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TFIID plays a key role in transcription initiation of RNA polymerase II preinitiation complex assembly. TFIID is comprised of the TATA box binding protein (TBP) and 14 TBP-associated factors (TAFs). A second set of transcriptional regulatory multiprotein complexes containing TAFs has been described (called SAGA, TFTC, STAGA, and PCAF/GCN5). Using matrix-assisted laser desorption ionization mass spectrometry, we identified a novel TFTC subunit, human TAF9Like, encoded by a TAF9 paralogue gene. We show that TAF9Like is a subunit of TFIID, and thus, it will be called TAF9b. TFIID and TFTC complexes in which both TAF9 and TAF9b are present exist. In vitro and in vivo experiments indicate that the interactions between TAF9b and TAF6 or TAF9 and TAF6 histone fold pairs are similar. We observed a differential induction of TAF9 and TAF9b during apoptosis that, together with their different ability to stabilize p53, points to distinct requirements for the two proteins in gene regulation. Small interfering RNA (siRNA) knockdown of TAF9 and TAF9b revealed that both genes are essential for cell viability. Gene expression analysis of cells treated with either TAF9 or TAF9b siRNAs indicates that the two proteins regulate different sets of genes with only a small overlap. Taken together, these data demonstrate that TAF9 and TAF9b share some of their functions, but more importantly, they have distinct roles in the transcriptional regulatory process.
Transcription initiation of protein-encoding genes by RNA polymerase II (Pol II) requires the transcription factor TFIID that is comprised of the TATA binding protein (TBP) and series of TBP-associated factors (TAFs) (1, 6, 57). In human HeLa cells, we showed that different human TFIID complexes containing or lacking TAF10 which exhibit functionally distinct properties exist (6, 7, 25). Cell type-specific TFIID complexes have been found to be composed of core TAFs and cell type-specific TAFs. TAF4b was found to be enriched in differentiated human B lymphocytes, and a unique TAF4b-containing TFIID was isolated from these cells (13). Moreover, during spermatogenesis, TAF7L-containing TFIID complexes have been found (48). Another set of human transcriptional regulatory multiprotein complexes containing TAFs are called TFTC, STAGA, or PCAF/GCN5 (6a, 36, 63). These complexes are functional homologues of the Saccharomyces cerevisiae SAGA complex, and all contain human homologues of the yeast histone acetyltransferase Gcn5 as well as a subset of SPT and ADA proteins, the 400-kDa TRRAP protein, and a number of TAFs (shared TAFs) also found in TFIID (36).
TAF9 was first identified as a TFIID subunit from multiple organisms: human (formerly called hTAFII31 or hTAFII32 [29, 35]), Drosophila melanogaster (formerly dTAFII40 [55]), and yeast (formerly yTaf17p [42]). Later, TAF9 was also identified as a component of different TBP-free TAF complexes containing the GCN5-type histone acetyltransferase, such as the yeast SAGA complex (19), the Drosophila TFTC complex (44), and human TFTC-type complexes (10, 36, 67). Drosophila TAF9 has also been described in the Polycomb group complex and the e(y)2 protein-containing complex (17, 50).
TAF9 was shown to interact directly with the tumor suppressor protein p53, the herpes simplex virus activator VP16, and the basal transcription factor TFIIB, and these interactions were suggested to be important for mediating transcriptional activation (29, 35, 55). TAF9 was shown to bind to the N-terminal region of p53, a region that is also required for interaction with the oncoprotein Mdm2. It has been shown that overexpression of TAF9 inhibits Mdm2-mediated ubiquitination of p53 and increases p53 levels and that TAF9-mediated p53 stabilization results in activation of p53-mediated transcriptional activity and leads to p53-dependent growth arrest in fibroblasts (9, 24).
Crystal structures demonstrate that the amino-terminal portions of Drosophila TAF9 and TAF6 adopt a canonical histone fold (HF) configuration consisting of two short α-helices flanking a long central α-helix. In the crystal structure, the dTAF9/TAF6 HF complex exists as a heterotetramer, resembling the (H3/H4)2 heterotetrameric core of the histone octamer, suggesting that TFIID may contain a histone octamer-like substructure (65). Furthermore, it was suggested that this histone octamer-like structure in TFIID may play a role in DNA wrapping and in the stable positioning of promoter DNA relative to TAFs (46). Our experiments localizing most of the yeast TAFs and TBP in TFIID (33, 34) showed that the nine TAFs, which contain the HF structural motif (15), are located in three distinct lobes of the TFIID structure. The distribution of these TAFs indicates that the previously reported pairwise interactions between HF-containing TAFs occur in the native TFIID complex. The TAF9-TAF6 pair was located in lobes A and B (34). The fact that most of the HF-containing TAF pairs have been found in two distinct lobes suggested that the organization of TFIID is more complex than originally thought and that there may be several histone octamer-like structures present in TFIID (34).
In the past decade, the role of different TAFs has been extensively investigated in a few model organisms. Several independent studies revealed that 3% to 60% of the yeast genes require individual TAFs for transcription depending on the TAF subunit, and in total, approximately 84% of yeast Pol II genes are TAF dependent (20, 53). Studies in several metazoan organisms revealed a wide but rarely universal requirement for TAFs. In Caenorhabditis elegans, individual TAF (i.e., TAF4, TAF5, TAF9, and TAF10) knockdowns by RNA interference affect embryo development and gene expression to different degrees, with TAF4 being required for essentially all early embryonic mRNA transcription, while TAF5, TAF9, and TAF10 show a more restricted requirement for embryonic transcription (58, 59). In Drosophila, TAF1 loss-of-function mutants affect several differentiation processes (62), and dTAF4 and dTAF6 affect Dorsal-mediated transcription (68), whereas dTAF6 seems to be broadly required for somatic and germ cell development (3). Also, in vertebrates, Pol II transcription displays a broad but not complete TAF dependency. In mice, TAF10 is required for early embryo development but not for the survival of trophoblast cells or the retinoic acid-mediated differentiation of F9 cells (38, 40). TAF4b knockout mice are characterized by female infertility (14). Moreover, the different enzymatic activity-containing functional domains of TAF1 modulate expression of a distinct subset of mammalian genes, suggesting that they are important for regulating largely nonoverlapping sets of genes (45).
Certain TAFs have paralogue genes, which often encode TAF-like (L) proteins with cell type- or tissue-specific expression patterns. The first TAF paralogue seems to have appeared in fission yeast where two genes encode TAF5 homologues, and both are present in the same TFIID complex (39). In C. elegans, Drosophila, and the human genome, several TAF paralogue genes encoding TAF-like proteins have been described (4, 16, 22, 57). Interestingly, the Drosophila homologues of TAF4 (Can), TAF5 (Nht), TAF6 (Mia), TAF8 (Sa), and TAF12 (Rye) are expressed exclusively in the testis and seem to regulate the testis-specific gene expression program in primary spermatocytes (21, 22). Moreover, Drosophila TAF10 homologues (TAF10 and TAF10b) are also differentially expressed during Drosophila embryogenesis (16). Also, a retrotranscribed and integrated copy of human TAF1 (TAF1L) and a homologue of TAF7 (TAF7L) are specifically expressed during male germ cell differentiation (48, 60). A human TAF9 paralogue gene was also identified (termed TAF9L), and it was shown that TAF9L can fully restore the function of chicken TAF9 in DT40 TAF9−/− cells, suggesting redundant function for these two proteins. Furthermore, human TAF9L was suggested to play a role in transcriptional repression and/or silencing (11). However, the association of TAF9L with TBP was not demonstrated.
Genome-wide expression analysis in Saccharomyces cerevisiae indicated that depletion of TAF9 causes a decrease in transcription of about 60 to 65% of the genes (41, 53). Thus, Saccharomyces cerevisiae TAF9 (scTAF9) seems to be broadly, but not universally, required for transcription in yeast. However, the fact that either targeted depletion of TAFs or different temperature-sensitive alleles of a single TAF can either result in broad effects on Pol II transcription (30, 49) or affect the transcription of only a subset of genes (20) has generated a yet-unresolved controversy concerning the true role of TAFs in regulation of transcription. Interestingly, depletion of chicken TAF9 DT40 cells resulted in no or only minor changes in total poly(A)+ mRNA transcription, suggesting that chicken TAF9 is not essential for general mRNA transcription in chicken DT40 cells (12).
Our recent identification of an alternatively spliced TAF6 transcript, which is induced upon different apoptotic stimuli and encodes for TAF6δ that is part of a TFIID-like complex lacking TAF9, showed that the TAF6δ interplay is an important component of a novel signaling pathway that acts as an interface between apoptotic signals and the Pol II transcriptional machinery (5, 18). The notion that TFIID function is important in the regulation of apoptosis was also supported by several previous reports which suggested that various TFIID subunits, including TAF9, may play a role in the regulation of apoptosis (2, 12, 38, 51, 61, 66).
In this study, we have identified TAF9L/DN7 as a bona fide subunit of both TFIID and TFTC, hereafter called TAF9b, and investigated the structural and functional similarities between human TAF9 and TAF9b. We show that TAF9 and TAF9b have specific as well as redundant roles in the regulation of Pol II transcription.
Plasmid constructions and cell transfections.The eukaryotic expression plasmids for TAF9, p53, and TAF6δ have been previously described (5); TAF9b cDNA has been provided by the IMAGE Consortium (accession number BC009566) and PCR amplified between nucleotides 23 and 778 with oligonucleotides containing EcoRI at 5′ and NotI at 3′ restriction sites and then cloned into the pXJ41 vector for mammalian expression and pTRIEX (Novagen) for bacterial and insect cell expression. Yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) TAF9b fusions were generated by subcloning TAF9b cDNA from pXJ41 using BamHI and EcoRI in BglII and EcoRI sites of pEYFP-C1 and pECFP-C1 (Clontech), respectively. Subcloning the NheI excised and blunted TAF6 and TAF9 cDNA fragments from pRSET vector (31) into the blunted BamHI in EcoRI sites of pEYFP-C1 and pECFP-C1 vectors, respectively, generated YFP- and CFP-TAF9 as well as YFP- and CFP-TAF6 fusions.
A total of 1.5 × 105 HeLa cells were transfected by using JetPEI (PolyTransfection, France) in 35-mm plates and harvested at the indicated time points after transfection (see below).
Immunization and antibody production.To generate the anti-TAF9 and TAF9b monoclonal antibodies (MAbs), peptides corresponding to amino acids 132 to 146 of hTAF9 [LQKKASTSAGRITV(C)] and amino acids 132 to 144 of hTAF9b [LIKKGPNQGRLVP(C)] (Fig. 1B, boxed area) were synthesized, coupled to ovalbumin as a carrier, and used for immunization of mice. Immunization and monoclonal antibody production were done essentially as described previously (7).
Immunoprecipitation and Western blot analysis.Routinely, proteins from 200 μg of nuclear extract (NE) were immunoprecipitated (IP) with 50 μl protein G-Sepharose (Pharmacia) and approximately 5 to 8 μg of the different antibodies (as indicated in the figures). Antibody-protein G-Sepharose-bound protein complexes were washed three times with IP buffer (25 mM Tris-HCl, pH 7.9, 10% [vol/vol] glycerol, 0.1% NP-40, 0.5 mM dithiothreitol, 5 mM MgCl2) containing 0.5 M KCl and twice with IP buffer containing 100 mM KCl. After washing, either proteins were eluted by an excess of the corresponding epitope peptide, or protein-G-antibody-bound proteins were directly boiled in sodium dodecyl sulfate (SDS) sample buffer and separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto nitrocellulose membrane, and probed with the indicated primary antibodies. Chemiluminescence detection was performed according to the manufacturer's instructions (Amersham). All the other antibodies used were previously described (5, 63). Monoclonal anti-β-actin antibody was purchased from Sigma (A5441).
Apoptosis assays.Apoptosis induction was obtained and measured as described previously (5) or by measuring sub-G1 DNA content with a Becton Dickinson FACScalibur.
Mass spectrometry.TFTC subunits were separated on a 10% SDS-PAGE gel. Protein bands were visualized by Coomassie G250 (Bio-Rad) staining, excised, and in-gel digested with trypsin (10). Peptide extracts (0.5 μl) were mixed with an equal volume of saturated α-cyano-4-hydroxycinnamic acid (LaserBio Labs) dissolved in 50% acetonitrile and applied to the target. Mass measurements were carried out on a Bruker Reflex IV matrix-assisted laser desorption-time of flight (MALDI-TOF) spectrometer in the positive-ion reflector mode. The acquisition mass range was 800 to 3,000 Da with the low-mass gate set at 700 Da. Internal calibration was performed using autolytic trypsin peptides (MH+ with m/z = 842.51, 2211.11, and 2807.47). Monoisotopic peptide masses were assigned manually using the Bruker X-TOF software. Database searches were performed using the Profound program (http://prowl.rockefeller.edu/) with the following parameters: NCBI database, proteins of human origin, molecular mass between 20 and 100 kDa, trypsin digestion with one missed cleavage allowed, cysteines modified by carbamidomethylation, methionine oxidation, and mass tolerance of 75 ppm.
