CD271 enrichment does not help isolating mesenchymal stromal cells from G-CSF-mobilized peripheral blood

Reports on the isolation of mesenchymal stromal cells (MSCs) from granulocyte colony stimulating factor mobilized peripheral blood (G-CSF-mobilized PB) using regular culturing techniques are controversial. Enrichment techniques such as CD133 isolation have increased the success rates. CD271 is a wellknown marker for enrichment of MSCs from bone marrow (BM). In the present study, we aimed to find out whether CD271 enrichment can help isolation of MSCs from G-CSF-mobiiized PB. Five G-CSF-mobilized PB samples were collected from the remnant parts of the bags used for BM transplantation. Five BM samples were used as the control. Mononuclear cells (MNCs) from both resources were collected and underwent magnetic sorting for CD271-positive cells. The isolated cells were cultured, undergoing flowcytometry and differentiation assays to determine if they fulfill MSCs characteristics. CD271-positive portion of G-CSF-mobilized PB did not yield any cell outgrowth but the BM counterpart could successfully form MSC colonies. Although the percentage of CD271⁺ cells showed no difference between BM-MNCs and G-CSF-mobilized PB-MNCs, hematopoietic markers such as CD45, CD34 and CD133 composed a higher percentage of CD271-positive cells in the G-CSF-tnobiiized PB group. Results obtained indicated that CD271 enrichment does not help isolation of MSCs from G-CSF-mobilized PB. In this source, almost all of the CD271⁺ cells are from hematopoietic origin and the frequency of MSCs is so low that possibly during the process of cell isolation most of them are lost and the isolation fails.     

KRT8, FAF1 and PTH1R gene polymorphisms are associated with leg weakness traits in pigs

The liability to lesions of dysfunctions of bone and joints in pigs, summarized as leg weakness and mostly expressed as osteochondrosis, is an animal welfare and economic issue in pig production. The objective of this study was to identify polymorphisms in the functional and positional candidate genes keratin 8 (KRT8), Fas-associated factor 1 (FAF1) and parathyroid hormone type I receptor (PTH1R) and to evaluate their association with leg weakness traits. Therefore, osteochondrosis lesions were scored in animals of a Duroc × Pietrain F2 population (DuPi; n = 310) and commercial herds of the breed Large White (n = 298). In addition, bone mineralization traits were observed in DuPi population. SNPs were identified in genes KRT8 (g.8,039G > A), FAF1 (g.380,914T > C) and PTH1R (c.1,672C > T). KRT8 showed significant association with bone mineral density and content (P ≤ 0.05). FAF1 was association with OC lesions score of all joints inspected (P ≤ 0.05). PTH1R showed significant dominance effects on OC lesion scores of the distal femur articular cartilage (P = 0.01) and epiphysis of the distal ulna (P = 0.05) as well as sums of scores of all joints (OCsum, P = 0.04) and assignment to groups of either severely or gently affected animals (OCcat, P = 0.01). This study reveals clear genetic-statistical evidence for a link of KRT8, FAF1 and PTH1R with some of leg weakness related traits in pigs.     

Size unlimited markerless deletions by a transconjugative plasmid-system in Bacillus licheniformis

Conjugative shuttle vectors of the pKVM series, based on an IncP transfer origin and the pMAD vector with a temperature sensitive replication were constructed to establish a markerless gene deletion protocol for Bacilli without natural competence such as the exoenzyme producer Bacillus licheniformis. The pKVM plasmids can be conjugated to strains of B. licheniformis and B. subtilis. For chromosomal gene deletion, regions flanking the target gene are fused and cloned in a pKVM vector prior to conjugative transfer from Escherichia coli to B. licheniformis. Appropriate markers on the vector backbone allow for the identification of the integration at the target locus and thereafter the vector excision, both events taking place via homologous recombination. The functionality of the deletion system was demonstrated with B. licheniformis by a markerless 939 bp in-frame deletion of the yqfD gene and the deletion of a 31 kbp genomic segment carrying a PBSX-like prophage.     

