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Research article | Open | Published: 24 August 2016
Characterization of transcription factor response kinetics in parallel
Betul Bilgin1,
Aritro Nath2,
Christina Chan1,3 &
S. Patrick Walton1
Transcription factors (TFs) are effectors of cell signaling pathways that regulate gene expression. TF networks are highly interconnected; one signal can lead to changes in many TF levels, and one TF level can be changed by many different signals. TF regulation is central to normal cell function, with altered TF function being implicated in many disease conditions. Thus, measuring TF levels in parallel, and over time, is crucial for understanding the impact of stimuli on regulatory networks and on diseases.
Here, we report the parallel analysis of temporal TF level changes due to multiple stimuli in distinct cell types. We have analyzed short-term dynamic changes in the levels of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), signal transducer and activator of transcription 3 (Stat3), cAMP response element-binding protein (CREB), glucocorticoid receptor (GR), and TATA binding protein (TBP), in breast and liver cancer cells after tumor necrosis factor-alpha (TNF-α) and palmitic acid (PA) exposure. In response to both stimuli, NF-kB and CREB levels were increased, Stat3 decreased, and TBP was constant. GR levels were unchanged in response to TNF-α stimulation and increased in response to PA treatment.
Our results show significant overlap in signaling initiated by TNF-α and by PA, with the exception that the events leading to PA-mediated cytotoxicity likely also include induction of GR signaling. These results further illuminate the dynamics of TF responses to cytokine and fatty acid exposure, while concomitantly demonstrating the utility of parallel TF measurement approaches in the analysis of biological phenomena.
Proper cellular responses to their microenvironments are crucial for cell and tissue homeostasis. Abnormal cellular signaling can lead to diseases such as cancer and diabetes [1–5]. Improved understanding of signaling has led to improvements in disease diagnosis, the understanding of developmental processes, and the engineering of artificial tissues [6–8]. In fact, recent advances in cell signaling pathway analysis have led to the advent of a new field of study, systems biology [9]. Systems biology relies on vast amounts of data that can be generated from the various "omics" techniques. These data represent the results of cell signal transduction and would be complemented by parallel measurements of the levels of transcription factors, the upstream mediators of cellular signaling.
Transcription factors (TFs) are responsible for altering the cell state in response to stimuli by changing the transcription rates of their targeted genes. They interact with specific sites on genomic DNA, often recruiting other co-factors to the location, resulting in activation or repression of the corresponding genes. Analysis of cell signaling responses to stimuli is complicated by the fact that TFs generally control several genes, most genes are controlled by multiple TFs, and any given stimulus can result in the activation of multiple TFs [10–12]. Moreover, TF levels must change in response to a stimulus but, in most cases, must then return to baseline levels to avoid long-term perturbation of cellular function [13, 14]. The TFs that control genes involved in the rapid response to stimuli are often present in the cytoplasm in an inactive state, translocated to the nucleus after activation, and then destroyed or trafficked out of the nucleus to halt their function [15–18]. Thus, tracking TF activities requires analysis across many dimensions, including activation status, subcellular location, and time. With an estimated 2000 TFs in humans, profiling the levels of active TFs is a dynamic and complex task.
To address this challenge, we and others are developing quantitative, sensitive, parallel techniques for measurement of TF levels [19–22]. We have previously demonstrated parallel measurements of TFs in extracts from breast cancer cells using our magnetic bead-based assay [23]. Our approach aims to simplify measuring TFs in parallel, by detecting TF-bound dsDNA, rather than the TF itself. In this way, a challenging task, parallel protein detection, is reformulated into a relatively straightforward task, parallel nucleic acid detection. In this study, we have expanded the number of TFs analyzed and applied our method to the kinetic analysis of cellular responses to multiple stimuli, specifically to cytokines (tumor necrosis factor-α, TNF-α) and saturated fatty acids (palmitic acid, PA).
DNA probe design and radiolabeling
Detailed design information and probe sequences are listed in the (Additional file 1: Table S1). ssDNAs were purchased from Integrated DNA Technologies (Coralville, IA). Probes were generated by hybridization of equimolar amounts of complementary ssDNAs in 1× STE buffer (10 mM Tris, 100 mM NaCl, and 1 mM EDTA), heating to 95 °C for 5 min, followed by incubation at room temperature for 1 h. After hybridization, dsDNA probes were 5′-radiolabeled with 10 pmoles of [γ-33P] ATP using T4 polynucleotide kinase (New England Biolabs, Ipswich, MA). Radiolabeled DNA probes were purified from unincorporated label with G-25 Sephadex columns (Roche Applied Science; Indianapolis, IN).
Our experiments were conducted with two different cell lines, HepG2 (human hepatocellular carcinoma, obtained from ATCC) and MDA-MB-231 (human breast adenocarcinoma, generously provided by Kathleen Gallo, Department of Physiology, Michigan State University). HepG2 cells were cultured in Dulbecco's Modified Eagle Medium (Life Technologies; Grand Island, NY) supplemented with 10 % fetal bovine serum (Life Technologies; Grand Island, NY), 100 μg/mL streptomycin (Life Technologies; Grand Island, NY) and 100 U/mL penicillin (Life Technologies; Grand Island, NY) in a humidified incubator at 37 °C and 5 % CO2. For TNF-α treatments, cells were grown to 90 % confluence in 6-well plates and then treated with 50 ng/ml recombinant human TNF-α (R&D Systems; Minneapolis, MN) for periods of 0.5, 1, 2, 4 and 24 h. Control cells received fresh media without TNF-α. For palmitic acid treatments, cells were grown to 90 % confluence in 6-well plates and then treated with media containing 0.7 mM palmitic acid (Sigma; St. Louis, MO) complexed with 2 % (w/v) fatty acid free BSA (US Biologicals; Salem, MA) for 0.5, 1, 2, 4, 16, and 24 h. Control cells were grown in media containing 2 % BSA for the same time periods.
MDA-MB-231 cells were cultured in Dulbecco's Modified Eagle Medium (Life Technologies; Grand Island, NY) with 10 % fetal bovine serum, 2 mM glutamine (Life Technologies; Grand Island, NY), 100 μg/mL streptomycin, and 100 U/mL penicillin. Cells were maintained in a humidified incubator at 37 °C and 10 % CO2 as described [24]. For TNF-α treatments, cells were grown to 90 % confluence in 6-well plates and then treated with 50 ng/ml recombinant human TNF-α for periods of 0.5, 1, 2, 4 and 24 h. Control cells received fresh media without TNF-α.
Nuclear extraction
For HepG2 cells, nuclear extraction was performed as described [25]. Briefly, after washing the cells with PBS, cells were resuspended and allowed to swell in five times the packed cell volume (PCV) of ice cold buffer (10 mM HEPES (pH = 7.9) (Sigma; St. Louis, MO), 10 mM KCl, 0.1 mM EDTA, freshly added protease inhibitors (complete mini EDTA free cocktail tablets, Roche), and phosphatase inhibitors (phosphatase inhibitor cocktail, Sigma)) for 15 min. After adding 10 % NP-40 solution to a final concentration of 0.5 % (v/v), cells were vortexed for 20 s, and nuclear pellets were collected by centrifugation at 13,000 g for 1 min at 4 °C. Nuclear pellets were washed three times with buffer (same as above) and resuspended in 1×PCV of ice cold buffer (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, freshly added protease inhibitors (complete mini EDTA free cocktail tablets, Roche), and phosphatase inhibitors (phosphatase inhibitor cocktail, Sigma)). Pellets were shaken for 15 min, and nuclear extracts were obtained by centrifugation at 13,000 g for 15 min at 4 °C. The total protein concentration in all extracts was measured by BCA Protein Assay kit (Thermo Scientific Pierce; Rockford, IL).
For MDA-MB-231 cells, nuclear extraction was performed as described [26]. Briefly, after washing the cells with PBS, the cells were trypsinized and allowed to swell in buffer (10 mM HEPES (pH = 8.0), 1.5 mM MgCl2, 10 mM KCl, protease inhibitor (complete mini EDTA free cocktail tablets, Roche)) on ice for 15 min. The cells were then lysed with 15 strokes of a 25-gauge, 5/8 inch needle, and the nuclear pellets were collected by centrifugation at 12,000 g for 15 min. Nuclear pellets were resuspended and incubated in a second buffer (20 mM HEPES (pH = 8.0), 1.5 mM MgCl2, 25 % glycerol, 420 mM NaCl, 0.2 mM EDTA (pH = 8.0), protease inhibitor) on ice for 30 min. After incubation, nuclear extracts (supernatants) were obtained following centrifugation at 12,000 g for 5 min.
It should be noted that complete separation of nuclear and cytoplasmic extracts is crucial for accurate TF analyses. High fidelity separations preclude latent TFs in the cytoplasmic fraction from giving a false positive signal for the nuclear extracts [27]. To assess nuclear/cytoplasmic separation, we conducted western blots for TBP (nuclear protein) and GAPDH (principally cytoplasmic protein) (Additional file 2: Figure S1).
TF biotinylation
