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1.  The pmr Gene, Encoding a Ca2+-ATPase, Is Required for Calcium and Manganese Homeostasis and Normal Development of Hyphae and Conidia in Neurospora crassa 
Eukaryotic Cell  2012;11(11):1362-1370.
The pmr gene is predicted to encode a Ca2+-ATPase in the secretory pathway. We examined two strains of Neurospora crassa that lacked PMR: the Δpmr strain, in which pmr was completely deleted, and pmrRIP, in which the gene was extensively mutated. Both strains had identical, complex phenotypes. Compared to the wild type, these strains required high concentrations of calcium or manganese for optimal growth and had highly branched, slow-growing hyphae. They conidiated poorly, and the shape and size of the conidia were abnormal. Calcium accumulated in the Δpmr strains to only 20% of the wild-type level. High concentrations of MnCl2 (1 to 5 mM) in growth medium partially suppressed the morphological defects but did not alter the defect in calcium accumulation. The Δpmr Δnca-2 double mutant (nca-2 encodes a Ca2+-ATPase in the plasma membrane) accumulated 8-fold more calcium than the wild type, and the morphology of the hyphae was more similar to that of wild-type hyphae. Previous experiments failed to show a function for nca-1, which encodes a SERCA-type Ca2+-ATPase in the endoplasmic reticulum (B. J. Bowman, S. Abreu, E. Margolles-Clark, M. Draskovic, and E. J. Bowman, Eukaryot. Cell 10:654-661, 2011). The pmrRIP Δnca-1 double mutant accumulated small amounts of calcium, like the Δpmr strain, but exhibited even more extreme morphological defects. Thus, PMR can apparently replace NCA-1 in the endoplasmic reticulum, but NCA-1 cannot replace PMR. The morphological defects in the Δpmr strain are likely caused, in part, by insufficient concentrations of calcium and manganese in the Golgi compartment; however, PMR is also needed to accumulate normal levels of calcium in the whole cell.
doi:10.1128/EC.00105-12
PMCID: PMC3486030  PMID: 22983986
2.  Role of Four Calcium Transport Proteins, Encoded by nca-1, nca-2, nca-3, and cax, in Maintaining Intracellular Calcium Levels in Neurospora crassa ▿ 
Eukaryotic Cell  2011;10(5):654-661.
We have examined the distribution of calcium in Neurospora crassa and investigated the role of four predicted calcium transport proteins. The results of cell fractionation experiments showed 4% of cellular calcium in mitochondria, approximately 11% in a dense vacuolar fraction, 40% in an insoluble form that copurifies with microsomes, and 40% in a high-speed supernatant, presumably from large vacuoles that had broken. Strains lacking NCA-1, a SERCA-type Ca2+-ATPase, or NCA-3, a PMC-type Ca2+-ATPase, had no obvious defects in growth or distribution of calcium. A strain lacking NCA-2, which is also a PMC-type Ca2+-ATPase, grew slowly in normal medium and was unable to grow in high concentrations of calcium tolerated by the wild type. Furthermore, when grown in normal concentrations of calcium (0.68 mM), this strain accumulated 4- to 10-fold more calcium than other strains, elevated in all cell fractions. The data suggest that NCA-2 functions in the plasma membrane to pump calcium out of the cell. In this way, it resembles the PMC-type enzymes of animal cells, not the Pmc1p enzyme in Saccharomyces cerevisiae that resides in the vacuole. Strains lacking the cax gene, which encodes a Ca2+/H+ exchange protein in vacuolar membranes, accumulate very little calcium in the dense vacuolar fraction but have normal levels of calcium in other fractions. The cax knockout strain has no other observable phenotypes. These data suggest that “the vacuole” is heterogeneous and that the dense vacuolar fraction contains an organelle that is dependent upon the CAX transporter for accumulation of calcium, while other components of the vacuolar system have multiple calcium transporters.
doi:10.1128/EC.00239-10
PMCID: PMC3127652  PMID: 21335528
3.  Structure and Distribution of Organelles and Cellular Location of Calcium Transporters in Neurospora crassa▿  
Eukaryotic Cell  2009;8(12):1845-1855.
