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Clinical Aspects
A Tri-Nucleotide Pattern in a 3' UTR Segment Affects The Activity of a Human Glucocorticoid Receptor Isoform
Green, Tajia L.; Leventhal, Stacey M.; Lim, Debora; Cho, Kiho; Greenhalgh, David G.
Shriners Hospitals for Children Northern California, Sacramento, California
Department of Surgery, University of California, Davis, Sacramento, California
Address reprint requests to David G. Greenhalgh, MD, Shriners Hospitals for Children Northern California, 2425 Stockton Blvd, Sacramento, CA 95817. E-mail: dggreenhalgh@ucdavis.edu
Received 27 July, 2016
Revised 16 August, 2016
Accepted 31 August, 2016
T.L.G., S.M.L., and D.L. contributed equally to this study.
This work was supported by Shriners of North America (grant #85230).
The authors report no conflicts of interest.
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (www.shockjournal.com).
Shock: February 2017 - Volume 47 - Issue 2 - p 148-157
doi: 10.1097/SHK.0000000000000750
We previously identified a truncated human glucocorticoid receptor (hGR) isoform of 118 amino acids, hGR-S1(-349A), that despite lacking the major functional domains, was more hyperactive after glucocorticoid treatment than the full-length receptor. Furthermore, its 3' untranslated region (UTR) was required. To dissect the underlying mechanisms for hyperactivity, a series of hGR isoforms with consecutive deletions in the 3' UTR were created to test their transactivation potential using reporter assays. The hGR-S1(-349A) isoform retaining 1303 bp of 3' UTR displayed unusually high activity with or without glucocorticoid stimulation. Unexpectedly, a complete loss of significant activity was observed with isoforms retaining 1293 bp or 1263 bp of 3' UTR. Analysis of the 20 bp region neighboring the 1293 bp site showed a pattern: 3'UTR termination at every third base pair in this region resulted in a loss of transactivation potential while the other sites retained hyperactivity with or without glucocorticoid stimulation. Variations in the activity of an hGR isoform, due to changes in the 3' UTR sequence configuration, may provide an important link in explaining inconsistent responses to glucocorticoid treatment in individuals and ultimately enable tailored, patient-specific care. Furthermore, understanding the mechanisms underlying the cyclic hyperactivity/loss of activity phenomenon may be a step toward identifying a novel mechanism of gene regulation.
Gene expression is highly regulated by epigenetic phenomena. The 3' untranslated regions (UTRs) of genes contribute significantly to that regulation by affecting mRNA stability, localization, and translation (1–4). There are several mechanisms through which the 3'UTR influences mRNA activity. AU-rich elements (ARE) are known to affect mRNA stability and microRNAs (miRNAs) bind to the mRNA and generally decrease expression (3, 5). The length of the 3'UTR, often regulated by alternative polyA sites, can also modulate mRNA expression (6). PolyA sites proximal to the stop codon tend to be preferred sites of regulation. They create shorter 3' UTR regions and usually result in greater protein expression (2, 7, 8). The increase in translation is possibly due to decreased availability of regulatory positions. Additionally, the 3' UTR secondary structure can also significantly influence mRNA translation (3).
Glucocorticoids, through binding to the glucocorticoid receptor, are major regulators of homeostasis. They are also used in the treatment of a wide variety of inflammatory conditions including septic shock. As such, securely controlling glucocorticoid receptor expression and activities are essential. The human glucocorticoid receptor (hGR) has an extensive 3' UTR region with multiple putative polyA signals (9, 10). During the initial identification of the hGR coding sequence, Hollenberg et al. (10) found that the proximal polyA signal at position 3101 (X03225) (corresponding to position 3963 of the current hGRα reference mRNA sequence NM_001018077) is the preferred site in a human placental cDNA library. It has also been found that an additional regulatory element, the class I ARE motif AUUUA, is overrepresented in the hGR 3' UTR (9, 11). There are 10 AUUUA sites in the 3' UTR of the biologically active splice variant, hGRα, and 4 sites in hGRβ, the major alternative splice variant. Three of the AUUUA motifs in hGRβ have been shown to be involved in hGRβ destabilization and for the AUUUA motif at position 3669, a naturally occurring A to G polymorphism has been linked to rheumatoid arthritis (12, 13). Schaaf and Cidlowski (13) also found that altering all 10 AUUUA sites in hGRα increased mRNA stability. Other important components in regulating hGR expression are the miRNA binding sites. The hGR 3' UTR, in general, has been predicted to have numerous miRNA sites (11). Two miRNAs, miR-18 and miR-124a, have been found to decrease protein expression and there is evidence that miR-124a may be involved in tissue-specific expression (14, 15). In a separate investigation in mice, miR-96, miR-101a, miR-142-3p, and miR-433 were individually able to decrease glucocorticoid receptor expression by up to 40% (11, 16).
An interesting hGR isoform has been identified in our laboratory, hGR-S1(-349A) (Fig. 1). This splice variant retains intron H between exons eight and nine and also has a deletion at position 349, resulting in a frame shift and early termination which generates a novel 3' UTR region (17). The putative protein has only 118 amino acids and lacks the transactivation, DNA binding, and ligand binding domains, which are the major functional domains. hGR-S1(-349A), however, has a hyperactive response to high doses of exogenous steroids in comparison to the reference hGRα. This hyperactivity is lost when the 3' UTR region is removed. In this study, we delved into the structure and function of the 3' UTR region to identify the component(s) contributing the hyperactivity of hGR-S1(-349A) and report an intriguing phenomenon relating to the 3' UTR length which results in an autonomous hyperactive function of hGR-S1(-349A).
Fig. 1:
Diagram of hGRα and hGR-S1(-349A).hGR consists of nine exons of which exons two through nine are translated. The biologically active isoform, hGRα, is 777 amino acids long and has a transactivation domain, DNA binding domain (DBD), hinge region (H), and ligand binding domain (LBD). hGR-S1(-349A) is a previously identified hyperactive isoform (17). hGR-S1(-349A) retains intron H between exons eight and nine, and has a single base pair deletion at position 349 of the coding sequence resulting in a frame shift and early termination. Its putative protein length is 118 amino acids. Arrows indicate primers used to amplify both isoforms. The stop codons and the -349A deletion are marked with vertical lines.
Identification, nomenclature, and construction of hGR recombinant isoforms
The discovery and identification of hGR-S1(-349A) (JN797823) was described earlier (17). The unique hGR isoform was isolated from one of 97 volunteers who were surveyed for variability in their receptors. The nomenclature of the isoforms was also discussed in the previous publication and was based on the published coding sequence of the most common hGR (hGRα). The reference hGRα mRNA sequence was obtained from the National Center for Biotechnology Informatics (NCBI) (NM_001018077). The term "hGR-S" signifies an alternative "splice" variant and the 1 signifies the first variant found in our laboratory. The term "-349A" signifies loss of an adenine (A) at site 349 of the coding sequence.
Polymerase chain reaction (PCR) was used to create the various isoforms. For the 3' UTR deletions, the hGR-S1(-349A) expression construct was used as the template and a forward primer upstream of the start codon (pc4A-1A) and a reverse primer specifically designed for the desired 3' UTR length was used. These isoforms were named hGR-S1(-349A) followed by their 3' UTR length. To create the isoforms lacking the H intron, PCR was used to amplify the first half from an isoform that contained the -349A deletion, and a back section from a different isoform without intron H. A second PCR reaction was then performed with the two sequences to concatenate the pieces into the desired single isoform. The complete list of primers used can be found in the Table, Supplementary Digital Content 1, http://links.lww.com/SHK/A480. The conjoined isoforms, as well as the other deletion isoforms that were created, were checked for accuracy by sequencing (MC Laboratories, South San Francisco, Calif or UCDNA Sequencing Facility, Davis, Calif). The hGR isoforms were cloned into the pcDNA4/HisMax vector (Life Technologies, Grand Island, NY) for functional analysis.
