Population Diversity of Yeasts and Lactic Acid Bacteria in Pig Feed Fermented with Whey, Wet Wheat Distillers' Grains, or Water at Different Temperatures

The diversity of populations of yeast and lactic acid bacteria (LAB) in pig feeds fermented at 10, 15, or 20°C was characterized by rRNA gene sequencing of isolates. The feeds consisted of a cereal grain mix blended with wet wheat distillers' grains (WWDG feed), whey (W feed), or tap water (WAT feed). Fermentation proceeded for 5 days without disturbance, followed by 14 days of daily simulated feed outtakes, in which 80% of the contents were replaced with fresh feed mixtures. In WWDG feed, Pichia galeiformis became the dominant yeast species, independent of the fermentation temperature and feed change. The LAB population was dominated by Pediococcus pentosaceus at the start of the fermentation period. After 3 days, the Lactobacillus plantarum population started to increase in feeds at all temperatures. The diversity of LAB increased after the addition of fresh feed components. In W feed, Kluyveromyces marxianus dominated, but after the feed change, the population diversity increased. With increasing fermentation temperatures, there was a shift toward Pichia membranifaciens as the dominant species. L. plantarum was the most prevalent LAB in W feed. The WAT feed had a diverse microbial flora, and the yeast population changed throughout the whole fermentation period. Pichia anomala was the most prevalent yeast species, with increasing occurrence at higher fermentation temperatures. Pediococcus pentosaceus was the most prevalent LAB, but after the feed change, L. plantarum started to proliferate. The present study demonstrates that the species composition in fermented pig feed may vary considerably, even if viable cell counts indicate stable microbial populations.     

PCR-mediated deletion of plasmid DNA

The PCR-mediated plasmid DNA deletion method is a simple approach to delete DNA sequences from plasmids using only one round of PCR, with two primers, and without ligation or purification prior to in vivo recombination. By using only PCR, the method is sequence independent and, as shown in this study, is applicable to various sizes of plasmids and deletions using minimal primer design.     

The identification of novel PLC-γ inhibitors using virtual high throughput screening

Phospholipase C-γ (PLC-γ) has been identified as a possible biological target for anticancer drug therapy but suitable inhibitors are lacking. Therefore, in order to identify active compounds (hits) virtual high throughput screening was performed. The crystal structure of the PLC-δ isoform was used as a model docking scaffold since no crystallographic data are available on its γ counterpart. A pilot screen was performed using ～9.2 × 10⁴ compounds, where the robustness of the methodology was tested. This was followed by the main screening effort where ～4.4 × 10⁵ compounds were used. In both cases, plausible compounds were identified (virtual hits) and a selection of these was experimentally tested. The most potent compounds were in the single digit micro-molar range as determined from the biochemical (Flashplate) assay. This translated into ～15 μM in a functional assay in cells. About 30% of the virtual hits showed activity against PLC-γ (IC₅₀ < 50 μM).     

Substrate-Induced Conformational Changes of the Tyrocidine Synthetase 1 Adenylation Domain Probed by Intrinsic Trp Fluorescence

Nonribosomal peptide synthetases (NRPS) are multifunctional proteins that catalyze the synthesis of the peptide products with enormous biological potential. The process of biosynthesis starts with the adenylation (A) domain, which during the catalytic cycle undergoes extensive structural rearrangements. In this paper, we present the first study of the tyrocidine synthetase 1 A-domain (TycA-A) fluorescence properties. The TycA-A protein contains five potentially fluorescent Trp residues at positions 227, 301, 323, 376 and 406. The contribution of each Trp to the TycA-A emission was determined using protein variants bearing single Trp to Phe substitutions. The accessibility of the Trp side chains during adenylation showed that only W227 is affected by substrate binding. The protein variant containing solely fluorescent W227 residue was constructed and further used as a probe to explore the binding effect of different non-cognate amino acid substrates. The results indicate a different accessibility of W227 residue in the presence of non-cognate amino acids, which might offer an explanation for the higher aminoacyl-adenenylate leakage. Overall, our results suggest that intrinsic tryptophan fluorescence could be used as a method to probe the effect of substrate binding on the local structure in NRPS adenylation domains.     

