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Item The GBT diffuse ionized gas survey (GDIGS): survey overview and first data release(American Astronomical Society, 2021-06) Anderson, L.D.; Luisi, Matteo; Liu, Bin; Wenger, Trey V.; Balser, Dana S.; Bania, Thomas M.; Haffner, L.M.; Linville, Dylan J.; Mascoop, J.L.The Green Bank Telescope (GBT) Diffuse Ionized Gas Survey (GDIGS) traces ionized gas in the Galactic midplane by measuring 4–8 GHz radio recombination line (RRL) emission. The nominal survey zone is 32.°3 > ℓ > − 5°, ∣b∣ < 0.°5, but coverage extends above and below the plane in select fields and additionally includes the areas around W47 (ℓ ≃ 37.°5) and W49 (ℓ ≃ 43°). GDIGS simultaneously observes 22 Hnα (15 usable), 25 Hnβ (18 usable), and 8 Hnγ RRLs (all usable), as well as multiple molecular line transitions (including those of H_2^13CO, H2CO, and CH3OH). Here, we describe the GDIGS survey parameters and characterize the RRL data, focusing primarily on the Hnα data. We produce sensitive data cubes by averaging the usable RRLs, after first smoothing to a common spectral resolution of 0.5 km s−1 and a spatial resolution of 2.′65 for Hnα, 2.′62 for Hnβ, and 2.′09 for Hnγ. The average spectral noise per spaxel in the Hnα data cubes is ∼10 mK (∼5 mJy beam−1). This sensitivity allows GDIGS to detect RRLs from plasma throughout the inner Galaxy. The GDIGS Hnα data are sensitive to emission measures EM ≳ 1100 cm^−6 pc, which corresponds to a mean electron density ⟨n_e⟩ ≳ 30 cm^−3 for a 1 pc path length or ⟨n_e〉 ≳ 1 cm^−3 for a 1 kpc path length.Item Social media enables people-centric climate action in the hard-to-decarbonise building sector(Springer Science and Business Media LLC, 2022-11-17) Debnath, Ramit; Bardhan, Ronita; Shah, Darshil U.; Mohaddes, Kamiar; Ramage, Michael H.; Alvarez, R. Michael; Sovacool, Benjamin K.The building and construction sector accounts for around 39% of global carbon dioxide emissions and remains a hard-to-abate sector. We use a data-driven analysis of global high-level climate action on emissions reduction in the building sector using 256,717 English-language tweets across a 13-year time frame (2009-2021). Using natural language processing and network analysis, we show that public sentiments and emotions on social media are reactive to these climate policy actions. Between 2009-2012, discussions around green building-led emission reduction efforts were highly influential in shaping the online public perceptions of climate action. From 2013 to 2016, communication around low-carbon construction and energy efficiency significantly influenced the online narrative. More significant interactions on net-zero transition, climate tech, circular economy, mass timber housing and climate justice in 2017-2021 shaped the online climate action discourse. We find positive sentiments are more prominent and recurrent and comprise a larger share of the social media conversation. However, we also see a rise in negative sentiment by 30-40% following popular policy events like the IPCC report launches, the Paris Agreement and the EU Green Deal. With greater online engagement and information diffusion, social and environmental justice topics emerge in the online discourse. Continuing such shifts in online climate discourse is pivotal to a more just and people-centric transition in such hard-to-decarbonise sectors.Item The influence of COVID-19 on dental school education in the United States: emerging and future challenges(Journal of Massachusetts Dental Society, 2020-11) Sonkar, Jyoti; Saxena, Debashree; Kong, CelesteCoronavirus (COVID-19) outbreak is a rapidly emerging infectious disease pandemic with the potential of transmission from animal-to-human and human-to-human which has grappled the current world into fear. Public health, trade, travel and numerous economic sectors have come to a standstill. Higher education has been struggling to adapt to the new rules and regulations proposed in response to the current dynamic situation. Per the Center for Disease Control, as of March 30, 2020, there are over 140,094 COVID-19 cases diagnosed in the United States with a total of 2,045 deaths. Due to the high-risk nature of dentistry, dental schools have been tasked with a challenging role of delegating a majority of their responsibilities remotely and shutting down non emergent patient care to safeguard their dental students, faculties and personnel. This is causing a delay in providing optimal dental care, especially to the aging population due to the magnitude of their susceptibility to the disease. As a teaching institution, the dental school must not only navigate through the challenge of managing and providing optimal patient care, but also address scholastic responsibilities. The aim of this article is to review and explore some of the emerging issues such as impact of virtual learning, increasing xenophobia, didactics and clinical protocols during and post lockdown, safety requirements and dynamic strategic policies safeguarding the mission and integrity of the dental schools during this hardship.Item Retrospective study to identify associations between clinician training and dental implant outcome and to compare the use of