We wanted to examine the cellular locations of four Neurospora crassa proteins that transport calcium. However, the structure and distribution of organelles in live hyphae of N. crassa have not been comprehensively described. Therefore, we made recombinant genes that generate translational fusions of putative organellar marker proteins with green or red fluorescent protein. We observed putative endoplasmic reticulum proteins, encoded by grp-78 and dpm, in the nuclear envelope and associated membranes. Proteins of the vacuolar membrane, encoded by vam-3 and vma-1, were in an interconnected network of small tubules and vesicles near the hyphal tip, while in more distal regions they were in large and small spherical vacuoles. Mitochondria, visualized with tagged ARG-4, were abundant in all regions of the hyphae. Similarly, we tagged the four N. crassa proteins that transport calcium with green or red fluorescent protein to examine their cellular locations. NCA-1 protein, a homolog of the SERCA-type Ca2+-ATPase of animal cells, colocalized with the endoplasmic reticulum markers. The NCA-2 and NCA-3 proteins are homologs of Ca2+-ATPases in the vacuolar membrane in yeast or in the plasma membrane in animal cells. They colocalized with markers in the vacuolar membrane, and they also occurred in the plasma membrane in regions of the hyphae more than 1 mm from the tip. The cax gene encodes a Ca2+/H+ exchange protein found in vacuoles. As expected, the CAX protein localized to the vacuolar compartment. We observed, approximately 50 to 100 μm from the tip, a few spherical organelles that had high amounts of tagged CAX protein and tagged subunits of the vacuolar ATPase (VMA-1 and VMA-5). We suggest that this organelle, not described previously in N. crassa, may have a role in sequestering calcium.
doi:10.1128/EC.00174-09
PMCID: PMC2794220  PMID: 19801418
4.  Selective criteria for the microbiological examination of faecal specimens. 
Journal of Clinical Pathology  1992;45(9):838-839.
To assess the effectiveness of predetermined investigation criteria for the examination of faecal samples from inpatients, cultured stool specimens were prospectively examined for Salmonella spp, Shigella spp, Campylobacter spp and Clostridium difficile, and screened microscopically for intestinal parasites. Out of a total of 505 specimens, 421 (83%) fulfilled the criteria for examination for C difficile, 254 (50%) for Salmonella spp, Shigella spp, and Campylobacter spp, and 87 (17%) for intestinal parasites. Isolation rates for these organisms in those groups of patients where examination was indicated were 22.5% for C difficile and 9.1% for Salmonella spp, Shigella spp, and Campylobacter spp; the detection rate for parasites was 3.5%. In those patients where the criteria did not suggest investigation, the isolation or detection rates were 3.6% for C difficile, 0% for Salmonella spp, Shigella spp, and Campylobacter spp, and 1.7% for intestinal parasites, suggesting that the use of predetermined investigation criteria was effective.
PMCID: PMC495122  PMID: 1401225
5.  Identification and properties of an ATPase in vacuolar membranes of Neurospora crassa. 
Journal of Bacteriology  1982;151(3):1326-1337.
Using a vacuolar preparation virtually free of contamination by other organelles, we isolated vacuolar membranes and demonstrated that they contain an ATPase. Sucrose density gradient profiles of vacuolar membranes show a single peak of ATPase activity at a density of 1.11 g/cm3. Comparison of this enzyme with the two well-studied proton-pumping ATPases of Neurospora plasma membranes and mitochondria shows that it is clearly distinct. The vacuolar membrane ATPase is insensitive to the inhibitors oligomycin, azide, and vanadate, but sensitive to N,N'-dicyclohexylcarbodiimide (Ki = 2 microM). It has a pH optimum of 7.5, requires a divalent cation (Mg2+ or Mn2+) for activity, and is remarkably unaffected (+/- 20%) by a number of monovalent cations, anions, and buffers. In its substrate affinity (Km for ATP = 0.2 mM), substrate preference (ATP greater than GTP, ITP greater than UTP greater than CTP), and loss of activity with repeated 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid washes, the vacuolar membrane ATPase resembles the F1F0 type of ATPase found in mitochondria and differs from the integral membrane type of ATPase in plasma membranes.
PMCID: PMC220411  PMID: 6213602
7.  CSF-1R inhibition alters macrophage polarization and blocks glioma progression 
Nature medicine  2013;19(10):10.1038/nm.3337.
