The chemical language of protein glycation.

Glycation is a non-enzymatic post-translational modification (PTM) that is correlated with many diseases, including diabetes, cancer and age-related disorders. Although recent work points to the importance of glycation as a functional PTM, it remains an open question whether glycation has a causal role in cellular signaling and/or disease development. In this Review, we contextualize glycation as a specific mechanism of carbon stress and consolidate what is known about advanced glycation end-product (AGE) structures and mechanisms. We highlight the current understanding of glycation as a PTM, focusing on mechanisms for installing, removing or recognizing AGEs. Finally, we discuss challenges that have hampered a more complete understanding of the biological consequences of glycation. The development of tools for predicting, modulating, mimicking or capturing glycation will be essential for interpreting a post-translational glycation network. Therefore, continued insights into the chemistry of glycation will be necessary to advance understanding of glycation biology.

Dual Noncanonical Amino Acid Incorporation Enabling Chemoselective Protein Modification at Two Distinct Sites in Yeast.

Incorporation of more than one noncanonical amino acid (ncAA) within a single protein endows the resulting construct with multiple useful features such as augmented molecular recognition or covalent cross-linking capabilities. Herein, for the first time, we demonstrate the incorporation of two chemically distinct ncAAs into proteins biosynthesized in Saccharomyces cerevisiae. To complement ncAA incorporation in response to the amber (TAG) stop codon in yeast, we evaluated opal (TGA) stop codon suppression using three distinct orthogonal translation systems. We observed selective TGA readthrough without detectable cross-reactivity from host translation components. Readthrough efficiency at TGA was modulated by factors including the local nucleotide environment, gene deletions related to the translation process, and the identity of the suppressor tRNA. These observations facilitated systematic investigation of dual ncAA incorporation in both intracellular and yeast-displayed protein constructs, where we observed efficiencies up to 6% of wild-type protein controls. The successful display of doubly substituted proteins enabled the exploration of two critical applications on the yeast surface─(A) antigen binding functionality and (B) chemoselective modification with two distinct chemical probes through sequential application of two bioorthogonal click chemistry reactions. Lastly, by utilizing a soluble form of a doubly substituted construct, we validated the dual incorporation system using mass spectrometry and demonstrated the feasibility of conducting selective labeling of the two ncAAs sequentially using a "single-pot" approach. Overall, our work facilitates the addition of a 22nd amino acid to the genetic code of yeast and expands the scope of applications of ncAAs for basic biological research and drug discovery.

Single-Dose Dexamethasone Is Not Inferior to 2 Doses in Mild to Moderate Pediatric Asthma Exacerbations in the Emergency Department.

OBJECTIVE: The purpose of this study was to compare the efficacy of a single dose of dexamethasone to 2 doses of dexamethasone in treating mild to moderate asthma exacerbations in pediatric patients. We anticipated that there would not be a difference in the rate of return visits to the emergency department (ED), urgent care, or primary care physician for continued asthma symptoms. METHODS: This was a prospective, randomized, single-center, unblinded, parallel-group randomized clinical trial of patients 2 to 20 years old presenting to a pediatric ED with mild to moderate asthma exacerbations. The patients were randomized to receive 1 or 2 doses of dexamethasone (0.6 mg/kg per dose, maximum of 16 mg). Telephone follow-up interviews were performed on the sixth day after ED visit. The primary outcome measures were return visits to either primary care physician or ED for continued asthma symptoms. Secondary outcomes were days of symptoms, missed school days, and adverse effects. RESULTS: Of the 318 children initially enrolled, 308 patients met the enrollment criteria. These patients were randomized into 2 groups. There were 116 patients in group 1 and 116 patients in group 2. There was no significant difference between groups regarding return visits (group 1, 12.1%; group 2, 10.3%; odds ratio [OR], 0.892 [95% confidence interval {CI}, 0.377-2.110]), days to symptom resolution (group 1, 2.4; group 2, 2.5; OR, 0.974 [95% 95% CI, 0.838-1.132]), missed school days (group 1, 47%; group 2, 51%; OR, 1.114 [95% CI, 0.613-2.023]), or vomiting (group 1, 8.6%; group 2, 3.4%; OR, 2.424 [95% CI, 0.637-9.228]). CONCLUSIONS: In this single-center, unblinded randomized trial of children and adolescents with mild to moderate acute exacerbations of asthma, there was no difference in the rate of return visits for continued or worsened symptoms between patients randomized to 1 or 2 doses of dexamethasone.

