The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering.

In this paper we investigate the use of classical fMRI Random Effect (RFX) group statistics when analyzing a very large cohort and the possible improvement brought from anatomical information. Using 1326 subjects from the IMAGEN study, we first give a global picture of the evolution of the group effect t-value from a simple face-watching contrast with increasing cohort size. We obtain a wide activated pattern, far from being limited to the reasonably expected brain areas, illustrating the difference between statistical significance and practical significance. This motivates us to inject tissue-probability information into the group estimation, we model the BOLD contrast using a matter-weighted mixture of Gaussians and compare it to the common, single-Gaussian model. In both cases, the model parameters are estimated per-voxel for one subgroup, and the likelihood of both models is computed on a second, separate subgroup to reflect model generalization capacity. Various group sizes are tested, and significance is asserted using a 10-fold cross-validation scheme. We conclude that adding matter information consistently improves the quantitative analysis of BOLD responses in some areas of the brain, particularly those where accurate inter-subject registration remains challenging.

Immuno-laser capture microdissection (immuno-LCM) enables highly selective retrieval of designated cell populations from their in situ locations in complex tissue like the brain. However, the amount of tissue acquired by immuno-LCM is extremely limited, and the RNA purification, amplification and labeling steps necessary for expression analysis by hybridization microarray are tedious and time consuming. This report therefore describes a protocol in which these RNA steps are eliminated altogether, yet allows for global gene profiling. Specifically, immuno-LCM tissue was solubilized and the extract directly subjected to reverse transcription to generate cDNA. Pre-amplification of cDNA was performed next, and then relative expression of 96 different immune-related genes simultaneously determined by quantitative real-time PCR using a microfluidic card TaqMan(®) Low Density Array (TLDA). This protocol was highly reproducible and extremely sensitive, demonstrating high correlation of raw Ct values among both technical and biological replicate samples when using only 1/32 of total pre-amplified cDNA obtained from as little as 500 LCM 'shots.' As this abridged protocol takes only approximately 7h from LCM tissue acquisition to analysis by TLDA, it can prove a very effective tool for both screening and validation purposes when investigating gene regulation in health and disease of the nervous system and other tissues.

Huntington's Disease (HD) is a neurodegenerative disease caused by a CAG triplet-repeat expansion-mutation in the Huntingtin gene. Subjects at risk for HD can be identified by genetic testing in the prodromal phase. Structural changes of basal-ganglia nuclei such as the caudate nucleus are well-replicated findings observable early in prodromal-HD subjects and may be preceded by distinct functional alterations of cortico-striatal circuits. This study aims to assess functional integrity of the motor system as a cortico-striatal circuit with particular clinical relevance in HD. Ten subjects in the prodromal phase of HD and ten matched controls were administered blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) at rest (3T). Functional connectivity was measured as synchrony of BOLD activity between the caudate nucleus and thirteen cortical brain regions (seeds). Basal-ganglia volumes were assessed as established markers of disease progression in prodromal-HD. Linear regression analysis was performed to test for a relationship between structural changes and group differences in functional connectivity. Prodromal-HD subjects showed reduced BOLD synchrony between two seeds in the premotor cortex (BA6) and the caudate nucleus. While similar effect sizes could be observed for reduced basal-ganglia volumes and differences in functional connectivity, coefficients of determination indicate a moderate relationship between functional connectivity and striatal atrophy. Our data show reduced cortico-striatal functional connectivity at rest in prodromal-HD and suggest a relation to early structural brain changes. Additional longitudinal studies are necessary to elucidate the temporal relationship between functional alterations and earliest structural brain changes in prodromal-HD.

Activin-A, a member of the TGF-β family, is known to be present in bone and cartilage. Although, involvement of the TGF-β family in chondrogenesis has been reported, the mechanism by which activin-A regulates chondrogenesis has not been fully elucidated. The aim of this study was to investigate the effects of activin-A on chondrocyte differentiation in vitro.

The aim of the current study was to follow on from previous findings that eye movements can have a causal influence on preference formation. Shimojo et al. (2003) previously found that faces that were presented for a longer duration in a two alternative forced choice task were more likely to be judged as more attractive. This effect only occurred when an eye movement was made towards the faces (with no effect when faces were centrally presented). The current study replicated Shimojo et al.'s (2003) design, whilst controlling for potential inter-stimuli interference in central presentations. As per previous findings, when eye movements were made towards the stimuli, faces that were presented for longer durations were preferred. However, faces that were centrally presented (thus not requiring an eye movement) were also preferred in the current study. The presence of an exposure duration effect for centrally presented faces casts doubt on the necessity of the eye movement in this decision making process and has implications for decision theories that place an emphasis on the role of eye movements in decision making.

