Spinocerebellar ataxia 3 (SCA3) is the most common autosomal dominant ataxia. The disease is caused by an expansion of a CAG-trinucelotide repeat region within the coding sequence of the ATXN3 gene, and this results in an expanded polyglutamine (polyQ) tract within the Ataxin-3 protein. The polyQ expansion leads to neuronal dysfunction and cell death. Here, we tested the ability of a number of proteins that interact with Ataxin-3 to modulate SCA3 pathogenicity using Drosophila. Of 10 candidates, we found four novel enhancers and one suppressor. The suppressor, PICK1 (Protein interacting with C kinase 1), is a transport protein that regulates the trafficking of ion channel subunits involved in calcium homeostasis to and from the plasma membrane. In line with calcium homeostasis being a potential pathway mis-regulated in SCA3, we also found that down-regulation of Nach, an acid sensing ion channel, mitigates SCA3 pathogenesis in flies. Modulation of PICK1 could be targeted in other neurodegenerative diseases, as the toxicity of SCA1 and tau was also suppressed when PICK1 was down-regulated. These findings indicate that interaction proteins may define a rich source of modifier pathways to target in disease situations.

Infection of cells with African swine fever virus (ASFV) can lead to the formation of zipper-like stacks of structural proteins attached to collapsed endoplasmic reticulum (ER) cisternae. We show that the collapse of ER cisternae observed during ASFV infection is dependent on the viral envelope protein, J13Lp. Expression of J13Lp alone in cells is sufficient to induce collapsed ER cisternae. Collapse was dependent on a cysteine residue in the N-terminal domain of J13Lp exposed to the ER lumen. Luminal collapse was also dependent on the expression of J13Lp within stacks of ER where antiparallel interactions between the cytoplasmic domains of J13Lp orientated N-terminal domains across ER cisternae. Cisternal collapse was then driven by disulphide bonds between N-terminal domains arranged in antiparallel arrays across the ER lumen. This provides a novel mechanism for biogenesis of modified stacks of ER present in cells infected with ASFV, and may also be relevant to cellular processes.

Chondroitin sulfate proteoglycans (CSPGs) present an inhibitory barrier to axonal growth and plasticity after trauma to the central nervous system. These extracellular and membrane bound molecules are altered after spinal cord injuries, but the magnitude, time course, and patterns of expression following contusion injury have not been fully described. Western blots and immunohistochemistry were combined to assess the expression of four classically inhibitory CSPGs, aggrecan, neurocan, brevican and NG2, at the lesion site and in distal segments of cervical and thoracic spinal cord at 3, 7, 14 and 28 days following a severe mid-thoracic spinal contusion. Total neurocan and the full-length (250 kDa) isoform were strongly upregulated both at the lesion epicenter and in cervical and lumbar segments. In contrast, aggrecan and brevican were sharply reduced at the injury site and were unchanged in distal segments. Total NG2 protein was unchanged across the injury site, while NG2+ profiles were distributed throughout the lesion site by 14 days post-injury (dpi). Far from the lesion, NG2 expression was increased at lumbar, but not cervical spinal cord levels. To determine if the robust increase in neurocan at the distal spinal cord levels corresponded to regions of increased astrogliosis, neurocan and GFAP immunoreactivity were measured in gray and white matter regions of the spinal enlargements. GFAP antibodies revealed a transient increase in reactive astrocyte staining in cervical and lumbar cord, peaking at 14 dpi. In contrast, neurocan immunoreactivity was specifically elevated in the cervical dorsal columns and in the lumbar ventral horn and remained high through 28 dpi. The long lasting increase of neurocan in gray matter regions at distal levels of the spinal cord may contribute to the restriction of plasticity in the chronic phase after SCI. Thus, therapies targeted at altering this CSPG both at and far from the lesion site may represent a reasonable addition to combined strategies to improve recovery after SCI.