Nuclear extracts were chemically biotinylated by EZ-Link-Iodoacetyl-PEG2-biotin (Thermo Scientific Pierce; Rockford, IL), according to the manufacturer's instructions. Nuclear extracts were mixed with EZ-Link-Iodoacetyl-PEG2-biotin in reaction buffer (50 mM Tris–HCl, 5 mM EDTA, pH 8.0) at RT for 90 min. Biotinylated TFs were purified with G-50 Sephadex columns (Roche Applied Science; Indianapolis, IN). Sephadex columns were washed three times with PBS prior to use.
Magnetic bead-based TF quantification
TFs were measured according to our established technique [23]. Biotin labeled TFs were immobilized on streptavidin-coated magnetic beads (Dynal/Invitrogen; Oslo, Norway) by incubation at RT for 20 min in 1× PBS. After applying a magnet, the supernatant was removed and the TF-bound beads recovered. Following three washes, the TF-loaded beads were mixed with dsDNA probes in binding buffer for 20 min at RT. The supernatant was collected by applying the magnet again, and the beads were washed twice with washing buffer (0.02 % Tween 20 in water) with the supernatants collected after each wash step.
Scintillation counting
For single TF measurements, the technique was performed using one radiolabeled dsDNA probe while all other probes were unlabeled. After binding to the TF-loaded beads and washes to remove non-specifically bound probes, the beads were resuspended in 50 μl of water and then mixed with 10 ml Safety Solve High Flash Point Scintillation Cocktail (Research Products International Corporation; Mount Prospect, IL). The signal from all fractions (supernatant, wash 1, wash 2, and beads) was measured, and the percentage of the signal retained on the beads was calculated. All signals were normalized to control to account for decay in the radiolabel on the probe.
Electrophoresis readout
For parallel TF measurements, dsDNA probes were bound to the TF-loaded beads. After washes, the beads were resuspended in 1×TBE and heated at 95 °C for 15 min to elute the retained DNA. A magnet was applied and the supernatant collected. Eluted DNA probes (1 μl of the 25 μl) were mixed with universal primers (300 nM) and amplified for 20 cycles with Taq DNA Polymerase (New England Biolabs; Ipswich, MA) in 50 μl reactions. The PCR program was: 95 °C for 30 s (melting), 61 °C for 30 s (annealing), and 72 °C for 10 s (extension). 12 μl of PCR product was mixed with 4 μl of gel loading buffer, and 14 μl was loaded onto native 4–12 % TBE gels. Gels were run at 300 V for 30 min on ice, stained with SYBR Gold (Invitrogen; Carlsbad, CA), and visualized in a ChemiDoc XRS System (Bio-Rad; Hercules, CA). Band intensities were quantified by QuantityOne software. For normalization, each signal was normalized to an internal standard included in each PCR reaction. The internal standard included the universal primer sites but did not contain a TF binding site. This accounted for run-to-run differences in PCR efficiency and gel exposure. After signals were normalized to the internal standard, the ratio of treated to control was calculated. We have previously validated the quantitative binding and release of probes from the TFs bound to the beads and all other steps prior to PCR [23].
TF measurements by EMSA
Biotinylated TFs were mixed with dsDNA probes (one probe radiolabeled, the rest unlabeled) in a 20 μl reaction volume of binding buffer (10 mM Tris–HCl, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 1 mM CaCl2, 0.2 mM KCl, 10 mM ZnCl2, 4 % glycerol, 20 mM acetic acid, 0.025 μg/μL poly (dI-dC)) for 30 min at RT. Biotinylated TF concentrations were measured by Bradford assay with 2 μg of protein loaded per assay. 15 μl of the binding reaction were mixed with 5 μl of gel loading buffer, and 18 μl was loaded onto native 4–12 % TBE gels. Gels were run at 300 V for 30 min on ice, dried, and detected by phosphorimaging using the Storm 860 (GE Healthcare; Pittsburgh, PA). Band intensities were quantified by QuantityOne software. A representative image is provided in the (Additional file 3: Figure S2).
All experiments were performed at least 3 times. For gel images, representative results are shown. All error bars show the mean +/− SD value of experiments performed. Two-way student t-test was used to evaluate statistical significance of values compared to control samples. The t-test was conducted as follows: a pooled variance was calculated for all time points for a given TF to account for the distribution of variances across the different samples. A t-statistic was calculated from the degrees of freedom for each TF and was used to build a 95 % confidence interval about the mean for each TF at each time point. Significance relative to normalized controls was determined by whether the value of 1 did (not significant) or did not (significant) fall within the 95 % confidence interval. This analysis was developed in consultation MSU's Center for Statistical Training and Consulting (CSTAT).
Having previously demonstrated applicability of our approach to parallel measurement of TFs [23], we sought to apply the technique to furthering our understanding of biological signaling kinetics while also demonstrating use of the technique for measuring a broader array of TFs in parallel. We chose to examine cytokine stimulation and fatty-acid exposure of cells as stimuli and designed our panel of TFs according to our expectations about which pathways could be activated by these stimuli. In addition, we tested our technique using extracts from two unique cell types to demonstrate the applicability of the method to different cell systems and to examine the commonalities and differences in response among the systems and stimuli.
TF measurements in MDA-MB-231 breast cancer cells stimulated with TNF-α
We first examined the changes in nuclear TF levels associated with TNF-α stimulation of MDA-MB-231 breast cancer cells. These experiments were intended to serve as an initial validation of our larger set of TF measurements. For these experiments, the levels of four different TFs were measured in parallel with respect to treatment time (Fig. 1). These TFs were nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), signal transducer and activator of transcription 3 (Stat3), glucocorticoid receptor (GR), and TATA binding protein (TBP). For measurements of individual TFs (in which we only tested NF-kB, Stat3, and TBP), our results showed an increase in nuclear NF-kB levels after TNF-α stimulation, with levels peaking at 1 h after stimulation (Fig. 1a). NF-kB levels returned to baseline after approximately 4 h, remaining at or near baseline levels for the remainder of the experiment. On the other hand, Stat3 levels decreased after 0.5 h, returning to baseline levels after 2 h. Our expected control TF, TBP, showed no significant change over the course of the experiment. To confirm our results, we performed electrophoretic mobility shift assays (EMSAs) on each of the samples. Agreement was seen between the EMSA (Fig. 1b) and measurements by our method (Fig. 1c) in the data trends (increases and decreases in TF levels) and generally in the magnitude of the changes, as well.
Quantification of TF levels in nuclear extracts of MDA-MB-231 cells after TNF-α stimulation. a Single TF detection by bead assay. The percentage of radiolabeled TF probe remaining on the beads (relative to signal that did not bind or was washed from the beads) was calculated. Fold changes relative to control are shown. (n = 3, * indicates p < 0.05). b Single TF detection by EMSA. The fractions of bound and unbound DNA probe were quantified, and the fraction of bound signal was calculated relative to the total signal from the lane. Fold changes relative to control are shown. (n = 3, * indicates p < 0.05). c Correlation between detection by bead assay and EMSA. The 1:1 line (blue) is included for reference. d Parallel TF detection by bead assay. Signals were normalized with respect to an internal standard and then the ratio with respect to control was calculated. (n = 3, * indicates p < 0.05). e Correlation of single and parallel bead assay measurements. The 1:1 line (blue) is included for reference
Our objective is to expand the technique to detection of hundreds of TFs in parallel with a high throughput technique such as parallel sequencing. For our current scale of 4 TFs in parallel, a PCR-based readout was applied as a proxy for such readouts. Each dsDNA probe was designed to include universal sense and antisense primer sequences and to yield a PCR product of unique length following amplification. To accomplish this, we included different numbers of binding sequence repeats for some TFs (Additional file 1: Table S1). In our experiments, eluted DNAs from magnetic beads were PCR amplified with universal primers for 20 cycles, to achieve a semi-quantitative PCR readout. For all TFs, our single protein measurements with EMSA and scintillation counting correlated well with the levels measured in parallel (Fig. 1d and e). In all detection techniques, NF-kB levels were increased within 0.5 h and returned to baseline after 4 h. In contrast, Stat3 levels decreased and recovered over the same time period. GR levels were unchanged with TNF-α treatment. This was our expectation based upon prior work in muscle cells [28]. The changes in NF-kB and STAT levels agree with previously published reports examining the responses of these TFs to TNF-α stimulation [29, 30], though these were measured at a single timepoint. Our kinetic data demonstrate that TF levels in the nucleus can change rapidly both in the initiation of the response and in the return to baseline levels.
TF measurements in HepG2 cells stimulated with TNF-α