Measurement of transactivation potentials of hGR isoforms
An HEK293 cell subclone, tsA201, was obtained from Dr. Daniel Feldman at Shriners Hospitals for Children Northern California (2003) and was used for transfection with plasmids containing individual hGR isoforms. The cells were validated by Short Tandem Repeat profiling (ATCC, Manassas, Va). For each experiment, 10,000 to 12,000 tsA201 cells, suspended in 100 μL of antibiotic-free Dulbecco's modified Eagle medium (Life Technologies) and supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, Ga), were seeded on day zero in a 96-well plate and then incubated at 37°C with 5% CO2. On day one, the cells were transfected with the hGR isoforms and a glucocorticoid response element (GRE)-luciferase reporter plasmid (PathDetect GRE Cis-Reporter Plasmid; Agilent Technologies, La Jolla, Calif) using Fugene 6 (Promega, Madison, Wis) per the manufacturer's protocol. On day two, the cells were treated with graded concentrations of hydrocortisone (10–2 μM to 1 μM) or vehicle (0.9% saline) for 24 h before measurement of activity. The hydrocortisone concentrations are based on previous experiments (17–19). Pharmaceutical grade hydrocortisone sodium succinate (Pfizer, New York, NY; clinical anti-inflammatory adult dosage, 15–240 mg; half-life, 8–12 h) was used for these assays. 1 μM mifepristone (RU486; Sigma, St. Louis, Mo) was also used as a glucocorticoid antagonist. 50 mM ethanol was used as the vehicle control. Transactivation potential was determined with a Luciferase Assay Kit (Agilent). A Perkin-Elmer MicroBeta Trilux (Perkin-Elmer, Waltham, Mass) was used to measure luminescence.
Western blot analysis of hGR isoforms
tsA201 cells transfected with recombinant hGR isoforms were lysed in ice-cold lysis buffer (Agilent) supplemented with cOmplete Protease Inhibitor Cocktail (Roche, Indianapolis, Ind) and the supernatants harvested. Extracted protein was run on a 4% to 20% Criterion gel (BioRad, Hercules, Calif). Separated proteins were transferred to a polyvinylidene difluoride Hybond-P membrane (GE Healthcare, Piscataway, NJ) and then blocked with 5% nonfat dry milk, washed, and incubated overnight with the HisG-HRP antibody (Life Technologies) in 5% milk in phosphate-buffered saline with 0.05% Tween-20. Protein visualization was performed via chemiluminescence using the ECL Plus Western Blot Detection System (GE Healthcare).
PCR quantitation
Total RNA was isolated from tsA201 cells transfected with recombinant hGR isoforms with an RNeasy Mini Kit (Qiagen, Valencia, Calif) followed by cDNA synthesis with QuantiTech Reverse Transcription Kit (Qiagen) with an oligo(dT) primer (pr49.3: GGCCACGCGTCGACTAGTACTTTTTTTTTTTTT). Real-time RT-PCR was then used to quantify the amount of recombinant hGR mRNA expression with Brilliant III Ultra-Fast SYBR QPCR (Agilent). This was performed using specific DNA oligonucleotide primers, pd4A-1D (GGATCCAGTGTGGTGGAATTCTGCA) and hGR 2–2I (CTGCTGTGGGAATCCCAGGTCA). A second PCR reaction with B2M was performed as an internal control (B2M-1A: CAGGTTTACTCACGTCATCCAG; B2M-2A: CAAAGTCACATGGTTCACACG).
For luciferase assays, all experiment samples were run in triplicate, except for the vehicle treatments that were run in duplicate, and each experiment repeated at least two times. After multiple experiments confirmed the data patterns, the data from multiple experiments were combined and normalized for presentation of figures and statistics. Values are expressed as means with error bars representing standard error of the mean (SEM). SEM values for the 3D graph are available in Table, Supplementary Digital Content 2, http://links.lww.com/SHK/A481. The results were compared by one-way ANOVA and significance was confirmed with a Tukey's post hoc test.
Retention of intron H and reference hGRα 3' UTR do not contribute to hyperactivity of hGR-S1(-349A)
In a previous work, we found that the deletion, frame shift, and early termination in hGR-S1(-349A) creates a unique new 2805 bp 3' UTR region. In addition, the newly formed 3' UTR of hGR-S1(-349A) is necessary for its hyperactivity (17). Within the 3' UTR are two unique components, the retained intron H and 303 base pairs (bp) of reference hGRα 3' UTR after the reference stop codon. To investigate whether or not these two additions were responsible for the unexpected augmentation in activity of hGR-S1(-349A), either the 303 bp of 3' UTR, intron H, or both were removed (Fig. 2A). The transactivation potential of these new constructs was tested after stimulation with graded doses of hydrocortisone. All three new constructs [without reference 3' UTR (-UTR), without intron H (-H), and without reference 3' UTR/intron H (-H/-UTR)] had an augmented, dose-dependent response to hydrocortisone (Fig. 2B). At 0.01 μM, there is minimal response; however, the activity of hGR-S1(-349A) without intron H (-H) is greater than the other constructs (P <0.01). At 0.1 μM of hydrocortisone, all constructs have significant activity although both constructs without intron H (-H and -H/UTR) had greater activity than either hGR-S1(-349A) or hGR-S1(-349A) without the 303 bp of 3' UTR (-UTR) (P <0.05). The greatest activity was at the highest concentration, 1 μM, and there was no significant difference in the transactivation potential between any of the altered constructs and the original hGR-S1(-349A).
Effect of intron H and reference hGRα 3' UTR on the transactivation potential of hGR-S1(-349A).A, Line diagrams of the new hGR-S1(-349A) variant isoforms in comparison to reference hGRα. For reference, the stop codon (dashed gray vertical line) and primers used for amplification (arrows) are identified. Due to the primer position there is an additional 303 bp of hGRα 3' UTR after the reference stop codon. The sequences for hGR-S1(-349A) and its variants, -UTR (removal of the 303 bp of reference 3' UTR), -H (the removal of intron H), and -H/UTR (the removal of 303 bp of reference 3'UTR and intron H) follow. The position of the -349A deletion and new stop codon (vertical lines) are indicated. For comparison, the position of the reference hGRα stop is shown (dashed gray vertical line). B, When stimulated with graded doses of hydrocortisone, all constructs have a hyperactive response at higher doses (0.1 μM and 1 μM). The -H construct has a significantly greater hyperactive response at 0.01 μM (**: P <0.01), and at 0.1 μM the responses of -H and -H/-UTR are both greater than hGR-S1(-349A) and -UTR (*: P <0.05). There is no significant difference in transactivation potential between hGR-S1(-349A) and its variants at 1 μM hydrocortisone. H indicates intron H; Hyd, hydrocortisone; UTR, 303 bp of reference 3' UTR. Data is a combination of seven experiments and is presented as means ± SEM.
1303 bp of 3' UTR in hGR-S1(-349A) contributes to an autonomous hyperactive response
Due to the deletion, frame shift, and early termination, hGR-S1(-349A) has a novel 2805 bp 3' UTR. To determine if there is an optimal 3' UTR length or a critical region necessary for the hyperactivity of hGR-S1(-349A), the 3' UTR of hGR-S1(-349A) was truncated in approximate 300 bp increments from the 3' end of the reference hGRα. The transactivation potential of the deletion constructs was then tested with graded doses of hydrocortisone (0.01 μM, 0.1 μM, and 1 μM) (Fig. 3 and see Table, Supplementary Digital Content 2, http://links.lww.com/SHK/A481, which lists SEM values). The response of hGRα and hGR-S1(-349A) confirmed previous results where both isoforms have dose-dependent responses to hydrocortisone with hGRα activity peaking at 0.1 μM and hGR-S1(-349A) displaying a hyperactive response that peaks at 1 μM (17). Removing just the first 300 bp of 3' UTR resulted in an almost complete loss of activity and this was the case with most of the other deletion constructs. Unexpectedly, the construct retaining 1303 bp of 3' UTR resulted in a transactivation potential even greater than hGR-S1(-349A). Even more interesting was the finding that the hyperactivity did not require hydrocortisone stimulation. Therefore, the hyperactivity was autonomous of steroid stimulation. The hGR-S1(-349A)(1303) isoform is more than 4.5 times greater than reference hGRα peaks at 0.1 μM of hydrocortisone. The hGR-S1(-349A)(1303) also has more than double the activity of hGR-S1(-349A) when it peaks at 1 μM of hydrocortisone.
Transactivation potentials of ∼300 bp serial 3' UTR deletions of hGR-S1(-349A).The 3'UTR of hGR-S1(-349A) was truncated by ∼300 bp increments and stimulated with graded doses of hydrocortisone (selected isoforms shown). Line drawings of each isoform are shown under the graph. As expected, there is a dose-dependent response by hGRα which peaks at 0.1 μM of hydrocortisone, and a dose-dependent response by hGR-S1(-349A) which peaks at 1 μM hydrocortisone. The transactivation potential of hGR-S1(-349A)(1303) is significantly greater than all other isoforms with saline vehicle or with hydrocortisone at all concentrations. The activities of all other isoforms are negligible, as are hGRα and hGR-S1(-349A) with the saline vehicle. Hyd indicates hydrocortisone. Data is a combination of two experiments and is presented as means. (See Table, Supplementary Digital Content 2, http://links.lww.com/SHK/A481, which lists SEM values).
