Pluripotent stem cell lines have been generated in several domestic animal species; however, these lines traditionally show poor self-renewal and differentiation. Using canine embryonic stem cell (cESC) lines previously shown to have sufficient self-renewal capacity and potency, we generated and compared canine neural stem cell (cNSC) lines derived by lineage selection with epidermal growth factor (EGF) or Noggin along the neural default differentiation pathway, or by directed differentiation with retinoic acid (RA)-induced floating sphere assay. Lineage selection produced large populations of SOX2+ neural stem/progenitor cell populations and neuronal derivatives while directed differentiation produced few and improper neuronal derivatives. Primary canine neural lines were generated from fetal tissue and used as a positive control for differentiation and electrophysiology. Differentiation of EGF- and Noggin-directed cNSC lines in N2B27 with low-dose growth factors (BDNF/NT-3 or PDGFαα) produced phenotypes equivalent to primary canine neural cells including 3CB2+ radial progenitors, MOSP+ glia restricted precursors, VIM+/GFAP+ astrocytes, and TUBB3+/MAP2+/NFH+/SYN+ neurons. Conversely, induction with RA and neuronal differentiation produced inadequate putative neurons for further study, even though appropriate neuronal gene expression profiles were observed by RT-PCR (including Nestin, TUBB3, PSD95, STX1A, SYNPR, MAP2). Co-culture of cESC-derived neurons with primary canine fetal cells on canine astrocytes was used to test functional maturity of putative neurons. Canine ESC-derived neurons received functional GABA(A)- and AMPA-receptor mediated synaptic input, but only when co-cultured with primary neurons. This study presents established neural stem/progenitor cell populations and functional neural derivatives in the dog, providing the proof-of-concept required to translate stem cell transplantation strategies into a clinically relevant animal model.

Caspase-9 is a component of the apoptosome that mediates cell death following release of cytochrome c from mitochondria. Inhibition of Caspase-9 with a dominant negative construct (Casp9DN) blocks apoptosome function, promotes viability and has been implicated in carcinogenesis. Inhibition of the apoptosome in vitro impairs mitochondrial function and promotes mitophagy. To examine whether inhibition of the apoptosome would enhance mitophagy and promote oncogenesis in vivo, transgenic mice were generated that express Casp9DN in the T cell lineage. The effects of Casp9DN on thymocyte viability, mitophagy and thymic tumor formation were examined. In primary thymocytes, Casp9DN delayed dexamethasone (Dex)-induced cell death, altered mitochondrial structure, and decreased oxidant production. Transmission electron microscopy (TEM) revealed that inhibition of the apoptosome resulted in structurally abnormal mitochondria that in some cases were engulfed by double-membrane structures resembling autophagosomes. Consistent with mitochondria being engulfed by autophagosomes (mitophagy), confocal microscopy showed colocalization of LC3-GFP and mitochondria. However, Casp9DN did not significantly accelerate T-cell lymphoma alone, or in combination with Lck-Bax38/1, or with Beclin 1+/- mice, two tumor-prone strains in which altered mitochondrial function has been implicated in promoting tumor development. In addition, heterozygous disruption of Beclin 1 had no effect on T-cell lymphoma formation in Lck-Bax38/1 mice. Further studies showed that Beclin 1 levels had no effect on Casp9DN-induced loss of mitochondrial function. These results demonstrate that neither inhibition of apoptosome function nor Beclin 1 haploinsufficiency accelerate T-cell lymphoma development in mice.

Phosphopeptide-binding domains mediate many vital cellular processes such as signal transduction and protein recognition. We studied three well-known domains important for signal transduction: BRCT repeats, WW domain and forkhead-associated (FHA) domain. The first two recognize both phosphothreonine (pThr) and phosphoserine (pSer) residues, but FHA has high specificity for pThr residues. Here we used molecular dynamics (MD) simulations to reveal how FHA exclusively chooses pThr and how BRCT and WW recognize both pThr/pSer. The work also investigated the energies and thermodynamic information of intermolecular interactions.

Pasteurella multocida causes disease in many host species throughout the world. In bovids, it contributes to bovine respiratory disease (BRD) and causes haemorrhagic septicaemia (HS). Previous studies have suggested that BRD-associated P. multocida isolates are of limited diversity. A multilocus sequence typing (MLST) scheme for P. multocida was used to determine whether the low levels of diversity reported are due to the limited discriminatory power of the typing method used, restricted sample selection or true niche association. Bovine respiratory isolates of P. multocida (n = 133) from the UK, the USA and France, collected between 1984 and 2008 from both healthy and clinically affected animals, were typed using MLST. Isolates of P. multocida from cases of HS, isolates from other host species and data from the MLST database were used as comparison.

