Metabolic perturbations of postnatal growth restriction and hyperoxia-induced pulmonary hypertension in a bronchopulmonary dysplasia model

Introduction

Neonatal pulmonary hypertension (PH) is a common manifestation of bronchopulmonary dysplasia (BPD) and contributes to increased morbidity and mortality of preterm birth. Postnatal growth restriction (PNGR) and hyperoxia are independent contributors to PH development, as indicated by our previous work in a rat model of BPD.

Objective

To explore the metabolic consequences of induction of PH with hyperoxia and PNGR in a rat model of BPD.

Methods

Sprague–Dawley rat pups (n?=?4/group) underwent three modes of PH induction: (1) growth restriction-induced by larger litter size; (2) hyperoxia-induced by 75% oxygen exposure; (3) combined growth restriction and hyperoxia. Primary metabolism, complex lipids, biogenic amines, and lipid mediators were characterized in plasma and lung tissue using GC- and LC-MS technologies.

Results

Specific to hyperoxic induction, pulmonary metabolomics suggested increased reactive oxygen species (ROS) generation as indicated by: (1) increased indicators of β-oxidation and mitochondrial respiration; (2) changes in ROS-sensitive pathway activity and metabolites including the polyol pathway and xanthine oxidase pathways, and reduced glutathione; (3) decreased plasmalogens. Unlike the lung, circulating metabolite changes were induction mode-specific or additive in the combined modes (e.g. 1) growth-restriction reduced phosphatidylcholine; (2) hyperoxia increased oxylipins and trimethylamine-N-oxide (TMAO); (3) additive effects on 3-hydroxybutyric acid and arginine.

Conclusion

The present study highlights the variety of metabolic changes that occur due to PNGR- and hyperoxia-induced PH, identifying numerous metabolites and pathways influenced by treatment-specific or combined effects. The rat model used in this study presents a robust means of uncovering the mechanisms that contribute to the pathology of PH.

     

Investigating the Role of 4‐Tert Butylpyridine in Perovskite Solar Cells

The majority of hole‐transporting layers used in n‐i‐p perovskite solar cells contain 4‐tert butylpyridine (tBP). High power‐conversion efficiencies and, in particular, good steady‐state performance appears to be contingent on the inclusion of this additive. On the quest to improve the steady state efficiencies of the carbon nanotube‐based hole‐transporter system, this study has found that the presence of tBP results in an extraordinary improvement in the performance of these devices. By deconstructing a prototypical device and investigating the effect of tBP on each individual layer, the results of this study indicate that this performance enhancement must be due to a direct chemical interaction between tBP and the perovskite material. This study proposes that tBP serves to p‐dope the perovskite layer and investigates this theory with poling and work function measurements.     

Epitope characterization of anti-JAM-A antibodies using orthogonal mass spectrometry and surface plasmon resonance approaches

Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells and associated with cancer progression. We present here the extensive characterization of immune complexes involving JAM-A antigen and three monoclonal antibodies (mAbs), including hz6F4-2, a humanized version of anti-tumoral 6F4 mAb identified by a functional and proteomic approach in our laboratory. A specific workflow that combines orthogonal approaches has been designed to determine binding stoichiometries along with JAM-A epitope mapping determination at high resolution for these three mAbs. Native mass spectrometry experiments revealed different binding stoichiometries and affinities, with two molecules of JAM-A being able to bind to hz6F4-2 and F11 Fab, while only one JAM-A was bound to J10.4. Surface plasmon resonance indirect competitive binding assays suggested epitopes located in close proximity for hz6F4-2 and F11. Finally, hydrogen-deuterium exchange mass spectrometry was used to precisely identify epitopes for all mAbs. The results obtained by orthogonal biophysical approaches showed a clear correlation between the determined epitopes and JAM-A binding characteristics, allowing the basis for molecular recognition of JAM-A by hz6F4-2 to be definitively established for the first time. Taken together, our results highlight the power of MS-based structural approaches for epitope mapping and mAb conformational characterization.     