FRET measurements.We used a Leica TCS SP2 (AOBS) confocal microscope operating with a 40-mW argon laser. The laser was tuned to lines of 458 nm and 514 nm. Cells were examined with a 63× 1.40-numerical aperture oil immersion objective and a ×2.5 zoom. Fluorescence resonance energy transfer (FRET) was measured using the acceptor photobleaching method (26). According to this procedure, if two proteins interact and FRET occurs, then photobleaching of the acceptor (YFP) should yield a significant increase in fluorescence of the donor (CFP). We bleached cells in the YFP channel by zooming in and scanning the cell of interest six times using the 514-nm argon laser line at 100% intensity. Before and after the bleaching, CFP and YFP images were collected to assess changes in donor fluorescence that corresponds specifically to the cell with the bleached acceptor. Images were collected at 16% of the 458-nm laser line intensity and 5% of the intensity of the 514-nm laser line. The gain of photon multipliers and the emission intervals were adjusted to eliminate cross talk between the CFP (470 to 500 nm) and YFP (530 to 600 nm) channels. First, we subtracted the background from both CFP and YFP signals measured. To calculate FRET efficiency, we used the EF = (Ipost − Ipre) × 100/Ipost, formula, where EF is the FRET efficiency and the Ipre and Ipost are the mean intensities measured using the Leica software in the CFP channel before and after photobleaching, respectively. As a control, we always performed similar calculations for the nonbleached cells of the image.
Reverse transcriptase PCR (RT-PCR).Total RNA was isolated with RNAsolv (Biofidal), precipitated, and quantified. One microgram was reverse transcribed with M-MLV reverse transcriptase (Sigma), and the TAF9- and TAF9b-specific PCRs were performed with the following oligonucleotides: at the 5′ end, GCC ACC TGA TAG ATA CTG CTT AAC, common to TAF9 and TAF9b; at the 3′ end for TAF9, GCT AGA TTA CAG ATT ATC ATA GTC; and at the 3′ end for TAF9b, GAG ATC TAA CAG TTT TAA CTG ACG. Other oligonucleotide sequences used in Fig. 8 are available upon request.
RNA interference.Double-stranded siRNAs were produced with a Silencer siRNA kit (Ambion) according to the manufacturer's instructions. Target sequences were obtained with the Dharmacon siDesign engine using the less-stringent parameters. A total of 1.5 × 105 HeLa cells were transfected by using Lipofectamine 2000 (Invitrogen) in 35-mm plates. Sequences of the specific siRNAs are available upon request.
Microarray analysis.A 25,000-gene human oligonucleotide microarray covering most of the known human transcripts has been used for transcriptome analysis. This oligonucleotide collection is a public resource (Réseau National des Génopoles, France, and MRC, United Kingdom). Information about these oligonucleotides is available at http://medcal.ipmc.cnrs.fr:8080/mediante. Oligonucleotides (synthesized by Proligo) were diluted to 50 μM in 50% dimethyl sulfoxide and 100 mM phosphate buffer and spotted using a μGridII arrayer (BioRobotics) onto amino silane-coated slides (UltraGAPS TM; Corning).
Total RNA from HeLa cells treated with siRNA targeting TAF9, TAF9b, or luciferase (Luc) was extracted (Trizol, Invitrogen). Total RNA (220 ng) was amplified by linear PCR, and the amplification products were labeled by Cy3 and Cy5 and purified using Nucleospin Extract II columns (Macherey Nagel). Labeled cDNAs were hybridized in a Discovery station using ChipHybe 80 hybridization buffer at 42°C for 8 h without any final stringency washes (Ventana Medical System hybridization automate, reagents, and microarray hybridization procedure). All the protocols used are available at http://www-microarrays.u-strasbg.fr/Chips/index.html. After slides scanning (ScanArray4000; Perkin-Elmer), the obtained TIF images were quantified using Imagene 5 (BioDiscovery), and the raw data were normalized using the Lowess method. For each different RNA sample, a flip-flop experiment was performed using the RNA obtained from luciferase RNA interference-treated HeLa cells as a reference. The normalized ratio of RNA sample to reference RNA (log2) was calculated, and this ratio was directly used for the comparison of different samples.
Identification of human TAF9b (formerly TAF9L) as a bona fide subunit of TFIID and TFTC.To further characterize TFTC subunits, the complex (63) was analyzed by MALDI mass spectrometry (10). As expected, a ∼35-kDa protein species was identified as TAF9 (previously called hTAFII31 or hTAFII32 [29, 35]) with a 41% coverage. However, MALDI peptide mass fingerprinting analysis showed that out of 16 peptides, 4 were TAF9 specific (Fig. 1A) and 9 were common with a human factor that has previously been named TAF9Like (TAF9L; GenBank accession number NP_057059) or neuronal cell death-related protein (DN7). TAF9 and TAF9L proteins are 82% identical (Fig. 1B) and encoded by two different human paralogue genes. Interestingly, accurate analysis of the TAF9 MALDI peptide mass fingerprinting also revealed the presence of three TAF9L-specific peptides (Fig. 1A). These results indicated that TAF9L comigrates with TAF9 and that TAF9L is a component of TFTC.
To confirm the presence of TAF9L in TFTC, we raised anti-peptide mouse MAbs against TAF9L (see Materials and Methods and Fig. 1B). In parallel, we also raised specific MAbs against hTAF9. When these antibodies were tested on recombinant TAF9 and TAF9L proteins, they showed high specificity and no cross-reaction (Fig. 1C). A further test on a HeLa NE prepared in the presence of phosphatase inhibitors showed that anti-TAF9 and anti-TAF9L antibodies recognized protein species migrating around 32 kDa, with a slightly different molecular weight (Fig. 1D). Moreover, the anti-TAF9L antibody recognized a single band, while the anti-TAF9 antibody recognized two bands (Fig. 1D). When phosphatase inhibitors were omitted during the NE preparation, the slower-migrating TAF9 band disappeared (data not shown), indicating a possible in vivo phosphorylation of TAF9.
We then verified the presence of TAF9L in highly purified human TFTC and tested whether it is present in TFIID complexes (63) by Western blot analysis using the anti-TAF9L-specific MAb. In good agreement with the MALDI spectrometry data, TAF9L was present in TFTC, and in addition, we demonstrate that it is also present in highly purified TFIID preparations (Fig. 2A). Thus, according to the novel TAF nomenclature (57), the TAF9L protein will hereafter be called TAF9b. Interestingly, both TAF9b and TAF9 were detected in either TFIID or TFTC preparations.
To prove unequivocally that TAF9b is a subunit of both TFTC and TFIID, the anti-TAF9b MAb has been used to immunoprecipitate proteins which associate with TAF9b in different multiprotein complexes (Fig. 2B). The analysis of the proteins which coimmunoprecipitated with TAF9b revealed that TFTC-specific subunits (i.e., GCN5 and TRRAP) and TFIID-specific subunits (TBP and TAF1) as well as common subunits (TAF5, TAF6, TAF9, and TAF10) associate with TAF9b. These results together show that TAF9b is a stable subunit of both TFTC and TFIID. The fact that TAF9b copurifies with TBP in an anti-TBP immunoprecipitation (Fig. 2A) and TBP coprecipitates with TAF9b (Fig. 2B) indicates that TAF9b is a bona fide TAF. Furthermore, the immunoprecipitation using an anti-TAF9b antibody also copurified TAF9 (Fig. 2B), even in the presence of ethidium bromide (data not shown), strongly suggesting that in HeLa cells, there are TAF-containing complexes (TFIID and/or TFTC) in which TAF9 and TAF9b are simultaneously present.
TAF9 and TAF9b are both coexpressed in multiple cell lines.Next, we investigated whether TAF9 and TAF9b are similarly expressed at either the RNA or protein level in different human cell lines. To determine the relative ratio of TAF9 and TAF9b mRNA, RT-PCRs were carried out by using a common 5′ and two different specific 3′ oligonucleotides which amplify either a 550-bp fragment on TAF9b cDNA or a 445-bp band on TAF9 cDNA. RT-PCR analysis revealed that both TAF9b and TAF9 were expressed at the RNA level in the different cell lines tested; however, expression levels and the ratio between the two mRNAs are variable from cell line to cell line (Fig. 3A). These findings were in good agreement with data from the Symatlas databank concerning TAF9 and TAF9b mRNA expression (http://symatlas.gnf.org/SymAtlas).
We then performed Western blot analysis on whole-cell extracts from certain cell lines tested by RT-PCR (Fig. 3B). In all of the tested protein extracts, both TAF9b and TAF9 were detected, and their ratio appeared to be relatively constant, with two exceptions. We found that in the two Ewing's sarcoma cell lines (RD-ES and COHIN), TAF9 is expressed at much lower levels than TAF9b (Fig. 3B, lanes 1 and 2). No clear correlation has been found between mRNA and protein levels detected, indicating that their expression is regulated at multiple steps (Fig. 3A and B). Surprisingly, the protein expression levels of different TAFs and TBP seem also to be highly variable between the different cell lines tested (Fig. 3B).
In vitro and in living cells, TAF9b interacts with TAF6 similarly to TAF9.TAF9 has been reported to interact in vitro with TAF6 (23) and TAF5 (54). To verify whether TAF9b interacts with these and other TAF subunits in a way similar to TAF9, TAF9b was coexpressed with a series of TAFs, including TAF5 and TAF6, in insect Sf9 cells using the baculovirus system (Fig. 4A). In parallel, TAF9 was also coexpressed with the same TAFs as a control. Using either TAF9- or TAF9b-specific antibodies to coimmunoprecipitate the interacting proteins, we show that TAF9b interacts with its putative histone fold dimerization partner, TAF6, and with the WD-40 repeat containing TAF5 similarly as TAF9 (Fig. 4A and B). Moreover, TFIID subunits which do not interact in vitro with TAF9 do not interact with TAF9b either (Fig. 4C and data not shown).
Next, we aimed to verify whether these in vitro TAF9b/TAF6 and TAF9/TAF6 interactions also exist in living cells. To this end, we used FRET and the acceptor photobleaching method. HeLa cells were transfected with constructs expressing the YFP-TAF6/CFP-TAF9, CFP-TAF6/YFP-TAF9, YFP-TAF6/CFP-TAF9b, and CFP-TAF6/YFP-TAF9b pairs, and mean FRET efficiencies were measured in 60 cells from three different experiments for each combination. Both the TAF6/TAF9 and TAF6/TAF9b interactions resulted in similar FRET efficiencies, at around 11% (Fig. 5B). These FRET efficiencies were the same when the two fluorophores were exchanged on the different pairs (Fig. 5B, compare C6 plus Y9b to C9b plus Y6 and C6 plus Y9 to C9 plusY6), indicating that the nature of the fluorescent protein tags did not influence the interactions. When measuring FRET with the control CFP/YFP pair, where the two fluorescent proteins are not supposed to interact, we obtained only 0.4% efficiency (Fig. 5B). In a further control experiment, when cells were transfected with the CFP-TAF6 construct alone, we obtained negative numbers that are the effect of photobleaching of the CFP fluorescence due to imaging (C6 in Fig. 5), pointing to the fact that the FRET efficiencies of the TAF6-TAF9 and TAF6-TAF9b pairs may have been underestimated. In contrast to the TAF6-TAF9 and TAF6-TAF9b pairs, the combinations among YFP-TAF6/CFP-TAF6,YFP-TAF6/CFP-TAF6/TAF9/TAF9b, CFP-TAF9b/YFP-TAF9b, CFP-TAF9/YFP-TAF9, and CFP-TAF9/YFP-TAF9b did not result in significant FRET efficiency (around 1 to 2%), suggesting that, at least with this method, no (TAF6-TAF9)2, (TAF6-TAF9b)2, or TAF62-TAF9-TAF9b heterotetramerstructures similar to that of histones H3-H4 can be observed. Note, however, that the absence of a FRET signal between two proteins does not necessarily mean that the proteins do not interact, because the orientation of the CFP or YFP tags on the TAFs may not be optimal for FRET detection in the case of the heterotetramer. In the latter experiments, when more than two proteins have been overexpressed, the third and fourth partners were expressed as nonfluorescent proteins (Fig. 5). These data together indicate that TAF6-TAF9 and TAF6-TAF9b pairs form in vivo.
TAF9 and TAF9b are differentially regulated during apoptosis induction.Since elevated levels of TAF9b RNA have been measured in apoptotic PC12 rat cells (2), we tested whether TAF9 or TAF9b protein levels are also induced by apoptotic stimuli in human HeLa cells. To induce apoptosis, VP16-etoposide or camptothecin was used, and induction was confirmed by testing p53 and TAF6δ levels by Western blot analysis (5) (Fig. 6A). In parallel, we have monitored whether we can detect an increase in TAF9 or TAF9b protein levels in these apoptotic cells (Fig. 6A). After both apoptotic stimuli, TAF9 protein expression levels increased about 6- to 8-fold, while TAF9b levels increased only about 1.5-fold (Fig. 6A and B), suggesting that their function is differentially required during apoptosis.
TAF9b overexpression has a weaker effect on p53 stabilization than TAF9.To further investigate functional differences between TAF9 and TAF9b, we used a previously described assay system where it was shown that overexpressed TAF9 is able to protect p53 from degradation due to the fact that it competes with MDM2 for p53 binding (9, 24). As shown in Fig. 6C, TAF9b overexpression has a weaker effect on p53 stabilization than TAF9 in MCF7 and HeLa cells (data not shown). These results further suggest that despite the 82% identity between these two proteins, they have different functions.
