Publications by authors named "Laura Neumann"

42 Publications

Dynamics and healing behavior of metallosupramolecular polymers.

Sci Adv 2021 Apr 28;7(18). Epub 2021 Apr 28.

Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700 Fribourg, Switzerland.

Self-healing or healable polymers can recuperate their function after physical damage. This process involves diffusion of macromolecules across severed interfaces until the structure of the interphase matches that of the pristine material. However, monitoring this nanoscale process and relating it to the mechanical recovery remain elusive. We report that studying diffusion across healed interfaces and a correlation of contact time, diffusion depth, and mechanical properties is possible when two metallosupramolecular polymers assembled with different lanthanoid salts are mended. The materials used display similar properties, while the metal ions can be tracked with high spatial resolution by energy-dispersive x-ray spectrum imaging. We find that healing actual defects requires an interphase thickness in excess of 100 nm, 10 times more than previously established for self-adhesion of smooth films of glassy polymers.
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http://dx.doi.org/10.1126/sciadv.abe4154DOI Listing
April 2021

Short P-Wave Duration is a Marker of Higher Rate of Atrial Fibrillation Recurrences after Pulmonary Vein Isolation: New Insights into the Pathophysiological Mechanisms Through Computer Simulations.

J Am Heart Assoc 2021 Jan 7;10(2):e018572. Epub 2021 Jan 7.

Division of Cardiology Cardiocentro Ticino Lugano Switzerland.

Background Short ECG P-wave duration has recently been demonstrated to be associated with higher risk of atrial fibrillation (AF). The aim of this study was to assess the rate of AF recurrence after pulmonary vein isolation in patients with a short P wave, and to mechanistically elucidate the observation by computer modeling. Methods and Results A total of 282 consecutive patients undergoing a first single-pulmonary vein isolation procedure for paroxysmal or persistent AF were included. Computational models studied the effect of adenosine and sodium conductance on action potential duration and P-wave duration (PWD). About 16% of the patients had a PWD of 110 ms or shorter (median PWD 126 ms, interquartile range, 115 ms-138 ms; range, 71 ms-180 ms). At Cox regression, PWD was significantly associated with AF recurrence (=0.012). Patients with a PWD <110 ms (hazard ratio [HR], 2.20; 95% CI, 1.24-3.88; =0.007) and patients with a PWD ≥140 (HR, 1.87, 95% CI, 1.06-3.30; =0.031) had a nearly 2-fold increase in risk with respect to the other group. In the computational model, adenosine yielded a significant reduction of action potential duration 90 (52%) and PWD (7%). An increased sodium conductance (up to 200%) was robustly accompanied by an increase in conduction velocity (26%), a reduction in action potential duration 90 (28%), and PWD (22%). Conclusions One out of 5 patients referred for pulmonary vein isolation has a short PWD which was associated with a higher rate of AF after the index procedure. Computer simulations suggest that shortening of atrial action potential duration leading to a faster atrial conduction may be the cause of this clinical observation.
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http://dx.doi.org/10.1161/JAHA.120.018572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955300PMC
January 2021

It is currently unknown whether SARS-CoV-2 is viable in semen or whether COVID-19 damages spermatozoa.

Andrology 2021 01 14;9(1):30-32. Epub 2020 Jun 14.

Department of Global Health, The George Washington University, Washington, DC, USA.

Research is needed to understand the presence of the SARS-CoV-2 virus in semen, sexual transmissibility, and impact on sperm quality. Several studies have examined men recovering from COVID-19, but large-scale community-based testing is needed to ascertain the effects on the male reproductive tract, and the potential for prolonged transmission.
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http://dx.doi.org/10.1111/andr.12831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7300609PMC
January 2021

Gut microbiome differences between wild and captive black rhinoceros - implications for rhino health.

Sci Rep 2019 05 28;9(1):7570. Epub 2019 May 28.

Smithsonian's National Zoo and Conservation Biology Institute, Front Royal, VA, USA.

A number of recent studies have shown the importance of the mammalian gut microbiome in host health. In the context of endangered species, a few studies have examined the relationship between the gut microbiome in wild versus captive populations due to digestive and other health issues. Unfortunately, the results seem to vary across taxa in terms of captive animals having higher, lower, or equivalent microbiome diversity relative to their wild counterparts. Here, we focus on the black rhinoceros as captive animals suffer from a number of potentially dietary related health effects. We compared gut microbiomes of wild and captive black rhinos to test for differences in taxonomic diversity (alpha and beta) and in functional diversity of the microbiome. We incorporated a more powerful metagenomic shotgun sequencing approach rather than a targeted amplification of the 16S gene for taxonomic assignment of the microbiome. Our results showed no significant differences in the alpha diversity levels between wild and captive black rhinos, but significant differences in beta diversity. We found that bacterial taxa traditionally associated with ruminant guts of domesticated animals had higher relative abundances in captive rhinos. Our metagenomic sequencing results suggest that unknown gut microbes of wild rhinos are being replaced by those found in conventional human-domesticated livestock. Wild rhinos have significantly different functional bacterial communities compared to their captive counterparts. Functional profiling results showed greater abundance of glycolysis and amino acid synthesis pathways in captive rhino microbiomes, representing an animal receiving sub-optimal nutrition with a readily available source of glucose but possibly an imbalance of necessary macro and micronutrients. Given the differences observed between wild and captive rhino gut microbiomes, we make a number of recommendations for potentially modifying captive gut microbiome to better reflect their wild counterparts and thereby hopefully improve overall rhino health in captivity.
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http://dx.doi.org/10.1038/s41598-019-43875-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538756PMC
May 2019

T Cell-Dependent Maturation of Pathogen-Specific Igs in the Antrum of Chronically -Infected Patients.

