GDF15/MIC1 and MMP9 Cerebrospinal Fluid Levels in Parkinson’s Disease and Lewy Body Dementia

Based on animal and ex-vivo experiments, Growth/Differentiation Factor-15 (GDF15, also called Macrophage Inhibitory Cytokine-1, MIC1), a member of the transforming growth factor-beta family, and Matrix Metalloproteinase-9 (MMP9), a member of the matrix metalloprotease family may be potential markers for Lewy body disorders, i.e. Parkinson’s disease with (PDD) and without dementia (PDND) and Lewy body dementia (DLB). GDF15 has a prominent role in development, cell proliferation, differentiation, and repair, whereas MMP9 degrades, as a proteolytic enzyme, components of the extracellular matrix. In this study, cerebrospinal fluid GDF15 and MMP9 levels of 59 PDND, 17 PDD and 23 DLB patients, as well as of 95 controls were determined, and associated with demographic, clinical and biochemical parameters. Our analysis confirmed the already described association of GDF15 levels with age and gender. Corrected GDF15 levels were significantly higher in PDD than in PDND patients, and intermediate in DLB patients. Within Lewy body disorders, GDF15 levels correlated positively with age at onset of Parkinsonism and dementia, Hoehn & Yahr stage and cerebrospinal fluid t-Tau and p-Tau levels, and negatively with the Mini Mental State Examination. Remarkably, it does not relevantly correlate with disease duration. MMP9 was not relevantly associated with any of these parameters. Cerebrospinal GDF15, but not MMP9, may be a potential marker of and in Lewy body disorders.     

Long-Term Survival of Salmonella enterica Serovar Typhimurium Reveals an Infectious State That Is Underrepresented on Laboratory Media Containing Bile Salts

Cells in desiccated Salmonella enterica serovar Typhimurium rdar (red, dry, and rough) morphotype colonies were examined for culturability and infectivity after 30 months. Culturability decreased only 10-fold; however, cells were underrepresented on Salmonella selective media containing bile salts. These cells were mildly attenuated compared to the infectivity of freshly grown cells but still able to cause systemic infections in mice.Salmonella enterica serovar Typhimurium (hereafter referred to as S. Typhimurium) is a gram-negative enteric pathogen of humans and other mammalian species. S. Typhimurium causes self-limiting gastroenteritis in humans and typhoid-like fever in mice (4). When propagated on the surface of nutrient-limited laboratory media, Salmonella cells form patterned colonies (1) involving the production of an extracellular matrix comprised of thin aggregative fimbriae (Tafi or curli) and cellulose (16, 21) and other polysaccharides. This rdar morphotype is characterized by the formation of red, dry, and rough colonies on solid agar medium containing Congo red as an indicator dye (8). Cells within rdar colonies are resistant to desiccation and commonly used disinfectants (14, 18), and the propensity to form the rdar morphotype is conserved throughout the salmonellae (7, 19). These findings, coupled with the observations that neither Tafi nor cellulose is required for virulence (13, 17), suggest that the rdar phenotype may contribute to the environmental survival of Salmonella (17).Previous experiments by our group established that approximately 10% (∼10⁸) of bacteria within a rdar morphotype colony were able to survive a 9-month period of starvation and desiccation (18). In this study, we examined survival after a longer time period of 30 months. S. Typhimurium ATCC 14028 cells were grown at 28°C for 4 days on tryptone agar, at which time colonies were detached from the agar surface and stored at room temperature in sterile, 24-well plates, as previously described (18). The results were compared to those for planktonic S. Typhimurium cells grown overnight in 1% tryptone and washed with distilled water prior to storage on plastic. Surprisingly, the number of viable cells recovered from rdar colonies after 30 months was similar to that obtained at the 9-month time point (Fig. ​(Fig.1).1). In contrast, planktonic S. Typhimurium cells exhibited a decrease in viability of 4 orders of magnitude within 14 days. However, if planktonic cells were stored in water or isotonic saline, the viability decreased by only 2 orders of magnitude after 84 days (Fig. ​(Fig.1).1). This finding indicated that desiccation rather than starvation was the primary cause of bacterial death under these conditions. The survival after 30 months of isogenic ΔcsgD mutant cells, which are deficient in extracellular matrix production (8, 18), was reduced 25-fold compared to that of wild-type cells (data not shown), confirming that cellulose and Tafi polymers have an important role in long-term survival. Together, these results indicated that extreme longevity of cells within rdar colonies is a morphotype-specific phenomenon rather than a general property of S. Typhimurium ATCC 14028.Open in a separate windowFIG. 1.Long-term survival of S. Typhimurium cells in rdar morphotype colonies or from planktonic culture under conditions of desiccation and nutrient absence. Approximately 10⁹ CFU of planktonic cells (▴) or cells within rdar colonies (•) were placed on sterile plastic, or planktonic cells were stored in sterile water (▪). At time points shown, cells were rehydrated, homogenized, and serially diluted before being grown on LB medium to enumerate the CFU. Data points represent averages and error bars show the standard deviations of the results from at least three biological samples.Under most laboratory conditions, S. Typhimurium is resistant to bile concentrations exceeding 60% (wt/vol) (3, 5, 6, 15); therefore, many selective media used in microbiological laboratories for culturing Salmonella contain bile salts. During storage on plastic with no nutrients or water, cells within rdar colonies became sensitive to sodium cholate and sodium deoxycholate (bile salts) over time (Table ​(Table1).1). As expected, freshly grown cells (exponential or stationary phase) did not display bile sensitivity (data not shown). The culturability of cells in 30-month-old colonies on media containing bile salts was significantly reduced compared to that of cells from 2-day-old or 2-week-old rdar colonies or colonies that were lyophilized for 1 week (Table ​(Table1).1). In contrast, culturability was not reduced to the same extent when aged cells were cultured on media that did not contain bile salts (brilliant green agar or XLD and LSA without bile salts [see Table ​Table11 for media]). Within diagnostic laboratories, bacterial isolates in environmental samples are commonly incubated in recovery broth prior to enumeration on selective medium (2). When cells derived from 30-month-old rdar colonies were incubated overnight in buffered peptone water, they recovered their resistance to bile salts (data not shown).

