g+) residue. Non-proline cis amide bonds can also be stabilized by C-H/O interactions.

Mechanistic Basis of the Cu(OAc)2 Catalyzed Azide-Ynamine (3 + 2) Cycloaddition Reaction.

The Cu-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is used as a ligation tool throughout chemical and biological sciences. Despite the pervasiveness of CuAAC, there is a need to develop more efficient methods to form 1,4-triazole ligated products with low loadings of Cu. In this paper, we disclose a mechanistic model for the ynamine-azide (3 + 2) cycloadditions catalyzed by copper(II) acetate. Using multinuclear nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy, and high-performance liquid chromatography analyses, a dual catalytic cycle is identified. First, the formation of a diyne species via Glaser-Hay coupling of a terminal ynamine forms a Cu(I) species competent to catalyze an ynamine-azide (3 + 2) cycloaddition. Second, the benzimidazole unit of the ynamine structure has multiple roles: assisting C-H activation, Cu coordination, and the formation of a postreaction resting state Cu complex after completion of the (3 + 2) cycloaddition. Finally, reactivation of the Cu resting state complex is shown by the addition of isotopically labeled ynamine and azide substrates to form a labeled 1,4-triazole product. This work provides a mechanistic basis for the use of mixed valency binuclear catalytic Cu species in conjunction with Cu-coordinating alkynes to afford superior reactivity in CuAAC reactions. Additionally, these data show how the CuAAC reaction kinetics can be modulated by changes to the alkyne substrate, which then has a predictable effect on the reaction mechanism.

A randomized phase III double-blind placebo-controlled trial of first-line chemotherapy and trastuzumab with or without bevacizumab for patients with HER2/neu-positive metastatic breast cancer: a trial of the ECOG-ACRIN Cancer Research Group (E1105).

BACKGROUND: In 2008, bevacizumab received accelerated Food and Drug Administration (FDA) approval for use in human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (MBC). Based on the pre-clinical and preliminary clinical activity of the trastuzumab and bevacizumab combination, ECOG-ACRIN E1105 trial was developed to determine if the addition of bevacizumab to a chemotherapy and trastuzumab combination for first-line therapy would improve progression-free survival (PFS) in patients with HER2-positive MBC. FINDINGS: 96 patients were randomized to receive standard first-line chemotherapy and trastuzumab with or without bevacizumab between November 2007 and October 2009, and 93 began protocol therapy. Induction therapy was given for 24 weeks, followed by maintenance trastuzumab with or without bevacizumab. 60% (56/93) began carboplatin and 74% (69/93) completed 6 cycles of induction therapy. Primary endpoint was PFS. Median PFS was 11.1 and 13.8 months for placebo and bevacizumab arms, respectively (hazard ratio [HR] 95%, Confidence Interval [Cl] for bevacizumab vs. placebo: 0.73 [0.43-1.23], p = 0.24), and at a median follow-up of 70.7 months, median survival was 49.1 and 63 months (HR [95% Cl] for OS: 1.09 [0.61-1.97], p = 0.75). The most common toxicities across both arms were neutropenia and hypertension, with left ventricular systolic dysfunction, fatigue, and sensory neuropathy reported more frequently with bevacizumab. CONCLUSIONS: In this trial, the addition of bevacizumab did not improve outcomes in patients with metastatic HER2-positive breast cancer. Although the trial was underpowered due to smaller than anticipated sample size, these findings corroborated other clinical trials during this time. CLINICAL TRIAL INFORMATION: NCT00520975.

Electronic Control of Polyproline II Helix Stability via the Identity of Acyl Capping Groups: the Pivaloyl Group Particularly Promotes PPII.