Convalescent plasma for pediatric patients with SARS-CoV-2-associated acute respiratory distress syndrome.

There are no proven safe and effective therapies for children who develop life-threatening complications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Convalescent plasma (CP) has demonstrated potential benefit in adults with SARS-CoV-2, but has theoretical risks.We present the first report of CP in children with life-threatening coronavirus disease 2019 (COVID-19), providing data on four pediatric patients with acute respiratory distress syndrome. We measured donor antibody levels and recipient antibody response prior to and following CP infusion. Infusion of CP was not associated with antibody-dependent enhancement (ADE) and did not suppress endogenous antibody response. We found CP was safe and possibly efficacious. Randomized pediatric trials are needed.

Differential Development of the Chordae Tendineae and Anterior Leaflet of the Bovine Mitral Valve.

There is increasing evidence that some adult mitral valve pathologies may have developmental origins involving errors in cell signaling and protein deposition during valvulogenesis. While early and late gestational stages are well-documented in zebrafish, chicks, and small mammalian models, longitudinal studies in large mammals with a similar gestational period to humans are lacking. Further, the mechanism of chordae tendineae formation and multiplication remains unclear. The current study presents a comprehensive examination of mitral anterior leaflet and chordae tendineae development in a bovine model (a large mammal with the same gestational period as humans). Remarkably distinct from small mammals, bovine development displayed early branched chordae, with increasing attachments only until birth, while the anterior leaflet grew both during gestation and postnatally. Chordae also exhibited accelerated collagen deposition, maturation, and crimp development during gestation. These findings suggest that the bovine anterior leaflet and chordae tendineae possess unique processes of development despite being a continuous collagenous structure and could provide greater insight into human valve development.

Sde Proteins Coordinate Ubiquitin Utilization and Phosphoribosylation to Establish and Maintain the Legionella Replication Vacuole.

The Legionella pneumophila Sde family of translocated proteins promotes host tubular endoplasmic reticulum (ER) rearrangements that are tightly linked to phosphoribosyl-ubiquitin (pR-Ub) modification of Reticulon 4 (Rtn4). Sde proteins have two additional activities of unclear relevance to the infection process: K63 linkage-specific deubiquitination and phosphoribosyl modification of polyubiquitin (pR-Ub). We show here that the deubiquitination activity (DUB) stimulates ER rearrangements while pR-Ub protects the replication vacuole from cytosolic surveillance by autophagy. Loss of DUB activity is tightly linked to lowered pR-Ub modification of Rtn4, consistent with the DUB activity fueling the production of pR-Ub-Rtn4. In parallel, phosphoribosyl modification of polyUb, in a region of the protein known as the isoleucine patch, prevents binding by the autophagy adapter p62. An inability of Sde mutants to modify polyUb results in immediate p62 association, a critical precursor to autophagic attack. The ability of Sde WT to block p62 association decays quickly after bacterial infection, as predicted by the presence of previously characterized L. pneumophila effectors that inactivate Sde and remove polyUb. In sum, these results show that the accessory Sde activities act to stimulate ER rearrangements and protect from host innate immune sensing in a temporal fashion.

Social opportunities and mate preference improve breeding success in Caribbean iguanas.

Conservation breeding of West Indian rock iguanas (Cyclura) has met with limited success historically. Many facilities witness high levels of aggression and mate incompatibility resulting in failed breeding introductions which often require animals to be separated. This may be due, in part, to lack of knowledge of how mate familiarity and preference affect reproductive outcomes in these species. We investigated whether social exposure during the pre-breeding season influenced copulation success, egg production, and breeding behaviors. Additionally, we examined whether mate preference, as determined by pre-mating dichotomous choice tests, increased these reproductive outcomes. Female rock iguanas that were socialized with males prior to breeding opportunities copulated with familiar males for longer periods of time than females that were not socialized. Socialization opportunities did not alter male reproductive success measurements or breeding behavior. Female rock iguanas introduced for mating to their preferred partners had a higher probability of successful copulations, higher average number of copulations, and less resting behavior during introductions than females mated to non-preferred males. Male mate preference had no effect on reproductive success measurements during mating introductions. These results indicate that socializing animals and providing mate choice opportunities increase breeding success of rock iguanas under managed care.

Dexamethasone versus methylprednisolone for critical asthma: A single center, open-label, parallel-group clinical trial.