To test the feasibility of our assay with different cell types, we measured TF levels in HepG2 cells. cAMP response element-binding protein (CREB) was included in this set of measurements. We and other have previously studied the effects of TNF-α on these TFs [31, 32]. As with the breast cancer cell experiments, individual TF levels were detected with scintillation counting and EMSA (Fig. 2a, b), while multiple TFs were detected by our parallel PCR readout (Fig. 2d). Results from the three different techniques correlated well, showing NF-kB and CREB levels increasing, with CREB peaking and returning to baseline later than NF-kB (Fig. 2c and e). Over a similar timeframe, Stat3 levels decreased and also returned to baseline. After 4 h, all measured TF levels returned to control levels. TBP and GR levels were unchanged during the measurement period. These results align with those from the breast cancer cell experiments (Fig. 1).
Quantification of TF levels in nuclear extracts of HepG2 cells after TNF-α stimulation. a Single TF detection by bead assay. The percentage of radiolabeled TF probe remaining on the beads (relative to signal that did not bind or was washed from the beads) was calculated. Fold changes relative to control are shown. (n = 3, * indicates p < 0.05). b Single TF detection by EMSA. The fractions of bound and unbound DNA probe were quantified, and the fraction of bound signal was calculated relative to the total signal from the lane. Fold changes relative to control are shown. (n = 3, * indicates p < 0.05). c Correlation between detection by bead assay and EMSA. The 1:1 line (blue) is included for reference. d Parallel TF detection by bead assay. Signals were normalized with respect to an internal standard and then the ratio with respect to control was calculated. (n = 3, * indicates p < 0.05). e Correlation of single and parallel bead assay measurements. The 1:1 line (blue) is included for reference
In hepatocytes, TNF-α has been shown to initiate several different responses, including cytotoxicity [33]. However, hepatocytes are capable of resisting the cytotoxic effects of TNF-α by activating NF-kB [34]. Additionally, recent studies have suggested that TNF-α signaling can activate the IKK/JNK pathway [32, 35], which, in turn, enhances CREB activity [36, 37]. Both NF-kB and CREB activate transcription of anti-apoptotic and cell proliferation genes [20, 38]. In line with this possible defensive response, we found rapid activation of both TFs by TNF-α.
The reduced nuclear levels of Stat3 may suggest a rapid export/degradation of nuclear Stat3 or a reduction in the rate of translocation from the cytoplasm to the nucleus. It is known that Stat3 localized to the cytoplasm may interact with protein kinase R (PKR) and inhibit its phosphorylation activity [39]. PKR inhibits translation initiation and induces apoptosis via the FADD-dependent Caspase 8 pathway [40]. Thus, lower levels of Stat3 in the nucleus, if accompanied by concomitant increases in cytoplasmic Stat3, may suggest a possible anti-apoptotic effect mediated by PKR repression.
For additional validation of our measured responses, we also compared the TF measurements to western blotting of each TF, with qualitative but not quantitative agreement (Additional file 4: Figure S3). Western blotting is a technique that measures the total quantity of TF protein, while our approach and EMSA specifically measure the quantity of active TF (as defined by its ability to bind its dsDNA recognition sequence). Since western blotting consistently showed elevated signals relative to the other approaches, this suggests that either TFs are being inactivated during sample preparation, which seems unlikely given the activity of the remaining proteins, or that a large fraction (~50 %) of the TF molecules that are present in the nucleus are inactive prior to destruction or trafficking out of the nucleus [41]. Our data also demonstrate how important kinetic measurements of TF levels can be to understanding biological processes. For our TFs, measurements at 24 h alone would have shown no response, hiding the early events in response to the stimulus that likely contribute to downstream changes in cellular function.
TF measurements in HepG2 cells stimulated with palmitic acid
In recent years, we and others have focused on the cytotoxic effects of FFAs on hepatocytes [42, 43]. However, the signaling pathways associated with palmitic acid (PA) exposure are not well-established. Thus, we wanted to address which TFs of our analytical set are involved in the cellular response to PA exposure. Prior work had identified a role for NF-kB and CREB [44]. Our particular interest in GR is two-fold. First, GR was shown to increase activated CREB levels [45], and CREB can act with GR to regulate genes where their binding sites are within 90 bp of each other [46]. As above, TF expression levels were measured by scintillation counting or EMSA for individual TFs, or PCR for TFs in parallel (Fig. 3) (A representative image of the parallel PCR readout is shown in the Additional file 5: Figure S4). Our data consistently show that NF-kB activity increased and reached a maximum at 30 min, then returned to baseline by 2 h, with a similar pattern observed with CREB. As with our TNF-α studies, nuclear Stat3 levels decreased by 30 min and returned to basal levels by 2 h. Importantly, GR levels were increased by PA treatment, peaking at 1 h. TBP levels were unchanged as expected.
Quantification of TF levels in nuclear extracts of HepG2 cells after palmitic acid treatment. a Single TF detection by bead assay. The percentage of radiolabeled TF probe remaining on the beads (relative to signal that did not bind or was washed from the beads) was calculated. Fold changes relative to control are shown. (n = 3, * indicates p < 0.05). b Single TF detection by EMSA. The fractions of bound and unbound DNA probe were quantified, and the fraction of bound signal was calculated relative to the total signal from the lane. Fold changes relative to control are shown. (n = 3, * indicates p < 0.05). c Correlation between detection by bead assay and EMSA. The 1:1 line (blue) is included for reference. d Parallel TF detection by bead assay. Signals were normalized with respect to an internal standard and then the ratio with respect to control was calculated. (n = 3, * indicates p < 0.05). e Correlation of single and parallel bead assay measurements. The 1:1 line (blue) is included for reference
These data show that HepG2 cells respond to PA treatment by dynamic changes in the trafficking of these TFs into or out of the nucleus. Of the TFs we measured, NF-kB was most rapidly activated, followed soon after by CREB and GR. The increased activities of NF-kB and CREB in response to elevated free fatty acids is in agreement with prior studies [44]. Given that elevated levels of PA are cytotoxic to HepG2 cells [47], this response suggests that NF-kB and CREB are activated to initiate an anti-apoptotic response to this stimulus, as in the TNF-α experiments.
Comparison of TNF-α and PA responses
The NF-kB, CREB and Stat3 responses of HepG2 cells to palmitic acid and TNF-α are similar, indicating that similar pathways are activated by each stimulus. It has already been shown that circulating and liver levels of TNF-α are elevated in non-alcoholic fatty liver disease (NAFLD) [48, 49]. Additionally, free fatty acids have been shown to elevate TNF-α expression [50]. It is unclear if TNF-α signaling is associated with the response to PA that we have measured, given the short time scales over which we detect significant changes in TF levels. It may be that the similarity in the responses is related to existing crosstalk between the two pathways.
Intriguingly, GR responds differently between the TNF-α and PA stimulated experiments. Currently, no data support a role of GR in mediating PA-induced cytotoxicity. However, it has been shown that elevated levels of glucocorticoids may be associated with pathogenesis of NAFLD [51]. Also, in previous studies, GR has been shown to significantly enhance activated CREB levels [44], with both being increased in our results. GR and CREB are known to synergistically activate expression of certain genes, e.g., phosphoenolpyruvate carboxykinase (PEPCK), somatostatin, and even GR itself [52, 53]. Importantly, activation of PEPCK by palmitic acid was observed with HepG2 cells previously [54]. It has also been shown that there is crosstalk between GR and CREB in regulating neuronal genes [55]. Taken together, our results and the literature suggest that genes regulated by GR (and perhaps particularly those co-regulated by CREB and GR) might be important in the cellular response to saturated fatty acid (palmitic acid) exposure. Stat3 may also respond differently to TNF-α and PA. While the early downregulation of Stat3 levels is consistent between the stimuli, in some of the TNF-α analyses, Stat3 levels at 4 h are slightly elevated (e.g., Figs. 1b, 2a). This may be a result of a feedback mechanism to restore Stat3 to basal levels. Additional timepoints between 4 h and 24 h may have clarified whether the observed increase in Stat3 was biologically meaningful.
Future development and experiments
In this work, we have shown that our assay can be applied to measure TF levels in parallel over time and is useful for the analysis of TFs involved in cytokine and fatty acid treatments of cultured cells. For both of our model systems, we used cancer cell lines with which we have considerable experience. We acknowledge that the responses we detected may not be universal across all human cell types but are nonetheless useful in demonstrating the ability of our method to detect changes in TF levels. We believe that our assay can be used to profile other TF pathways and to study early cellular responses.