J Immunol 2019 07 17;203(1):208-215. Epub 2019 May 17.

Medizinische Klinik für Gastroenterologie, Infektiologie und Rheumatologie, Charité - Universitätsmedizin Berlin, 12203 Berlin, Germany.

Mucosal plasma cells (PC) and Ig production are essential to fend pathogens and to maintain mucosal homeostasis. In human infection, mucosal PC express inducible NO synthase (iNOS), which positively correlates with clearance of experimental human infection. To characterize Ig genes and specificities of antral mucosal iNOS and iNOS PC in infection, we sequenced rearranged Ig genes from single cell-sorted PC from biopsy specimens of chronically infected patients and analyzed them with respect to their molecular features. The binding specificity of individual PC's Ig was determined following recombinant expression. We identified high rates of somatic hypermutations, especially targeting RGYW/WRCY hotspot motifs in the individual Ig genes, indicating T cell-dependent maturation. For seven of 14 recombinantly expressed Ig, Ag specificity could be determined. Two clones reacted to proteins, and five were found to be polyreactive against LPSs, dsDNA, and ssDNA. All specific Ig originated from iNOS PC. -specific Ig are encoded by V and J family genes previously shown to be also used in rearranged Ig loci of MALT B cell lymphomas. In summary, mucosal iNOS PC producing -specific Ig accumulate in infection and appear to be a product of T cell-dependent B cell maturation. Moreover, the Ig's molecular features partly resembled that of MALT B cell lymphoma Ig genes, suggestive of a mechanism in which a progressive molecular evolution of pathogen-specific B cells to MALT B cell lymphoma occurs.
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http://dx.doi.org/10.4049/jimmunol.1900074DOI Listing
July 2019

Healing of Polymeric Solids by Supramolecular Means.

Chimia (Aarau) 2019 Apr;73(4):277-282

Adolphe Merkle Institute (AMI), Polymer Chemistry and Materials, University of Fribourg, Chemin des Verdiers 4, CH-1700 Fribourg;, Email:

Equipping a polymeric material with the ability to heal an inflicted damage is a crucial advantage for many applications. The incorporation of reversible and dynamic supramolecular interactions into polymeric systems has proven to be a promising route towards such materials. In this article, recent developments in the field of healable materials are highlighted with a particular focus on the design principles, driving forces, and mechanisms that allow healing to occur.
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http://dx.doi.org/10.2533/chimia.2019.277DOI Listing
April 2019

Functional Polymers Through Mechanochemistry.

Chimia (Aarau) 2019 Feb;73(1):7-11

While coupling mechanical and chemical processes is widespread in living organisms, the idea to harness the mechanically induced dissociation of weak covalent and non-covalent bonds to create artificial materials that respond to mechanical stimulation has only recently gained attention. Here we summarize our activities that mainly revolve around the exploitation of non-covalent interactions in (supramolecular) polymeric materials with the goal to translate mechanical stresses into useful, pre-defined events. Focusing on mechano- chromic polymers that alter their optical absorption or fluorescence properties, several new operating principles, mechanosensitive entities, and materials systems were developed. Such materials are expected to be useful for technical applications that range from the detection of very small forces in biological systems to the monitoring of degradation processes and damage in coatings and structural objects.
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http://dx.doi.org/10.2533/chimia.2019.7DOI Listing
February 2019

A General Protein Glycosylation Gene Cluster Encodes the Species-Specific Glycan of the Oral Pathogen : -Glycan Biosynthesis and Immunological Implications.

Front Microbiol 2018 28;9:2008. Epub 2018 Aug 28.

NanoGlycobiology Unit, Department of NanoBiotechnology, Universität für Bodenkultur Wien, Vienna, Austria.

The cell surface of the oral pathogen is heavily glycosylated with a unique, complex decasaccharide that is -glycosidically linked to the bacterium's abundant surface (S-) layer, as well as other proteins. The S-layer glycoproteins are virulence factors of and there is evidence that protein glycosylation underpins the bacterium's pathogenicity. To elucidate the protein -glycosylation pathway, genes suspected of encoding pathway components were first identified in the genome sequence of the ATCC 43037 type strain, revealing a 27-kb gene cluster that was shown to be polycistronic. Using a gene deletion approach targeted at predicted glycosyltransferases (Gtfs) and methyltransferases encoded in this gene cluster, in combination with mass spectrometry of the protein-released glycans, we show that the gene cluster encodes the species-specific part of the ATCC 43037 decasaccharide and that this is assembled step-wise on a pentasaccharide core. The core was previously proposed to be conserved within the phylum, to which is affiliated, and its biosynthesis is encoded elsewhere on the bacterial genome. Next, to assess the prevalence of protein glycosylation among sp., the publicly available genome sequences of six strains were compared, revealing gene clusters of similar size and organization as found in the ATCC 43037 type strain. The corresponding region in the genome of a periodontal health-associated isolate showed a different gene composition lacking most of the genes commonly found in the pathogenic strains. Finally, we investigated whether differential cell surface glycosylation impacts 's overall immunogenicity. Release of proinflammatory cytokines by dendritic cells (DCs) upon stimulation with defined Gtf-deficient mutants of the type strain was measured and their T cell-priming potential post-stimulation was explored. This revealed that the glycan is pivotal to modulating DC effector functions, with the -specific glycan portion suppressing and the pentasaccharide core activating a Th17 response. We conclude that complex protein glycosylation is a hallmark of pathogenic strains and propose it as a valuable target for the design of novel antimicrobials against periodontitis.
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http://dx.doi.org/10.3389/fmicb.2018.02008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120980PMC
August 2018