The type II polyproline helix (PPII) is a fundamental secondary structure of proteins, important in globular proteins, in intrinsically disordered proteins, and at protein-protein interfaces. PPII is stabilized in part by n→π* interactions between consecutive carbonyls, via electron delocalization between an electron-donor carbonyl lone pair (n) and an electron-acceptor carbonyl (π*) on the subsequent residue. We previously demonstrated that changes to the electronic properties of the acyl donor can predictably modulate the strength of n→π* interactions, with data from model compounds, in solution in chloroform, in the solid state, and computationally. Herein, we examined whether the electronic properties of acyl capping groups could modulate the stability of PPII in peptides in water. In X-PPGY-NH2 peptides (X=10 acyl capping groups), the effect of acyl group identity on PPII was quantified by circular dichroism and NMR spectroscopy. Electron-rich acyl groups promoted PPII relative to the standard acetyl (Ac-) group, with the pivaloyl and iso-butyryl groups most significantly increasing PPII. In contrast, acyl derivatives with electron-withdrawing substituents and the formyl group relatively disfavored PPII. Similar results, though lesser in magnitude, were also observed in X-APPGY-NH2 peptides, indicating that the capping group can impact PPII conformation at both proline and non-proline residues. The pivaloyl group was particularly favorable in promoting PPII. The effects of acyl capping groups were further analyzed in X-DfpPGY-NH2 and X-ADfpPGY-NH2 peptides, Dfp=4,4-difluoroproline. Data on these peptides indicated that acyl groups induced order Piv- > Ac- > For-. These results suggest that greater consideration should be given to the identity of acyl capping groups in inducing structure in peptides.

Consistent changes in muscle phenotype and mitochondrial abundance underlie dive performance across multiple lineages of diving ducks.

Diving animals must sustain high muscle activity with finite oxygen (O2) to forage underwater. Studies have shown that some diving mammals exhibit changes in the metabolic phenotype of locomotory muscles compared to non-divers, but the pervasiveness of such changes across diving animals is unclear, particularly among diving birds. Here, we examine whether changes in muscle phenotype and mitochondrial abundance are associated with dive capacity across 17 species of ducks from three distinct evolutionary clades (tribes) in the subfamily Anatinae - the longest diving sea ducks, the mid-tier diving pochards, and the non-diving dabblers. In the gastrocnemius (the primary swimming and diving muscle), mitochondrial volume density in both oxidative and glycolytic fiber types were 70% and 30% higher in sea ducks compared to dabblers, respectively. These differences were associated with preferential proliferation of the subsarcolemmal subfraction, the mitochondria adjacent to the cell membrane and nearest to capillaries, relative to the intermyofibrillar subfraction. Capillary density and capillary-to-fiber ratio were positively correlated with mitochondrial volume density, with no variation in the density of oxidative fiber types across tribes. In the pectoralis, sea ducks had greater abundance of oxidative fiber types than dabblers, whereas pochards were intermediate between the two. These data suggest that skeletal muscles of sea ducks have a heightened capacity for aerobic metabolism and an enhanced ability to utilize O2 stores in the blood and muscle while diving.

Identification and transcriptomic assessment of latent profile pediatric septic shock phenotypes.

BACKGROUND: Sepsis poses a grave threat, especially among children, but treatments are limited owing to heterogeneity among patients. We sought to test the clinical and biological relevance of pediatric septic shock subclasses identified using reproducible approaches. METHODS: We performed latent profile analyses using clinical, laboratory, and biomarker data from a prospective multi-center pediatric septic shock observational cohort to derive phenotypes and trained a support vector machine model to assign phenotypes in an internal validation set. We established the clinical relevance of phenotypes and tested for their interaction with common sepsis treatments on patient outcomes. We conducted transcriptomic analyses to delineate phenotype-specific biology and inferred underlying cell subpopulations. Finally, we compared whether latent profile phenotypes overlapped with established gene-expression endotypes and compared survival among patients based on an integrated subclassification scheme. RESULTS: Among 1071 pediatric septic shock patients requiring vasoactive support on day 1 included, we identified two phenotypes which we designated as Phenotype 1 (19.5%) and Phenotype 2 (80.5%). Membership in Phenotype 1 was associated with ~ fourfold adjusted odds of complicated course relative to Phenotype 2. Patients belonging to Phenotype 1 were characterized by relatively higher Angiopoietin-2/Tie-2 ratio, Angiopoietin-2, soluble thrombomodulin (sTM), interleukin 8 (IL-8), and intercellular adhesion molecule 1 (ICAM-1) and lower Tie-2 and Angiopoietin-1 concentrations compared to Phenotype 2. We did not identify significant interactions between phenotypes, common treatments, and clinical outcomes. Transcriptomic analysis revealed overexpression of genes implicated in the innate immune response and driven primarily by developing neutrophils among patients designated as Phenotype 1. There was no statistically significant overlap between established gene-expression endotypes, reflective of the host adaptive response, and the newly derived phenotypes, reflective of the host innate response including microvascular endothelial dysfunction. However, an integrated subclassification scheme demonstrated varying survival probabilities when comparing patient endophenotypes. CONCLUSIONS: Our research underscores the reproducibility of latent profile analyses to identify pediatric septic shock phenotypes with high prognostic relevance. Pending validation, an integrated subclassification scheme, reflective of the different facets of the host response, holds promise to inform targeted intervention among those critically ill.

