Porencephaly is a neurological disorder characterized by fluid-filled cysts or cavities in the brain that often cause hemiplegia. It has been suggested that porencephalic cavities result from focal cerebral degeneration involving hemorrhages. De novo or inherited heterozygous mutations in COL4A1, which encodes the type IV α1 collagen chain that is essential for structural integrity for vascular basement membranes, have been reported in individuals with porencephaly. Most mutations occurred at conserved Gly residues in the Gly-Xaa-Yaa repeats of the triple-helical domain, leading to alterations of the α1α1α2 heterotrimers. Here we report on two individuals with porencephaly caused by a heterozygous missense mutation in COL4A2, which encodes the type IV α2 collagen chain. Mutations c.3455G>A and c.3110G>A, one in each of the individuals, cause Gly residues in the Gly-Xaa-Yaa repeat to be substituted as p.Gly1152Asp and p.Gly1037Glu, respectively, probably resulting in alterations of the α1α1α2 heterotrimers. The c.3455G>A mutation was found in the proband's mother, who showed very mild monoparesis of the left upper extremity, and the maternal elder uncle, who had congenital hemiplegia. The maternal grandfather harboring the mutation is asymptomatic. The c.3110G>A mutation occurred de novo. Our study confirmed that abnormalities of the α1α1α2 heterotrimers of type IV collagen cause porencephaly and stresses the importance of screening for COL4A2 as well as for COL4A1.

Collagen, type IV, alpha 1 (COL4A1) and alpha 2 (COL4A2) form heterotrimers and are abundant components of basement membranes, including those of the cerebral vasculature. COL4A1 mutations are an increasingly recognized cause of multisystem disorders, including highly penetrant cerebrovascular disease and intracerebral hemorrhage (ICH). Because COL4A1 and COL4A2 are structurally and functionally associated, we hypothesized that variants in COL4A2 would also cause ICH. We sequence COL4A2 in 96 patients with ICH and identify three rare, nonsynonymous coding variants in four patients that are not present in a cohort of 144 ICH-free individuals. All three variants change evolutionarily conserved amino acids. Using a cellular assay, we show that these putative mutations cause intracellular accumulation of COL4A1 and COL4A2 at the expense of their secretion, which supports their pathogenecity. Furthermore, we show that Col4a2 mutant mice also have completely penetrant ICH and that mutations in mouse and human lead to retention of COL4A1 and COL4A2 within the endoplasmic reticulum (ER). Importantly, two of the three putative mutations found in patients trigger ER stress and activate the unfolded protein response. The identification of putative COL4A2 mutations that might contribute to ICH in human patients provides insight into the pathogenic mechanisms of this disease. Our data suggest that COL4A2 mutations impair COL4A1 and COL4A2 secretion and can also result in cytotoxicity. Finally, our findings suggest that, collectively, mutations in COL4A1 and COL4A2 contribute to sporadic cases of ICH.

The mechanisms regulating the differential selenium (Se)-dependent stability of selenoprotein mRNAs are partially characterized. To further study the Se-dependent regulation of selenoproteins, we developed a novel chemiluminescent reporter to monitor the steady-state mRNA level of an artificial selenoprotein. Our reporter is a fusion of the Renilla luciferase gene and of the β-globin gene, but contains features required for incorporation of selenocysteine (SEC), namely, a UGA-SEC codon and a 3' untranslated region RNA stem loop called a SEC incorporation sequence (SECIS). At various levels of Se, the activity of reporters containing GPX1 or GPX4 SECIS elements is proportional to the steady-state mRNA level of the reporter construct and reflects the level of the corresponding endogenous mRNA. In a reporter containing a UGA codon and a functional GPX1 SECIS, Se-dependent nonsense-mediated decay (NMD) occurred in the cytoplasm, as opposed to the more typical nuclear location. To validate the reporter system, we used genetic and pharmacologic approaches to inhibit or promote NMD. Modulation of UPF1 by siRNA, overexpression, or by inhibition of SMG1 altered NMD in this system. Our reporter is derived from a Renilla luciferase reporter gene fused to an intron containing B-globin gene and is subject to degradation by NMD when a stop codon is inserted before the second intron.

