Treatment with inhaled aerosolised ethanol reduces viral load and potentiates macrophage responses in an established influenza mouse model.

AIM: Treatment options for viral lung infections are currently limited. We aimed to explore the safety and efficacy of inhaled ethanol in an influenza-infection mouse model. MATERIALS AND METHODS: In a safety and tolerability experiment, 80 healthy female BALB/c mice (20 per group) were exposed to nebulized saline (control) or three concentrations of ethanol (40/60/80% ethanol v/v in water) for 3x30-minute periods, with a two-hour break between exposures. In a separate subsequent experiment, 40 Female BALB/c mice were nasally inoculated with 104.5 plaque-forming units of immediate virulence "Mem71" influenza. Infection was established for 48-h before commencing treatment in 4 groups of 10 mice with either nebulized saline (control) or one of 3 different concentrations of ethanol (40/60/80% ethanol v/v in water) for 3x30-minute periods daily over three consecutive days. In both experiments, mouse behavior, clinical scores, weight change, bronchoalveolar lavage cell viability, cellular composition, and cytokine levels, were assessed 24-h following the final exposure, with viral load also assessed after the second experiment. RESULTS: In uninfected BALB/c mice, 3x30-minute exposures to nebulized 40%, 60%, and 80% ethanol resulted in no significant differences in mouse weights, cell counts/viability, cytokines, or morphometry measures. In Mem71-influenza infected mice, we observed a dose-dependent reduction in viral load in the 80%-treated group and potentiation of macrophage numbers in the 60%- and 80%-treated groups, with no safety concerns. CONCLUSIONS: Our data provides support for inhaled ethanol as a candidate treatment for respiratory infections.

An infant mouse model of influenza-driven nontypeable Haemophilus influenzae colonization and acute otitis media suitable for preclinical testing of novel therapies.

Nontypeable Haemophilus influenzae (NTHi) is a major otitis media (OM) pathogen, with colonization a prerequisite for disease development. Most acute OM is in children <5 years old, with recurrent and chronic OM impacting hearing and learning. Therapies to prevent NTHi colonization and/or disease are needed, especially for young children. Respiratory viruses are implicated in driving the development of bacterial OM in children. We have developed an infant mouse model of influenza-driven NTHi OM, as a preclinical tool for the evaluation of safety and efficacy of clinical therapies to prevent NTHi colonization and the development of OM. In this model, 100% of infant BALB/cARC mice were colonized with NTHi, and all developed NTHi OM. Influenza A virus (IAV) facilitated the establishment of dense (1 × 105 CFU/mL) and long-lasting (6 days) NTHi colonization. IAV was essential for the development of NTHi OM, with 100% of mice in the IAV/NTHi group developing NTHi OM compared with 8% of mice in the NTHi only group. Histological analysis and cytokine measurements revealed that the inflammation observed in the middle ear of the infant mice with OM reflected inflammation observed in children with OM. We have developed the first infant mouse model of NTHi colonization and OM. This ascension model uses influenza-driven establishment of OM and reflects the clinical pathology of bacterial OM developing after a respiratory virus infection. This model provides a valuable tool for testing therapies to prevent or treat NTHi colonization and disease in young children.

Fe protein docking transduces conformational changes to MoFe nitrogenase active site in a nucleotide-dependent manner.

The reduction of dinitrogen to ammonia catalyzed by nitrogenase involves a complex series of events, including ATP hydrolysis, electron transfer, and activation of metal clusters for N2 reduction. Early evidence shows that an essential part of the mechanism involves transducing information between the nitrogenase component proteins through conformational dynamics. Here, millisecond time-resolved hydrogen-deuterium exchange mass spectrometry was used to unravel peptide-level protein motion on the time scale of catalysis of Mo-dependent nitrogenase from Azotobacter vinelandii. Normal mode analysis calculations complemented this data, providing insights into the specific signal transduction pathways that relay information across protein interfaces at distances spanning 100 Å. Together, these results show that conformational changes induced by protein docking are rapidly transduced to the active site, suggesting a specific mechanism for activating the metal cofactor in the enzyme active site.