Immunocompromised-Associated Pediatric Acute Respiratory Distress Syndrome: Experience From the 2016/2017 Pediatric Acute Respiratory Distress Syndrome Incidence and Epidemiology Prospective Cohort Study.

OBJECTIVES: To characterize immunocompromised-associated pediatric acute respiratory distress syndrome (I-PARDS) and contrast it to PARDS. DESIGN: This is a secondary analysis of the 2016-2017 PARDS incidence and epidemiology (PARDIE) study, a prospective observational, cross-sectional study of children with PARDS. SETTING: Dataset of 145 PICUs across 27 countries. PATIENTS: During 10 nonconsecutive weeks (from May 2016 to June 2017), data about immunocompromising conditions (ICCs, defined as malignancy, congenital/acquired immunodeficiency, posttransplantation, or diseases requiring immunosuppression) were collected. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Of 708 subjects, 105 (14.8%) had ICC. Before the development of I-PARDS, those with ICC were more likely to be hospitalized (70% vs. 35%, p < 0.001), have more at-risk for PARDS ( p = 0.046), and spent more hours at-risk (20 [interquartile range, IQR: 8-46] vs. 11 [IQR: 4-33], [ p = 0.002]). Noninvasive ventilation (NIV) use was more common in those with ICC ( p < 0.001). Of those diagnosed with PARDS on NIV ( n = 161), children with ICC were more likely to be subsequently intubated ( n = 28/40 [70%] vs n = 53/121 [44%], p = 0.004). Severe PARDS was more common (32% vs 23%, p < 0.001) in I-PARDS. Oxygenation indices were higher at diagnosis and had less improvement over the first 3 days of PARDS ( p < 0.001). Children with I-PARDS had greater nonpulmonary organ dysfunction. Adjusting for Pediatric Risk of Mortality IV and oxygenation index, children with I-PARDS had a higher severity of illness-adjusted PICU mortality (adjusted hazard ratio: 3.0 [95% CI, 1.9-4.7] p < 0.001) and were less likely to be extubated alive within 28 days (subdistribution hazard ratio: 0.47 [95% CI, 0.31-0.71] p < 0.001). CONCLUSIONS: I-PARDS is a unique subtype of PARDS associated with hospitalization before diagnosis and increased: time at-risk for PARDS, NIV use, hypoxia, nonpulmonary organ dysfunction, and mortality. The opportunity for early detection and intervention seems to exist. Dedicated study in these patients is imperative to determine if targeted interventions will benefit these unique patients with the ultimate goal of improving outcomes.

Biomarker Assessment of a High-Risk, Data-Driven Pediatric Sepsis Phenotype Characterized by Persistent Hypoxemia, Encephalopathy, and Shock.