TAF9 and TAF9b are essential for cell viability.Previously, it was shown that TAF9b (formerly TAF9L) is essential for cell growth in human cells (11) and that TAF9 depletion leads to apoptotic cell death in chicken cells (12). To further characterize the functional differences between human TAF9 and TAF9b during normal cell growth and in the same system, we specifically knocked down either TAF9 or TAF9b expression by using siRNAs. When TAF9 or TAF9b siRNAs were transfected in HeLa cells, we obtained a specific reduction of the targeted mRNAs and the consequent protein levels without affecting the expression of the other homologue (Fig. 7A and B). Moreover, the almost complete elimination of one or the other factor resulted in a significant induction (about threefold) of apoptosis over control siRNA transfection (Fig. 7B). When the number of cells was scored in samples which had been treated with either control, TAF9, TAF9b, or TAF9 plus TAF9b siRNAs, we observed that 24 h after transfection, the TAF9b siRNA reduced the growing cell population by 54% (Fig. 7C). This reduction was almost as efficient as the TAF9 and TAF9b siRNAs together (60%), while the TAF9 siRNA had only a mild effect (25%) on cell survival (Fig. 7C). However, at the 72-h time point, both TAF9 and TAF9b siRNAs induced apoptosis in a similar way compared to the control (58%). Interestingly, at this time point, the TAF9 and TAF9b siRNAs together had a stronger effect (70%) on cell death than the individual siRNAs alone (Fig. 7C). These results suggest that both genes are essential for cell viability, and the results obtained with the double TAF9 and TAF9b siRNA treatment suggest that their roles are only partially redundant.
TAF9 and TAF9b have only minimal overlapping functions.To further investigate gene regulation by TAF9 and TAF9b, we performed gene expression profiling in siRNA knockdown cells. HeLa cells were treated, in triplicate, with control Luc siRNA, TAF9 siRNA, or TAF9b siRNA. The treatment was carried out for 30 h to be able to analyze the expression of genes, which are primarily affected by the knockdowns, and to obtain good quality RNA that is not affected by the induced cell death (Fig. 7). Total RNA was extracted, retrotranscribed, labeled, and used to probe an oligonucleotide-based microarray representing 25,000 genes (see Materials and Methods). In order to reduce false positives due to oscillations of weak signals, we qualified only those genes whose average signal intensities of Cy3 and Cy5 as log2 were over 10 in the luciferase samples as "present." After this passage, we were able to qualify 5,974 genes as "present." We then compared TAF9 siRNA- and TAF9b siRNA-treated samples to Luc siRNA-treated samples in order to obtain up- and down-regulated genes and to eliminate genes that were not specifically affected by the siRNA treatment. Out of 5,974 "present" genes, a total of 1,081 were affected (18%). Out of this total, 222 genes (3.7%) were affected by the TAF9 knockdown; 689 genes (11.6%) were affected by the TAF9b knockdown, and 160 genes (2.7%) were common between the TAF9 and TAF9b knockdowns. Out of all 387 TAF9 knockdown-affected genes, 177 (2.9%) were up-regulated and 210 (3.6%) were down-regulated; out of the 854 TAF9b knockdown-affected genes, 150 (2.5%) were up-regulated, whereas 704 (11.8%) were down-regulated. Affected genes do not fall into any specific category or pathway, suggesting that TAF9 and TAF9b are not restricted to any specific gene expression program and that they rather function in a more general manner. The two most interesting groups of the genes are those whose expression is only either TAF9 or TAF9b dependent. We chose some of these genes to perform quantitative RT-PCR (qRT-PCR) (Fig. 8B) to validate the microarray results. The qRT-PCR data confirmed the microarray data (Fig. 8B), further suggesting that only TAF9- or TAF9b-regulated genes exist and that the two proteins regulate only partially overlapping transcription programs. In agreement with several previous studies (11, 20, 27, 28, 32, 53), our results also demonstrate that TAF depletion (either TAF9 or TAF9b) causes both up- and down-regulation of gene expression.
TAFs have been identified in biochemically stable complexes with TBP, whereas TAF-like (TAFL) proteins are those which are present in multiprotein complexes without TBP or which were not yet shown to be associated in stable complexes with TBP (57). In our study, we demonstrate that the previously described TAF9L factor (11) is a bona fide member of the TAF family since it is associates with TBP in TFIID complexes. Thus, according to the existing nomenclature, it is called TAF9b. In contrast to most of the other known TAF homologues, which usually replace their counterparts during defined spatiotemporal developmental events (14, 21, 48, 60), TAF9b is ubiquitously expressed, together with TAF9, in all cell types tested. We show here that in the same cell type, TAF-containing complexes in which TAF9 and TAF9b are simultaneously present exist. In the light of this finding and despite the high degree of identity between the two proteins, it is surprising that their functions are only partially overlapping (see below).
How are TAF9 and TAF9b positioned in the TFIID/TFTC structure?Immunoprecipitations using anti-TAF9b antibodies demonstrated that at least some of the TAF9b-containing complexes also contain TAF9. These data suggest that in certain TAF-containing complexes, both proteins can be present and that their presence is not mutually exclusive in TFIID and TFTC complexes. We have shown previously that in Saccharomyces cerevisiae TFIID, of which the genome contains only one TAF9 gene, TAF9 can be present in two copies, one in lobe A and one in lobe B (34). Moreover, in the SAGA complex, which is the yeast counterpart of the human TFTC-type complexes, TAF9 seems to be present in two copies as well, one in domain II and another one in domain IV (56, 64). These findings open the interesting possibility that in mammals, where two TAF9 paralogue genes exist in the different genomes, TAF9 would be present in lobe A (or domain II) and TAF9b would be present in lobe B (or domain IV) of TFIID (of TFTC), or vice versa. However, it is also conceivable that in mammalian cells, different TFIID and TFTC complexes may exist that contain (i) only TAF9, (ii) only TAF9b, (iii) TAF9 in lobe A (or domain II) and TAF9b in lobe B (or domain IV in TFTC), and (iv) TAF9b in lobe A (or domain II) and TAF9 in lobe B (domain IV in TFTC). Despite our efforts to identify the precise location of TAF9 and TAF9b in human TFIID using electron microscopy, we could not distinguish among the above-mentioned four possibilities. Concerning the overall structure of the different TAF-containing complexes, our FRET experiments seem to exclude the formation of a heterotetramer between TAF6-TAF9 and/or TAF6-TAF9b in living cells, suggesting that in TFIID and TFTC complexes, TAF6-TAF9 and TAF6-TAF9b exist as heterodimers in the distinct lobes. Nevertheless, in the future, it will be important to understand how the incorporation of the TAF9/TAF6 and TAF9b/TAF6 heterodimers in the different domains of the distinct TAF complexes is regulated in spite of the high degree of identity between TAF9 and TAF9b (82%). As TAF9 was found to be phosphorylated (reference 37 and this study), it is possible that the incorporation of TAF9 (and TAF9b) in the different domains of the complexes is regulated by distinct posttranslational modifications. If TAF9 and TAF9b are present in different complexes or domains of the same complex, the above-described functional differences between TAF9 and TAF9b suggest that the complexes or domains containing one or the other protein will have distinct functions.
As in the in vitro and in vivo interaction tests, where TAF9 and TAF9b interacted with their partners (TAF6 and TAF5) in a comparable way, it is conceivable that TAF9/TAF6 and TAF9b/TAF6 heterodimers will have very similar interactions within the different lobes or domains of the TAF-containing complexes. However, they may have differential interactions outside of the respective complexes with general transcription factors, transcription activators, and/or promoter sequences. The Drosophila HF pair TAF9/TAF6 has been shown to bind to downstream promoter elements (8). More recently, the DNA-binding domain of human TAF9 has been assigned to an evolutionarily conserved region (between amino acids 70 and 147 of TAF9), and it was shown that the HF domain-mediated interaction between TAF9 and TAF6 enhanced the DNA binding activity of this heterodimer (52). Thus, in the future, it will be important to test whether TAF9b-containing complexes would have a different DNA binding specificity than that of TAF9-containing complexes.
TAF-containing complexes with only TAF9 or only TAF9b.The fact that the ratio between endogenous TAF9 and TAF9b is far from being 1:1 in Ewing's sarcoma cell lines suggests that at least in these cells, different TAF-containing complexes exist, and those which contain both TAF9 and TAF9b are less abundant than those which contain only TAF9b. In addition, TAFs and TBP do not show a uniform expression pattern in different cells but are expressed at very different levels in distinct cell types or tissues (references 40 and 47 and this study). Thus, our results are in good agreement with recent data suggesting that TFIID and TFTC are not unique complexes but rather a family of complexes that can be modulated depending the transcriptional requirements of the different cell types that compose an organism (5, 6, 43).
Eighty-two percent identical TAF homologues with only partially overlapping functions.Interestingly, two TAF9 paralogue genes were found only in mammalian genomes and not in fully sequenced invertebrate or other vertebrate genomes, i.e., Drosophila, zebra fish, Xenopus laevis, and chicken (our unpublished observations). At present, however, it is difficult to determine when the TAF9 gene duplication occurred and whether it is a mammal-specific event or it appeared at an earlier vertebrate evolutionary stage. The general rule for the retention of a duplicated gene copy is either divergence in expression pattern and/or differences in functions. As TAF9 and TAF9b are coexpressed in the different cell lines tested, it would rather suggest that the duplication of the TAF9 gene in mammals served a new function. TAF9 and TAF9b have been the subject of different evolutionary pressures, and this led to a series of mutations in TAF9b which resulted in partially novel functions. While the TAF9 functions seem to be conserved from yeast to humans, TAF9b might have evolved to serve functions that may tolerate more diversity, suggesting that TAF9b is not necessarily involved in exactly the same regulatory pathways as TAF9.
Nevertheless, the relatively high similarity between human TAF9 and TAF9b would suggest rather redundant functions. However, when the different results obtained with these two proteins are compared, and in agreement with a new function that was retained following gene duplication in mammals, the following important functional differences were found: (i) distinct apoptotic stimuli do not induce the expression of the two proteins to the same levels, although they induce similar levels of apoptosis, suggesting that they are differentially required during induction of apoptotic pathways (Fig. 6A); (ii) exogenous expression of the two proteins does not protect p53 in the same way from Mdm2-mediated degradation (Fig. 6B); (iii) knocking down either TAF9 or TAF9b led to cell death, but the double knockdown has a stronger effect than the individual knockdowns alone (Fig. 7); and (iv) the analysis of genes affected by the two different knockdowns revealed that only a rather limited subset of genes is regulated by both TAF9 and TAF9b (Fig. 8). All these differences together indicate that the two proteins do not fulfill identical roles in the cell but also serve different functions.
Knockdown of TAF9 and TAF9b revealed that they are both indispensable for human HeLa cell survival and that each plays an essential role (Fig. 7). Gene expression profiling revealed that the two proteins control the expression of different sets of genes and that, surprisingly, there is only a small subset of genes which are regulated by both TAF9 and TAF9b. Whether the different functions of TAF9 and TAF9b reflect their distinct (i) structural interactions within TFIID and/or TFTC complexes, (ii) interactions with transcription factors outside of the complexes, and/or (iii) DNA binding properties has to be further investigated. Unfortunately, our antibodies against TAF9 and TAF9b do not work in chromatin IP and thus do not allow us to verify whether the identified TAF9- or TAF9b-dependent genes are indeed bound by only one or both factors. The finding that there are genes regulated only by TAF9 or TAF9b suggests that in HeLa cells, TAF9 cannot structurally and/or functionally replace TAF9b and vice versa. Surprisingly, human TAF9b (formerly hTAF9L) could restore the viability of the chicken DT40 TAF9−/− cells (11), suggesting that in chicken cells, only one TAF9 gene exists and that at this stage of evolution, TAF9 and TAF9b functions were not yet separated. Our results also demonstrate that TAF depletion (either TAF9 or TAF9b) causes both up- and down-regulation of gene expression, further suggesting that TAFs can act as both positive and negative regulatory factors. This finding together with other TAF knockdown studies carried out mainly in yeast (11, 20, 27, 28, 32, 53) raise an interesting hypothesis that TAFs, when binding to promoters in the different TAF-containing complexes (TFIID and TFTC type), would be involved at certain promoters as repressors and at others as positive cofactors of transcription.
We are grateful to M. Oulad-Abdelghani for generating specific TAF9 and TAF9b antibodies, to E. Martinez and R. G. Roeder for the polyclonal antibody that recognizes both TAF9 and TAF9b, to I. Kolb-Cheynel for help with the recombinant baculoviruses, to P. Schultz and C. Ruhlman for the EM study, to A. Jànoshàzi for training to do the FRET experiments, and to T. Hilton, M. Demény, S. Brancorsini, and N. Clarke for critical reading of the manuscript. We also thank the IGBMC cell culture facility for providing cells.
M.F. is supported by a European Community Marie Curie individual fellowship (MEIF-CT-2003-501146), and E.S. is supported by an EMBO long-term fellowship. This work was supported by funds from INSERM, CNRS, Hôpital Universitaire de Strasbourg, Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale, the Font Nationale de La Science ACI, INTAS (01-0211), and European Community RTN (HPRN-CT-2000-00087, HPRN-CT-2000-00088, and HPRN-CT-2004-504228), STREP (LSHG-CT-2004-502950), and AICR (03-084) grants.
- Received 16 February 2005.
- Accepted 4 March 2005.
- American Society for Microbiology