Previously we showed that the cellular protein P58(IPK) contributes to viral protein synthesis by decreasing the activity of the anti-viral protein, PKR. Here, we constructed a mathematical model to examine the P58(IPK) pathway and investigated temporal behavior of this biological system. We find that influenza virus infection results in the rapid activation of P58(IPK) which delays and reduces maximal PKR and eIF2α phosphorylation, leading to increased viral protein levels. We confirmed that the model could accurately predict viral and host protein levels at extended time points by testing it against experimental data. Sensitivity analysis of relative reaction rates describing P58(IPK) activity and the downstream proteins through which it functions helped identify processes that may be the most beneficial targets to thwart virus replication. Together, our study demonstrates how computational modeling can guide experimental design to further understand a specific metabolic signaling pathway during viral infection in a mammalian system.

Zn²⁺ is an important cofactor for insulin biosynthesis and storage in pancreatic β-cells. Correspondingly, polymorphisms in the SLC30A8 gene, encoding the secretory granule Zn²⁺ transporter ZnT8, are associated with type 2 diabetes risk. Using a genetically engineered (FRET)-based sensor (eCALWY-4), we show here that elevated glucose time-dependently increases free cytosolic Zn²⁺ ([Zn²⁺](cyt)) in mouse pancreatic β-cells. These changes become highly significant (853 ± 96 pm versus 452 ± 42 pm, p < 0.001) after 24 h and are associated with increased expression of the Zn²⁺ importer family members Slc39a6, Slc39a7, and Slc39a8, and decreased expression of metallothionein 1 and 2. Arguing that altered expression of the above genes is not due to altered [Zn²⁺](cyt), elevation of extracellular (and intracellular) [Zn²⁺] failed to mimic the effects of high glucose. By contrast, increases in intracellular cAMP prompted by 3-isobutyl-1-methylxanthine and forskolin partially mimicked the effects of glucose on metallothionein, although not ZiP, gene expression. Modulation of intracellular Ca²⁺ and insulin secretion with pharmacological agents (tolbutamide and diazoxide) suggested a possible role for changes in these parameters in the regulation of Slc39a6 and Slc39a7 but not Slc39a8, nor metallothionein expression. In summary, 1) glucose induces increases in [Zn²⁺](cyt), which are then likely to facilitate the processing and/or the storage of insulin and its cocrystallization with Zn²⁺, and 2) these increases are associated with elevated expression of zinc importers. Conversely, a chronic increase in [Zn²⁺](cyt) following sustained hyperglycemia may contribute to β-cell dysfunction and death in some forms of diabetes.

Sugar transport is critical for normal plant development and stress responses. However, functional evidence for the roles of monosaccharide transporters in rice (Oryza sativa) has not previously been presented. In this study, reversed genetics was used to identify OsGMST1 as a member of the monosaccharide transporter family in rice. The predicted 481 amino acid protein has the typical features of a sugar transporter in the plastid glucose transporter subfamily consistent with reduced monosaccharide accumulation in plants with reduced OsGMST1 expression. OsGMST1-green fluorescent protein is localized to the Golgi apparatus. OsGMST1 expression is induced by salt treatment and reduced expression confers hypersensitivity to salt stress in rice. OsGMST1 may play a direct or an indirect role in tolerance to salt stress in rice.

It is generally accepted that soluble N-ethylmaleimide-sensitive factor attachment protein receptors mediate the docking and fusion of transport intermediates with target membranes. Our research identifies Caenorhabditis elegans homologue of synaptosomal-associated protein 29 (SNAP-29) as an essential regulator of membrane trafficking in polarized intestinal cells of living animals. We show that a depletion of SNAP-29 blocks yolk secretion and targeting of apical and basolateral plasma membrane proteins in the intestinal cells and results in a strong accumulation of small cargo-containing vesicles. The loss of SNAP-29 also blocks the transport of yolk receptor RME-2 to the plasma membrane in nonpolarized oocytes, indicating that its function is required in various cell types. SNAP-29 is essential for embryogenesis, animal growth, and viability. Functional fluorescent protein-tagged SNAP-29 mainly localizes to the plasma membrane and the late Golgi, although it also partially colocalizes with endosomal proteins. The loss of SNAP-29 leads to the vesiculation/fragmentation of the Golgi and endosomes, suggesting that SNAP-29 is involved in multiple transport pathways between the exocytic and endocytic organelles. These observations also suggest that organelles comprising the endomembrane system are highly dynamic structures based on the balance between membrane budding and fusion and that SNAP-29-mediated fusion is required to maintain proper organellar morphology and functions.