Substance use disorders (SUD) are a major public health problem. Attention deficit hyperactivity disorder (ADHD) is a comorbid condition associated with both onset and prognosis of SUD. Prevalence estimates of ADHD in SUD vary significantly.

Brain-derived neurotrophic factor (BDNF) plays an important role in neuronal plasticity, learning, and memory. Levels of BDNF and its main receptor TrkB (TrkB.TK) have been reported to be decreased while the levels of the truncated TrkB (TrkB.T1) are increased in Alzheimer's disease. We show here that incubation with amyloid-β increased TrkB.T1 receptor levels and decreased TrkB.TK levels in primary neurons. In vivo, APPswe/PS1dE9 transgenic mice (APdE9) showed an age-dependent relative increase in cortical but not hippocampal TrkB.T1 receptor levels compared with TrkB.TK. To investigate the role of TrkB isoforms in Alzheimer's disease, we crossed AP mice with mice overexpressing the truncated TrkB.T1 receptor (T1) or the full-length TrkB.TK isoform. Overexpression of TrkB.T1 in APdE9 mice exacerbated their spatial memory impairment while the overexpression of TrkB.TK alleviated it. These data suggest that amyloid-β changes the ratio between TrkB isoforms in favor of the dominant-negative TrkB.T1 isoform both in vitro and in vivo and supports the role of BDNF signaling through TrkB in the pathophysiology and cognitive deficits of Alzheimer's disease.

A motor unit consists of a motoneurone and the multiple muscle fibres that it innervates, and forms the final neural pathway that influences movement. Discharge of motor units is altered (decreased discharge rate and/or cessation of firing; and increased discharge rate and/or recruitment of new units) during matched-force contractions with pain. This is thought to be mediated by nociceptive (pain) input on motoneurones, as demonstrated in animal studies. It is also possible that motoneurone excitability is altered by pain related descending inputs, that these changes persist after noxious stimuli cease, and that direct nociceptive input is not necessary to induce pain related changes in movement. We aimed to determine whether anticipation of pain (descending pain related inputs without nociceptor discharge) alters motor unit discharge, and to observe motor unit discharge recovery after pain has ceased. Motor unit discharge was recorded with fine-wire electrodes in the quadriceps of 9 volunteers. Subjects matched isometric knee-extension force during anticipation of pain (anticipation: electrical shocks randomly applied over the infrapatellar fat-pad); pain (hypertonic saline injected into the fat-pad); and 3 intervening control conditions. Discharge rate of motor units decreased during pain (P<.001) and anticipation (P<.01) compared with control contractions. De-recruitment of 1 population of units and new recruitment of another population were observed during both anticipation and pain; some changes in motor unit recruitment persisted after pain ceased. This challenges the fundamental theory that pain-related changes in muscle activity result from direct nociceptor discharge, and provides a mechanism that may underlie long-term changes in movement/chronicity in some musculoskeletal conditions.

Advances in our understanding of sensory-motor integration suggest a unique role of the frontal lobe circuits in cognition and behaviour. Long-range afferent connections convey higher order sensory information to the frontal cortex, which in turn responds to internal and external stimuli with flexible and adaptive behaviour. Long-range connections from and to frontal lobes have been described in detail in monkeys but little is known about short intralobar frontal connections mediating local connectivity in humans. Here we used spherical deconvolution diffusion tractography and post-mortem dissections to visualize the short frontal lobe connections of the human brain. We identified three intralobar tracts connecting: i) posterior Broca's region with supplementary motor area (SMA) and pre-supplementary motor area (pre-SMA) (i.e., the frontal 'aslant' tract - FAT); ii) posterior orbitofrontal cortex with anterior polar region (i.e., fronto-orbitopolar tract - FOP); iii) posterior pre-central cortex with anterior prefrontal cortex (i.e., the frontal superior longitudinal - FSL faciculus system). In addition more complex systems of short U-shaped fibres were identified in the regions of the central, pre-central, perinsular and fronto-marginal sulcus (FMS). The connections between Broca and medial frontal areas (i.e. FAT) and those between the hand-knob motor region and post-central gyrus (PoCG) were found left lateralized in a group of twelve healthy right-handed subjects. The existence of these short frontal connections was confirmed using post-mortem blunt dissections. The functional role of these tracts in motor learning, verbal fluency, prospective behaviour, episodic and working memory is discussed. Our study provides a general model for the local connectivity of the frontal lobes that could be used as an anatomical framework for studies on lateralization and future clinical research in neurological and psychiatric disorders.

