How I maximised my training during the COVID-19 pandemic.

The COVID-19 pandemic was declared in March 2020 and London maternity units were among the first in the United Kingdom to report maternal infection and vertical transmission. To manage resources, over half of all Obstetrics and Gynaecology trainees were redeployed to support front-line specialities such as Core Medicine and Accident and Emergency. The vignettes in this article illustrate how three trainees maximised their limited training opportunities in the face of exceptional disruption, lack of surgical training opportunities and workload pressures.

Should wrong extraction site be classed as a never event?

NHS England has set out plans for combatting the rise in never events in the realm of dentistry. This Opinion article investigates the root cause of never events, whether a wrong site surgery should be deemed a never event, and whether the approaches adopted to reduce risk exposure, such as local safety standards for invasive procedures (LocSSIPS), are helping or hindering safe medical care.

The efficacy of student-centered instruction in supporting science learning.

Transforming science learning through student-centered instruction that engages students in a variety of scientific practices is central to national science-teaching reform efforts. Our study employed a large-scale, randomized-cluster experimental design to compare the effects of student-centered and teacher-centered approaches on elementary school students' understanding of space-science concepts. Data included measures of student characteristics and learning and teacher characteristics and fidelity to the instructional approach. Results reveal that learning outcomes were higher for students enrolled in classrooms engaging in scientific practices through a student-centered approach; two moderators were identified. A statistical search for potential causal mechanisms for the observed outcomes uncovered two potential mediators: students' understanding of models and evidence and the self-efficacy of teachers.

Probing the specifics of substrate binding for cytochrome c oxidase: a computer assisted approach.

A deficiency of cytochrome c oxidase (COX) is associated with a number of diseases but details of the enzyme's mechanism of action especially the interaction with its substrate, ferrocytochrome c, remain unclear. It is known that the transfer of electrons from ferrocytochrome c to COX is facilitated by the formation of enzyme-substrate (ES) complexes which are stabilized by intermolecular salt bridges, however the identity of residues participating in the salt bridges remains obscure. Using the published structures of the two proteins, computer simulations were employed to model their interactions and to attempt to identify residues that participate in intermolecular salt bridges. The simulation process was guided in the main by cross-linking studies which proposed that Lys-13 of cytochrome c is paired with Asp-158 of COX. The initial enzyme-substrate complex, created by computer assisted manipulation of the two structures exhibited three salt bridges; following the application of energy minimization procedures, the number of salt bridges increased to seven and there were twenty-four intermolecular hydrogen bonds. The salt bridges emanated from: Glu-119 and Asp-221 of subunit I; Glu-114, Asp-115 and Asp-158 of subunit II and Asp-73 and Glu-78 of subunit VIb. These were paired with Lys-87, 8, 25, 27, 13, 22 and 100 respectively of cytochrome c. These results suggest that subunits I, II and VIb play direct roles in substrate binding. The results also suggest that hydrogen bonds contribute significantly to the stability of the ES-complex.

Early electron transfer in cytochrome c oxidase occurs by a chymotrypsin type relay.

Evidence suggests that when ferrocytochrome c (the substrate) reduces cytochrome c oxidase (COX), electrons from the former enter the latter via Trp-104. What is still to be determined is the method by which electrons are transferred from ferrocytochrome c to Trp-104 and the method by which electrons arriving at Trp-104 are moved on to Cu(A), the first of the enzyme's four redox centres to be reduced. To shed light on this process, we used the computer to create and analyse an enzyme-substrate complex formed from the published structure of the two proteins. It was found that the front haem edge of ferrocytochrome c was in close proximity to Trp-104 of COX and that inclusive of Trp-104, only nine amino acid residues from COX lie along a broad channel stretching from Trp-104 to the enzyme's Cu(A) centre. Six of the nine residues, Trp-104, Tyr-105, His-102 Trp-106, Asp-158 and Glu-198, had the ideal chemical properties and were properly aligned to facilitate electron transfer. Here we propose that the reduction of Trp-104 and the subsequent reduction of Cu(A) occur by a hydride/hydrogen ion relay system similar to that seen at the active site of chymotrypsin.

Cytochrome c/cytochrome c oxidase interaction. Direct structural evidence for conformational changes during enzyme turnover.

