Developing A Clinical Support Tool of Suboptimal Caregiver Behaviours During Vaccination: Preliminary Validation of the Opportunities to Understand Childhood Hurt Inoculation (OUCHI) Tool

Infant distress regulation is heavily contingent on sensitive caregiver soothing, which is important for child socioemotional development. Routine vaccinations provide clinicians the opportunity to support caregiver soothing across childhood. However, there is no clinical tool or norms to identify dyads that may need support in this important area. The object of the present thesis was to develop a clinical support tool highlighting caregiver behaviours that increase infant distress during vaccination the OUCHI Tool (Opportunities to Understand Childhood Hurt Inoculation Tool), and establish its preliminary psychometric properties (i.e., reliability and validity). The tool was developed and validated by synergizing extensive research experience in vaccination behaviours, clinician expertise in everyday practice, and archival vaccination data (n = 537). Our findings showed interrater reliability, test-retest reliability, and ecological, content, face, convergent, and divergent validity. The OUCHI Tool is a promising tool that can help integrate infant mental health practices during pediatric well-baby visits

Learning environment and attitudes among mathematics students with specific learning disabilities in self-contained and inclusion classes

I investigated attitudes and classroom environments among 242 eight-grade students with specific learning disabilities in inclusion classes, general-education students in inclusion classes, and students with specific learning disabilities in self-contained classes in Florida. Students with specific learning disabilities in integrated settings had higher scores than students with disabilities in separate settings, especially for task orientation and enjoyment. In integrated classes, general-education student had higher scores than students with specific learning disabilities

Sequence analysis of cytochrome bd oxidase suggests a revised topology for subunit I

AbstractNumerous sequences of the cytochrome bd quinol oxidase (cytochrome bd) have recently become available for analysis. The analysis has revealed a small number of conserved residues, a new topology for subunit I and a phylogenetic tree involving extensive horizontal gene transfer. There are 20 conserved residues in subunit I and two in subunit II. Algorithms utilizing multiple sequence alignments predicted a revised topology for cytochrome bd, adding two transmembrane helices to subunit I to the seven that were previously indicated by the analysis of the sequence of the oxidase from E. coli. This revised topology has the effect of relocating the N-terminus and C-terminus to the periplasmic and cytoplasmic sides of the membrane, respectively. The new topology repositions I-H19, the putative ligand for heme b595, close to the periplasmic edge of the membrane, which suggests that the heme b595/heme d active site of the oxidase is located near the outer (periplasmic) surface of the membrane. The most highly conserved region of the sequence of subunit I contains the sequence GRQPW and is located in a predicted periplasmic loop connecting the eighth and ninth transmembrane helices. The potential importance of this region of the protein was previously unsuspected, and it may participate in the binding of either quinol or heme d. There are two very highly conserved glutamates in subunit I, E99 and E107, within the third transmembrane helix (E. coli cytochrome bd-I numbering). It is speculated that these glutamates may be part of a proton channel leading from the cytoplasmic side of the membrane to the heme d oxygen-reactive site, now placed near the periplasmic surface. The revised topology and newly revealed conserved residues provide a clear basis for further experimental tests of these hypotheses. Phylogenetic analysis of the new sequences of cytochrome bd reveals considerable deviation from the 16sRNA tree, suggesting that a large amount of horizontal gene transfer has occurred in the evolution of cytochrome bd

Functional importance of Glutamate-445 and Glutamate-99 in proton-coupled electron transfer during oxygen reduction by cytochrome bd from Escherichia coli

The recent X-ray structure of the cytochrome bd respiratory oxygen reductase showed that two of the three heme components, heme d and heme b, have glutamic acid as an axial ligand. No other native heme proteins are known to have glutamic acid axial ligands. In this work, site-directed mutagenesis is used to probe the roles of these glutamic acids, E445 and E99 in the E. coli enzyme. It is concluded that neither glutamate is a strong ligand to the heme Fe and they are not the major determinates of heme binding to the protein. Although very important, neither glutamate is absolutely essential for catalytic function. The close interactions between the three hemes in cyt bd result in highly cooperative properties. For example, mutation of E445, which is near heme d, has its greatest effects on the properties of heme b and heme b. It is concluded that 1) O binds to the hydrophilic side of heme d and displaces E445; 2) E445 forms a salt bridge with R448 within the O binding pocket, and both residues play a role to stabilize oxygenated states of heme d during catalysis; 3) E445 and E99 are each protonated accompanying electron transfer to heme d and heme b, respectively; 4) All protons used to generate water within the heme d active site come from the cytoplasm and are delivered through a channel that must include internal water molecules to assist proton transfer: [cytoplasm] → E107 → E99 (heme b) → E445 (heme d) → oxygenated heme d

Evolution of the cytochrome-bd type oxygen reductase superfamily and the function of cydAA in Archaea

Cytochrome bd-type oxygen reductases (cytbd) belong to one of three enzyme superfamilies that catalyze oxygen reduction to water. They are widely distributed in Bacteria and Archaea, but the full extent of their biochemical diversity is unknown. Here we used phylogenomics to identify 3 families and several subfamilies within the cytbd superfamily. The core architecture shared by all members of the superfamily consists of four transmembrane helices that bind two active site hemes, which are responsible for oxygen reduction. While previously characterized cytochrome bd-type oxygen reductases use quinol as an electron donor to reduce oxygen, sequence analysis shows that only one of the identified families has a conserved quinol binding site. The other families are missing this feature, suggesting that they use an alternative electron donor. Multiple gene duplication events were identified within the superfamily, resulting in significant evolutionary and structural diversity. The CydAA’ cytbd, found exclusively in Archaea, is formed by the co-association of two superfamily paralogs. We heterologously expressed CydAA’ from Caldivirga maquilingensis and demonstrated that it performs oxygen reduction with quinol as an electron donor. Strikingly, CydAA’ is the first isoform of cytbd containing only b-type hemes shown to be active when isolated, demonstrating that oxygen reductase activity in this superfamily is not dependent on heme d

Transmembrane proton translocation by cytochrome c oxidase

AbstractRespiratory heme-copper oxidases are integral membrane proteins that catalyze the reduction of molecular oxygen to water using electrons donated by either quinol (quinol oxidases) or cytochrome c (cytochrome c oxidases, CcOs). Even though the X-ray crystal structures of several heme-copper oxidases and results from functional studies have provided significant insights into the mechanisms of O2-reduction and, electron and proton transfer, the design of the proton-pumping machinery is not known. Here, we summarize the current knowledge on the identity of the structural elements involved in proton transfer in CcO. Furthermore, we discuss the order and timing of electron-transfer reactions in CcO during O2 reduction and how these reactions might be energetically coupled to proton pumping across the membrane

Structures of the intermediates in the catalytic cycle of mitochondrial cytochrome c oxidase

Cytochrome c oxidase is the terminal complex of the respiratory chains in the mitochondria of nearly all eu-karyotes. It catalyzes the reduction of molecular O-2 to water using electrons from the respiratory chain, delivered via cytochrome c on the external surface of the inner mitochondrial membrane. The protons required for water formation are taken from the matrix side of the membrane, making catalysis vectorial. This vectorial feature is further enhanced by the fact that the redox catalysis is coupled to the translocation of protons from the inside to the outside of the inner mitochondrial membrane. We are dealing with a molecular machine that converts redox free energy into a protonmotive force (pmf). Here, we review the current extensive knowledge of the structural changes in the active heme-copper site that accompany catalysis, based on a large variety of time-resolved spectroscopic experiments, X-ray and cryoEM structures, and advanced computational chemistry.Peer reviewe