Characterization of choline uptake in Trypanosoma brucei procyclic and bloodstream forms

Choline is an essential nutrient for eukaryotic cells, where it is used as precursor for the synthesis of choline-containing phospholipids, such as phosphatidylcholine (PC). According to published data, Trypanosoma brucei parasites are unable to take up choline from the environment but instead use lyso-phosphatidylcholine as precursor for choline lipid synthesis. We now show that T. brucei procyclic forms in culture readily incorporate [3H]-labeled choline into PC, indicating that trypanosomes express a transporter for choline at the plasma membrane. Characterization of the transport system in T. brucei procyclic and bloodstream forms shows that uptake of choline is independent of sodium and potassium ions and occurs with a Km in the low micromolar range. In addition, we demonstrate that choline uptake can be blocked by the known choline transport inhibitor, hemicholinium-3, and by synthetic choline analogs that have been established as anti-malarials. Together, our results show that T. brucei parasites express an uptake system for choline and that exogenous choline is used for PC synthesis.

Whole genome methylation array analysis reveals new aspects in Balkan endemic nephropathy etiology

Background Balkan endemic nephropathy (BEN) represents a chronic progressive interstitial nephritis in striking correlation with uroepithelial tumours of the upper urinary tract. The disease has endemic distribution in the Danube river regions in several Balkan countries. DNA methylation is a primary epigenetic modification that is involved in major processes such as cancer, genomic imprinting, gene silencing, etc. The significance of CpG island methylation status in normal development, cell differentiation and gene expression is widely recognized, although still stays poorly understood. Methods We performed whole genome DNA methylation array analysis on DNA pool samples from peripheral blood from 159 affected individuals and 170 healthy individuals. This technique allowed us to determine the methylation status of 27 627 CpG islands throughout the whole genome in healthy controls and BEN patients. Thus we obtained the methylation profile of BEN patients from Bulgarian and Serbian endemic regions. Results Using specifically developed software we compared the methylation profiles of BEN patients and corresponding controls and revealed the differently methylated regions. We then compared the DMRs between all patient-control pairs to determine common changes in the epigenetic profiles. SEC61G, IL17RA, HDAC11 proved to be differently methylated throughout all patient-control pairs. The CpG islands of all 3 genes were hypomethylated compared to controls. This suggests that dysregulation of these genes involved in immunological response could be a common mechanism in BEN pathogenesis in both endemic regions and in both genders. Conclusion Our data propose a new hypothesis that immunologic dysregulation has a place in BEN etiopathogenesis. Keywords: Epigenetics; Whole genome array analysis; Balkan endemic nephropathy

Cone Beam and Micro-Computed Tomography Validation of Manual Array Insertion for Minimally Invasive Cochlear Implantation

Delivering cochlear implants through a minimally invasive tunnel (1.8 mm in diameter) from the mastoid surface to the inner ear is referred to as direct cochlear access (DCA). Based on cone beam as well as micro-computed tomography imaging, this in vitro study evaluates the feasibility and efficacy of manual cochlear electrode array insertions via DCA. Free-fitting electrode arrays were inserted in 8 temporal bone specimens with previously drilled DCA tunnels. The insertion depth angle, procedural time, tunnel alignment as well as the inserted scala and intracochlear trauma were assessed. Seven of the 8 insertions were full insertions, with insertion depth angles higher than 520°. Three cases of atraumatic scala tympani insertion, 3 cases of probable basilar membrane rupture and 1 case of dislocation into the scala vestibuli were observed (1 specimen was damaged during extraction). Manual electrode array insertion following a DCA procedure seems to be feasible and safe and is a further step toward clinical application of image-guided otological microsurgery.

Gene array of primary human osteoblasts exposed to enamel matrix derivative in combination with a natural bone mineral