Subsequently, to determine if the classical hGR pathway plays a role in signaling for these two hyperactive hGR isoforms, hGR-S1(-349A) and hGR-S1(-349A)(1303) were treated with 1 μM of the glucocorticoid antagonist, mifepristone (RU486), before being stimulated with 1 μM of hydrocortisone. Mifepristone is able to effectively and significantly decrease the transactivation potentials of both constructs, indicating that the two isoforms interact with exogenous hydrocortisone for their activity (Fig. 4).
Transactivation potentials of hGR-S1(-349A) and hGR-S1(-349A)(1303) with mifepristone treatment.Before being stimulated with 1 μM of hydrocortisone, hGR-S1(-349A) and hGR-S1(-349A)(1303) were treated with 1 μM of mifepristone or ethanol vehicle. The transactivation potentials of both isoforms were effectively blocked by mifepristone treatment. Data is a combination of two experiments and is presented as means ± SEM.
Incremental deletions of the 3' UTR surrounding hGR-S1(-349A)(1303) reveals a peak in activity with 1303 bp of 3' UTR
To further dissect and possibly pinpoint a critical region of the 3' UTR region involved in the autonomous hyperactivity seen in the hGR-S1(-349A)(1303) isoform, the 3' UTR region was dissected to look at an area 50 bp up- and down-stream of 1303 bp by adding or removing 3' UTR in ∼10 bp increments, creating constructs with 1342 bp to 1253 bp of 3' UTR (Fig. 5A). When stimulated with graded doses of hydrocortisone in the luciferase assay, we found that the 3' UTR regions surrounding hGR-S1(-349A)(1303) also have autonomous hyperactivity that is not dependent on hydrocortisone stimulation (Fig. 5, B and C). The amount of activation is directly dependent on 3' UTR length and activity peaks with 1303 bp of 3' UTR. The pattern was the same with all concentrations of hydrocortisone examined. Interestingly, an anomaly occurs with 1293 bp and 1263 bp of 3' UTR in that there is virtually a complete loss of activity for both isoforms with vehicle and with hydrocortisone.
Transactivation potentials of ∼10 bp serial deletions of 3' UTR surrounding hGR-S1(-349A)(1303).A, Line diagrams depict the constructs altering the 3' UTR in ∼10 bp increments surrounding hGR-S1(-349A)(1303), resulting in UTR lengths from 1342 bp to 1253 bp. The 3' UTR of all new isoforms terminate in what would correspond to Exon 5 of hGRα. B, Transactivation potential of hGR-S1(-349A)(1342) through hGR-S1(-349A)(1253) without hydrocortisone (only 1 μM saline shown) revealed the transactivation potential is directly related to length of 3' UTR with a peak in activity at hGR-S1(-349A)(1303). C, Transactivation potentials of the isoforms with hydrocortisone (only 1 μM shown) revealed that, similar to the vehicle controls, transactivation potentials are directly related to 3' UTR length with activity peaking at hGR-S1(-349A)(1303). There is an unexpected loss of activity at 1293 bp and 1263 bp either with or without hydrocortisone stimulation. Data is a combination of four experiments and is presented as means ± SEM.
Deletion of the 3' UTR in 1 bp increments surrounding hGR-S1(-349A)(1293) reveals a 3 bp cyclical loss in activity
To further investigate the inexplicable loss of activity of the 1293 bp 3' UTR construct, an additional set of 3' UTR deletion constructs was created in the 20 bp region surrounding hGR-S1(-349A)(1293) in increments of 1 bp (from 1302 bp to 1284 bp) (Fig. 6A). Luciferase assay results reveal a very interesting pattern. At all concentrations of vehicle and hydrocortisone in this deletion series, there is a loss of activity when the 3' UTR ends at every third nucleotide while all other sites maintain the autonomous hyperactive activity (Fig. 6, B and C). This pattern of loss in activity continued until the 1263 bp position where the second drop in activity was seen. Additionally, investigation of the 3' UTR region upstream of hGR-S1(-349A)(1303), from 1303 to 1313 showed that the pattern continued (Fig. 7).
Transactivation potentials of 1 bp serial deletions of 3' UTR surrounding hGR-S1(-349A)(1293).A, Line diagrams depicting the constructs altering the 3'UTR of hGR-S1(-349A)(1293) in 1 bp increments (1303 bp to 1283 bp of 3' UTR). B, Transactivation potential of hGR-S1(-349A)(1303) through hGR-S1(-349A)(1283) without hydrocortisone (only 1 μM saline shown) shows a cyclic loss of activity every third base pair. C, Similar to vehicle controls, the transactivation potential of the constructs after treatment with graded doses of hydrocortisone (only 1 μM shown) has a cyclic loss of activity every third base pair. Data is a combination of four experiments and is presented as means ± SEM.
Transactivation potentials of 1 bp serial deletions of 3' UTR upstream of hGR-S1(-349A)(1303).A, Line diagrams depicting the constructs altering the 3'UTR upstream of hGR-S1(-349A)(1303) in 1 bp increments (1313 bp to 1303 bp of 3' UTR). B, Transactivation potential of hGR-S1(-349A)(1313) through hGR-S1(-349A)(1303) without hydrocortisone (only 1 μM saline shown) shows the continued cyclic loss of activity at every third base pair. C, The transactivation potential of the isoforms after treatment with graded doses of hydrocortisone (only 1 μM shown) also has a cyclic loss of activity every third base pair. Data is a combination of four experiments and is presented as means ± SEM.
Subsequently, the transcript and protein expression of selected hGR-S1(-349A) deletion constructs was examined to determine if a difference in expression led to the decrease in activity. The constructs from 1302 to 1300 and 1287 to 1285 were transfected into tsA201 cells alongside hGR-S1(-349A)(1303). The expression of all isoforms was compared by Western blot. The luciferase assay confirmed a loss of activity and the Western blot showed that the 1302 and 1287 isoforms expressed proteins that were similar in size and amount in comparison to all the other deletion isoforms (Fig. 8, A and B). Additionally, real-time RT-PCR was used to contrast the expression of hGR-S1(-349A)(1303) and hGR-S1(-349A)(1293), an active and nonactive isoform, respectively (Fig. 8C). The real-time RT-PCR showed that there was also no difference in mRNA expression between the active and inactive isoforms.
No difference in expression of inactive isoforms.A, Western blot for hGR-S1(-349A)(1303) with two sets of 3' UTR deletion isoforms that have loss of activity, 1302 to 1300 UTR and 1287 to 1285 UTR. All isoforms express the same ∼18 kd putative protein and there is no difference in expression between the autonomous hyperactive isoforms and the inactive isoforms. B, Corresponding luciferase data for same isoforms treated with 1 μM hydrocortisone confirms no activity at 1302 UTR and 1287 UTR. Experiment was performed once as a confirmation and is presented as means ± SEM. C. Real-time RT-PCR for hGR-S1(-349A)(1303), an active isoform, and hGR-S1(-349A)(1293), an inactive isoform showed no difference in mRNA expression. Data is a combination of two experiments and is presented as means ± SEM.
Screening for homology of the active 1303 3' UTR sequence in other genes
Since there was such a profound influence of the 1303 bp 3' UTR of the hGR-S1(-349A) on activity, a 250 bp region overlapping the 3' end of hGR-S1(-349A)(1303) was aligned against the other members of the nuclear hormone receptor family. It was found that this region occurs in approximately the same area in all of the receptors with ∼50% identity (Fig. 9A). This region was BLAST analyzed against the NCBI database for its presence in other genes and matched to the androgen, mineralocorticoid, and progesterone receptors in humans and other species (Fig. 9B).
Homology of hGR-S1(-349A)(1303) active region UTR to other nuclear receptors.A, Alignment results of a 250 bp region of 3' UTR (shaded box) surrounding the end of hGR-S1(-349A)(1303) with the nuclear hormone receptors: androgen receptor (AR), estrogen receptor-α (ERα), mineralocorticoid receptor (MR), and progesterone receptor (PR). The 250 bp region aligned in the same area in all the receptors from the end of the hinge to the start of the ligand binging domain with coverage of 252 bp to 268 bp and approximately 50% identity (gray shaded region). D indicates DNA binding domain; H, hinge region; LBD, ligand binding domain; TD, transactivation domain. B, The 250 bp region was also subjected to a BLASTn analysis. The results for each of the nuclear receptors are shown.