Describing and understanding behavioral responses to multiple stressors and multiple stimuli

Understanding the effects of environmental change on natural ecosystems is a major challenge, particularly when multiple stressors interact to produce unexpected “ecological surprises” in the form of complex, nonadditive effects that can amplify or reduce their individual effects. Animals often respond behaviorally to environmental change, and multiple stressors can have both population‐level and community‐level effects. However, the individual, not combined, effects of stressors on animal behavior are commonly studied. There is a need to understand how animals respond to the more complex combinations of stressors that occur in nature, which requires a systematic and rigorous approach to quantify the various potential behavioral responses to the independent and interactive effects of stressors. We illustrate a robust, systematic approach for understanding behavioral responses to multiple stressors based on integrating schemes used to quantitatively classify interactions in multiple‐stressor research and to qualitatively view interactions between multiple stimuli in behavioral experiments. We introduce and unify the two frameworks, highlighting their conceptual and methodological similarities, and use four case studies to demonstrate how this unification could improve our interpretation of interactions in behavioral experiments and guide efforts to manage the effects of multiple stressors. Our unified approach: (1) provides behavioral ecologists with a more rigorous and systematic way to quantify how animals respond to interactions between multiple stimuli, an important theoretical advance, (2) helps us better understand how animals behave when they encounter multiple, potentially interacting stressors, and (3) contributes more generally to the understanding of “ecological surprises” in multiple stressors research.     

Bursaphelenchus anatolius n. sp. (Nematoda: Parasitaphelenchidae), an Associate of Bees in the Genus Halictus

Bursaphelenchus anatolius n. sp., a phoretic associate of Halictus bees from Ankara, Turkey, is described and illustrated. Bursaphelenchus anatolius n. sp. is closest to B. kevini, which is phoretically associated with Halictus bees from the Pacific Northwest. Bursaphelenchus anatolius n. sp. and B. kevini appear to be sister taxa based upon several shared morphological features, similar life histories involving phoresy with soil-dwelling Halictus bees, and molecular analysis of the near-full-length small subunit rDNA, D2D3 expansion segments of the large subunit rDNA, and partial mitochondrial DNA COI. Bursaphelenchus anatolius n. sp. can be differentiated from all other species of Bursaphelenchus based upon spicule morphology. The paired spicules are uniquely shaped and ventrally recurved, and both B. anatolius n. sp. and B. kevini possess extending flaps that open when the spicules are protracted beyond the cloaca. Population growth of B. anatolius n. sp. was measured at 23 °C in the laboratory on cultures of the fungus Monilinia fructicola grown on lactic acid-treated, 5% glycerol-supplemented potato dextrose agar. Nematode population densities rapidly increased from 110 to about 110,000/9-cm-diam. dish within 21 days.     

An evaluation of PCR methods to detect strains of Mycoplasma fermentans.

A panel of 30 putative Mycoplasma fermentans strains, isolated from various sources including human, ovine and cell lines, were tested by a previously described polymerase chain reaction (PCR) to confirm their identity by amplification of a conserved 206 bp region of the insertion sequence IS1550. In addition, the application of another PCR based on the major part of the IS1550 element showed one or two products of different length (1144 and 1341 bp) enabling M. fermentans strains to be divided into two types designated as Type A and Type B. A PCR, which amplifies the macrophage activating lipopeptide gene (malp), supported the identification of all the strains as M. fermentans. Thirteen other species of Mycoplasma from human sources gave negative results in these tests, with the exception of Mycoplasma orale, which was detected by both IS1550-PCRs based on the major part and the conserved 206 bp region of the IS1550 element. This study suggests that all M. fermentans isolates possess both the IS1550 element and the malp gene. In contrast to the IS1550, the malp gene is shown to be species-specific and the use of a malp PCR described here could prove to be a useful adjunct to IS1550 detection as confirmation of the presence of M. fermentans in clinical material.     

Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis

Wolbachia pipientis are obligate endosymbionts that infect a wide range of insect and other arthropod species. They act as reproductive parasites by manipulating the host reproduction machinery to enhance their own transmission. This unusual phenotype is thought to be a consequence of the actions of secreted Wolbachia proteins that are likely to contain disulfide bonds to stabilize the protein structure. In bacteria, the introduction or isomerization of disulfide bonds in proteins is catalyzed by Dsb proteins. The Wolbachia genome encodes two proteins, α-DsbA1 and α-DsbA2, that might catalyze these steps. In this work we focussed on the 234 residue protein α-DsbA1; the gene was cloned and expressed in Escherichia coli, the protein was purified and its identity confirmed by mass spectrometry. The sequence identity of α-DsbA1 for both dithiol oxidants (E. coli DsbA, 12%) and disulfide isomerases (E. coli DsbC, 14%) is similar. We therefore sought to establish whether α-DsbA1 is an oxidant or an isomerase based on functional activity. The purified α-DsbA1 was active in an oxidoreductase assay but had little isomerase activity, indicating that α-DsbA1 is DsbA-like rather than DsbC-like. This work represents the first successful example of the characterization of a recombinant Wolbachia protein. Purified α-DsbA1 will now be used in further functional studies to identify protein substrates that could help explain the molecular basis for the unusual Wolbachia phenotypes, and in structural studies to explore its relationship to other disulfide oxidoreductase proteins.