Glioblastoma multiforme (GBM) comprises several molecular subtypes including proneural GBM. Most therapeutic approaches targeting glioma cells have failed. An alternative strategy is to target cells in the glioma microenvironment, such as tumor-associated macrophages and microglia (TAMs). Macrophages depend upon colony stimulating factor (CSF)-1 for differentiation and survival. A CSF-1R inhibitor was used to target TAMs in a mouse proneural GBM model, which dramatically increased survival, and regressed established tumors. CSF-1R blockade additionally slowed intracranial growth of patient-derived glioma xenografts. Surprisingly, TAMs were not depleted in treated mice. Instead, glioma-secreted factors including GM-CSF and IFN-γ facilitated TAM survival in the context of CSF-1R inhibition. Alternatively activated/ M2 macrophage markers decreased in surviving TAMs, consistent with impaired tumor-promoting functions. These gene signatures were associated with enhanced survival in proneural GBM patients. Our results identify TAMs as a promising therapeutic target for proneural gliomas, and establish the translational potential of CSF-1R inhibition for GBM.
doi:10.1038/nm.3337
PMCID: PMC3840724  PMID: 24056773
8.  Metallic Nickel Nanoparticles May Exhibit Higher Carcinogenic Potential than Fine Particles in JB6 Cells 
PLoS ONE  2014;9(4):e92418.
While numerous studies have described the pathogenic and carcinogenic effects of nickel compounds, little has been done on the biological effects of metallic nickel. Moreover, the carcinogenetic potential of metallic nickel nanoparticles is unknown. Activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) have been shown to play pivotal roles in tumor initiation, promotion, and progression. Mutation of the p53 tumor suppressor gene is considered to be one of the steps leading to the neoplastic state. The present study examines effects of metallic nickel fine and nanoparticles on tumor promoter or suppressor gene expressions as well as on cell transformation in JB6 cells. Our results demonstrate that metallic nickel nanoparticles caused higher activation of AP-1 and NF-κB, and a greater decrease of p53 transcription activity than fine particles. Western blot indicates that metallic nickel nanoparticles induced a higher level of protein expressions for R-Ras, c-myc, C-Jun, p65, and p50 in a time-dependent manner. In addition, both metallic nickel nano- and fine particles increased anchorage-independent colony formation in JB6 P+ cells in the soft agar assay. These results imply that metallic nickel fine and nanoparticles are both carcinogenetic in vitro in JB6 cells. Moreover, metallic nickel nanoparticles may exhibit higher carcinogenic potential, which suggests that precautionary measures should be taken in the use of nickel nanoparticles or its compounds in nanomedicine.
doi:10.1371/journal.pone.0092418
PMCID: PMC3972196  PMID: 24691273
9.  Memory, Mood, and Vitamin D in Persons with Parkinson’s Disease 
Journal of Parkinson's disease  2013;3(4):547-555.
Background
Research in recent years has suggested a role of vitamin D in the central nervous system. The final converting enzyme and the vitamin D receptor are found throughout the human brain. From animal studies vitamin D appears important in neurodevelopment, up-regulation of neurotrophic factors, stabilization of mitochondrial function, and antioxidation.
Objective
To examine the relationship between serum vitamin D and neuropsychiatric function in persons with Parkinson’s disease (PD).
Methods
This is an add-on study to a longitudinal study following neuropsychiatric function in persons with PD. Baseline neuropsychiatric performance and serum 25-hydroxyvitamin D were examined for 286 participants with PD. Measures of global cognitive function (MMSE, MOCA, Mattis Dementia Scale), verbal memory (Hopkins Verbal Learning Test), fluency (animals, vegetables, and FAS words), visuospatial function (Benton Line Orientation), executive function (Trails Making Test and Digit-Symbol Substitution), PD severity (Hoehn & Yahr and Unified Parkinson’s Disease Rating Scale) and depression (Geriatric Depression Scale (GDS)) were administered. Multivariate linear regression assessed the association between vitamin D concentration and neuropsychiatric function, in the entire cohort as well as the non-demented and demented subsets.
Results
Using a multivariate model, higher vitamin concentrations were associated with better performance on numerous neuropsychiatric tests in the non-demented subset of the cohort. Significant associations were specifically found between vitamin D concentration and verbal fluency and verbal memory (t = 4.31, p < 0.001 and t = 3.04, p = 0.0083). Vitamin D concentrations also correlated with depression scores (t =−3.08, p = 0.0083) in the non-demented subset.
Conclusions
Higher plasma vitamin D is associated with better cognition and better mood in this sample of PD patients without dementia. Determination of causation will require a vitamin D intervention study.
doi:10.3233/JPD-130206
PMCID: PMC3966187  PMID: 24081441
Parkinson’s disease; vitamin D; dementia; depression; cognition
10.  Antiproliferative and Antiplasmodial Dimeric Phloroglucinols from Mallotus oppositifolius from the Madagascar Dry Forest1# 
Journal of natural products  2013;76(3):388-393.