BACKGROUND: Evidence for the use of dexamethasone for pediatric critical asthma is limited. We sought to compare the clinical efficacy and safety of dexamethasone versus methylprednisolone among children hospitalized in the pediatric intensive care unit (PICU) for critical asthma. METHODS: A prospective, single center, open-label, two-arm, parallel-group, nonrandomized trial among children ages 5-17 years hospitalized within the PICU from April 2019 to December 2021 for critical asthma consented to receive methylprednisolone (standard care) or dexamethasone (intervention) at a 2:1 allocation ratio, respectively. The intervention arm received intravenous dexamethasone 0.25 mg/kg/dose (max: 15 mg/dose) every 6 h for 48 h and the standard care arm intravenous methylprednisolone 1 mg/kg/dose every 6 h (max dose: 60 mg/dose) for 5 days. Study endpoints were clinical efficacy (i.e., length of stay [LOS], continuous albuterol duration, and a composite of adjunctive asthma interventions) and safety (i.e., corticosteroid-related adverse events). RESULTS: Ninety-two participants were analyzed of whom 31 were allocated to the intervention arm and 61 the standard care arm. No differences in demographics, clinical characteristics, or acute/chronic asthma severity indices were observed. Regarding efficacy and safety endpoints, no differences in hospital LOS, continuous albuterol duration, adjunctive asthma intervention rates, or corticosteroid-related adverse events were noted. Compared to the intervention arm, participants in the standard care arm more frequently were prescribed corticosteroids at discharge (72% vs. 13%, p < 0.001). CONCLUSIONS: Among children hospitalized for critical asthma, dexamethasone appears safe and warrants further investigation to fully assess clinical efficacy and potential advantages over commonly applied agents such as methylprednisolone.

Role of familiarity and preference in reproductive success in ex situ breeding programs.

Success of captive-breeding programs centers on consistent reproduction among captive animals. However, many individuals do not reproduce even when they are apparently healthy and presented with mates. Mate choice can affect multiple parameters of reproductive success, including mating success, offspring production, offspring survival, and offspring fecundity. We investigated the role of familiarity and preference on reproductive success of female Columbia Basin pygmy rabbits (Brachylagus idahoensis) as measured by litter production, litter size, average number of young that emerged from the burrow, and average number of young that survived to 1 year. We conducted these studies on pygmy rabbits at the Oregon Zoo (Portland, Oregon, U.S.A.) and Washington State University (Pullman, Washington, U.S.A.) from February to June 2006, 2007, and 2008. Before mating, we housed each female adjacent to 2 males (neighbors). Female preference for each potential mate was determined on the basis of behavioral interactions observed and measured between the rabbits. We compared reproductive success between females mated with neighbor and non-neighbor males and between females mated with preferred and nonpreferred males. Our findings suggest that mating with a neighbor compared with a non-neighbor and mating with a preferred neighbor compared with a nonpreferred neighbor increased reproductive success in female pygmy rabbits. Litter production, average number of young that emerged, and average number of young that survived to 1 year were higher in rabbits that were neighbors before mating than in animals who were not neighbors. Pairing rabbits with a preferred partner increased the probability of producing a litter and was significantly associated with increased litter size. In captive breeding programs, mates are traditionally selected on the basis of genetic parameters to minimize loss of genetic diversity and inbreeding coefficients. Our results suggest that integrating genetic information with social dynamics and behavioral measures of preference may increase the reproductive output of the pygmy rabbit captive-breeding program. Our findings are consistent with the idea that allowing mate choice and familiarity increase the reproductive success of captive-breeding programs for endangered species.

Natriuretic Peptide Screening for Primary Prevention or Early Detection of Heart Failure: A Pharmacist-Driven Team-Based Approach.

Background: Emerging data indicates that natriuretic peptide biomarker-based screening and early intervention could prevent left ventricular dysfunction or new-onset heart failure (HF). The 2017 update of the American College of Cardiology/ American Heart Association/Heart Failure Society of America guideline for managing HF provides a IIa recommendation for natriuretic peptide biomarker screening followed by a team-based approach for preventing HF. Observations: Clinical pharmacists worked collaboratively with a cardiology specialist and primary care practitioners to establish a protocol to identify patients at risk for HF. Patients with hypertension and/or type 2 diabetes mellitus (T2DM) and without a history of HF with N-terminal pro-B-type natriuretic peptide > 125 pg/mL received follow-up from clinical pharmacists, including initiation and/or adjustment of reninangiotensin system inhibitors, discussion of echocardiogram, and comprehensive disease state management of hypertension, T2DM, atherosclerotic cardiovascular disease risk reduction, oral nonsteroidal anti-inflammatory drug reduction, and tobacco cessation. Conclusions: By using natriuretic peptide screening, clinical pharmacists were able to identify patients with hypertension and/or T2DM who were at higher risk for HF and provide comprehensive medication management.