OBJECTIVES: Identification of children with sepsis-associated multiple organ dysfunction syndrome (MODS) at risk for poor outcomes remains a challenge. We sought to the determine reproducibility of the data-driven "persistent hypoxemia, encephalopathy, and shock" (PHES) phenotype and determine its association with inflammatory and endothelial biomarkers, as well as biomarker-based pediatric risk strata. DESIGN: We retrained and validated a random forest classifier using organ dysfunction subscores in the 2012-2018 electronic health record (EHR) dataset used to derive the PHES phenotype. We used this classifier to assign phenotype membership in a test set consisting of prospectively (2003-2023) enrolled pediatric septic shock patients. We compared profiles of the PERSEVERE family of biomarkers among those with and without the PHES phenotype and determined the association with established biomarker-based mortality and MODS risk strata. SETTING: Twenty-five PICUs across the United States. PATIENTS: EHR data from 15,246 critically ill patients with sepsis-associated MODS split into derivation and validation sets and 1,270 pediatric septic shock patients in the test set of whom 615 had complete biomarker data. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The area under the receiver operator characteristic curve of the modified classifier to predict PHES phenotype membership was 0.91 (95% CI, 0.90-0.92) in the EHR validation set. In the test set, PHES phenotype membership was associated with both increased adjusted odds of complicated course (adjusted odds ratio [aOR] 4.1; 95% CI, 3.2-5.4) and 28-day mortality (aOR of 4.8; 95% CI, 3.11-7.25) after controlling for age, severity of illness, and immunocompromised status. Patients belonging to the PHES phenotype were characterized by greater degree of systemic inflammation and endothelial activation, and were more likely to be stratified as high risk based on PERSEVERE biomarkers predictive of death and persistent MODS. CONCLUSIONS: The PHES trajectory-based phenotype is reproducible, independently associated with poor clinical outcomes, and overlapped with higher risk strata based on prospectively validated biomarker approaches.

Life-Threatening Complications of Influenza vs Coronavirus Disease 2019 (COVID-19) in US Children.

BACKGROUND: Clinical differences between critical illness from influenza infection vs coronavirus disease 2019 (COVID-19) have not been well characterized in pediatric patients. METHODS: We compared demographics, clinical characteristics, and outcomes of US children (aged 8 months to 17 years) admitted to the intensive care or high-acuity unit with influenza or COVID-19. Using mixed-effects models, we assessed the odds of death or requiring life support for influenza vs COVID-19 after adjustment for age, sex, race and Hispanic origin, and underlying conditions including obesity. RESULTS: Children with influenza (n = 179) were younger than those with COVID-19 (n = 381; median, 5.2 years vs 13.8 years), less likely to be non-Hispanic Black (14.5% vs 27.6%) or Hispanic (24.0% vs 36.2%), and less likely to have ≥1 underlying condition (66.4% vs 78.5%) or be obese (21.4% vs 42.2%), and a shorter hospital stay (median, 5 days vs 7 days). They were similarly likely to require invasive mechanical ventilation (both 30.2%), vasopressor support (19.6% and 19.9%), or extracorporeal membrane oxygenation (2.2% and 2.9%). Four children with influenza (2.2%) and 11 children with COVID-19 (2.9%) died. The odds of death or requiring life support in children with influenza vs COVID-19 were similar (adjusted odds ratio, 1.30; 95% confidence interval, .78-2.15; P = .32). CONCLUSIONS: Despite differences in demographics and clinical characteristics of children with influenza or COVID-19, the frequency of life-threatening complications was similar. Our findings highlight the importance of implementing prevention measures to reduce transmission and disease severity of influenza and COVID-19.

Patterns of antibiotic use, pathogens, and prediction of mortality in hospitalized neonates and young infants with sepsis: A global neonatal sepsis observational cohort study (NeoOBS).