OBJECTIVES The application of an enamel matrix derivative (EMD) for regenerative periodontal surgery has been shown to promote formation of new cementum, periodontal ligament, and alveolar bone. In intrabony defects with a complicated anatomy, the combination of EMD with various bone grafting materials has resulted in additional clinical improvements, but the initial cellular response of osteoblasts coming in contact with these particles have not yet been fully elucidated. The objective of the present study was to evaluate the in vitro effects of EMD combined with a natural bone mineral (NBM) on a wide variety of genes, cytokines, and transcription factors and extracellular matrix proteins on primary human osteoblasts. MATERIAL AND METHODS Primary human osteoblasts were seeded on NBM particles pre-coated with versus without EMD and analyzed for gene differences using a human osteogenesis gene super-array (Applied Biosystems). Osteoblast-related genes include those transcribed during bone mineralization, ossification, bone metabolism, cell growth and differentiation, as well as gene products representing extracellular matrix molecules, transcription factors, and cell adhesion molecules. RESULTS EMD promoted gene expression of various osteoblast differentiation markers including a number of collagen types and isoforms, SMAD intracellular proteins, osteopontin, cadherin, alkaline phosphatase, and bone sialoprotein. EMD also upregulated a variety of growth factors including bone morphogenetic proteins, vascular endothelial growth factors, insulin-like growth factor, transforming growth factor, and their associated receptor proteins. CONCLUSION The results from the present study demonstrate that EMD is capable of activating a wide variety of genes, growth factors, and cytokines when pre-coated onto NBM particles. CLINICAL RELEVANCE The described in vitro effects of EMD on human primary osteoblasts provide further biologic support for the clinical application of a combination of EMD with NBM particles in periodontal and oral regenerative surgery.

Insights into the regulation of GPEET procyclin during differentiation from early to late procyclic forms of Trypanosoma brucei.

The procyclic form of Trypanosoma brucei colonises the gut of its insect vector, the tsetse fly. GPEET and EP procyclins constitute the parasite's surface coat at this stage of the life cycle, and the presence or absence of GPEET distinguishes between early and late procyclic forms, respectively. Differentiation from early to late procyclic forms in vivo occurs in the fly midgut and can be mimicked in culture. Our analysis of this transition in vitro delivered new insights into the process of GPEET repression. First, we could show that parasites followed a concrete sequence of events upon triggering differentiation: after undergoing an initial growth arrest, cells lost GPEET protein, and finally late procyclic forms resumed proliferation. Second, we determined the stability of both GPEET and EP mRNA during differentiation. GPEET mRNA is exceptionally stable in early procyclic forms, with a half-life >6h. The GPEET mRNA detected in late procyclic form cultures is a mixture of transcripts from both bona fide late procyclic forms and GPEET-positive 'laggard' parasites present in these cultures. However, its stability was clearly reduced during differentiation and in late procyclic form cultures. Alternatively processed GPEET transcripts were enriched in samples from late procyclic forms, suggesting that altered mRNA processing might contribute to repression of GPEET in this developmental stage. In addition, we detected GPEET transcripts with non-templated oligo(U) tails that were enriched in late procyclic forms. To the best of our knowledge, this is the first study reporting a uridylyl-tailed, nuclear-encoded mRNA species in trypanosomatids or any other protozoa.

Concept, design and implementation of a cardiovascular gene-centric 50 k SNP array for large-scale genomic association studies.

A wealth of genetic associations for cardiovascular and metabolic phenotypes in humans has been accumulating over the last decade, in particular a large number of loci derived from recent genome wide association studies (GWAS). True complex disease-associated loci often exert modest effects, so their delineation currently requires integration of diverse phenotypic data from large studies to ensure robust meta-analyses. We have designed a gene-centric 50 K single nucleotide polymorphism (SNP) array to assess potentially relevant loci across a range of cardiovascular, metabolic and inflammatory syndromes. The array utilizes a "cosmopolitan" tagging approach to capture the genetic diversity across approximately 2,000 loci in populations represented in the HapMap and SeattleSNPs projects. The array content is informed by GWAS of vascular and inflammatory disease, expression quantitative trait loci implicated in atherosclerosis, pathway based approaches and comprehensive literature searching. The custom flexibility of the array platform facilitated interrogation of loci at differing stringencies, according to a gene prioritization strategy that allows saturation of high priority loci with a greater density of markers than the existing GWAS tools, particularly in African HapMap samples. We also demonstrate that the IBC array can be used to complement GWAS, increasing coverage in high priority CVD-related loci across all major HapMap populations. DNA from over 200,000 extensively phenotyped individuals will be genotyped with this array with a significant portion of the generated data being released into the academic domain facilitating in silico replication attempts, analyses of rare variants and cross-cohort meta-analyses in diverse populations. These datasets will also facilitate more robust secondary analyses, such as explorations with alternative genetic models, epistasis and gene-environment interactions.