MATLAB with SAS(International Journal of Implant Dentistry, 2019-08) Sonkar, Jyoti; Maney, Pooja; Yu, Qingzhao; Palaiologou, AngelaBackground: The aim of this study was to identify any associations between predictor variables, mainly clinician training and dental implant outcome, among the residents in different departments and to compare statistical analysis with the use of MATLAB R2017a™ to SAS version 9.4. Methods: Dental records were reviewed from January 1, 2011, to December 31, 2015. Two thousand forty-eight dental implants were placed on 471 patients seen by residents from the departments of Periodontics, Prosthodontics, and Oral and Maxillofacial Surgery (OMFS) at Louisiana State University Health Sciences Center School of Dentistry. The following parameters were investigated by means of multilevel logistic regression analysis: demographics, implant parameters, department, and residents’ year of training. Results: A total of 1449 implants were included in the study. Overall, within a 1–5-year time period, 1343 (92.6%) implants had survived and 106 (7.4%) implants failed. Discipline (p = 0.0004), residents’ year of training (p < 0.0001), and implant systems (p = 0.0024) showed significant associations with implant outcome. Periodontics had a survival rate of 94.14% followed by Prosthodontics (91.48%) and OMFS (89.64%). The survival rates of implants by year of training were as follows: third-year Periodontics and OMFS (94.20%), second-year (89.38%), and first-year (88.6%). Conclusion: The level and type of clinician training had an impact on implant outcome in different residency programs. Further studies will be necessary to identify the reasons for the differences in implant failure rates.Item How do we treat dental patients under influence of marijuana?(2020) Grover, Simran; Sonkar, Jyoti; Kong, Celeste V.BACKGROUND Marijuana is the third most widely used illicit substance in the United States. In the past 20 years, its use has increased 30-fold; It is estimated that 22.2 million Americans of age 12 years and older report current marijuana use.1 Massachusetts reported that 45% of adults between the age of 18-25 years used marijuana along with 22% increase in marijuana consumption in 2017 after legalization.2 This review explores the latest trends in the use of marijuana and reviews oral health implications and guidelines for treating dental patients under the influence. Dental patient on marijuana use are high and impaired to provide informed consent, these patients are most often noncompliant, long term treatment prognosis are questionable. Such patients also often seek cosmetic dental treatment, such as veneers and whitening, due to these unaesthetic dental complications; this represents another opportunity for the dentist to discuss suspected substance misuse, provide appropriate referrals for treatment, and encourage cessation of use as part of the treatment process prior to initiating any cosmetic treatments that may otherwise fail.Item Identification of pseudolysin (lasB) as an aciduric gluten-degrading enzyme with high therapeutic potential for celiac disease(Wolters Kluwer Health, Inc., 2015-06) Wei, Guoxian; Tian, Na; Valery, Adriana C.; Zhong, Yi; Schuppan, Detlef; Helmerhorst, Eva J.OBJECTIVES: Immunogenic gluten proteins implicated in celiac disease (CD) largely resist degradation by human digestive enzymes. Here we pursued the isolation of gluten-degrading organisms from human feces, aiming at bacteria that would digest gluten under acidic conditions, as prevails in the stomach. METHODS: Bacteria with gluten-degrading activities were isolated using selective gluten agar plates at pH 4.0 and 7.0. Proteins in concentrated bacterial cell sonicates were separated by diethylaminoethanol chromatography. Enzyme activity was monitored with chromogenic substrates and gliadin zymography. Elimination of major immunogenic gluten epitopes was studied with R5 and G12 enzyme-linked immunosorbent assays. RESULTS: Gliadin-degrading enzyme activities were observed for 43 fecal isolates, displaying activities in the ~150-200 and <50 kDa regions. The active strains were identified as Pseudomonas aeruginosa. Gliadin degradation in gel was observed from pH 2.0 to 7.0. Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis identified the enzyme as pseudolysin (lasB), a metalloprotease belonging to the thermolysin (M4) family proteases. Its electrophoretic mobility in SDS-polyacrylamide gel electrophoresis and gliadin zymogram gels was similar to that of a commercial lasB preparation, with tendency of oligomerization. Pseudolysin eliminated epitopes recognized by the R5 antibody, while those detected by the G12 antibody remained intact, despite destruction of the nearby major T-cell epitope QPQLPY. CONCLUSIONS: Pseudolysin was identified as an enzyme cleaving gluten effectively at extremely low as well as near-neutral pH values. The potential to degrade gluten during gastric transport opens possibilities for its application as a novel therapeutic agent for the treatment of CD.Item Pharmaceutically modified subtilisins withstand acidic conditions and effectively degrade gluten in vivo(Nature Publishing Group, 2019-05-16) Darwish, Ghassan; Helmerhorst, Eva