The pandemic of HIV-1 has continued for decades, yet there remains no licensed vaccine. Previous research has demonstrated the effectiveness of a multi-envelope, multi-vectored HIV-1 vaccine in a macaque-SHIV model, illustrating a potential means of combating HIV-1. Specifically, recombinant DNA, vaccinia virus (VV) and purified protein (DVP) delivery systems were used to vaccinate animals with dozens of antigenically distinct HIV-1 envelopes for induction of immune breadth. The vaccinated animals controlled disease following challenge with a heterologous SHIV. This demonstration suggested that the antigenic cocktail vaccine strategy, which has succeeded in several other vaccine fields (e.g. pneumococcus), might also succeed against HIV-1. The strategy remains untested in an advanced clinical study, in part due to safety concerns associated with the use of replication-competent VV. To address this concern, we designed a macaque study in which psoralen/ultraviolet light-inactivated VV (UV VV) was substituted for replication-competent VV in the multi-envelope DVP protocol. Control animals received a vaccine encompassing no VV, or no vaccine. All VV vaccinated animals generated an immune response toward VV, and all vaccinated animals generated an immune response toward HIV-1 envelope. After challenge with heterologous SHIV 89.6P, animals that received replication-competent VV or UV VV experienced similar outcomes. They exhibited reduced peak viral loads, maintenance of CD4+ T cell counts and improved survival compared to control animals that received no VV or no vaccine; there were 0/15 deaths among all animals that received VV and 5/9 deaths among controls. Results define a practical means of improving VV safety, and encourage advancement of a promising multi-envelope DVP HIV-1 vaccine candidate.

In the vast majority of eukaryotic organisms, the mitochondrial cox2 gene encodes subunit II of cytochrome c oxidase (COX2). However, in some lineages including legumes and chlorophycean algae, the cox2 gene migrated to the nucleus. Furthermore, in chlorophycean algae, this gene was split in two different units. Thereby the COX2 subunit is encoded by two independent nuclear genes, cox2a and cox2b, and mitochondria have to import the cytosol-synthesized COX2A and COX2B subunits and assemble them into the cytochrome c oxidase complex. In the chlorophycean algae Chlamydomonas reinhardtii and Polytomella sp., the COX2A precursor exhibits a long (130-140 residues), cleavable mitochondrial targeting sequence (MTS). In contrast, COX2B lacks an MTS, suggesting that mitochondria use different mechanisms to import each subunit. Here, we explored the in vitro import processes of both, the Polytomella sp. COX2A precursor and the COX2B protein. We used isolated, import-competent mitochondria from this colorless alga. Our results suggest that COX2B is imported directly into the intermembrane space, while COX2A seems to follow an energy-dependent import pathway, through which it finally integrates into the inner mitochondrial membrane. In addition, the MTS of the COX2A precursor is eliminated. This is the first time that the in vitro import of split COX2 subunits into mitochondria has been achieved.

Bone destruction is a hallmark of multiple myeloma and affects more than 80% of patients. However, current therapy is unable to completely cure and/or prevent bone lesions. Although it is accepted that myeloma cells mediate bone destruction by inhibition of osteoblasts and activation of osteoclasts, the underlying mechanism is still poorly understood. This study demonstrates that constitutive activation of p38 mitogen-activated protein kinase in myeloma cells is responsible for myeloma-induced osteolysis. Our results show that p38 is constitutively activated in most myeloma cell lines and primary myeloma cells from patients. Myeloma cells with high/detectable p38 activity, but not those with low/undetectable p38 activity, injected into severe combined immunodeficient (SCID) or SCID-hu mice caused bone destruction. Inhibition or knockdown of p38 in human myeloma reduced or prevented myeloma-induced osteolytic bone lesions without affecting tumor growth, survival, or homing to bone. Mechanistic studies showed that myeloma cell p38 activity inhibited osteoblastogenesis and bone formation and activated osteoclastogenesis and bone resorption in myeloma-bearing SCID mice. This study elucidates a novel molecular mechanism-activation of p38 signaling in myeloma cells-by which myeloma cells induce osteolytic bone lesions, and indicates that targeting myeloma cell p38 may be a viable approach to treating or preventing myeloma bone disease.