We recognize that we only measured a limited number of TFs in parallel and only over a 24 h time period. Subsequent work and redesign of the technique will focus on inclusion of late-acting TFs, whose expression level changes would be expected after 8 h. Complementing our analytical approach with chromatin immunoprecipitation (ChIP), either for individual TFs or in parallel, would further our understanding of the downstream signaling that results from the nuclear TFs we measure. This combination of early-acting and late-acting TFs would considerably enhance the information provided by the technique in analyzing biological systems. Combined with other parallel analytical techniques, our approach would contribute to a more complete picture of TF regulation and signaling in response to a variety of stimuli.
We have dynamically measured TF levels in response to cytokine and palmitic acid treatments using our previously developed assay. With our assay, NF-kB, CREB, Stat3, GR, and TBP levels were successfully analyzed over 24 h. Our assay can be further improved for profiling larger sets of TFs and could contribute to explorations of cellular regulatory mechanisms, TF pathway mapping, and modeling of dynamic signaling networks.
BSA:
CREB:
cAMP response element-binding protein
CSTAT:
Center for statistical training and consulting
DTT:
Dithiothreitol
EDTA:
EMSA:
Electrophoretic mobility shift assay
FFA:
Free fatty acid
GR:
Glucocorticoid receptor
HEPES:
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HepG2:
Human hepatocellular carcinoma
MUCI:
Michigan universities commercialization initiative
NAFLD:
Non-alcoholic fatty liver disease
NF-kB:
Nuclear factor kappa-light-chain-enhancer of activated B cells
PA:
PBS:
Phosphate-buffered saline
PCV:
Packed cell volume
PEPCK:
Phosphoenolpyruvate carboxykinase
PKR:
Protein kinase R
Stat3:
Signal transducer and activator of transcription 3
STE:
Sodium Chloride-Tris-EDTA
TBE:
Tris-Borate-EDTA
TATA binding protein
TNF-α:
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Liu X, Fagotto F. A method to separate nuclear, cytosolic, and membrane-associated signaling molecules in cultured cells. Sci Signal. 2011;4(203):l2.
Dekelbab BH, Witchel SF, DeFranco DB. TNF-α and glucocorticoid receptor interaction in L6 muscle cells: a cooperative downregulation of myosin heavy chain. Steroids. 2007;72:705–12.
Nikolaidou-Neokosmidou V, Zannis VI, Kardassis D. Inhibition of hepatocyte nuclear factor 4 transcriptional activity by the nuclear factor kappaB pathway. Biochem J. 2006;398(3):439–50.
Regueira T, Lepper PM, Brandt S, Ochs M, Vuda M, Takala J, Jakob SM, Djafarzadeh S. Hypoxia inducible factor-1 alpha induction by tumour necrosis factor-alpha, but not by toll-like receptor agonists, modulates cellular respiration in cultured human hepatocytes. Liver Int. 2009;29(10):1582–92.
Schwabe RF, Brenner DA. Selective inhibition of NF-kB activation by the flavonoid hepatoprotector silymarin in HepG2: Evidence for different activating pathways. Am J Physiol Gastrointest Liver Physiol. 2006;290(4):G583–9.
Wullaert A, Heyninck K, Beyaert R. Mechanisms of crosstalk between TNF-induced NF-kappaB and JNK activation in hepatocytes. Biochem Pharmacol. 2006;72(9):1090–101.
Ding WX, Yin XM. Dissection of the multiple mechanisms of TNF-alpha-induced apoptosis in liver injury. J Cell Mol Med. 2004;8:445–54.
Tilg H, Diehl AM. Cytokines in alcoholic and nonalcoholic steatohepatitis. N Engl J Med. 2000;343:1467–76.
Zhan S, Rockey DC. Tumor necrosis factor α stimulates endothelin-1 synthesis in rat hepatic stellate cells in hepatic wound healing through a novel IKK/JNK pathway. Exp Cell Res. 2011;317(7):1040–1048S.
Johnson GL. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science. 2002;298(5600):1911–2.
Clarke DL, Clifford RL, Jindarat S, Proud D, Pang LH, Belvisi M, Knox AJ. TNF alpha and IFN gamma synergistically enhance transcriptional activation of CXCL10 human airway smoth muscle cells via Stat-1, NF-kappa B and the transcriptional coactivator CREB-binding protein. J Biol Chem. 2010;285(38):29101–10.
Park JM, Greten FR, Wong A. Signaling pathways and genes that inhibit pathogen-induced macrophage apoptosis- CREB and NF-kappa B as key regulators. Immunity. 2005;23(3):319–29.
Shen S, Niso-Santano M, Adjemian S, Takehara T, Malik SA, Minoux H, Souquere S, Mariño G, Lachkar S, Senovilla L, Galluzzi L, Kepp O, Pierron G, Maiuri MC, Hikita H, Kroemer R, Kroemer G. Cytoplasmic STAT3 represses autophagy by inhibiting PKR activity. Mol Cell. 2012;48(5):667–80.
Gil J, Esteban M. Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): Mechanism of action. Apoptosis. 2000;5:107–14.
Darnell JE. Transcription factors as target for cancer therapy. Nat Rev. 2002;2:740–9.
Li Z, Yang SX, Mittal S, et al. A hierarchical approach employing metabolic and gene expression profiles to identify the pathways that confer cytotoxicity in HepG2 cells. BMC Syst Biol. 2007;1.
Wang X, Nath A, Yang X, Portis A, et al. Synergy analysis reveals association between insulin signaling and desmoplakin expression in palmitate treated HepG2 cells. PLoS One. 2011;6:e28138.
Cho H, Wu M, Zhang L, Thompson R, Nath A, Chan C. Signaling dynamics of palmitate-induced ER stress responses mediated by ATF4 in HepG2 Cells. BMC Syst Biol. 2013;7:9.
Zhu XO, Yang Z, Guo CM, Ni XT, et al. Paradoxical stimulation of Cyclooxygenase2 expression by glucocorticoids via a cyclic AMP response element in human amnion fibroblasts. Mol Endocrinol. 2009;23:1839–49.
Imai E, Miner JN, Mitchell JA, Yamamoto KR, et al. Glucocorticoid receptor-cAMP response element-binding protein interaction and the response of the phosphoenolpyruvate carboxykinase gene to glucocorticoids. J Biol Chem. 1993;268:5353–6.
Unger RH, Orci L. Lipoapoptosis: its mechanism and its diseases. Biochim Biophys Acta Mol Cell Biol Lipids. 2002;1585:202–12.
Bugianesi E. Non-alcoholic steatophepatitis and cancer. Clin Liver Dis. 2007;11(1):191–207.
Valenti L, et al. Tumor necrosis factor α promoter polymorphisms and insulin resistance in nonalcoholic fatty liver disease. Gastroenterology. 2002;122(2):274–80.
Feldstein AE, Werneburg NW, Canbay A, Guicciardi ME, et al. Free fatty acids promote hepatic lipotoxicity by stimulating TNF-α expression via a lysosomal pathway. Hepatology. 2004;40:185–94.
Vegiopoulos A, Herzig S. Glucocorticoids, metabolism and metabolic diseases. Mol Cell Endocrinol. 2007;275(1–2):43–61.
Liu JL, Papachristou DN, Patel YC. Glucocorticoids activate somatostatin gene transcription through co-operative interaction with the cyclic AMP signalling pathway. Biochem J. 1994;301:863–9.
Govindan MV. Recruitment of cAMP-response element-binding protein and histone deacetylase has opposite effects on glucocorticoid receptor gene transcription. J Biol Chem. 2010;285:4489–510.
Gao D, Nong S, Huang X, Lu Y, et al. The effects of palmitate on hepatic insulin resistance Are mediated by NADPH oxidase 3-derived reactive oxygen species through JNK and p38 MAPK pathways. J Biol Chem. 2010;285:29965–73.
Focking M, Holker I, Trapp T. Chronic glucocorticoid receptor activation impairs CREB transcriptional activity in clonal neurons. Biochem Biophys Res Commun. 2003;304:720–3.
We thank the members of the Cellular and Biomolecular Laboratory for their advice and support. Assistance with the statistical analyses was provided by MSU's Center for Statistical Training and Consulting (CSTAT).
Financial support for this work was provided in part by Michigan State University, the National Science Foundation (CBET 0941055), the National Institutes of Health (GM079688, RR024439, GM089866, DK081768, DK088251), the Michigan Universities Commercialization Initiative (MUCI), and the Center for Systems Biology. The funding bodies were not involved in design of the studies; collection, analysis, nor interpretation of the data; nor writing of the manuscript.
The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.
BB, AN, CC and SPW designed the studies. BB and AN performed the experiments. All authors analyzed the data and wrote, read, and approved the final manuscript.
Department of Chemical Engineering and Materials Science, Michigan State University, 428 S. Shaw Lane, Room 3249, Engineering Building, East Lansing, MI, 48824-1226, USA
Betul Bilgin
, Christina Chan
& S. Patrick Walton
Genetics Program, Michigan State University, East Lansing, MI, 48824, USA
Aritro Nath
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, 48824, USA
Christina Chan
Search for Betul Bilgin in:
Search for Aritro Nath in:
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Correspondence to S. Patrick Walton.
Sequences and designs of DNA probes and PCR primers. Complementary sequences of universal primers to DNA probes are underlined. Repeats of TF binding sites are shown in design with the repeat of the same color. (DOCX 523 kb)
Representative western blots showing separation of nuclear (lanes 1–6 from left) and cytoplasmic (lanes 7–12) fractions. Detection was for either TBP (top) or GAPDH (bottom). (DOCX 677 kb)
Representative image of EMSA detection of TF binding. Replicate experiments for detection of CREB in TNF-α treated (50 ng/ml) HepG2 cells. Lanes 1, 7 – No TNF-α; Lanes 2, 8–0.5 h; Lanes 3, 9–1 h; Lanes 4, 10–2 h; Lanes 5, 11–4 h; Lanes 6, 12–24 h. (DOCX 1 mb)
Western blot analysis of NF-kB levels in TNF-α treated HepG2 cells. Representative western blot image and quantification of NF-kB levels from multiple western blots. (DOCX 606 kb)
Parallel TF detection by bead assay. Representative image of a parallel PCR readout (PA treated HepG2 samples). (DOCX 580 kb)
Palmitic acid treatment
HepG2 cells
MDA-MB-231 cells


