Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, <Emphasis Type="Italic">in vitro</Emphasis>effects on cartilage metabolism and intra-articular levels after injection

Introduction  

Intraarticular administration of autologous conditioned serum (ACS) recently demonstrated some clinical effectiveness in treatment of osteoarthritis (OA). The current study aims to evaluate the in vitro effects of ACS on cartilage proteoglycan (PG) metabolism, its composition and the effects on synovial fluid (SF) cytokine levels following intraarticular ACS administration.     

Sampling Protein Form and Function with the Atomic Force Microscope

To study the structure, function, and interactions of proteins, a plethora of techniques is available. Many techniques sample such parameters in non-physiological environments (e.g. in air, ice, or vacuum). Atomic force microscopy (AFM), however, is a powerful biophysical technique that can probe these parameters under physiological buffer conditions. With the atomic force microscope operating under such conditions, it is possible to obtain images of biological structures without requiring labeling and to follow dynamic processes in real time. Furthermore, by operating in force spectroscopy mode, it can probe intramolecular interactions and binding strengths. In structural biology, it has proven its ability to image proteins and protein conformational changes at submolecular resolution, and in proteomics, it is developing as a tool to map surface proteomes and to study protein function by force spectroscopy methods. The power of AFM to combine studies of protein form and protein function enables bridging various research fields to come to a comprehensive, molecular level picture of biological processes. We review the use of AFM imaging and force spectroscopy techniques and discuss the major advances of these experiments in further understanding form and function of proteins at the nanoscale in physiologically relevant environments.To understand biological processes at the molecular level it is essential to identify the involved proteins and proteinaceous assemblies, to characterize their structure and function, and to unravel their interplay with other proteins and molecules (1). Techniques like x-ray crystallography, electron microscopy, nuclear magnetic resonance spectroscopy, and mass spectrometry have contributed massively to elucidate such protein properties. These techniques can easily sample the properties of a large ensemble of proteins; however, they require subjecting the sample to harsh treatments such as drying, crystallizing, or vaporizing in vacuum, thereby limiting the range of measurable dynamical properties of the sample. One powerful method that permits the investigation of molecules in their native physiological buffer condition is atomic force microscopy (AFM)¹ (2). An atomic force microscope is a microscope and force spectrometer at the same time. The imaging resolution of the atomic force microscope is comparable with that of electron microscopes, and it has the special capability to image samples in a variety of environments such as in vacuum, air, or liquid, which therefore enables studying biological specimens in their native environments (i.e. in buffer solutions) (3, 4). In addition, its ability to “touch” the sample gives it the advantage to manipulate single particles/molecules and probe their mechanical properties (5–8). However, AFM force spectroscopy is currently a technique with rather fast pulling and pushing speeds, thereby often operating out of equilibrium conditions. Improvements with ultrastable atomic force microscopes are underway to tackle this problem with promising results (9, 10). Furthermore, AFM is not well suited to apply and resolve forces at the single piconewton range due to large size tips and relatively stiff cantilevers. The issue of nonspecificity of the tip interaction with the sample is also of concern, especially in pulling experiments that require the capability to accurately recognize and select the appropriate molecule or point of interest. The current introduction of carbon nanotube tips can address the former issue (11, 12), whereas techniques in chemical functionalization can provide directed tip specificity and recognition capability (13–18), thereby further improving and widening the applicability of AFM in the future. In addition, the coupling of the atomic force microscope to fluorescence microscopes further enhances its versatility by adding (single molecule) fluorescence imaging to the AFM imaging capability (19–21), and the development of high speed systems makes it possible for AFM to probe fast dynamics of various biological processes (22–26).The applicability of AFM in proteomics is diverse and includes the characterization of the cell surface proteome (for a recent review, see Ref. 27), label-free detection and counting of single proteins (28, 29), and force spectroscopy measurements of binding and unbinding events (30, 31). In structural biology, AFM has shown to be a powerful tool for high resolution imaging of proteins in near native conditions (3, 6) and structural studies of supramolecular assemblies like protein filaments and viruses by nanoindentation methods (32, 33). These experiments show the potential of AFM to study both “form” and “function” of proteins, thereby resolving questions in proteomics and structural biology quasi-simultaneously. In the following, we will explain the principles of atomic force microscopy and its different operation modes and finally discuss examples of imaging, nanoindentation, and protein (un)binding and unfolding studies using AFM.     

Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h(-1) g [dry weight](-1)) and ethanol production (0.29 g h(-1) g [dry weight](-1)) and a high ethanol yield (0.43 g g(-1)) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.     