J.; Schuppan, Detlef; Oppenheim, Frank G.; Wei, GuoxianDetoxification of gluten immunogenic epitopes is a promising strategy for the treatment of celiac disease. Our previous studies have shown that these epitopes can be degraded in vitro by subtilisin enzymes derived from Rothia mucilaginosa, a natural microbial colonizer of the oral cavity. The challenge is that the enzyme is not optimally active under acidic conditions as encountered in the stomach. We therefore aimed to protect and maintain subtilisin-A enzyme activity by exploring two pharmaceutical modification techniques: PEGylation and Polylactic glycolic acid (PLGA) microencapsulation. PEGylation of subtilisin-A (Sub-A) was performed by attaching methoxypolyethylene glycol (mPEG, 5 kDa). The PEGylation protected subtilisin-A from autolysis at neutral pH. The PEGylated Sub-A (Sub-A-mPEG) was further encapsulated by PLGA. The microencapsulated Sub-A-mPEG-PLGA showed significantly increased protection against acid exposure in vitro. In vivo, gluten immunogenic epitopes were decreased by 60% in the stomach of mice fed with chow containing Sub-A-mPEG-PLGA (0.2mg Sub-A/ g chow) (n=9) compared to 31.9 % in mice fed with chow containing unmodified Sub-A (n=9). These results show that the developed pharmaceutical modification can protect Sub-A from auto-digestion as well as from acid inactivation, thus rendering the enzyme more effective for applications in vivo.Item Risk of Tooth Loss After Cigarette Smoking Cessation(Centers for Disease Control and Prevention, 2006-9-15) Krall Kaye, Elizabeth; Dietrich, Thomas; Nunn, Martha E.; Garcia, Raul I.INTRODUCTION. Little is known about the effect of cigarette smoking cessation on risk of tooth loss. We examined how risk of tooth loss changed with longer periods of smoking abstinence in a prospective study of oral health in men. METHODS. Research subjects were 789 men who participated in the Veterans Administration Dental Longitudinal Study from 1968 to 2004. Tooth status and smoking status were determined at examinations performed every 3 years, for a maximum follow-up time of 35 years. Risk of tooth loss subsequent to smoking cessation was assessed sequentially at 1-year intervals with multivariate proportional hazards regression models. Men who never smoked cigarettes, cigars, or pipes formed the reference group. Hazard ratios were adjusted for age, education, total pack-years of cigarette exposure, frequency of brushing, and use of floss. RESULTS. The hazard ratio for tooth loss was 2.1 (95% confidence interval [CI], 1.5-3.1) among men who smoked cigarettes during all or part of follow-up. Risk of tooth loss among men who quit smoking declined as time after smoking cessation increased, from 2.0 (95% CI, 1.4-2.9) after 1 year of abstinence to 1.0 (95% CI, 0.5-2.2) after 15 years of abstinence. The risk remained significantly elevated for the first 9 years of abstinence but eventually dropped to the level of men who never smoked after 13 or more years. CONCLUSION. These results indicate that smoking cessation is beneficial for tooth retention, but long-term abstinence is required to reduce the risk to the level of people who have never smoked.Item Activin A expression regulates multipotency of mesenchymal progenitor cells(BioMed Central, 2010-5-4) Djouad, Farida; Jackson, Wesley M.; Bobick, Brent E.; Janjanin, Sasa; Song, Yingjie; Huang, George T.J.; Tuan, Rocky S.INTRODUCTION. Bone marrow (BM) stroma currently represents the most common and investigated source of mesenchymal progenitor cells (MPCs); however, comparable adult progenitor or stem cells have also been isolated from a wide variety of tissues. This study aims to assess the functional similarities of MPCs from different tissues and to identify specific factor(s) related to their multipotency. METHODS. For this purpose, we directly compared MPCs isolated from different adult tissues, including bone marrow, tonsil, muscle, and dental pulp. We first examined and compared proliferation rates, immunomodulatory properties, and multidifferentiation potential of these MPCs in vitro. Next, we specifically evaluated activin A expression profile and activin A:follistatin ratio in MPCs from the four sources. RESULTS. The multidifferentiation potential of the MPCs is correlated with activin A level and/or the activin A:follistatin ratio. Interestingly, by siRNA-mediated activin A knockdown, activin A was shown to be required for the chondrogenic and osteogenic differentiation of MPCs. These findings strongly suggest that activin A has a pivotal differentiation-related role in the early stages of chondrogenesis and osteogenesis while inhibiting adipogenesis of MPCs. CONCLUSIONS. This comparative analysis of MPCs from different tissue sources also identifies bone marrow-derived MPCs as the most potent MPCs in terms of multilineage differentiation and immunosuppression, two key requirements in cell-based regenerative medicine. In addition, this study implicates the significance of activin A as a functional marker of MPC identity.Item Discovery of a Novel and Rich Source of Gluten-Degrading Microbial Enzymes in the Oral Cavity(Public Library of Science, 2010-10-11) Helmerhorst, Eva