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.

While grammatical aspects of language are preserved, executive deficits are prominent in Lewy body spectrum disorder (LBSD), including Parkinson's disease (PD), Parkinson's dementia (PDD) and dementia with Lewy bodies (DLB). We examined executive control during sentence processing in LBSD by assessing temporary structural ambiguities. Using an on-line word detection procedure, patients heard sentences with a syntactic structure that has high-compatibility or low-compatibility with the main verb's statistically preferred syntactic structure, and half of the sentences were lengthened strategically between the onset of the ambiguity and its resolution. We found selectively slowed processing of lengthened ambiguous sentences in the PDD/DLB subgroup. This correlated with impairments on measures of executive control. Regression analyses related the working memory deficit during ambiguous sentence processing to significant cortical thinning in frontal and parietal regions. These findings emphasize the role of prefrontal disease in the executive limitations that interfere with processing ambiguous sentences in LBSD.

Cellular responses following low-dose irradiation have been widely debated. Several studies have revealed detrimental effects of low-dose irradiation; however, some studies have shown contrasting results. Moreover, the effects of periapical irradiation on osteoblastic cells have not yet been revealed. Therefore, in this study, we tested the hypothesis that low-dose dental irradiation of osteoblastic cells reduces reactive oxygen species (ROS) production and leads to increased cellular proliferation and high-dose dental irradiation of osteoblastic cells increases ROS production and leads to cellular apoptosis.

Expression of viral proteins causes important epigenetic changes leading to abnormal cell growth. Whether viral proteins directly target histone methyltransferases (HMTs), a key family enzyme for epigenetic regulation, and modulate their enzymatic activities remains elusive. Here we show that the E6 proteins of both low-risk and high-risk human papillomavirus (HPV) interact with three coactivator HMTs, CARM1, PRMT1 and SET7, and downregulate their enzymatic activities in vitro and in HPV-transformed HeLa cells. Furthermore, these three HMTs are required for E6 to attenuate p53 transactivation function. Mechanistically, E6 hampers CARM1- and PRMT1-catalyzed histone methylation at p53-responsive promoters, and suppresses the binding of p53 to chromatinized DNA independently of E6-mediated p53 degradation. p53 pre-methylated at lysine-372 (p53K372 mono-methylation) by SET7 protects p53 from E6-induced degradation. Consistently, E6 downregulates p53K372 mono-methylation and thus reduces p53 protein stability. As a result of the E6-mediated inhibition of HMT activity, expression of p53 downstream genes is suppressed. Together, our results not only reveal a clever approach for the virus to interfere with p53 function, but also demonstrate the modulation of HMT activity as a novel mechanism of epigenetic regulation by a viral oncoprotein.

Electro- and magnetoencephalography (EEG/MEG) are the means to investigate the dynamics of neuronal activity non-invasively in the human brain. However, both EEG and MEG are also sensitive to non-neural sources, which can severely complicate the interpretation. The saccadic spike potential (SP) at saccade onset has been identified as a particularly problematic artifact in EEG because it closely resembles synchronous neuronal gamma band activity. While the SP and its confounding effects on EEG have been thoroughly characterized, the corresponding artifact in MEG, the saccadic spike field (SF), has not been investigated. Here we provide a detailed characterization of the SF. We simultaneously recorded MEG, EEG, gaze position and electrooculogram (EOG). We compared the SF in MEG for different saccade sizes and directions and contrasted it with the well-known SP in EEG. Our results reveal a saccade amplitude and direction dependent, lateralized saccadic spike artifact, which was most prominent in the gamma frequency range. The SF was strongest at frontal and temporal sensors but unlike the SP in EEG did not contaminate parietal sensors. Furthermore, we observed that the source configurations of the SF were comparable for regular and miniature saccades. Using distributed source analysis we identified the sources of the SF in the extraocular muscles. In summary, our results show that the SF in MEG closely resembles neuronal activity in frontal and temporal sensors. Our detailed characterization of the SF constitutes a solid basis for assessing possible saccadic spike related contamination in MEG experiments.