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In situ synthesis of FeP-decorated Ti–Fe2O3: an effective strategy to improve the interfacial charge transfer in the photoelectrochemical water oxidation reaction†
Modifying inexpensive cocatalysts on semiconductors to accelerate the charge transfer at the photoanode/electrolyte interface, which possess good charge transfer in the bulk photoanode simultaneously, is priority among priorities in constructing low-cost and efficient PEC solar energy conversion devices. However, it is often difficult to satisfy outstanding charge transfer both in the bulk and at the photoanode/electrolyte interface when integrating cocatalysts with semiconductors. In our work, we synthesized FeP-decorated Ti–Fe2O3in situ through simple low-temperature phosphidation, which possessed good interfacial contact between Ti–Fe2O3 and FeP. The results demonstrated that the in situ deposition of FeP not only reduced the charge transfer resistance in the bulk photoanode by improving the electrical conductivity, but also accelerated the charge transfer at the photoanode/electrolyte interface. Based on the above advantages, the novel FeP/Ti–Fe2O3 exhibited splendid photocurrent density as high as 3.9 mA cm−2 at 1.23 V vs. RHE; meanwhile, the onset potential of FeP/Ti–Fe2O3 shifted cathodically to 0.88 V vs. RHE in comparison with that of Ti–Fe2O3 (0.98 V vs. RHE). This work paves a simple path to construct a cocatalyst-modified photoanode with excellent interfacial charge transfer and offers a low-cost and promising cocatalyst that can be applied in the photoelectrochemical water oxidation reaction.