J.; Zamakhchari, Maram; Schuppan, Detlef; Oppenheim, Frank G.BACKGROUND. Celiac disease is a T cell mediated-inflammatory enteropathy caused by the ingestion of gluten in genetically predisposed individuals carrying HLA-DQ2 or HLA-DQ8. The immunogenic gliadin epitopes, containing multiple glutamine and proline residues, are largely resistant to degradation by gastric and intestinal proteases. Salivary microorganisms however exhibit glutamine endoprotease activity, discovered towards glutamine- and proline-rich salivary proteins. The aim was to explore if gliadins can serve as substrates for oral microbial enzymes. METHODOLOGY/PRINCIPAL FINDINGS. Proteolytic activity in suspended dental plaque was studied towards a) gliadin-derived paranitroanilide(pNA)-linked synthetic enzyme substrates b) a mixture of natural gliadins and c) synthetic highly immunogenic gliadin peptides (33-mer of a2-gliadin and 26-mer of ?-gliadin). In addition, gliadin zymography was conducted to obtain the approximate molecular weights and pH activity profiles of the gliadin-degrading oral enzymes and liquid iso-electric focusing was performed to establish overall enzyme iso-electric points. Plaque bacteria efficiently hydrolyzed Z-YPQ-pNA, Z-QQP-pNA, Z-PPF-pNA and Z-PFP-pNA, with Z-YPQ-pNA being most rapidly cleaved. Gliadin immunogenic domains were extensively degraded in the presence of oral bacteria. Gliadin zymography revealed that prominent enzymes exhibit molecular weights >70 kD and are active over a broad pH range from 3 to 10. Liquid iso-electric focusing indicated that most gliadin-degrading enzymes are acidic in nature with iso-electric points between 2.5 and 4.0. CONCLUSIONS/SIGNIFICANCE. This is the first reported evidence for gluten-degrading microorganisms associated with the upper gastro-intestinal tract. Such microorganisms may play a hitherto unappreciated role in the digestion of dietary gluten and thus protection from celiac disease in subjects at risk.Item The Jacob2 Lectin of the Entamoeba Histolytica Cyst Wall Binds Chitin and Is Polymorphic(Public Library of Science, 2010-7-20) Ghosh, Sudip K.; Van Dellen, Katrina L.; Chatterjee, Anirban; Dey, Tuli; Haque, Rashidul; Robbins, Phillips W.; Samuelson, JohnBACKGROUND. The infectious and diagnostic form of Entamoeba histolytica (Eh), cause of amebic dysentery and liver abscess, is the quadranucleate cyst. The cyst wall of Entamoeba invadens (Ei), a model for Eh, is composed of chitin fibrils and three sets of chitin-binding lectins that cross-link chitin fibrils (multivalent Jacob lectins), self-aggregate (Jessie lectins), and remodel chitin (chitinase). The goal here was to determine how well the Ei model applies to Entamoeba cysts from humans. METHODS/RESULTS. An Eh Jacob lectin (EhJacob2) has three predicted chitin-binding domains surrounding a large, Ser-rich spacer. Recombinant EhJacob2 made in transfected Eh trophozoites binds to particulate chitin. Sequences of PCR products using primers flanking the highly polymorphic spacer of EhJacob2 may be used to distinguish Entamoeba isolates. Antibodies to the EhJacob2, EhJessie3, and chitinase each recognize cyst walls of clinical isolates of Entamoeba. While numerous sera from patients with amebic intestinal infections and liver abscess recognize recombinant EhJacob1 and EhJessie3 lectins, few of these sera recognize recombinant EhJacob2. CONCLUSIONS/SIGNIFICANCE. The EhJacob2 lectin binds chitin and is polymorphic, and Jacob2, Jessie3, and chitinase are present in cyst walls of clinical isolates of Entamoeba. These results suggest there are substantial similarities between cysts of the human pathogen (Eh) and the in vitro model (Ei), even though there are quantitative and qualitative differences in their chitin-binding lectins. Author SummaryFor many years, we and others have used cysts of Entamoeba invadens (Ei), a reptilian parasite, to model the infectious and diagnostic cysts of the human pathogen Entamoeba histolytica (Eh). The Ei cyst wall is composed of chitin fibrils, as well as Jacob and Jessie lectins that have unique chitin-binding domains. Our recent results suggest a "wattle and daub" model of the Ei cyst wall, where the wattle or sticks (chitin fibrils bound by multivalent Jacob lectins) is constructed prior to the addition of the mortar or daub (self-aggregating Jessie3 lectins). Here we "humanize" the Ei model of the cyst wall with four findings. First, a recombinant Eh Jacob2 lectin, which has three predicted chitin-binding domains surrounding a large spacer domain, binds chitin beads. Second, polymorphisms in the spacer domain of EhJacob2 discriminate clinical isolates of Entamoeba. Third, chitinase, Jacob2 lectin, and Jessie3 lectin are present in cyst walls of clinical isolates of Entamoeba. Finally, numerous sera from patients infected with Entamoeba recognize recombinant Eh Jacob1 and Jessie3 lectins.Item Toll-Like Receptor-2 Mediates Diet and/or Pathogen Associated Atherosclerosis: Proteomic Findings(Public Library of Science, 2008-9-12) Madan, Monika; Amar, SalomonBACKGROUND. Accumulating evidence implicates a fundamental link between the immune system and atherosclerosis. Toll-like receptors are principal sensors of the innate immune system. Here