Many studies of genes focus on polymorphisms within the coding region or promoters in the variable 5' UTR (20–22). The role of the 3' UTR in gene activity, especially in mutations that result in truncated proteins, has often been underappreciated (23). In the hGR, there have been reports of both activation and repression of activity in truncated versions of the receptor, which can be dependent on cell type (19, 23, 24).
Usually, single-nucleotide alterations that result in early termination lead to a decrease in activity, which is a logical expectation for a truncated protein. In our case, we found that hGR-S1(-349A) has an unexpected hyperactive effect with steroid treatment even though the truncated protein lacks many of the known functional components (ligand binding, DNA binding, and a significant portion of the transactivation domains). The 3'UTR is also required since activity is almost nonexistent when using an isoform without the 3' UTR region (17). Furthermore, the hyperactivity of this truncated isoform is blocked by mifepristone, indicating that the classical glucocorticoid receptor pathway is still somehow involved in its function. This opens the door to possible alternative methods of action that have yet to be explored. It is conceivable that the shortened protein acts as a cofactor to augment the activity of the full-sized hGR. It is possible that during periods of profound stress, such as after a major burn injury, a splice variant, like the hGR-S1(-349A), is produced and results in an increased sensitivity of hGRα to endogenous corticosteroids. This cofactor, in essence, "supercharges" the patient's stress response to allow him or her to handle such a severe insult. We are currently evaluating hGR expression in patients with severe burns to determine whether this variant is present. To date, we have found the -349A deletion in four patients and all subjects express an isoform retaining intron H.
Furthermore, the hGR-S1(-349A)(1303) isoform, which has a 3'UTR length of 1303 bp and is hyperactive with or without the addition of steroids, may conceivably act like an oncogene in that it has lost any negative feedback and is constantly "turned on" (8). It is unlikely, however, to expect that a variant such as this would be found in nature under normal physiologic conditions since overexpression of the glucocorticoid response would lead to a severe case of Cushing syndrome that would most likely be fatal in utero. Cushing syndrome results in the overproduction of cortisol and has profound effects on the untreated patient. This hypothesis could be tested in knockout studies but it is unlikely that continuous expression of the GR without steroid binding would lead to a viable organism.
There have also been reports that many genes express alternative UTRs (25). In studies that have examined the effects of the 3' UTR in the reference hGR, three areas of interest have become the focus as possible regulatory regions: AU- or GU-rich elements, miRNAs, and polyadenylation sites (3). The 1303 bp 3' UTR of hGR-S1(-349A)(1303) was examined for these elements (RegRNA 2, http://regrna2.mbc.nctu.edu.tw/) (26). This analysis identified no AU- or GU-rich elements or polyadenylation sites; however, three putative miRNA sites (distance from the hGRα stop codon: 233 bp, hsa-miR-4667-5p; 377 bp, hsa-miR-4669; 907 bp, hsa-miR-4793-5p) were identified. These three miRNA target sites were initially found in a screening study but not much is known about their activities (27).
Most miRNAs act by reducing the stability of the mRNA or by repression of protein production. Western blot and real-time PCR data, however, indicated that there is no difference in mRNA or protein expression among the hGR-S1(-349A) isoforms with variant 3' UTR (Fig. 8). Therefore, there is a small possibility these miRNAs sites might be involved in the hyperactivity, but they have not been examined.
Analysis of the deletion isoforms near the 1303 3' UTR region revealed an unusual pattern in which there was a drop-off in activity at every third nucleotide (Fig. 10). The reason for the drop-off in activity remains unclear. In the three-nucleotide codon, the first two positions are important in determining coding specificity. Thus, it could be speculated that this type of nucleotide hierarchy partitioning may factor in the observed trinucleotide pattern of activity loss.
Fig. 10:
Illustration of loss of function at every third nucleotide of the 3' UTR for hGR-S1(-349A)(1303) to hGR-S1(-349A)(1253).Illustration of nucleotide and relative activity at each 3' UTR position, 1303 to 1253, in grayscale, with greatest activity in black to no activity in white for 1 μM. Saline is on the top half and hydrocortisone is on the bottom half. Loss of activity is continued at every third position throughout the series. Hyd indicates hydrocortisone; Sal, saline.
The hGR-S1(-349A)(1303) 3' UTR was surveyed for open reading frames (ORFs). A significant ORF was found that begins at the same position as the hGR-D alternative translation isoforms and terminates using a stop codon in the vector backbone (28). Due to the truncation and use of the vector sequence, the 3' end of this transcript would be drastically different from the previously reported hGR-D isoforms. A preliminary study indicated that a putative protein may be produced from this start codon, and it will be important to confirm the expression and analyze possible functions.
Since the hGR is a member of the family of closely related nuclear receptors, this phenomenon may also be reflected in the other steroid hormone receptors (progesterone, mineralocorticoid, and androgen). Since the ∼1303 bp region of the hGR-S1(-349A) UTR would correspond to the reference hGRα transactivation site τ2, this would be a well-conserved area. Therefore, the findings from this study may have direct applicability to the other members of this receptor family.
While determining how a truncated hGR isoform, hGR-S1(-349A), becomes hyperactive in response to steroids, we identified a region of the 3'UTR that confers autonomous and profound hyperactivity. Even more interesting is the finding that this 3'UTR region is present in other nuclear receptors. This section of the nuclear receptors may be a major regulator of many nuclear receptor activities. It is well known that there are variable responses to steroid treatments for many diseases. Some patients respond to normal doses of steroids while other patients are "non-responders" who either do not respond or need higher doses for a response. It was clearly illustrated in the sepsis studies by Annane et al. (29) that some patients are "responders" and others are "non-responders" when tested for their response to adrenocorticotropic hormone. Our hypothesis is that variations in the hGR may explain these differential responses. Therefore, elucidating the mechanism behind this ordered anomaly may prove valuable in understanding the complex layers of gene regulation, as well as variations in patient response to glucocorticoid treatment.
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3' untranslated region (UTR); cyclic activity; glucocorticoid receptor; hyperactivity; truncated isoform
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If you have a small garden, you know the challenge of fitting all you want to grow in the space you actually have.
I used to have a large backyard with a vegetable garden that grew bigger each year. Then we moved. Our new house has lots of lovely features, but on our tree-covered lot, sunny space ideal for vegetables is at a premium.
As I have built and planted and planned for our small-space garden over the last two years, I have learned a few things to make the most of a small gardening space.
1. Plant things you can't buy easily. This was a hard one for me to learn, because I wanted to grow everything myself. But with my newly limited garden space, I realized it just wasn't possible. I first eliminated things that were easily available locally. Red and yellow onions? I could buy them at the farmers market. Rainbow swiss chard? Definitely something to grow myself. I also try to grow as much as I can from the "dirty dozen" list, since organic produce is hard to come by in my area.
2. Grow up. You can gain lots of garden space by training vine plants such as melons, cucumbers, and squash to grow up, instead of out. We are blessed with a fence at our house that does this job well, but building a simple trellis would also work. As your vine starts to grow, coax it up by gently wrapping it around the fence. Soon enough it will continue the path up on its own.
3. Look for compact varieties of your favorite plants. I love growing my own zucchini but the plants can become monstrous. Last year I switched varieties to one known for smaller, bushier plants. I got the same yield of fruit, but was able to plant four plants in a space that would normally have been a squish for three, and was able to plant herbs in around them as well. Many plants also have dwarf varieties which take up less room, but often have smaller fruit, so be sure to read descriptions carefully.
4. Practice succession planting. This means plant one thing, harvest it, and then plant something else in its place. This works particularly well if you have a long growing season, but even in my very short season I am able to plant a second crop after harvesting radishes and peas.
6. Don't take seed spacing too seriously. If I followed the proper seed spacing on each seed packet, I wouldn't have near the harvests that I do in my small garden. It can be a balancing act between too squished and extra room, so err on the side of caution your first year, and make note of where you can squeeze in something else the following year. Tomatoes are the one plant I don't crowd as it makes them more prone to disease.
7. Practice companion planting. Some plants naturally grow well together, and some plans can actually impede the growth of another. Plan your garden to make the most of beneficial relationships. For instance, you can grow radishes among your carrots, as your radishes will be harvested long before the carrot roots start to really develop. Planting lettuce under tomato plants can act like a natural mulch around the base of the plant and the leaves of the tomato plant shade the lettuce from the hot summer sun.
8. Use containers. Container planting is a great way to extend your garden space. Just remember to keep them well-watered. Containers are especially great for things that tend to take over a garden, like mints, lemon balm, and Jerusalem artichokes. The yield may not be as big in a pot, but it's worth it to keep these plants contained!
Do you have a small garden? What do you do to make the most of the space you have?
I have a container (square foot garden) on our flat kitchen roof, where there's plenty of sun.