subset: commoncrawl


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Salt Lake City,Ut,
Member Since:
Feb 11, 2010
Goal Type:
Running Accomplishments:
I had some success in high school and college. Winner 1985 Rod Dixon Run
Had a fair amount of success as a Masters runner for most of my 40s.
Short-Term Running Goals:
Have fun, not get fat, stay fit.
Long-Term Running Goals:
Keep running in some fashion.
I teach AP European History and other courses at Alta High School. I coached the track and cross country teams at Alta for 16 years.
Married, two kids - Abby and Andy
My Twitter @murphy_rob
Great workout today. Four x 1 mile in 5:30, 5:30, 5:27, 5:24 with 800 meters of easy jogging between each.


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THE HEADLINE: Juniata's starters and bench players broke out offensively in their 88-58 victory over Wilkes University on Wednesday, Nov. 30.
21 Katy Leamer
42 Kelly Leamer
FIRST HALF: In a fast-paced first half, the Eagles played strong defense and aggressive offense to gain and maintain the lead. Down 5-2 in the first two minutes of the game, Anstine (York, Pa./Dallastown) and Atkinson (Lancaster, Pa./Lancaster Catholic) contributed to take the lead; they did not trail again.
Atkinson led the team with 11 points in the first half. Kelly Leamer (Altoona, Pa./Bellwood-Antis) and Haley Myers (Huntingdon, Pa./Juniata Valley) followed with nine. Myers shot 1.000 on her field goal attempts and behind the three-point line.
With the full team effort, Juniata took a 48-28 lead into the second half.
SECOND HALF: Bench players, Myers and Bri Wasser (Ebensburg, Pa./Central Cambria) stood out, as they added to Juniata's growing score. By the end of the third quarter, Myers and Wasser contributed 14 points in their time off the bench. Freshman Veronica Cosmopolis (Lima, Peru/Colegio Carmelitas) made a long three-point jumper, putting the Eagles ahead 73-46 going into the final ten minutes.
The Eagles did not let their guard down in the fourth quarter and they were able to finished the game ahead 30 points, 88-58.
KEY PERFORMANCES: Wasser led the team in scoring and set a new career high with 16 total points. She was five for her nine field goal attempts and four-for-four on the foul line. She sank two threes, one in the first quarter and the other in the third.
Myers could not miss during her minutes in the first half, tallying nine points. On her first two attempts in the second half, she swished a jumper and a three.
Anstine showed her strength in the paint yet again with nine rebounds, five defensive and four offensive, including one put-back.
IN THE BOX: Juniata shot 50.8% (32-63) from the field, while Wilkes shot 35.3% (18-51). The Eagles earned 24 points off of turnovers and 20 second-chance points.
POST-GAME REMARKS: Head coach Danny Young-Uhrich was happy with the win tonight over Wilkes, noting that they accomplished a few of their goals they set for the contest. However, she knows there is still work to be done. "I know I'm not feeling a sense of complacency...I don't want the girls to be satisfied with anything," she said.
WHAT'S NEXT?: Juniata men and women are traveling to Scranton on Saturday, Dec. 3 to compete in a doubleheader. The women tip off against the reigning conference champions at 2 p.m.; the men follow at 4 p.m.


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QMV is privileged to be nominated in Super Review's inaugural Superannuation Consultancy of the Year alongside Deloitte and Rice Warner. The winner will be announced at the Super Fund of the Year Awards on 15 August 2019.Read More
Many data remediation programs are only started after an issue being brought to light by a customer or group of customers: often upon investigation, this gives rise to a slew of other issues that may have been impacting thousands of customers across several years.Read More
QMV has moved into new Sydney offices situated on Clarence Street. Our permanent base in Sydney is fast approaching twelve months and we sincerely thank QMV's valued clients and friends for their backing.Read More
The superannuation industry should prepare for an "open super" regime in the next few years, in light of current moves towards "open data" and "open banking" and the fundamental shift in the balance of power in the information economy, says Jonathan Steffanoni, principal consultant – legal and risk at QMV.Read More
A continued regulatory focus on member outcomes will dominate the superannuation industry in 2018 and contribute to the continued consolidation of funds, says Mr Jonathan Steffanoni, Principal Consultant, Legal & Risk, QMV.Read More
Data errors are costing the superannuation industry, and its customers, millions of dollars every year with poor data spreading like a disease through the system, says Mark Vaughan, managing director of QMV.Read More
Superannuation consulting and technology firm QMV has appointed Mark Vaughan as managing director, replacing Michael Quinn who has held the role since March 2014. QMV provides independent consulting services and technology systems to superannuation funds, trustees, administrators and wealth management organisations.Read More
I really enjoyed the opportunity to speak at ASFA's Spotlight on Member Engagement Forum held this week in Sydney. My task was to discuss how trustee approaches to member engagement would be influenced by the rise ofRead More


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Foreign Minister Gebran Bassil. (The Daily Star/ File)
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Free Patriotic Movement President Gebran Bassil Friday said that his party rejected permanent settlement of refugees in Lebanon, saying it would undermine Lebanon's independence.As a result of the heavy Syrian bombardment, Aoun, who was then a military commander, fled to the French Embassy before the Syrian army reached the palace.Aoun returned to Lebanon in May 2005, a few weeks after Syria withdrew its army from Lebanon under local and international pressures, ending nearly three decades of its domination of the country.


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gustavo0Q needed a new logo design and created a contest on 99designs.
A winner was selected from 73 designs submitted by 27 freelance designers.
We are a modern lawtech (startup in the legal industry) in Brazil that aims to change the way lawsuits reports are presented. We present the information of lawsuits with simple language and great visual appeal, so that anyone, even without legal knowledge, can understand the progress of the lawsuits.
Most of our clients are in-house attorneys from corporate legal departments, who often have to provide to the directors of the company informations about the lawsuits status. With our system they are able to easily show to the "non-legal people" of the company, in a gamified and visual manner, all the information needed.
The in-house attorney simply have to provide us with the numbers of their lawsuits and we do all the hard work to collect data with our bots and team, to present the information.
Some keywords that guide our product: simplicity, minimalism, visual, little information, predictability, flow, gamification, zoom, drill down.
To give you a better idea of how the system works, I am attaching some screenshots. Each screen is like a user click, which goes deeper into the information. The layout was inspired by the Apple Watch layout.
Find out how a design contest works or learn more about our logo design services.