Importance of codB for new codA-based markerless gene deletion in Gluconobacter strains

For the detailed molecular analysis, genomic modification, and application of acetic acid bacteria such as Gluconobacter in biotechnological processes, a simple markerless deletion system is essential. The available methods have either low efficiencies or their applicability is restricted to strains containing an upp mutation. We now developed a method based on counterselection by cytosine deaminase, encoded by the codA gene from Escherichia coli, in the presence of the fluorinated pyrimidine analogue 5-fluorocytosine (FC). The codA-encoded enzyme converts nontoxic FC to toxic 5-fluorouracil, which is channeled into the metabolism by the uracil phosphoribosyltransferase, encoded by the chromosomal upp gene of Gluconobacter. We found that the presence of E. coli codB, encoding a cytosine permease, was needed for a high efficiency of gene deletion. The system is applicable in wild-type strains because no preceding deletions are required. Based on the fact that a codA gene is absent and an upp gene is present in almost all acetic acid bacteria sequenced so far, the method should also be applicable for other genera of the Acetobacteraceae.     

Whole-body scanning PCR; a highly sensitive method to study the biodistribution of mRNAs,noncoding RNAs and therapeutic oligonucleotides

Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides. Screening for novel delivery methods with unique tissue-homing properties requires a rapid, sensitive, flexible and unbiased technique able to visualize the in vivo biodistribution of these oligonucleotides. Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image. This platform was designed to be compatible with a novel RT-qPCR assay for the detection of siRNAs and with an assay suitable for the detection of heavily chemically modified oligonucleotides, which we termed Chemical-Ligation qPCR (CL-qPCR). In addition to this, the platform can also be used to investigate the global expression of endogenous mRNAs and non-coding RNAs. Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.     

Babo1, formerly Vop1 and Cop1/2, is no eyespot photoreceptor but a basal body protein illuminating cell division in Volvox carteri

In photosynthetic organisms many processes are light dependent and sensing of light requires light‐sensitive proteins. The supposed eyespot photoreceptor protein Babo1 (formerly Vop1) has previously been classified as an opsin due to the capacity for binding retinal. Here, we analyze Babo1 and provide evidence that it is no opsin. Due to the localization at the basal bodies, the former Vop1 and Cop1/2 proteins were renamed V.c. Babo1 and C.r. Babo1. We reveal a large family of more than 60 Babo1‐related proteins from a wide range of species. The detailed subcellular localization of fluorescence‐tagged Babo1 shows that it accumulates at the basal apparatus. More precisely, it is located predominantly at the basal bodies and to a lesser extent at the four strands of rootlet microtubules. We trace Babo1 during basal body separation and cell division. Dynamic structural rearrangements of Babo1 particularly occur right before the first cell division. In four‐celled embryos Babo1 was exclusively found at the oldest basal bodies of the embryo and on the corresponding d‐roots. The unequal distribution of Babo1 in four‐celled embryos could be an integral part of a geometrical system in early embryogenesis, which establishes the anterior–posterior polarity and influences the spatial arrangement of all embryonic structures and characteristics. Due to its retinal‐binding capacity, Babo1 could also be responsible for the unequal distribution of retinoids, knowing that such concentration gradients of retinoids can be essential for the correct patterning during embryogenesis of more complex organisms. Thus, our findings push the Babo1 research in another direction.     

Time-dependent alterations of cerebral proteins following short-term normobaric hyperoxia