I live in a row home in "Suburbia" with a back yard that is primarily a concrete patio, with 7′ privacy fences all around and a detached garage in the back, which leaves only a few small spots that enjoy full sun, so I realized that it was really important to only grow things that I love and will use. I've been slowly giving away the "builder landscaping" plants that came with our new build, and creating spaces wherever I can find them. This year, I've utilized my 2 large planters that were my containers for "4th floor dirt therapy" in our condo, and planted blueberry plants in them, my actual garden spot has rainbow chard, rainbow carrots, and sugar snap peas, and I'm creating a separate, slightly raised area for my bush beans, since they took over my whole plot last summer. My pots will be one with a mix of lettuces, one or two for herbs I use regularly, and one (hopefully) with "mosquito repellent" plants for bug control. I hope to get some hanging baskets to hang from the fences to attract pollinators, as well as add some colour to our backyard. My other issue with my small space is that I have 3 kids under 5, so it also needs to be a functional play space for them. It's definitely a work in progress! Thanks for the great tips! I'll be trying a couple of them out this year.
Many plants will grow well with light shade like very tall pine trees. Some of these are chard, lettuce…also, plant 2rows close together then a space to walk. Easier to pick and less weed space. Make sure to get your soil tested and amend it for optimal growth. For tomatoes, peppers and many other plants stagger planting them in beds. Less space for weeds and nothing would grow in the empty corners anyway.
Thanks for the tips, Cheryl! When I used to have garden beds, instead of the boxes, I utilized stagger planting as well!


text:
'A high-grade, twisted abuser': Don Burke a sexual harasser and bully, claims series of women
By Kate McClymont
November 26, 2017 — 10.05pm
For almost two decades Don Burke was one of the most powerful men in Australia's entertainment industry. His popular gardening program Burke's Backyard was a ratings juggernaut for the Nine Network until it was unceremoniously axed in 2004.
But now a major Fairfax Media/ABC investigation can reveal that behind the scenes those who worked with Burke claim he was a "psychotic bully", a "misogynist" and a "sexual predator" who indecently assaulted, sexually harassed and bullied a string of female employees.
"He was a vile, vile human being," said Bridget Ninness, a former producer on Burke's Backyard, who later launched legal action against Burke for psychological abuse. "He was lewd and he was crude" and his constant talk of sex was "designed to confront you and to demean you", she said.
Louise Langdon, a former researcher, was subjected to ongoing harassment by Burke which included trying to remove her top, and on another occasion Burke "put his foot into my rear end, with the view to checking the tone of my ... my backside". He then stated that the firmness of her "backside" wasn't "up to scratch".
A series of women have accused Don Burke of sexually harassing them.
"I loathed him, he was just a pig. He was lecherous and sinister," said another former researcher who alleges she was indecently assaulted by Burke.
Even David Leckie, the former chief executive of the Nine Network, said he wasn't surprised to receive our call about Burke. "I've been trying to think of Harvey Weinstein-type people [in Australia] and the only one I can ever come up with is Burke. He was a horrible, horrible man," he said.
Unlike Weinstein, Burke is not accused of rape, but the more than 50 people interviewed during this investigation have made serious allegations about Burke's actions.
"Don Burke was a disgrace because of his behaviour internally and externally," said Sam Chisholm, Leckie's predecessor at Nine.
Louise Langdon, a former researcher, says she was subjected to ongoing harassment by Don Burke. Credit:ABC
Chisholm, a stalwart of the industry, also described Burke as "terrible grub". Asked if he felt the network had done enough to rein in Burke's behaviour, Chisholm said he didn't know.
Did anyone ever come to him personally about Burke? "Probably they did, but I don't know. It's a long time ago," he said.
"He was a horrible, horrible man": Former Nine chief David Leckie on Don Burke. Credit:Sasha Woolley
"I think the public that have loved him should know the real Don Burke," said another former highly placed Nine executive. "If Harvey Weinstein's been outed, Don needs to be outed."
However, in a written response, Burke angrily denied comparisons to Weinstein. "I loathe the reported behaviour of Mr Weinstein and hope that the legal system deals with him in such a way as to prevent this happening again.
Don Burke was "a disgrace" and a "terrible grub", says Sam Chisholm, David Leckie's predecessor at Nine Network. Credit:Bob Pearce
Suddenly he made this move and grabbed me, grabbed me hard on the breast. This was not a clumsy, oafish move. It was a calculated action. That is what was scary
A former researcher
"The bitter irony is that I have had a life-long opposition to sexism and misogyny. Burke's Backyard was a lone bastion of anti-misogyny since its inception in 1987," said Burke.
"He is a high-grade, twisted abuser," said Ms Ninness, who eventually settled out of court with Burke's company over "sustained and systemic psychological abuse" in the workplace.
Don Burke's program Burke's Backyard was a ratings juggernaut for Channel Nine before it was cancelled in 2004.
Burke was "two people", explained a former male crew member. There was the genial gardening guru on camera who was "very, very good at what he did". But off camera the real Burke was described by many of those interviewed as a psychopathic narcissist.
"He was an absolute sexual predator. He was a bully. He was horrible to people in the office. He would often have women in tears. He used to take great delight in it. It was like sport to him," said the former male crew member.
When journalist Tracey Spicer announced that she was investigating the behaviour of powerful Australian men in the media in the wake of America's now infamous sex scandal involving film mogul Harvey Weinstein, she was inundated with reports. One name kept recurring – Don Burke.
Among those who came forward was Ms Langdon, now a psychologist who lives in the United States. In 1987 Ms Langdon, then 27, was working as broadcaster Alan Jones' personal assistant when she landed a job as a weekend producer for Burke's gardening program on radio 2UE. She later became a researcher at Burke's Backyard.
"I was told before I went to meet Don that he was sleazy and to be careful," she said. "I was quite shocked about that because the only Don Burke that I knew was the affable Friday night Burke's Backyard gardener.
"So I thought, 'Oh well, how bad can it be?"'
As it turned out, nothing could have prepared Ms Langdon for what was to come.
"Dealing with Don Burke was an endurance test in terms of his persistence in commentary about anything sexual," she said.
On one occasion, while the pair was working at the radio station, Burke insisted she watch a video. To her horror it was a video showing a woman having sex with a donkey.
Ms Langdon was upset and disgusted. "Don was getting so much pleasure from seeing my reaction to it ... I was just, just ... speechless," she said. "I was really overcome."
Louise Langdon when she worked with Don Burke at 2UE.
Burke also subjected Ms Langdon to harassing behaviour – flicking her bra straps and lifting up her shirt to see the colour of her underwear. During a work trip in the Northern Territory he allegedly tried to remove her top.
"I was sitting next to Don on the bus and he decided that it was OK for him to put his hands on my T-shirt and try and pull my bra strap, my bra off and try to somehow remove my clothing. He was trying to take my top off," Ms Langdon said.
Another researcher, who asked not to be named, also recalled being warned about working for Burke. Her job interview was at night at Burke's home, which is on acreage at Kenthurst, a semi-rural suburb in Sydney's Hills district.
The taxi driver expressed concern because he had heard things about Burke and insisted on waiting for her.
During the interview Burke said, "Our nickname for the receptionist is 'the fat c---', so make sure you call her that".
"I think he was testing me to see whether I would be shocked," the woman recalled.
At the time the woman, who was in her late 20s, thought the interview was being held at night because Burke was very busy. But she later came to realise "it was all about power and control ... It was in his own environment so he could control it. It puts you at that disadvantage."
About a month after she started work as a researcher, the woman was having a cigarette on the rooftop garden at the Crows Nest office of Burke's production company, CTC (Cut The Crap) Productions.
The researcher said Burke had been telling her about a cocktail party he was going to that evening. "'You know what I love about cocktail parties?' he mused. 'It's the name tags. I get to grab women's tits while pretending I can't read their name."'
Burke was already standing uncomfortably close to her during the conversation when "suddenly he made this move and grabbed me, grabbed me hard on the breast. This was not a clumsy, oafish move. It was a calculated action. That is what was scary. It was premeditated. I jumped back. He sneered. 'You've got small tits, no one would want to touch your tits'."
"The rooftop incident never occurred," said Burke, who also denied all of Ms Langdon's allegations.
In his written response, Burke denied "absolutely" claims he was bully. Because he was a "perfectionist" and demanded excellence in his production company, "a small number" of former employees were dismissed either for underperformance or misbehaviour. Some of these people "still bear a strong grudge against me", he said.
