Release of plasmid DNA from intravascular stents coated with ultrathin multilayered polyelectrolyte films

Materials that permit control over the release of DNA from the surfaces of topologically complex implantable devices, such as intravascular stents, could contribute to the development of new approaches to the localized delivery of DNA. We report the fabrication of ultrathin, multilayered polyelectrolyte films that permit both the immobilization and controlled release of plasmid DNA from the surfaces of stainless steel intravascular stents. Our approach makes use of an aqueous-based, layer-by-layer method for the assembly of nanostructured thin films consisting of alternating layers of plasmid DNA and a hydrolytically degradable polyamine. Characterization of coated stents using scanning electron microscopy (SEM) demonstrated that stents were coated uniformly with an ultrathin film ca. 120 nm thick that adhered conformally to the surfaces of stent struts. These ultrathin films did not crack, peel, or delaminate substantially from the surface after exposure to a range of mechanical challenges representative of those encountered during stent deployment (e.g., balloon expansion). Stents coated with eight bilayers of degradable polyamine and a plasmid encoding enhanced green fluorescent protein (EGFP) sustained the release of DNA into solution for up to four days when incubated in phosphate buffered saline at 37 degrees C, and coated stents were capable of mediating the expression of EGFP in a mammalian cell line without the aid of additional transfection agents. The approach reported here could, with further development, contribute to the development of localized gene-based approaches to the treatment of cardiovascular diseases or related conditions.     

Identification of genetic factors influencing salt stress tolerance in white clover (Trifolium repens L.) by QTL analysis

Allotetraploid (2n = 4x = 32) white clover (Trifolium repens L.) is the most commonly cultivated legume component of temperate pastures, sown in swards with a companion grass species. Genetic control of growth performance of white clover on saline land is highly important for dairy industries, due to increasing soil salinity problems. The objective of this study was to identify quantitative trait loci (QTLs) for salinity tolerance in terms of vegetative growth under stress. Two parental genetic maps consisting of 213 and 159 marker loci and spanning 1,973.0 and 1,837.6 cM, respectively, were constructed using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers from a two-way pseudo-test cross F₁ population derived from pair-crossing of the Haifa₂ and LCL₂ genotypes. A total of 8 unique genomic regions on 8 linkage groups (LGs) of the Haifa₂ parental map and 6 unique regions on 5 LGs in the LCL₂ parental map were associated with plant growth under salt stress and relative growth under stress, as compared to control conditions. The results of this study indicate that salt tolerance in white clover is controlled by multiple QTLs, some at common locations, but each of limited magnitude. Location of these QTLs provides the genetic basis and potential for pyramiding of salt tolerance genes in breeding improvement.     

Tertiary Structure and Characterization of a Glycoside Hydrolase Family 44 Endoglucanase from Clostridium acetobutylicum