We previously identified FOXF1 as a potential tumor suppressor gene with an essential role in preventing DNA rereplication to maintain genomic stability, which is frequently inactivated in breast cancer through the epigenetic mechanism. Here we further addressed the role of the p53-p21(WAF1) checkpoint pathway in DNA rereplication induced by silencing of FOXF1. Knockdown of FOXF1 by small interference RNA (siRNA) rendered colorectal p53-null and p21(WAF1)-null HCT116 cancer cells more susceptible to rereplication and apoptosis than the wild-type parental cells. In parental HCT116 cells with a functional p53 checkpoint, the p53-p21(WAF1) checkpoint pathway was activated upon FOXF1 knockdown, which was concurrent with suppression of the CDK2-Rb cascade and induction of G(1) arrest. In contrast, these events were not observed in FOXF1-depleted HCT116-p53-/- and HCT116-p21-/- cells, indicating that the p53-dependent checkpoint function is vital for inhibiting CDK2 to induce G(1) arrest and protect cells from rereplication. The pharmacologic inhibitor (caffeine) of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) protein kinases abolished activation of the p53-p21(WAF1) pathway upon FOXF1 knockdown, suggesting that suppression of FOXF1 function triggered the ATM/ATR-mediated DNA damage response. Cosilencing of p53 by siRNA synergistically enhanced the effect of FOXF1 depletion on the stimulation of DNA rereplication and apoptosis in wild-type HCT116. Finally, we show that FOXF1 expression is predominantly silenced in breast and colorectal cancer cell lines with inactive p53. Our study demonstrated that the p53-p21(WAF1) checkpoint pathway is an intrinsically protective mechanism to prevent DNA rereplication induced by silencing of FOXF1.

Over the past 50 years, the ovary development has been subject of fewer studies as compare to the male pathway. Nevertheless due to the advancement of genetics, mouse ES cells and the development of genetic models, studies of ovarian differentiation was boosted. This review emphasizes some of new progresses in the research field of the mammalian ovary differentiation that have occurred in recent years with focuses of the period around prophase I of meiosis and of recent roles of small non-RNAs in the ovarian gene expression.

The production of recombinant transmembrane proteins is due to their biochemical properties often troublesome and time consuming. Here the prokaryotic expression and purification of the transmembrane envelope proteins of the feline and primate foamy viruses using a screening assay for optimisation of expression in 96 deep well plates is described. Testing simultaneously various bacterial strains, media, temperatures, inducer concentrations and different transformants, conditions for an about twentyfold increased production were quickly determined. These small scale test conditions could be easily scaled up, allowing purification of milligram amounts of recombinant protein. Proteins with a purity of about 95% were produced using a new purification protocol, they were characterised by gel filtration and circular dichroism and successfully applied in immunological assays screening for foamy virus infection and in immunisation studies. Compared to the previously described protocol (M. Mühle, A. Bleiholder, S. Kolb, J. Hübner, M. Löchelt, J. Denner, Immunological properties of the transmembrane envelope protein of the feline foamy virus and its use for serological screening, Virology 412 (2011) 333-340), proteins with similar characteristics but about thirtyfold increased yields were obtained. The screening and production method presented here can also be applied for the production of transmembrane envelope proteins of other retroviruses, including HIV-1.

The Poisson process is an often employed model for the activity of neuronal populations. It is known, though, that superpositions of realistic, non- Poisson spike trains are not in general Poisson processes, not even for large numbers of superimposed processes. Here we construct superimposed spike trains from intracellular in vivo recordings from rat neocortex neurons and compare their statistics to specific point process models. The constructed superimposed spike trains reveal strong deviations from the Poisson model. We find that superpositions of model spike trains that take the effective refractoriness of the neurons into account yield a much better description. A minimal model of this kind is the Poisson process with dead-time (PPD). For this process, and for superpositions thereof, we obtain analytical expressions for some second-order statistical quantities-like the count variability, inter-spike interval (ISI) variability and ISI correlations-and demonstrate the match with the in vivo data. We conclude that effective refractoriness is the key property that shapes the statistical properties of the superposition spike trains. We present new, efficient algorithms to generate superpositions of PPDs and of gamma processes that can be used to provide more realistic background input in simulations of networks of spiking neurons. Using these generators, we show in simulations that neurons which receive superimposed spike trains as input are highly sensitive for the statistical effects induced by neuronal refractoriness.