The muscle growth in mammals is regulated by several growth factors including myostatin (MSTN), a member of the transforming growth factor-beta (TGF-beta) superfamily. To date, it is unknown in fish whether MSTN could have any effect on proliferation or differentiation of myogenic cells. Using culture of trout satellite cells, we showed that mstn1a and mstn1b mRNA are expressed in myoblasts and that their expression decreased in differentiating myoblasts. We also demonstrated that a treatment with huMSTN decreased the proliferation of IGF1-stimulated myoblasts in a dose-dependent manner. By contrast, treatment of myoblasts with 100 nM of huMSTN for three days, did not affect the percentage of positive cells for myogenin neither the percentage of nuclei in myosin positive cells. Moreover, our results clearly indicated that huMSTN treatment had no effect on MyoD and myogenin protein levels, which suggests that huMSTN did not strongly affect MyoD activity. In conclusion, we showed that huMSTN inhibited proliferation but not differentiation of trout myoblasts, probably resulting from a lack of huMSTN effect on MyoD activity. Altogether, these results show high interspecies differences in the function of MSTN.

Erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor, has been shown to have benefits for non-small cell lung cancer and pancreatic cancer patients; however, almost all patients develop progressive disease during the therapy. On the other hand, it has been reported that a tumor continues to express epidermal growth factor receptor even after developing progressive disease. To demonstrate the clinical relevance of erlotinib treatment after progressive disease, we investigated whether continuous administration of erlotinib in combination with chemotherapy has a useful effect on progressive disease development during erlotinib treatment. For this purpose, we examined the antitumor effect of a combination therapy of a chemotherapeutic agent with erlotinib using two types of erlotinib-resistant tumor xenograft models: a non-small cell lung cancer model, in which EBC-1, H1975 and HCC827TR3 tumors were implanted, and an HPAC pancreatic cancer cell xenograft which generates erlotinib-resistant tumors in vivo. As a result, the combination therapy showed a significantly higher antitumor activity compared with chemomonotherapy in all xenograft models except the H1975 xenografts. Furthermore, erlotinib alone suppressed the phosphorylation of epidermal growth factor receptor in HPAC tumors and the two non-small cell lung cancer cell lines other than H1975. Therefore, combination therapy which uses erlotinib can be considered effective if epidermal growth factor receptor phosphorylation is inhibited by erlotinib, even in erlotinib-resistant tumor xenograft models. Our results suggest that the continuous inhibition of epidermal growth factor receptor phosphorylation by erlotinib after progressive disease enhances the antitumor activity of chemotherapy.

In this paper, we present an extensive performance evaluation of a novel source localization algorithm, Champagne. It is derived in an empirical Bayesian framework that yields sparse solutions to the inverse problem. It is robust to correlated sources and learns the statistics of non-stimulus-evoked activity to suppress the effect of noise and interfering brain activity. We tested Champagne on both simulated and real M/EEG data. The source locations used for the simulated data were chosen to test the performance on challenging source configurations. In simulations, we found that Champagne outperforms the benchmark algorithms in terms of both the accuracy of the source localizations and the correct estimation of source time courses. We also demonstrate that Champagne is more robust to correlated brain activity present in real MEG data and is able to resolve many distinct and functionally relevant brain areas with real MEG and EEG data.

Toxoplasma gondii is an obligate intracellular protozoan parasite in which 36 predicted Hsp40 family members were identified by searching the T. gondii genome. The predicted protein sequence from the gene ID TGME49_065310 showed an amino acid sequence and domain structure similar to Saccharomyces cerevisiae Sis1. TgSis1 did not show differences in its expression profile during alkaline stress by microarray analysis. Furthermore, TgSis1 showed to be a cytosolic Hsp40 which co-immunoprecipitated with T. gondii Hsp70 and Hsp90. Structural modeling of the TgSis1 peptide binding fragment revealed structural and electrostatic properties different from the experimental model of human Sis1-like protein (Hdj1). Based on these differences; we propose that TgSis1 may be a potentially attractive drug target for developing a novel anti-T. gondii therapy.