we report an assessment of the role of the TLR2 pathway in atherosclerosis associated with a high-fat diet and/or bacteria in ApoE+/- mice. METHODS AND RESULTS. To explore the role of TLR2 in inflammation- and infection-associated atherosclerosis, 10 week-old ApoE+/--TLR2+/+, ApoE+/--TLR2+/- and ApoE+/--TLR2-/- mice were fed either a high fat diet or a regular chow diet. All mice were inoculated intravenously, once per week for 24 consecutive weeks, with 50 μl live Porphyromonas gingivalis (P.g) (107 CFU) or vehicle (normal saline). Animals were euthanized 24 weeks after the first inoculation. ApoE+/--TLR2+/+ mice showed a significant increase in atheromatous lesions in proximal aorta and aortic tree compared to ApoE+/--TLR2+/- and ApoE+/--TLR2-/- mice for all diet conditions. They also displayed profound changes in plaque composition, as evidenced by increased macrophage infiltration and apoptosis, increased lipid content, and decreased smooth muscle cell mass, all reflecting an unstable plaque phenotype. SAA levels from ApoE+/--TLR2+/+ mice were significantly higher than from ApoE+/--TLR2+/- and ApoE+/--TLR2-/- mice. Serum cytokine analysis revealed increased levels of pro-inflammatory cytokines in ApoE+/--TLR2+/+ mice compared to ApoE+/--TLR2+/- and TLR2-/- mice, irrespective of diet or bacterial challenge. ApoE+/--TLR2+/+ mice injected weekly for 24 weeks with FSL-1 (a TLR2 agonist) also demonstrated significant increases in atherosclerotic lesions, SAA and serum cytokine levels compared to ApoE+/--TLR2-/- mice under same treatment condition. Finally, mass-spectrometry (MALDI-TOF-MS) of aortic samples analyzed by 2-dimentional gel electrophoresis differential display, identified 6 proteins upregulated greater than 2-fold in ApoE+/--TLR2+/+ mice fed the high fat diet and inoculated with P.g compared to any other group. CONCLUSION. Genetic deficiency of TLR2 reduces diet- and/or pathogen-associated atherosclerosis in ApoE+/- mice, along with differences in plaque composition suggesting greater structural stability while TLR-2 ligand-specific activation triggers atherosclerosis. The present data offers new insights into the pathophysiological pathways involved in atherosclerosis and paves the way for new pharmacological interventions aimed at reducing atherosclerosis.Item Role of Esrrg in the Fibrate-Mediated Regulation of Lipid Metabolism Genes in Human ApoA-I Transgenic Mice(Nature Publishing Group, 2009-12-01) Sanoudou, D.; Duka, A.; Drosatos, K.; Hayes, K. C.; Zannis, V. I.We have used a new ApoA-I transgenic mouse model to identify by global gene expression profiling, candidate genes that affect lipid and lipoprotein metabolism in response to fenofibrate treatment. Multilevel bioinformatical analysis and stringent selection criteria (2-fold change, 0% false discovery rate) identified 267 significantly changed genes involved in several molecular pathways. The fenofibrate-treated group did not have significantly altered levels of hepatic human APOA-I mRNA and plasma ApoA-I compared with the control group. However, the treatment increased cholesterol levels to 1.95-fold mainly due to the increase in high-density lipoprotein (HDL) cholesterol. The observed changes in HDL are associated with the upregulation of genes involved in phospholipid biosynthesis and lipid hydrolysis, as well as phospholipid transfer protein. Significant upregulation was observed in genes involved in fatty acid transport and β-oxidation, but not in those of fatty acid and cholesterol biosynthesis, Krebs cycle and gluconeogenesis. Fenofibrate changed significantly the expression of seven transcription factors. The estrogen receptor-related gamma gene was upregulated 2.36-fold and had a significant positive correlation with genes of lipid and lipoprotein metabolism and mitochondrial functions, indicating an important role of this orphan receptor in mediating the fenofibrate-induced activation of a specific subset of its target genes.Item Identification of Positive Regulators of the Yeast Fps1 Glycerol Channel(Public Library of Science, 2009-11-26) Beese, Sara E.; Negishi, Takahiro; Levin, David E.The yeast Fps1 protein is an aquaglyceroporin that functions as the major facilitator of glycerol transport in response to changes in extracellular osmolarity. Although the High Osmolarity Glycerol pathway is thought to have a function in at least basal control of Fps1 activity, its mode of regulation is not understood. We describe the identification of a pair of positive regulators of the Fps1 glycerol channel, Rgc1 (Ypr115w) and Rgc2 (Ask10). An rgc1/2Δ mutant experiences cell wall stress that results from osmotic pressure associated with hyper-accumulation of glycerol. Accumulation of glycerol in the rgc1/2Δ mutant results from a defect in Fps1 activity as evidenced by suppression of the defect through Fps1 overexpression, failure to release glycerol upon hypo-osmotic shock, and resistance to arsenite, a toxic metalloid that enters the cell through Fps1. Regulation of Fps1 by Rgc1/2 appears to be indirect; however, evidence is presented supporting the view that Rgc1/2 regulate Fps1 channel activity, rather than its expression, folding, or localization. Rgc2 was phosphorylated