Cellular aging leads to functional heterogeneity of hematopoietic stem cells: a modeling perspective

Hematopoietic stem cells (HSCs) are the source for the life-long supply of functional cells in peripheral blood while they simultaneously maintain their own reserve pool. However, there is accumulating evidence that HSCs are themselves subject to quantitative and qualitative exhaustion. Although several processes linked to mitotic activity can potentially account for the observed aging phenomena (e.g., DNA damage, telomere shortening, epigenetic modification), a precise understanding of HSC exhaustion is still missing. It is particularly unclear how individual aging processes on the single-cell level translate on the phenotypic level of the overall tissue and whether there is a functional implication of an age-structured HSC population. We address these issues by applying a novel mathematical model of HSC organization in which division-specific, cumulative alterations of stem cell quality determine the phenotypic and functional appearance of the overall cell population. Adapting the model to a number of basic experimental findings, we quantify the level of additional heterogeneity that is introduced by a population of individually aging cells. Based on this model, we are able to conclude that division-dependent processes of cellular aging explain a wide range of phenomena on HSC exhaustion and that HSC aging needs to be considered as a highly heterogeneous process. We furthermore report that functional heterogeneity between young and old HSCs appears closely similar to the phenomena described for long- and short-term repopulating cells. We speculate whether differential, division-coupled stem cell aging introduces an intra-animal variability that also accounts for heterogeneity with respect to the repopulation ability of HSCs.     

Phylogenetic and gene-centric metagenomics of the canine intestinal microbiome reveals similarities with humans and mice

This study is the first to use a metagenomics approach to characterize the phylogeny and functional capacity of the canine gastrointestinal microbiome. Six healthy adult dogs were used in a crossover design and fed a low-fiber control diet (K9C) or one containing 7.5% beet pulp (K9BP). Pooled fecal DNA samples from each treatment were subjected to 454 pyrosequencing, generating 503 280 (K9C) and 505 061 (K9BP) sequences. Dominant bacterial phyla included the Bacteroidetes/Chlorobi group and Firmicutes, both of which comprised ∼35% of all sequences, followed by Proteobacteria (13–15%) and Fusobacteria (7–8%). K9C had a greater percentage of Bacteroidetes, Fusobacteria and Proteobacteria, whereas K9BP had greater proportions of the Bacteroidetes/Chlorobi group and Firmicutes. Archaea were not altered by diet and represented ∼1% of all sequences. All archaea were members of Crenarchaeota and Euryarchaeota, with methanogens being the most abundant and diverse. Three fungi phylotypes were present in K9C, but none in K9BP. Less than 0.4% of sequences were of viral origin, with >99% of them associated with bacteriophages. Primary functional categories were not significantly affected by diet and were associated with carbohydrates; protein metabolism; DNA metabolism; cofactors, vitamins, prosthetic groups and pigments; amino acids and derivatives; cell wall and capsule; and virulence. Hierarchical clustering of several gastrointestinal metagenomes demonstrated phylogenetic and metabolic similarity between dogs, humans and mice. More research is required to provide deeper coverage of the canine microbiome, evaluate effects of age, genetics or environment on its composition and activity, and identify its role in gastrointestinal disease.     

Diagnostic application of padlock probes--multiplex detection of plant pathogens using universal microarrays

Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray detection. Since target recognition is separated from downstream processing, PLPs enable the development of flexible and extendable diagnostic systems, targeting diverse organisms. To adapt padlock technology for diagnostic purposes, we optimized PLP design to ensure high specificity and eliminating ligation on non-target sequences under real-world assay conditions. We designed and tested 11 PLPs to target various plant pathogens at the genus, species and subspecies levels, and developed a prototype PLP-based plant health chip. Excellent specificity was demonstrated toward the target organisms. Assay background was determined for each hybridization using a no-target reference sample, which provided reliable and sensitive identification of positive samples. A sensitivity of 5 pg genomic DNA and a dynamic range of detection of 100 were observed. The developed multiplex diagnostic system was validated using genomic DNAs of characterized isolates and artificial mixtures thereof. The demonstrated system is adaptable to a wide variety of applications ranging from pest management to environmental microbiology.     