The murine vasorin (Vasn) gene, initially known as Slit-like 2, encodes a transmembrane protein that shares structural similarities with the eponymous Slit proteins. However, whether it also shares functional similarities with these large secreted proteins remains to be elucidated. Here, we report expression of Vasn during embryonic and fetal development of the mouse using whole-mount in situ hybridization (WISH) and histochemical detection of β-galactosidase expressed from a targeted Vasn(lacZ) knock-in allele. Comparison of whole-mount staining patterns of both approaches showed identical expression domains, confirming that Vasn promoter-driven β-galactosidase expression faithfully reflects endogenous Vasn expression. Vasn is highly expressed in vascular smooth muscle cells (hence the name), a finding consistent with a previous report on its human homolog VASN, whose extracellular domain was shown to function as a TGF-β trap (Ikeda et al., 2004). Most striking, however, is Vasn's prominent expression in the developing skeletal system, starting as early as the first mesenchymal condensations appear. Moreover, distinct expression domains outside the bones, e.g., in the developing kidneys and lungs, suggest further roles for this gene in the mouse. Recently, it was shown that mitochondria-localized Vasn protects cells from TNFα- and hypoxia-induced apoptosis, and partial deletion of the Vasn coding sequence leads to increased sensitivity of hepatocytes to TNFα-induced apoptosis (Choksi et al., 2011). By providing a first comprehensive analysis of the Vasn expression pattern during mouse embryonic development, our study will help to further elucidate its biological functions.

The mouse posterior primitive streak at neural plate/headfold stages (NP/HF, ~7.5 dpc-8 dpc) represents an optimal window from which hemangioblasts can be isolated. We performed immunohistochemistry on this domain using established monoclonal antibodies for proteins that affect blood and endothelial fates. We demonstrate that HoxB4 and GATA1 are the first set of markers that segregate independently to endothelial or blood populations during NP/HF stages of mouse embryonic development. In a subset of cells, both proteins are co-expressed and immunoreactivities appear mutually excluded within nuclear spaces. We searched for this particular state at later sites of hematopoietic stem cell emergence, viz., the aorta-gonad-mesonephros (AGM) and the fetal liver at 10.5-11.5 dpc, and found that only a rare number of cells displayed this character. Based on this spatial-temporal argument, we propose that the earliest blood progenitors emerge either directly from the epiblast or through segregation within the allantoic core domain (ACD) through reduction of cell adhesion and pSmad1/5 nuclear signaling, followed by a stochastic decision toward a blood or endothelial fate that involves GATA1 and HoxB4, respectively. A third form in which binding distributions are balanced may represent a common condition shared by hemangioblasts and HSCs. We developed a heuristic model of hemangioblast maturation, in part, to be explicit about our assumptions.

Unlike humans, some vertebrate animals are able to completely regenerate damaged appendages and other organs. For example, adult zebrafish will regenerate the complex structure of an amputated caudal fin to a degree that the original and replacement fins are indistinguishable. The blastema, a mass of cells that uniquely forms following appendage amputation in regenerating animals, is the major source of regenerated tissue. However, the cell lineage(s) that contribute to the blastema and their ultimate contribution(s) to the regenerated fin have not been definitively characterized. It has been suggested that cells near the amputation site dedifferentiate forming multipotent progenitors that populate the blastema and then give rise to multiple cell types of the regenerated fin. Other studies propose that blastema cells are non-uniform populations that remain restricted in their potential to contribute to different cell lineages. We tested these models by using inducible Cre-lox technology to generate adult zebrafish with distinct, isolated groups of genetically labeled cells within the caudal fin. We then tracked populations of several cell types over the entire course of fin regeneration in individual animals. We found no evidence for the existence of multipotent progenitors. Instead, multiple cell types, including epidermal cells, intra-ray fibroblasts, and osteoblasts, contribute to the newly regenerated tissue while remaining highly restricted with respect to their developmental identity. Our studies further demonstrate that the regenerating fin consists of many repeating blastema "units" dedicated to each fin ray. These blastemas each have an organized structure of lineage restricted, dedifferentiated cells that cooperate to regenerate the caudal fin.

The significance of IL-6 production in tuberculosis is yet to be fully elucidated, although it is known for quite some time that IL-6 interferes with IFN-γ induced signal. In order to know which cellular process induced by IFN-γ is actually counteracted by IL-6, we studied the role of IL-6 on IFN-γ induced autophagy formation in virulent Mycobacterium tuberculosis infection in THP-1 cells, since it is well characterized that induction of autophagy by IFN-γ eliminates intracellular mycobacterium by overcoming the phagosome maturation block imposed by bacilli. We report here that IL-6 inhibits both IFN-γ and starvation induced autophagy in M. tuberculosis H37Rv infected cells. M. tuberculosis H37Rv infection results in time dependent production of IL-6 in THP-1 cells and neutralization of this endogenous IL-6 by anti-IL-6 antibody significantly enhances the IFN-γ mediated killing of the intracellular bacteria. IL-6 time dependently lowers Atg12-Atg5 complex and therefore inhibits autophagosome biogenesis rather than autophagolysosome formation. IL-6 also affects IFN-γ mediated stimulation of mTOR, p-38 and JNK pathways. These results clearly indicate that virulent mycobacteria strategically upregulate IL-6 production to combat innate immunity.