Efficient and prolonged human cystic fibrosis transmembrane conductance regulator (hCFTR) expression is a major goal for cystic fibrosis (CF) lung therapy. A hCFTR expression plasmid was optimized as a payload for compacted DNA nanoparticles formulated with polyethylene glycol (PEG)-substituted 30-mer lysine peptides. A codon-optimized and CpG-reduced hCFTR synthetic gene (CO-CFTR) was placed in a polyubiquitin C expression plasmid. Compared to hCFTR complementary DNA (cDNA), CO-CFTR produced a ninefold increased level of hCFTR protein in transfected HEK293 cells and, when compacted as DNA nanoparticles, produced a similar improvement in lung mRNA expression in Balb/c and fatty acid binding protein promoter (FABP) CF mice, although expression duration was transient. Various vector modifications were tested to extend duration of CO-CFTR expression. A novel prolonged expression (PE) element derived from the bovine growth hormone (BGH) gene 3' flanking sequence produced prolonged expression of CO-CFTR mRNA at biologically relevant levels. A time course study in the mouse lung revealed that CO-CFTR mRNA did not change significantly, with CO-CFTR/mCFTR geometric mean ratios of 94% on day 2, 71% on day 14, 53% on day 30, and 14% on day 59. Prolonged CO-CFTR expression is dependent on the orientation of the PE element and its transcription, is not specific to the UbC promoter, and is less dependent on other vector backbone elements.

Protein interacting specifically with Tc10, PIST, is a Golgi-associated sorting protein involved in regulating cell-surface targeting of plasma membrane receptors. The present study provides the first comprehensive description of PIST distribution in the mammalian central nervous system and of its subcellular localization by immunocytochemistry. PIST is distributed widely throughout the neuraxis, predominantly associated with neuronal cell bodies and dendrites. In hippocampal neurons, in vitro and in situ, PIST displayed a patchy subcellular distribution in an area surrounding the nucleus and extending into one of the major dendrites. By colocalization with the trans-Golgi marker TGN38, we were able to show that PIST is associated largely but not exclusively with the trans-Golgi network in central neurons. High or moderate to high levels of PIST-like immunoreactivity were found in cortical areas, in particular in layer V of the neocortex. The motor cortex was most strongly labeled. Also, the piriform and insular cortices displayed strong PIST labeling. In the hippocampus, CA2 but not CA1 or CA3 pyramidal cells displayed strong PIST-labeling, extending into their apical dendrites. In the thalamus, ventrolateral and laterodorsal nuclei were most strongly stained, whereas in the hypothalamus the supraoptic nucleus stood out with strong immunoreactivity. Strikingly, in the brainstem all cranial nerve motor nuclei were PIST-positive at varying levels, which is in keeping with the prominent expression of PIST in forebrain motor areas. This selective distribution of PIST suggests that the protein serves distinctive roles in specific neuronal populations, establishing functionally distinct zones, for instance, in the hippocampus.

The brain seeks to combine related inputs from different senses (e.g., hearing and vision), via multisensory integration. Temporal information can indicate whether stimuli in different senses are related or not. A recent human fMRI study (Noesselt et al. [2007]: J Neurosci 27:11431-11441) used auditory and visual trains of beeps and flashes with erratic timing, manipulating whether auditory and visual trains were synchronous or unrelated in temporal pattern. A region of superior temporal sulcus (STS) showed higher BOLD signal for the synchronous condition. But this could not be related to performance, and it remained unclear if the erratic, unpredictable nature of the stimulus trains was important. Here we compared synchronous audiovisual trains to asynchronous trains, while using a behavioral task requiring detection of higher-intensity target events in either modality. We further varied whether the stimulus trains had predictable temporal pattern or not. Synchrony (versus lag) between auditory and visual trains enhanced behavioral sensitivity (d') to intensity targets in either modality, regardless of predictable versus unpredictable patterning. The analogous contrast in fMRI revealed BOLD increases in several brain areas, including the left STS region reported by Noesselt et al. [2007: J Neurosci 27:11431-11441]. The synchrony effect on BOLD here correlated with the subject-by-subject impact on performance. Predictability of temporal pattern did not affect target detection performance or STS activity, but did lead to an interaction with audiovisual synchrony for BOLD in inferior parietal cortex.