In yeast the Golgi-associated retrograde protein (GARP) complex is required for tethering of endosome-derived transport vesicles to the late Golgi. It consists of four subunits--Vps51p, Vps52p, Vps53p, and Vps54p--and shares similarities with other multimeric tethering complexes, such as the conserved oligomeric Golgi (COG) and the exocyst complex. Here we report the functional characterization of the GARP complex in the nematode Caenorhabditis elegans. Furthermore, we identified the C. elegans Vps51 subunit, which is conserved in all eukaryotes. GARP mutants are viable but show lysosomal defects. We show that GARP subunits bind specific sets of Golgi SNAREs within the yeast two-hybrid system. This suggests that the C. elegans GARP complex also facilitates tethering as well as SNARE complex assembly at the Golgi. The GARP and COG tethering complexes may have overlapping functions for retrograde endosome-to-Golgi retrieval, since loss of both complexes leads to a synthetic lethal phenotype.

Integrating individual cell movements to create tissue-level shape change is essential to building an animal. We explored mechanisms of adherens junction (AJ):cytoskeleton linkage and roles of the linkage regulator Canoe/afadin during Drosophila germband extension (GBE), a convergent-extension process elongating the body axis. We found surprising parallels between GBE and a quite different morphogenetic movement, mesoderm apical constriction. Germband cells have an apical actomyosin network undergoing cyclical contractions. These coincide with a novel cell shape change--cell extension along the anterior-posterior (AP) axis. In Canoe's absence, GBE is disrupted. The apical actomyosin network detaches from AJs at AP cell borders, reducing coordination of actomyosin contractility and cell shape change. Normal GBE requires planar polarization of AJs and the cytoskeleton. Canoe loss subtly enhances AJ planar polarity and dramatically increases planar polarity of the apical polarity proteins Bazooka/Par3 and atypical protein kinase C. Changes in Bazooka localization parallel retraction of the actomyosin network. Globally reducing AJ function does not mimic Canoe loss, but many effects are replicated by global actin disruption. Strong dose-sensitive genetic interactions between canoe and bazooka are consistent with them affecting a common process. We propose a model in which an actomyosin network linked at AP AJs by Canoe and coupled to apical polarity proteins regulates convergent extension.

Biochemical studies suggest that caspase activity is required for a functional mitotic checkpoint (MC) and mitotic slippage. To test this directly, we followed nontransformed human telomerase immortalized human retinal pigment epithelia (RPE-1) cells through mitosis after inhibiting or depleting selected caspases. We found that inhibiting caspases individually, in combination, or in toto did not affect the duration or fidelity of mitosis in otherwise untreated cells. When satisfaction of the MC was prevented with 500 nM nocodazole or 2.5 μM dimethylenastron (an Eg5 inhibitor), 92-100% of RPE-1 cells slipped from mitosis in the presence of pan-caspase inhibitors or after simultaneously depleting caspase-3 and -9, and they did so with the same kinetics (~21-22 h) as after treatment with nocodazole or Eg5 inhibitors alone. Surprisingly, inhibiting or depleting caspase-9 alone doubled the number of nocodazole-treated, but not Eg5-inhibited, cells that died in mitosis. In addition, inhibiting or depleting caspase-9 and -3 together accelerated the rate of slippage ~40% (to ~13-15 h). Finally, nocodazole-treated cells that recently slipped through mitosis in the presence or absence of pan-caspase inhibitors contained numerous BubR1 foci in their nuclei. From these data, we conclude that caspase activity is not required for a functional MC or for mitotic slippage.