Systemic humoral immunity in beef bulls following therapeutic vaccination against Tritrichomonas foetus.

Vet Parasitol 2018 May 31;255:69-73. Epub 2018 Mar 31.

Department of Wildlife, Ecology, and Conservation, University of Florida Institute of Food and Agricultural Sciences, Range Cattle Research and Education Center, 3401 Experiment Station, Ona, FL, 33865, United States. Electronic address:

The utility of therapeutic vaccination of bulls against Tritrichomonas foetus has been advocated in previous studies, but anecdotal reports suggest this practice does not clear infections and may additionally confound diagnostic testing by reducing parasite burdens below detectable limits. The objective of this study was to characterize the systemic humoral immune response to therapeutic vaccination in T. foetus-infected bulls over a period of four months using an indirect ELISA and to compare the dynamics of this response to culture and PCR results to establish the existence of a relationship (or lack thereof) between immunization and infection status. A study population of 4- to 6-year-old T. foetus-infected beef bulls (n = 20) was divided equally into a treatment group and a control group. The treatment group received two doses of commercially prepared whole cell killed vaccine 2 weeks apart while the control group received injections of vaccine diluent. Blood samples were collected at each injection and at 4 subsequent dates every 4 weeks thereafter (i.e. 0, 2, 6, 10, 14, and 18 wks) to measure IgG and IgG antibody subisotype response via an indirect ELISA. Preputial smegma samples were collected at the four monthly intervals following vaccination for diagnosis of infection via InPouch™ culture, Modified Diamond's Medium (MDM) culture, and PCR. Humoral response for both IgG isotypes from week 2 through week 18 were significantly increased in vaccinates compared to controls. No significant decrease in infection prevalence was detected in the treatment group for any of the diagnostic methods used. The apparent lack of pathogen clearance during a stimulated immune response suggests that therapeutic vaccination may not be a useful T. foetus management practice.
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http://dx.doi.org/10.1016/j.vetpar.2018.03.028DOI Listing
May 2018

Adductor muscle thickness of the thumb: A new and reliable parameter for nutritional assessment of pediatric inpatients.

Clin Nutr 2019 04 16;38(2):891-896. Epub 2018 Feb 16.

Graduate Program in Child and Adolescent Health, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil.

Background & Aims: The adductor pollicis muscle thickness (APMT) is a promising method for evaluation of muscle loss and, consequently, malnutrition in adult and elderly patients. However, to date, there have been no studies of its applicability to the pediatric population. Within this context, we sought to evaluate the association of APMT with anthropometric variables, body mass index (BMI), pediatric Subjective Global Assessment (SGA) of nutrition, nutritional screening, and clinical outcomes in hospitalized pediatric patients.

Methods: This was a cross-sectional study of inpatients aged 4-8.9 years, recruited via convenience sampling from a pediatric hospital in Porto Alegre, Rio Grande do Sul, Brazil. Data collection took place between December 2014 and February 2016. Patients admitted to the intensive care unit, those unable to feed orally, and those with cerebral palsy or Down syndrome were excluded from the study. General and socioeconomic information was collected and the SGA Ped and STRONGkids were administered at hospital admission. Clinical data were collected from the electronic medical record. Anthropometric parameters and APMT were measured by properly calibrated examiners. Data analysis was carried out in SPSS version 21.0. The significance level was set at 5%.

Results: The sample consisted of 447 patients. Most (55.9%) were male; the mean age was 6.2 ± 1.4 years. Low APMT was significantly associated with underweight, short stature, low body fat percentage, and poor muscle reserve (p < 0.001). There were also significant associations of moderate and severe malnutrition (assessed by the SGA Ped) and high nutritional risk (assessed by the STRONGkids instrument) with reduced APMT (p < 0.001). Regarding clinical outcomes, a longer hospital stay was observed in patients with reduced APMT (p = 0.001). A receiver operating characteristic (ROC) curve, plotted considering the SGA Ped as the gold standard, suggested APMT cutoff points of 10.2 mm for boys and 9.5 mm for girls. Stratification by age yielded APMT cutoff points of 9.8 mm for boys younger than 6 years and 10.2 mm for those older than 6 years, and 9.2 mm and 9.8 mm for girls younger and older than 6 years, respectively.

Conclusion: The APMT is an efficient parameter for the detection of malnutrition in hospitalized pediatric patients.
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http://dx.doi.org/10.1016/j.clnu.2018.02.010DOI Listing
April 2019

Mucosal Inducible NO Synthase-Producing IgA+ Plasma Cells in Helicobacter pylori-Infected Patients.