Application of platform process development approaches to the manufacturing of Mabcalin™ bispecifics.

Bispecific biotherapeutics offer potent and highly specific treatment options in oncology and immuno-oncology. However, many bispecific formats are prone to high levels of aggregation and instability, leading to prolonged development timelines, inefficient manufacturing, and high costs. The novel class of Mabcalin™ molecules consist of Anticalin® proteins fused to an IgG and are currently being evaluated in pre-clinical and clinical studies. Here, we describe a robust high-yield manufacturing platform for these therapeutic fusion proteins providing data up to commercially relevant scales. A platform upstream process was established for one of the Mabcalin bispecifics and then applied to other clinically relevant drug candidates with different IgG target specificities. Process performance was compared in 3 L bioreactors and production was scaled-up to up to 1000 L for confirmation. The Mabcalin proteins' structural and biophysical similarities enabled a downstream platform approach consisting of initial protein A capture, viral inactivation, mixed-mode anion exchange polishing, second polishing by cation exchange or hydrophobic interaction chromatography, viral filtration, buffer exchange and concentration by ultrafiltration/diafiltration. All three processes met their target specifications and achieved comparable clearance of impurities and product yields across scales. The described platform approach provides a fast and economic path to process confirmation and is well comparable to classical monoclonal antibody approaches in terms of costs and time to clinic.

Comparative polar and lipid plasma metabolomics differentiate KSHV infection and disease states.

BACKGROUND: Kaposi sarcoma (KS) is a neoplastic disease etiologically associated with infection by the Kaposi sarcoma-associated herpesvirus (KSHV). KS manifests primarily as cutaneous lesions in individuals due to either age (classical KS), HIV infection (epidemic KS), or tissue rejection preventatives in transplantation (iatrogenic KS) but can also occur in individuals, predominantly in sub-Saharan Africa (SSA), lacking any obvious immune suppression (endemic KS). The high endemicity of KSHV and human immunodeficiency virus-1 (HIV) co-infection in Africa results in KS being one of the top 5 cancers there. As with most viral cancers, infection with KSHV alone is insufficient to induce tumorigenesis. Indeed, KSHV infection of primary human endothelial cell cultures, even at high levels, is rarely associated with long-term culture, transformation, or growth deregulation, yet infection in vivo is sustained for life. Investigations of immune mediators that distinguish KSHV infection, KSHV/HIV co-infection, and symptomatic KS disease have yet to reveal consistent correlates of protection against or progression to KS. In addition to viral infection, it is plausible that pathogenesis also requires an immunological and metabolic environment permissive to the abnormal endothelial cell growth evident in KS tumors. In this study, we explored whether plasma metabolomes could differentiate asymptomatic KSHV-infected individuals with or without HIV co-infection and symptomatic KS from each other. METHODS: To investigate how metabolic changes may correlate with co-infections and tumorigenesis, plasma samples derived from KSHV seropositive sub-Saharan African subjects in three groups, (A) asymptomatic (lacking neoplastic disease) with KSHV infection only, (B) asymptomatic co-infected with KSHV and HIV, and (C) symptomatic with clinically diagnosed KS, were subjected to analysis of lipid and polar metabolite profiles RESULTS: Polar and nonpolar plasma metabolic differentials were evident in both comparisons. Integration of the metabolic findings with our previously reported KS transcriptomics data suggests dysregulation of amino acid/urea cycle and purine metabolic pathways, in concert with viral infection in KS disease progression. CONCLUSIONS: This study is, to our knowledge, the first to report human plasma metabolic differentials between in vivo KSHV infection and co-infection with HIV, as well as differentials between co-infection and epidemic KS.

Element distribution in electrochemically treated mine wastewater for efficient resource recovery and water treatment: A pilot study.