The acanthodian<Emphasis Type="Italic">Machaeracanthus</Emphasis> from the Lower Devonian Hunsrück Slate of the Hunsrück region (Germany)

Two recently collected slabs from the Lower Devonian Hunsrück Slate of Bundenbach, Hunsrück region, Germany, with spines of the acanthodianMachaeracanthus hunsrueckianum n. sp. are described. Both are associations of large and small spines and are the first to show groupings of symmetrical pairs; the spines are not homologous with those of other acanthodians. A pair of small spines ofMachaeracanthus peracutus Newberry, 1857 from the Karschheck quarry near Oberkirn, Hunsrück region, Germany, is articulated with the pectoral girdle and is the first such complex to be described. The only spines whichMachaeracanthus appears to have had were a pair of large and small pectoral spines on each side of the body. These spines could have helped to prevent the fish from sinking into the mud while resting on the sea floor.     

Bone regeneration during distraction osteogenesis: mechano-regulation by shear strain and fluid velocity

Corroboration of mechano-regulation algorithms is difficult, partly because repeatable experimental outcomes under a controlled mechanical environment are necessary, but rarely available. In distraction osteogenesis (DO), a controlled displacement is used to regenerate large volumes of new bone, with predictable and reproducible outcomes, allowing to computationally study the potential mechanisms that stimulate bone formation. We hypothesized that mechano-regulation by octahedral shear strain and fluid velocity can predict the spatial and temporal tissue distributions seen during experimental DO. Variations in predicted tissue distributions due to alterations in distraction rate and frequency could then also be studied. An in vivo ovine tibia experiment evaluating bone-segment transport (distraction, 1 mm/day) over an intramedullary nail was used for comparison. A 2D axisymmetric finite element model, with a geometry originating from the experimental data, was created and included into a previously developed model of tissue differentiation. Cells migrated and proliferated into the callus, differentiating into fibroblasts, chondrocytes or osteoblasts, dependent on the biophysical stimuli. Matrix production was modelled with an osmotic swelling model to allow tissues to grow at individual rates. The temporal and spatial tissue distributions predicted by the computational model agreed well with those seen experimentally. In addition, it was observed that decreased distraction rate (0.5 mm/d vs. 0.25 mm/d) increased the overall time needed for complete bone regeneration, whereas increased distraction frequency (0.5 mm/12 h vs. 0.25 mm/6 h) stimulated faster bone regeneration, as found in experimental findings by others. Thus, the algorithm regulated by octahedral shear strain and fluid velocity was able to predict the bone regeneration patterns dependent on distraction rate and frequency during DO.     

Characterization of articular cartilage by combining microscopic analysis with a fibril-reinforced finite-element model

Load-bearing characteristics of articular cartilage are impaired during tissue degeneration. Quantitative microscopy enables in vitro investigation of cartilage structure but determination of tissue functional properties necessitates experimental mechanical testing. The fibril-reinforced poroviscoelastic (FRPVE) model has been used successfully for estimation of cartilage mechanical properties. The model includes realistic collagen network architecture, as shown by microscopic imaging techniques. The aim of the present study was to investigate the relationships between the cartilage proteoglycan (PG) and collagen content as assessed by quantitative microscopic findings, and model-based mechanical parameters of the tissue. Site-specific variation of the collagen network moduli, PG matrix modulus and permeability was analyzed. Cylindrical cartilage samples (n=22) were harvested from various sites of the bovine knee and shoulder joints. Collagen orientation, as quantitated by polarized light microscopy, was incorporated into the finite-element model. Stepwise stress-relaxation experiments in unconfined compression were conducted for the samples, and sample-specific models were fitted to the experimental data in order to determine values of the model parameters. For comparison, Fourier transform infrared imaging and digital densitometry were used for the determination of collagen and PG content in the same samples, respectively. The initial and strain-dependent fibril network moduli as well as the initial permeability correlated significantly with the tissue collagen content. The equilibrium Young's modulus of the nonfibrillar matrix and the strain dependency of permeability were significantly associated with the tissue PG content. The present study demonstrates that modern quantitative microscopic methods in combination with the FRPVE model are feasible methods to characterize the structure-function relationships of articular cartilage.     