Wendy Dent was employed as an entertainer when she met Burke at the Melbourne Garden Show in 1995. Burke came over, kneeled before the 21-year-old who was wearing a fairy costume, and asked to be granted a wish. In front of the crew and onlookers, Burke opened his eyes and said, "It didn't work. You've still got your clothes on."
Wendy Dent encountered Don Burke while working as a children's entertainer. Credit:ABC
When Burke encouraged her to come to Sydney to audition for his gardening show, Ms Dent recalled a crew member taking her aside to apologise for Burke's "sleazy comment" and also warning her to be careful of him.
Months later, when she had moved to Sydney, Ms Dent took up Burke's offer to audition for the show. Over the phone Burke was flattering, telling Ms Dent she had "real charisma" and great potential. "You definitely have got what it takes for TV," he told her and, what's more, he was "the No. 1 in the business" and he could make it happen.
He said "well, you'll have to audition, but you'll have to be topless".
As a result, she decided she wanted no part of this "sleazy slummy industry".
"These men become dream killers and I lost a career," she said.
Male crew members spoken to by the joint Fairfax Media/ABC investigation confirmed Burke's obsession with breasts and sex.
One former crew member recalled Burke spotting a group of schoolgirls, aged around 14, holding a puppy.
"He stopped and chatted to them and said, 'Do you want me to show you how to hold the puppy properly?"'
Because it was the legendary Don Burke, one of the girls said, "Yeah, that'd be great."
"He showed them how to nurse it close to their chest, keep it nice and warm," the crew member recalled. "When they walked away he exclaimed: 'Give me three!'
"I said, 'three what?' He boasted that he had stroked her nipple three times while showing her how to hold the puppy," said the crew member.
Other crew members talked about Burke's voracious sexual appetite while on the road. The lure of Burke being a national prime-time celebrity meant women of all shapes, sizes and ages were happy to go off with him. "It was pretty much anything with a pulse," said one of his crew.
There were occasions when Burke's behaviour with women worried the crew. The most notorious occasion was referred to by several people as the "Newcastle incident", which involved a young woman who had joined Burke and the crew for drinks after filming that day in Newcastle. As the other crew members retired to bed, Burke was seen leading the unenthusiastic, protesting woman towards the lift.
No one knew what occurred behind Burke's closed door on this occasion or on other occasions but his constant bragging about his exploits the following morning made his crew cringe.
"Don would always come back the next morning and say, 'Oh my God, it was fantastic ... once she got up there it was on for young and old'," said one former crew member.
Another former Nine producer recalled a troubling incident involving Burke. The network had organised the season launch on a boat to celebrate the start of the 1989 season. One of those aboard was an 18-year-old woman who was about to launch her career on a new Nine program.
The young woman had too much to drink so the producer, one of the few who had not been drinking, was asked by a publicity head to escort the woman back to her hotel.
With the assistance of Mike Willesee's driver, he got the woman to the foyer of her hotel only to find that she'd lost her room key. After getting another one from reception, they arrived to find the door to her room slightly ajar. "Sitting in the shadows – in the darkened room, with no lights – was the hunched-over figure of Don Burke," said the producer. "I said, 'What the f--- are you doing here'?"
"Don tried telling me that she'd given him her room key while on the boat so he could teach her 'presentation skills' back in the room," said the producer. After arguing with the network's star for an hour, the producer finally got him to leave the hotel room.
"This was predatory behaviour ... Any normal person would have left straight away, but Don was determined to stay there and do God-knows-what to this girl," he said.
The following day the producer informed executives and the publicity department of Burke's behaviour but no action was taken. When he got back to his desk there were two messages. One was from the young woman thanking him for looking after her.
The other was from Burke. It read: "Thanks very much for saving my bacon last night, mate."
"I remember going, 'You f---er, you f---er!' I'd never come across anything like it and I had done television for years."
Burke denied that the incident occurred.
Despite numerous complaints to Nine executives, both male and female employees were told to soldier on. "There was an institutionalised acceptance of his behaviour and it was actually not just an acceptance, it was an institutionalised enabling," said researcher Louise Langdon.
Leckie confirms that there were complaints made about Burke but suggested he heard them second-hand. "I am not going to mention any specific girls or anything like that," he said.
For one young television writer, the comments Burke made about a young female relative were the last straw. It was the late 1980s and the reporter was 21 when she first went off to interview one of Nine's biggest stars.
Burke continually interrupted the interview with lewd comments such as "I bet you're a demon f--k". The next year she was reluctant to interview him again, "but he was a very, very big star back there and I think that's what people need to take into account".
The second year Burke not only invaded her personal space but made suggestions as to what sexual positions she might enjoy. He also openly stared at and commented about her breasts. It was as though his body language was "I am staring at your boobs and I don't care if you notice, that's what I'm going to do", she said.
Her innocuous question about garden gnomes led to a vulgar comment from Burke about the size of his "cock". Her third encounter with Burke, an interview at his Kenthurst home, was her last.
When Burke said he had bought a horse for a young relative "because I love watching her rub her c--t on its back," the reporter snapped the tape off.
"I was absolutely and utterly repulsed by the man, I felt compromised, I felt violated, I just felt disgusted.
"I took the tape recording to the head of publicity at the time and said I want action. And the next day I received a bunch of flowers and that was the end of it."
There was no apology from Burke and no action was taken against Burke, who told Fairfax Media and the ABC that the story was a "total fabrication".
In 2004 Burke was unceremoniously dumped by the Nine Network after more than 17 years hosting Burke's Backyard.
Over those years countless employees allegedly suffered from Burke's bullying, lewd behaviour and sexual harassment. The network's failure to do anything to rein in Burke's behaviour leaves a bitter taste for many. "Every single person in management ... has known about Don Burke. Every male manager. There is not one that does not know," said a former Nine staffer.
Even the male managers found his incredible ego and narcissistic behaviour impossible to deal with, said the staffer.
"But in terms of the sexual harassment stuff, they didn't really give a damn. He was too popular, he was just too popular a celebrity," said one former senior employee at the network.
Another long-term male producer on Burke's Backyard said that management told staff "to suck it up because it was the No. 1 rating show, the cash cow for Channel Nine".
"Lots of women I know just left. There was a huge staff turnover," he said. "He had the power, the profile and the tacit backing of Channel Nine."
However, the Nine Network refused to accept any responsibility for Burke's behaviour. Questioned about claims that the network turned a blind eye to complaints and failed to take any action to protect their staff from Burke, Nine issued a statement saying: "Burke's Backyard was a production of CTC Productions and they employed and managed all staff."
However, some of Burke's worst behaviour allegedly occurred while the program was produced at Nine. In late 1991 Burke took over the production of his popular gardening program.
Nine also said they could find no records of complaints or payouts to any women in relation to Burke's behaviour.
One woman who did complain paid a very high price.
"He got off on terrorising [female researchers]. Women were his playthings and he loved seeing them shocked by his behaviour and language," said producer Bridget Ninness, who worked at Burke's Backyard for more than seven years from 1990.
Bridget Ninness worked as a producer for Credit:ABC
On her first overseas trip as a reporter, Burke turned to her and said that if everything did not go smoothly, "I'm going to rip your f---ing head off and shit down your throat".
Ms Ninness was so nervous and upset she vomited. When she complained to the head of news and current affairs Peter Meakin, she alleges he said she needed to have "broad shoulders".
Years later her colleagues at Burke's Backyard encouraged her to take legal action against Burke for the stress and psychological abuse she suffered at his hands. But when she did, Ms Ninness found herself totally alone.
She said David Leckie warned her against the legal action, saying: "We don't want to have to crush you." This was denied by Leckie who said "honestly that is not my speech". He was adamant that the complaints he heard about Burke's behaviour were second-hand.
Similarly Meakin did not recall Ms Ninness's complaint. "I never received any official complaints, written or verbal, that I can recall about his conduct.
"There was gossip about inappropriate language and he was incredibly demanding. If someone fell short of the mark, he would excoriate them," said Meakin, who is now at Channel Ten.
"There was absolutely no care whatsoever for the consequences – the psychological damage, the physical damage," said Ms Ninness. She still suffers post-traumatic stress disorder.
"Twenty years later we are saying actually our lives do matter, our mental health does matter," she said.
"This is unfair and unworthy journalism" from a "small clique of malcontents" who were fired, said Burke.
He enclosed an email from former reporter Jackie French, now a children's author, saying that in the decade she worked closely with Burke she never saw "any hint of sexual harassment, nor heard any gossip".
Michael Freedman, Burke's former CEO, said: "I never observed any sexual harassment. Don's not the kind of person to engage in that type of behaviour."
According to Tracey Spicer, this is a seminal moment for women both in the workplace and society.
