Mesenchymal stem cells in regenerative medicine: Opportunities and challenges for articular cartilage and intervertebral disc tissue engineering

Defects of load‐bearing connective tissues such as articular cartilage and intervertebral disc (IVD) can result from trauma, degenerative, endocrine, or age‐related disease. Current surgical and pharmacological options for the treatment of arthritic rheumatic conditions in the joints and spine are ineffective. Cell‐based surgical therapies such as autologous chondrocyte transplantation (ACT) have been in clinical use for cartilage repair for over a decade but this approach has shown mixed results. This review focuses on the potential of mesenchymal stem cells (MSCs) as an alternative to cells derived from patient tissues in autologous transplantation and tissue engineering. Here we discuss the prospects of using MSCs in regenerative medicine and summarize the advantages and disadvantages of these cells in articular cartilage and IVD tissue engineering. We discuss the conceptual and practical difficulties associated with differentiating and pre‐conditioning MSCs for subsequent survival in a physiologically harsh extracellular matrix, an environment that will be highly hypoxic, acidic, and nutrient deprived. Implanted MSCs will be exposed to traumatic physical loads and high levels of locally produced inflammatory mediators and catabolic cytokines. We also explore the potential of culture models of MSCs, fully differentiated cells and co‐cultures as “proof of principle” ethically acceptable “3Rs” models for engineering articular cartilage and IVD in vitro for the purpose of replacing the use of animals in arthritis research. J. Cell. Physiol. 222:23–32, 2010. © 2009 Wiley‐Liss, Inc.     

Physiology and Bioenergetics of [NiFe]-Hydrogenase 2-Catalyzed H2-Consuming and H2-Producing Reactions in Escherichia coli

Escherichia coli uptake hydrogenase 2 (Hyd-2) catalyzes the reversible oxidation of H₂ to protons and electrons. Hyd-2 synthesis is strongly upregulated during growth on glycerol or on glycerol-fumarate. Membrane-associated Hyd-2 is an unusual heterotetrameric [NiFe]-hydrogenase that lacks a typical cytochrome b membrane anchor subunit, which transfers electrons to the quinone pool. Instead, Hyd-2 has an additional electron transfer subunit, termed HybA, with four predicted iron-sulfur clusters. Here, we examined the physiological role of the HybA subunit. During respiratory growth with glycerol and fumarate, Hyd-2 used menaquinone/demethylmenaquinone (MQ/DMQ) to couple hydrogen oxidation to fumarate reduction. HybA was essential for electron transfer from Hyd-2 to MQ/DMQ. H₂ evolution catalyzed by Hyd-2 during fermentation of glycerol in the presence of Casamino Acids or in a fumarate reductase-negative strain growing with glycerol-fumarate was also shown to be dependent on both HybA and MQ/DMQ. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Hyd-2-dependent H₂ evolution from glycerol, indicating the requirement for a proton gradient. In contrast, CCCP failed to inhibit H₂-coupled fumarate reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H₂ oxidation or H₂ evolution in whole cells, reversible H₂-dependent reduction of viologen dyes still occurred. Finally, hydrogen-dependent dye reduction by Hyd-2 was reversibly inhibited in extracts derived from cells grown in H₂ evolution mode. Our findings suggest that Hyd-2 switches between H₂-consuming and H₂-producing modes in response to the redox status of the quinone pool. Hyd-2-dependent H₂ evolution from glycerol requires reverse electron transport.     

Identification of ELF3 as an early transcriptional regulator of human urothelium

Despite major advances in high-throughput and computational modelling techniques, understanding of the mechanisms regulating tissue specification and differentiation in higher eukaryotes, particularly man, remains limited. Microarray technology has been explored exhaustively in recent years and several standard approaches have been established to analyse the resultant datasets on a genome-wide scale. Gene expression time series offer a valuable opportunity to define temporal hierarchies and gain insight into the regulatory relationships of biological processes. However, unless datasets are exactly synchronous, time points cannot be compared directly.     

Pyrogenic carbon capture and storage

The growth of biomass is considered the most efficient method currently available to extract carbon dioxide from the atmosphere. However, biomass carbon is easily degraded by microorganisms releasing it in the form of greenhouse gases back to the atmosphere. If biomass is pyrolyzed, the organic carbon is converted into solid (biochar), liquid (bio‐oil), and gaseous (permanent pyrogas) carbonaceous products. During the last decade, biochar has been discussed as a promising option to improve soil fertility and sequester carbon, although the carbon efficiency of the thermal conversion of biomass into biochar is in the range of 30%–50% only. So far, the liquid and gaseous pyrolysis products were mainly considered for combustion, though they can equally be processed into recalcitrant forms suitable for carbon sequestration. In this review, we show that pyrolytic carbon capture and storage (PyCCS) can aspire for carbon sequestration efficiencies of >70%, which is shown to be an important threshold to allow PyCCS to become a relevant negative emission technology. Prolonged residence times of pyrogenic carbon can be generated (a) within the terrestrial biosphere including the agricultural use of biochar; (b) within advanced bio‐based materials as long as they are not oxidized (biochar, bio‐oil); and (c) within suitable geological deposits (bio‐oil and CO₂ from permanent pyrogas oxidation). While pathway (c) would need major carbon taxes or similar governmental incentives to become a realistic option, pathways (a) and (b) create added economic value and could at least partly be implemented without other financial incentives. Pyrolysis technology is already well established, biochar sequestration and bio‐oil sequestration in soils, respectively biomaterials, do not present ecological hazards, and global scale‐up appears feasible within a time frame of 10–30 years. Thus, PyCCS could evolve into a decisive tool for global carbon governance, serving climate change mitigation and the sustainable development goals simultaneously.     