in response to stresses that lead to regulation of Fps1. This stress-induced phosphorylation was partially dependent on the Hog1 MAPK. Hog1 was also required for basal phosphorylation of Rgc2, suggesting a mechanism by which Hog1 may regulate Fps1 indirectly. Author Summary When challenged by changes in extracellular osmolarity, many fungal species regulate their intracellular glycerol concentration to modulate their internal osmotic pressure. Maintenance of osmotic homeostasis prevents either cellular collapse under hyper-osmotic stress or cell rupture under hypo-osmotic stress. In baker's yeast, the Fps1 glycerol channel functions as the main vent for glycerol. Proper regulation of Fps1 is critical to the maintenance of osmotic homeostasis. In this study, we identify a pair of proteins (Rgc1 and Rgc2) that function as positive regulators of Fps1 activity. Their absence results in hyper-accumulation of glycerol and consequent cell lysis due to impaired Fps1 channel activity. Additionally, we found that these glycerol channel regulators function between the Hog1 (High Osmolarity Glycerol response) signaling kinase and Fps1, defining a signaling pathway for control of glycerol efflux. Because members of the Rgc1/2 family are found among pathogenic fungal species, but not in humans, they represent potentially attractive targets for antifungal drug development.Item Evidence for a "Wattle and Daub" Model of the Cyst Wall of Entamoeba(Public Library of Science, 2009-7-3) Chatterjee, Anirban; Ghosh, Sudip K.; Jang, Ken; Bullitt, Esther; Moore, Landon; Robbins, Phillips W.; Samuelson, JohnThe cyst wall of Entamoeba invadens (Ei), a model for the human pathogen Entamoeba histolytica, is composed of fibrils of chitin and three chitin-binding lectins called Jacob, Jessie3, and chitinase. Here we show chitin, which was detected with wheat germ agglutinin, is made in secretory vesicles prior to its deposition on the surface of encysting Ei. Jacob lectins, which have tandemly arrayed chitin-binding domains (CBDs), and chitinase, which has an N-terminal CBD, were each made early during encystation. These results are consistent with their hypothesized roles in cross-linking chitin fibrils (Jacob lectins) and remodeling the cyst wall (chitinase). Jessie3 lectins likely form the mortar or daub of the cyst wall, because 1) Jessie lectins were made late during encystation; 2) the addition to Jessie lectins to the cyst wall correlated with a marked decrease in the permeability of cysts to nucleic acid stains (DAPI) and actin-binding heptapeptide (phalloidin); and 3) recombinant Jessie lectins, expressed as a maltose-binding proteins in the periplasm of Escherichia coli, caused transformed bacteria to agglutinate in suspension and form a hard pellet that did not dissociate after centrifugation. Jessie3 appeared as linear forms and rosettes by negative staining of secreted recombinant proteins. These findings provide evidence for a "wattle and daub" model of the Entamoeba cyst wall, where the wattle or sticks (chitin fibrils likely cross-linked by Jacob lectins) is constructed prior to the addition of the mortar or daub (Jessie3 lectins). Author SummaryParasitic protists, which are spread by the fecal-oral route, have cyst walls that resist environmental insults (e.g. desiccation, stomach acids, bile, etc.). Entamoeba histolytica, the cause of amebic dysentery and liver abscess, is the only protist characterized to date that has chitin in its cyst wall. We have previously characterized Entamoeba chitin synthases, chitinases, and multivalent chitin-binding lectins called Jacob. Here we present evidence that the Entamoeba Jessie3 lectin contributes to the mortar or daub, which makes the cyst wall impenetrable to small molecules. First, the Jessie3 lectin was made after chitin and Jacob lectins had already been deposited onto the surface of encysting Entamoeba. Second, cysts became impenetrable to small molecules at the same time that Jessie3 was deposited into the wall. Third, recombinant Jessie3 lectins self-aggregated and caused transfected bacteria to agglutinate. These results suggest a "wattle and daub" model of the Ei cyst wall, where the wattle or sticks (chitin fibrils likely cross-linked by Jacob lectins) is constructed prior to the addition of the mortar or daub (Jessie3 lectins).Item A new brief measure of oral quality of life(Centers for Disease Control and Prevention, 2008-3-15) Kressin, Nancy R.; Jones, Judith A.; Orner, Michelle B.; Spiro, AvronINTRODUCTION. We developed a brief measure of the impact of oral conditions on individual functioning and well-being, known as oral quality of life. METHODS. Among older male veterans (N = 827) and community dental patients (N = 113), we administered surveys consisting of extant oral quality of life items, using clinical dental data from the veteran samples. We assigned each oral quality of life item to a theoretical dimension, conducted an iterative series of multitrait scaling analyses to examine the item-fit with the dimensions, reduced the number of items, and examined the psychometric characteristics of new scales and their association with clinical indices. RESULTS. We developed two brief oral quality of life