Homologous recombination (HR) is a major mechanism utilized to repair blockage of DNA replication forks. Here, we report that a sister chromatid exchange (SCE) generated by crossover-associated HR efficiently occurs in response to replication fork stalling before any measurable DNA double-strand breaks (DSBs). Interestingly, SCE produced by replication fork collapse following DNA DSBs creation is specifically suppressed by ATR, a central regulator of the replication checkpoint. BRCA1 depletion leads to decreased RPA2 phosphorylation (RPA2-P) following replication fork stalling but has no obvious effect on RPA2-P following replication fork collapse. Importantly, we found that BRCA1 promotes RAD51 recruitment and SCE induced by replication fork stalling independent of ATR. In contrast, BRCA1 depletion leads to a more profound defect in RAD51 recruitment and SCE induced by replication fork collapse when ATR is depleted. We concluded that BRCA1 plays a dual role in two distinct HR-mediated repair upon replication fork stalling and collapse. Our data established a molecular basis for the observation that defective BRCA1 leads to a high sensitivity to agents that cause replication blocks without being associated with DSBs, and also implicate a novel mechanism by which loss of cell cycle checkpoints promotes BRCA1-associated tumorigenesis via enhancing HR defect resulting from BRCA1 deficiency.

It is generally accepted that functionally important RNA structure is more conserved than sequence due to compensatory mutations that may alter the sequence without disrupting the structure. For small RNA molecules sequence-structure relationships are relatively well understood. However, structural bioinformatics of mRNAs is still in its infancy due to a virtual absence of experimental data. This report presents the first quantitative assessment of sequence-structure divergence in the coding regions of mRNA molecules based on recently published transcriptome-wide experimental determination of their base paring patterns. Structural resemblance in paralogous mRNA pairs quickly drops as sequence identity decreases from 100% to 85-90%. Structures of mRNAs sharing sequence identity below roughly 85% are essentially uncorrelated. This outcome is in dramatic contrast to small functional non-coding RNAs where sequence and structure divergence are correlated at very low levels of sequence similarity. The fact that very similar mRNA sequences can have vastly different secondary structures may imply that the particular global shape of base paired elements in coding regions does not play a major role in modulating gene expression and translation efficiency. Apparently, the need to maintain stable three-dimensional structures of encoded proteins places a much higher evolutionary pressure on mRNA sequences than on their RNA structures.

Bacterial nucleoid associated proteins play a variety of roles in genome maintenance and dynamics. Their involvement in genome packaging, DNA replication and transcription are well documented but it is still unclear whether they play any specific roles in genome repair. We discovered that untwisting of the DNA double helix by bacterial non-specific DNA binding proteins stimulates the activity of a repair endonuclease of the Nth/MutY family involved in abasic site removal during base excision repair. The essential Bacillus subtilis primosomal gene dnaD, coding for a protein with DNA-untwisting activity, is in the same operon with nth and the promoter activity of this operon is transiently stimulated by H(2)O(2). Consequently, dnaD mRNA levels persist high upon treatment with H(2)O(2) compared to the reduced mRNA levels of the other essential primosomal genes dnaB and dnaI, suggesting that DnaD may play an important role in DNA repair in addition to its essential role in replication initiation. Homologous Nth repair endonucleases are found in nearly all organisms, including humans. Our data have wider implications for DNA repair as they suggest that genome associated proteins that alter the superhelicity of the DNA indirectly facilitate base excision repair mediated by repair endonucleases of the Nth/MutY family.