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For aspiring business professionals, it is very necessary to earn a Master in Business Administration (MBA) degree, as it happens to be the most sought-after qualification of recent times. Several business schools are merging all over the world to offer MBA flexible courses, making it easier for individuals who work during the day to take classes and earn degrees. However, if you decide to opt for a business school MBA, it is essential to obtain the degree from a reputed institution, as it will boost your career development plan.Business School MBA: An Insight
You can begin your search for an MBA institute with the aid of various directories, which will point to several colleges and suggest the most appropriate names, based on your requirements, specifications and qualifications. Several websites are also dedicated for these purposes and they facilitate the search by location, degree and subject. They can also provide information on financial resources.
Another important addition in this field is the online business school for MBA. Online education brings dynamic, time efficient and extremely streamlined MBA programs and aspirants can benefit from online education in spite of living anywhere in the world. Most of these schools are partners with reputable business schools, and they offer engaging and highly focused e-learning programs for bachelors, associate's, master's and doctorate degrees. Owing to this flexibility, online business schools are gaining increasing popularity.
MBA programs come in several forms like distance-learning MBAs, executive MBAs, accelerated MBAs, full-time and part-time MBAs. Most MBA programs put special emphasis on work experience. The specialization can be obtained on varied subjects like Finance, HR, Accounting, Insurance, Information System, Administration, Project Management, etc.The Admission Procedure And The Course
To gain admission in a business school MBA program it is imperative to score well on the GMAT (Graduate Management Admission Test) exam and to have a high GPA (grade point average). The candidate also needs to write a strong essay on the application, have work experience, good reference and should be able to impress the interview team during the personal interview phase. The character of a candidate (adjusted by active participation in extra curricular activities and community service) is also very important.
Once admitted, the students are endowed with practical management skills while sharpening their decision making, enhancing their creativity and developing business ethics so as to take up leadership in business environments. Reputed business schools, impart advanced management knowledge (pertaining to professional business designation) to its students and at the same time they are given chance to gather in-depth dedicated knowledge in a selected field. These top business schools strike a perfect balance between the practical knowledge and theory for all-round development of professionals with adequate business expertise and managerial skills.The Ranking Of The Schools
Since MBA is the most happening and in-demand course of study right now, there is a general consensus to rank the schools and universities who offer the MBA programs. Till date, Harvard happens to top the list with its special "case method" learning process, which brings reality into the classroom. Next comes the Wharton Business School, with the world's largest and most cited faculty making all the difference with their innovative teaching method. The Kellogg School of Management with its ever changing curriculum stands third, whereas Stanford School of Business ranks fourth. The latter emphasizes four key factors of general management namely leadership, entrepreneurship, global awareness and social innovation. Apart from these, the Michigan Ross School of Business, Duke University, University of Phoenix, Nan Yang Business School and the Australian University deserve special mention.
So, waste no time; enroll in a business school MBA program and develop your business proficiency and managerial skills.
The number of applicants for JD/MBA joint degree is rising rapidly. But before joining the program..
The MBA (Master's of Business Administration) degree added to your resume would definitely make a..
International MBA program can well be said to be the direct consequence of the globalization of business...
The MBA degree that holds assurances for a high profile, high paying job, is among the most coveted..
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text: Mr. and Mrs. Daniel Pelczynski of Depew celebrated their 50th wedding anniversary with a dinner hosted by their children in Protocol Restaurant, Amherst. Mr. Pelczynski and Marie Piacente were married Sept. 16, 1961, in St. Francis of Assisi Catholic Church, Buffalo. He is a retired auto worker for General Motors, with 41 years of service. The couple has four children and four grandchildren.


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The Three-Month Tithe Challenge
Countless people experience God's blessings when they tithe, but often the first step is the hardest one to take. The Three-Month Tithe Challenge is a money-back guarantee of sorts. Essentially, it's a contract based on God's promises in Malachi 3:10-11.
Here's our commitment to you. If you tithe for three months and God doesn't hold true to His promises, we will refund 100% of your tithe.
I understand this form must be completed and received by the Sacred Church Team prior to the beginning of the Three-Month Tithe Challenge Period.
I understand my household qualifies for participation because we have not been tithing for the last six months.
I understand I cannot seek a refund prior to the end of the Three-Month Tithe Challenge Period, and that I cannot seek a refund for any contributions made prior to the beginning of the Three-Month Tithe Challenge Period.
I understand any request for refund must be received by the Sacred Church Team within 30 days of the end of the Three-Month Tithe Challenge Period.
I understand if paid at the physical Sacred Church building, my tithe must be paid by check, or by completed offering envelope, so that my tithe can be properly credited.
I understand I must log in prior to paying my tithe online, so that my tithe can be properly credited.
I would like to test God's faithfulness by accepting the Three-Month Tithe Challenge. I agree that for the three-month period, my household will contribute to God, through Sacred Church a tithe equal to 10% of our income. At the end of the three-month period, if I am not convinced of God's faithfulness to bless my life as a result of my obedience to His Word, then I will be entitled to request a refund of the full amount of contributions made during that three-month period.
By clicking on the link, you are agreeing to the terms and beginning the Three-Month Tithe Challenge. If you have any questions, please email firstname.lastname@example.com.
Whoever finds God, finds life!
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Post # 1
- niko88
- 1 year ago
Hi, may be my phone but the first picture doesn't seem to have uploaded?
niko88 : I can see a faint line! When will you take another test?
Yes I can see a faint line!
I see a line
I definitely see a faint pink line whoot whoot!!
I see a line!
Thanks everyone , should I test tomorrow or wait a few days for a stronger line ?


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Womens Capelli New York mid calf rain boots have a fabric lining to keep your feet warm and dry and a traction outsole. Heel height: 1.
These Womens Capelli mid calf rain boots are solid black with black trim and red lining. Black side buckle and back pull loop for easy on and off. Durable, top quality material. Height: 10.25.
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text: Syrian state television says Syrian regime forces have regained "full control" of the largest rebel-held area in eastern Aleppo on Saturday. Government forces carried out a ground and air assault on the Hanano housing district early Saturday after rebels spent days defending it in fierce fighting. Rebels say the capture of Hanano is meant to split their stronghold into two in Aleppo and give regime forces line-of-fire control, according to Agence France-Presse. While rebel sources said fighting was ongoing in the area, a Syrian state TV broadcast from the scene of fighting said the Syrian army had retaken the area and secured the safe passage of at least 150 people out of the housing district. Syrian forces were now busy de-mining the area and checking it for explosives, the report said. At least 212 civilians have been killed in the 12 days since air strikes were renewed against Aleppo, according to the Syrian Observatory for Human Rights.


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Maria Island
Connecting Hobart to Tasmania's stunning East Coast. Our return shuttle bus service will provide transport for guests on our Cruise & Walk day tour and provide connections to the Maria Island ferry.
October & May (Fri, Sat, Sun)
November to April (Daily)
Morning Timetable:
Departs 7:10am
The Pickled Frog
(281 Liverpool Street, Hobart)
Departs 7:20am
Tasmanian Travel & Information Centre
(Cnr Davey & Elizabeth Streets, Hobart)
Departs 7:40am
Hobart International Airport
Departs 7:50am
Sorell, Velvet Cafe
(21 Gordon St, Sorell)
Arrives 8:40am
Triabunna marina
Afternoon Timetable:
Departs 5:10pm
Triabunna marina
Arrives 6:00pm
Sorell, Velvet Cafe
(21 Gordon St, Sorell)
Arrives 6:10pm
Hobart International Airport
Arrives 6:25pm
Tasmanian Travel & Information Centre
(Cnr Davey & Elizabeth Streets, Hobart)
Arrives 6:40pm
The Pickled Frog
(281 Liverpool Street, Hobart)
Adults (16+): $30
Children (3-15): $15
Infant (0-2): $0
Same Day Return
Adults (16+): $40
Children (3-15): $25
Infant (0-2): $0
Luggage & Freight
Each adult passenger will be allocated 1 small day bag that they can carry onto the bus (to be stowed at their feet). Additional luggage will be charged at the following rates. Please break down additional luggage items that combine to the equivalent of 60L. Fees are set for each travel day.
Additional luggage (up to 60L): $5
Bikes: $10
Heavy freight (20kg-30kg): $15
Heavy freight (+30kg): $20
Kayaks: $15
Select Shuttle Bus below in the products list.
© Copyright East Coast Cruises


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Hi There! Is this Your First Time?
Did you know if you Register you have access to free search tools including the ability to save listings and property searches? Did you know that you can bypass the search altogether and have listings sent directly to your email address? Check out our how-to page for more info.Arterra Realty - The fastest growing real estate company in Michigan
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0000 Noble Rd, Addison Twp
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Essential Information
- Price$850,000
- Acres123.59
- TypeVacant Land
- StatusActive
- Lot Description537x798x2417x1849
Additional Information
- ZoningAgricultural, Residential
Community Information
- Address0000 Noble Rd
- Area02051-Addison Twp
- CityAddison Twp
- CountyOakland
- StateMI
- Zip Code48371
School Information
- DistrictOxford
Listing Details
- Listing OfficeWillowdale Realty & Dev
IDX provided courtesy of Realcomp II Ltd., via Arterra Realty and REALCOMP, Copyright 2019 Realcomp II Ltd. Shareholders. IDX information is provided exclusively for consumers' personal, noncommercial use and may not be used for any purpose other than to identify prospective properties consumers may be interested in purchasing. The accuracy of all information, regardless of source, is not guaranteed or warranted. All information should be independently verified.