BACKGROUND: There is limited data on antibiotic treatment in hospitalized neonates in low- and middle-income countries (LMICs). We aimed to describe patterns of antibiotic use, pathogens, and clinical outcomes, and to develop a severity score predicting mortality in neonatal sepsis to inform future clinical trial design. METHODS AND FINDINGS: Hospitalized infants <60 days with clinical sepsis were enrolled during 2018 to 2020 by 19 sites in 11 countries (mainly Asia and Africa). Prospective daily observational data was collected on clinical signs, supportive care, antibiotic treatment, microbiology, and 28-day mortality. Two prediction models were developed for (1) 28-day mortality from baseline variables (baseline NeoSep Severity Score); and (2) daily risk of death on IV antibiotics from daily updated assessments (NeoSep Recovery Score). Multivariable Cox regression models included a randomly selected 85% of infants, with 15% for validation. A total of 3,204 infants were enrolled, with median birth weight of 2,500 g (IQR 1,400 to 3,000) and postnatal age of 5 days (IQR 1 to 15). 206 different empiric antibiotic combinations were started in 3,141 infants, which were structured into 5 groups based on the World Health Organization (WHO) AWaRe classification. Approximately 25.9% (n = 814) of infants started WHO first line regimens (Group 1-Access) and 13.8% (n = 432) started WHO second-line cephalosporins (cefotaxime/ceftriaxone) (Group 2-"Low" Watch). The largest group (34.0%, n = 1,068) started a regimen providing partial extended-spectrum beta-lactamase (ESBL)/pseudomonal coverage (piperacillin-tazobactam, ceftazidime, or fluoroquinolone-based) (Group 3-"Medium" Watch), 18.0% (n = 566) started a carbapenem (Group 4-"High" Watch), and 1.8% (n = 57) a Reserve antibiotic (Group 5, largely colistin-based), and 728/2,880 (25.3%) of initial regimens in Groups 1 to 4 were escalated, mainly to carbapenems, usually for clinical deterioration (n = 480; 65.9%). A total of 564/3,195 infants (17.7%) were blood culture pathogen positive, of whom 62.9% (n = 355) had a gram-negative organism, predominantly Klebsiella pneumoniae (n = 132) or Acinetobacter spp. (n = 72). Both were commonly resistant to WHO-recommended regimens and to carbapenems in 43 (32.6%) and 50 (71.4%) of cases, respectively. MRSA accounted for 33 (61.1%) of 54 Staphylococcus aureus isolates. Overall, 350/3,204 infants died (11.3%; 95% CI 10.2% to 12.5%), 17.7% if blood cultures were positive for pathogens (95% CI 14.7% to 21.1%, n = 99/564). A baseline NeoSep Severity Score had a C-index of 0.76 (0.69 to 0.82) in the validation sample, with mortality of 1.6% (3/189; 95% CI: 0.5% to 4.6%), 11.0% (27/245; 7.7% to 15.6%), and 27.3% (12/44; 16.3% to 41.8%) in low (score 0 to 4), medium (5 to 8), and high (9 to 16) risk groups, respectively, with similar performance across subgroups. A related NeoSep Recovery Score had an area under the receiver operating curve for predicting death the next day between 0.8 and 0.9 over the first week. There was significant variation in outcomes between sites and external validation would strengthen score applicability. CONCLUSION: Antibiotic regimens used in neonatal sepsis commonly diverge from WHO guidelines, and trials of novel empiric regimens are urgently needed in the context of increasing antimicrobial resistance (AMR). The baseline NeoSep Severity Score identifies high mortality risk criteria for trial entry, while the NeoSep Recovery Score can help guide decisions on regimen change. NeoOBS data informed the NeoSep1 antibiotic trial (ISRCTN48721236), which aims to identify novel first- and second-line empiric antibiotic regimens for neonatal sepsis. TRIAL REGISTRATION: ClinicalTrials.gov, (NCT03721302).

Consistent changes in muscle metabolism underlie dive performance across multiple lineages of diving ducks.

Diving animals must sustain high activity with limited O2 stores to successfully capture prey. Studies suggest that increasing body O2 stores supports breath-hold diving, but less is known about metabolic specializations that underlie underwater locomotion. We measured maximal activities of 10 key enzymes in locomotory muscles (gastrocnemius and pectoralis) to identify biochemical changes associated with diving in pathways of oxidative and substrate-level phosphorylation and compared them across three groups of ducks-the longest diving sea ducks (eight spp.), the mid-tier diving pochards (three spp.) and the non-diving dabblers (five spp.). Relative to dabblers, both diving groups had increased activities of succinate dehydrogenase and cytochrome c oxidase, and sea ducks further showed increases in citrate synthase (CS) and hydroxyacyl-CoA dehydrogenase (HOAD). Both diving groups had relative decreases in capacity for anaerobic metabolism (lower ratio of lactate dehydrogenase to CS), with sea ducks also showing a greater capacity for oxidative phosphorylation and lipid oxidation (lower ratio of pyruvate kinase to CS, higher ratio of HOAD to hexokinase). These data suggest that the locomotory muscles of diving ducks are specialized for sustaining high rates of aerobic metabolism, emphasizing the importance of body O2 stores for dive performance in these species.

Macular Pigment Optical Density and Skin Carotenoids in a Childhood Sample.