J Immunol 2016 09 25;197(5):1801-8. Epub 2016 Jul 25.

Medical Clinic I, Gastroenterology, Infectious Diseases and Rheumatology, Charité-University Medicine Berlin, 12203 Berlin, Germany;

The mucosal immune system is relevant for homeostasis, immunity, and also pathological conditions in the gastrointestinal tract. Inducible NO synthase (iNOS)-dependent production of NO is one of the factors linked to both antimicrobial immunity and pathological conditions. Upregulation of iNOS has been observed in human Helicobacter pylori infection, but the cellular sources of iNOS are ill defined. Key differences in regulation of iNOS expression impair the translation from mouse models to human medicine. To characterize mucosal iNOS-producing leukocytes, biopsy specimens from H. pylori-infected patients, controls, and participants of a vaccination trial were analyzed by immunohistochemistry, along with flow cytometric analyses of lymphocytes for iNOS expression and activity. We newly identified mucosal IgA-producing plasma cells (PCs) as one major iNOS(+) cell population in H. pylori-infected patients and confirmed intracellular NO production. Because we did not detect iNOS(+) PCs in three distinct infectious diseases, this is not a general feature of mucosal PCs under conditions of infection. Furthermore, numbers of mucosal iNOS(+) PCs were elevated in individuals who had cleared experimental H. pylori infection compared with those who had not. Thus, IgA(+) PCs expressing iNOS are described for the first time, to our knowledge, in humans. iNOS(+) PCs are induced in the course of human H. pylori infection, and their abundance seems to correlate with the clinical course of the infection.
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http://dx.doi.org/10.4049/jimmunol.1501330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4991246PMC
September 2016

Supramolecular polymers for organocatalysis in water.

Org Biomol Chem 2015 Jul;13(28):7711-9

Laboratory for Macromolecular and Organic Chemistry, Institute for Complex Molecular Sciences, TU Eindhoven, PO Box 513, 5600 MB Eindhoven, The Netherlands.

A water-soluble benzene-1,3,5-tricarboxamide (BTA) derivative that self-assembles into one-dimensional, helical, supramolecular polymers is functionalised at the periphery with one L-proline moiety. In water, the BTA-derivative forms micrometre long supramolecular polymers, which are stabilised by hydrophobic interactions and directional hydrogen bonds. Furthermore, we co-assemble a catalytically inactive, but structurally similar, BTA with the L-proline functionalised BTA to create co-polymers. This allows us to assess how the density of the L-proline units along the supramolecular polymer affects its activity and selectivity. Both the supramolecular polymers and co-polymers show high activity and selectivity as catalysts for the aldol reaction in water when using p-nitrobenzaldehyde and cyclohexanone as the substrates for the aldol reaction. After optimisation of the reaction conditions, a consistent conversion of 92 ± 7%, deanti of 92 ± 3%, and eeanti of 97 ± 1% are obtained with a concentration of L-proline as low as 1 mol%.
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http://dx.doi.org/10.1039/c5ob00937eDOI Listing
July 2015

Lymphatic fluid for the detection of Mycobacterium avium subsp. paratuberculosis in cows by PCR, compared to fecal sampling and detection of antibodies in blood and milk.

Vet Microbiol 2014 Aug 29;172(1-2):301-8. Epub 2014 May 29.

Food Animal Reproduction and Medicine Service, Department of Large Animal Clinical Sciences, University of Florida, Gainesville, FL, USA.

Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), can cause considerable economic losses in affected herds. Early diagnosis of JD is hampered by the chronic nature of the disease with a slow subclincal progression. The aim of the present study was to challenge the hypothesis that lymphatic fluid is of diagnostic value in the early stages of the disease. Lymphatic fluid from 122 animals was collected and tested for MAP by nested PCR for IS900 and compared to the results of testing for MAP in feces (culture), blood and milk (ELISA) in 110 of these samples. MAP was detected by PCR in 27.1% of the lymph samples. Agreement between the tests was poor: 6.9% of the lymph positive cows were also positive in all other tests applied, and 69.0% had negative results in fecal culture, blood and milk ELISA. Resampling of 25 cows after 8 to 12 and 16 to 20 months revealed 20.0% lymph positive animals at the first, 5.5% at the second and 27.8% at the third sampling, respectively. Only one cow showed positive lymph-PCR results at more than one sampling date. Lymph-positive cows had a 7.2 times greater likelihood of being culled within 8 to 12 months after sampling, compared to negative cows, mainly due to other health issues than JD. It can be concluded, that lymphatic fluid might be promising for the detection of early MAP-infection in cows, but further studies to elucidate the potential of this diagnostic approach are needed.
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http://dx.doi.org/10.1016/j.vetmic.2014.05.022DOI Listing
August 2014

Reduced quenching and extraction time for mammalian cells using filtration and syringe extraction.

J Biotechnol 2014 Jul 29;182-183:97-103. Epub 2014 Apr 29.