The feasibility of recovering major and critical elements from acid mine drainage using a pilot-scale electrochemical reactor (ECR) was investigated by assessing elements concentration and species distribution in the liquid and solid phase (sludge) in multistage tests. These were carried out at different electrical currents (18-22 amps) and thus, pH (8-12). The results showed that major metals Al, Cu and Fe were removed from the liquid phase at pH 5.9 while remaining the majority of Zn (57.2 ppm). On the other hand, at pH 7, the effluent was mainly composed of Mn (7.3 ppm). These results were confirmed by the simulation results using the PHREEQC model, which also identified the main chemical species in solution and the precipitates formed after the treatment (oxyhydroxides/sulfates/oxides). The ECR treatment led to sludges with targeted critical elements, some up to 20 times (Co, Be and Sb) higher than their earth's crustal abundance. At pH 10, rare earth elements in the sludge targeted Ce, followed by Nd and La. This study is one of the few studies carrying a detailed analysis of the behavioural distribution pattern of these elements at each pH, which opens the door for the potential of recovering these elements.

Genome of Paspalum vaginatum and the role of trehalose mediated autophagy in increasing maize biomass.

A number of crop wild relatives can tolerate extreme stress to a degree outside the range observed in their domesticated relatives. However, it is unclear whether or how the molecular mechanisms employed by these species can be translated to domesticated crops. Paspalum (Paspalum vaginatum) is a self-incompatible and multiply stress-tolerant wild relative of maize and sorghum. Here, we describe the sequencing and pseudomolecule level assembly of a vegetatively propagated accession of P. vaginatum. Phylogenetic analysis based on 6,151 single-copy syntenic orthologues conserved in 6 related grass species places paspalum as an outgroup of the maize-sorghum clade. In parallel metabolic experiments, paspalum, but neither maize nor sorghum, exhibits a significant increase in trehalose when grown under nutrient-deficit conditions. Inducing trehalose accumulation in maize, imitating the metabolic phenotype of paspalum, results in autophagy dependent increases in biomass accumulation.

Structural Dynamics and Activity of B19V VP1u during the pHs of Cell Entry and Endosomal Trafficking.

Parvovirus B19 (B19V) is a human pathogen that is the causative agent of fifth disease in children. It is also known to cause hydrops in fetuses, anemia in AIDS patients, and transient aplastic crisis in patients with sickle cell disease. The unique N-terminus of Viral Protein 1 (VP1u) of parvoviruses, including B19V, exhibits phospholipase A2 (PLA2) activity, which is required for endosomal escape. Presented is the structural dynamics of B19V VP1u under conditions that mimic the pHs of cell entry and endosomal trafficking to the nucleus. Using circular dichroism spectroscopy, the receptor-binding domain of B19V VP1u is shown to exhibit an α-helical fold, whereas the PLA2 domain exhibits a probable molten globule state, both of which are pH invariant. Differential scanning calorimetry performed at endosomal pHs shows that the melting temperature (Tm) of VP1u PLA2 domain is tuned to body temperature (37 °C) at pH 7.4. In addition, PLA2 assays performed at temperatures ranging from 25-45 °C show both a temperature and pH-dependent change in activity. We hypothesize that VP1u PLA2 domain differences in Tm at differing pHs have enabled the virus to "switch on/off" the phospholipase activity during capsid trafficking. Furthermore, we propose the environment of the early endosome as the optimal condition for endosomal escape leading to B19V infection.

Exacerbation of chronic cigarette-smoke induced lung disease by rhinovirus in mice.