BACKGROUND: Carotenoids are plant pigments with light filtering and antioxidant properties that deposit in human tissues, including retina and skin. Descriptive characteristics and covariates of carotenoid status in macula and skin have been examined in adults; however, similar studies in children are limited. Thus, this study aimed to delineate how factors of age, sex, race, weight status, and dietary carotenoid intake relate to macular and skin carotenoids in children. METHODS: Children (7-13 y, N = 375) completed heterochromatic flicker photometry to assess macular pigment optical density (MPOD). Participants underwent anthropometrics to measure weight status (BMI percentile [BMI%]), and parent/guardian provided demographic information. Subsample data were available for skin carotenoids (N = 181), assessed using reflection spectroscopy, and dietary carotenoids (N = 101) using the Block Food Frequency Questionnaire. Relationships between skin and macular carotenoids were assessed using partial Pearson's correlations controlling for age, sex, race, and BMI%. Relationships between dietary carotenoids and macular and skin carotenoids were assessed using stepwise linear regression including age, sex, race, and BMI% in the model. RESULTS: Mean MPOD was 0.56 ± 0.22 and skin carotenoid score was 282 ± 94.6. There was no significant correlation between MPOD and skin carotenoids (r = 0.02, P = 0.76). BMI% was negatively associated with skin (stdß = -0.42, P < 0.001), but not macular carotenoids (stdß = -0.04, P = 0.70). Neither MPOD nor skin carotenoids were associated with age, sex, or race (all P > 0.10). MPOD was positively associated with energy-adjusted reported lutein + zeaxanthin intake (stdß = 0.27, P = 0.01). Skin carotenoids were positively associated with energy-adjusted reported carotenoid intake (stdß = 0.26, P = 0.01). CONCLUSIONS: The mean MPOD values in children were higher than what has been reported in adult populations. Previous studies in adult samples report an average MPOD of 0.21. Although macular and skin carotenoids were not related, they were associated with dietary carotenoids relevant to the respective tissues; however, skin carotenoids may be more susceptible negative influence from higher weight status.

Adaptive increases in respiratory capacity and O2 affinity of subsarcolemmal mitochondria from skeletal muscle of high-altitude deer mice.

Aerobic energy demands have led to the evolution of complex mitochondrial reticula in highly oxidative muscles, but the extent to which metabolic challenges can be met with adaptive changes in physiology of specific mitochondrial fractions remains unresolved. We examined mitochondrial mechanisms supporting adaptive increases in aerobic performance in deer mice (Peromyscus maniculatus) adapted to the hypoxic environment at high altitude. High-altitude and low-altitude mice were born and raised in captivity, and exposed as adults to normoxia or hypobaric hypoxia (12 kPa O2 for 6-8 weeks). Subsarcolemmal and intermyofibrillar mitochondria were isolated from the gastrocnemius, and a comprehensive substrate titration protocol was used to examine mitochondrial physiology and O2  kinetics by high-resolution respirometry and fluorometry. High-altitude mice had greater yield, respiratory capacity for oxidative phosphorylation, and O2 affinity (lower P50 ) of subsarcolemmal mitochondria compared to low-altitude mice across environments, but there were no species difference in these traits in intermyofibrillar mitochondria. High-altitude mice also had greater capacities of complex II relative to complexes I + II and higher succinate dehydrogenase activities in both mitochondrial fractions. Exposure to chronic hypoxia reduced reactive oxygen species (ROS) emission in high-altitude mice but not in low-altitude mice. Our findings suggest that functional changes in subsarcolemmal mitochondria contribute to improving aerobic performance in hypoxia in high-altitude deer mice. Therefore, physiological variation in specific mitochondrial fractions can help overcome the metabolic challenges of life at high altitude.

Inter-individual variation in mitochondrial phosphorylation efficiency predicts growth rates in ectotherms at high temperatures.