Sufficient oxygenation is indispensable for cognitive performance in mammals. In order to assure adequate oxygenation and to prevent hypoxia in medicine or aviation, different approaches of oxygen delivery are realized. With regard to hyperoxia, it is well known that it increases the risk of tissue toxicity and inflammation by generating radical oxygen species. However, this impact of hyperoxia on the expression of specific brain proteins has not been evaluated in detail yet. The present study analyzes time-dependent changes in protein expression in rat brain after a short-term exposure to normobaric hyperoxia. Thirty-six Wistar rats were randomly assigned to six different groups, three normobaric hyperoxia (NH) groups or three normobaric normoxia (NN) groups, each consisting of n = 6 animals. NH animals were exposed to 100% oxygen, NN rats to 21% oxygen, each group for 3 h. One group of NH and one group of NN were killed immediately after the 3 h, one group each after 3 days and one group each after 7 days. Rat brains were removed for analysis and whole brain detergent protein lysates were separated via two-dimensional gel electrophoresis followed by subsequent identification of protein expression alterations by peptide mass fingerprinting using mass spectrometry. Also, a functional network mapping and molecular pathway analysis were carried out. Statistical analysis was performed using analysis of variance (ANOVA) with Bonferroni correction using P < 0.01. Physiological parameters of the animals did not differ significantly between the two groups except for partial oxygen pressure (580 vs. 89 mmHg; P < 0.05). The expression of nine proteins was found to be significantly altered (five up-regulated: GOT1, CCT2, TCP1, G6PD, and ALB; four down-regulated: PEBP1, PRDX2, ENO1, and MDH1). IPA generated a network with eight focus proteins associated with pathways in “cell death, cancer, and signalling”. Although hyperoxia was normobaric and induced for only 3 h, significant changes in brain protein expression were detectable immediately after the 3 h, after 3 days, as well as after 7 days. This may indicate effects on brain protein expression take place in the rat brain following a relatively short period of hyperoxia.     

The AQEM/STAR taxalist – a pan-European macro-invertebrate ecological database and taxa inventory

The European list of aquatic macro-invertebrate taxa, and its associated ecological database, originated within the context of the AQEM project and have been extended during the STAR project. The AQEM/STAR taxalist is a product of co-operation between applied freshwater ecologists and scientists from different zoological fields, applied partners and the administration. The basic idea is that a sound understanding of benthic invertebrate ecology is a prerequisite for the implementation of a biological approach to aquatic ecosystem management in Europe. The database has been generated under the management of BOKU (University of Natural Resources and Applied Life Sciences, Vienna) and UDE (University of Duisburg-Essen) and provides an important means of standardisation and unification of ecological classifications in Europe. This paper outlines the aims for setting up the AQEM/STAR macro-invertebrate taxalist and autecological database and provides a current summary of the numbers of aquatic orders, families, species, and species occurrences in 14 European countries. The number of available and applicable assignments of taxa to each ecological parameter is summarised and examples are given for different parameters and taxonomic groups. Gaps in the autecological information are identified and discussed. Besides its ecological relevance, the operational character of this database is underlined by the fact that it provides the associated taxon codes for each of five different European assessment systems for nearly 10,000 European macro-invertebrate taxa.     

A novel partial agonist of peroxisome proliferator-activated receptor-gamma (PPARgamma) recruits PPARgamma-coactivator-1alpha, prevents triglyceride accumulation, and potentiates insulin signaling in vitro

Partial agonists of peroxisome proliferator-activated receptor-gamma (PPARgamma), also termed selective PPARgamma modulators, are expected to uncouple insulin sensitization from triglyceride (TG) storage in patients with type 2 diabetes mellitus. These agents shall thus avoid adverse effects, such as body weight gain, exerted by full agonists such as thiazolidinediones. In this context, we describe the identification and characterization of the isoquinoline derivative PA-082, a prototype of a novel class of non-thiazolidinedione partial PPARgamma ligands. In a cocrystal with PPARgamma it was bound within the ligand-binding pocket without direct contact to helix 12. The compound displayed partial agonism in biochemical and cell-based transactivation assays and caused preferential recruitment of PPARgamma-coactivator-1alpha (PGC1alpha) to the receptor, a feature shared with other selective PPARgamma modulators. It antagonized rosiglitazone-driven transactivation and TG accumulation during de novo adipogenic differentiation of murine C3H10T1/2 mesenchymal stem cells. The latter effect was mimicked by overexpression of wild-type PGC1alpha but not its LXXLL-deficient mutant. Despite failing to promote TG loading, PA-082 induced mRNAs of genes encoding components of insulin signaling and adipogenic differentiation pathways. It potentiated glucose uptake and inhibited the negative cross-talk of TNFalpha on protein kinase B (AKT) phosphorylation in mature adipocytes and HepG2 human hepatoma cells. PGC1alpha is a key regulator of energy expenditure and down-regulated in diabetics. We thus propose that selective recruitment of PGC1alpha to favorable PPARgamma-target genes provides a possible molecular mechanism whereby partial PPARgamma agonists dissociate TG accumulation from insulin signaling.