text:
County Map of Virginia and West Virginia
Map is among the earliest to distinguish West Virginia from Virginia.
Map of Greenbrier County, W. Va. (reproduction)
Map shows towns and geographic features of Greenbrier County, West Virginia. Reproduction was created in 1973.
Map of the Lands of the Flat-Top Land Trust
Map shows lands of the Flat-top Land Trust, with a key identifying the various divisions of that land.
Dates: March 1888
State of West Virginia and Part of Maryland Coal Fields and Producing Districts
Map shows coal fields and producing districts in West Virginia and part of Maryland.
West Virginia -- Maps 9
Maryland -- Maps 1
Flat-top Land Trust 1
Gamble, W. H. (William H.) 1
Greenbrier Historical Society 1
Handley, J. O. 1
Harrison, H. H. 1
Lesher, C. E. (Carl Eugene), b.1885 1
Mitchell, S. Augustus (Samuel Augustus), 1792-1868 1

redpajama_set_name: RedPajamaCommonCrawl


text:
Thousands of Iraqis Flee Heavy Fighting in Western Mosul
By Lisa Schlein
GENEVA - The United Nations reports more than 190,000 people — over half of them children — have fled Mosul since Iraqi government forces began their offensive on October 17 to retake the city from Islamic State insurgents.
A significant increase in the number of people fleeing western Mosul was reported last week, as fighting between Iraqi soldiers and Islamic State militants intensified. The U.N. estimates about 30,000 people have become displaced, half of them children.
Bastien Vigneau is the U.N. Children's Fund Emergency Coordinator for Mosul operations in Iraq. Speaking by telephone from Baghdad, he says the children and their families have been arriving in desperate condition at a camp for displaced people in Hamam al-Alil, which is 20 kilometers south of Mosul.
Displaced Iraqis, who fled fighting between Iraqi security forces and Islamic State militants, gather to receive humanitarian aid at a camp for internally displaced people in Hamam al-Alil, some 10 kilometers south of Mosul, Iraq, March 2, 2017.
"Basically, the children and the families that we meet in Hamam al-Alil are most likely coming by buses," Vigneau said. "This is organized by the military. And I have seen, for example, I was there yesterday, they really carry the bare minimum. They seem to be quite exhausted. I would say they have been in this situation for 2½ years and extremely frightened, of course, by the operations happening."
Vigneau says the new arrivals describe conditions in Mosul as dire and getting worse. He says they report that one kilo of rice costs $60, with food, water, medicine and fuel are running low.
While up to 750,000 people in the city remain inaccessible to humanitarians, it is possible to reach some that are trapped there, according to Vigneau.
He says UNICEF, the World Food Program, and the U.N. Population Fund are planning an aid mission to west Mosul this weekend. They will deliver a month's worth of food, water, dried biscuits and other relief to some 4,000 families, including 12,000 children.
For security reasons, Vigneau did not name the neighborhoods that will receive aid.
Mosul Victims Treated for Chemical Agents as Displacement Accelerates
The number of civilians escaping the fighting in Mosul has increased significantly as battles intensify between U.S.-backed Iraqi forces and Islamic State militants, and some have been exposed to chemical agents, the Red Cross said on Friday. Iraqi armed forces meanwhile said they had captured another district as they push toward the densely packed old city center where the fighting is expected to become tougher. Among casualties in the past 48 hours, five…
Mosul's Wealthy Mourn, Rebuild in Wake of Destruction
Once this was an elegant residential street next to a small park and across from an imposing home belonging to the family of the governor of Mosul, Iraq. Nearby was a Turkish consulate, and the roads were freshly paved.When Islamic State militants took over in 2014, they promised the local people — many of them business people, engineers and doctors — better governance, education, electricity and water. And despite the quiet wealth of this district, resentment of the…
By Heather Murdock
'I've Never Seen Such War' - 90-year-old Rescued From Mosul
Ninety-year-old Khatla Ali Abdallah has survived decades of turbulence in northern Iraq, but the frail grandmother who fled the battle for Mosul this week says the fighting there is the worst she has ever seen. Carried across the desert by her grandsons, under sniper and mortar fire, she was one of thousands who braved the difficult and dangerous journey out of Islamic State's shrinking stronghold in the west of the city. "I'm a 90-year-old woman…
New Wave of Families Flee Fierce Mosul Battles
"When Islamic State militants took over, we buried our AK-47's in the garden," said Khalil Hamad, a father of five, at the newest refugee camp surrounding Mosul, Iraq, on Tuesday. Nearly 8,000 people had fled to the camp since it opened Monday afternoon, and busloads of families continued to stream in. Islamic State militants were going house to house demanding that residents move deeper into Mosul, and many families, like Hamad's, risked…
Wed, 03/01/2017 - 04:30 PM
Lisa Schlein
Shoura - An Experiment in Reconciliation in Post-Islamic State Iraq
VOA News About Iran


Knight Commander of the Most Excellent Order of the British Empire, a grade within the British order of chivalry.
Knowledge-based economy, an economic construct in which economic benefit is primarily derived from the use of knowledge to more effectively produce products and services.
Knowledge-based engineering, an engineering approach based on the use of electronic knowledge models.


text:
How to Decide Between a Facebook Live, Google Hangout, Zoom Or Telephone Town Hall
Telephone Town Halls, Telephone Town Halls - What Are They
One thing that events streamed on Facebook Live, Google Hangout or Zoom have in common is that they all depend on people remembering to sign on.
Sounds simple enough. But in practice, only your most committed supporters or donors will keep these appointments. It is just human nature. Moreover, the capacities of these platforms are in the hundreds of participants — not the thousands.
If you want to talk to a lot of people at once, or you want to reach out to people who are not in your inner circle – think voters or potential supporters — you need a Telephone Town Hall.
Now, decision makers don't need to choose between Facebook Live, Google Hangout, Zoom or Telephone Town Halls. There is a middle ground that has the benefits of both.
A hybrid event has a Telephone Town Hall connect with voters or potentials supports the way it always has – those called receive an automated message asking them to simply stay on the line to participate.
Alongside the Telephone Town Hall the organizers set up one of the video streaming platforms and have all their speakers log on. Stones' Phones then takes the audio from the Telephone Town Hall and pushes it to the livestream.
While capturing a passive audience through the phone, these events also have an audience of supporters who have actively tuned into the livestream of the video, either through Zoom, Google Hangout or Facebook Live.
"These events have a bigger audience," said Will Wrigley, Strategist at Stones' Phones. "They reach more people because it's pushed out on the phones and online simultaneously. A lot of people will never see an ad for your virtual event online, but they will pick up their phone."
The hybrid Telephone Town Halls and livestream work like this:
Those watching the livestream can submit their questions digitally through the platform's comments section, while those on the phones can submit the question to the screener who then relays it to the moderator.
"The only thing you miss from a traditional Telephone Town Hall is the live asking of the question," said Wrigley.
While the technology to support these events isn't new, their popularity and users' desire to merge the two is growing during the pandemic. "Now it's really come to the forefront as something that campaigns and organizations really want to do because their audiences are all at home, and they're doing video chats for work or staying in touch with family, so the familiarity is there," said Wrigley.
In fact, other practitioners in the campaign industry are also observing increased response rates to phones outreach. Democratic pollster John Anzalone put it this way to the Washington Post: "It's like it's the 1990s and everyone is basically an old woman in rural Iowa picking up their landlines."
For those planning to host an event, here are a few things to remember:
Have a lively moderator with a nicely staged background. Her or his voice will keep the telephone audience engaged, and good lighting and staging helps for the video audience.
Take questions from both the Telephone Town Hall participants and the live stream folks. Remember your live stream folks may be more knowledgeable about your effort or are already supporters, the folks on the Telephone Town Hall need to be persuaded – so don't preach to the smaller streaming audience and forget the people you need to bring to your side.
Scheduling is key for reaching a wide audience. If your event is local, consider a time between 6 and 8 p.m. in your time zone. If your event is national, consider a time around 7:30-8 p.m. EST to engage audiences on both coasts.
PrevPreviousWhat Is a Telephone Town Hall?
NextTelephone Town Halls Strengthen Donor Relationships Even More During The PandemicNext
The Importance of Scripting for Mail-in-Ballots
This November millions of Americans are more hesitant to vote in person due to the pandemic and a desire to keep their loved ones
Stones' Phones	September 12, 2020
Telephone Town Halls Strengthen Donor Relationships Even More During The Pandemic
In this time of uncertainty, we are all trying to maintain our connections. Whether with family, friends, or work, we are often relying on virtual
Stones' Phones	June 2, 2020
How To Do Them
They Work
Patch Through
Story Capture
Manual Texting
Auto Calls
Live Calls


I saw this old well the other day and although the metalwork is rusted and the rope looks dry as dust, clearly there's water there….just look at that abundance of foliage.
I've got an old well in my garden too. It's a long way down to the water but plants push their way out of the mouth of the well all year around.
Sometimes I think we take plants for granted. Look how they can take root and flourish in the least likely situations. They can spread their seeds far and wide, take root in pretty nutrition-deficient environments, capture carbon and oxygen from the air, soak up the sun's rays and with a little water added can make themselves big and beautiful.
Human technologies just can't match them.


text:
//
// Generated file, do not edit! Created by nedtool 5.0 from messages/DVCast.msg.
//

#ifndef __DVCAST_M_H
#define __DVCAST_M_H

#include <omnetpp.h>

// nedtool version check
#define MSGC_VERSION 0x0500
#if (MSGC_VERSION!=OMNETPP_VERSION)
#    error Version mismatch! Probably this file was generated by an earlier version of nedtool: 'make clean' should help.
#endif

// cplusplus {{
#include "veins/modules/messages/WaveShortMessage_m.h"
#include "veins/base/utils/Coord.h"
// }}

/**
 * Class generated from <tt>messages/DVCast.msg:38</tt> by nedtool.
 * <pre>
 * //
 * // TODO generated message class
 * //
 * packet DVCast extends WaveShortMessage
 * {
 *     int id;
 *     Coord roi_up;
 *     Coord roi_down;
 *     double angle;
 * }
 * </pre>
 */
class DVCast : public ::WaveShortMessage
{
  protected:
    int id;
    Coord roi_up;
    Coord roi_down;
    double angle;

  private:
    void copy(const DVCast& other);

  protected:
    // protected and unimplemented operator==(), to prevent accidental usage
    bool operator==(const DVCast&);

  public:
    DVCast(const char *name=nullptr, int kind=0);
    DVCast(const DVCast& other);
    virtual ~DVCast();
    DVCast& operator=(const DVCast& other);
    virtual DVCast *dup() const {return new DVCast(*this);}
    virtual void parsimPack(omnetpp::cCommBuffer *b) const;
    virtual void parsimUnpack(omnetpp::cCommBuffer *b);

    // field getter/setter methods
    virtual int getId() const;
    virtual void setId(int id);
    virtual Coord& getRoi_up();
    virtual const Coord& getRoi_up() const {return const_cast<DVCast*>(this)->getRoi_up();}
    virtual void setRoi_up(const Coord& roi_up);
    virtual Coord& getRoi_down();
    virtual const Coord& getRoi_down() const {return const_cast<DVCast*>(this)->getRoi_down();}
    virtual void setRoi_down(const Coord& roi_down);
    virtual double getAngle() const;
    virtual void setAngle(double angle);
};

inline void doParsimPacking(omnetpp::cCommBuffer *b, const DVCast& obj) {obj.parsimPack(b);}
inline void doParsimUnpacking(omnetpp::cCommBuffer *b, DVCast& obj) {obj.parsimUnpack(b);}