TABLE 1.

Relative recovery of S. Typhimurium ATCC 14028 on common Salmonella selective media

Release and uptake of volatile inorganic and organic gases through the snowpack at Niwot Ridge,Colorado

Whole air drawn from four heights within the high elevation (3,340 m asl), deep, winter snowpack at Niwot Ridge, Colorado, were sampled into stainless steel canisters, and subsequently analyzed by gas chromatography for 51 volatile inorganic and organic gases. Two adjacent plots with similar snow cover were sampled, one over bare soil and a second one from within a snow-filled chamber where Tedlar/Teflon-film covered the ground and isolated it from the soil. This comparison allowed for studying effects from processes in the snowpack itself versus soil influences on the gas concentrations and fluxes within and through the snowpack. Samples were also collected from ambient air above the snow surface for comparison with the snowpack air. Analyzed gas species were found to exhibit three different kinds of behavior: (1) One group of gases, i.e., carbon dioxide (CO₂), chloroform (CHCl₃), dimethylsulfide (CH₃)₂S, carbondisulfide (CS₂), and dichlorobromomethane (CHBrCl₂), displayed higher concentrations inside the snow, indicating a formation of these species and release into the atmosphere. (2) A second group of compounds, including carbon monoxide (CO), carbonyl sulfide (COS), the hydrocarbons methane, ethane, ethyne, benzene, and the halogenated compounds methylchloride (CH₃Cl), methylbromide (CH₃Br), dibromomethane (CH₂Br₂), bromoform (CHBr₃), tetrachloromethane (CCl₄), CFC-11, CFC-12, HCFC-22, CFC-113, 1,2-dichloroethane, methylchloroform, HCFC-141b, and HCFC-142b, were found at lower concentrations in the snow, indicating that the snow and/or soil constitute a sink for these gases. (3) For 21 other gases absolute concentrations, respectively concentration gradients, were too low to unequivocally identify their uptake or release behavior. For gases listed in the first two groups, concentration gradients were incorporated into a snowpack gas diffusion model to derive preliminary estimates of fluxes at the snow-atmosphere interface. The snowpack gradient flux technique was found to offer a highly sensitive method for the study of these surface gas exchanges. Microbial activities below this deep, winter snowpack appear to be the driving mechanism behind these gas sources and sinks. Flux results were applied to a simple box model to assess the potential contribution of the snowpack uptake rates to atmospheric lifetimes of these species.     