Telomeres are specialized nucleoprotein structures, which protect chromosome ends and have been implicated in the ageing process. Telomere shortening has been shown to contribute to a persistent DNA damage response (DDR) during replicative senescence, the irreversible loss of division potential of somatic cells. Similarly, persistent DDR foci can be found in stress-induced senescence, although their nature is not understood. Here we show, using immuno-fluorescent in situ hybridization and ChIP, that up to half of the DNA damage foci in stress-induced senescence are located at telomeres irrespective of telomerase activity. Moreover, live-cell imaging experiments reveal that all persistent foci are associated with telomeres. Finally, we report an age-dependent increase in frequencies of telomere-associated foci in gut and liver of mice, occurring irrespectively of telomere length. We conclude that telomeres are important targets for stress in vitro and in vivo and this has important consequences for the ageing process.

Neuronal injury elicits potent cellular responses from glia, but molecular pathways modulating glial activation, phagocytic function and termination of reactive responses remain poorly defined. Here we show that positive or negative regulation of glial responses to axon injury is molecularly encoded by unique isoforms of the Drosophila melanogaster engulfment receptor Draper. Draper-I promotes engulfment of axonal debris through an immunoreceptor tyrosine-based activation motif (ITAM). In contrast, Draper-II, an alternative splice variant, potently inhibits glial engulfment function. Draper-II suppresses Draper-I signaling through a previously undescribed immunoreceptor tyrosine-based inhibitory motif (ITIM)-like domain and the tyrosine phosphatase Corkscrew (Csw). Intriguingly, loss of Draper-II-Csw signaling prolongs expression of glial engulfment genes after axotomy and reduces the ability of glia to respond to secondary axotomy. Our work highlights a novel role for Draper-II in inhibiting glial responses to neurodegeneration, and indicates that a balance of opposing Draper-I and Draper-II signaling events is essential to maintain glial sensitivity to brain injury.

Regulation of bone density is partly dependent upon steroid hormones, with estrogens playing an important role. Inactive conjugated estrogens may serve as precursors to active estrogens, especially in post-menopausal women, via steroid sulfatase, which converts conjugated estrogens into unconjugated estrogens. The purpose of this study was to characterize steroid sulfatase in the MC3T3-E1 mouse pre-osteoblastic cell line. Enzyme conversion assays were performed on whole MC3T3-E1 cells in culture and on microsomes prepared by differential centrifugation. (3)H-E(1)S and (3)H-DHEAS were used as tracers, and radioinert E(1)S and DHEAS were used as substrate. Whole cells and microsomes exhibited steroid sulfatase activity, which was blocked by the specific inhibitor estrone-3-O-sulfamate (EMATE). The K(m) of steroid sulfatase in microsomes averaged 83 μM when using E(1)S as substrate and 64 μM when using DHEAS. Western blotting of MC3T3-E1 microsomes for steroid sulfatase was performed, after SDS-PAGE, using an antibody generated against a peptide based on a conserved region of steroid sulfatase. Western blotting revealed three bands of cross-reactivity, ranging from 50 to 79 kDa. Reverse transcriptase polymerase chain reaction (RT-PCR), using specific primers, resulted in a single cDNA band of the expected size (100 bp) and sequence, indicating the presence of steroid sulfatase mRNA. Growth assays revealed that the MC3T3-E1 cells were stimulated by estradiol-17β, and also by estrone sulfate and DHEAS, revealing that the cells can use steroid sulfatase to produce active estrogens. Furthermore, growth of these cells in the presence of estradiol, estrone and estrone sulfate was inhibited by the estrogen receptor blocker ICI 182,780, indicating that stimulation of cell growth is mediated by the estrogen receptor. In our studies, four lines of evidence (enzyme activity, immunoassay, RT-PCR and growth assays) demonstrated the presence of steroid sulfatase in mouse MC3T3-E1 bone cells. The existence of steroid sulfatase in these pre-osteoblastic cells, along with the ability of sulfated steroids to promote their growth, suggest the possibility that this enzyme is involved in regulation of bone density in mice.

