Serial optical coherence microscopy for label-free volumetric histopathology

The observation of histopathology using optical microscope is an essential procedure for examination of tissue biopsies or surgically excised specimens in biological and clinical laboratories. However, slide-based microscopic pathology is not suitable for visualizing the large-scale tissue and native 3D organ structure due to its sampling limitation and shallow imaging depth. Here, we demonstrate serial optical coherence microscopy (SOCM) technique that offers label-free, high-throughput, and large-volume imaging of ex vivo mouse organs. A 3D histopathology of whole mouse brain and kidney including blood vessel structure is reconstructed by deep tissue optical imaging in serial sectioning techniques. Our results demonstrate that SOCM has unique advantages as it can visualize both native 3D structures and quantitative regional volume without introduction of any contrast agents

Quality of life: international and domestic students studying medicine in New Zealand

International students form a significant proportion of students studying within universities in Western countries. The quality of life perceptions of international medical students in comparison with domestic medical students has not been well documented. There is some evidence to suggest that international medical students may have different educational and social experiences in relation to their domestic peers. This study investigates the levels of quality of life experienced by international and domestic students studying medicine. A total of 548 medical students completed the abbreviated version of the World Health Organization Quality of Life questionnaire. The focus of the analysis was to evaluate differences between international and domestic students in their early clinical years. The responses were analysed using multivariate analysis of variance methods. International medical students are experiencing lower social and environmental quality of life compared with domestic peers. International medical students in New Zealand have expressed quality of life concerns, which likely have an impact on their academic achievement, feelings of wellness, acculturation, and social adaptation. The findings reinforce the need for creating stronger social networks and accessible accommodation, as well as developing systems to ensure safety, peer mentorship and student support.published_or_final_versio

Control of substrate access to the active site in methane monooxygenase

Methanotrophs consume methane as their major carbon source and have an essential role in the global carbon cycle by limiting escape of this greenhouse gas to the atmosphere. These bacteria oxidize methane to methanol by soluble and particulate methane monooxygenases (MMOs). Soluble MMO contains three protein components, a 251-kilodalton hydroxylase (MMOH), a 38.6-kilodalton reductase (MMOR), and a 15.9-kilodalton regulatory protein (MMOB), required to couple electron consumption with substrate hydroxylation at the catalytic diiron centre of MMOH. Until now, the role of MMOB has remained ambiguous owing to a lack of atomic-level information about the MMOH–MMOB (hereafter termed H–B) complex. Here we remedy this deficiency by providing a crystal structure of H–B, which reveals the manner by which MMOB controls the conformation of residues in MMOH crucial for substrate access to the active site. MMOB docks at the α[subscript 2]β[subscript 2] interface of α[subscript 2]β[subscript 2]γ[subscript 2] MMOH, and triggers simultaneous conformational changes in the α-subunit that modulate oxygen and methane access as well as proton delivery to the diiron centre. Without such careful control by MMOB of these substrate routes to the diiron active site, the enzyme operates as an NADH oxidase rather than a monooxygenase. Biological catalysis involving small substrates is often accomplished in nature by large proteins and protein complexes. The structure presented in this work provides an elegant example of this principle.National Institute of General Medical Sciences (U.S.) (Grant GM 32114

Effect of carbon starvation on toluene degradation activity by toluene monooxygenase-expressing bacteria

Subsurface bacteria commonly exist in a starvation state with only periodic exposure to utilizable sources of carbon and energy. In this study, the effect of carbon starvation on aerobic toluene degradation was quantitatively evaluated with a selection of bacteria representing all the known toluene oxygenase enzyme pathways. For all the investigated strains, the rate of toluene biodegradation decreased exponentially with starvation time. First-order deactivation rate constants for TMO-expressing bacteria were approximately an order of magnitude greater than those for other oxygenase-expressing bacteria. When growth conditions (the type of growth substrate and the type and concentration of toluene oxygenase inducer) were varied in the cultures prior to the deactivation experiments, the rate of deactivation was not significantly affected, suggesting that the rate of deactivation is independent of previous substrate/inducer conditions. Because TMO-expressing bacteria are known to efficiently detoxify TCE in subsurface environments, these findings have significant implications for in situ TCE bioremediation, specifically for environments experiencing variable growth-substrate exposure conditions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45353/1/10532_2005_Article_9014.pd

