A novel dermal tissue construct: Development and in vitro characterization

A dermal tissue construct composed of human dermal fibroblasts and a chitosan sponge has been developed, targeted towards the treatment of diabetic nonhealing ulcers. The construct has been designed in a way that the dermal fibroblasts are arranged as a three‐dimensional sheet adhered entirely on one side of the chitosan sponge. This design would allow maximal diffusion of growth factors from the cells to the wound bed when the construct is applied on the wound with the cellular sheet side making contact with the wound bed. The diffusion of secreted growth factors would take place directly from cells to the wound bed without being impeded by a matrix. The cells are present at a high density in the dermal construct, which would aid in accelerated wound healing. The construct has a porous chitosan sponge base, which would allow gas exchange, and renders the dermal construct very flexible so that it would take the shape of the wound contours well, while having mechanical integrity. The viability of cells in the construct is greater than 90%. The dermal construct produces a high amount of vascular endothelial growth factor, from 42 ng to 31 ng in 24 h. The construct also produces high amounts of Interleukin‐8 (IL‐8), from 375 ng to 1065 ng in the first 24 h. Both VEGF and IL‐8 have important roles in the healing of chronic diabetic ulcers. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Urban riparian systems function as corridors for both native and invasive plant species

Riparian areas are often the only green areas left in urban and suburban landscapes, providing opportunities for conservation and connectivity of both aquatic and terrestrial organisms. While city planners and land managers often tout the importance of riparian networks for these uses, it is not well established if urban riparian plant communities are actually functioning as connected assemblages. Furthermore, urban riparian zones are well known to be highly invaded by non-native plant species and may be functioning to increase the spread of non-native species across the landscape. Here we examine connectivity of plant assemblages in riparian networks within an extensively urbanized landscape. We sampled riparian plant communities at 13 sites along three second-order streams of the Rahway River watershed, New Jersey. We also characterized propagule dispersal at each site by sampling litter packs on the river banks five times between March–October 2011 and identifying germinants from litter packs after cold stratification. Species turnover of both riparian and litter vegetation was more strongly associated with flow distance, particularly for native species, indicating that riverine systems are important for promoting connectivity of native plant assemblages in urban landscapes. However, non-native germinants significantly dominated propagule dispersal along the stream reaches, particularly early in the growing season, suggesting spread utilizing the river system and preemption may be an important mechanism for invasion success in this system. Our data show that management of invasive species should be planned and implemented at the watershed scale to reduce spread via the river system.     

Rhizosphere competent <Emphasis Type="Italic">Pantoea agglomerans</Emphasis> enhances maize (<Emphasis Type="Italic">Zea mays</Emphasis>) and chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) growth,without altering the rhizosphere functional diversity

Plant growth promoting Pantoea agglomerans NBRISRM (NBRISRM) was able to produce 60.4 μg/ml indole acetic acid and solubilize 77.5 μg/ml tri-calcium phosphate under in vitro conditions. Addition of 2% NaCl (w/v) in the media induced the IAA production and phosphate solubilization by 11% and 7%, respectively. For evaluating the plant growth promotory effect of NBRISRM inoculation a micro plot trial was conducted using maize and chickpea as host plants. The results revealed significant increase in all growth parameters tested in NBRISRM inoculated maize and chickpea plants, which were further confirmed by higher macronutrients (N, P and K) accumulation as compared to un-inoculated controls. Throughout the growing season of maize and chickpea, rhizosphere population of NBRISRM were in the range 10⁷–10⁸ CFU/g soil and competing with 10⁷–10⁹ CFU/g soil with heterogeneous bacterial population. Functional richness, diversity, and evenness were found significantly higher in maize rhizosphere as compared to chickpea, whereas NBRISRM inoculation were not able to change it, in both crops as compared to their un-inoculated control. To the best of our knowledge this is first report where we demonstrated the effect of P. agglomerans strain for improving maize and chickpea growth without altering the functional diversity.     

Mitochondrial aconitase knockdown attenuates paraquat-induced dopaminergic cell death via decreased cellular metabolism and release of iron and H₂O₂