In many species, including Syrian hamsters, the generation of male reproductive behavior depends critically on the perception of female odor cues from conspecifics in the environment. The behavioral response to these odors is mediated by a network of steroid-sensitive ventral forebrain nuclei including the medial amygdala (MA), posterior bed nucleus of the stria terminalis (BNST) and medial preoptic area (MPOA). Previous studies have demonstrated that each of these three nuclei is required for appropriate sexual behavior and that MA preferentially sends female odor information directly to BNST and MPOA. It is unknown, however, how the functional connections between MA and BNST and/or MPOA are organized to generate different aspects of reproductive behavior. Therefore, the following experiments used the asymmetrical pathway lesion technique to test the role of the functional connections between MA and BNST and/or MPOA in odor preference and copulatory behaviors. Lesions that functionally disconnected MA from MPOA eliminated copulatory behavior but did not affect odor preference. In contrast, lesions that functionally disconnected MA from BNST eliminated preference for volatile female odors but did not affect preference for directly contacted odors or copulatory behavior. These results therefore demonstrate a double dissociation in the functional connections required for attraction to volatile sexual odors and copulation and, more broadly, suggest that appetitive and consummatory reproductive behaviors are mediated by distinct neural pathways.

Cardiac remodeling and hypertrophy are the pathological consequences of cardiovascular disease and are correlated with its associated mortality. Activity of the transcription factor NF-κB is increased in the diseased heart; however, our present understanding of how the individual subunits contribute to cardiovascular disease is limited. We assign a new role for the c-Rel subunit as a stimulator of cardiac hypertrophy and fibrosis. We discovered that c-Rel-deficient mice have smaller hearts at birth, as well as during adulthood, and are protected from developing cardiac hypertrophy and fibrosis after chronic angiotensin infusion. Results of both gene expression and cross-linked chromatin immunoprecipitation assay analyses identified transcriptional activators of hypertrophy, myocyte enhancer family, Gata4, and Tbx proteins as Rel gene targets. We suggest that the p50 subunit could limit the prohypertrophic actions of c-Rel in the normal heart, because p50 overexpression in H9c2 cells repressed c-Rel levels and the absence of cardiac p50 was associated with increases in both c-Rel levels and cardiac hypertrophy. We report for the first time that c-Rel is highly expressed and confined to the nuclei of diseased adult human hearts but is restricted to the cytoplasm of normal cardiac tissues. We conclude that c-Rel-dependent signaling is critical for both cardiac remodeling and hypertrophy. Targeting its activities could offer a novel therapeutic strategy to limit the effects of cardiac disease.

Fetal cattle B-cell development proceeds via a pre-B cell stage that is characterized by the expression of surrogate light chain and recombination activation genes. In this paper, we identify a new member of bovine pre-B lymphocyte genes, VPREB2. Using RT-qPCR, we assess the expression of VPREB2 and three other surrogate light chain genes as well as RAG1 and RAG2 in fetal and adult cattle tissues. The absence of VPREB1, IGLL1, RAG1 and RAG2 expression in adult tissues and the lack of B-lymphoid differentiation in adult bone marrow - OP9 stromal cell co-culture, suggest a decline of B lymphopoiesis in adult cattle. The marked differences in the expression profiles of VPREB2 and VPREB3 in comparison to those of VPREB1, IGLL1 and RAGs suggest that the biological roles of VPREB2 and VPREB3 are unrelated to the pre-B cells.

Pseudomonas stutzeri, isolated from crude oil-contaminated soil, was used to degrade diesel oil. Of three surfactants, 120 mg rhamnolipids 1(-1) significantly increased degradation of diesel oil giving 88% loss after 14 days compared to 54% loss without the surfactant. The system with rhamnolipids was characterised by relatively high particle homogeneity. However, the addition of saponins to diesel oil caused the cells to aggregate (the polydispersity index: 0.542) and the biodegradation of diesel oil was only 46%. The cell yield was 0.22 g l(-1).