Restructuring of Pancreatic Islets and Insulin Secretion in a Postnatal Critical Window

Function and structure of adult pancreatic islets are determined by early postnatal development, which in rats corresponds to the first month of life. We analyzed changes in blood glucose and hormones during this stage and their association with morphological and functional changes of alpha and beta cell populations during this period. At day 20 (d20), insulin and glucose plasma levels were two- and six-fold higher, respectively, as compared to d6. Interestingly, this period is characterized by physiological hyperglycemia and hyperinsulinemia, where peripheral insulin resistance and a high plasmatic concentration of glucagon are also observed. These functional changes were paralleled by reorganization of islet structure, cell mass and aggregate size of alpha and beta cells. Cultured beta cells from d20 secreted the same amount of insulin in 15.6 mM than in 5.6 mM glucose (basal conditions), and were characterized by a high basal insulin secretion. However, beta cells from d28 were already glucose sensitive. Understanding and establishing morphophysiological relationships in the developing endocrine pancreas may explain how events in early life are important in determining adult islet physiology and metabolism

Long-Range Intra-Protein Communication Can Be Transmitted by Correlated Side-Chain Fluctuations Alone

Allosteric regulation is a key component of cellular communication, but the way in which information is passed from one site to another within a folded protein is not often clear. While backbone motions have long been considered essential for long-range information conveyance, side-chain motions have rarely been considered. In this work, we demonstrate their potential utility using Monte Carlo sampling of side-chain torsional angles on a fixed backbone to quantify correlations amongst side-chain inter-rotameric motions. Results indicate that long-range correlations of side-chain fluctuations can arise independently from several different types of interactions: steric repulsions, implicit solvent interactions, or hydrogen bonding and salt-bridge interactions. These robust correlations persist across the entire protein (up to 60 Å in the case of calmodulin) and can propagate long-range changes in side-chain variability in response to single residue perturbations

Role of Visible Light-Activated Photocatalyst on the Reduction of Anthrax Spore-Induced Mortality in Mice

BACKGROUND: Photocatalysis of titanium dioxide (TiO(2)) substrates is primarily induced by ultraviolet light irradiation. Anion-doped TiO(2) substrates were shown to exhibit photocatalytic activities under visible-light illumination, relative environmentally-friendly materials. Their anti-spore activity against Bacillus anthracis, however, remains to be investigated. We evaluated these visible-light activated photocatalysts on the reduction of anthrax spore-induced pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Standard plating method was used to determine the inactivation of anthrax spore by visible light-induced photocatalysis. Mouse models were further employed to investigate the suppressive effects of the photocatalysis on anthrax toxin- and spore-mediated mortality. We found that anti-spore activities of visible light illuminated nitrogen- or carbon-doped titania thin films significantly reduced viability of anthrax spores. Even though the spore-killing efficiency is only approximately 25%, our data indicate that spores from photocatalyzed groups but not untreated groups have a less survival rate after macrophage clearance. In addition, the photocatalysis could directly inactivate lethal toxin, the major virulence factor of B. anthracis. In agreement with these results, we found that the photocatalyzed spores have tenfold less potency to induce mortality in mice. These data suggest that the photocatalysis might injury the spores through inactivating spore components. CONCLUSION/SIGNIFICANCE: Photocatalysis induced injuries of the spores might be more important than direct killing of spores to reduce pathogenicity in the host

Clinical and Immunological Phenotype of Patients With Primary Immunodeficiency Due to Damaging Mutations in NFKB2