We investigated the interaction between cytochrome c oxidase and its substrate cytochrome c by catalyzing the covalent linkage of the two proteins to yield 1 : 1 covalent enzyme-substrate complexes under conditions of low ionic strength. In addition to the 'traditional' oxidized complex formed between oxidized cytochrome c and the oxidized enzyme we prepared complexes under steady-state reducing conditions. Whereas for the 'oxidized' complex cytochrome c became bound exclusively to subunit II of the enzyme, for the 'steady-state' complex cytochrome c became bound to subunit II and two low molecular mass subunits, most likely VIb and IV. For both complexes we investigated: (a) the ability of the covalently bound cytochrome c to relay electrons into the enzyme, and (b) the ability of the covalently bound enzyme to catalyze the oxidation of unbound (exogenous) ferrocytochrome c. Steady-state spectral analysis (400-630 nm) combined with stopped-flow studies, confirmed that the bound cytochrome c mediated the efficient transfer of electrons from the reducing agent ascorbate to the enzyme. In the case of the latter, the half life for the ascorbate reduction of the bound cytochrome c and that for the subsequent transfer of electrons to haem a were both < 5 ms. In contrast the covalent complexes, when reduced, were found to be totally unreactive towards oxidized cytochrome c oxidase confirming that the previously observed reduction of haem a within the complexes occurred via intramolecular rather than intermolecular electron transfer. Additionally, stopped-flow analysis at 550 nm showed that haem a within both covalent complexes catalyzed the oxidation of exogenous ferrocytochrome c: The second order rate constant for the traditional complex was 0.55x10(6) m(-1) x s(-1) while that for the steady-state was 0.27x10(6) m(-1) x s(-1). These values were approximately 25-50% of those observed for 1 : 1 electrostatic complexes of similar concentrations. These results combined with those of the ascorbate and the electrophoresis studies suggest that electrons are able to enter cytochrome c oxidase via two independent pathways. We propose that during enzyme turnover the enzyme cycles between two conformers, one with a substrate binding site at subunit II and the other along the interface of subunits II, IV and VIb. Structural analysis suggests that Glu112, Glu113, Glu114 and Asp125 of subunit IV and Glu40, Glu54, Glu78, Asp35, Asp49, Asp73 and Asp74 of subunit VIb are residues that might possibly be involved.

Investigating the enzyme-substrate interactions of cytochrome c oxidase: a computer-assisted approach [abstract]

Although the enzyme cytochrome c oxidase (COX) is critical to respiration and has been studied extensively, the interactions between this enzyme and its substrate cytochrome c are still not very well understood. We employed a computer assisted approach to study these interactions. We used the Swiss-pdb v 2.5 computer programme, which measures the percentage accessibility of residues and the online server ANOLEA, which calculates the non-local energy of residues in a polypeptide chain, to analyze the respective molecular structures of cytochrome c and COX. The accessibility studies showed that, compared to the oxidized, the reduced form of cytochrome c normally had the greater proportion of more highly accessible residues: for reduced cytochrome c, 7 of the 8 residues exhibiting 55 percent accessibility were lysines. Interestingly enough, lysine 13, shown by other studies to be important for substrate binding, was not significantly accessible. For COX, neither asparate 158 nor glutamate 198 residues, reported to be important for substrate binding and catalysis, were significantly accessible either. The energy studies showed that whereas oxidized cytochrome c was a stable structure of low energy, approximatley 81 percent of the protein was reduced. For COX, a few small regions, including 4 residues in the vicinity of CuA, which functions as the enzyme's electron entry port, were of high energy. Since lysine 13, aspartate 158 and glutamate 198 are known to play important roles in enzyme-substrate interactions, it must be that these residues become more accessible when the two proteins interact. The results of the accessibility studies therefore appear to suggest that COX employs a mix of induced-fit and strain mechanisms when it binds substrate. On the basis of the energy studies, we conclude that the structure of reduced cytochrome c (the substrate) resembles a high energy transition-state intermediate and that when this protein binds and reduces oxidized COX, the structures of both proteins are stabilized. (AU)

Cytochrome c oxidase: structural evidence for conformational switching between two substrate binding sites [abstract]

Cytochrome c oxidase, the final member of the electron transport chain is critical to aerobic respiration and the absence, deficiency or malnutrition of this enzyme causes a number of myopathies and other diseases, some of which are fatal. In spite of its importance, the enzyme is still not well understood and whether one or two binding sites for its substrate cytochrome c remains unresolved. In an attempt to answer this question, we prepared 1:1 covalent enzyme-substrate complexes under conditions of low ionic strength. In addition to the `traditional' complex formed between oxidized cytochrome c and the oxidized enzyme, we prepared a new complex under `steady-state' reducing conditions. For both complexes, we investigated the ability of the covalently bound enzyme to catalyze the oxidation of unbound (exogenous) ferrocytochrome c. Whereas for the `traditional' oxidized complex, cytochrome c became bound exclusively to subunit II of the enzyme, for the `steady-state'complex, cytochrome c became bound to subunits II, VIa and VIb. Steady-state spectral analysis (400-630nm), combined with Stopped-Flow studies, confirmed that the bound cytochrome c mediated the efficient transfer of electrons from the reducing agent ascorbate to the enzyme. Additionally, pre-steady state analysis at 550nm showed haem a within both covalent complexes catalyzed the oxidation of exogenous ferrocytochroms c. The second order rate constant for these reactions was approximately 25-50 percent of those observed for controls. Our results suggests (i) that electrons are able to enter cytochrome c oxidase via two independent pathways and (ii) that during enzyme turnover the enzyme cycles between two conformers, one of with a substrate binding site at subunit II and the other wth a site at subunits VIa and VIb. Structural analysis suggests that Glu 43, Asp 64 and Glu 83 of subunit VIa and Asp 73, Asp 74 and Glu 78 of subunit VIb are residues that might possibly be involved at the latter site. (AU)

Cytochrome-c detection: a diagnostic marker for myocardial infarction.