A gene encoding a glycoside hydrolase family 44 (GH44) protein from Clostridium acetobutylicum ATCC 824 was synthesized and transformed into Escherichia coli. The previously uncharacterized protein was expressed with a C-terminal His tag and purified by nickel-nitrilotriacetic acid affinity chromatography. Crystallization and X-ray diffraction to a 2.2-Å resolution revealed a triose phosphate isomerase (TIM) barrel-like structure with additional Greek key and β-sandwich folds, similar to other GH44 crystal structures. The enzyme hydrolyzes cellotetraose and larger cellooligosaccharides, yielding an unbalanced product distribution, including some glucose. It attacks carboxymethylcellulose and xylan at approximately the same rates. Its activity on carboxymethylcellulose is much higher than that of the isolated C. acetobutylicum cellulosome. It also extensively converts lichenan to oligosaccharides of intermediate size and attacks Avicel to a limited extent. The enzyme has an optimal temperature in a 10-min assay of 55°C and an optimal pH of 5.0.Thirteen glycoside hydrolase (GH) families, each having members related to each other by amino acid sequence, contain enzymes that hydrolyze cellulose and/or cellooligosaccharides (4; http://www.cazy.org). Among them is GH family 44 (GH44), most of whose enzymes are endoglucanases (EGs). In general, EGs are more active on longer rather than on shorter chains and are more likely to attack bonds in the interiors of carbohydrate chains than those near their termini.With one exception, GH44 enzymes are produced by bacteria, both aerobic and anaerobic. At present, 29 amino acid sequences of GH44 members have been determined (4). Often they are combined with other GHs in multienzyme proteins (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Structural organization of genes coding for GH44 CDs, excluding GH44 members with only a signal peptide and a CD. The gene encoding O. terrae''s GH44 member produces a 920-residue protein whose domain structure is unclassified. The sequence was searched against the Pfam library, and a low E-value, 1.22 × 10⁻²², was found for a PKD (polycystic kidney disease)-type domain. Ig, immunoglobulin.Not all of these GH44 enzymes have been produced in vitro, and those that have been produced have only been partially characterized. Experimental results indicate that GH44 enzymes exclusively cleave β-1,4 bonds between glucosyl and xylosyl residues and that they have different abilities to attack xylan, lichenan, and different cellulose forms, such as Avicel, acid-swollen cellulose, and carboxymethyl cellulose (CMC), with the presence of a carbohydrate-binding module (CBM) allowing higher activity on solid cellulose. They appear to be inactive on short oligosaccharides, like p-nitrophenyl (PNP)-β-glucopyranoside, PNP-β-cellobioside, and PNP-β-xylopyranoside.Most GH families containing cellulases have at least one member with a known tertiary structure. That was not true of GH44 until Kitago et al. (15) published six different crystal structures of an EG, CelJ, from Clostridium thermocellum. Three of the crystal structures are of the wild-type enzyme, and the other three are of the E186Q mutant, with each form being both unliganded and complexed with cellopentaose or cellohexaose. The enzyme uses a retaining mechanism, with Glu186 being the proton donor/acceptor and Glu359 being the nucleophile. Subsites −4 to −1 of the wild-type enzyme hold cellotetraose. When the E186Q mutant is soaked with cellopentaose or cellohexaose, different-length cellooligosaccharides are complexed in its subsites −4 to +5.A second tertiary structure from an unidentified bacterium is similar to that from C. thermocellum (23). The enzyme, CelM2, is a triose phosphate isomerase (TIM)-like (β,α)₈ barrel with a β-sandwich domain. It also has Glu221 and Glu393 as the catalytic proton donor/acceptor and nucleophile, respectively. These two residues are located approximately 4 Å apart from one another, similar to the catalytic residues of CelJ.The present work concerns the GH44 putative EG from Clostridium acetobutylicum ATCC 824, a Gram-positive, mesophilic, anaerobic, solvent-producing bacterium. This organism and other solvent-producing Clostridium strains cannot grow on cellulose as a sole carbon source, but the first can produce EGs, mainly extracellular, when grown on glucose, xylose, mannose, and cellobiose (18). Nearly all of the same strains can grow on larch wood xylan, but C. acetobutylicum ATCC 824 can do this only when cultured in a chemostat, where it produces xylanase activity (19).Genomic sequencing has found the gene CAC0915, which putatively encodes a fusion protein consisting of a signal peptide, a GH44 catalytic domain (CD), and a type I dockerin but no CBM in C. acetobutylicum ATCC 824 (25). This putative protein, CAC0915, has 606 amino acids for a calculated molecular mass of 66.8 kDa (25). The same project found genes for many other cellulases and xylanases. In fact, the complete coding for a cellulosome similar to those in the cellulolytic species Clostridium cellulovorans and Clostridium cellulolyticum appears to be present in C. acetobutylicum ATCC 824 (25), and a cellulosome is produced, but its cellulolytic activity is very low (28). Schwarz et al. (29) have hypothesized that C. acetobutylicum has repressed cellulosome expression and cellulolytic activity during evolution since it can grow on simpler substrates, including starch, oligosaccharides, and monosaccharides.This article reports the phylogenetic tree of the GH44 enzymes and the production, purification, and subsequent structural and kinetic characterization of C. acetobutylicum GH44 EG. This protein apparently had not been observed in isolated form before this project.     

Epidemiology of diabetes mellitus among First Nations and non-First Nations adults

Background

First Nations people in Canada experience a disproportionate burden of type 2 diabetes mellitus. To increase our understanding of this evolving epidemic, we compared the epidemiology of diabetes between First Nations and non-First Nations adults in Saskatchewan from 1980 to 2005.

Methods

We used administrative databases to perform a population-based study of diabetes frequency, incidence and prevalence in adults by ethnic background, year, age and sex.

Results

We identified 8275 First Nations and 82 306 non-First Nations people with diabetes from 1980 to 2005. Overall, the incidence and prevalence of diabetes were more than 4 times higher among First Nations women than among non-First Nations women and more than 2.5 times higher among First Nations men than among non-First Nations men. The number of incident cases of diabetes was highest among First Nations people aged 40–49 , while the number among non-First Nations people was greatest in those aged 70 or more years. The prevalence of diabetes increased over the study period from 9.5% to 20.3% among First Nations women and from 4.9% to 16.0% among First Nations men. Among non-First Nations people, the prevalence increased from 2.0% to 5.5% among women and from 2.0% to 6.2% among men. By 2005, almost 50% of First Nations women and more than 40% of First Nations men aged 60 or older had diabetes, compared with less than 25% of non-First Nations men and less than 20% of non-First Nations women aged 80 or older.