The neuropeptide galanin and its three receptor subtypes (GalR1-3) are expressed in the central amygdala (CeA), a brain region involved in stress- and anxiety-related behaviors, as well as alcohol dependence. Galanin also has been suggested to play a role in alcohol intake and alcohol dependence. We examined the effects of galanin in CeA slices from wild-type and knockout (KO) mice deficient of GalR2 and both GalR1 and GalR2 receptors. Galanin had dual effects on gamma-aminobutyric acid (GABA)-ergic transmission, decreasing the amplitudes of pharmacologically isolated GABAergic inhibitory postsynaptic potentials (IPSPs) in over half of CeA neurons but augmenting IPSPs in the others. The increase in IPSP size was absent after superfusion of the GalR3 antagonist SNAP 37889, whereas the IPSP depression was absent in CeA neurons of GalR1 × GalR2 double KO and GalR2 KO mice. Paired-pulse facilitation studies showed weak or infrequent effects of galanin on GABA release. Thus, galanin may act postsynaptically through GalR3 to augment GABAergic transmission in some CeA neurons, whereas GalR2 receptors likely are involved in the depression of IPSPs. Co-superfusion of ethanol, which augments IPSPs presynaptically, together with galanin caused summated effects of ethanol and galanin in those CeA neurons showing galanin-augmented IPSPs, suggesting the two agents act via different mechanisms in this population. However, in neurons showing IPSP-diminishing galanin effects, galanin blunted the ethanol effects, suggesting a preemptive effect of galanin. These findings may increase understanding of the complex cellular mechanisms that underlie the anxiety-related behavioral effects of galanin and ethanol in CeA.

Influenza surveillance is important to identify circulating, emerging/reemerging strains and unusual epidemiological trends. With these objectives, a multisite human influenza surveillance network was initiated in India in 2004.

The T lineage glycoprotein CD6 is generally considered to be a costimulator of T-cell activation. Here, we demonstrate that CD6 significantly reduces early and late T-cell responses upon superantigen stimulation or TCR triggering by Abs. Measuring calcium mobilization in single cells responding to superantigen, we found that human T cells expressing rat CD6 react significantly less well compared with T cells not expressing the exogenous receptor. When the cytoplasmic domain of rat CD6 was removed, calcium responses were recovered, indicating that the inhibitory properties of CD6 are attributable to its cytoplasmic domain. Calcium responses, and also late indicators of T-cell activation such as IL-2 release, were also diminished in TCR-activated Jurkat cells expressing human CD6, compared with CD6-deficient cells or cells expressing a cytoplasmic deletion mutant of human CD6. Similarly, calcium signals triggered by anti-CD3 were enhanced in human T lymphocytes following morpholino-mediated suppression of CD6 expression. Finally, the proliferation of T lymphocytes was increased when the CD6-CD166 interaction was blocked with anti-CD166 Abs, but inhibited when anti-CD6 Abs were used. Our data suggest that CD6 is a signaling attenuator whose expression alone, i.e. in the absence of ligand engagement, is sufficient to restrain signaling in T cells.

Study of activity of cloned promoters in slow-growing Mycobacterium tuberculosis during long-term growth conditions in vitro or inside macrophages, requires a genome-integration proficient promoter probe vector, which can be stably maintained even without antibiotics, carrying a substrate-independent, easily scorable and highly sensitive reporter gene. In order to meet this requirement, we constructed pAKMN2, which contains mycobacterial codon-optimized gfp(m) (2+) gene, coding for GFP(m) (2+) of highest fluorescence reported till date, mycobacteriophage L5 attP-int sequence for genome integration, and a multiple cloning site. pAKMN2 showed stable integration and expression of GFP(m) (2+) from M. tuberculosis and M. smegmatis genome. Expression of GFP(m) (2+), driven by the cloned minimal promoters of M. tuberculosis cell division gene, ftsZ (MtftsZ), could be detected in the M. tuberculosis/pAKMN2-promoter integrants, growing at exponential phase in defined medium in vitro and inside macrophages. Stable expression from genome-integrated format even without antibiotic, and high sensitivity of detection by flow cytometry and fluorescence imaging, in spite of single copy integration, make pAKMN2 useful for the study of cloned promoters of any mycobacterial species under long-term in vitro growth or stress conditions, or inside macrophages.

We report on a 64 year-old woman presenting with Epilepsia Partialis Continua (EPC) affecting the left hand since the age of 24 without neurological deficit. Structural MRI showed a region of focal cortical dysplasia (FCD) over the right central gyrus and lesions in the mesial frontal and occipital cortex secondary to perinatal hypoxic injury. Ictal spike haemodynamic mapping using simultaneous EEG-fMRI revealed significant BOLD signal changes prominent in the region of FCD (larger cluster), occipital cortex (global statistical maximum), prefrontal cortex and cerebellum. The cluster over FCD was in good agreement with the result of EEG source analysis. Our findings provide an interesting illustration of the ability of EEG-fMRI to reveal epileptogenic networks confirming the intrinsic epileptogenic properties of dysplastic neurons.