Bioassay-guided fractionation of an ethanol extract of the leaves and inflorescence of Mallotus oppositifolius collected in Madagascar led to the isolation of the two new bioactive dimeric phloroglucinols, mallotojaponins B (1) and C (2), together with the known mallotophenone (3). The structures of the new compounds were determined on the basis of spectroscopic evidence, including their 1- and 2D-NMR spectra, mass spectrometry, and an X-ray crystal structure. Compounds 1 and 2 showed potent antimalarial activity against chloroquine-resistant Plasmodium falciparum, with IC50 values of 0.75 ± 0.30 and 0.14 ± 0.04 μM, while 3 was inactive in this assay. Compounds 1–3 also displayed strong antiproliferative activity against the A2780 human ovarian cancer cell line (IC50 1.10 ± 0.05, 1.3 ± 0.1 and 6.3 ± 0.4 μM, respectively).
doi:10.1021/np300750q
PMCID: PMC3606680  PMID: 23286240
11.  Emotion-based learning: insights from the Iowa Gambling Task 
Interest in the cognitive and/or emotional basis of complex decision-making, and the related phenomenon of emotion-based learning, has been heavily influenced by the Iowa Gambling Task. A number of psychological variables have been investigated as potentially important in understanding emotion-based learning. This paper reviews the extent to which humans are explicitly aware of how we make such decisions; the biasing influence of pre-existing emotional labels; and the extent to which emotion-based systems are anatomically and functionally independent of episodic memory. Review of literature suggests that (i) an aspect of conscious awareness does appear to be readily achieved during the IGT, but as a relatively unfocused emotion-based “gut-feeling,” akin to intuition; (ii) Several studies have manipulated the affective pre-loading of IGT tasks, and make it clear that such labeling has a substantial influence on performance, an experimental manipulation similar to the phenomenon of prejudice. (iii) Finally, it appears that complex emotion-based learning can remain intact despite profound amnesia, at least in some neurological patients, a finding with a range of potentially important clinical implications: in the management of dementia; in explaining infantile amnesia; and in understanding of the possible mechanisms of psychotherapy.
doi:10.3389/fpsyg.2014.00162
PMCID: PMC3968745
emotion-based learning; intuition; prejudice; psychotherapy; episodic-memory
12.  Rtp801, a suppressor of mTOR signaling, is an essential mediator of cigarette smoke – induced pulmonary injury and emphysema 
Nature medicine  2010;16(7):767-773.
Rtp801, a stress – related protein triggered by adverse environmental conditions, inhibits mTOR and enhances oxidative stress – dependent cell death. We postulated that Rtp801 acts as potential amplifying switch in the development of cigarette smoke – induced lung injury, leading to emphysema. Rtp801 was overexpressed in human emphysematous lungs and in lungs of mice exposed to cigarette smoke. The upregulation of Rtp801 expression by cigarette smoke in the lung relied on oxidative stress – dependent activation of the CCAAT response element. Rtp801 was necessary and sufficient for NF – κ B activation in cultured cells and, when forcefully expressed in mouse lungs, it promoted NF – kB activation, alveolar inflammation, oxidative stress, and apoptosis of alveolar septal cells. On the other hand, Rtp801 − / − mice were markedly protected against acute cigarette smoke – induced lung injury, partly via increased mTOR signaling, and, when exposed chronically, against emphysema. Our data support the notion that Rtp801 may represent an important molecular sensor and mediator of lung injury to cigarette smoke.
doi:10.1038/nm.2157
PMCID: PMC3956129  PMID: 20473305
Rtp801; cigarette smoke; oxidative stress; apoptosis; inflammation; NF –κB; rapamycin
13.  Countering Countermeasures: Detecting Identity Lies by Detecting Conscious Breakthrough 
PLoS ONE  2014;9(3):e90595.
One major drawback of deception detection is its vulnerability to countermeasures, whereby participants wilfully modulate their physiological or neurophysiological response to critical guilt-determining stimuli. One reason for this vulnerability is that stimuli are usually presented slowly. This allows enough time to consciously apply countermeasures, once the role of stimuli is determined. However, by increasing presentation speed, stimuli can be placed on the fringe of awareness, rendering it hard to perceive those that have not been previously identified, hindering the possibility to employ countermeasures. We tested an identity deception detector by presenting first names in Rapid Serial Visual Presentation and instructing participants to lie about their own identity. We also instructed participants to apply a series of countermeasures. The method proved resilient, remaining effective at detecting deception under all countermeasures.
doi:10.1371/journal.pone.0090595
PMCID: PMC3946631  PMID: 24608749
14.  Global Genome Response of Escherichia coli O157∶H7 Sakai during Dynamic Changes in Growth Kinetics Induced by an Abrupt Downshift in Water Activity 
PLoS ONE  2014;9(3):e90422.