Non-canonical NF-κB-pathway signaling is integral in immunoregulation. Heterozygous mutations in NFKB2 have recently been established as a molecular cause of common variable immunodeficiency (CVID) and DAVID-syndrome, a rare condition combining deficiency of anterior pituitary hormone with CVID. Here, we investigate 15 previously unreported patients with primary immunodeficiency (PID) from eleven unrelated families with heterozygous NFKB2-mutations including eight patients with the common p.Arg853* nonsense mutation and five patients harboring unique novel C-terminal truncating mutations. In addition, we describe the clinical phenotype of two patients with proximal truncating mutations. Cohort analysis extended to all 35 previously published NFKB2-cases revealed occurrence of early-onset PID in 46/50 patients (mean age of onset 5.9 years, median 4.0 years). ACTH-deficiency occurred in 44%. Three mutation carriers have deceased, four developed malignancies. Only two mutation carriers were clinically asymptomatic. In contrast to typical CVID, most patients suffered from early-onset and severe disease manifestations, including clinical signs of T cell dysfunction e.g., chronic-viral or opportunistic infections. In addition, 80% of patients suffered from (predominately T cell mediated) autoimmune (AI) phenomena (alopecia > various lymphocytic organ-infiltration > diarrhea > arthritis > AI-cytopenia). Unlike in other forms of CVID, auto-antibodies or lymphoproliferation were not common hallmarks of disease. Immunophenotyping showed largely normal or even increased quantities of naïve and memory CD4+ or CD8+ T-cells and normal T-cell proliferation. NK-cell number and function were also normal. In contrast, impaired B-cell differentiation and hypogammaglobinemia were consistent features of NFKB2-associated disease. In addition, an array of lymphocyte subpopulations, such as regulatory T cell, Th17-, cTFH-, NKT-, and MAIT-cell numbers were decreased. We conclude that heterozygous damaging mutations in NFKB2 represent a distinct PID entity exceeding the usual clinical spectrum of CVID. Impairment of the non-canonical NF-κB pathways affects function and differentiation of numerous lymphocyte-subpopulations and thus causes a heterogeneous, more severe form of PID phenotype with early-onset. Further characteristic features are multifaceted, primarily T cell-mediated autoimmunity, such as alopecia, lymphocytic organ infiltration, and in addition frequently ACTH-deficiency

Measured and Simulated Nitrous Oxide Emissions from Ryegrass- and Ryegrass/White Clover-Based Grasslands in a Moist Temperate Climate

There is uncertainty about the potential reduction of soil nitrous oxide (N2O) emission when fertilizer nitrogen (FN) is partially or completely replaced by biological N fixation (BNF) in temperate grassland. The objectives of this study were to 1) investigate the changes in N2O emissions when BNF is used to replace FN in permanent grassland, and 2) evaluate the applicability of the process-based model DNDC to simulate N2O emissions from Irish grasslands. Three grazing treatments were: (i) ryegrass (Lolium perenne) grasslands receiving 226 kg FN ha−1 yr−1 (GG+FN), (ii) ryegrass/white clover (Trifolium repens) grasslands receiving 58 kg FN ha−1 yr−1 (GWC+FN) applied in spring, and (iii) ryegrass/white clover grasslands receiving no FN (GWC-FN). Two background treatments, un-grazed swards with ryegrass only (G–B) or ryegrass/white clover (WC–B), did not receive slurry or FN and the herbage was harvested by mowing. There was no significant difference in annual N2O emissions between G–B (2.38±0.12 kg N ha−1 yr−1 (mean±SE)) and WC-B (2.45±0.85 kg N ha−1 yr−1), indicating that N2O emission due to BNF itself and clover residual decomposition from permanent ryegrass/clover grassland was negligible. N2O emissions were 7.82±1.67, 6.35±1.14 and 6.54±1.70 kg N ha−1 yr−1, respectively, from GG+FN, GWC+FN and GWC-FN. N2O fluxes simulated by DNDC agreed well with the measured values with significant correlation between simulated and measured daily fluxes for the three grazing treatments, but the simulation did not agree very well for the background treatments. DNDC overestimated annual emission by 61% for GG+FN, and underestimated by 45% for GWC-FN, but simulated very well for GWC+FN. Both the measured and simulated results supported that there was a clear reduction of N2O emissions when FN was replaced by BNF