Determination of interproton distances from NOESY spectra in the active site of paramagnetic metalloenzymes: cyanide-inhibited horseradish peroxidase

Two dimensional (2D) nuclear Overhauser effect (NOE) or NOESY experiments are performed on cyanide inhibited horseradish peroxidase in order to assess the prospects for obtaining quantitative interproton distances for the hyperfine shifted and paramagnetically relaxed active site signals in an intermediate sized (-44 kDa) paramagnetic metalloenzyme. This protein represents an ideal test case for such experiments because a series of structurally defined proton pairs on the heme and axial His 170 have been previously assigned. The relaxation properties of hyperfine shifted signals relevant to the experimental setup of 2D experiments and interpretation of both 1D and 2D NOE data are also investigated. NOESY spectra as a function of mixing time show that quantitative rise curves can be obtained that clearly differentiate between primary and secondary NOES even among the most strongly relaxed protons, but this requires very short mixing times in the range 0.5-3.0 ms. The sensitivity of the weak cross peaks at these short mixing times is improved by the relatively rapid pulse repetition rate and concommitant increase in the number of scans allowed by the rapid relaxation of active site protons. The paramagnetic relaxation influence, as well as the size of the protein, results in rise curves that are linear to only 1.5 ms for geminal protons and to only 3-5 ms for more weakly dipolar coupled proton pairs. However, the cross peak intensities in the linear region are shown to yield cross relaxation rates and internuclear distances for a series of assigned and orientationally invariant proton pairs that are in good agreement with their known distances. The patterns of NOESY rise curves are used both to determine the orientations of one propionate and both vinyl groups relative to the heme and to show that the axial His exhibits an orientation relative to its helix that is similar but not identical to that in crystallographically characterized cytochrome c peroxidase. Selective and nonselective 1D as well as 2D selective relaxation rate measurements for hyperfine shifted signals show that only selective or intrinsic relaxation rates can be used to optimize the setup of NOESY experiments and interpret 1D NOE data. The results of the study indicate that NOESY spectra can be expected to yield valuable quantitative structural information on the hyperfine shifted active site residues in a variety of cyanide-inhibited heme peroxidases

Feedback and focus: Exploring post-secondary students’ perceptions of feedback, mindfulness, and stress

Addressing feedback-associated stress as a barrier to learning is increasingly relevant to student success and well-being. Mindfulness practices support stress management for students during the academic feedback process. Even if students receive high-quality feedback, the receiving end of feedback can be stressful, perhaps raising feelings of anxiety, confusion, or inadequacy. Feedback literacy and mindfulness practices complement one another. Mindfulness can potentially support feedback literacy by focusing one’s attention on the tasks needed to address feedback, instead of being distracted by emotions triggered by feedback. This study, comprised of an online survey (n = 237) and focus groups (n = 6), assesses post-secondary students’ perceptions concerning feedback literacy, mindfulness, and stress, and their thoughts about digital mindfulness tools intended to support students experiencing feedback-associated stress. Recruitment of students was from courses in Health Sciences, Medical Sciences, Media Studies, and Law. The survey data demonstrate that students with greater mindfulness have significantly greater feedback literacy as well as lower stress. Focus group data shows that a broad range of affective and behavioral responses are shaped by students’ perceptions of their abilities, circumstances, and feedback itself. Although students expressed familiarity with mindfulness practices, few considered explicitly linking mindfulness to their feedback process. Nevertheless, students expressed interest regarding the development of digital mindfulness tools to alleviate feedback-associated stress and offered recommendations for implementation

Passatempo Virus, a Vaccinia Virus Strain, Brazil

Passatempo virus was isolated during a zoonotic outbreak. Biologic features and molecular characterization of hemagglutinin, thymidine kinase, and vaccinia growth factor genes suggested a vaccinia virus infection, which strengthens the idea of the reemergence and circulation of vaccinia virus in Brazil. Molecular polymorphisms indicated that Passatempo virus is a different isolate

Brazilian Vaccinia Viruses and Their Origins

Genetic diversity enables this virus to persist in Brazil and other parts of the world

Vaccinia Virus Infection in Monkeys, Brazilian Amazon

To detect orthopoxvirus in the Brazilian Amazon, we conducted a serosurvey of 344 wild animals. Neutralizing antibodies against orthopoxvirus were detected by plaque-reduction neutralizing tests in 84 serum samples. Amplicons from 6 monkey samples were sequenced. These amplicons identified vaccinia virus genetically similar to strains from bovine vaccinia outbreaks in Brazil

Monkeypox Transmission and Pathogenesis in Prairie Dogs

During May and June 2003, the first cluster of human monkeypox cases in the United States was reported. Most patients with this febrile vesicular rash illness presumably acquired the infection from prairie dogs. Monkeypox virus was demonstrated by using polymerase chain reaction in two prairie dogs in which pathologic studies showed necrotizing bronchopneumonia, conjunctivitis, and tongue ulceration. Immunohistochemical assays for orthopoxviruses demonstrated abundant viral antigens in surface epithelial cells of lesions in conjunctiva and tongue, with less amounts in adjacent macrophages, fibroblasts, and connective tissues. Viral antigens in the lung were abundant in bronchial epithelial cells, macrophages, and fibroblasts. Virus isolation and electron microscopy demonstrated active viral replication in lungs and tongue. These findings indicate that both respiratory and direct mucocutaneous exposures are potentially important routes of transmission of monkeypox virus between rodents and to humans. Prairie dogs offer insights into transmission, pathogenesis, and new vaccine and treatment trials because they are susceptible to severe monkeypox infection