scales, one consisting of 12 items and the other of 6, the latter a subset of the former. Each demonstrated sound psychometric properties and was sensitive to clinical indices. CONCLUSION. The two brief oral quality of life scales can be used to assess the population-based impact of oral conditions as well as outcomes of dental care.Item Giardia Cyst Wall Protein 1 Is a Lectin That Binds to Curled Fibrils of the GalNAc Homopolymer(Public Library of Science, 2010-8-19) Chatterjee, Aparajita; Carpentieri, Andrea; Ratner, Daniel M.; Bullitt, Esther; Costello, Catherine E.; Robbins, Phillips W.; Samuelson, JohnThe infectious and diagnostic stage of Giardia lamblia (also known as G. intestinalis or G. duodenalis) is the cyst. The Giardia cyst wall contains fibrils of a unique β-1,3-linked N-acetylgalactosamine (GalNAc) homopolymer and at least three cyst wall proteins (CWPs) composed of Leu-rich repeats (CWPLRR) and a C-terminal conserved Cys-rich region (CWPCRR). Our goals were to dissect the structure of the cyst wall and determine how it is disrupted during excystation. The intact Giardia cyst wall is thin (~400 nm), easily fractured by sonication, and impermeable to small molecules. Curled fibrils of the GalNAc homopolymer are restricted to a narrow plane and are coated with linear arrays of oval-shaped protein complex. In contrast, cyst walls of Giardia treated with hot alkali to deproteinate fibrils of the GalNAc homopolymer are thick (~1.2 µm), resistant to sonication, and permeable. The deproteinated GalNAc homopolymer, which forms a loose lattice of curled fibrils, is bound by native CWP1 and CWP2, as well as by maltose-binding protein (MBP)-fusions containing the full-length CWP1 or CWP1LRR. In contrast, neither MBP alone nor MBP fused to CWP1CRR bind to the GalNAc homopolymer. Recombinant CWP1 binds to the GalNAc homopolymer within secretory vesicles of Giardia encysting in vitro. Fibrils of the GalNAc homopolymer are exposed during excystation or by treatment of heat-killed cysts with chymotrypsin, while deproteinated fibrils of the GalNAc homopolymer are degraded by extracts of Giardia cysts but not trophozoites. These results show the Leu-rich repeat domain of CWP1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. During excystation, host and Giardia proteases appear to degrade bound CWPs, exposing fibrils of the GalNAc homopolymer that are digested by a stage-specific glycohydrolase. Author SummaryWhile the walls of plants and fungi contain numerous sugar homopolymers (cellulose, chitin, and β-1,3-glucans) and dozens of proteins, the cyst wall of Giardia is relatively simple. The Giardia wall contains a unique homopolymer of β-1,3-linked N-acetylgalactosamine (GalNAc) and at least three cyst wall proteins (CWPs), each of which is composed of Leu-rich repeats and a C-terminal Cys-rich region. The three major discoveries here are: 1) Fibrils of the GalNAc homopolymer are curled and form a lattice that is compressed into a narrow plane by bound protein in intact cyst walls. 2) Leu-rich repeats of CWP1 form a novel lectin domain that is specific for fibrils of the GalNAc homopolymer, which can be isolated by methods used to deproteinate fungal walls. 3) A cyst-specific glycohydrolase is able to degrade deproteinated fibrils of the GalNAc homopolymer. We incorporate these findings into a new curled fiber and lectin model of the intact Giardia cyst wall and a protease and glycohydrolase model of excystation.Item Bioinformatics Analysis of Macrophages Exposed to Porphyromonas gingivalis: Implications in Acute vs. Chronic Infections(Public Library of Science, 2010-12-23) Yu, Wen-Han; Hu, Han; Zhou, Qingde; Xia, Yu; Amar, SalomonBACKGROUND. Periodontitis is the most common human infection affecting tooth-supporting structures. It was shown to play a role in aggravating atherosclerosis. To deepen our understanding of the pathogenesis of this disease, we exposed human macrophages to an oral bacteria, Porphyromonas gingivalis (P. gingivalis), either as live bacteria or its LPS or fimbria. Microarray data from treated macrophages or control cells were analyzed to define molecular signatures. Changes in genes identified in relevant pathways were validated by RT-PCR. METHODOLOGY/PRINCIPAL FINDINGS. We focused our analysis on three important groups of genes. Group PG (genes differentially expressed by live bacteria only); Group LFG (genes differentially expressed in response to exposure to LPS and/or FimA); Group CG (core gene set jointly activated by all 3 stimulants). A total of 842 macrophage genes were differentially expressed in at least one of the three conditions compared to naïve cells. Using pathway analysis, we found that group CG activates the initial phagocytosis process and induces genes relevant to immune response, whereas group PG can de-activate the phagocytosis process associated with phagosome-lysosome fusion. LFG mostly affected RIG-I-like receptor signaling pathway. CONCLUSION/SIGNIFICANCE. In light of the fact that acute infections involve live bacteria while chronic infections involve a combination of live bacteria and their byproducts, group PG could represent acute P. gingivalis infection while group LFG could represent chronic