A significant proportion of chronic obstructive pulmonary disease exacerbations are strongly associated with rhinovirus infection (HRV). In this study, we combined long-term cigarette smoke exposure with HRV infection in a mouse model. Our aim was to better understand the effects of HRV infection on such exacerbations, using a realistic method for generating a COPD-like phenotype. After 12-weeks of cigarette smoke exposure, adult female BALB/c mice were infected with HRV-1A and three days later we assessed a range of outcomes including lung volume and function, collected lung tissue for measurement of viral titre, bronchoalveolar lavage for assessment of pulmonary inflammation and levels of key mediators, and fixed lungs for stereological structural analyses. Cigarette smoke exposure alone significantly increased total cells and macrophages, and reduced MIP-2 in bronchoalveolar lavage. HRV-1A infection alone increased neutrophilic inflammation, IP-10 and total protein in lavage and also increased specific airway resistance measured at functional residual capacity. Cigarette smoke and HRV-1A together impacted various lung structural parameters including increasing stereological lung volume. Our results show that long-term cigarette smoke exposure and HRV-1A infection both individually impact respiratory outcomes and combine to alter aspects of lung structure in a mouse model, thus providing insight into the development of future mechanistic studies and appropriate interventions in human disease.

Previous Influenza Infection Exacerbates Allergen Specific Response and Impairs Airway Barrier Integrity in Pre-Sensitized Mice.

In this study we assessed the effects of antigen exposure in mice pre-sensitized with allergen following viral infection on changes in lung function, cellular responses and tight junction expression. Female BALB/c mice were sensitized to ovalbumin and infected with influenza A before receiving a second ovalbumin sensitization and challenge with saline, ovalbumin (OVA) or house dust mite (HDM). Fifteen days post-infection, bronchoalveolar inflammation, serum antibodies, responsiveness to methacholine and barrier integrity were assessed. There was no effect of infection alone on bronchoalveolar lavage cellular inflammation 15 days post-infection; however, OVA or HDM challenge resulted in increased bronchoalveolar inflammation dominated by eosinophils/neutrophils or neutrophils, respectively. Previously infected mice had higher serum OVA-specific IgE compared with uninfected mice. Mice previously infected, sensitized and challenged with OVA were most responsive to methacholine with respect to airway resistance, while HDM challenge caused significant increases in both tissue damping and tissue elastance regardless of previous infection status. Previous influenza infection was associated with decreased claudin-1 expression in all groups and decreased occludin expression in OVA or HDM-challenged mice. This study demonstrates the importance of the respiratory epithelium in pre-sensitized individuals, where influenza-infection-induced barrier disruption resulted in increased systemic OVA sensitization and downstream effects on lung function.

The Best of Both Worlds: Eliminating Creatine Kinase-Muscle/Brain (CK-MB) Testing in the Emergency Department Leads to Lower Costs Without Missed Clinical Diagnoses.

The 2014 American Heart Association/American College of Cardiology (AHA/ACC) clinical guidelines recommend cardiac troponin as a superior biomarker to creatine kinase (CK) and creatine kinase-muscle/brain (CK-MB) for the detection of acute coronary syndrome (ACS), namely myocardial infarction and unstable angina. In April 2018, our Emergency Department (ED) transitioned from using standard troponin to using high-sensitivity troponin T, and adopted a clinical guideline consistent with the AHA/ACC. The guideline recommended high-sensitivity troponin T without CK/CK-MB testing in the majority of clinical situations, limiting CK/CK-MB testing to two specific clinical cases: 1) estimated glomerular filtration rate (eGFR) value <15 mL/min, or 2) recent acute coronary syndrome (ACS) event. Per our ED's policy, a "negative" troponin T was defined as being below the limit of detection (LOD) (i.e., <6 ng/L); such a value obtained at least 3 hours after symptom onset "ruled out" an ACS event and did not require a repeat troponin. The goal of this retrospective study was to determine whether the guideline limiting CK-MB testing missed clinically-significant cardiac outcomes (ACS or new diagnosis of coronary artery disease [CAD]) or was associated with mortality. Pre-implementation data (July 1, 2017 - December 31, 2017) was compared with post-implementation data (July 1, 2018 - December 31, 2018). After guideline introduction, CK/CK-MB ordering decreased by nearly 90%, while troponin ordering increased by nearly 20%, likely due to the introduction in June 2018 of high-sensitivity troponin T, which yielded numerous intermediate/indeterminate-range results that prompted repeat testing. Fewer than 1.5% of patients with a "negative" troponin (below the LOD) and a "positive" CK-MB (above the upper limit of normal [ULN]) had ACS or new-diagnosis CAD; patients with either diagnosis did not expire during their hospital stay or within 30 days of their index visit. CK-MB Index, which has a higher specificity than CK, only found ACS or new CAD among 0.8% of positive results. Considering both decreased CK/CK-MB and increased troponin ordering, the net annual direct cost savings in cardiac biomarker testing was extrapolated to $12,700. Had our institution not transitioned to higher cost high-sensitivity troponin ($2.054/unit) from standard troponin ($1.65/unit), and had the rate of troponin ordering increased solely proportionate to the rate of ED visit increase (2% year-over-year) rather than increase nearly 20% (likely due to the transition to high-sensitivity troponin), then the total six-month direct costs on troponin testing would have been $14,632 instead of $21,267.12, and annual direct cost savings would have been $18,945.80 instead of $12,700. The new ED clinical guideline did not result in a significant number of missed ACS or new-diagnosis CAD, and was associated with direct cost savings. These savings probably underestimate total savings, as the reduced number of "false-positive" CK-MB results likely prevented additional costs, such as hospitalization, specialty consultation, coronary calcium CT, echocardiogram, cardiac stress test, and coronary artery catheterization.