The present study was undertaken to investigate growth kinetics and time-dependent change in global expression of Escherichia coli O157∶H7 Sakai upon an abrupt downshift in water activity (aw). Based on viable count data, shifting E. coli from aw 0.993 to aw 0.985 or less caused an apparent loss, then recovery, of culturability. Exponential growth then resumed at a rate characteristic for the aw imposed. To understand the responses of this pathogen to abrupt osmotic stress, we employed an integrated genomic and proteomic approach to characterize its cellular response during exposure to a rapid downshift but still within the growth range from aw 0.993 to aw 0.967. Of particular interest, genes and proteins with cell envelope-related functions were induced during the initial loss and subsequent recovery of culturability. This implies that cells undergo remodeling of their envelope composition, enabling them to adapt to osmotic stress. Growth at low aw, however, involved up-regulating additional genes and proteins, which are involved in the biosynthesis of specific amino acids, and carbohydrate catabolism and energy generation. This suggests their important role in facilitating growth under such stress. Finally, we highlighted the ability of E. coli to activate multiple stress responses by transiently inducing the RpoE and RpoH regulons to control protein misfolding, while simultaneously activating the master stress regulator RpoS to mediate long-term adaptation to hyperosmolality. This investigation extends our understanding of the potential mechanisms used by pathogenic E. coli to adapt, survive and grow under osmotic stress, which could potentially be exploited to aid the selection and/or development of novel strategies to inactivate this pathogen.
doi:10.1371/journal.pone.0090422
PMCID: PMC3940904  PMID: 24594867
15.  Development and Pilot Evaluation of Native CREST – a Cancer Research Experience and Student Training Program for Navajo Undergraduate Students 
The Mayo Clinic Cancer Center and Diné College received funding for a 4-year collaborative P20 planning grant from the National Cancer Institute in 2006. The goal of the partnership was to increase Navajo undergraduates’ interest in and commitment to biomedical coursework and careers, especially in cancer research. This paper describes the development, pilot testing and evaluation of Native CREST (Cancer Research Experience & Student Training), a 10-week cancer research training program providing mentorship in a Mayo Clinic basic science or behavioral cancer research lab for Navajo undergraduate students. Seven Native American undergraduate students (5 females, 2 males) were enrolled during the summers of 2008 - 2011. Students reported the program influenced their career goals and was valuable to their education and development. These efforts may increase the number of Native American career scientists developing and implementing cancer research, which will ultimately benefit the health of Native American people.
doi:10.1007/s13187-012-0417-1
PMCID: PMC3537881  PMID: 23001889
16.  Highly predictive ligand-based pharmacophore and homology models of ABHD6 
Chemical biology & drug design  2012;81(3):382-388.
α/β-hydrolase domain-containing 6 (ABHD6) represents a potentially attractive therapeutic target for indirectly potentiating 2-arachidonoylglycerol signaling, however, the enzyme is currently largely uncharacterized. Here we describe a five element, ligand-based pharmacophore model along with a refined homology model of ABHD6. Following a virtual screen of a modest database, both the pharmacophore and homology models were found to be highly predictive, preferentially identifying ABHD6 inhibitors over druglike noninhibitors. The models yield insight into the features required for optimal ligand binding to ABHD6 and the atomic structure of the binding site. In combination the two models should be very helpful not only in high throughput virtual screening, but also in lead optimization, and will facilitate the development of novel, selective ABHD6 inhibitors as potential drugs.
doi:10.1111/cbdd.12086
PMCID: PMC3573238  PMID: 23110439
17.  Patient-derived Models of Human Breast Cancer: Protocols for In vitro and In vivo Applications in Tumor Biology and Translational Medicine 
Research models that replicate the diverse genetic and molecular landscape of breast cancer are critical for developing the next generation therapeutic entities that can target specific cancer subtypes. Patient-derived tumorgrafts, generated by transplanting primary human tumor samples into immune-compromised mice, are a valuable method to model the clinical diversity of breast cancer in mice, and are a potential resource in personalized medicine. Primary tumorgrafts also enable in vivo testing of therapeutics and make possible the use of patient cancer tissue for in vitro screens. Described in this unit are a variety of protocols including tissue collection, biospecimen tracking, tissue processing, transplantation, and 3-dimensional culturing of xenografted tissue, that enable use of bona fide uncultured human tissue in designing and validating cancer therapies.