ACIB GmbH, Austrian Centre of Industrial Biotechnology, Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria. Electronic address:

In order to preserve the in vivo metabolite levels of cells, a quenching protocol must be quickly executed to avoid degradation of labile metabolites either chemically or biologically. In the case of mammalian cell cultures cultivated in complex media, a wash step previous to quenching is necessary to avoid contamination of the cell pellet with extracellular metabolites, which could distort the real intracellular concentration of metabolites. This is typically achieved either by one or multiple centrifugation/wash steps which delay the time until quenching (even harsh centrifugation requires several minutes for processing until the cells are quenched) or filtration. In this article, we describe and evaluate a two-step optimized protocol based on fast filtration by use of a vacuum pump for quenching and subsequent extraction of intracellular metabolites from CHO (Chinese hamster ovary) suspension cells, which uses commercially available components. The method allows transfer of washed cells into liquid nitrogen within 10-15s of sampling and recovers the entire extraction solution volume. It also has the advantage to remove residual filter filaments in the final sample, thus preventing damage to separation columns during subsequent MS analysis. Relative to other methods currently used in the literature, the resulting energy charge of intracellular adenosine nucleotides was increased to 0.94 compared to 0.90 with cold PBS quenching or 0.82 with cold methanol/AMBIC quenching.
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http://dx.doi.org/10.1016/j.jbiotec.2014.04.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4071440PMC
July 2014

NBDE Part II practice analyses: an overview.

J Dent Educ 2013 Dec;77(12):1566-80

2286 University Drive, Naperville, IL 60565;

The Standards for Educational and Psychological Testing emphasize the importance of documenting and describing the procedures followed in developing valid test content. As a result, the Joint Commission on National Dental Examinations, the testing agency responsible for administering the National Board Dental Examination Part I and Part II, routinely communicates information about the validity of Part II to dental schools and other communities of interest. Since 2000, the content of Part II has been validated through the use of three practice analyses. This article provides an overview of these practice analyses, including procedures and findings. In general, the findings confirm that the content of Part II is valid in determining the qualifications of individuals seeking dental licensure.
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December 2013

Multiple checkpoints for the expression of the chloroplast-encoded splicing factor MatK.

Plant Physiol 2013 Dec 30;163(4):1686-98. Epub 2013 Oct 30.

Institute for Theoretical Biology, Charité-Universitätsmedizin Berlin, D-10115 Berlin, Germany.

The chloroplast genome of land plants contains only a single gene for a splicing factor, Maturase K (MatK). To better understand the regulation of matK gene expression, we quantitatively investigated the expression of matK across tobacco (Nicotiana tabacum) development at the transcriptional, posttranscriptional, and protein levels. We observed striking discrepancies of MatK protein and matK messenger RNA levels in young tissue, suggestive of translational regulation or altered protein stability. We furthermore found increased matK messenger RNA stability in mature tissue, while other chloroplast RNAs tested showed little changes. Finally, we quantitatively measured MatK-intron interactions and found selective changes in the interaction of MatK with specific introns during plant development. This is evidence for a direct role of MatK in the regulation of chloroplast gene expression via splicing. We furthermore modeled a simplified matK gene expression network mathematically. The model reflects our experimental data and suggests future experimental perturbations to pinpoint regulatory checkpoints.
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http://dx.doi.org/10.1104/pp.113.227579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3850197PMC
December 2013

Protein O-glucosylation in Lactobacillus buchneri.

Glycoconj J 2014 Feb 27;31(2):117-31. Epub 2013 Oct 27.

Department of NanoBiotechnology, NanoGlycobiology unit, Universität für Bodenkultur Wien, Muthgasse 11, 1190, Vienna, Austria,

Based on the previous demonstration of surface (S-) layer protein glycosylation in Lactobacillus buchneri 41021/251 and because of general advantages of lactic acid bacteria for applied research, protein glycosylation in this bacterial species was investigated in detail. The cell surface of L. buchneri CD034 is completely covered with an oblique 2D crystalline array (lattice parameters, a = 5.9 nm; b = 6.2 nm; γ ~ 77°) formed by self-assembly of the S-layer protein SlpB. Biochemical and mass spectrometric analyses revealed that SlpB is the most abundant protein and that it is O-glycosylated at four serine residues within the sequence S(152)-A-S(154)-S(155)-A-S(157) with, on average, seven Glc(α1-6) residues, each. Subcellular fractionation of strain CD034 indicated a sequential order of SlpB export and glucosylation as evidenced by lack of glucosylation of cytosolic SlpB. Protein glycosylation analysis was extended to strain L. buchneri NRRL B-30929 where an analogous glucosylation scenario could be detected, with the S-layer glycoprotein SlpN containing an O-glycosylation motif identical to that of SlpB. This corroborates previous data on S-layer protein glucosylation of strain 41021/251 and let us propose a species-wide S-layer protein O-glucosylation in L. buchneri targeted at the sequence motif S-A-S-S-A-S. Search of the L. buchneri genomes for the said glucosylation motif revealed one further ORF, encoding the putative glycosyl-hydrolase LbGH25B and LbGH25N in L. buchneri CD034 and NRRL B-30929, respectively, for which we have indications of a glycosylation comparable to that of the S-layer proteins. These findings demonstrate the presence of a distinct protein O-glucosylation system in Gram-positive and beneficial microbes.
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http://dx.doi.org/10.1007/s10719-013-9505-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4396861PMC
February 2014

Expression of human butyrylcholinesterase with an engineered glycosylation profile resembling the plasma-derived orthologue.