Mouse Lung Structure and Function after Long-Term Exposure to an Atmospheric Carbon Dioxide Level Predicted by Climate Change Modeling.

BACKGROUND: Climate change models predict that atmospheric carbon dioxide [CO2] levels will be between 700 and 900 ppm within the next 80 y. Despite this, the direct physiological effects of exposure to slightly elevated atmospheric CO2 (as compared with ∼410 ppm experienced today), especially when exposures extend from preconception to adulthood, have not been thoroughly studied. OBJECTIVES: In this study we aimed to assess the respiratory structure and function effects of long-term exposure to 890 ppm CO2 from preconception to adulthood using a mouse model. METHODS: We exposed mice to CO2 (∼890 ppm) from prepregnancy, through the in utero and early life periods, until 3 months of age, at which point we assessed respiratory function using the forced oscillation technique, and lung structure. RESULTS: CO2 exposure resulted in a range of respiratory impairments, particularly in female mice, including higher tissue elastance, longer chord length, and lower lung compliance. Importantly, we also assessed the lung function of the dams that gave birth to our experimental subjects. Even though these mice had been exposed to the same level of increased CO2 for a similar amount of time (∼8wk), we measured no impairments in lung function. This suggests that the early life period, when lungs are undergoing rapid growth and development, is particularly sensitive to CO2. DISCUSSION: To the best of our knowledge, this study, for the first time, shows that long-term exposure to environmentally relevant levels of CO2 can impact respiratory function in the mouse. https://doi.org/10.1289/EHP7305.

Novel phenolic antimicrobials enhanced activity of iminodiacetate prodrugs against biofilm and planktonic bacteria.

Prodrugs are pharmacologically attenuated derivatives of drugs that undergo bioconversion into the active compound once reaching the targeted site, thereby maximizing their efficiency. This strategy has been implemented in pharmaceuticals to overcome obstacles related to absorption, distribution, and metabolism, as well as with intracellular dyes to ensure concentration within cells. In this study, we provide the first examples of a prodrug strategy that can be applied to simple phenolic antimicrobials to increase their potency against mature biofilms. The addition of (acetoxy)methyl iminodiacetate groups increases the otherwise modest potency of simple phenols. Biofilm-forming bacteria exhibit a heightened tolerance toward antimicrobial agents, thereby accentuating the need for new antibiotics as well as those, which incorporate novel delivery strategies to enhance activity toward biofilms.

Staged electrochemical treatment guided by modelling allows for targeted recovery of metals and rare earth elements from acid mine drainage.