Following a myocardial infarction (MI) cells die or are damaged and their contents leak into the blood circulation, resulting in elevated serum levels of various enzymes, proteins, and organic molecules. Over the past few decades, it has become standard practice to employ the detection of these elevated substances as markers for the confirmation of MIs and to monitor MI patients' response to treatment. Although it has previously been shown that cytochrome-c, a small respiratory protein, is among those elevated, the lack of a suitable detection system has prevented its routine use in the diagnosis of MIs. We present a preliminary study in which chemiluminescence was employed to detect elevated levels of cytochrome-c in the serum of MI patients. The technique, which is specific for c-type proteins, is approx 30 times more sensitive than the traditional Coomassie blue stain and can detect as little as 0.03 microg of protein. It also has potential for diagnostic use in other diseases that are characterized by mitochondrial damage.

Identification and characterization of a recombinant metallothionein protein from a marine alga, Fucus vesiculosus.

A cDNA library was constructed from macroalgae adapted to prolonged elevated environmental copper levels. To investigate the possible existence of a metallothionein (MT) gene, the library was screened with degenerate probes designed using plant MT cysteine-rich motifs. A gene was identified (1229 bp) with a putative open reading frame (204 bp) encoding a 67-amino-acid protein exhibiting several characteristic features of MT proteins, including 16 cysteine residues (24%) and only one aromatic residue. Although the protein sequence showed high identity with plant and invertebrate MTs, it contained a unique 'linker' region (14 amino acid residues) between the two putative metal-binding domains which contained no cysteine residues. This extended linker is larger than the tripeptide found in archetypal vertebrate MTs, but does not conform either with the 40-amino-acid linkers commonly found in plant MT sequences. An S-peptide Fucus MT fusion protein expressed in Escherichia coli exhibited a relative molecular mass of approximately 14 kDa. The recombinant fusion bound seven Cd ions, of which 50% were dissociated at pH 4.1. Under anaerobic conditions, the Cd ions were displaced by Cu(I), which associated with the protein at a ratio of 13:1. Laboratory exposure of F. vesiculosus to elevated copper resulted in induction of the MT gene. Thus this paper describes, for the first time, an MT gene identified from macroalgae which is induced by copper exposure and whose encoded protein product binds cadmium and copper.

Cytochrome-c oxidase isolated from the brain of swayback-diseased sheep displays unusual structure and uncharacteristic kinetics.

Swayback disease, a neurodegenerative disorder of lambs, and Menkes disease, the human equivalent, are caused by a deficiency of dietary copper. Reports of low enzymic activity suggest that several copper-containing enzymes, including cytochrome-c oxidase (COX), may influence the progress of these diseases. To investigate its role in the development of neurodegenerative disorders, in particular swayback disease, we isolated COX from the brains and livers of swayback-diseased lambs. Comparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with densitometric analysis revealed that whereas the structure of COX from the liver of diseased animals was normal, the corresponding brain enzyme was subunits II-, III-, and IV-deficient; the deficiency was 55, 30, and 65% respectively. The activities of liver and brain COX from normal and diseased lambs were compared by polarographic assay at low ionic strength. Whereas the enzyme from normal brains and both forms of the liver enzyme yielded characteristic biphasic Eadie-Hofstee plots, the brain enzyme from diseased animals displayed a single phase with a K(m) of 4.7 +/- 2.4 x 10(-6) M: the K(m) values of COX from the normal brain were 12 +/- 2.5 x 10(-6) and 5.5 +/- 0.5 x 10(-7) M. We conclude that the altered enzyme structure accounts for the uncharacteristic kinetics and low activity we have observed for the isolated brain enzyme. We also conclude that the altered enzyme structure partly accounts for the low oxidase activity and decreased ATP synthesis that has been widely reported for brain tissue from swayback-diseased animals. We postulate that the subunit deficiency probably results from incomplete crosslinking between the subunits and the membrane, and predict that similar structural and kinetic factors may also account for low COX activity in Menkes disease.

The cellular basis for the relative resistance of parvalbumin and calretinin immunoreactive neocortical neurons to the pathology of Alzheimer's disease.