Isolation and characterization of cellulose-degrading bacteria from the deep subsurface of the Homestake gold mine,Lead, South Dakota,USA

The present study investigated the cultivable mesophilic (37°C) and thermophilic (60°C) cellulose-degrading bacterial diversity in a weathered soil-like sample collected from the deep subsurface (1.5 km depth) of the Homestake gold mine in Lead, South Dakota, USA. Chemical characterization of the sample by X-ray fluorescence spectroscopy revealed a high amount of toxic heavy metals such as Cu, Cr, Pb, Ni, and Zn. Molecular community structures were determined by phylogenetic analysis of 16S rRNA gene sequences retrieved from enrichment cultures growing in presence of microcrystalline cellulose as the sole source of carbon. All phylotypes retrieved from enrichment cultures were affiliated to Firmicutes. Cellulose-degrading mesophilic and thermophilic pure cultures belonging to the genera Brevibacillus, Paenibacillus, Bacillus, and Geobacillus were isolated from enrichment cultures, and selected cultures were studied for enzyme activities. For a mesophilic isolate (DUSELG12), the optimum pH and temperature for carboxymethyl cellulase (CMCase) were 5.5 and 55°C, while for a thermophilic isolate (DUSELR7) they were 5.0 and 75°C, respectively. Furthermore, DUSELG12 retained about 40% CMCase activity after incubation at 60°C for 8 h. Most remarkably, thermophilic isolate, DUSELR7 retained 26% CMCase activity at 60°C up to a period of 300 h. Overall, the present work revealed the presence of different cellulose-degrading bacterial lineages in the unique deep subsurface environment of the mine. The results also have strong implications for biological conversion of cellulosic agricultural and forestry wastes to commodity chemicals including sugars.     

FLU, a negative feedback regulator of tetrapyrrole biosynthesis, is physically linked to the final steps of the Mg(++)-branch of this pathway

Regulation of tetrapyrrole biosynthesis in higher plants has been attributed to negative feedback control. Two effectors of feedback inhibition have been identified, heme and the FLU protein. Inhibition by heme implicates the Fe-branch via regulation of the initial step of tetrapyrrole synthesis. In the present work a FLU-containing chloroplast membrane complex was identified, that besides FLU comprises the four enzymes catalyzing the final steps of chlorophyll synthesis. The results support the notion that FLU links chlorophyll synthesis and the target of feedback control, glutamyl-tRNA reductase, thereby allowing also the Mg-branch to control the initial step of tetrapyrrole synthesis.     

Thrombin-dependent NF-{kappa}B activation and monocyte/endothelial adhesion are mediated by the CARMA3·Bcl10·MALT1 signalosome

Thrombin is a potent modulator of endothelial function and, through stimulation of NF-κB, induces endothelial expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). These cell surface adhesion molecules recruit inflammatory cells to the vessel wall and thereby participate in the development of atherosclerosis, which is increasingly recognized as an inflammatory condition. The principal receptor for thrombin on endothelial cells is protease-activated receptor-1 (PAR-1), a member of the G protein-coupled receptor superfamily. Although it is known that PAR-1 signaling to NF-κB depends on initial PKC activation, the subsequent steps leading to stimulation of the canonical NF-κB machinery have remained unclear. Here, we demonstrate that a complex of proteins containing CARMA3, Bcl10, and MALT1 links PAR-1 activation to stimulation of the IκB kinase complex. IκB kinase in turn phosphorylates IκB, leading to its degradation and the release of active NF-κB. Further, we find that although this CARMA3·Bcl10·MALT1 signalosome shares features with a CARMA1-containing signalosome found in lymphocytes, there are significant differences in how the signalosomes communicate with their cognate receptors. Specifically, whereas the CARMA1-containing lymphocyte complex relies on 3-phosphoinositide-dependent protein kinase 1 for assembly and activation, the CARMA3-containing endothelial signalosome functions completely independent of 3-phosphoinositide-dependent protein kinase 1 and instead relies on β-arrestin 2 for assembly. Finally, we show that thrombin-dependent adhesion of monocytes to endothelial cells requires an intact endothelial CARMA3·Bcl10·MALT1 signalosome, underscoring the importance of the signalosome in mediating one of the most significant pro-atherogenic effects of thrombin.     

Fibroblast growth factor receptors 1 and 2 in keratinocytes control the epidermal barrier and cutaneous homeostasis

Fibroblast growth factors (FGFs) are master regulators of organogenesis and tissue homeostasis. In this study, we used different combinations of FGF receptor (FGFR)-deficient mice to unravel their functions in the skin. Loss of the IIIb splice variants of FGFR1 and FGFR2 in keratinocytes caused progressive loss of skin appendages, cutaneous inflammation, keratinocyte hyperproliferation, and acanthosis. We identified loss of FGF-induced expression of tight junction components with subsequent deficits in epidermal barrier function as the mechanism underlying the progressive inflammatory skin disease. The defective barrier causes activation of keratinocytes and epidermal γδ T cells, which produce interleukin-1 family member 8 and S100A8/A9 proteins. These cytokines initiate an inflammatory response and induce a double paracrine loop through production of keratinocyte mitogens by dermal cells. Our results identify essential roles for FGFs in the regulation of the epidermal barrier and in the prevention of cutaneous inflammation, and highlight the importance of stromal–epithelial interactions in skin homeostasis and disease.     