Acid mine drainage (AMD) is a challenge for current and legacy mining operations worldwide given its potential to severely harm ecosystems and communities if inadequately managed. Treatment costs for AMD are amongst the highest in the industrial wastewater treatment sector, with limited sustainable options available to date. This work demonstrates a novel chemical-free approach to tackle AMD, whereby staged electrochemical neutralisation is employed to treat AMD and concomitantly recover metals as precipitates. This approach was guided by physico-chemical modelling and tested on real AMD from two different legacy mine sites in Australia, and compared against conventional chemical-dosing-based techniques using hydrated lime (Ca(OH)2) and sodium hydroxide (NaOH). The electrochemical treatment demonstrated the same capacity than Ca(OH)2 to neutralise AMD and remove sulfates, and both were significantly better than NaOH. However, the electrochemical approach produced less voluminous and more easily settleable sludge than Ca(OH)2. Moreover, the staged treatment approach demonstrated the potential to produce metal-rich powdered solids with a targeted composition, including rare earth elements and yttrium (REY). REY were recovered in concentrations up to 0.1% of the total solids composition, illustrating a new avenue for AMD remediation coupled with the recovery of critical metals.

The cumulative effect of inflammation and infection on structural lung disease in early cystic fibrosis.

INTRODUCTION: Pulmonary inflammation and infection are important clinical and prognostic markers of lung disease in cystic fibrosis (CF). However, whether in young children they are transient findings or have cumulative, long-term impacts on respiratory health is largely unknown. We aimed to determine whether their repeated detection has a deleterious effect on structural lung disease. METHODS: All patients aged <6 years with annual computed tomography (CT) and bronchoalveolar lavage (BAL) were included. Structural lung disease on CT (%Disease) was determined using the PRAGMA-CF (Perth-Rotterdam Annotated Grid Morphometric Analysis for CF) method. The number of times free neutrophil elastase (NE) and infection were detected in BAL were counted, to determine cumulative BAL history. Linear mixed model analysis, accounting for repeat visits and adjusted for age, was used to determine associations. RESULTS: 265 children (683 scans) were included for analysis, with BAL history comprising 1161 visits. %Disease was significantly associated with the number of prior NE (0.31, 95% CI 0.09-0.54; p=0.007) but not infection (0.23, 95% CI -0.01-0.47; p=0.060) detections. Reference equations were determined. CONCLUSIONS: Pulmonary inflammation in surveillance BAL has a cumulative effect on structural lung disease extent, more so than infection. This provides a strong rationale for therapies aimed at reducing inflammation in young children.

The Role of Mass Spectrometry in Structural Studies of Flavin-Based Electron Bifurcating Enzymes.

For decades, biologists and biochemists have taken advantage of atomic resolution structural models of proteins from X-ray crystallography, nuclear magnetic resonance spectroscopy, and more recently cryo-electron microscopy. However, not all proteins relent to structural analyses using these approaches, and as the depth of knowledge increases, additional data elucidating a mechanistic understanding of protein function is desired. Flavin-based electron bifurcating enzymes, which are responsible for producing high energy compounds through the simultaneous endergonic and exergonic reduction of two intercellular electron carriers (i.e., NAD+ and ferredoxin) are one class of proteins that have challenged structural biologists and in which there is great interest to understand the mechanism behind electron gating. A limited number of X-ray crystallography projects have been successful; however, it is clear that to understand how these enzymes function, techniques that can reveal detailed in solution information about protein structure, dynamics, and interactions involved in the bifurcating reaction are needed. In this review, we cover a general set of mass spectrometry-based techniques that, combined with protein modeling, are capable of providing information on both protein structure and dynamics. Techniques discussed include surface labeling, covalent cross-linking, native mass spectrometry, and hydrogen/deuterium exchange. We cover how biophysical data can be used to validate computationally generated protein models and develop mechanistic explanations for regulation and performance of enzymes and protein complexes. Our focus will be on flavin-based electron bifurcating enzymes, but the broad applicability of the techniques will be showcased.