#endif // ifndef __DVCAST_M_H

redpajama_set_name: RedPajamaGithub


text:
Tertiary Education Quality and Standards Agency Annual Report 2019-20
Response to COVID-19 pandemic
At the onset of the COVID-19 pandemic in March 2020, TEQSA quickly mobilised support for the Australian higher education sector. The agency deployed a number of initiatives, including:
streamlining assessment processes for Graduate Certificate and Undergraduate Certificate programs to support providers to offer these online short courses to students
refunding and waiving eligible fees paid by providers as part of the Australian Government's Higher Education Relief Package
liaising with local and international peak bodies to stay informed of the evolving situation, domestically and internationally
continuing to update TEQSA's website with information and resources to support the sector.
A dedicated online learning good practice website was the first key resource developed to support providers in making the rapid transition to online delivery as a result of the pandemic restrictions. The resources made available covered a range of topics related to online learning, from getting started and enabling staff to work online, to supporting student experiences and assessment integrity. From its launch in April 2020 to June 2020, the website was visited more than 17 300 times. More information about the website is available at www.teqsa.gov.au/online-learning-good-practice.
The COVID-19 pandemic had a significant effect on TEQSA's work practices, with staff moving to working from home arrangements in March 2020. Measures such as lessening the administrative burden of regulation on providers through granting extensions of registration and accreditation periods and focusing on individual provider support, directly and through liaison with peak bodies, were also implemented. For more information about adjustments TEQSA has made to support staff see Section 4: Human resources.
Due to the impact of the COVID-19 pandemic, the 2020 survey of stakeholders was not conducted and instead a series of focus groups with provider peak bodies were held to gauge stakeholder views of TEQSA's performance during 2019-20. As a consequence, some aspects of TEQSA's performance were not able to be measured.
Introductory statement
Australian National Audit Office performance audit
https://www.transparency.gov.au/annual-reports/tertiary-education-quality-and-standards-agency/reporting-year/2019-20-9
TEQSA Annual Report 2019-20
Accessing this report online
Section 1: Review by the accountable authority
Review by the accountable authority
Section 2: Agency overview
About TEQSA
Section 3: Performance review
TEQSA's objectives 2019-23
Performance against objectives
Analysis of TEQSA's financial performance
Section 4: Management and accountability
Section 5: Financial report
Independent Auditor's Report
Statement by the accountable authority and Chief Financial Officer
Statement of Comprehensive Income
Statement of Changes in Equity
Administered Schedule of Comprehensive Income
Administered Schedule of Assets and Liabilities
Administered Reconciliation Schedule
Administered Cash Flow Statement
Notes to and forming part of the financial statements
Section 6: Appendices
Appendix A: Legislative framework
Appendix B: Summary of resources
Appendix C: Staffing profile
Appendix D: Freedom of Information
Appendix E: Ecologically-sustainable development and environmental performance
Appendix F: Advertising and market research
Appendix G: Workplace health and safety
Appendix H: Complaints handling
Appendix I: Disability reporting
Appendix J: Corrections to previous annual report
Section 7: Indices and References
Compliance index


Your network is the backbone of your business. Just think about all the ways you rely on your IT tools. You can't afford downtime. You can't even afford inefficiency. A sluggish network means lower productivity—and lower profits.
That's why a technology assessment is so critical.
We'll analyze your IT infrastructure from top to bottom and provide you with a thorough report. If there are opportunities for improvement, we'll let you know. There's no reason to put up with poor performance.
What do you get with a technology assessment?
Why do you need a technology assessment?
You can't improve without first knowing where you are. An in-depth technology assessment gives you the ability to be deliberate and calculated as you plan for future growth. After all, it's not enough to have a functional IT network today. You have to plan for tomorrow, as well.
When your network goes down, you lose money. The best way to avoid downtime is to catch problems before they start. A tech assessment does just that.
Constant computer issues kill morale like nothing else. Keep your staff productive and happy with a stable network that works the way it should.
Best-in-class technology changes all the time. A thorough technology assessment will help you decide what to upgrade to remain competitive.
How does a technology assessment work?
We'll take a look at your current equipment to get a full picture of what you're working with right now. We'll also ask some strategic questions to identify any pain points. Then we'll use our expertise to determine the best tech options to address shortcomings and misfires.
Getting a technology assessment doesn't mean your IT infrastructure is below par. It just means you're serious about being the best you can possibly be.
We'll find the top opportunities for improvement and hand off our recommendations. What you do from there is entirely up to you.


Amazing Wedding Bracelet Jewelry!. Gorgeous, Stunning, Breath taking awesome Pearls with rose gold roundels and pave balls embedded with Zircon all over, a Unique and Scintillating Wedding Bracelet for Mother of Groom, Mother of Bride, Grandma of Groom, Grandma of bride, Nana of Groom, Nana of Bride, Bridesmaid, Matron of honor, Maid of honor gift.
If you wish to order for kids like daughter, daughter of bride, daughter of groom, flower girl, junior bridesmaid or mini bride then please mention the age of the child before check out of the order. Thank You!
Please See options to purchase and select the charm of your choice.
So beautiful and arrived very quickly! 💞 I love it, more beautiful than the pictures!


text:
Q: INNER JOIN SQL ORDER BY NOT WORKING WORKING!
SELECT s.*, COUNT(rs.id_segreto) as tot
FROM Segreti s INNER JOIN
     Reazioni_Segreti rs
     ON s.id = rs.id_segreto
WHERE rs.tipo_reazione = 'mi_piace'
GROUP BY s.id
ORDER BY tot DESC
LIMIT 10;

But it print me only 1 row.
$rs_result = mysql_query($sql);
while ($row = mysql_fetch_array($rs_result, MYSQL_ASSOC)) {
echo $row['id']."<br>";
}

Why happened? What i wrong?

A: This is a syntax that MySQL allows, but the SQL standard does not -- nor do other databases.  You have a multitude of ungrouped fields in theSELECT, but the COUNT() turns this into an aggregation query.
With no group by, such a query always returns exactly one row.
I would also recommend using table aliases:
SELECT s.*, COUNT(rs.id_segreto) as tot
FROM Segreti s INNER JOIN
     Reazioni_Segreti rs
     ON s.id = rs.id_segreto
WHERE rs.tipo_reazione = 'mi_piace'
GROUP BY s.id
ORDER BY tot DESC
LIMIT 10;

Note that the use of s.id (rather than listing all the columns) in the GROUP BY is allowed -- even under ANSI -- assuming that id is defined to be unique in the table.

redpajama_set_name: RedPajamaStackExchange


text:
package Tetris::Shape;

use strict;
use Data::Dumper;
use Class::Accessor;

use fields qw(ths);
use base qw( Class::Accessor );
my @accessors = keys %Tetris::Shape::FIELDS;

__PACKAGE__->mk_accessors( @accessors );

sub new {

   my $class = shift;

   my $data = shift;

   my $self = fields::new($class);

   return $self;

}


text:
package me.kenzierocks.autoergel.osadata.data.value.mutable;

import java.util.Set;

import me.kenzierocks.autoergel.osadata.data.value.immutable.ImmutableSetValue;

/**
 * Represents a type of {@link CollectionValue} backed by a {@link Set}. The
 * reasoning is that a {@link Set} retains no ordering of the elements it
 * contains.
 *
 * @param <E>
 *            The type of elements supported
 */
public interface SetValue<E>
        extends CollectionValue<E, Set<E>, SetValue<E>, ImmutableSetValue<E>> {

}


James Nyhan married Catherine Lordan in Cork @1854-55, probably Dunmanway. They had several children before emigrating to the USA, where they settled in Chicago. SOME of the children are John, Mary,1856; Catherine,1859; Ellen, 1861; Thomas, 1863; Julia, 1864; Patrick, 1866.I believe Nora was another.
Parish of St Patrick (in Dunmanway)records for most of these kids are on the way to me; however,the marriage was not found, nor was the birth of John. Does anyone have any info? He was my direct ancestor and this record is needed.


I have a survey with general likert scale questions and then an open ended question. Every survey respondent has a unique ID which is a mix of letters and numbers.
I've built a codeframe for the open ended question to summarise the insights. So let's say the question is "What did you like about your experience today?" And say the respondent answered "The customer service was wonderful and the turn-around time was fast!". Each survey respondent is allocated two response options - that is, we record two coded responses per person as appropriate. In this example, the individual will be allocated code "1" for "Excellent / good customer service" and code "3" for "Good / fast turnaround time".
I've then created a separate excel spreadsheet and reshaped the data, and in this table each survey respondent has two rows - giving an answer (or a blank) to each of the two response fields.
In Tableau, I then join this spreadsheet to the master spreadsheet (containing the likert scale questions) using a left join. I then easily created a summary table showing the % who mentioned each code for my open ended question.
The problem is that there has been an unexpected knock-on effect on some calculations on my likert scale questions.
Since creating this join, I've found this calculated field is now double counting records which have reshaped data, and as a result my outputs from the calculation are wrong. I keep trying to allocate the "COUNTD" function but it's not making a difference. How do I adjust the If/then statement to only calculate on unique survey IDs ?
Any insights would be gratefully received.
There are a few ways to work around the problem of duplicated rows in "stacked" data.
For views that don't include the multi-response questions, you can add a filter to the worksheet: response_row=1, preferably as a context filter. Context filters are evaluated before sets and other regular filters; right click the filter > Add to Context.
Need to remember to add the filter.
You'd use your original dataset as is, and the new dataset when you need the multi-response questions in the view.
You could either 1) create a full data set with both types of questions, which will make it easier to put both types of questions in the view 2) create a data set with just the multi-response questions (safer, but more difficult to blend).
Need to maintain two data sources.
IF FIRST()==0 with addressing / compute using set to respondent ID, partitioned by question ID and all other dimensions.
You don't have to modify your data source.
I usually go with the second option, and use this data source only on the views with multi-response questions. I don't trust myself to remember that I need to add filters, especially if I haven't opened the workbook in six months.
On the other hand, if you have just a few measures / calculated fields you're using, the first option can work well.
Jim I am sincerely grateful for your advice - thank you ! I think I might go with the first option given I've only got a couple of calculated fields. You've reminded me that I should have also mentioned I don't have duplicated records for every single survey respondent. This is because I'm combining data from a 8 different surveys into one master file, and the open ended question was only asked in 6 of those surveys. More specifically, only 2/3 of survey respondents have multiple rows. But I can work around this in excel using an if / then statement to determine the appropriate text in the new "response_row" field. I'll let you know how I go - and thank you again.