doi:10.1002/0471141755.ph1423s60
PMCID: PMC3630511  PMID: 23456611
Breast cancer; Tumorgraft; Organoid; 3D Culture; Matrigel; EHS matrix; Biospecimen; Tissue repository; Tissue collection; Xenograft
18.  Small RNA Profiling Reveals Phosphorus Deficiency as a Contributing Factor in Symptom Expression for Citrus Huanglongbing Disease 
Molecular Plant  2013;6(2):301-310.
SUMMARY
We identified several HLB-induced citrus small RNAs that can be potentially developed into early diagnostic markers of HLB. Induction of miR399 by Las led to the discovery that HLB-positive trees suffer from phosphorus starvation. Applying phosphorus solutions help reduce HLB symptoms.
Huanglongbing (HLB) is a devastating citrus disease that is associated with bacteria of the genus ‘Candidatus Liberibacter’ (Ca. L.). Powerful diagnostic tools and management strategies are desired to control HLB. Host small RNAs (sRNA) play a vital role in regulating host responses to pathogen infection and are used as early diagnostic markers for many human diseases, including cancers. To determine whether citrus sRNAs regulate host responses to HLB, sRNAs were profiled from Citrus sinensis 10 and 14 weeks post grafting with Ca. L. asiaticus (Las)-positive or healthy tissue. Ten new microRNAs (miRNAs), 76 conserved miRNAs, and many small interfering RNAs (siRNAs) were discovered. Several miRNAs and siRNAs were highly induced by Las infection, and can be potentially developed into early diagnosis markers of HLB. miR399, which is induced by phosphorus starvation in other plant species, was induced specifically by infection of Las but not Spiroplasma citri that causes citrus stubborn—a disease with symptoms similar to HLB. We found a 35% reduction of phosphorus in Las-positive citrus trees compared to healthy trees. Applying phosphorus oxyanion solutions to HLB-positive sweet orange trees reduced HLB symptom severity and significantly improved fruit production during a 3-year field trial in south-west Florida. Our molecular, physiological, and field data suggest that phosphorus deficiency is linked to HLB disease symptomology.
doi:10.1093/mp/sst002
PMCID: PMC3716302  PMID: 23292880
Huanglongbing; small RNA; miRNA399; disease diagnosis; phosphorus deficiency.
19.  The Genetics of Canadian Type 3 von Willebrand Disease (VWD): Further Evidence for Co-dominant Inheritance of Mutant Alleles 
Summary
Background
Type 3 von Willebrand disease (VWD) is the most severe form of the disease and is classically inherited in an autosomal recessive fashion.
Objectives
The aim of the current study was to investigate the molecular pathogenesis of a Canadian cohort of type 3 VWD patients.
Patients/Methods
34 families comprised of 100 individuals were investigated. Phenotypic data, including bleeding scores (BS), von Willebrand factor (VWF) laboratory values, and anti-VWF inhibitor status were included as well as sequence analysis.
Results
We identified 31 different mutations (20 novel): 8 frameshift, 5 splice site, 9 nonsense, 1 gene conversion, 6 missense, and 2 partial gene deletion mutations. The majority of mutations identified were in the propeptide (42%); index cases (IC) with these mutations exhibited more severe bleeding (BS=22) than those with mutations elsewhere in VWF (BS=13). 62 of 68 (91%) mutant alleles were identified. Twenty-nine IC (85%) had a VWF null genotype identified; 17 homozygous, 12 compound heterozygous. In five IC (15%), two mutant VWF alleles were not identified to explain the type 3 VWD phenotype. In four ICs only one mutant VWF allele was identified and in one IC no mutant VWF alleles were identified.
Conclusions
We have investigated the molecular pathogenesis of a Canadian cohort of type 3 VWD patients. Obligate carriers are not phenotypically silent in the Canadian population; 48% have been diagnosed with type 1 VWD. In ~50% of families in this study the inheritance pattern for type 3 VWD is co-dominant and not recessive.
doi:10.1111/jth.12130
PMCID: PMC3904644  PMID: 23311757
obligate carrier; type 3; VWD; VWF
20.  How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase 
Biochemical Journal  2014;458(Pt 3):449-458.
Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydro-genase was observed to comprise associated large and small subunits. The structure indicated that His229 from the large subunit was close to the proximal [4Fe–3S] cluster in the small subunit. In addition, His229 was observed to lie close to a buried glutamic acid (Glu73), which is conserved in oxygen-tolerant hydrogenases. His229 and Glu73 of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His229 or Glu73 with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that His229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73could play a supporting role in fine-tuning the chemistry of His229 to enable this function.
A hydrogenase consists of two subunits: a large and a small subunit. In the present study, amino acids from the large subunit were found to influence a cofactor in the small subunit, such that they help to confer oxygen-tolerance to the enzyme.
doi:10.1042/BJ20131520
PMCID: PMC3940037  PMID: 24428762
hydrogen metabolism; iron–sulphur cluster [NiFe]-hydrogenase; oxygen-tolerance; protein film electrochemistry (PFE); Salmonella enterica; BV, Benzyl Viologen; IMAC, immobilized metal-ion-affinity chromatography; PFE, protein film electrochemistry; scc, standard cubic cm; SHE, standard H2 electrode; TM, transmembrane domain
21.  Redox Initiation of Bulk Thiol-Ene Polymerizations 
Polymer chemistry  2012;4(4):1167-1175.
The unique formation-structure-property attributes and reaction behavior of the thiol-ene “click” reaction have been explored extensively for photochemically and thermally initiated reactions but have been much less explored for redox initiation. Therefore, the objective of this work is to characterize fully the impact of the initiation system, monomer structure, degree of functionalization, and inhibitor level on the redox-mediated thiol-ene polymerization rate and behavior. Moreover, this study confirms the ability of redox initiation to achieve full conversion of desired thiol-ene “click” products for small molecules in solution. For the multifunctional thiol-ene systems, polymerization rate was shown to be comparable to photo- and thermally initiated systems, but with the additional advantages of unlimited depth of cure and mild reaction conditions. Additionally, the network properties of the redox-initiated thiol-ene systems were on par with a photocured material formulated with identical monomers and radical initiating potential. Lastly, control over the polymerization rate and preceding induction period was garnered from the concentration of inhibitor included in the reaction mixture. The mechanism of action of quinone inhibition in redox-mediated thiol-ene polymerizations is shown to depend on both the presence of an aniline reducing agent and the concentration of inhibitor, with quinone concentrations in great excess of oxidizing agent concentrations actually leading to heightened polymerization rates when aniline is present.
doi:10.1039/C2PY20843A
PMCID: PMC3616679  PMID: 23565125
22.  The long term immunological response of swine after two exposures to a salmon thrombin and fibrinogen hemostatic bandage 
Experimental salmon thrombin/fibrinogen dressings have been shown to provide effective hemostasis in severe hemorrhage situations. The hypothesis for this study was that swine would still remain healthy without coagulopathy six months after exposure to salmon thrombin/fibrinogen dressings. Initial exposure was by insertion of the salmon dressing into the peritoneal cavity. Three months after the initial exposure, the same animals were subjected to two full thickness dermal wounds on the dorsal surface. One wound was bandaged with the salmon thrombin/fibrinogen bandage and the other wound was dressed with a standard bandage. The animals were monitored for an additional three months. Blood was drawn every 14 days over the six months for immunological and coagulation function analysis. All of the animals (8 pigs) remained healthy during the six month period and the dermal wounds healed without incidence. Lymph nodes and spleen showed signs of normal immune response and Western blots showed development of antibodies against salmon fibrinogen, but none of the animals made antibodies that recognized any species of thrombin. Coagulation parameters (fibrinogen concentration, thrombin time, PT and aPTT) and hematological parameters remained normal over the course of the study when compared to initial values of the subject swine.
doi:10.1016/j.biologicals.2010.07.001
PMCID: PMC3930570  PMID: 20705479
Salmon; Hemostasis; Coagulation; Antibodies; Thrombin; Fibrinogen; Bandage
23.  The Plasma Membrane Proton Pump PMA-1 Is Incorporated into Distal Parts of the Hyphae Independently of the Spitzenkörper in Neurospora crassa 
Eukaryotic Cell  2013;12(8):1097-1105.