General Description of Partly Meromictic Hypertrophic Lake Verevi,its Ecological Status,Changes during the Past Eight Decades,and Restoration Problems

The present study describes generally the ecosystem of Lake Verevi while more detailed approaches are presented in the same issue. The main task of the article is to estimate long-term changes and find the best method for the restoration of good ecological status. Lake Verevi (surface 12.6 ha, mean depth 3.6 m, maximum depth 11 m, drainage area 1.1 km², water exchange 0.63-times per year) is a hypertrophic hard-water lake located in town Elva (6400 inhabitants). Long-term complex limnological investigations have taken place since 1929. The lake has been contaminated by irregular discharge of urban wastewaters from oxidation ponds since 1978, flood from streets, and infiltrated waters from the surrounding farms. The so-called spring meromixis occurred due to extremely warm springs in recent years. The index value of buffer capacity of Lake Verevi calculated from natural conditions is on the medium level. Water properties were analysed according to the requirements of the EU Water Framework Directive. According to the classification, water quality as a long-term average of surface layers is moderate-good, but the water quality of bottom layers is bad. Values in deeper layers usually exceed 20–30 times the calculated reference values by Vighi and Chiaudani’s model. Naturally, at the beginning of the 20th century the limnological type of the lake was moderately eutrophic. During the 1980s and 1990s the ecosystem was out of balance by abiotic characteristics as well as by plankton indicators. Rapid fluctuations of species composition and abundance can be found in recent years. Seasonal variations are considerable and species composition differs remarkably also in the water column. The dominating macrophyte species vary from year to year. Since the annual amount of precipitation from the atmosphere onto the lake surface is several times higher, the impact of swimmers could be considered irrelevant. Some restoration methods were discussed. The first step, stopping external pollution, was completed by damming the inlet. Drainage (siphoning) of the hypolimnetic water is discussed. Secondary pollution occurs because Fe:P values are below the threshold. The authors propose to use phosphorus precipitation and hypolimnetic aeration instead of siphoning.     

Bacterial delivery of functional messenger RNA to mammalian cells

The limited access to the nuclear compartment may constitute one of the major barriers after bacteria-mediated expression plasmid DNA delivery to eukaryotic cells. Alternatively, a self-destructing Listeria monocytogenes strain was used to release translation-competent mRNA directly into the cytosol of epithelial cells, macrophages and human dendritic cells. Enhanced green fluorescent protein (EGFP)-encoding mRNA, adapted for translation in mammalian cells by linking an IRES element to the 5'-end of the egfp coding sequence, was produced by T7 RNA polymerase in the carrier bacteria upon entry into the cytosol where the mRNA is efficiently released from the lysed bacteria and immediately translated in eukaryotic host cells. Besides the much earlier expression of EGFP being detectable already 4 h after infection, the number of EGFP expressing mammalian cells obtained with this novel RNA delivery technique is comparable to or - especially in phagocytic cells - even higher than that obtained with the expression plasmid DNA delivery strategy. Accordingly, bacteria-mediated delivery of ovalbumin-encoding mRNA to macrophages resulted in efficient antigen processing and presentation in vitro indicating that this approach may also be adapted for the in vivo delivery of antigen-encoding mRNA leading to a more efficient immune response when applied to vaccine development.     

A novel MET-interacting protein shares high sequence similarity with RanBPM, but fails to stimulate MET-induced Ras/Erk signaling

MET is a receptor protein tyrosine kinase for hepatocyte growth factor, a multifunctional cytokine controlling cell growth, morphogenesis, and motility. In our previous study, RanBPM/RanBP9, whose name originated from its ability to interact with Ran, was identified as a MET-interacting protein. RanBPM/RanBP9 activates the Ras/Erk signaling pathway by serving as an adaptor protein of MET to recruit Sos. In this study, we identify a protein sharing a high amino acid sequence identity with RanBPM/RanBP9, especially in its SPRY domain, the region responsible for MET binding. This protein lacks the N-terminal poly-proline and poly-glutamine (Poly-PQ) stretch present in RanBPM/RanBP9 and has less homology with RanBPM/RanBP9 in its mid-region. We subsequently named this protein RanBP10 after demonstrating its interaction with Ran. We show that, like RanBPM/RanBP9, RanBP10 interacts with the tyrosine kinase domain of MET via its SPRY domain and these two proteins can compete with each other to bind to MET. Interestingly, unlike RanBPM/RanBP9, overexpression of RanBP10 cannot induce Erk1/2 phosphorylation and serum response element-luciferase (SRE-LUC) reporter gene expression. More importantly, co-transfection of RanBPM/RanBP9 and RanBP10 significantly represses SRE-LUC reporter gene expression induced by overexpression of RanBPM/RanBP9. Additional binding assays demonstrate that RanBP10 fails to interact with Sos, which explains its inability to activate the Ras/Erk pathway. Furthermore, we show that the N-terminus of RanBPM/RanBP9 with the Poly-PQ stretch is required for recruiting Sos and a truncated RanBPM/RanBP9 lacking this region fails to recruit Sos, indicating that the functional difference between RanBP10 and RanBPM/RanBP9 lies in their sequence difference in their N-termini.     