Biotechnol J 2014 Apr 8;9(4):501-10. Epub 2013 Nov 8.

Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.

Human butyrylcholinesterase (BChE) is considered a candidate bioscavenger of nerve agents for use in pre- and post-exposure treatment. However, the presence and functional necessity of complex N-glycans (i.e. sialylated structures) is a challenging issue in respect to its recombinant expression. Here we transiently co-expressed BChE cDNA in the model plant Nicotiana benthamiana with vectors carrying the genes necessary for in planta protein sialylation. Site-specific sugar profiling of secreted recombinant BChE (rBChE) collected from the intercellular fluid revealed the presence of mono- and di-sialylated N-glycans, which largely resembles to the plasma-derived orthologue. Attempts to increase that sialylation content of rBChE by the over-expression of an additional glycosylation enzyme that generates branched N-glycans (i.e. β1,4-N-acetylglucosaminyl-transferase IV), allowed the production of rBChE decorated with tri-sialylated structures (up to 70%). Sialylated and non-sialylated plant-derived rBChE exhibited functional in vitro activity comparable to that of its commercially available equine-derived counterpart. These results demonstrate the ability of plants to generate valuable proteins with designed sialylated glycosylation profiles optimized for therapeutic efficacy. Moreover, the efficient synthesis of carbohydrates present only in minute amounts on the native protein (tri-sialylated N-glycans) facilitates the generation of a product with superior efficacies and/or new therapeutic functions.
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http://dx.doi.org/10.1002/biot.201300229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3975692PMC
April 2014

Generation of biologically active multi-sialylated recombinant human EPOFc in plants.

PLoS One 2013 25;8(1):e54836. Epub 2013 Jan 25.

Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.

Hyperglycosylated proteins are more stable, show increased serum half-life and less sensitivity to proteolysis compared to non-sialylated forms. This applies particularly to recombinant human erythropoietin (rhEPO). Recent progress in N-glycoengineering of non-mammalian expression hosts resulted in in vivo protein sialylation at great homogeneity. However the synthesis of multi-sialylated N-glycans is so far restricted to mammalian cells. Here we used a plant based expression system to accomplish multi-antennary protein sialylation. A human erythropoietin fusion protein (EPOFc) was transiently expressed in Nicotiana benthamiana ΔXTFT, a glycosylation mutant that lacks plant specific N-glycan residues. cDNA of the hormone was co-delivered into plants with the necessary genes for (i) branching (ii) β1,4-galactosylation as well as for the (iii) synthesis, transport and transfer of sialic acid. This resulted in the production of recombinant EPOFc carrying bi- tri- and tetra-sialylated complex N-glycans. The formation of this highly complex oligosaccharide structure required the coordinated expression of 11 human proteins acting in different subcellular compartments at different stages of the glycosylation pathway. In vitro receptor binding assays demonstrate the generation of biologically active molecules. We demonstrate the in planta synthesis of one of the most complex mammalian glycoforms pointing to an outstanding high degree of tolerance to changes in the glycosylation pathway in plants.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0054836PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3555983PMC
July 2013

"Cross-glycosylation" of proteins in Bacteroidales species.

Glycobiology 2013 May 19;23(5):568-77. Epub 2012 Dec 19.

Department of NanoBiotechnology, NanoGlycobiology Unit, Universität für Bodenkultur Wien, Muthgasse 11, 1190 Vienna, Austria.

While it is now evident that the two Bacteroidales species Bacteroides fragilis and Tannerella forsythia both have general O-glycosylation systems and share a common glycosylation sequon, the ability of these organisms to glycosylate a protein native to the other organism has not yet been demonstrated. Here, we report on the glycosylation of heterologous proteins between these two organisms. Using genetic tools previously developed for Bacteroides species, two B. fragilis model glycoproteins were expressed in the fastidious anaerobe T. forsythia and the attachment of the known T. forsythia O-glycan to these proteins was demonstrated by liquid chromatography electrospray ionization tandem mass spectrometry. Likewise, two predominant T. forsythia glycoproteins were expressed in B. fragilis and glycosylation with the B. fragilis O-glycan was confirmed. Purification of these proteins from B. fragilis allowed the preliminary characterization of the previously uncharacterized B. fragilis protein O-glycan. Based on mass spectrometric data, we show that the B. fragilis protein O-glycan is an oligosaccharide composed of nine sugar units. Compositional and structural similarities with the T. forsythia O-glycan suggest commonalities in their biosynthesis. These data demonstrate the feasibility of exploiting these organisms for the design of novel glycoproteins.
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http://dx.doi.org/10.1093/glycob/cws172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3608352PMC
May 2013

Characterization of α-l-Iduronidase (Aldurazyme®) and its complexes.

J Proteomics 2013 Mar 28;80:26-33. Epub 2012 Sep 28.