There is increasing evidence that aquatic ectotherms are especially vulnerable to global warming since their metabolic demands increase with ambient temperature while water-oxygen content decreases. The possible role of shrinking aerobic scope in limiting performance has been much discussed; however, less attention has been given to whether tissue-level changes in the efficiency of oxygen usage occur at elevated temperatures. Here, we show that this varies widely among individuals, with consequences for performance. We examined the inter-individual variation in growth rate and mitochondrial function from white muscle and liver of brown trout (Salmo trutta) acclimated to either high (19.5°C) or near-optimal temperature (12°C). Liver (but not muscle) mitochondria showed a positive relationship between growth rate and maximal oxidative phosphorylation at both temperatures, and a negative relationship between growth rate and ROS release. There was a positive correlation in both tissues between individual mitochondrial phosphorylation efficiency and growth rate, but only at 19.5°C. In this representative of aquatic ectotherms, an individual's liver mitochondrial efficiency thus seems to dictate its capacity to grow at elevated temperatures. This suggests that individual heterogeneity in cellular function may cause variation in the thermal limits of aquatic ectotherms and could adversely affect wild populations in warming environments.

Synthesis and conformational preferences of peptides and proteins with cysteine sulfonic acid.

Cysteine sulfonic acid (Cys-SO3H; cysteic acid) is an oxidative post-translational modification of cysteine, resulting from further oxidation from cysteine sulfinic acid (Cys-SO2H). Cysteine sulfonic acid is considered an irreversible post-translational modification, which serves as a biomarker of oxidative stress that has resulted in oxidative damage to proteins. Cysteine sulfonic acid is anionic, as a sulfonate (Cys-SO3-; cysteate), in the ionization state that is almost exclusively present at physiological pH (pKa ∼ -2). In order to understand protein structural changes that can occur upon oxidation to cysteine sulfonic acid, we analyzed its conformational preferences, using experimental methods, bioinformatics, and DFT-based computational analysis. Cysteine sulfonic acid was incorporated into model peptides for α-helix and polyproline II helix (PPII). Within peptides, oxidation of cysteine to the sulfonic acid proceeds rapidly and efficiently at room temperature in solution with methyltrioxorhenium (MeReO3) and H2O2. Peptides containing cysteine sulfonic acid were also generated on solid phase using trityl-protected cysteine and oxidation with MeReO3 and H2O2. Using methoxybenzyl (Mob)-protected cysteine, solid-phase oxidation with MeReO3 and H2O2 generated the Mob sulfone precursor to Cys-SO2- within fully synthesized peptides. These two solid-phase methods allow the synthesis of peptides containing either Cys-SO3- or Cys-SO2- in a practical manner, with no solution-phase synthesis required. Cys-SO3- had low PPII propensity for PPII propagation, despite promoting a relatively compact conformation in ϕ. In contrast, in a PPII initiation model system, Cys-SO3- promoted PPII relative to neutral Cys, with PPII initiation similar to Cys thiolate but less than Cys-SO2- or Ala. In an α-helix model system, Cys-SO3- promoted α-helix near the N-terminus, due to favorable helix dipole interactions and favorable α-helix capping via a sulfonate-amide side chain-main chain hydrogen bond. Across all peptides, the sulfonate side chain was significantly less ordered than that of the sulfinate. Analysis of Cys-SO3- in the PDB revealed a very strong propensity for local (i/i or i/i + 1) side chain-main chain sulfonate-amide hydrogen bonds for Cys-SO3-, with >80% of Cys-SO3- residues exhibiting these interactions. DFT calculations conducted to explore these conformational preferences indicated that side chain-main chain hydrogen bonds of the sulfonate with the intraresidue amide and/or with the i + 1 amide were favorable. However, hydrogen bonds to water or to amides, as well as interactions with oxophilic metals, were weaker for the sulfonate than the sulfinate, due to lower charge density on the oxygens in the sulfonate.

Interventions, Barriers, and Proposed Solutions Associated With the Implementation of a Protocol That Uses Clinical Decision Support and a Stress Biomarker Test to Identify ICU Patients at High-Risk for Drug Associated Acute Kidney Injury.