Stereotypic behaviour predicts reproductive performance and litter sex ratio in giant pandas.

Breeding and welfare problems confront many conservation breeding programs. Stereotypies-repetitive, unvarying, functionless behaviours -are common abnormal behaviours that often arise in suboptimal conditions. While the role of stereotypies in welfare assessment is well studied, few investigations address the relationship between stereotypic behaviour and reproduction. We examined the correlation between stereotypic behaviour and reproductive performance in 101 giant pandas (Ailuropoda melanoleuca). High stereotyping males copulated more and produced more cubs, suggesting that highly sexually motivated males were prone to stereotypy but also had high reproductive competence. Female stereotypies were negatively associated with all reproductive measures closely tied to behavioural competence: high stereotyping females were less likely to copulate, less likely to mother-rear cubs, and-probably a result of poor maternal care-had lower cub survival. However, females that exhibited stereotypies were more likely to produce a cub, suggesting stereotypies are tied to behavioural but not physiological competence. High stereotyping female pandas also displayed strong and consistent bias toward production of female offspring while paternal relationship to sex allocation was the reverse. These results are consistent with stress-mediated sex allocation theory. Our findings raise concern about differential reproductive success among high and low stereotyping pandas, and possible genetic adaptation to captivity.

A new longitudinal variation in the structure of collagen fibrils and its relationship to locations of mechanical damage susceptibility.

The hierarchical architecture of the collagen fibril is well understood, involving non-integer staggering of collagen molecules which results in a 67 nm periodic molecular density variation termed D-banding. Other than this variation, collagen fibrils are considered to be homogeneous at the micro-scale and beyond. Interestingly, serial kink structures have been shown to form at discrete locations along the length of collagen fibrils from some mechanically overloaded tendons. The formation of these kinks at discrete locations along the length of fibrils (discrete plasticity) may indicate pre-existing structural variations at a length scale greater than that of the D-banding. Using a high velocity nanomechanical mapping technique, 25 tendon collagen fibrils, were mechanically and structurally mapped along 10 µm of their length in dehydrated and hydrated states with resolutions of 20 nm and 8 nm respectively. Analysis of the variation in hydrated indentation modulus along individual collagen fibrils revealed a micro-scale structural variation not observed in the hydrated or dehydrated structural maps. The spacing distribution of this variation was similar to that observed for inter-kink distances seen in SEM images of discrete plasticity type damage. We propose that longitudinal variation in collagen fibril structure leads to localized mechanical susceptibility to damage under overload. Furthermore, we suggest that this variation has its origins in heterogeneous crosslink density along the length of collagen fibrils. The presence of pre-existing sites of mechanical vulnerability along the length of collagen fibrils may be important to biological remodeling of tendon, with mechanically-activated sites having distinct protein binding capabilities and enzyme susceptibility.

Suppressed plasmablast responses in febrile infants, including children with Kawasaki disease.

BACKGROUND: Kawasaki disease (KD), the leading cause of acquired heart disease in children, primarily affects infants and toddlers. Investigations on immune responses during KD are hampered by a limited understanding of normal immune responses in these ages. It's well known that Infants have poorer vaccine responses and difficulty with maintaining prolonged serum immunity, but there are few studies on human infants detailing immune deficiencies. Limited studies propose an inability to maintain life-long bone marrow plasma cells. Plasmablasts are a transitional cell form of B cells that lead to long-term Plasma cells. Plasmablasts levels rise in the peripheral blood after exposure to a foreign antigen. In adult studies, these responses are both temporally and functionally well characterized. To date, there have been few studies on plasmablasts in the predominant age range of KD. METHODS: Children presenting to an urban pediatric emergency room undergoing laboratory evaluation, who had concern of KD or had fever and symptoms overlapping those of KD, were recruited. Peripheral blood mononuclear cells were isolated and evaluated utilizing flow cytometry with specific B cell markers from 18 KD subjects and 69 febrile controls. RESULTS: Plasmablast numbers and temporal formation are similar between infectious disease controls and KD subjects. In both groups, infants have diminished plasmablast responses compared to older children. CONCLUSION: In this single-time point survey, infants have a blunted peripheral plasmablast response. Overall, similar plasmablast responses in KD and controls support an infectious disease relationship to KD. Future time-course studies of plasmablasts in infants are warranted as this phenomenon may contribute to observed immune responses in this age group.