Four novel yeasts from decaying organic matter: Blastobotrys robertii sp. nov., Candida cretensis sp. nov., Candida scorzettiae sp. nov. and Candida vadensis sp. nov

Four novel yeast species are described, two from decaying mushrooms, viz. Candida cretensis and Candida vadensis, and two from rotten wood, viz. Blastobotrys robertii and Candida scorzettiae. Accession numbers for the CBS and ARS Culture Collections, and GenBank accession numbers for the D1/D2 domains of the large subunit of ribosomal DNA are: B. robertii CBS 10106^T, NRRL Y-27775, DQ839395; C. cretensis CBS 9453^T, NRRL Y-27777, AY4998861 and DQ839393; C. scorzettiae CBS 10107^T, NRRL Y-27665, DQ839394; C. vadensis CBS 9454^T, NRRL Y-27778, AY498863 and DQ839396. The GenBank accession number for the ITS region of C. cretensis is AY498862 and that for C. vadensis is AY498864. C. cretensis was the only species of the four that displayed fermentative activity. All four type strains grew on n-hexadecane. C. scorzettiae is the only one of the new species that assimilates some phenolic compounds, viz. 3-hydroxy derivatives of benzoic, phenylacetic and cinnamic acids, but not the corresponding 4-hydroxy acids. This is indicative of an operative gentisate pathway.     

Metabolite transport across the peroxisomal membrane

In recent years, much progress has been made with respect to the unravelling of the functions of peroxisomes in metabolism, and it is now well established that peroxisomes are indispensable organelles, especially in higher eukaryotes. Peroxisomes catalyse a number of essential metabolic functions including fatty acid beta-oxidation, ether phospholipid biosynthesis, fatty acid alpha-oxidation and glyoxylate detoxification. The involvement of peroxisomes in these metabolic pathways necessitates the transport of metabolites in and out of peroxisomes. Recently, considerable progress has been made in the characterization of metabolite transport across the peroxisomal membrane. Peroxisomes posses several specialized transport systems to transport metabolites. This is exemplified by the identification of a specific transporter for adenine nucleotides and several half-ABC (ATP-binding cassette) transporters which may be present as hetero- and homo-dimers. The nature of the substrates handled by the different ABC transporters is less clear. In this review we will describe the current state of knowledge of the permeability properties of the peroxisomal membrane.     

Impact of climatic variability on parameters used in typology and ecological quality assessment of surface waters—implications on the Water Framework Directive

In most cases the negative impacts of climate change to aquatic ecosystems cannot be mitigated by measures in the river basin management. Ignoring climate change by the Water Framework Directive may have strong implications for the typology and quality assessment systems used for water bodies. As a result of climate change, water bodies, especially those located near the type boundaries may change their type. Compared to typology characteristics, water quality parameters are even more labile and may be easily affected by climate change. The paper exemplifies that the anticipated deterioration of water quality within the time frame relevant for WFD implementation may be large enough to endanger the fulfillment of the set water quality objectives. The review of the river basin characterization every six years, as required by the WFD, might also include re-evaluation of reference conditions according to the changes observed at pristine reference sites. As a consequence, the restoration targets (i.e., the good ecological status) would also need to be evaluated periodically.     

Red blood cell folate vitamer distribution in healthy subjects is determined by the methylenetetrahydrofolate reductase C677T polymorphism and by the total folate status

BACKGROUND: Red blood cells (RBCs) represent a storage pool for folate. In contrast to plasma, RBC folate can appear in different biochemical isoforms. So far, only the methylenetetrahydrofolate reductase (MTHFR) 677 TT genotype has been identified as a determinant of RBC folate vitamer distribution. OBJECTIVE: The purpose of this study is to identify clinical and biochemical determinants of RBC folate vitamer distribution in healthy subjects. DESIGN: In an observational study, 109 subjects, aged 18 to 65 years, were studied. Red blood cell folate vitamers were analyzed using a liquid chromatography-tandem mass spectrometry method. Other variables recorded included vitamin B(2), B(6) and B(12) status, homocysteine, plasma and RBC S-adenosylhomocysteine and S-adenosylmethionine, renal function and the MTHFR C677T polymorphism. RESULTS: The MTHFR C677T genotype was the dominant determinant of nonmethylfolate accumulation. The median (range) nonmethylfolate/total folate ratio was 0.58% (0-12.2%) in the MTHFR CC group (n=55), 0.99% (0-14.3%) in the CT group (n=39) and 30.3% (5.7-73.3%) in the TT genotype group (n=15), P<.001. The 95th percentile for the nonmethylfolate/total folate ratio was 2.8% for the CC group, 9.1% for the CT group and 73.3% for the TT group. In the CC and CT genotype subjects, the T-allele and total folate status were positively and independently correlated with nonmethylfolate accumulation, but the degree of nonmethylfolate accumulation in these subjects was usually minor compared with those with the TT genotype. None of the other studied variables was associated with nonmethylfolate accumulation. CONCLUSIONS: The MTHFR C677T genotype is the dominant determinant of nonmethylfolate accumulation in RBCs. In addition, high total folate status may contribute to minor to moderate nonmethylfolate accumulation in MTHFR CC and CT subjects.