Spicer said a generation of women had been lost to the media and entertainment industry "because they were simply sick of being groped and grabbed" while trying to do their job.
"We've put up with sexual harassment and indecent assault for so long and finally we're able to say, 'Enough is enough, let's change the structures within the workplace so women can feel safe'," she said.
Additional reporting by Michael Evans, Lorna Knowles and Alison Branley
The ABC's 7.30 on Monday will be broadcasting interviews with Burke's accusers.
Do you know more? Email to kmcclymont@fairfaxmedia.com.au
Kate McClymont
Kate McClymont is an investigative journalist at The Sydney Morning Herald.
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text:
In Zygarde we trust
By Chris Priestman
New Pokémon have leaked online following the arrival of Japanese magazine CoroCoro's latest issue.
The Pokémon revealed are to appear in the upcoming Pokemon XY & Z anime.
The one most worth looking at is Pokémon X and Y's third Legendary, Zygarde, and it has new forms.
It now has five forms, in fact: a blob, a cell, a dog, the snake-like one we're used to, and a new Mega Evolution. They're called Core, Cell, 10%, 50%, and Perfect Formes, respectively.
We're expecting another Pokémon game to be announced for 2016 at some point. And many have speculated that it'll be "Pokémon Z" (following X and Y) due to this upcoming anime.
With Zygarde being developed in this way it seems more likely that the next game will revolve around it.
The other Pokémon shown in the leaked images of the magazine is less exciting.
It's a new colour variation of frog-ninja Greninja that is oddly similar to Ash's clothing. I guess


text:
NHS might fund the war memorial relocation
Denis Kenyon, Rosalind Willatts, Mark Robinson (chairman) and Brian Tanner (Civic Society) inside the Cottage Hospital War Memorial. PICTURE: ANDREW CARPENTER
The NHS has not ruled out funding, or helping to fund, the re-siting of Market Harborough's listed War Memorial, the Mail can reveal.
When asked directly by the Mail if the NHS would pay for the relocation of the Grade II listed monument, a spokesman for NHS Property Services said: "It's not 'yes' or 'no' at the moment.
"We're looking at various options with the Memorial Group itself, with Historic England and with the local council."
The town's unique First World War Memorial Portico is currently located at the front of the Cottage Hospital in Coventry Road.
It and much of the hospital behind it were paid for by public subscription in the 1920s. The site was later inherited by the NHS.
But as we reported in the Mail last week, when the new St Luke's Hospital on Leicester Road is completed, the Coventry Road site is likely to be sold by the NHS.
A new town-based committee is looking to move the monument to the Memorial Gardens in town.
Mark Robinson, chair of the group, said: "There are issues to resolve ... including financing the cost."
This week a spokesman for NHS Property Services said: "We recognise the importance of the listed War Memorial to local people and have held preliminary discussions with the planning authority and the war memorial group to talk about the options.
"We are seeking expert advice from specialist heritage organisations to help inform our proposals and will continue to work with local partners as the project progresses."
He conceded that the site was probably worth more to the NHS without a War Memorial which any future developer would have to deal with.
The War Memorial contains the names of 1,655 Market Harborough people who fought in the First World War.
County show returning to Market Harborough this summer


The application process for Year 7 enrolment is determined by the Victorian Department of Education and is initially undertaken by the primary school your child attends during Grade 6. It is not possible to apply for a place in Year 7 before your child is in Grade 6. Families residing interstate and overseas when their child is in Grade 6 are advised to contact the school to make a direct application.
Frankston High School has a designated neighbourhood zone and receives a substantial number of out of zone applications for Year 7 placements each year. It is not possible to successfully place all out- of- zone applicants.
* If your child lives outside the Frankston High School designated zone, an Out Of Zone Application Form, must be submitted to the primary school with the Application for Enrolment form. Out Of Zone Applications can be obtained from our information sessions.
The primary school will forward applications to Frankston High School. In August the primary school will notify parents/guardians of the results of their application. If your child does not attend a Government Primary School, they may not receive the Application for Enrolment (Year 7) form. Please request this form from your child's primary school.
Please note that Frankston High School is not able to disclose the results of the application process to parents/guardians prior to being informed of the results by their primary school in August.
Each year student enrol from other schools in Years 8 – 12. If a student resides outside the zone and is interested in enroling at Frankston High School it is recommended that contact is made with the school to explore enrolment opportunities.
If you are considering enrolment from an interstate or international destination please contact the school.
For any queries regarding enrolment please contact the school via telephone (03) 9783 7955.


You've now added Museum of Old and New Art to your itinerary. Kick off your visit on the 5th (Wed): examine the collection at Museum of Old and New Art and then take your sightseeing to a higher altitude at Mount Wellington.
To find traveler tips, reviews, and more tourist information, use the Hobart trip itinerary maker tool .
February in Hobart sees daily highs of 26°C and lows of 14°C at night. Finish your sightseeing early on the 6th (Thu) so you can drive to Coles Bay.
Start off your visit on the 6th (Thu): let the kids burn off some steam at Melshell Oysters and then kick back and relax at Spiky Beach Conservation Area. On the 7th (Fri), you'll have a packed day of sightseeing: enjoy the sand and surf at Raspins Beach, snap pictures at Spiky Bridge, do a tasting at Gala Estate Vineyard, then examine the collection at Tasmanian Wool Centre., and finally learn about winemaking at Spring Vale Wines.
For ratings, other places to visit, maps, and more tourist information, you can read our Swansea travel planner.
Traveling by car from Hobart to Swansea takes 2 hours. In February, daytime highs in Swansea are 26°C, while nighttime lows are 14°C. Wrap up your sightseeing on the 8th (Sat) to allow time to travel to Mount William National Park.
Cranbrook, Raspins Beach, Tasmanian Wool Centre.
Bay of Fires Conservation Area has been added to your itinerary. On the 8th (Sat), kick back and relax at Bay of Fires Conservation Area.
For reviews, other places to visit, ratings, and other tourist information, read our Mount William National Park trip itinerary maker website .
Explore your travel options from Coles Bay to Mount William National Park with the Route module. Finish your sightseeing early on the 9th (Sun) so you can travel to Beauty Point.
Beauty Point is a town by the Tamar River, in the north-east of Tasmania, Australia. Your day-by-day itinerary now includes Narawntapu National Park. On the 9th (Sun), explore the wealth of natural beauty at Narawntapu National Park.
For maps, more things to do, other places to visit, and more tourist information, go to the Beauty Point trip itinerary maker .
The Route module can help you plan travel from Mount William National Park to Beauty Point. In February, plan for daily highs up to 28°C, and evening lows to 13°C. Cap off your sightseeing on the 10th (Mon) early enough to go by car to Cradle Mountain-Lake St. Clair National Park.
Your day-by-day itinerary now includes Shadow Lake Circuit Walk as well as The Overland Track. On the 10th (Mon), hike along The Overland Track. Get ready for a full day of sightseeing on the 11th (Tue): hike along Shadow Lake Circuit Walk.
For reviews, where to stay, photos, and other tourist information, read our Cradle Mountain-Lake St. Clair National Park trip itinerary planning tool .
Traveling by car from Beauty Point to Cradle Mountain-Lake St. Clair National Park takes 3.5 hours. On the 12th (Wed), wrap the sightseeing up by early afternoon so you can travel to Eaglehawk Neck.
You've added Three Capes Track, Devil's Kitchen, Tasman Arch, and other attractions to your itinerary. Kick off your visit on the 12th (Wed): head outdoors with Tasman Blowhole, then get outside with Tasman Arch, and then head outdoors with Devil's Kitchen. On your second day here, trek along Three Capes Track.
To find where to stay, maps, and tourist information, you can read our Eaglehawk Neck online sightseeing planner .
The Route module can help you plan travel from Cradle Mountain-Lake St. Clair National Park to Eaglehawk Neck. In February, plan for daily highs up to 26°C, and evening lows to 14°C. Finish your sightseeing early on the 13th (Thu) so you can drive to Hobart.
Your day-by-day itinerary now includes Salamanca Market as well as Royal Tasmanian Botanical Gardens. On the 14th (Fri), take in nature's colorful creations at Royal Tasmanian Botanical Gardens and then shop like a local with Salamanca Market.
To find where to stay, more things to do, other places to visit, and more tourist information, go to the Hobart day trip planner.
You can drive from Eaglehawk Neck to Hobart in 1.5 hours. Finish up your sightseeing early on the 14th (Fri) so you can travel back home.