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Q: Calculate the integral $\int_{[0,1]\times[0,1]}\frac{1}{(1-xy)^a}dydx$ for $a>0$ I'm having trouble with the integral $\int_{[0,1]\times[0,1]}\frac{1}{(1-xy)^a}dydx$ for the case in which $a\neq{1}$. For the case in which $a=1$ it was not difficult to calculate the integral and I got the convergent series $\sum_{n=1}^\infty\frac{1}{n^2}$.
Now, for the case in which $a>0$ and $a\neq{1}$, first, I calculated the integral $\int_0^1\frac{1}{(1-xy)^a}dy$ and by making a change of variable I got the result $\frac{-1}{x}$($\frac{(1-x)^{1-a}}{1-a}$ - $\frac{1}{1-a}$) which can be simplified to $\frac{1-(1-x)^{1-a}}{x(1-a)}$ (I'm almost sure this is right, but if anyone finds my calculation of this integral with respecto to $y$ is wrong, let me know).
So, then I would have to calculate the integral $\int_o^1\frac{1-(1-x)^{1-a}}{x(1-a)}dx$ and this is where I'm stuck. Maybe I should express one of the expressions in the integrand as a series? Or is there any easier approach to the problem? Any suggestions would be appreciated.

A: Given the integral you have reached,
$$ \frac{1}{1-a}\int_0^1 \frac{1-(1-x)^{1-a}}{x} \, dx, $$
if you substitute $t=1-x$, you obtain
$$ \frac{1}{1-a}\int_0^1 \frac{1-t^{1-a}}{1-t} \, dt = \frac{1}{1-a} H_{1-a},  $$
in fact by the definition of the harmonic numbers for $a<1$. This looks like a cop-out (because it is), but it comes from one of the ways of defining the digamma function.


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James Gunn's GUARDIANS OF THE GALAXY VOL. 2
May 24, 2017 Frank Mengarelli	Leave a comment
James Gunn doesn't quite surpass the first Guardians film with his followup, but there is more than enough to love from a sequel that stands monumentally taller than any other Marvel film (save for the first).
Gunn is such a remarkable auteur; his use of seminal popular music, blended with his not only perfect casting of genre actors but knowing how to use them, is what keeps this Guardians film from being a rehash of the first.
The story, while at times has too many plot points running at once, stands on its own, and is not reliant upon any other arc within the Marvel Cinematic Universe. That is incredibly refreshing. The film is about Star-Lord and his father Ego, played by Kurt Russell who turns in yet another fantastic performance.
Guardians 2 does use a few conventional gimmicks: the token Stan Lee cameo that has worn out its effectiveness sixteen movies ago, and an opening scene with a CGI de-age character which actually works well. Aside from that, and a second act that drags its feet slightly, the film is a lot of fun and you'll be smiling and laughing through the entire film. Heck, you may even tear up during a few moments.
What's very disappointing about this film, is the incredible missed opportunity of reuniting onscreen Kurt Russell and Sylvester Stallone. Call me shallow, but that's a moment a lot of us were hoping for going into this film, knowing the kind of genre respect and sensibilities that Gunn has as a filmmaker, it is kind of a shock that this didn't happen.
Speaking of Stallone, seeing him in a film like this is an absolute joy. He doesn't have a whole lot to do in the film, he's mainly being setup for an expanded role in future Marvel films, but you can tell he's having a lot of fun. Towards the beginning of the film, he shares a scene with Michael Rooker, and anyone who loves CLIFFHANGER will stand up and fist pump in the theatre.
Perhaps the best, and most effective part of the film isn't the special effects (which are brilliant), or the genre actor cameos (which is even more brilliant), but a scene between Star-Lord and his Ego, as they discuss The Looking Glass' hit song, BRANDY. It's a very sweet and emotional moment between a father and son and showcases the star power that Russell brings to the role.
There are a plethora of scene stealing moments. The opening scene, the opening credits, the musical numbers, Baby Groot, Awesome Mix Tape Vol. 2, Michael Rooker – like I said, this film may not be as good as the first, but it's an awesome experience and do yourself a favor and run the theatre to go see it.
What's almost hard to understand is how Marvel allows Gunn to make non-templated films that are a part of the MCU, yet really have nothing to do with any of these silly "phases". The two Guardians films are different, they don't fit inside of Marvel's box of conventionality. They take place within a world where Gunn has the absolute freedom to do whatever he wants, and that in itself is a feat that is a cause for celebration, and very much leaves you looking forward to the next Guardians film.
Baby GrootBradley CooperChris PrattDave BautistaDraxEgogrootGuardians of the GalaxyGuardians of the Galaxy Vol. 2James GunnKurt RussellMantisMarvelMCUMichael RookerMichelle YeohMiley CyrusRocketStar-LordSylvester StalloneVing RhamesYonduZoe Saldana
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8 incredible weekend trips from Madrid
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Just Travelling Through
Hi! My name's Stuart. Just a normal lad from Yorkshire who travels around the world. I'm here to share my experiences on the road from cities and countries around the world, including some less well-known places off the beaten path.
Six continents down (so far!) and a few years living in Spain. I don't tend to stay in one place for too long. As the name says... Just Travelling Through
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It turns out sunrises in Santander across the bay
Jerez, just a short trip from Cadiz, was a much be
There's some narrow streets around Vejer de la Fro
The bright white houses all over Vejer create a st
Of the many famous White Villages of Andalucía, V
Things to do in Cadiz (save for later) At the ver
The top of the Tavira Tower give some of the best
Along Cadiz's seafront lives a colony of stray cat
The view from one of Cadiz's castles to the other.
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I've been using Windows 8 as a dual-boot option on my iMac for a while. When Windows 8.1 came out, I was pretty excited, as Peter Bright's review on Ars Technica was quite positive about it.
Unfortunately, I was getting errors doing the upgrade it directly from the Microsoft Store, eventually getting Error 0x0073B92 - 0x20009. I was a little stumped about how to get around this.
The solution was actually simple: disconnect all of your USB devices during the installation. (Internet searching seems to indicate that this is related to a problem with a second hard drive).
This entry was posted in Using software and tagged Microsoft, operating system, Upgrade, Windows 8, Windows 8.1 on December 5, 2013 by irwinhkwan.


Erin Mahone is a wife, mother of three, writer and storyteller. She is the author of #IfYouCouldSeeMe: Life, Motherhood, and the Pursuit of Sanity. She presents a one-woman show about life, laughter, mental illness and love, titled "It Runs in the Family," and invites others to share their stories.


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So keep in mind that on this single day we flew over three very magical areas around Page and also hiked two of them. As you might imagine, we have some pix we like from that day. Here are just a few of the ones I like a lot from our hike through Lower Antelope Canyon, though none really begin to capture what it looks like much less the impact of being there.
It's quite challenging to get pix that capture the range of light, from deep shadows to bright sunlight. But this one looking up the canyon from the entrance area near the top of the stairs gives at least an impression. It looked pretty mysterious and deep, and a few people worried aloud about claustrophobia and how narrow it might be in places. Here's a look down into the entrance, with the bottom of the metal stairs visible.
Descending was quite enchanting, and I noticed that nearly everyone grew quietly reverent. After setting foot on the sandy bottom, everyone seemed to me quite relaxed with any misgivings completely forgotten. Though it's quite deep, the bottom is mostly much wider than the top. The walls are luminous with light reflected down the narrow upper channels making the bottom seem almost lit from within.
It's a dream cave. Light, dry, airy, smelling of clean sand, and delightful to touch. Also a photographer's dream, with so many views like this. Most people in our small group were snapping a lot, but a few just strolled in silence and some like me did both.
Most of us moved very slowly, because around every turn you could get very different and evocative perspectives just by turning your head or moving a few inches. We were art lovers in an exceptional gallery.
Shifting your perspective would also make the camera see the same spot differently, due to how the settings would change. This is basically the same spot, for example.
In a few places you could catch glimpses of the sky, and tilting the camera straight up could give the impression of looking horizontally at a mountain range with sunlight falling on some rock faces and reflected light bringing a glow to others.
Some of the shapes were just so magical. I don't recall now whether this was looking up, down or through the passage.
It's interesting to me how taking pix both puts me more in the moment, and less. In looking for and snapping pix, the eye Notices and Attends more. Pix also provide an instant window back into the Moment, even years later. Yet I was always glad to lower the camera, breathe it all in, caress the walls with hand and eye, allow imagination to blossom, and even close my eyes. Thank you, Zubair, for placing this hour into the earth and out of time high on our agenda for the trip.


"On the extended Rutgers-Delaware rule for MR suspensions under magneti" by Weihua Li, Yongbo Yang et al.
Chen, G., Li, L., Yang, Y. and Li, W. 2006, ''On the extended Rutgers-Delaware rule for MR suspensions under magnetic fields'', International Journal of Modern Physics B, vol. 20, no. 5, pp. 579-592.