The vulnerability of nerve cells to the neurofibrillary pathology of Alzheimer's disease (AD) may be determined by the presence within them of certain cytoskeletal proteins. Fluorescence multiple labeling was used to assess the vulnerability of two separate subpopulations of nonpyramidal neurons in the superior frontal gyrus, distinguished by their content of the calcium-binding proteins parvalbumin (PV) and calretinin (CR), to the neuropathology of AD. In AD, counterstaining PV- and CR-labeled sections with thioflavine S demonstrated that the great majority of these cells did not contain neurofibrillary tangles, except for the large CR-immunoreactive neurons in layer I. This latter group of cells was also characterized as containing neurofilament (NF) triplet proteins, whereas other CR-labeled cortical neurons were not immunoreactive for NF. There was also a small AD-related increase in the proportion of PV-labeled cells showing NF protein immunoreactivity (1-9% of the total population in AD cases compared to 0-0.4% in non-AD cases), which likewise may be linked to the susceptibility of a minute proportion (0-0.7%) of these neurons to form neurofibrillary tangles in AD. These data are further evidence that the presence of NF in cortical nerve cells is linked to their vulnerability to the pathological process underlying AD.

Dystrophic neurite formation associated with age-related beta amyloid deposition in the neocortex: clues to the genesis of neurofibrillary pathology.

The formation of dystrophic neurites associated with beta amyloid plaques in Alzheimer's disease (AD) appears to involve a transformation of normal neuronal cytoskeletal proteins. In order to investigate what may be the earliest neuronal changes associated with the development of dystrophic neurites, we have examined the neurochemical profile of abnormal neuritic processes associated with the beta amyloid deposition in non-AD, aged cases. In all non-AD individuals demonstrating some degree of beta amyloid deposition in the superior frontal gyrus, clustered swollen and ring-like structures, located principally in layers II and III, were labeled with antibodies to phosphorylated and nonphosphorylated domains of the middle and high molecular weight neurofilament subunits. These abnormal neurites were not immunolabeled for tau or ubiquitin or stained with thioflavine S. Double labeling for neurofilaments and thioflavine S confirmed that these clusters of dystrophic neurites were associated with plaque-like deposits. These results show that anatomically and neurochemically specific forms of dystrophic neurites can occur in non-AD cases that contain beta amyloid deposition. If these abnormal neurites correspond to an immature form of the dystrophic neurites found in the neuritic plaques of Alzheimer's disease, then neurofibrillary pathology associated with this disease may begin with an initial misprocessing and accumulation of neurofilament proteins. Furthermore, these data are consistent with the proposal that the development of neurofibrillary pathology may begin with neurofilamentous hypertrophy in damaged distal processes followed by reactive changes in the cell bodies of origin of these fibers involving cytoskeletal alterations that ultimately lead to neurofibrillary tangle formation.

Dental family stress and coping patterns.

This exploratory study of 28 married male dentists and their families was designed to gain an understanding about the stressors that dentists and their spouses experience, the life events and family strains they incur, the behavioral coping patterns they utilize, and their psychosocial characteristics. The study found that although stable dental families did encounter a significant number of stressors arising from both the dental practice and the family, they maintained their sense of balance through strong family coping skills and family resources. The effect of the dentist's office-related stress was directly felt in the family, especially by the spouse. Strong coping patterns resulted when dentists and spouses maintained a balance of time and responsibility, satisfaction in work and family activity, regular communication, sharing of decision making, good physical health, and the inclusion of an active exercise program within multiple demands on their time.

High density lipoprotein cholesterol and other serum lipids in New Zealand Maoris.

The pattern of fasting serum lipids, with emphasis on high density lipoprotein cholesterol, and the relationship of the lipids with each other and with other risk factors is examined in a population based sample of New Zealand Maoris. There are no sex differences in the distribution of total cholesterol and cholesterol fractions but triglycerides are higher in men. High density lipoprotein cholesterol levels are lower in Maoris than reported in other populations. High density lipoprotein cholesterol is negatively correlated with low density lipoprotein cholesterol but not associated with total cholesterol. High density lipoprotein cholesterol is negatively correlated with body mass index and in men high density lipoprotein cholesterol levels are higher in current alcohol drinkers. The possible relationship between the low levels of high density lipoprotein cholesterol and the high risk of coronary heart disease in Maoris requires investigation.

High-density-lipoprotein cholesterol and other serum lipids in a New Zealand biracial adolescent sample. The Wairoa College survey.

Among New Zealand adolescents, Maoris have lower serum high-density-lipoprotein cholesterol levels and higher serum triglyceride levels than non-Maoris. Boys have lower high-density-lipoprotein cholesterol and triglyceride levels than girls. Low-density-lipoprotein cholesterol levels do not show sex/race differences. These findings are reflected in an excess of hyperlipidaemia type IV in Maori girls. There may be a relation between these lipid distributions and the excess Maori mortality, especially among females, from ischaemic heart-disease.