Hydrogen Deuterium Exchange Mass Spectrometry of Oxygen Sensitive Proteins.

The protocol detailed here describes a way to perform hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) on oxygen sensitive proteins. HDX-MS is a powerful tool for studying the protein structure-function relationship. Applying this technique to anaerobic proteins provides insight into the mechanism of proteins that perform oxygen sensitive chemistry. A problem when using HDX-MS to study anaerobic proteins is that there are many parts that require constant movement into and out of an anaerobic chamber. This can affect the seal, increasing the likelihood of oxygen exposure. Exposure to oxygen causes the cofactors bound to these proteins, a common example being FeS clusters, to no longer interact with the amino acid residues responsible for coordinating the FeS clusters, causing loss of the clusters and irreversible inactivation of the protein. To counteract this, a double vial system was developed that allows the preparation of solutions and reaction mixtures anaerobically, but also allows these solutions to be moved to an aerobic environment while shielding the solutions from oxygen. Additionally, movement isn't limited like it is in an anaerobic chamber, ensuring more consistent data, and fewer errors during the course of the reaction.

Foetal growth restriction in mice modifies postnatal airway responsiveness in an age and sex-dependent manner.

Epidemiological studies demonstrate an association between intrauterine growth restriction (IUGR) and asthma; however the underlying mechanism is unknown. We investigated the impact of maternal hypoxia-induced IUGR on airway responsiveness in male and female mice during juvenility and adulthood. Pregnant BALB/c mice were housed under hypoxic conditions for gestational days 11-17.5 and then returned to normoxic conditions for the remainder of pregnancy. A control group was housed under normoxic conditions throughout pregnancy. Offspring were studied at 2 weeks (juveniles) and 8 weeks (adults), where lung volume was assessed by plethysmography, airway responsiveness to methacholine determined by the forced oscillation technique and lungs fixed for morphometry. IUGR offspring were lighter at birth, exhibited "catch-up growth" by 2 weeks, but were again lighter in adulthood. IUGR males were "hyper-responsive" at 2 weeks and "hypo-responsive" as adults, in contrast with IUGR females who were hyper-responsive in adulthood. IUGR males had increased inner and total wall thickness at 2 weeks which resolved by adulthood, while airways in IUGR females were structurally normal throughout life. There were no differences in lung volume between Control and IUGR offspring at any age. Our data demonstrate changes in airway responsiveness as a result of IUGR that could influence susceptibility to asthma development and contribute to sexual dimorphism in asthma prevalence which switches from a male dominated disease in early life to a female dominated disease in adulthood.

H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle.

Recent investigations into ferredoxin-dependent transhydrogenases, a class of enzymes responsible for electron transport, have highlighted the biological importance of flavin-based electron bifurcation (FBEB). FBEB generates biomolecules with very low reduction potential by coupling the oxidation of an electron donor with intermediate potential to the reduction of high and low potential molecules. Bifurcating systems can generate biomolecules with very low reduction potentials, such as reduced ferredoxin (Fd), from species such as NADPH. Metabolic systems that use bifurcation are more efficient and confer a competitive advantage for the organisms that harbor them. Structural models are now available for two NADH-dependent ferredoxin-NADP+ oxidoreductase (Nfn) complexes. These models, together with spectroscopic studies, have provided considerable insight into the catalytic process of FBEB. However, much about the mechanism and regulation of these multi-subunit proteins remains unclear. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and statistical coupling analysis (SCA), we identified specific pathways of communication within the model FBEB system, Nfn from Pyrococus furiosus, under conditions at each step of the catalytic cycle. HDX-MS revealed evidence for allosteric coupling across protein subunits upon nucleotide and ferredoxin binding. SCA uncovered a network of co-evolving residues that can provide connectivity across the complex. Together, the HDX-MS and SCA data show that protein allostery occurs across the ensemble of iron­sulfur cofactors and ligand binding sites using specific pathways that connect domains allowing them to function as dynamically coordinated units.