BACKGROUND: Damage biomarkers are helpful in early identification of patients who are at risk of developing acute kidney injury (AKI). Investigations are ongoing to identify the optimal role of stress/damage biomarkers in clinical practice regarding AKI risk prediction, surveillance, diagnosis, and prognosis. OBJECTIVE: To determine the impact of utilizing a clinical decision support system (CDSS) to guide stress biomarker testing in intensive care unit (ICU) patients at risk for drug-induced acute kidney injury (D-AKI). METHODS: A protocol was designed utilizing a clinical decision support system (CDSS) alert to identify patients that were ordered 3 or more potentially nephrotoxic medications, suggesting risk for progressing to AKI from nephrotoxic burden. Once alerted to these high-risk patients, the pharmacist determined if action was needed by ordering a stress biomarker test, tissue inhibitor of metalloproteinase-2-insulin-like growth factor-binding protein 7 (TIMP-2•IGFBP7). If the biomarker test result was elevated, the pharmacist provided nephrotoxin stewardship recommendations to the team. Pharmacists recorded the response to the clinical decision support alert, ordering, and interpreting the TIMP-2•IGFBP7, and information regarding clinical interventions. An alert in conjunction with TIMP-2•IGFBP7 as a strategy for AKI risk prediction and stimulant for patient care management was assessed. In addition, barriers and solutions to protocol implementation were evaluated. RESULTS: There were 394 total activities recorded by pharmacists for 345 unique patients. Ninety-three (93/394; 23.6%) actionable alerts resulted in a TIMP-2•IGFBP7 test being ordered. Thirty-one TIMP-2•IGFBP7 results were >0.3 (31/81; 38.3%), suggesting a high-risk of progression to AKI, which prompted 191 pharmacist/team interventions. On average, there were 1.64 interventions per patient in the low-risk patients, 3.43 in high-risk patients, and 3.75 in the highest-risk patients. CONCLUSION AND RELEVANCE: Stress biomarkers can be used in conjunction with CDSS alerts to affect therapeutic decisions in ICU patients at high-risk for D-AKI.

Prognostic and predictive value of endothelial dysfunction biomarkers in sepsis-associated acute kidney injury: risk-stratified analysis from a prospective observational cohort of pediatric septic shock.

BACKGROUND: Sepsis-associated acute kidney injury (SA-AKI) is associated with high morbidity, with no current therapies available beyond continuous renal replacement therapy (CRRT). Systemic inflammation and endothelial dysfunction are key drivers of SA-AKI. We sought to measure differences between endothelial dysfunction markers among children with and without SA-AKI, test whether this association varied across inflammatory biomarker-based risk strata, and develop prediction models to identify those at highest risk of SA-AKI. METHODS: Secondary analyses of prospective observational cohort of pediatric septic shock. Primary outcome of interest was the presence of ≥ Stage II KDIGO SA-AKI on day 3 based on serum creatinine (D3 SA-AKI SCr). Biomarkers including those prospectively validated to predict pediatric sepsis mortality (PERSEVERE-II) were measured in Day 1 (D1) serum. Multivariable regression was used to test the independent association between endothelial markers and D3 SA-AKI SCr. We conducted risk-stratified analyses and developed prediction models using Classification and Regression Tree (CART), to estimate risk of D3 SA-AKI among prespecified subgroups based on PERSEVERE-II risk. RESULTS: A total of 414 patients were included in the derivation cohort. Patients with D3 SA-AKI SCr had worse clinical outcomes including 28-day mortality and need for CRRT. Serum soluble thrombomodulin (sTM), Angiopoietin-2 (Angpt-2), and Tie-2 were independently associated with D3 SA-AKI SCr. Further, Tie-2 and Angpt-2/Tie-2 ratios were influenced by the interaction between D3 SA-AKI SCr and risk strata. Logistic regression demonstrated models predictive of D3 SA-AKI risk performed optimally among patients with high- or intermediate-PERSEVERE-II risk strata. A 6 terminal node CART model restricted to this subgroup of patients had an area under the receiver operating characteristic curve (AUROC) 0.90 and 0.77 upon tenfold cross-validation in the derivation cohort to distinguish those with and without D3 SA-AKI SCr and high specificity. The newly derived model performed modestly in a unique set of patients (n = 224), 84 of whom were deemed high- or intermediate-PERSEVERE-II risk, to distinguish those patients with high versus low risk of D3 SA-AKI SCr. CONCLUSIONS: Endothelial dysfunction biomarkers are independently associated with risk of severe SA-AKI. Pending validation, incorporation of endothelial biomarkers may facilitate prognostic and predictive enrichment for selection of therapeutics in future clinical trials among critically ill children.