Assessing soil biological characteristics: a comparison of bulk soil community DNA-, PLFA-, and Biolog-analyses

Soil microbiological analyses may serve as a means for assessing soil characteristics. Standard microbiological culture-techniques, however, leave over 90% of the microorganisms in the environment unaccounted for. Several more recently developed analytical techniques such as DNA, phospholipid fatty acid (PLFA), and community level substrate utilization (CLSU) Ængerprints allow for more detailed analyses of soil microbial communities. We applied analyses of (1) community DNA with PCR and restriction fragment length polymorphism (RFLP), (2) community PLFAs with gas chromatography and mass spectrometry, and (3) CLSU with Biologe gram-negative-plates, to evaluate the biological characteristics of three soils used in pesticide degradation studies. Each of these methods analyzes a different aspect of soil microbial characteristics. A protocol was developed for the statistical comparison and combination of the data from all the analyses, thus allowing for a polyphasic approach to biological soil characterization. We found that all three methods yielded highly reproducible results for each soil and allowed to distinguish the soils based on the structures of speciÆc gene- and PLFA-pools as well as on CLSU Ængerprints. Not all methods, however, revealed the same relative similarities of the three soils based on cluster analysis of the biological characteristics. These results demonstrate the value of comparative data analyses and indicate that biological soil characterization needs to be interpreted with caution if it is performed with one method only. (C) 2001 Elsevier Science Ltd

Modeling Esophagitis Using Human Three-Dimensional Organotypic Culture System

Esophagitis, whether caused by acid reflux, allergic responses, graft-versus-host disease, drugs, or infections, is a common condition of the gastrointestinal tract affecting nearly 20% of the US population. The instigating agent typically triggers an inflammatory response. The resulting inflammation is a risk factor for the development of esophageal strictures, Barrett esophagus, and esophageal adenocarcinoma. Research into the pathophysiology of these conditions has been limited by the availability of animal and human model systems. Three-dimensional organotypic tissue culture (OTC) is an innovative three-dimensional multicellular in vitro platform that recapitulates normal esophageal epithelial stratification and differentiation. We hypothesized that this platform can be used to model esophagitis to better understand the interactions between immune cells and the esophageal epithelium. We found that human immune cells remain viable and respond to cytokines when cultured under OTC conditions. The acute inflammatory environment induced in the OTC significantly affected the overlying epithelium, inducing a regenerative response marked by increased cell proliferation and epithelial hyperplasia. Moreover, oxidative stress from the acute inflammation induced DNA damage and strand breaks in epithelial cells, which could be reversed by antioxidant treatment. These findings support the importance of immune cell-mediated esophageal injury in esophagitis and confirms the utility of the OTC platform to characterize the underlying molecular events in esophagitis

A tissue engineering approach for periodontal regeneration based on a biodegradable double-layer scaffold and adipose-derived stem cells

Human and canine periodontium are often affected by an inflammatory pathology called periodontitis, which isassociated with severe damages across tissues, namely, in the periodontal ligament, cementum, and alveolarbone. However, the therapies used in the routine dental practice, often consisting in a combination of differenttechniques, do not allow to fully restore the functionality of the periodontium. Tissue Engineering (TE) appearsas a valuable alternative approach to regenerate periodontal defects, but for this purpose, it is essential todevelop supportive biomaterial and stem cell sourcing/culturing methodologies that address the complexity ofthe various tissues affected by this condition. The main aim of this work was to study the in vitro functionalityof a newly developed double-layer scaffold for periodontal TE. The scaffold design was based on a combinationof a three-dimensional (3D) fiber mesh functionalized with silanol groups and a membrane, both made of ablend of starch and poly-e-(caprolactone). Adipose-derived stem cells (canine adipose stem cells [cASCs]) wereseeded and cultured onto such scaffolds, and the obtained constructs were evaluated in terms of cellularmorphology, metabolic activity, and proliferation. The osteogenic potential of the fiber mesh layer functionalizedwith silanol groups was further assessed concerning the osteogenic differentiation of the seeded andcultured ASCs. The obtained results showed that the proposed double-layer scaffold supports the proliferationand selectively promotes the osteogenic differentiation of cASCs seeded onto the functionalized mesh. Thesefindings suggest that the 3D structure and asymmetric composition of the scaffold in combination with stemcells may provide the basis for developing alternative therapies to treat periodontal defects more efficiently.The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no REGPOT-CT2012-316331-POLARIS. J.F.R. acknowledges the Portuguese Foundation for Science and Technology (FCT) for his PhD scholarship (SFRH/BD/44143/2008)