Mitochondrial oxidative stress is a contributing factor in the etiology of numerous neuronal disorders. However, the precise mechanism(s) by which mitochondrial reactive oxygen species modify cellular targets to induce neurotoxicity remains unknown. In this study, we determined the role of mitochondrial aconitase (m-aconitase) in neurotoxicity by decreasing its expression. Incubation of the rat dopaminergic cell line, N27, with paraquat (PQ(2+) ) resulted in aconitase inactivation, increased hydrogen peroxide (H(2) O(2) ) and increased ferrous iron (Fe(2+) ) at times preceding cell death. To confirm the role of m-aconitase in dopaminergic cell death, we knocked down m-aconitase expression via RNA interference. Incubation of m-aconitase knockdown N27 cells with PQ(2+) resulted in decreased H(2) O(2) production, Fe(2+) accumulation, and cell death compared with cells expressing basal levels of m-aconitase. To determine the metabolic role of m-aconitase in mediating neuroprotection, we conducted a complete bioenergetic profile. m-Aconitase knockdown N27 cells showed a global decrease in metabolism (glycolysis and oxygen consumption rates) which blocked PQ(2+) -induced H(+) leak and respiratory capacity deficiency. These findings suggest that dopaminergic cells are protected from death by decreasing release of H(2) O(2) and Fe(2+) in addition to decreased cellular metabolism.     

Allergenic pollen in the atmosphere of Rohtak city,Haryana (India): a pioneer study

An attempt has been made in the present study to assess the allergenicity of dominant pollen types recorded from the atmosphere of Rohtak city. Skin prick test was performed with the antigenic extracts of 22 pollen types on 150 local patients who visited Asthma Clinic, University of Health Sciences, Rohtak. Markedly positive skin reactions (2+ and above) varied from 4.6 to 20.6 % to various pollen antigens. Cenchrus ciliaria (20.6 %), Zea mays (20 %) and Pennisetum typhoides (19.3 %) were the pollen allergens exhibiting maximum sensitivity. Antigenic extract of Cassia occidentalis, Cynodon dactylon and Ricinus communis showed marked skin reactivity in 18.6 % of patients. Prosopis juliflora, Chenopodium murale, Amaranthus spinosus, Cassia fistula and Cassia siamea showed 2+ and above reactions in 16.6, 15.3, 14.6 and 14.0 % of the local patients, respectively. Least reactivity (4.6 %) was shown to the antigenic extract of Cyperus rotundus. Out of 52 sera screened for the presence of specific IgE antibodies against different antigenic extracts, only 5.5 % showed >60 % binding. About 30 % and above binding was shown to the antigenic extracts of Z. mays, A. spinosus, R. communis and Xanthium strumarium. The concordance between positive skin reaction and serum-specific IgE antibodies ranged from 15 to 69 %.     

Digital Genotyping of Macrosatellites and Multicopy Genes Reveals Novel Biological Functions Associated with Copy Number Variation of Large Tandem Repeats

Tandem repeats are common in eukaryotic genomes, but due to difficulties in assaying them remain poorly studied. Here, we demonstrate the utility of Nanostring technology as a targeted approach to perform accurate measurement of tandem repeats even at extremely high copy number, and apply this technology to genotype 165 HapMap samples from three different populations and five species of non-human primates. We observed extreme variability in copy number of tandemly repeated genes, with many loci showing 5–10 fold variation in copy number among humans. Many of these loci show hallmarks of genome assembly errors, and the true copy number of many large tandem repeats is significantly under-represented even in the high quality ‘finished’ human reference assembly. Importantly, we demonstrate that most large tandem repeat variations are not tagged by nearby SNPs, and are therefore essentially invisible to SNP-based GWAS approaches. Using association analysis we identify many cis correlations of large tandem repeat variants with nearby gene expression and DNA methylation levels, indicating that variations of tandem repeat length are associated with functional effects on the local genomic environment. This includes an example where expansion of a macrosatellite repeat is associated with increased DNA methylation and suppression of nearby gene expression, suggesting a mechanism termed “repeat induced gene silencing”, which has previously been observed only in transgenic organisms. We also observed multiple signatures consistent with altered selective pressures at tandemly repeated loci, suggesting important biological functions. Our studies show that tandemly repeated loci represent a highly variable fraction of the genome that have been systematically ignored by most previous studies, copy number variation of which can exert functionally significant effects. We suggest that future studies of tandem repeat loci will lead to many novel insights into their role in modulating both genomic and phenotypic diversity.     

A Novel Method to Evaluate the Community Built Environment Using Photographs – Environmental Profile of a Community Health (EPOCH) Photo Neighbourhood Evaluation Tool

Background

Previous research has shown that environments with features that encourage walking are associated with increased physical activity. Existing methods to assess the built environment using geographical information systems (GIS) data, direct audit or large surveys of the residents face constraints, such as data availability and comparability, when used to study communities in countries in diverse parts of the world. The aim of this study was to develop a method to evaluate features of the built environment of communities using a standard set of photos. In this report we describe the method of photo collection, photo analysis instrument development and inter-rater reliability of the instrument.