Evaluating the relative status of health and safety programs for minority academic and research institutions

A variety of occupational hazards are indigenous to academic and research institutions, ranging from traditional life safety concerns, such as fire safety and fall protection, to specialized occupational hygiene issues such as exposure to carcinogenic chemicals, radiation sources, and infectious microorganisms. Institutional health and safety programs are constantly challenged to establish and maintain adequate protective measures for this wide array of hazards. A unique subset of academic and research institutions are classified as historically Black universities which provide educational opportunities primarily to minority populations. State funded minority schools receive less resources than their non-minority counterparts, resulting in a reduced ability to provide certain programs and services. Comprehensive health and safety services for these institutions may be one of the services compromised, resulting in uncontrolled exposures to various workplace hazards. Such a result would also be contrary to the national health status objectives to improve preventive health care measures for minority populations.^ To determine if differences exist, a cross-sectional survey was performed to evaluate the relative status of health and safety programs present within minority and non-minority state-funded academic and research institutions. Data were obtained from direct mail questionnaires, supplemented by data from publicly available sources. Parameters for comparison included reported numbers of full and part-time health and safety staff, reported OSHA 200 log (or equivalent) values, and reported workers compensation experience modifiers. The relative impact of institutional minority status, institution size, and OSHA regulatory environment, was also assessed. Additional health and safety program descriptors were solicited in an attempt to develop a preliminary profile of the hazards present in this unique work setting.^ Survey forms were distributed to 24 minority and 51 non-minority institutions. A total of 72% of the questionnaires were returned, with 58% of the minority and 78% of the non-minority institutions participating. The mean number of reported full-time health and safety staff for the responding minority institutions was determined to be 1.14, compared to 3.12 for the responding non-minority institutions. Data distribution variances were stabilized using log-normal transformations, and although subsequent analysis indicated statistically significant differences, the differences were found to be predicted by institution size only, and not by minority status or OSHA regulatory environment. Similar results were noted for estimated full-time equivalent health and safety staffing levels. Significant differences were not noted between reported OSHA 200 log (or equivalent) data, and a lack of information provided on workers compensation experience modifiers prevented comparisons on insurance premium expenditures. Other health and safety program descriptive information obtained served to validate the study's presupposition that the inclusion criteria would encompass those organizations with occupational risks from all four major hazard categories. Worker medical surveillance programs appeared to exist at most institutions, but the specific tests completed were not readily identifiable.^ The results of this study serve as a preliminary description of the health and safety programs for a unique set of workplaces have not been previously investigated. Numerous opportunities for further research are noted, including efforts to quantify the relative amount of each hazard present, the further definition of the programs reported to be in place, determination of other means to measure health outcomes on campuses, and comparisons among other culturally diverse workplaces. ^