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Ahrobiznes-TSK Romny (ukr. Футбольний клуб «Агробізнес-TSK» Ромни, Futbolnyj Kłub "Ahrobiznes-TSK" Romny) – ukraiński klub piłkarski, mający siedzibę w miasteczku Romny w obwodzie połtawskim.

Historia
Chronologia nazw:
 2014—...: Ahrobiznes-TSK Romny (ukr.«Агробізнес-TSK» Ромни)

Klub piłkarski Ahrobiznes-TSK został założony w miejscowości Romny w roku 2014. Głównym sponsorem jest miejscowe przedsiębiorstwo o tej samej nazwie "Ahrobiznes-TSK", które specjalizuje się w uprawie zbóż i roślin przemysłowych. W 2014 zespół startował w mistrzostwach obwodu połtawskiego.

W 2015 klub debiutował w rozgrywkach Pucharu Ukrainy wśród drużyn amatorskich.

Latem 2016 otrzymał prawo gry w rozgrywkach Pucharu Ukrainy.

Sukcesy

Trofea międzynarodowe
Nie uczestniczył w rozgrywkach europejskich (stan na 31-05-2015).

Trofea krajowe

 Amatorski Puchar Ukrainy:
 półfinalista: 2015
 Mistrzostwo obwodu połtawskiego:
 mistrz: 2014, 2015
 Puchar obwodu połtawskiego:
 zdobywca: 2014, 2015
 Superpuchar obwodu połtawskiego:
 zdobywca: 2014

Stadion
Klub rozgrywa swoje mecze domowe na stadionie im. Wołodymyra Okіpnogo w Romnach, który może pomieścić 3000 widzów.

Trenerzy
 201?–...:  Wałerij Kliszczenko

Zobacz też
 Ełektron Romny

Przypisy

Bibliografia

Kluby piłkarskie w obwodzie połtawskim
Kluby piłkarskie założone w 2014
Romny

redpajama_set_name: RedPajamaWikipedia


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West Island BlogNews from the west island of montreal
CTV's 6 Pm News Pre-Empted Due NFL Schedule
Riviere-Des-Prairies-Pointe-Aux-Trembles Shooting Leaves One Dead
Montreal's Tampa Bay To Unveil Team Graphic When Views Peak During...
CTV's 6 pm News has been pre-empted owing to the NFL football, although viewers will still get updates relating to top headlines of the day. Viewers can tune int the...
A man sustained fatal gunshot injuries last Sunday evening a shooting in the Riviere-des-Prairies-aux-Trembles, Montréal. According to police reports, the man 33, was found on the ground, dead, some minutes...
Montreal's Tampa Bay To Unveil Team Graphic When Views Peak During Playoffs
Montreal's baseball fans should expect a surprise that will be displayed on the right field wall at the Tropicana as the MLB play-offs go in which the Tampa Bays...
Quebec Legislators Call Out English Speaking Children, Blames Their Lack Of French Fluency On...
Quebec's school children were the centre of bill 96 tough exchanges last Thursday over provincial language laws. English speaking children were lambasted over what legislators called their lack of...
Quebec Enacts Law Prohibiting Anti-Vaccine Protests Near Schools, Hospitals
After only a handful of hours of deliberations, the National Assembly swiftly passed Bill 105 into law prohibiting anti-vaccine demonstrations nears schools and hospitals. The new law prescribes a...
Bonuses Set Up For Health Workers In Understaffed Public Network
Quebec health system is in the middle of s staffing crisis and is planning to cut cheques to its health workers in order to bolster system that is under...
Two Montreal Schools Closed due to COVID-19 Outbreak
Two Elementary Schools in Montreal were closed this week as a result of a COVID-19 outbreak. The first confirmed closure was that of Saint-Odile Elementary, on Depatie Street, Ahuntsic-Cartierville. All...
Quebec Plans To Table Anti-Vax Protestors Bill Thursday, One MNA Likely To Stay Out
Quebec's Francois Legault noted the province plans to table a bill this Thursday that will fine anti-vax protestors who are found causing disturbance or blocking access to schools and...
Bloc Quebecois Secure Two More Seats, Ballot Counts In One Riding Still Too Close...
Ruth Brosseau's return to politics after a two-year hiatus farming may have to wait a while longer. The last mail-in ballots have been counted and the results favour the...
Quebec Might Enact Law To Deter Anti-Vax Protesters From Blocking Access To Schools, Hospitals
Premier Francois Legault stated announced Tuesday that it is not acceptable for protester against the COVID-19 vaccines to block access to schools and hospitals, adding that the province might...
A new casino for real high rollers December 27, 2021 1:41 am
Manitoban with Several Chemical Sensitivities Wants Broader Exemptions, Alternatives to Vaccine Passports September 27, 2021 6:01 am
14-Year-Old Boy Dead Following Collision in East Gwillimbury, Ontario September 27, 2021 5:59 am
Elective Surgeries on Hold Due to Oxygen Problem at Stanton Territorial Hospital September 27, 2021 5:58 am
CTV's 6 Pm News Pre-Empted Due NFL Schedule September 27, 2021 5:56 am


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Awards Show
Nicki Minaj's Team Is Furious After Singer Is Snubbed From Being Nominated At The Grammy Awards
Awards ShowNicki Minaj
Nicki Minaj's team have vented out on Twitter after finding out she had been snubbed from being nominated at next year's Grammy Awards. The singer, who achieved a platinum selling album with Pink Friday: Roman Reloaded earlier this year, was said to have been upset that producers had not taken her success since last year's Grammys into consideration. But it seemed like no one was more upset than her manager, Cortez Bryant who explained how he felt about it on Twitter.
Bryant was happy to hear Drake and Lil Wayne – who are all under the same label – had received several nominations in the rap categories, but Nicki failed to make the cut and some people are saying it's due to her spat with pop singer Mariah Carey two months ago. Though her platinum selling second studio album went platinum, the reviews were terrible and producers of the Grammys take this kind of information into consideration.
Cortez had written on the social networking site:
"Congrats to @LilTunechi and @Drake and @Hit_Boy on their Grammy Noms butttt….. They was on some bulls**t for not nominating @NICKIMINAJ."
Nicki shouldn't be too upset because Justin Bieber and One Direction were also avoided from next year's nominations. While Scooter has already expressed his feelings about the decision for Bieber not to be nominated for any of the awards, Simon Cowell has said that his boy band are not bothered by it at all, adding that they will most likely make the cut next year.
Cowell told MTV News:
"You know what's more important? Fans, record sales, I've always said that. Maybe next year they'll get something. Trust me, they're not complaining."
Louis Tomlinson and Danielle Campbell Dating: Romantic Ice Cream Date – Baby-Mama…
Nicki Minaj Brother Jelani Maraj Arrested In Nassau County For Raping A Child
Zayn Malik Finally Discusses Quitting One Direction And First Solow Album – Listen To…
Louis Tomlinson DNA Test Drama: Pregnant Baby-Mama Briana Jungwirth Reportedly Slept…


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Working with Dean Hoyle at Huddersfield Town: Jan Siewert inspiration for Championship
It would be great to follow David Wagner and Daniel Farke in winning promotion
Town head coach Jan Siewert speaks with Jurgen Klopp before the Liverpool match
Jan Siewert says he is enjoying working with Dean Hoyle at Huddersfield Town and gaining inspiration for the Championship challenge to come.
The 36-year-old is bidding against Manchester United to avoid a club record nine successive league defeat in what is Town's final home match of their two-season first Premier League advanture.
He admits, however, that working towards next season's campaign began the minute relegation was confirmed at Crystal Palace with still six matches to play.
And, having noted his Borussia Dortmund II predecessors David Wagner and Daniel Farke (at Norwich City) have both won promotion to the top flight in England, Siewert is keen to add his name to the list.
"It would be great if I'm the next one, so I am looking forward to it," said Siewert, who took over from Wagner at Town back in January, since when the club have picked up three points from 42.
"I signed a two-and-a-half-year contract so it was important for me to have a long-term view when I came in, and it was important for me to learn about the players in each game.
"I have to have a view on each player and learn in the remaining games."
Town play United on Sunday and Southampton the following Sunday before heading into the summer break.
They have announced a pre-season training camp in Austria in preparation for the big Championship kick-off on the first weekend of August.
"After relegation was confirmed we could put even more work into our plans, look at the Championship, going to more games and making observations live," revealed Siewert, in an interview with Sky Sports.
"It's obviously important to know more about the league for the next year and to be prepared for it.
"The grand plan is making the right decisions and seeing my face on the pitch with the players.
"This means my idea, my identity, my behaviour and my attitude. This is what I am really keen on and I'm going for it."
Faces in the crowd: can you spot yourself in the Town end at Anfield?
It's anticipated there will be a big turnover of players this summer as Town re-shape the playing roster on a lower budget.
Siewert says players wanting to leave is normal following a relegation campaign.
"Each player experienced that it wasn't a good season, so they have their ideas now and we have our ideas as a club - and this all comes together now and, at the end, we will have a solution for it," he added.
"This is why I like the club because it is prepared and always was.
"It was always an honest conversation about the things which could happen and so it was honest for me. That's why I signed here.
"I liked the way the chairman was and the way the people who are responsible here were, so this was important.
"We have a long-term view and to go through this process keeps you together. This is important for me."
Look back on Huddersfield Town's famous 2-1 win over Manchester United in 2017
As Siewert revealed in his press conference before the Liverpool game, he is in regular contract with Hoyle and delighted the chairman is now getting over the illness with prevented being so hands-on this season as he has been in the past.
"It was also not easy for me, but I am in touch with him more often and for me it's good to see that he is getting healthier and that he is vivid," said Siewert of the club owner.
"We always were in touch, and we take care for each other. This links us."
Dean Hoyle