In mammals, silencing of one of the two X chromosomes in female cells compensates for the different number of X chromosomes between the sexes. The noncoding Xist RNA initiates X chromosome inactivation. Xist spreads from its transcription site over the X chromosome territory and triggers the formation of a repressive chromatin domain. To understand localization of Xist over one X chromosome we aimed to develop a system for investigating Xist in living cells. Here we report successful visualization of transgenically expressed MS2-tagged Xist in mouse embryonic stem cells. Imaging of Xist during an entire cell cycle shows that Xist spreads from a single point to a steady state when the chromosome is covered with a constant amount of Xist. Photobleaching experiments of the established Xist cluster indicate that chromosome-bound Xist is dynamic and turns over on the fully Xist covered chromosome. It appears that in interphase the loss of bound Xist and newly produced Xist are in equilibrium. We also show that the turnover of bound Xist requires transcription, and Xist binding becomes stable when transcription is inhibited. Our data reveal a strategy for visualizing Xist and indicate that spreading over the chromosome might involve dynamic binding and displacement.

Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (also known as NR5A2) regulates bile acid biosynthesis. Structural studies have identified phospholipids as potential LRH-1 ligands, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine (DLPC)) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver-specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signalling pathway that regulates bile acid metabolism and glucose homeostasis.

Cajanus cajan, a tropical shrub, serves as source of food and traditional medicines. The evaluation of aqueous and ethanol extracts for activity against measles virus and toxicity to embryonated chicken eggs was carried out in this study. In vivo and in vitro assay techniques using embryonated chicken eggs and tissue culture (Hep-2 cell lines) as media for both virus cultivation and anti-virus assay showed that a hot-water extract yielded higher activity against measles virus. The hot-water extract of the stem yielded a Log(2) titre of 0.1 for the in vivo assay and an inhibition of cytopathic effect (CPE) in Hep-2 cells by 100% for the in vitro assay. At all concentrations of the extracts, there was a lowering of virus concentration (p = 0.05), indicated by hemagglutination (HA) titration, which is the advantage of HA titration over the tissue culture technique using CPE. This study validates embryonated chicken eggs as suitable media for anti-virus assay and the use of C. cajan in the treatment of some diseases of viral origin.

Genes of the Major Histocompatibility Complex (MHC) are very popular genetic markers among evolutionary biologists because of their potential role in pathogen confrontation and sexual selection. However, MHC genotyping still remains challenging and time-consuming in spite of substantial methodological advances. Although computational haplotype inference has brought into focus interesting alternatives, high heterozygosity, extensive genetic variation and population admixture are known to cause inaccuracies. We have investigated the role of sample size, genetic polymorphism and genetic structuring on the performance of the popular Bayesian PHASE algorithm. To cover this aim, we took advantage of a large database of known genotypes (using traditional laboratory-based techniques) at single MHC class I (N = 56 individuals and 50 alleles) and MHC class II B (N = 103 individuals and 62 alleles) loci in the lesser kestrel Falco naumanni.

Gene-gene interaction in genetic association studies is computationally intensive when a large number of SNPs are involved. Most of the latest Central Processing Units (CPUs) have multiple cores, whereas Graphics Processing Units (GPUs) also have hundreds of cores and have been recently used to implement faster scientific software. However, currently there are no genetic analysis software packages that allow users to fully utilize the computing power of these multi-core devices for genetic interaction analysis for binary traits.

Adenoviruses force quiescent cells to re-enter the cell cycle to replicate their DNA, and for the most part, this is accomplished after they express the E1A protein immediately after infection. In this context, E1A is believed to inactivate cellular proteins (e.g., p130) that are known to be involved in the silencing of E2F-dependent genes that are required for cell cycle entry. However, the potential perturbation of these types of genes by E1A relative to their functions in regulatory networks and canonical pathways remains poorly understood.

Genotyping analysis using capillary DNA sequencing with fluorescently labeled primer pairs obtained by polymerase chain reaction (PCR) is widely used, but is expensive. The post-PCR labeling method using fluorescently labeled short oligonucleotides and nested PCR of the amplified product obtained from unlabeled primer pairs is a simple and inexpensive alternative. However, previously reported protocols often produced spurious peaks or inconsistent amplification under multiplexed analysis as a result of simultaneous progress of both the amplification and labeling reactions and local homology of the attached tag sequence.

Charging for tuberculosis (TB) treatment could reduce completion rates, particularly in the poor. We identified and synthesised studies that measure costs of TB treatment, estimates of adherence and the potential impact of charging on treatment completion in China.

Alkyl hydroperoxidase activity provides an important antioxidant defense for bacterial cells. The catalytic mechanism requires two peroxidases, AhpC and AhpD, where AhpD plays the role of an essential adaptor protein.