Characterization of osteopontin charge forms produced by osteoblasts

Osteopontin (OPN) is a highly-phosphorylated extracellular matrix protein localized in bone, kidney, placenta, T-lymphocytes, macrophages, smooth muscle of the vascular system, milk, urine, and plasma. In ROS 17/2.8 osteoblast-like osteosarcoma cells, 1,25-dihydroxyvitamin D3 [1,25(OH)2D 3] regulates OPN at the transcriptional level resulting in increased steady state mRNA levels and increased production of OPN protein, maximal at 48 hours. Using ROS 17/2.8 cells as an osteoblast model, OPN was purified from culture medium after three hour treatments of either vehicle (ethanol) or 1,25(OH)2D3 via barium citrate precipitation followed by immunoaffinity chromatography. ^ Here, further evidence of regulation of OPN by 1,25(OH)2D 3 at the posttranslational level is presented. Prior to the up-regulation of OPN at the transcriptional level, 1,25(OH)2D3 induces a shift in OPN isoelectric point (pI) detected on two-dimensional gels from pI 4.6 to pI 5.1. Loading equal amounts of [32P]-labeled OPN recovered from ROS 17/2.8 cells exposed to 1,25(OH)2D3 or vehicle alone for three hours reveals that the shift from pI 4.6 to 5.1 is the result of reduced phosphorylation. Using structural analogs to 1,25(OH) 2D3, analog AT [25-(OH)-16-ene-23-yne-D3], which triggers Ca2+ influx through voltage sensitive Ca2+ channels but does not bind to the vitamin D receptor, mimicked the OPN pI shift while analog BT [1,25(OH)2-22-ene-24-cyclopropyl-D 3], which binds to the vitamin D receptor but does not allow Ca 2+ influx, did not. Inclusion of the Ca2+ channel blocker nifedipine also blocks the charge shift conversion of OPN. Further analysis of the signaling pathway initiated by 1,25(OH)2D3 reveals that inhibition of the cyclic 3′,5′ -adenosine monophosphate-dependent kinase, protein kinase A, or inhibition of the cyclic 3′,5′-guanine monophosphate-dependent kinase, protein kinase G, also prevents the charge shift conversion. ^ Isolation of OPN from rat femurs and tibiae provides evidence for the existence of these two OPN charge forms in vivo, evidenced by differential migration on isoelectric focusing gels and sodium dodecyl sulfate-polyacrylamide gels. Peptide sequencing of rat long bone fractions revealed the presence of a presumed dentin specific protein, dentin matrix protein-1 (DMP-1). Western blot analysis confirmed the existence of DMP-1 in these fractions. ^ Using the OPN charge forms in functional assays, it was determined that the charge forms have differential roles in both cell surface and mineralization functions. In cell attachment assays and Ca2+ influx assays using PC-3 prostate cancer cells, the pI 5.1 charge form of OPN was found to permit binding and increase intracellular Ca2+ concentrations of PC-3 cells. The increase in intracellular Ca2+ concentration was found to be integrin αvβ3-dependent. In mineralization assays, the pI 4.6 charge form of OPN promoted hydroxyapatite formation, while the pI 5.1 charge form had improved Ca2+ binding ability. ^ In conclusion, these findings suggest that 1,25(OH) 2D3 regulates OPN not only at the transcriptional level, but also plays a role in determination of the OPN phosphorylation state. The latter involves a short term (less than three hours) treatment and is associated with membrane-initiated Ca2+ influx. Functional assays utilizing the two OPN charge forms reveal the dependence of OPN post-translational state on its function. ^

STABILITY AND FOLDING OF MUTANT FORMS OF ISO-1 CYTOCHROME-C FROM SACCHAROMYCES CEREVISIAE

Partially functional forms of iso-1-cytochrome c from Saccharomyces cerevisiae were obtained by replacements of the evolutionarily conserved proline 71 with valine, isoleucine and threonine (Ernst et.al.,1985). Pro-71 lies at the juncture of two short helical regions and is believed to be important for proper local polypeptide chain folding within the iso-1-cytochrome c structure.^ To study folding in the absence of intermolecular disulfide dimer formation the free sulfhydryl group of Cys-102 was modified in both wild type and mutant proteins with an alkylating reagent, methyl methanethiosulfonate. Spectral analysis of the wild type and mutant proteins shows that the native-like functional (or partially functional) folded structure of cytochrome c is retained in the chemically modified derivatives. The replacement of Pro-71 with valine, isoleucine or threonine reduces the intensity of the 696 nm absorbance band which is an indicator of the Met-80 ligation to the heme. Thermal stability and guanidine hydrochloride unfolding studies of the mutant proteins shows a destabilization of the protein as a result of mutation. The degree of destabilization depends on the chemical nature of the substituent amino acid in the mutant protiens.^ Kinetics of folding/unfolding reactions of the proteins were monitored by fluorescence changes using stopped flow mixing to obtain guanidine hydrochloride concentration jumps ending below, within, and above the transition zone. The replacement of Pro-71 alters the rate on one of the fastest phases, $\tau\sb3$, while the two other phases, $\tau\sb1$ & $\tau\sb2$, remain the same.^ Slow refolding kinetic studies indicate that replacement of Pro-71 does not completely eliminate the absorbance or fluorescence detected slow phases leading to the conclusion that Pro-71 is not involved in the generation of the slow phases in the folding kinetics of iso-1-cytochrome c.^ The alkaline conformational change involving the disappearance of the 696 nm absorbance band occurs with increasing pH in the alkaline pH region (Davis et al., 1974). The apparent pK of this conformational change in mutant proteins is shifted as much as two pH units compared to wild type. The equilibrium and kinetic data of alkaline transition for the wild type follows a simple mechanism proposed by Davis et al., (1974) for horse heart cytochrome c. A more complex mechanism is proposed for the behavior of the mutant proteins. ^

Brain-derived neurotrophic factor protects against multiple forms of brain injury in bacterial meningitis