ISCOM vaccines induce a balanced Th1/Th2 response, long-lasting antibody responses and cytotoxic T lymphocytes. The mode of action for the adjuvant component, the ISCOM-Matrix, is known to some extent but questions remain regarding its mechanism of action. The Affymetrix GeneChip® Porcine Genome Array was applied to study the global transcriptional response to ISCOM-Matrix in pigs at the injection site and in the draining lymph node 24h after i.m. injection. Gene enrichment analysis revealed inflammation, innate immunity and antigen processing to be central in the ISCOM-Matrix response. At the injection site, 594 genes were differentially expressed, including up-regulation of the cytokines osteopontin (SPP1), IL-10 and IL-18 and the chemokines CCL2, CCL19 and CXCL16. Of the 362 genes differentially expressed in the lymph node, IL-1β and CXCL11 were up-regulated whereas IL18, CCL15 and CXCL12 were down-regulated. ISCOM-Matrix also modulated genes for pattern recognition receptors at the injection site (TLR2, TLR4, MRC1, PTX3, LGALS3) and in the lymph node (TLR4, RIG-I, MDA5, OAS1, EIF2AK2, LGALS3). A high proportion of up-regulated interferon-regulated genes indicated an interferon response. Thus, several genes, genetic pathways and biological processes were identified that are likely to shape the early immune response elicited by ISCOM-based vaccines.

Investigations have characterized addictive drug-induced developmental cardiovascular malformation in human, non-human primate and rodent. However, the underlying mechanism of malformation caused by drugs during pregnancy is still largely unknown, and preventive and therapeutic measures have been lacking. Using 1H NMR spectroscopy, we profiled the metabolites from human embryo endothelial cells exposed to methamphetamine (METH) and quantified a total of 226 peaks. We identified 11 metabolites modified robustly and found that taurine markedly increased. We then validated the hypothesis that this dramatic increase in taurine could attribute to its effect in inhibiting METH-induced developmental angiogenesis defect. Taurine supplement showed a more significant potential than other metabolites in protecting against METH-induced injury in endothelial cells. Taurine strongly attenuated METH-induced inhibition of proliferation and migration in endothelial cells. Furthermore, death rate and vessel abnormality of zebrafish embryos treated with METH were greatly reversed by taurine. In addition, taurine supplement caused a rapid decrease in reactive oxygen species generation and strongly attenuated the excitable arise of antioxidase activities in the beginning of METH exposure prophase. Dysregulations of NF-κB, p-ERK as well as Bax, which reflect apoptosis, cell cycle arrest and oxidative stress in vascular endothelium, were blocked by taurine. Our results provide the first evidence that taurine prevents METH-caused developmental angiogenesis defect through antioxidant mechanism. Taurine could serve as a potential therapeutic or preventive intervention of developmental vascular malformation for the pregnant women with drug use.

Alzheimer's disease (AD) is characterized by progressive memory loss due to extracellular senile plaques and intracellular neurofibrillary tangles. The toxic β-amyloid (Aβ) aggregates that form in AD can induce the overproduction of reactive oxygen species (ROS), nitric oxide (NO), and proinflammatory cytokines. These Aβ aggregates likely play a pivotal role in the onset and progression of AD. Reducing Aβ generation, inhibiting Aβ toxicity, and improving Aβ clearance are promising therapeutic strategies for AD. The present paper is the first to reveal a heptapeptide (XD4) isolated from a Ph.D.-C7C library through phage display that significantly inhibited Aβ cytotoxicity, increased the microglial phagocytosis of Aβ, decreased the Aβ-induced generation of ROS and NO, and attenuated the disequilibrium of calcium homeostasis in vitro. Remarkably, XD4 also attenuated memory deficits in β-amyloid precursor protein/presenilin 1 (APPswe/PS1dE9) transgenic mice, and reduced amyloid plaque burden and Aβ40/42 levels. The results of the present study indicate that this peptide, which specifically targets Aβ, may be a promising new therapy for patients exhibiting cognitive impairment and increased Aβ burden.

Limiting the development of secondary damage represents one of the major goals of neuroprotective therapies after spinal cord injury. Here, we demonstrate that specific JNK inhibition via a single intraperitoneal injection of the cell permeable peptide D-JNKI1 6h after lesion improves locomotor recovery assessed by both the footprint and the BMS tests up to 4 months post-injury in mice. JNK inhibition prevents c-jun phosphorylation and caspase-3 cleavage, has neuroprotective effects and results in an increased sparing of white matter at the lesion site. Lastly, D-JNKI1 treated animals show a lower increase of erythrocyte extravasation and blood brain barrier permeability, thus indicating protection of the vascular system. In total, these results clearly point out JNK inhibition as a promising neuroprotective strategy for preventing the evolution of secondary damage after spinal cord injury.