Most models for fungal growth have proposed a directional traffic of secretory vesicles to the hyphal apex, where they temporarily aggregate at the Spitzenkörper before they fuse with the plasma membrane (PM). The PM H+-translocating ATPase (PMA-1) is delivered via the classical secretory pathway (endoplasmic reticulum [ER] to Golgi) to the cell surface, where it pumps H+ out of the cell, generating a large electrochemical gradient that supplies energy to H+-coupled nutrient uptake systems. To characterize the traffic and delivery of PMA-1 during hyphal elongation, we have analyzed by laser scanning confocal microscopy (LSCM) strains of Neurospora crassa expressing green fluorescent protein (GFP)-tagged versions of the protein. In conidia, PMA-1-GFP was evenly distributed at the PM. During germination and germ tube elongation, PMA-1-GFP was found all around the conidial PM and extended to the germ tube PM, but fluorescence was less intense or almost absent at the tip. Together, the data indicate that the electrochemical gradient driving apical nutrient uptake is generated from early developmental stages. In mature hyphae, PMA-1-GFP localized at the PM at distal regions (>120 μm) and in completely developed septa, but not at the tip, indicative of a distinct secretory route independent of the Spitzenkörper occurring behind the apex.
doi:10.1128/EC.00328-12
PMCID: PMC3754540  PMID: 23729384
24.  Genetic assessment of a summer chum salmon metapopulation in recovery 
Evolutionary Applications  2013;7(2):266-285.
Programs to rebuild imperiled wild fish populations often include hatchery-born fish derived from wild populations to supplement natural spawner abundance. These programs require monitoring to determine their demographic, biological, and genetic effects. In 1990s in Washington State, the Summer Chum Salmon Conservation Initiative developed a recovery program for the threatened Hood Canal summer chum salmon Evolutionarily Significant Unit (ESU) (the metapopulation) that used in-river spawners (wild fish) for each respective supplementation broodstock in six tributaries. Returning spawners (wild-born and hatchery-born) composed subsequent broodstocks, and tributary-specific supplementation was limited to three generations. We assessed impacts of the programs on neutral genetic diversity in this metapopulation using 16 microsatellite loci and a thirty-year dataset spanning before and after supplementation, roughly eight generations. Following supplementation, differentiation among subpopulations decreased (but not significantly) and isolation by distance patterns remained unchanged. There was no decline in genetic diversity in wild-born fish, but hatchery-born fish sampled in the same spawning areas had significantly lower genetic diversity and unequal family representation. Despite potential for negative effects from supplementation programs, few were detected in wild-born fish. We hypothesize that chum salmon natural history makes them less vulnerable to negative impacts from hatchery supplementation.
doi:10.1111/eva.12118
PMCID: PMC3927888  PMID: 24567747
conservation; hatchery impacts; microsatellites; population structure; recovery; salmonids; supplementation
25.  MicroRNA-34c is associated with emphysema severity and modulates SERPINE1 expression 
BMC Genomics  2014;15:88.
Background
MicroRNAs (MiRNA) are small non-coding RNAs that regulate gene expression. The aim of this study was to identify miRNAs differentially expressed between mild and moderately emphysematous lung, as well as their functional target mRNAs. Resected lung from patients with COPD undergoing lung cancer surgery was profiled using miRNA (Agilent Human miRNA profiler G4470 V1.01) and mRNA (OperonV2.0) microarrays. Cells of lung origin (BEAS-2B and HFL1) were profiled using mRNA microarrays (Illumina HumanHT-12 V3) after in vitro manipulation.
Results
COPD patients had mean (SD) age 68 (6) years, FEV1 72 (17)% predicted and gas transfer (KCO) 70 (10)% predicted. Five miRNAs (miR-34c, miR-34b, miR-149, miR-133a and miR-133b) were significantly down-regulated in lung from patients with moderate compared to mild emphysema as defined by gas transfer (p < 0.01). In vitro upregulation of miR-34c in respiratory cells led to down-regulation of predicted target mRNAs, including SERPINE1, MAP4K4, ZNF3, ALDOA and HNF4A. The fold change in ex-vivo expression of all five predicted target genes inversely correlated with that of miR-34c in emphysematous lung, but this relationship was strongest for SERPINE1 (p = 0.05).
Conclusion
Differences in miRNA expression are associated with emphysema severity in COPD patients. MiR-34c modulates expression of its putative target gene, SERPINE1, in vitro in respiratory cell lines and ex vivo in emphysematous lung tissue.
doi:10.1186/1471-2164-15-88
PMCID: PMC3922660  PMID: 24479666
Chronic obstructive pulmonary disease; microRNA; miR-34c; Microarray

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