Observational study of Interleukin-21 (IL-21) does not distinguish Kawasaki disease from other causes of fever in children.

BACKGROUND: Kawasaki disease (KD) is a febrile childhood vasculitis of unknown etiology. The diagnosis is highly concerning as over a quarter of children who fail to receive timely treatment with intravenous immunoglobulin (IVIG) will develop coronary aneurysms. Diagnosis relies on proper symptomatology and is supported by non-specific markers of inflammation. Previous studies have identified elevated plasma levels of interleukin-21 (IL-21) as a sensitive and specific biomarker in KD. The aim of this study is to assess the validity of IL-21 as a diagnostic biomarker for KD in febrile children in North America. METHODS: Plasma samples were collected from children who presented to an urban Emergency Department in North America. IL-21 levels were measured using commercial ELISA kits in 12 KD versus 60 controls subjects. RESULTS: Our study shows that IL-21 levels were non-specifically elevated across all febrile children, irrespective of KD diagnosis. Length of fever prior to sample collection does not correlate with IL-21 levels. Other inflammatory markers and laboratory values were also compared to IL-21 and show no significant correlation. CONCLUSIONS: Since IL-21 is elevated non-specifically in this cohort, our data supports that IL-21 is not an appropriate biomarker for diagnosis of KD in North American pediatric populations.

Peptide YY(3-36) inhibits morning, but not evening, food intake and decreases body weight in rhesus macaques.

Peptide YY(3-36) [PYY(3-36)] is a hormone that is released after meal ingestion that is currently being investigated for the treatment of obesity; however, there are conflicting reports of the effects of PYY(3-36) on energy balance in rodent models. To shed light on this controversy, we studied the effect of PYY(3-36) on food intake and body weight in a nonhuman primate. Intravenous PYY(3-36) infusions before a morning meal transiently suppressed the rate of food intake but did not suppress the evening meal or 24-h intake. Twice-daily or continuous intravenous PYY(3-36) infusions to supraphysiological levels (levels that exceeded normal physiological levels) again suppressed the rate of feeding for the morning but not the evening meal. Twice-daily intravenous PYY(3-36) infusions for 2 weeks significantly decreased body weight in all test animals (average weight loss 1.9%) without changing insulin response to glucose infusion. These results show that endogenous PYY(3-36) may alter morning but not evening meal intake, and supraphysiological doses are required for effective suppression of food intake.

The effect of subtalar joint position on dorsiflexion of the ankle/rearfoot versus midfoot/forefoot during gastrocnemius stretching.

BACKGROUND: Limited ankle joint dorsiflexion passive range of motion (PROM) has been associated with common chronic lower extremity conditions, and clinicians often instruct patients in stretching exercises to increase dorsiflexion. However, little is known about how subtalar joint (STJ) position affects dorsiflexion at the midfoot/forefoot versus ankle/rearfoot during gastrocnemius stretching. The purpose of this study was to determine if more dorsiflexion occurs at the ankle/rearfoot and less at the midfoot/forefoot during gastrocnemius stretching with the STJ positioned in supination versus pronation. METHODS: In this repeated measures design, 27 participants (23 females, 4 males; mean age = 31.3 years, SD = 10.7) with current or recent history of lower extremity chronic conditions and less than 10 degrees ankle dorsiflexion measured with the knee in extension on the involved side(s) performed five 30-second gastrocnemius stretching trials in pronation and supination on each side in a randomly determined sequence. A 7-camera Vicon Motion Analysis System and an AMTI force plate were used to measure midfoot/forefoot dorsiflexion, ankle/rearfoot dorsiflexion, knee extension, and normalized vertical ground reaction force. RESULTS: Two-way repeated measures ANOVA revealed a significant increase in midfoot/forefoot dorsiflexion when stretching in pronation compared to supination (P < .001). ANOVAs also demonstrated significantly more extension of the knee when stretching in supination compared to pronation (P < .001), and increased normalized vertical ground reaction force when stretching in supination compared to pronation (P = .032). With the numbers available, no significant difference in ankle/rearfoot dorsiflexion when stretching in supination compared to pronation could be detected (P > .05). CONCLUSION: Gastrocnemius stretching in pronation resulted in more dorsiflexion at the midfoot/forefoot than stretching in supination. CLINICAL RELEVANCE: Clinicians may want to consider STJ position during gastrocnemius stretching to either facilitate or limit recruitment of dorsiflexion motion at the midfoot/forefoot.