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Our artists
bringing together talented artisans and artists from across the Southern Highlands.
About The SHAC
see artisans and artists at work in their studios creating their works
What's On
From exhibitions to workshops and more, find out what's happening at The Shac.
Artisan Rental Opportunities
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About us
We are an established family run consultancy business specialising in sewer and pipe renovation. We're able to offer unrivaled support to our clients, from conception & structural design through to installation. All this backed up with training and technical support on all of our products. C J Kelly International Ltd are sole representative in the UK for some of the world's leading trenchless technology companies including, KOB Brawoliner, MC Construction Chemicals, HÄRKE & Picote Solutions, UK service centre which allows us to offer a long lasting rehabilitation solution for our customers our products also comply with WRC and iKT European standards.
We supply some of the UK and Ireland's top drainage companies with sewer renovation materials. Our client base includes JAF Services (Dyno Rod), MainDrain Ltd (Metro Rod), Drainway, DalRod, Crowley services, SDI Unistride LTD, McAlilister Bros LTD and a multitude of other satisfied customers across the UK. We are also proud members of the Pipeline Industries Guild & UKSTT.
Gary and the team at West Sussex Drains recently took on a challenging project with their new Picote Maxi miller, removing hard fat deposits from a 4" PVC pipe within a food processing facility.
Germany-based OSSCAD GmbH & Co. KG specialises in the design, development, support and technical consulting for optical sensor technologies. The company places a special emphasis on the ongoing development and promotion of its products in the area of CIPP (cured-in-place pipe) rehabilitation and monitoring of buried energy cables. In 2017 OSSCAD will celebrate its 10th anniversary.
As part of its product range OSSCAD has developed the CMS measuring system for the monitoring of the liner curing process during pipeline rehabilitation operations of both sewer and drinking (potable) water pipes. The CMS system consists of a CMS measuring instrument (laser radar), a fibre optic cable (LWL cable) as a line-shaped temperature sensor (CMS sensor cable) and the CMS application software (VISCOM liner) for the evaluation and visualisation of the curing temperature measuring data.
The BRAWOLINER® 3D lining system was recently utilised on a very challenging sewer rehabilitation project which was part of the on-going £1 million Torbay Sewer Rehabilitation Program being undertaken by South West Water (SWW) in association with its consulting engineer AECOM. The main contractor for the works was Balfour Beatty Utility Solutions Ltd (BBUSL).
In an 'old town' area of Torquay on the English Riviera, a section of clayware sewer pipeline running beneath steep steps (with 85 steps) connecting Melville Street and Rock Road was found to be in need of renovation after CCTV inspection and analysis. These steps are believed to be the steepest in the town.
Originally, the proposed solution was to replace the sewer using the traditional 'open cut' technique. The steps are a maximum of 2 m in width and run adjacent to dwellings on the one side and a substantial retaining wall on the other. The sewer is at a depth of between 2 m and 2.5 m. An inspection of the site showed that to open cut the section would require structural surveys to be undertaken on the properties and wall adjacent to these steps together with specialist designed excavation and shoring techniques to safeguard the structures stability during the excavation operation. Therefore, the client sought a trenchless rehabilitation solution that would eliminate the need to open cut the pipeline.
Whilst this might sound an easy option given the number of renovation systems currently available, practically this was far from the truth.
It is widely known that a significant proportion of East Anglia relies for its existence on the effective operation of widespread land drainage networks. Without these networks much of the region would not be able to function in the way that it does and the population would be hard pressed to continue their lifestyles in the way they do in the modern world due to the high ground water levels.
Much of the drainage to keep ground water levels in check is achieved using local pumping stations that take water from the land drainage network and pump it into local rivers and drainage channels that remove the water off the land and out to sea so keeping the local area from flooding. These pumping stations are under the control of the Internal Drainage Boards (IDB's).
However, when undertaking rehabilitation of a pumping station at a site near Kings Lynn, a reverse syphon carrying water from the pump station to the main drainage channels that fed the local estuary, was found to have failed and there were fears that water normally carried by the syphon away from its local pumping station would cause problems.
The failure of the syphon meant that water from the pumping station was not, as would normally be the case, all removed from the network but that it was being returned back into and potentially undermining the pumping station due to leakage in the syphon. This was also causing the river bank near the pumping station to erode. It was causing problems in the pump station as water was recycling, meaning the pump station was working significantly harder than it should, incurring extra cost and potentially could lead to significant flooding problems for the area.


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I obtained the Bfa in Film yield from Nyu's Tisch School of the Arts. As man who has also attended Usc's Film School as well as having taken classes at Ucla's Film Program, I view I could offer some good perspective of what I view of each and start with the one I spent the most time at: Nyu.
The Location
Nyu's film school is placed in Greenwich community on the island of Manhattan in New York City. Going to school in such an urban location offers both positives and negatives. On a inevitable note, New York City is your campus and therefore you are in the mix of all the limitless cafes, stores, notable locations, iconic neighborhoods and curious New York personalities that the city is notable for.
On the negative side, also, New York City is your campus so you don't have the 'protected campus experience' you would have at a school with a concluded off dedicated campus. In my view I viewed living, taking classes and development films in Nyc to be a mammoth advantage. Where else would you get such great inspiration and material to draw from than in one of the greatest cities in the world. The only negative would be if you wanted to get out of the city to shoot or just to get some quiet away from the hustle of the city. It's hard to keep a car in the city without spending a fortune but subways and trains can take you just about in any place you need to go affordably.
The Curriculum
Nyu Film School's curriculum consists of classes in all the major areas of the filmmaking process. There are classes in sound, editing, writing, television technology and ultimately group classes where you make films together in small crews. Generally, as you move through the schedule the projects you work on become more complex. In beginning film classes you might make black and white 8mm or short videos.
As you expand you make longer and more complicated projects and can choose between experimental type or report films. Ordinarily students are given a inevitable amount of tape or film stock and the basic equipment such as lights and dollies that one would need. If a learner wishes to make something that requires more complicated props and/or equipment then they need to contribute the funding themselves.
Generally, every learner that wants to can direct their own films with other students acting as crew members on their films and vice versa. Nyu has a good option of equipment. It is not all the time the latest technology and technology changes so frequently, especially when it comes to video, but in normal you won't be lacking for anything. They have ample editing facilities running from old steenbeck for film splicing to computer systems for use of final draft and adobe premier. Again, if a learner wishes to shoot more footage than the school is willing to contribute film or video for the learner can pay for added developing, etc.
The Professors
Generally I found the professors to be competent with a few 'exceptional' ones thrown in. They are all working professionals with some more thriving than others. In some classes you also have guest lecturers and occasionally notable alumni will teach a class such as Spike Lee or Woody Allen. Each week the school hosts film screenings and the director or producers of the film will show up to discuss with the class and take questions about how the film was put together and the history of development the film. These are great opportunities to network with thriving alumni and often previous graduates of the schedule show up to watch these screenings so they are a great way to make contacts.
After Graduation.
Where I felt the Nyu Film schedule fell a wee short was in vocation planning and direction. They didn't dedicate a whole lot of time to teaching you how to build a vocation after you graduated. This is one of the flaws I have found in most film programs. Luckily, Nyu has so many graduates that work in the industry that if you are proactive you should precisely be able to find thriving graduates willing to sit down and speak with you about your 'after graduation' game plan.
In recent years the school has been good at building up its active alumni society on both the East and West coasts so either you conclude to be based in New York or Los Angeles you can apply this reserved supply to originate a network for yourself which is very important. Overall, I found the Nyu Film School palpate to be a good one for those wanting a generalized instruction in different aspects of filmmaking before embarking on a career.


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Adjusting CS in shared custody when one parent has no income
View Single Post
Senior Member
Join Date: Feb 2012
Posts: 353
Hi everyone I have some questions about this as it relates to my situation.
1) Currently I have access 5 out of every 14 days (36%) but with the next sitting of my settlement conference should be going up to 6/14 putting me into a shared physical custody situation.
2) Problem is that my X has no income to report on line 150 other than UCCB ($1200) so I would still be paying the full table amount (whose model literally assumes that the paying parent has no child related expenses) when I get to shared custody.
3) I am having huge difficulties financially (LOC maxed, CC getting there, regular expenses exceed my income every month) and am contemplating asking for an adjustment in CS.
Am I...
a) better off trying to seek an adjustment through asking for an imputed income for voluntary unemployment (my ex has a university Degree but has not attempted to gain employment, or taken any steps to update her skills to find employment at all since our separation in May 2011) or...
b) look for some acknowledgement of the increased costs of maintaining two households under section 9 b) of the guidelines?
"9. Where a parent or spouse exercises a right of access to, or has physical custody of, a child for not less than 40 per cent of the time over the course of a year, the amount of the order for the support of a child must be determined by taking into account,
(a) the amounts set out in the applicable tables for each of the parents or spouses;
(b) the increased costs of shared custody arrangements; and
(c) the condition, means, needs and other circumstances of each parent or spouse and of any child for whom support is sought. O. Reg. 391/97, s. 9."
c) do nothing and hope she gets a job eventually and continue to pay full CS (and 98% of section 7, but that is another story altogether).
Any advise or experience in this area would be helpful.
Thanks in advance.
Last edited by Mess; 12-04-2012 at
. Reason: Merged identical thread
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Description of courses:
Mindfulness Based Stress Reduction (MBSR) Course and Mindful Self Compassion (MSC )Course
\\\'Free to be\\\' mindfulness retreat day with yoga including lunch at farmacy
Date & Time: 26th January 2017, 9.30am - 5pm Cost: £175 Location:The Garden Studio
The day includes yoga and mindfulness practices, a lunch at Farmacy with a selection of their signature Earth Bowls and a cold pressed juice and a Closing Tea. There will also be Sweaty Betty goodie bags, kindly donated by our neighbourhood Sweaty Betty Store on Ledbury Road.
In many ways, yoga and mindfulness are very similar, sharing the end goal of achieving a calm state of mind.
Both practices enable us to develop healthier minds that are more focused, happy and calm.
26 Newton Road
Notting Hill
Ms Rosemary Sandham
What qualifications or training do you hold?
I completed the Mindful Self Compassion Teachers Training with Germer and Neff at Bangor University in 2014. I also completed the Frantic World Teachers Training in 2014 with The Oxford Centre For Mindfulness
If you teach MBCT, what professional mental health training do you hold?
Diploma in Person Centred Counselling Metanoia Institute 200 Diploma in Person Centred Counselling Metanoia Institute 1999 BSc reflective Therapeutic Practice Metanoia Institute 2010 Qualified Mindfulness Teacher - Mindfulness Based Approaches (MBSR/MBCT, Mindfulness & Therapy, Mindfulness and Compassion training) - Bangor University
How do you continually ensure your knowledge and skills are up-to-date?
Ongoing training I also keep up to date with new publications including books, journals, articles and research.
Do you adhere to appropriate professional and ethical boundaries and frameworks in your work as a mindfulness-based teacher?