Recorded electric potentials and magnetic fields due to cortical electrical activity have spatial spread even if their underlying brain sources are focal. Consequently, as a result of source cancellation, loss in signal amplitude and reduction in the effective signal-to-noise ratio can be expected when distributed sources are active simultaneously. Here we investigate the cancellation effects of EEG and MEG through the use of an anatomically correct forward model based on structural MRI acquired from 7 healthy adults. A boundary element model (BEM) with four compartments (brain, cerebrospinal fluid, skull and scalp) and highly accurate cortical meshes (~300,000 vertices) were generated. Distributed source activations were simulated using contiguous patches of active dipoles. To investigate cancellation effects in both EEG and MEG, quantitative indices were defined (source enhancement, cortical orientation disparity) and computed for varying values of the patch radius as well as for automatically parcellated gyri and sulci. Results were calculated for each cortical location, averaged over all subjects using a probabilistic atlas, and quantitatively compared between MEG and EEG. As expected, MEG sensors were found to be maximally sensitive to signals due to sources tangential to the scalp, and minimally sensitive to radial sources. Compared to EEG, however, MEG was found to be much more sensitive to signals generated antero-medially, notably in the anterior cingulate gyrus. Given that sources of activation cancel each other according to the orientation disparity of the cortex, this study provides useful methods and results for quantifying the effect of source orientation disparity upon source cancellation.

Expression of c-Myb is required for normal hematopoiesis and for proliferation of myeloid leukemia blasts and a subset of T-cell leukemia, but its role in B-cell leukemogenesis is unknown. We tested the role of c-Myb in p190(BCR/ABL)-dependent B-cell leukemia in mice transplanted with p190(BCR/ABL)-transduced marrow cells with a c-Myb allele (Myb(f/d)) and in double transgenic p190(BCR/ABL)/Myb(w/d) mice. In both models, loss of a c-Myb allele caused a less aggressive B-cell leukemia. In p190(BCR/ABL)-expressing human B-cell leukemia lines, knockdown of c-Myb expression suppressed proliferation and colony formation. Compared with c-Myb(w/f) cells, expression of Bmi1, a regulator of stem cell proliferation and maintenance, was decreased in pre-B cells from Myb(w/d) p190(BCR/ABL) transgenic mice. Ectopic expression of a mutant c-Myb or Bmi1 enhanced the proliferation and colony formation of Myb(w/d) p190(BCR/ABL) B-cells; by contrast, Bmi1 downregulation inhibited colony formation of p190(BCR/ABL)-expressing murine B cells and human B-cell leukemia lines. Moreover, c-Myb interacted with a segment of the human Bmi1 promoter and enhanced its activity. In blasts from 19 Ph(1) adult acute lymphoblastic leukemia patients, levels of c-Myb and Bmi1 showed a positive correlation. Together, these findings support the existence of a c-Myb-Bmi1 transcription-regulatory pathway required for p190(BCR/ABL) leukemogenesis.

Hyperhomocysteinemia, a risk factor for cardiovascular disorder, obesity, and type 2 diabetes, is prevalent among Indians who are at high risk of these metabolic disorders. We evaluated association of common variants of genes involved in homocysteine metabolism or its levels with type 2 diabetes, obesity, and related traits in North Indians. We genotyped 90 variants in initial phase (2.115 subjects) and replicated top signals in an independent sample set (2.085 subjects). The variant MTHFR-rs1801133 was the top signal for association with type 2 diabetes (OR = 0.78 (95%  CI = 0.67-0.92), P = 0.003) and was also associated with 2 h postload plasma glucose (P = 0.04), high-density lipoprotein cholesterol (P = 0.004), and total cholesterol (P = 0.01) in control subjects. These associations were neither replicated nor significant after meta-analysis. Studies involving a larger study population and different ethnic groups are required before ruling out the role of these important candidate genes in type 2 diabetes, obesity, and related traits.