BACKGROUND Brain-derived neurotrophic factor (BDNF) blocks activation of caspase-3, reduces translocation of apoptosis-inducing factor (AIF), attenuates excitotoxicity of glutamate, and increases antioxidant enzyme activities. The mechanisms of neuroprotection suggest that BDNF may be beneficial in bacterial meningitis. METHODS To assess a potentially beneficial effect of adjuvant treatment with BDNF in bacterial meningitis, 11-day-old infant rats with experimental meningitis due to Streptococcus pneumoniae or group B streptococci (GBS) were randomly assigned to receive intracisternal injections with either BDNF (3 mg/kg) or equal volumes (10 mu L) of saline. Twenty-two hours after infection, brains were analyzed, by histomorphometrical examination, for the extent of cortical and hippocampal neuronal injury. RESULTS Compared with treatment with saline, treatment with BDNF significantly reduced the extent of 3 distinct forms of brain cell injury in this disease model: cortical necrosis in meningitis due to GBS (median, 0.0% [range, 0.0%-33.7%] vs. 21.3% [range, 0.0%-55.3%]; P<.03), caspase-3-dependent cell death in meningitis due to S. pneumoniae (median score, 0.33 [range, 0.0-1.0] vs. 1.10 [0.10-1.56]; P<.05), and caspase-3-independent hippocampal cell death in meningitis due to GBS (median score, 0 [range, 0-2] vs. 0.88 [range, 0-3.25]; P<.02). The last form of injury was associated with nuclear translocation of AIF. CONCLUSION BDNF efficiently reduces multiple forms of neuronal injury in bacterial meningitis and may hold promise as adjunctive therapy for this disease.

Bacterial meningitis causes two distinct forms of cellular damage in the hippocampal dentate gyrus in infant rats.

Bacterial meningitis causes neurological sequelae in up to 50% of survivors. Two pathogens known for their propensity to cause severe neurological damage are Streptococcus pneumoniae and group B streptococci. Some forms of neuronal sequelae, such as learning and memory deficits, have been associated with neuronal injury in the hippocampus. To learn more about hippocampal injury in meningitis, we performed a comparative study in bacterial meningitis due to S. pneumoniae and group B streptococcus, in which 11-day-old infant rats were infected intracisternally with either of the two pathogens. Histopathological examination of the neuronal injury in the dentate gyrus of the hippocampus showed that S. pneumoniae caused predominantly classical apoptotic cell death. Cells undergoing apoptosis were located only in the subgranular zone and stained positive for activated caspase-3 and TUNEL. Furthermore, dividing progenitor cells seemed particularly sensitive to this form of cell death. Group B streptococcus was mainly responsible for a caspase-3-independent (and TUNEL-negative) form of cell death. Compared with the morphological features found in apoptosis (e.g., apoptotic bodies), this form of neuronal death was characterized by clusters of uniformly shrunken cells. It affected the dentate gyrus throughout the blade, showing no preferences for immature or mature neurons. Thus, depending on the infecting agent, bacterial meningitis causes two distinct forms of cell injury in the dentate gyrus.

Observing Biogeochemical Cycles at Global Scales With Profiling Floats and Gliders Prospects for a Global Array

Chemical and biological sensor technologies have advanced rapidly in the past five years. Sensors that require low power and operate for multiple years are now available for oxygen, nitrate, and a variety of bio-optical properties that serve as proxies for important components of the carbon cycle (e.g., particulate organic carbon). These sensors have all been deployed successfully for long periods, in some cases more than three years, on platforms such as profiling floats or gliders. Technologies for pH, pCO(2), and particulate inorganic carbon are maturing rapidly as well. These sensors could serve as the enabling technology for a global biogeochemical observing system that might operate on a scale comparable to the current Argo array. Here, we review the scientific motivation and the prospects for a global observing system for ocean biogeochemistry.

Antenna Simulations and Measurements of Focal Plane Array Facet Reflectors

We present a conceptual prototype model of a focal plane array unit for the STEAMR instrument, highlighting the challenges presented by the required high relative beam proximity of the instrument and focus on how edge-diffraction effects contribute to the array's performance. The analysis was carried out as a comparative process using both PO & PTD and MoM techniques. We first highlight general differences between these computational techniques, with the discussion focusing on diffractive edge effects for near-field imaging reflectors with high truncation. We then present the results of in-depth modeling analyses of the STEAMR focal plane array followed by near-field antenna measurements of a breadboard model of the array. The results of these near-field measurements agree well with both simulation techniques although MoM shows slightly higher complex beam coupling to the measurements than PO & PTD.