Metabolic Features of Protochlamydia amoebophila Elementary Bodies – A Link between Activity and Infectivity in Chlamydiae

The Chlamydiae are a highly successful group of obligate intracellular bacteria, whose members are remarkably diverse, ranging from major pathogens of humans and animals to symbionts of ubiquitous protozoa. While their infective developmental stage, the elementary body (EB), has long been accepted to be completely metabolically inert, it has recently been shown to sustain some activities, including uptake of amino acids and protein biosynthesis. In the current study, we performed an in-depth characterization of the metabolic capabilities of EBs of the amoeba symbiont Protochlamydia amoebophila. A combined metabolomics approach, including fluorescence microscopy-based assays, isotope-ratio mass spectrometry (IRMS), ion cyclotron resonance Fourier transform mass spectrometry (ICR/FT-MS), and ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was conducted, with a particular focus on the central carbon metabolism. In addition, the effect of nutrient deprivation on chlamydial infectivity was analyzed. Our investigations revealed that host-free P. amoebophila EBs maintain respiratory activity and metabolize D-glucose, including substrate uptake as well as host-free synthesis of labeled metabolites and release of labeled CO₂ from ¹³C-labeled D-glucose. The pentose phosphate pathway was identified as major route of D-glucose catabolism and host-independent activity of the tricarboxylic acid (TCA) cycle was observed. Our data strongly suggest anabolic reactions in P. amoebophila EBs and demonstrate that under the applied conditions D-glucose availability is essential to sustain metabolic activity. Replacement of this substrate by L-glucose, a non-metabolizable sugar, led to a rapid decline in the number of infectious particles. Likewise, infectivity of Chlamydia trachomatis, a major human pathogen, also declined more rapidly in the absence of nutrients. Collectively, these findings demonstrate that D-glucose is utilized by P. amoebophila EBs and provide evidence that metabolic activity in the extracellular stage of chlamydiae is of major biological relevance as it is a critical factor affecting maintenance of infectivity.     

Molecular AFM imaging of Hsp70-1A association with dipalmitoyl phosphatidylserine reveals membrane blebbing in the presence of cholesterol

Hsp70-1A—the major stress-inducible member of the HSP70 chaperone family—is being implicated in cancer diseases with the development of resistances to standard therapies. In normal cells, the protein is purely cytosolic, but in a growing number of tumor cells, a significant fraction can be identified on to the cell surface. The anchoring mechanism is still under debate, as Hsp70-1A lacks conventional signaling sequences for translocation from the cytosol to exoplasmic leaflet of the plasma membrane and common membrane binding domains. Recent reports propose a lipid-mediated anchoring mechanism based on a specific interaction with charged, saturated lipids such as dipalmitoyl phosphatidylserine (DPPS). Here, we prepared planar supported lipid bilayers (SLBs) to visualize the association of Hsp70-1A directly and on the single molecule level by atomic force microscopy (AFM). The single molecule sensitivity of our approach allowed us to explore the low concentration range of 0.05 to 1.0 μg/ml of Hsp70-1A which was not studied before. We compared the binding of the protein to bilayers with 20% DPPS lipid content both in the absence and presence of cholesterol. Hsp70-1A inserted exclusively into DPPS domains and assembled in clusters with increasing protein density. A critical density was reached for incubation with 0.5 μg/ml (7 nM); at higher concentrations, membrane defects were observed that originated from cluster centers. In the presence of cholesterol, this critical concentration leads to the formation of membrane blebs, which burst at higher concentrations supporting a previously proposed non-classical pathway for the export of Hsp70-1A by tumor cells. In the discussion of our data, we attempt to link the lipid-mediated plasma membrane localization of Hsp70-1A to its potential involvement in the development of resistances to radiation and chemotherapy based on our own findings and the current literature.     

Generic quality of life assessment in dementia patients: a prospective cohort study

Background  

Quality of life (QoL) is increasingly used to characterize the impact of disease and the efficacy of interventions.     

Molecular basis for the catalytic inactivity of a naturally occurring near-null variant of human ALOX15

Mammalian lipoxygenases belong to a family of lipid-peroxidizing enzymes, which have been implicated in cardiovascular, hyperproliferative and neurodegenerative diseases. Here we report that a naturally occurring mutation in the hALOX15 gene leads to expression of a catalytically near-null enzyme variant (hGly422Glu). The inactivity may be related to severe misfolding of the enzyme protein, which was concluded from CD-spectra as well as from thermal and chemical stability assays. In silico mutagenesis experiments suggest that most mutations at hGly422 have the potential to induce sterical clash, which might be considered a reason for protein misfolding. hGly422 is conserved among ALOX5, ALOX12 and ALOX15 isoforms and corresponding hALOX12 and hALOX5 mutants also exhibited a reduced catalytic activity. Interestingly, in the hALOX5 Gly429Glu mutants the reaction specificity of arachidonic acid oxygenation was shifted from 5S- to 8S- and 12R-H(p)ETE formation. Taken together, our data indicate that the conserved glycine is of functional importance for these enzyme variants and most mutants at this position lose catalytic activity.     

Dandelion diaspore dispersal: frictional anisotropy of cypselae of Taraxacum officinale enhances their interlocking with the soil

Plant and Soil - Green roofs are important novel urban ecosystems, but their shallow substrates can create plant water deficits in dry climates. Physiological approaches can improve green roof...