This study aimed to investigate phenotype of RP105(-) B cell subsets in patients with systemic lupus erythematosus (SLE). Flow cytometry was used for phenotyping RP105-negaive B cell subsets. Based on CD19, RP105, and CD138 expression, RP105(-) B cells consist of at least 5 subsets of late B cells, including CD19(+)RP105(int), CD19(+) RP105(-), CD19(low) RP105(-) CD138(-), CD19(low) RP105(-)CD138(int), and CD19(low) RP105(-) CD138(++) B cells. Especially, CD19(+)RP105(int) and CD19(low) RP105(-)CD138(int) B cells are significantly larger than other RP105(-) B cell subsets in SLE. By comparison of RP105(-) B cell subsets between patients with SLE and normal subjects, these subsets were detectable even in normal subjects, but the percentages of RP105(-) B cell subsets were significantly larger in SLE. The phenotypic analysis of RP105(-) B cell subsets suggests dysregulation of later B cell subsets in SLE and may provide new insights into understanding regulation of B cells in human SLE.

Colonization of the airways by filamentous fungi can occur in asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis. A recent study found IgE sensitization to Aspergillus fumigatus to be associated with reduced lung function. Significantly higher rates of A. fumigatus were detected in sputum from asthmatics sensitized to this fungus compared to non-sensitized asthmatics. The rate of positive cultures was far higher than equivalent historical samples analysed by the local clinical laboratory following protocols recommended by the UK Health Protection Agency (HPA). This study compares the HPA procedure with our sputum processing method, whereby sputum plugs are separated from saliva and aliquots of approximately 150 mg are inoculated directly onto potato dextrose agar. A total of 55 sputum samples from 41 patients with COPD were analyzed, comparing fungal recovery of five dilutions of sputa on two media. Isolation of A. fumigatus in culture was significantly higher using the research approach compared to the HPA standard method for mycological investigations (P < 0.001). There was also a significant difference in the recovery rate of A. fumigatus (P < 0.05) between media. This highlights the need for a standardized approach to fungal detection which is more sensitive than the method recommended by the HPA.

Schizophrenia is a complex and devastating mental disorder of unknown etiology. Hypofunction of N-methyl-D-aspartate (NMDA) receptors are implicated in the disorder, since phencyclidine (PCP) and other NMDA receptor antagonists mimic schizophrenia-like symptoms in humans and animals so well. Moreover, genetic linkage and post mortem studies strongly suggest a role for altered neuregulin 1 (Nrg1)/erbB4 signaling in schizophrenia pathology. This study investigated the relationship between the NMDA receptor and Nrg1 signaling pathways using the perinatal PCP animal model. Rats (n=5/group) were treated with PCP (10 mg/kg) or saline on postnatal days (PN) 7, 9 and 11 and were sacrificed on PN12, 5 weeks and 20 weeks for biochemical analyses. Western blotting was used to determine total and phosphorylated levels of proteins involved in NMDA receptor/Nrg1 signaling in the prefrontal cortex and hippocampus. In the cortex, PCP treatment altered Nrg1/erbB4 expression levels throughout development, including decreased Nrg1 and erbB4 at PN12 (-25-30%; p<0.05); increased erbB4 and p-erbB4 (+18-27%; p<0.01) at 5 weeks; and decreased erbB4 and p-erbB4 (-16-18%; p<0.05) along with increased Nrg1 (+33%; p<0.01) at 20 weeks. In the hippocampus, levels of Nrg1/erbB4 were largely unaffected apart from a significant decrease in p-erbB4 at 20 weeks (-13%; p<0.001); however NMDA receptor subunits and PSD-95 showed increases at PN12 and 5 weeks (+20-32%; p<0.05), and decreases at 20 weeks (-22-29%; p<0.05). This study shows that NMDA receptor antagonism early in development can have long term effects on Nrg1/erbB4 expression which could be important in understanding pathological processes which might be involved in schizophrenia.