Retroviral-based gene therapy with cyclooxygenase-2 promotes the union of bony callus tissues and accelerates fracture healing in the rat

BACKGROUND: An in vivo gene therapy strategy was developed to accelerate bone fracture repair. METHODS: Direct injection of a murine leukemia virus-based vector targeted transgene expression to the proliferating periosteal cells arising shortly after fracture. Cyclooxygenase-2 (Cox-2) was selected because the transgene for its prostaglandin products that promote angiogenesis, bone formation and bone resorption, are all required for fracture healing. The human (h) Cox-2 transgene was modified to remove AU-rich elements in the 3'-untranslated region and to improve protein translation. RESULTS: In vitro studies revealed robust and sustained Cox-2 protein expression, prostaglandin E(2) and alkaline phosphatase production in rat bone marrow stromal cells and osteoblasts transgenic for the hCox-2 gene. In vivo studies in the rat femur fracture revealed that Cox-2 transgene expression produced bony union of the fracture by 21 days post-fracture, a time when cartilage persisted within the fracture tissues of control animals and approximately 1 week earlier than the healing normally observed in this model. None of the ectopic bone formation associated with bone morphogenetic protein gene therapy was observed. CONCLUSIONS: This study represents the first demonstration that a single local application of a retroviral vector expressing a single osteoinductive transgene consistently accelerated fracture repair.     

Characterization of interactions of adapter protein RAPL/Nore1B with RAP GTPases and their role in T cell migration

Using a model of integrin-triggered random migration of T cells, we show that stimulation of LFA-1 integrins leads to the activation of Rap1 and Rap2 small GTPases. We further show that Rap1 and Rap2 have distinct roles in adhesion and random migration of these cells and that an adapter protein from the Ras association domain family (Rassf), RAPL, has a role downstream of Rap2 in addition to its link to Rap1. Further characterization of the RAPL protein and its interactions with small GTPases from the Ras family shows that RAPL forms more stable complexes with Rap2 and classical Ras proteins compared with Rap1. The different interaction pattern of RAPL with Rap1 and Rap2 is not affected by the disruption of the C-terminal SARAH domain that we identified as the alpha-helical region responsible for RAPL dimerization in vitro and in cells. Based on mutagenesis and three-dimensional modeling, we propose that interaction surfaces in RAPL-Rap1 and RAPL-Rap2 complexes are different and that a single residue in the switch I region of Rap proteins (residue 39) contributes considerably to the different kinetics of these protein-protein interactions. Furthermore, the distinct role of Rap2 in migration of T cells is lost when this critical residue is converted to the residue present in Rap1. Together, these observations suggest a wider role for Rassf adapter protein RAPL and Rap GTPases in cell motility and show that subtle differences between highly similar Rap proteins could be reflected in distinct interactions with common effectors and their cellular function.     

Laboratory contamination over time during low‐biomass sample analysis

Bacteria are not only ubiquitous on earth but can also be incredibly diverse within clean laboratories and reagents. The presence of both living and dead bacteria in laboratory environments and reagents is especially problematic when examining samples with low endogenous content (e.g., skin swabs, tissue biopsies, ice, water, degraded forensic samples or ancient material), where contaminants can outnumber endogenous microorganisms within samples. The contribution of contaminants within high‐throughput studies remains poorly understood because of the relatively low number of contaminant surveys. Here, we examined 144 negative control samples (extraction blank and no‐template amplification controls) collected in both typical molecular laboratories and an ultraclean ancient DNA laboratory over 5 years to characterize long‐term contaminant diversity. We additionally compared the contaminant content within a home‐made silica‐based extraction method, commonly used to analyse low endogenous content samples, with a widely used commercial DNA extraction kit. The contaminant taxonomic profile of the ultraclean ancient DNA laboratory was unique compared to modern molecular biology laboratories, and changed over time according to researcher, month and season. The commercial kit also contained higher microbial diversity and several human‐associated taxa in comparison to the home‐made silica extraction protocol. We recommend a minimum of two strategies to reduce the impacts of laboratory contaminants within low‐biomass metagenomic studies: (a) extraction blank controls should be included and sequenced with every batch of extractions and (b) the contributions of laboratory contamination should be assessed and reported in each high‐throughput metagenomic study.     