Interpretation

First Nations adults are experiencing a diabetes epidemic that disproportionately affects women during their reproductive years. This ethnicity-based pattern suggests diverse underlying mechanisms that may include differences in the diabetogenic impact of gestational diabetes.The global epidemic of type 2 diabetes mellitus disproportionately affects indigenous and developing populations.¹ Although genotypic variants related to energy balance may underlie this epidemic,² the rapid emergence of type 2 diabetes in genetically diverse populations worldwide is most likely caused by environmental factors. Increasing rates of type 2 diabetes among Canada’s First Nations people, for example, parallel an epidemic of overweight and obesity that has coincided with socio-cultural disruption and a loss of traditional lifestyles.³In Saskatchewan in 1937, diabetes was not detected among the 1500 First Nations people who underwent a tuberculosis survey.⁴ By 1990, almost 10% of the province’s First Nations adults had diabetes;⁵ by 2006, the proportion was over 20%,⁶ while it remained at about 6% in the general population.^5,6 Although an increased prevalence of diabetes among First Nations people has also been documented in other Canadian provinces,³ only recently have consistent diabetes case definitions applied to health care system administrative databases been used to compare differences between large populations of First Nations and non-First Nations people.^7–9We sought to describe the epidemiology of diabetes in Saskatchewan from 1980 to 2005. We reasoned that finding ethnicity-based differences in trends and patterns of type 2 diabetes over the longest period reported for a Canadian jurisdiction would help to clarify the underlying mechanisms behind known disparities and translate into more effective diabetes prevention and management initiatives.     

The obscure events contributing to the evolution of an incipient sex chromosome in Populus: a retrospective working hypothesis

Genetic determination of gender is a fundamental developmental and evolutionary process in plants. Although it appears that dioecy in Populus is genetically controlled, the precise gender-determining systems remain unclear. The recently released second draft assembly and annotated gene set of the Populus genome provided an opportunity to revisit this topic. We hypothesized that over evolutionary time, selective pressure has reformed the genome structure and gene composition in the peritelomeric region of the chromosome XIX, which has resulted in a distinctive genome structure and cluster of genes contributing to gender determination in Populus trichocarpa. Multiple lines of evidence support this working hypothesis. First, the peritelomeric region of the chromosome XIX contains significantly fewer single nucleotide polymorphisms than the rest of Populus genome and has a distinct evolutionary history. Second, the peritelomeric end of chromosome XIX contains the largest cluster of the nucleotide-binding site?Cleucine-rich repeat (NBS?CLRR) class of disease resistance genes in the entire Populus genome. Third, there is a high occurrence of small microRNAs on chromosome XIX, which is coincident to the region containing the putative gender-determining locus and the major cluster of NBS?CLRR genes. Further, by analyzing the metabolomic profiles of floral bud in male and female Populus trees using a gas chromatography-mass spectrometry, we found that there are gender-specific accumulations of phenolic glycosides. Taken together, these findings led to the hypothesis that resistance to and regulation of a floral pathogen and gender determination coevolved, and that these events triggered the emergence of a nascent sex chromosome. Further studies of chromosome XIX will provide new insights into the genetic control of gender determination in Populus.     

Porewater Stoichiometry of Terminal Metabolic Products, Sulfate, and Dissolved Organic Carbon and Nitrogen in Estuarine Intertidal Creek-bank Sediments

Porewater equilibration samplers were used to obtain porewater inventories of inorganic nutrients (NH₄⁺, NO_x, PO₄³⁻), dissolved organic carbon (DOC) and nitrogen (DON), sulfate (SO₄²⁻), dissolved inorganic carbon (DIC), hydrogen sulfide (H₂S), chloride (Cl⁻), methane (CH₄) and reduced iron (Fe²⁺) in intertidal creek-bank sediments at eight sites in three estuarine systems over a range of salinities and seasons. Sulfate reduction (SR) rates and sediment particulate organic carbon (POC) and nitrogen (PON) were also determined at several of the sites. Four sites in the Okatee River estuary in South Carolina, two sites on Sapelo Island, Georgia and one site in White Oak Creek, Georgia appeared to be relatively pristine. The eighth site in Umbrella Creek, Georgia was directly adjacent to a small residential development employing septic systems to handle household waste. The large data set (>700 porewater profiles) offers an opportunity to assess system-scale patterns of porewater biogeochemical dynamics with an emphasis on DOC and DON distributions. SO₄²⁻ depletion (SO₄²⁻)_Dep was used as a proxy for SR, and (SO₄²⁻)_Dep patterns agreed with measured (³⁵S) patterns of SR. There were significant system-scale correlations between the inorganic products of terminal metabolism (DIC, NH₄⁺ and PO₄³⁻) and (SO₄²⁻)_Dep, and SR appeared to be the dominant terminal carbon oxidation pathway in these sediments. Porewater inventories of DIC and (SO₄²⁻)_Dep indicate a 2:1 stoichiometry across sites, and the C:N ratio of the organic matter undergoing mineralization was between 7.5 and 10. The data suggest that septic-derived dissolved organic matter with a C:N ratio below 6 fueled microbial metabolism and SR at a site with development in the upland. Seasonality was observed in the porewater inventories, but temperature alone did not adequately describe the patterns of (SO₄²⁻)_Dep, terminal metabolic products (DIC, NH₄⁺, PO₄³⁻), DOC and DON, and SR observed in this study. It appears that production and consumption of labile DOC are tightly coupled in these sediments, and that bulk DOC is likely a recalcitrant pool. Preferential hydrolysis of PON relative to POC when overall organic matter mineralization rates were high appears to drive the observed patterns in POC:PON, DOC:DON and DIC:DIN ratios. These data, along with the weak seasonal patterns of SR and organic and inorganic porewater inventories, suggest that the rate of hydrolysis limits organic matter mineralization in these intertidal creek-bank sediments.     