Transcriptome Analysis of MSC and MSC-Derived Osteoblasts on Resomer? LT706 and PCL: Impact of Biomaterial Substrate on Osteogenic Differentiation

Background

Mesenchymal stem cells (MSC) represent a particularly attractive cell type for bone tissue engineering because of their ex vivo expansion potential and multipotent differentiation capacity. MSC are readily differentiated towards mature osteoblasts with well-established protocols. However, tissue engineering frequently involves three-dimensional scaffolds which (i) allow for cell adhesion in a spatial environment and (ii) meet application-specific criteria, such as stiffness, degradability and biocompatibility.

Methodology/Principal Findings

In the present study, we analysed two synthetic, long-term degradable polymers for their impact on MSC-based bone tissue engineering: PLLA-co-TMC (Resomer® LT706) and poly(ε-caprolactone) (PCL). Both polymers enhance the osteogenic differentiation compared to tissue culture polystyrene (TCPS) as determined by Alizarin red stainings, scanning electron microscopy, PCR and whole genome expression analysis. Resomer® LT706 and PCL differ in their influence on gene expression, with Resomer® LT706 being more potent in supporting osteogenic differentiation of MSC. The major trigger on the osteogenic fate, however, is from osteogenic induction medium.

Conclusion

This study demonstrates an enhanced osteogenic differentiation of MSC on Resomer® LT706 and PCL compared to TCPS. MSC cultured on Resomer® LT706 showed higher numbers of genes involved in skeletal development and bone formation. This identifies Resomer® LT706 as particularly attractive scaffold material for bone tissue engineering.     

The chloroplast division mutant caa33 of Arabidopsis thaliana reveals the crucial impact of chloroplast homeostasis on stress acclimation and retrograde plastid-to-nucleus signaling

Retrograde plastid-to-nucleus signaling tightly controls and coordinates the nuclear and plastid gene expression that is required for plastid biogenesis and chloroplast activity. As chloroplasts act as sensors of environmental changes, plastid-derived signaling also modulates stress responses of plants by transferring stress-related signals and altering nuclear gene expression. Various mutant screens have been undertaken to identify constituents of plastid signaling pathways. Almost all mutations identified in these screens target plastid-specific but not extraplastidic functions. They have been suggested to define either genuine constituents of retrograde signaling pathways or components required for the synthesis of plastid signals. Here we report the characterization of the constitutive activator of AAA-ATPase (caa33) mutant, which reveals another way of how mutations that affect plastid functions may modulate retrograde plastid signaling. caa33 disturbs a plastid-specific function by impeding plastid division, and thereby perturbing plastid homeostasis. This results in preconditioning plants by activating the expression of stress genes, enhancing pathogen resistance and attenuating the capacity of the plant to respond to plastid signals. Our study reveals an intimate link between chloroplast activity and the susceptibility of the plant to stress, and emphasizes the need to consider the possible impact of preconditioning on retrograde plastid-to-nucleus signaling.     

Small-scale habitat use of black grouse (Tetrao tetrix L.) and rock ptarmigan (Lagopus muta helvetica Thienemann) in the Austrian Alps

We investigated the small-scale habitat use of two grouse species, black grouse (Tetrao tetrix L.) and rock ptarmigan (Lagopus muta helvetica Thienemann) in a study area in the Austrian Central Alps in summer. To build habitat suitability models, we applied multiple logistic regression using presence–absence data from fieldwork as the response variable and a set of habitat characteristics as explanatory variables, respectively. To gain a better understanding of the mechanisms that drive habitat selection, we tested for two-way interaction terms before excluding any variables from the initial variable set. Four explanatory variables significantly contributed to the black grouse model: dwarf shrub cover, dwarf shrub height, patchiness and ant hills. The final model for rock ptarmigan contained three explanatory variables: dwarf shrub cover, rock cover and dwarf shrub height. Most notably, the interaction terms dwarf shrub cover × patchiness in the black grouse model and dwarf shrub cover × dwarf shrub height, rock cover × dwarf shrub height in the rock ptarmigan model point out trade-off mechanisms between food, cover and overview providing features. Thus, our models do not only identify the parameters that mainly drive habitat selection, but also deepen our understanding about the causal relationships between these factors. Therefore, the information gained in this study allows for a deduction of appropriate habitat management strategies and supports conservation efforts of local stakeholders.