It is unclear whether people with and without diabetes equally benefitted from reductions in cardiovascular disease (CVD). We aimed to compare recent trends in hospital admission rates for angina, acute myocardial infarction (AMI), stroke, percutaneous coronary intervention (PCI), and coronary artery bypass graft (CABG) among people with and without diabetes in England.

We previously reported that miR-1 is among the most consistently down-regulated miRs in primary human prostate tumors. In this follow-up study, we further corroborated this finding in an independent data set and made the novel observation that miR-1 expression is further reduced in distant metastasis and is a candidate predictor of disease recurrence. Moreover, we performed in vitro experiments to explore the tumor suppressor function of miR-1. Cell-based assays showed that miR-1 is epigenetically silenced in human prostate cancer. Overexpression of miR-1 in these cells led to growth inhibition and down-regulation of genes in pathways regulating cell cycle progression, mitosis, DNA replication/repair and actin dynamics. This observation was further corroborated with protein expression analysis and 3'-UTR-based reporter assays, indicating that genes in these pathways are either direct or indirect targets of miR-1. A gene set enrichment analysis revealed that the miR-1-mediated tumor suppressor effects are globally similar to those of histone deacetylase inhibitors. Lastly, we obtained preliminary evidence that miR-1 alters the cellular organization of F-actin and inhibits tumor cell invasion and filipodia formation. In conclusion, our findings indicate that miR-1 acts as a tumor suppressor in prostate cancer by influencing multiple cancer-related processes and by inhibiting cell proliferation and motility.

The Yin Yang 1 (YY1) transcription factor is a master regulator of development, essential for early embryogenesis and adult tissues formation. YY1 is the mammalian orthologue of Pleiohomeotic, one of the transcription factors that binds Polycomb DNA response elements in Drosophila melanogaster and mediates Polycomb group proteins (PcG) recruitment to DNA. Despite several publications pointing at YY1 having a similar role in mammalians, others showed features of YY1 that are not compatible with PcG functions. Here, we show that, in mouse Embryonic Stem (ES) cells, YY1 has genome-wide PcG-independent activities while it is still stably associated with the INO80 chromatin-remodeling complex, as well as with novel RNA helicase activities. YY1 binds chromatin in close proximity of the transcription start site of highly expressed genes. Loss of YY1 functions preferentially led to a down-regulation of target genes expression, as well as to an up-regulation of several small non-coding RNAs, suggesting a role for YY1 in regulating small RNA biogenesis. Finally, we found that YY1 is a novel player of Myc-related transcription factors and that its coordinated binding at promoters potentiates gene expression, proposing YY1 as an active component of the Myc transcription network that links ES to cancer cells.

Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF). In contrast to natural ceramides, PyrCer promotes phosphorylation of splicing factors. Exons that are regulated by PyrCer have in common suboptimal splice sites, are unusually short and share two 4-nt motifs, GAAR and CAAG. They are dependent on PSF/SFPQ, whose phosphorylation is regulated by PyrCer. Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity.

Peptide recognition domains and transcription factors play crucial roles in cellular signaling. They bind linear stretches of amino acids or nucleotides, respectively, with high specificity. Experimental techniques that assess the binding specificity of these domains, such as microarrays or phage display, can retrieve thousands of distinct ligands, providing detailed insight into binding specificity. In particular, the advent of next-generation sequencing has recently increased the throughput of such methods by several orders of magnitude. These advances have helped reveal the presence of distinct binding specificity classes that co-exist within a set of ligands interacting with the same target. Here, we introduce a software system called MUSI that can rapidly analyze very large data sets of binding sequences to determine the relevant binding specificity patterns. Our pipeline provides two major advances. First, it can detect previously unrecognized multiple specificity patterns in any data set. Second, it offers integrated processing of very large data sets from next-generation sequencing machines. The results are visualized as multiple sequence logos describing the different binding preferences of the protein under investigation. We demonstrate the performance of MUSI by analyzing recent phage display data for human SH3 domains as well as microarray data for mouse transcription factors.