A transmembrane osteoclastic protein-tyrosine phosphatase regulates osteoclast activity in part by promoting osteoclast survival through c-Src-dependent activation of NFkappaB and JNK2

This study evaluated the effects of overexpression of wild-type (WT) or phosphatase-deficient (PD) mutant of an osteoclastic protein-tyrosine phosphatase (PTP-oc) in RAW/C4 cells. Osteoclast-like cells derived from WT-PTP-oc overexpressing clones increased, while those derived from PD-PTP-oc expressing clones decreased, their resorption activity. WT-PTP-oc clones had lower apoptosis, lower caspase 3/7 activity, reduced c-Src tyr-527 phosphorylation (PY527) and IkappaBalpha cellular levels, and increased NFkappaB activation and JNK phosphorylation. Overexpression of PD-PTP-oc or PTP-oc siRNA treatment increased apoptosis, caspase 3/7 activity, PY527 and IkappaBalpha levels, and decreased NFkappaB and JNK2 activation. Inhibition of the c-Src kinase blocked the PTP-oc-mediated NFkappaB and JNK2 activation. Blocking the NFkappaB activation had no effect on the JNK2 activation. Inhibiting the NFkappaB and/or JNK2 pathway prevented the PTP-oc-mediated reduction in apoptosis. In conclusion, PTP-oc activates osteoclast activity in part by promoting osteoclast survival through the PTP-oc-mediated c-Src-dependent activation of NFkappaB and JNK2.     

Role of phospholipase Cgamma1 in cell spreading requires association with a beta-Pix/GIT1-containing complex, leading to activation of Cdc42 and Rac1

The significance of multiprotein signaling complexes in cell motility is becoming increasingly important. We have previously shown that phospholipase Cgamma1 (PLCgamma1) is critical for integrin-mediated cell spreading and motility (N. Jones et al., J. Cell Sci. 118:2695-2706, 2005). In the current study we show that, on a basement membrane-type matrix, PLCgamma1 associates with the adaptor protein GIT1 and the Rac1/Cdc42 guanine exchange factor beta-Pix; GIT1 and beta-Pix form tight complexes independently of PLCgamma1. The association of PLCgamma1 with the complex requires both GIT1 and beta-Pix and the specific array region (gammaSA) of PLCgamma1. Mutations of PLCgamma1 within the gammaSA region reveal that association with this complex is essential for the phosphorylation of PLCgamma1 and the progression to an elongated morphology after integrin engagement. Short interfering RNA (siRNA) depletion of either beta-Pix or GIT1 inhibited cell spreading in a fashion similar to that seen with siRNA against PLCgamma1. Furthermore, siRNA depletion of PLCgamma1, beta-Pix, or GIT1 inhibited Cdc42 and Rac1 activation, while constitutively active forms of Cdc42 or Rac1, but not RhoA, were able to rescue the elongation of these cells. Signaling of the PLCgamma1/GIT1/beta-Pix complex to Cdc42/Rac1 was found to involve the activation of calpains, calcium-dependent proteases. Therefore, we propose that the association of PLCgamma1 with complexes containing GIT1 and beta-Pix is essential for its role in integrin-mediated cell spreading and motility. As a component of this complex, PLCgamma1 is also involved in the activation of Cdc42 and Rac1.