Molecular and biochemical characterisation of a novel mutation in POLG associated with Alpers syndrome

<p>Abstract</p> <p>Background</p> <p>DNA polymerase γ (<it>POLG</it>) is the only known mitochondrial DNA (mtDNA) polymerase. It mediates mtDNA replication and base excision repair. Mutations in the <it>POLG </it>gene lead to reduction of functional mtDNA (mtDNA depletion and/or deletions) and are therefore predicted to result in defective oxidative phosphorylation (OXPHOS). Many mutations map to the polymerase and exonuclease domains of the enzyme and produce a broad clinical spectrum. The most frequent mutation p.A467T is localised in the linker region between these domains. In compound heterozygote patients the p.A467T mutation has been described to be associated amongst others with fatal childhood encephalopathy. These patients have a poorer survival rate compared to homozygotes.</p> <p>Methods</p> <p>mtDNA content in various tissues (fibroblasts, muscle and liver) was quantified using quantitative PCR (qPCR). OXPHOS activities in the same tissues were assessed using spectrophotometric methods and catalytic stain of BN-PAGE.</p> <p>Results</p> <p>We characterise a novel splice site mutation in <it>POLG </it>found <it>in trans </it>with the p.A467T mutation in a 3.5 years old boy with valproic acid induced acute liver failure (Alpers-Huttenlocher syndrome). These mutations result in a tissue specific depletion of the mtDNA which correlates with the OXPHOS-activities.</p> <p>Conclusions</p> <p>mtDNA depletion can be expressed in a high tissue-specific manner and confirms the need to analyse primary tissue. Furthermore<it>, POLG </it>analysis optimises clinical management in the early stages of disease and reinforces the need for its evaluation before starting valproic acid treatment.</p

One More Piece in the VACV Ecological Puzzle: Could Peridomestic Rodents Be the Link between Wildlife and Bovine Vaccinia Outbreaks in Brazil?

BACKGROUND: Despite the fact that smallpox eradication was declared by the World Health Organization (WHO) in 1980, other poxviruses have emerged and re-emerged, with significant public health and economic impacts. Vaccinia virus (VACV), a poxvirus used during the WHO smallpox vaccination campaign, has been involved in zoonotic infections in Brazilian rural areas (Bovine Vaccinia outbreaks - BV), affecting dairy cattle and milkers. Little is known about VACV's natural hosts and its epidemiological and ecological characteristics. Although VACV was isolated and/or serologically detected in Brazilian wild animals, the link between wildlife and farms has not yet been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we describe for the first time, to our knowledge, the isolation of a VACV (Mariana virus - MARV) from a mouse during a BV outbreak. Genetic data, in association with biological assays, showed that this isolate was the same etiological agent causing exanthematic lesions observed in the cattle and human inhabitants of a particular BV-affected area. Phylogenetic analysis grouped MARV with other VACV isolated during BV outbreaks. CONCLUSION/SIGNIFICANCE: These data provide new biological and epidemiological information on VACV and lead to an interesting question: could peridomestic rodents be the link between wildlife and BV outbreaks

Cytocompatibility, degradation, mechanical property retention and ion release profiles for phosphate glass fibre reinforced composite rods

Fibre reinforced composites have recently received much attention as potential bone fracture fixation applications. Bioresorbable composites based on poly lactic acid (PLA) and phosphate based glass fibre were investigated according to ion release, degradation, biocompatibility and mechanical retention profiles. The phosphate based glass fibres used in this study had the composition of 40P2O5-24MgO-16CaO-16Na2O-4Fe2O3 in mol% (P40). The degradation and ion release profiles for the composites showed similar trends with the amount of sodium and orthophosphate ions released being greater than the other cations and anions investigated. This was attributed to low Dietzal's field strength for the Na(+) in comparison with Mg(2+) and Ca(2+) and breakdown of longer chain polyphosphates into orthophosphate ions. P40 composites exhibited good biocompatibility to human mesenchymal stem cells (MSCs), which was suggested to be due to the low degradation rate of P40 fibres. After 63 days immersion in PBS at 37 °C, the P40 composite rods lost ~1.1% of mass. The wet flexural, shear and compressive strengths for P40 UD rods were ~70%, ~80% and ~50% of their initial dry values after 3 days of degradation, whereas the flexural modulus, shear and compressive strengths were ~70%, ~80%, and ~65% respectively. Subsequently, the mechanical properties remained stable for the duration of the study at 63 days. The initial decrease in mechanical properties was attributed to a combination of the plasticisation effect of water and degradation of the fibre-matrix interface, with the subsequent linear behaviour being attributed to the chemical durability of P40 fibres. P40 composite rods showed low degradation and ion release rates, good biocompatibility and maintained mechanical properties similar to cortical bone for the duration of the study. Therefore, P40 composite rods have huge potential as resorbable intramedullary nails or rods