Toll-like receptor (TLR) signaling is linked to autophagy that facilitates elimination of intracellular pathogens. However, it is largely unknown whether autophagy controls TLR signaling. Here, we report that poly(I:C) stimulation induces selective autophagic degradation of the TLR adaptor molecule TRIF and the signaling molecule TRAF6, which is revealed by gene silencing of the ubiquitin-editing enzyme A20. This type of autophagy induced formation of autophagosomes and could be suppressed by an autophagy inhibitor and lysosomal inhibitors. However, this autophagy was not associated with canonical autophagic processes, including involvement of Beclin-1 and conversion of LC3-I to LC3-II. Through screening of TRIF-interacting 'autophagy receptors' in human cells, we identified that NDP52 mediated the selective autophagic degradation of TRIF and TRAF6 but not TRAF3. NDP52 was polyubiquitinated by TRAF6 and was involved in aggregation of TRAF6, which may result in the selective degradation. Intriguingly, only under the condition of A20 silencing, NDP52 could effectively suppress poly(I:C)-induced proinflammatory gene expression. Thus, this study clarifies a selective autophagic mechanism mediated by NDP52 that works downstream of TRIF-TRAF6. Furthermore, although A20 is known as a signaling fine-tuner to prevent excess TLR signaling, it paradoxically downregulates the fine-tuning effect of NDP52 on TLR signaling.

TFIIH is a multisubunit factor essential for transcription initiation and promoter escape of RNA polymerase II and for the opening of damaged DNA double strands in nucleotide excision repair (NER). In this study, we have analyzed at which step of the transcription cycle TFIIH is essential for transcription by RNA polymerase I. We demonstrate that TFIIH associates with the rDNA promoter and gene-internal sequences and leaves the rDNA promoter in a complex with RNA polymerase I after start of transcription. Moreover, mutations in the TFIIH subunits XPB and XPD found in Cockayne syndrome impair the interaction of TFIIH with the rDNA, but do not influence initiation complex formation or promoter escape of RNA polymerase I, but preclude the productivity of the enzyme by reducing transcription elongation in vivo and in vitro. Our results implicate that reduced RNA polymerase I transcription elongation and ribosomal stress could be one factor contributing to the Cockayne syndrome phenotype.

Propolis is a resinous product collected by honey bees. It was also reported that propolis has a wide variety of biological actions, including antimicrobial activity and antioxidant, anti-inflammatory, and suppressive effects of dioxin toxicity activities. The aim of this study was to compare the in vitro cytotoxic activities of green propolis (G12) and red propolis (G13) in human leukemia cells. These cells were incubated with different concentrations of propolis and 48 hours after the IC(50) was calculated for each cell. The results showed that the red propolis has cytotoxic effect in vitro higher than green propolis. Red propolis was showed to be cytostatic in K562 cells and caused the same amount of apoptosis as its control Gleevec. In conclusion, these results showed that red propolis is more cytotoxic than the green propolis in a variety of human cell lines of leukemia. Red propolis may contain drugs capable of inhibiting cancer cell growth. Therefore, further isolation of respective chemical ingredients from the red propolis (G13) for identification of the activities is necessary.

In Drosophila multiple circadian oscillators and behavioral rhythms are known to exist, yet most previous studies that attempted to understand circadian entrainment have focused on the activity/rest rhythm and to some extent the adult emergence rhythm. Egg laying behavior of Drosophila females also follows circadian rhythmicity and has been seen to deviate substantially from the better characterized rhythms in a few aspects. Here we report the findings of our study aimed at evaluating how circadian egg laying rhythm in fruit flies Drosophila melanogaster entrains to time cues provided by light and temperature. Previous studies have shown that activity/rest rhythm of flies entrains readily to light/dark (LD) and temperature cycles (TC). Our present study revealed that egg laying rhythm of a greater percentage of females entrains to TC compared to LD cycles. Therefore, in the specific context of our study this result can be taken to suggest that egg laying clocks of D. melanogaster entrains to TC more readily than LD cycles. However, when TC were presented along with out-of-phase LD cycles, the rhythm displayed two peaks, one occurring close to lights-off and the other near the onset of low temperature phase, indicating that upon entrainment by TC, LD cycles may be able to exert a greater influence on the phase of the rhythm. These results suggest that temperature and light associatively entrain circadian egg laying clocks of Drosophila.