Methods/Principal Findings

A minimum of 5 photos were taken per community in 86 communities in 5 countries according to a standard set of instructions from a designated central point of each community by researchers at each site. A standard pro forma derived from reviewing existing instruments to assess the built environment was developed and used to score the characteristics of each community. Photo sets from each community were assessed independently by three observers in the central research office according to the pro forma and the inter-rater reliability was compared by intra-class correlation (ICC). Overall 87% (53 of 60) items had an ICC of ≥0.70, 7% (4 of 60) had an ICC between 0.60 and 0.70 and 5% (3 of 60) items had an ICC ≤0.50.

Conclusions/Significance

Analysis of photos using a standardized protocol as described in this study offers a means to obtain reliable and reproducible information on the built environment in communities in very diverse locations around the world. The collection of the photographic data required minimal training and the analysis demonstrated high reliability for the majority of items of interest.     

Subversion of Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins

Pathogenic microbes have evolved sophisticated molecular strategies to subvert host defenses. Here we show that virulent bacteria interfere directly with Toll-like receptor (TLR) function by secreting inhibitory homologs of the Toll/interleukin-1 receptor (TIR) domain. Genes encoding TIR domain containing-proteins (Tcps) were identified in Escherichia coli CFT073 (TcpC) and Brucella melitensis (TcpB). We found that TcpC is common in the most virulent uropathogenic E. coli strains and promotes bacterial survival and kidney pathology in vivo. In silico analysis predicted significant tertiary structure homology to the TIR domain of human TLR1, and we show that the Tcps impede TLR signaling through the myeloid differentiation factor 88 (MyD88) adaptor protein, owing to direct binding of Tcps to MyD88. Tcps represent a new class of virulence factors that act by inhibiting TLR- and MyD88-specific signaling, thus suppressing innate immunity and increasing virulence.     

Oxidative dealkylation of a hindered phenol catalyzed by copper (II) bis benzimidazole diamide complex

The oxidative dealkylation of 2,4,6-tri-tert-butylphenol (TTBP) has been investigated using molecular oxygen and [Cu(NO₃(GBHA)](NO₃) as catalyst, where GBHA is N,N′-bis((benzimidazol-2-yl)methyl)hexanediamide [(a) M. Gupta, P. Mathur, R.J. Butcher, Inorg. Chem. 40 (2001) 878; (b) M. Gupta, S.K. Das, P. Mathur, A.W. Cordes, Inorg. Chim. Acta 353 (2003) 197; (c) S. Tehlan, M.S. Hundal, P. Mathur, Inorg. Chem. 43 (2004) 6589; (d) F. Afreen, P. Mathur, A. Rheingold, Inorg. Chim. Acta 358 (2005) 1125.]. X-ray structural characterization of complex [Cu(NO₃)(GBHA)](NO₃) · CH₃OH confirms that the Cu (II) ion is in a distorted square pyramidal geometry (τ = 0.168). The TTBP oxidation reaction proceeds via tri-tert-butylphenoxyl radical producing two products 2,6-di-tert-butyl-1,4-benzoquinone (A) and 4,6-di-tert-butyl-1,2-benzoquinone (B). Both A and B have been well characterized by ¹H NMR, ¹³C NMR, UV-Vis and mass data.     

Immunization with recombinant adenovirus synthesizing the secretory form of Japanese encephalitis virus envelope protein protects adenovirus-exposed mice against lethal encephalitis

Replication-defective recombinant adenoviruses (RAds) were constructed that synthesized the pre-membrane and envelope (E) proteins of Japanese encephalitis virus (JEV). Recombinant virus RAdEa synthesized Ea, the membrane-anchored E protein, and RAdEs synthesized Es, the secretory E protein. Compared with RAdEs, RAdEa replicated poorly in HEK 293A cells and synthesized lower amounts of E protein. Oral immunization of mice with RAds generated low titers of anti-JEV antibodies that had little JEV neutralizing activity. Intra-muscular (IM) immunization of mice with either RAd generated high titers of anti-JEV antibodies. Interestingly, RAdEa induced only low titers of JEV neutralizing antibodies. Titers were significantly higher in case of RAdEs immunization. Splenocytes from mice immunized IM with RAds secreted large amounts of interferon-γ and moderate amounts of interleukin-5 in the presence of JEV and showed cytotoxic activity against JEV-infected cells. Naïve mice immunized IM with RAdEs showed complete protection against a lethal dose of JEV given intra-cerebrally. In order to study the effect of the pre-existing adenovirus 5 (Ad5) immunity on the outcome of the RAdEs immunization, mice were exposed to Ad5 through IM or intra-nasal (IN) routes before immunization with RAdEs. Mice exposed to Ad5 through the IN route, when immunized with RAdEs given IM, or those exposed to Ad5 through the IM route, when immunized with RAdEs given IN, were completely protected against lethal JEV challenge.