Clindamycin is neuroprotective in experimental Streptococcus pneumoniae meningitis compared with ceftriaxone

In animal models of Streptococcus pneumoniae meningitis, rifampin is neuroprotective in comparison to ceftriaxone. So far it is not clear whether this can be generalized for other protein synthesis-inhibiting antimicrobial agents. We examined the effects of the bactericidal protein synthesis-inhibiting clindamycin (n = 12) on the release of proinflammatory bacterial components, the formation of neurotoxic compounds and neuronal injury compared with the standard therapy with ceftriaxone (n = 12) in a rabbit model of pneumococcal meningitis. Analysis of the CSF and histological evaluation were combined with microdialysis from the hippocampal formation and the neocortex. Compared with ceftriaxone, clindamycin reduced the release of lipoteichoic acids from the bacteria (p = 0.004) into the CSF and the CSF leucocyte count (p = 0.011). This led to lower extracellular concentrations of hydroxyl radicals (p = 0.034) and glutamate (p = 0.016) in the hippocampal formation and a subsequent reduction of extracellular glycerol levels (p = 0.018) and neuronal apoptosis in the dentate gyrus (p = 0.008). The present data document beneficial effects of clindamycin compared with ceftriaxone on various parameters linked with the pathophysiology of pneumococcal meningitis and development of neuronal injury. This study suggests neuroprotection to be a group effect of bactericidal protein synthesis-inhibiting antimicrobial agents compared with the standard therapy with beta-lactam antibiotics in meningitis.     

Evaluation of nutrient digestibility and fecal characteristics of exotic felids fed horse‐ or beef‐based diets: use of the domestic cat as a model for exotic felids

The objective of this study was to determine the effects of feeding commercially available beef‐ and horse‐based diets on nutrient digestibility and fecal characteristics of large captive exotic felids and domestic cats. Four species of large exotic felids including cheetahs, Malayan tigers, jaguars, and Amur tigers, and domestic cats were utilized in a crossover design. Raw meat diets included a beef‐based diet (57% protein; 28% fat) and a horse‐based diet (51% protein; 30% fat). All cats were acclimated to the diet for 16 days followed by a 4 day collection period, where total feces, including one fresh sample, were collected. All feces were scored on collection. Intake did not differ due to diet, but fecal output was greater when cats consumed the horse‐based diet. Total tract apparent dry matter (DM) digestibility was higher (P<0.05) and organic matter (OM) and crude protein (CP) digestibilities were lower (P<0.05) when cats were fed the beef‐based diet compared with the horse‐based diet. CP digestibility was similar in domestic cats and cheetahs, and greater (P<0.05) than Amur tigers. Fecal scores were lower and fecal DM was greater (P<0.05) when cats consumed the horse‐based diet compared with the beef‐based diet. Domestic cats had lower (P<0.05) fecal ammonia concentrations compared with all other species. Fecal ammonia concentrations were lowest (P<0.05) when cats were fed the horse‐based diet. Fecal total short‐chain fatty acid (SCFA), branched‐chain fatty acid (BCFA), and butyrate concentrations were higher (P<0.05) when cats consumed the beef‐based diet. Our results suggest that the domestic cat serves as an appropriate model for large exotic felid species, but differences among the species exist. Decreased nutrient digestibility by tigers and jaguars should be considered when developing feeding recommendations for these species based on domestic cat data. Zoo Biol 29:432–448, 2010. © 2009 Wiley‐Liss, Inc.