Department of Pediatrics, Medical University of Vienna, Vienna, Austria. Electronic address:

Alpha-l-Iduronidase(IDUA) was the first enzyme replacement therapy approved for mucopolysaccharidosis type I and the corresponding recombinant protein drug, Aldurazyme®, is commercially available. In the frame of gel-based mass spectrometrical characterization of protein drugs, we intended to identify protein sequence and possible protein modifications. Moreover, we were interested in which aggregation/complex form Aldurazyme® would exist, which complexes were enzymatically active and in which form the naturally occurring enzyme would be present in the brain. Aldurazyme® was run on 2DE gel electrophoresis, spots were excised, in-gel digested with several proteases and identified by nano-LC-ESI-MS/MS on an ion trap. IDUA-activity was determined by a fluorometric principle. Blue-native gel electrophoresis with subsequent immunoblotting was carried out to show the presence of protein complexes. The protein was unambiguously identified by 100% sequence coverage; several amino acid substitutions were detected and protein modifications were novel phosphorylations on S59 and S482, histidine methylation at H572 and provide evidence for already known N-glycosylations. Four Aldurazyme® complexes that all were enzymatically active, were observed while a single complex was observed for the physiologically occurring IDUA in the brain. The findings are relevant for understanding chemistry, physiology, pharmacology and medicine of IDUA, design of further and interpretation of previous work.
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http://dx.doi.org/10.1016/j.jprot.2012.09.022DOI Listing
March 2013

Myrosinases TGG1 and TGG2 from Arabidopsis thaliana contain exclusively oligomannosidic N-glycans.

Phytochemistry 2012 Dec 23;84:24-30. Epub 2012 Sep 23.

Department of Applied Genetics and Cell Biology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.

In all eukaryotes N-glycosylation is the most prevalent protein modification of secretory and membrane proteins. Although the N-glycosylation capacity and the individual steps of the N-glycan processing pathway have been well studied in the model plant Arabidopsis thaliana, little attention has been paid to the characterization of the glycosylation status of individual proteins. We report here the structural analysis of all N-glycans present on the endogenous thioglucoside glucohydrolases (myrosinases) TGG1 and TGG2 from A. thaliana. All nine glycosylation sites of TGG1 and all four glycosylation sites of TGG2 are occupied by oligomannosidic structures with Man₅GlcNAc₂ as the major glycoform. Analysis of the oligomannosidic isomers from wild-type plants and mannose trimming deficient mutants by liquid chromatography with porous graphitic carbon and mass spectrometry revealed that the N-glycans from both myrosinases are processed by Golgi-located α-mannosidases.
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http://dx.doi.org/10.1016/j.phytochem.2012.08.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494833PMC
December 2012

Engineering of sialylated mucin-type O-glycosylation in plants.

J Biol Chem 2012 Oct 4;287(43):36518-26. Epub 2012 Sep 4.

Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna Austria.

Proper N- and O-glycosylation of recombinant proteins is important for their biological function. Although the N-glycan processing pathway of different expression hosts has been successfully modified in the past, comparatively little attention has been paid to the generation of customized O-linked glycans. Plants are attractive hosts for engineering of O-glycosylation steps, as they contain no endogenous glycosyltransferases that perform mammalian-type Ser/Thr glycosylation and could interfere with the production of defined O-glycans. Here, we produced mucin-type O-GalNAc and core 1 O-linked glycan structures on recombinant human erythropoietin fused to an IgG heavy chain fragment (EPO-Fc) by transient expression in Nicotiana benthamiana plants. Furthermore, for the generation of sialylated core 1 structures constructs encoding human polypeptide:N-acetylgalactosaminyltransferase 2, Drosophila melanogaster core 1 β1,3-galactosyltransferase, human α2,3-sialyltransferase, and Mus musculus α2,6-sialyltransferase were transiently co-expressed in N. benthamiana together with EPO-Fc and the machinery for sialylation of N-glycans. The formation of significant amounts of mono- and disialylated O-linked glycans was confirmed by liquid chromatography-electrospray ionization-mass spectrometry. Analysis of the three EPO glycopeptides carrying N-glycans revealed the presence of biantennary structures with terminal sialic acid residues. Our data demonstrate that N. benthamiana plants are amenable to engineering of the O-glycosylation pathway and can produce well defined human-type O- and N-linked glycans on recombinant therapeutics.
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http://dx.doi.org/10.1074/jbc.M112.402685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3476317PMC
October 2012

Validating the standard for the National Board Dental Examination Part II.

J Dent Educ 2012 May;76(5):540-4

R&D/Psychometrics, Department of Testing Services, American Dental Association, Chicago, IL 60611, USA.

As part of the overall exam validation process, the Joint Commission on National Dental Examinations periodically reviews and validates the pass/fail standard for the National Board Dental Examination (NBDE), Parts I and II. The most recent standard-setting activities for NBDE Part II used the Objective Standard Setting method. This report describes the process used to set the pass/fail standard for the 2009 exam. The failure rate on the NBDE Part II increased from 5.3 percent in 2008 to 13.7 percent in 2009 and then decreased to 10 percent in 2010. This article describes the Objective Standard Setting method and presents the estimated probabilities of classification errors based on the beta binomial mathematical model. The results show that the probability of correct classifications of candidate performance is very high (0.97) and that probabilities of false negative and false positive errors are very small (.03 and <0.001, respectively). The low probability of classification errors supports the conclusion that the pass/fail score on the NBDE Part II is a valid guide for making decisions about candidates for dental licensure.
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May 2012

Assessing context effects on test validity of the National Board Dental Examination Part I.

J Dent Educ 2012 Apr;76(4):395-406

Department of Testing Services, American Dental Association, Chicago, IL 60611-2637, USA.