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Samsung has surprised us all by showing off its next smartwatch
Samsung is soon going to announce its Gear S2 smartwatch. But, just recently it made an appearance.
It was seen in a duo of official promo images. Samsung surprised everyone by teasing the Gear S2 smartwatch.
It made an appearance at the end of the Galaxy Note5 and Galaxy S6 edge+ official announcement.
The model appears to be a far cry from the original Gear S. Samsung's new promo gave a shot to the stylish and trendy look along with its new products.
The model appears to be wearing a very stylish smartwatch with white and black wristband. Gear S2 has the round watch face, new user interface and a very sleek metal body.
The watch has the new UI design which clearly shows that it is not an Android Wear device. It consists of a circle of apps arranged around the face.
It is generally expected that it runs on Samsung's Tizen OS rather than Android Wear, like the original Gear S. All we were able to saw today were the hardware and watch face.
Apart from this, Samsung teased a round watch in February. But that watch was assumed to be an Android device.
Samsung Gear S2 will make its official debut during IFA in Berlin next month. The event is scheduled to be held on 3rd September.


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The Strange and Unusual: Our Favorite Tim Burton Films
With the premiere of Dumbo, we've decided to go back and pick some of our favorite films from Tim Burton, spanning over thirty years. Take a look at our picks below:
Batman (1989)
In what is arguably Tim Burton's most significant step as a director, 1989's Batman was a rare accomplishment in that it used the "weird" sensibility that Burton had excelled in with Pee Wee's Big Adventure and Beetlejuice and forged it with a blockbuster template. As a kid, I was more eager to see Batman than any other film because of the marketing – the cropped logo with the gold and black colors was so iconic that it almost eclipses the film itself. The anticipation of the release date, June 19th, is still seared into my memory. I will admit that once I saw the movie, I was more entranced with the tone and the look of the film – the idea that a comic book movie could work dramatically while still maintaining a high level of spectacle was a new frontier and Burton should get loads of credit for that achievement. The performances by Michael Keaton and Jack Nicholson were also strokes of genius, telling the audience that this isn't going to be disposable fluff, this is going to something different. While other films in the franchise (namely its extremely underrated sequel, Batman Returns) took the character to greater heights, Batman set the tone and with its massive success, paved the way for the modern day comic-book blockbuster. - Joey Aucoin
Beetlejuice (1988)
Burton's sophomore follow-up is one of the most wildly original films he's ever created. It's hard to believe this film isn't an adaptation or remake. The film's characters are one of its best features. Without Beetlejuice, we would not have our goth icon Lydia Deetz, played to perfection by a young Winona Ryder, who people still quote 30 years later. And then there is Michael Keaton, in his earliest most recognizable role as Betelgeuse, a character you are meant to abhor, but is somehow beloved. Everything about this film is unique. Its clever use of postmodern grotesque, carnival style aesthetics, all cut with an amazing sense of morbid humor. In Beetlejuice's opening scene, a terrifyingly large spider climbs over a country home, only to be gently picked up like it wasn't a threat at all, lifted away from a hand-made miniature town. This use of pseudo-horror camera tricks lay the foundation for the rest of the film's more shocking gags, which include human deformation, jokes about suicide ("little accident"), and a dead chain smoker who is, "thinking about cutting back." Laughter takes away from the macabre, making it easy for those watching to not think too hard about what was just said. Beetlejuice is Burton at his very best. - Sara Sorrentino
Ed Wood (1994)
Any other filmmaker could have turned a biopic about Ed Wood—the so-called "Worst Director of All Time" who populated his b-movies with has-beens, charlatans, and misfits—into a sideshow, but who loves a misfit more than Tim Burton? Ed Wood is a glorious tribute to the act of movie-making, money or talent be damned. Featuring the best performance of Johnny Depp's long-departed "indie golden boy" years, along with crisp black and white cinematography from Stefan Czapsky, Ed Wood is the kind of passion project only Burton could make. Though it drapes a pink angora sweater over the grimier aspects of the director's life (and invents an inspirational encounter with Wood's hero Orson Welles), the movie's can-do gumption overwhelms any nitpicking over historical accuracy. And yet, there's a dark tenderness at its core for Wood and his friend, the drug-addicted former Dracula, Bela Lugosi (Oscar-winning Martin Landau). They were dreamers who reached for success, grabbed it briefly, and then tumbled hard back to Earth, though ultimately both achieved a strange sort of immortality. One can easily imagine Tim Burton echoing Wood's words: "This is the one I'll be remembered for." - Kayleigh Hearn
Sleepy Hollow (1999)
Before the dark times, before the Apes, Tim Burton once again dazzled audiences with his own personal stamp on the Hammer Horror genre. 1999 brought us the delightfully gruesome Sleepy Hollow featuring longtime collaborator Johnny Depp as the tortured Ichabod Crane and Christopher Walken as the mute Headless Horseman. A visual feast thanks to its Oscar-winning production design and Emmanuel Lubezki's top-tier cinematography, Burton's horror picture certainly doesn't hold back when it comes to scares and gore. Although a low-key romance between Depp and Christina Ricci's characters, Sleepy Hollow wears Burton's horror influences on its sleeve. From the stacked supporting cast featuring Martin Landau, Michael Gough, Ian McDiarmid, and Michael Gambon, to the many twists and turns in relation to the plot, to the thrilling, gore-soaked climax, this is Hammer in all but name. Burton has always had a knack for getting studios to bite when it comes to his outlandish ideas, from the gothic atmosphere of Batman Returns to the big budget farce of Mars Attacks!, and Sleepy Hollow is no different. He got a studio to make a classic horror movie in the modern age and pulled it off exceedingly well. With beheadings and impaling galore it's a high point for both Burton and the genre as a whole, and it's also this writer's personal favorite horror film. - Matt Curione
Batman Returns (1992)
Wild stories like Batman Returns need a wild director, and Tim Burton's take on the Caped Crusader will always be one of my favorites. The sequel to Batman (1989) is outlandish at times but that's the way a comic book film should be. In Batman Returns, the Penguin (Danny DeVito) wants revenge on Gotham after his parents abandoned him, and plans to destroy all firstborn sons in Gotham. Meanwhile we're introduced to the slimy Max Shreck (Christopher Walken), who also wants to take over Gotham, and a sassy Selina Kyle/Catwoman (Michelle Pfeiffer) who wants revenge on Shreck for trying to murder her. All of this is happening while our obvious Batman (Michael Keaton) is trying to save the day. I was only 5 when Batman Returns came out, but I vividly remember seeing this movie for the first time and loving it ever since. Selina Kyle was my personal favorite. She stuck up for herself and survived everything that was thrown at her, and had a KILLER costume to boot. Even to this day seeing Catwoman stand in her window with the neon pink radiating from her room gives me so much joy. Batman Returns is a staple of my youth and I have all the thanks to Tim Burton for that. - Rachael Hauschild
Charlie and the Chocolate Factory (2005)
When Tim Burton stormed onto the scene of Warner Brothers' Willy Wonka update, everything about it changed. The names in consideration for portraying the signature fictional creation of author Roald Dahl (pronounced Roo-all) ranged from Bill Murray to Adam Sandler to Will Smith. But when Burton's long-time collaborator Johnny Depp stepped into the shoes of Wonka, he vowed to work with Burton to create a unique take on the character made famous by Gene Wilder. The result was a critically lauded but popularly divisive remake that, to this day, few people regard as a worthy discussion point for the director's filmography.
However, with the release of Dumbo this month, I invite you take a step back and look at this fourteen-year-old entry as one of Burton's weirdest, warmest, and most ambitious works. Burton's choice of stylish, colorful, and offbeat cartoonish imagery makes this a sumptuous feast of a film, but would it be as quirky without Depp as Wonka? Probably not. This is has always been one of my favorite Depp roles. Not that it's one of his best, but it's one of the strangest things I've ever seen on a screen. His makeup and actions blend into Burton's atmosphere, yet feeling distant from Wilder's interpretation, to the extent that it feels like an entirely different character. And yet, by the time the credits roll, I always move past the initial shock of seeing the pale, lipstick smeared Depp, and end up falling in love with his character; a more personal and intimate Wonka than portrayed in '71, especially with the addition of Christopher Lee as Wonka's father in the third act. The film is not without flaws. It's a tad too long, a bit too stylish for its own good, and most of the performances lack anything worth writing home about, and Deep Roy as the Oompa Loompas was a colossal misfire. And yet, this remains one of my favorite feel-good pleasures. It's a movie I can always go back to to uplift my spirits. It's warm, inviting, funny, sweet, and visually appealing, even if to a fault. Tim Burton's Charlie and the Chocolate Factory, for me, always wins the war over which Willy Wonka adaptation is better, even though both are lovable in their own special ways. - Ben Lane
Pee-wee's Big Adventure (1985)
Pee-wee's Big Adventure is a bit of an odd entry in Tim Burton's filmography, as it doesn't really have the signature style of the rest of his work. The comedy of the character of Pee-wee is much louder and more brazen than the winking, more subtle humor of Burton's later work. Even the visual design of sets and camerawork is totally different than his very next film, Beetlejuice. However, this doesn't come across like some form of erasure of a director's distinct voice, but rather, just a knowing and selfless step back out of the spotlight for another to shine. Burton and Herman are coexisting, perfectly symbiotic, onscreen. When Burton gets his moments to shine through, like the iconic Large Marge scene, he's showing off the best side of his early directing style, using practical effects for chilling results that work right alongside all the laughs. Still, the real star of the picture is the boyish, oddly charming personality of Pee-wee Herman and his mischievous adventures. Pee-wee is not only a force of nature to be reckoned with throughout the story, but also such an indelible part of the film's DNA that Burton's involvement can feel a little eclipsed. - Callie Smith


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Blacked – Evelyn Claire, Jason Luv Room For One More (01.03.2018)
It is the last day before Evelyn Claire's parents are back from vacation, so she has decided to turn things up. After meeting two hot guys at a party the night before, Evelyn and her friend have invited them over to carry on the fun. When she disappears into the bedroom with one of the boys, her friend decides to leave. Before Evelyn knows it, there are three at this private party and that suits her just fine.