P. gingivalis infection. Group CG may be associated with core immune pathways, triggered irrespective of the specific stimulants and indispensable to mount an appropriate immune response. Implications in acute vs. chronic infection are discussed.Item Trichomonas Transmembrane Cyclases Result from Massive Gene Duplication and Concomitant Development of Pseudogenes(Public Library of Science, 2010-8-3) Cui, Jike; Das, Suchismita; Smith, Temple F.; Samuelson, JohnBACKGROUND. Trichomonas vaginalis has an unusually large genome (∼160 Mb) encoding ∼60,000 proteins. With the goal of beginning to understand why some Trichomonas genes are present in so many copies, we characterized here a family of ∼123 Trichomonas genes that encode transmembrane adenylyl cyclases (TMACs). METHODOLOGY/PRINCIPAL FINDINGS. The large family of TMACs genes is the result of recent duplications of a small set of ancestral genes that appear to be unique to trichomonads. Duplicated TMAC genes are not closely associated with repetitive elements, and duplications of flanking sequences are rare. However, there is evidence for TMAC gene replacements by homologous recombination. A high percentage of TMAC genes (∼46%) are pseudogenes, as they contain stop codons and/or frame shifts, or the genes are truncated. Numerous stop codons present in the genome project G3 strain are not present in orthologous genes of two other Trichomonas strains (S1 and B7RC2). Each TMAC is composed of a series of N-terminal transmembrane helices and a single C-terminal cyclase domain that has adenylyl cyclase activity. Multiple TMAC genes are transcribed by Trichomonas cloned by limiting dilution. CONCLUSIONS/SIGNIFICANCE. We conclude that one reason for the unusually large genome of Trichomonas is the presence of unstable families of genes such as those encoding TMACs that are undergoing massive gene duplication and concomitant development of pseudogenes. Author SummaryTrichomonas vaginalis is the only medically important protist (single-cell eukaryote) that is sexually transmitted. The ∼160-Mb Trichomonas genome contains more predicted protein-encoding genes (∼60,000) than the human genome. To begin to understand why there are so many copies of some genes, we chose here to study a large family of genes encoding unique transmembrane cyclases. Our most important results include the following. More than 100 transmembrane cyclase genes do not result from chromosomal duplications, because for the most part only the coding regions of the genes, rather than flanking sequences, are duplicated. Almost half of the transmembrane cyclase genes are pseudogenes, and these pseudogenes are polymorphic among laboratory strains of Trichomonas. Messenger RNAs for numerous transmembrane cyclases are expressed simultaneously, and representative cyclase domains have adenylyl cyclase activity. In summary, the large family of Trichomonas genes encoding transmembrane adenylyl cyclases results from massive gene duplication and concomitant development of pseudogenes.Item Immune response of macrophages from young and aged mice to the oral pathogenic bacterium Porphyromonas gingivalis(BioMed Central, 2010-11-29) Shaik-Dasthagirisaheb, Yazdani B.; Kantarci, Alpdogan; Gibson, Frank C.Periodontal disease is a chronic inflammatory gum disease that in severe cases leads to tooth loss. Porphyromonas gingivalis (Pg) is a bacterium closely associated with generalized forms of periodontal disease. Clinical onset of generalized periodontal disease commonly presents in individuals over the age of 40. Little is known regarding the effect of aging on inflammation associated with periodontal disease. In the present study we examined the immune response of bone marrow derived macrophages (BMM) from young (2-months) and aged (1-year and 2-years) mice to Pg strain 381. Pg induced robust expression of cytokines; tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10, chemokines; neutrophil chemoattractant protein (KC), macrophage colony stimulating factor (MCP)-1, macrophage inflammatory protein (MIP)-1α and regulated upon activation normal T cell expressed and secreted (RANTES), as well as nitric oxide (NO, measured as nitrite), and prostaglandin E2 (PGE2) from BMM of young mice. BMM from the 2-year age group produced significantly less TNF-α, IL-6 and NO in response to Pg as compared with BMM from 2-months and 1-year of age. We did not observe any difference in the levels of IL-1β, IL-10 and PGE2 produced by BMM in response to Pg. BMM from 2-months and 1-year of age produced similar levels of all chemokines measured with the exception of MCP-1, which was reduced in BMM from 1-year of age. BMM from the 2-year group produced significantly less MCP-1 and MIP-1α compared with 2-months and 1-year age groups. No difference in RANTES production was observed between age groups. Employing a Pg attenuated mutant, deficient in major fimbriae (Pg DPG3), we observed reduced ability of the mutant to stimulate inflammatory mediator expression from BMMs as compared to Pg 381, irrespective of age. Taken together these results support senescence as an important facet of the reduced immunological response observed by BMM of aged host to the periodontal pathogen Pg.