In support of actions taken by the Joint Commission on National Dental Examinations, two changes--the inclusion of testlet items and the random presentation of items in an interdisciplinary format--were made to enhance the test validity of the National Board Dental Examination Part I in 2007. As a result, the examination was changed from a conjunctive to a comprehensive format. It was assumed that validity would be enhanced with regard to the examination's internal structure, while not disturbing item performance and examinee score. This study of the results found that 1) three underlying variables were extracted from the conjunctive Part I but only two underlying variables from the comprehensive Part I and 2) the differences in item performance and examinee score were generally small in effect size across formats. Factor analyses revealed that Part I was more discipline-sensitive for the conjunctive format but more item format-sensitive for the comprehensive format. The revision of Part I changed the nature of the examination from a discipline-based format to a more clinically relevant, interdisciplinary format, a favorable outcome anticipated by the Joint Commission. The results of this study provide evidence supporting the validity of the revised Part I examination for its intended purpose in the licensure process.
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April 2012

The collection of lymphatic fluid from the bovine udder and its use for the detection of Mycobacterium avium subsp. paratuberculosis in the cow.

J Vet Diagn Invest 2012 Jan 6;24(1):23-31. Epub 2011 Dec 6.

Clinic for Ruminants, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria.

The objective of the current study was to evaluate the feasibility of lymph collection from the bovine udder and to investigate if the lymphatic fluid might be of diagnostic value in cows infected with Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of paratuberculosis. Lymph fluid collection was attempted from 58 cows, and the reactions of the cows as well as the level of difficulty of the procedure were recorded in 56 animals. Lymph samples (51 in total) were tested for the presence of MAP by nested polymerase chain reaction. Collection of the lymphatic fluid caused no or mild signs of discomfort in 94.6% of the cows; in 51.8% of cows, lymphatic fluid was attained on the first attempt, while sample collection was unsuccessful in 12.1%. Mycobacterium avium subsp. paratuberculosis was detected in 43.1% of all lymph samples. The bacterium was present in 66.7% of cows with clinical Johne's disease, in 42.8% of asymptomatic cows with a positive or suspicious enzyme-linked immunosorbent assay (ELISA) result in blood, and in 38.7% of cows with a negative ELISA result in blood. The present study shows that the procedure was well tolerated by most cows and can easily be performed on farm. The current report of the isolation of MAP from lymph fluid suggests that the present approach could be used for the early detection of Johne's disease in cattle.
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http://dx.doi.org/10.1177/1040638711425943DOI Listing
January 2012

Generalizability analyses of NBDE Part II.

Eval Health Prof 2012 Jun 7;35(2):169-81. Epub 2011 Dec 7.

Department of Testing Services, American Dental Association, Chicago, IL 60611, USA.

This research applied generalizability theory to assess the effect of varying the number of cases and items nested within cases on generalizability of scores on Part II of the National Board Dental Examinations (NBDE Part II). In this research, sources of error were defined. Measurement conditions were classified. Error variances and generalizability coefficients for different conditions were computed. The data analyzed were the item responses of 1,535 candidates enrolled in accredited dental education programs who all took the same test form in 2007. Results showed that using more cases of fewer items might lead to a greater increase in generalizability than using more items per case. Other practical considerations such as time and cost constraints must be taken into account when applying the results of this research in other testing situations.
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http://dx.doi.org/10.1177/0163278711425382DOI Listing
June 2012

An investigation of the relationship between fecal and rumen bacterial concentrations in sheep.

Zoo Biol 2008 Mar;27(2):100-8

Department of Animal Sciences, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio.

With no acceptable method for collecting fresh rumen fluid from zoo ruminants, it was proposed that fecal bacterial concentrations may be correlated with rumen bacteria. If so, fecal bacterial concentrations could be used to study both the effects of diet on rumen bacteria as well as rumen abnormalities. Total and cellulolytic bacterial concentrations were determined in whole rumen contents and feces of sheep using a most-probable-number (MPN) assay. In a Latin square design, four crossbred ewes were fed diets of 100% long or chopped orchardgrass hay (OH) and 60% ground or whole shelled corn plus 40% chopped OH. In a second trial, the sheep were fed a pelleted complete feed at varying levels of intake i.e., control at 2.0% of body weight and at 1.8, 1.6, and 1.2% of body weight. Higher total rumen bacterial concentrations (P<0.01) were found on the high concentrate diets as compared with the high forage diets. Grinding the corn also increased total bacterial concentrations (P<0.05). Fecal concentrations of total bacteria were higher (P<0.01) with the high concentrate diets. Chopping the forage decreased the concentration of fecal cellulolytic bacteria (P<0.05) but had no effect on their concentration in the rumen. An inverse linear relationship (P<0.01) was observed between total bacterial concentrations in the feces and diet intake. Although relationships were observed between the rumen and feces for total and cellulolytic bacterial concentrations, they were dependent on diet, particle size, and level of intake. Thus, fecal bacterial concentrations cannot be used to reliably predict rumen bacterial concentrations. Zoo Biol 27:100-108, 2008. (c) 2008 Wiley-Liss, Inc.
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http://dx.doi.org/10.1002/zoo.20166DOI Listing
March 2008

Dental student assessment toolbox.

J Dent Educ 2009 Jan;73(1):12-35

American Dental Association, Chicago, IL 60611-2637, USA.

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January 2009