Establishing a Statistical Link between Network Oscillations and Neural Synchrony

Pairs of active neurons frequently fire action potentials or “spikes” nearly synchronously (i.e., within 5 ms of each other). This spike synchrony may occur by chance, based solely on the neurons’ fluctuating firing patterns, or it may occur too frequently to be explicable by chance alone. When spike synchrony above chances levels is present, it may subserve computation for a specific cognitive process, or it could be an irrelevant byproduct of such computation. Either way, spike synchrony is a feature of neural data that should be explained. A point process regression framework has been developed previously for this purpose, using generalized linear models (GLMs). In this framework, the observed number of synchronous spikes is compared to the number predicted by chance under varying assumptions about the factors that affect each of the individual neuron’s firing-rate functions. An important possible source of spike synchrony is network-wide oscillations, which may provide an essential mechanism of network information flow. To establish the statistical link between spike synchrony and network-wide oscillations, we have integrated oscillatory field potentials into our point process regression framework. We first extended a previously-published model of spike-field association and showed that we could recover phase relationships between oscillatory field potentials and firing rates. We then used this new framework to demonstrate the statistical relationship between oscillatory field potentials and spike synchrony in: 1) simulated neurons, 2) in vitro recordings of hippocampal CA1 pyramidal cells, and 3) in vivo recordings of neocortical V4 neurons. Our results provide a rigorous method for establishing a statistical link between network oscillations and neural synchrony.     

Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis

We report here a high-throughput method for the modification of bacterial artificial chromosomes (BACs) that uses a novel two-plasmid approach. In this protocol, a vector modified in our laboratory to hold an R6Kγ origin of replication and a marker recombination cassette is inserted into a BAC in a single recombination step. Temporal control of recombination is achieved through the use of a second plasmid, pSV1.RecA, which possesses a recombinase gene and a temperature-sensitive origin of replication. This highly efficient protocol has allowed us to successfully modify more than 2,000 BACs, from which over 1,000 BAC transgenic mice have been generated. A complete cycle from BAC choice to embryo implantation takes about 5 weeks. Marker genes introduced into the mice include EGFP and EGFP-L10a. All vectors used in this project can be obtained from us by request, and the EGFP reporter mice are available through the Mutant Mouse Regional Resource Center (NINDS/GENSAT collection). CNS anatomical expression maps of the mice are available to the public at http://www.gensat.org/.     

The 1.9 Å Structure of Human α-N-Acetylgalactosaminidase: The Molecular Basis of Schindler and Kanzaki Diseases

α-N-acetylgalactosaminidase (α-NAGAL; E.C. 3.2.1.49) is a lysosomal exoglycosidase that cleaves terminal α-N-acetylgalactosamine residues from glycopeptides and glycolipids. In humans, a deficiency of α-NAGAL activity results in the lysosomal storage disorders Schindler disease and Kanzaki disease. To better understand the molecular defects in the diseases, we determined the crystal structure of human α-NAGAL after expressing wild-type and glycosylation-deficient glycoproteins in recombinant insect cell expression systems. We measured the enzymatic parameters of our purified wild-type and mutant enzymes, establishing their enzymatic equivalence. To investigate the binding specificity and catalytic mechanism of the human α-NAGAL enzyme, we determined three crystallographic complexes with different catalytic products bound in the active site of the enzyme. To better understand how individual defects in the α-NAGAL glycoprotein lead to Schindler disease, we analyzed the effect of disease-causing mutations on the three-dimensional structure.