text:
NewsNational Politics
Pelosi appoints Rep. Liz Cheney to House committee that will investigate Jan. 6 Capitol riot
House Republican Conference chair Rep. Liz Cheney, R-Wyo., with House Minority Whip Steve Scalise, R-La., at right, talks to reporters during a news conference at the Capitol in Washington, Wednesday, Feb. 13, 2019. (AP Photo/J. Scott Applewhite)
House Speaker Nancy Pelosi on Thursday said she was appointing Rep. Liz Cheney, R-Wyoming, to serve on the select committee that will investigate the Jan. 6 riot at the U.S. Capitol, according to USA Today and the Associated Press.
Cheney will be the only Republican serving on the committee. Other members include Reps. Zoe Lofgren, Adam Schiff, Pete Aguilar, Stephanie Murphy, Jamie Raskin and Elaine Luria.
Rep. Bennie Thompson, D-Mississippi, will serve as the chairman of the committee.
Pelosi announced she was commissioning the Democrat-backed report earlier this week. The move comes several weeks after Republicans in the Senate blocked an effort to establish a bipartisan committee to investigate the riot, similar to how Congress handled an investigation into the Sept. 11 terrorist attacks.
Cheney's comes months after the House Republican caucus removed the Wyoming representative from her leadership position earlier this year. She had previously served as the chair of the House Republican conference.
Cheney has been an outspoken critic of President Donald Trump and his efforts to overturn the results of the 2020 election.
In a statement released Thursday, Cheney said she was "honored" to serve on the committee.
"What happened on January 6th can never happen again. Those who are responsible for the attack need to be held accountable and this select committee will fulfill that responsibility in a professional, expeditious, and non-partisan manner," she said. "Our oath to the Constitution, our commitment to the rule of law, and the preservation of the peaceful transfer of power must always be above partisan politics."


text:
Amt Naumburg steht für:

 Amt Naumburg (Hochstift Naumburg-Zeitz)
 Amt Naumburg (Markgrafschaft Baden)
 Amt Naumburg (Grafschaft Hanau-Münzenberg)
 Amt Naumburg (Erzstift Mainz)
 Amt Naumburg (Kurfürstentum Hessen)

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On Monday February 12th Bishop David Zubick visited Nazareth Prep. The Bishop celebrated mass at the campus chapel with the NCCP staff and students. His sermon focused on the importance of maintaining Christian faith and values in the face of public opposition.
After mass, the Bishop toured the school and visited several classrooms. He was very impressed with the innovative atmosphere of the classrooms as well as student engagement.
The visit ended with Bishop Zubick meeting with NCCP faculty to discuss the rapid growth of the school and the deeper learning philosophy.


Your technician will perform a thorough inspection of your home and any bed bug activity. This inspection will focus on areas where bed bugs normally are found or where you suspect they're active, such as beds, bed linens, bedroom furniture and clothing, and pictures or other wall decorations.
After the inspection, the technician will remove any visible bed bugs and apply a customized treatment to all areas where they are or may be present.
The product(s) applied during this initial treatment will quickly kill all adult bed bugs in the treated area(s).
This first treatment normally gets rid of most bed bugs in an area.
After two weeks, a technician will perform a second inspection and service.
At this point the existing bed bugs will be taken care of and no new ones should be hatching. If you see any activity during the 30 day window after the second treatment, simply let us know and we'll treat your home again at no charge to make sure you're free from bed bugs.


text:
GBSD GN&C Principal Embedded Software Engineer at Northrop Grumman Corporation in Roy, UT
Northrop Grumman Space Systems is seeking a GBSD GN&C Principal Embedded Software Engineer. This position will be located in Roy, UT with 50% travel to El Segundo, CA through Q4 2021 with 10% travel afterwards, and will support the Ground Based Strategic Deterrent (GBSD) program. Learn more about the GBSD program here.
What you'll get to do:
Design, develop, document, test and debugshard real-time embedded software containing logical and mathematical solutions.
Initial work will focus on developing test software for an embedded computer.
Conduct multidisciplinary research and collaborate with equipment designers and/or hardware engineers in the planning, design, development, and utilization of embedded systems for safety and mission critical software.
Collaborate with a team of software engineers in an Agile environment to meet critical milestones.
Ensure program mandated software standards and compliance criteria are met. Understands and can develop in a continuous integration tool chain meeting technical and compliance requirements.
As a full-time employee of Northrop Grumman Space Systems, you are eligible for our robust benefits package including:
- Medical, Dental & Vision coverage
- 401k
- Educational Assistance
- Employee Assistance Programs & Work/Life Solutions
- Paid Time Off
- Health & Wellness Resources
- Employee Discounts
This position's standard work schedule is a 9/80. The 9/80 schedule allows employees who work a nine-hour day Monday through Thursday to take every other Friday off.
UTSpace
About Northrop Grumman Corporation
Northrop Grumman Corporation is an American global aerospace and defense technology company. With 90,000 employees and an annual revenue in excess of $30 billion, it is one of the world's largest weapons manufacturers and military technology providers. The firm ranks No. 96 on the 2020 Fortune 500 list of America's largest corporations. Northrop Grumman and its industry partners have won the Collier Trophy eight times, most recently for developing the X-47B, the first unmanned, autonomous air system to operate from an aircraft carrier. In 2004, its subsidiary Scaled Composites won the trophy for SpaceShipOne, the first privately financed, built, and flown space vehicle. Northrop Grumman currently leads the development of the B-21 Raider, a long-range, stealth strategic bomber that can drop conventional and nuclear weapons; it will replace Northrop's own B-2 Spirit, the world's only known stealth bomber. Among its other current projects are development and production of the James Webb Space Telescope, an orbiting observatory slated for launch in 2021; and production of the solid rocket boosters for NASA's Space Launch System program. It was the sole bidder on the Air Force's Ground Based Strategic Deterrent program, which aims to develop and build a new intercontinental ballistic missile.
Learn More About Northrop Grumman Corporation
More Jobs at Northrop Grumman Corporation
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If good lawyering skills are essentials for an advocate, mooting during law school is the greatest asset that a student can inculcate. Here, at Symbiosis Law School, Pune, we understand this need to encourage mooting and aspire to create such a healthy pool of young lawyers. The Student Bar Association (SBA) is the institution's student committee entrusted with fostering, administering and facilitating mooting activities and upholding the benchmark of excellence that Symbiosis Law School, Pune has set for itself.
SBA serves to develop future mooters, as well as an interest in mooting. In pursuance of this, the SBA follows a policy of encouraging participation in mooting from all batches, including the first years. To allow them to compete on an equal footing, the SBA organizes extensive workshops and demonstrations to enable them to learn the process of mooting from the very basics to the most intricate details. Further, the SBA arranges for interactive sessions with experienced mooters and litigators from previous batches to guide the present mooters in a better manner. The SBA also organizes 'Symbiosis Law School, Pune - Internal Moot Elimination' during the year which puts senior batches against the junior batches and the 'Novice Moot Elimination' to encourage the first years to take up mooting, and subsequently represent the college in numerous moot court, mock trials, judgment writing, client counseling and negotiation competitions.
Students who represent the college in the national and international mooting circuit are trained and assisted by not only the SBA members, but also by experts in the various fields of law. Workshops are conducted regularly to hone the oratory and presentation skills of the students. Memorials are reviewed and mock pleadings are conducted by both faculty and senior mooters to ensure that every team which represents the college has been given optimum training.
The institution along with the SBA has taken huge strides in providing impetus for the development of the competitions both at national and international level i.e. Symbiosis Law School, Pune - National ADR Tournament and Symbiosis Law School, Pune - International Criminal Trial Advocacy Competition.
The zeal which the students have towards mooting and the support offered by our faculty are reflected in our results. In the past, our students have represented and won laurels in prestigious competitions such as Philip. C. Jessup International Law Moot Court Competition 2017, Indian National Rounds, 18th D. M. Harish Memorial GLC International Moot Court Competition, 2017, International Rounds of 2nd Prof. N.R. Madhava Menon SAARC Mooting Competition 2017, Willem C. Vis (East) Moot Court Competition 2017, 3rd GNLU Moot on Securities and Investment Law.
We have also secured First Rank in March, 2017 in the Mooting Premiere League (MPL). Symbiosis Law School, Pune was the only private university to ever achieve First Rank in the MPL history. Present MPL Rank is 6th.


text:
Top Md. Court Throws Out Appeal by Prisoner Facing Death Penalty
Posted on Wed 04/13/2011 by d3admin
… Judges clear path for trial in 2006 homicide …
baltimoresun.com
By Andrea F. Siegel, The Baltimore Sun
6:57 PM EDT, April 12, 2011
Less than a week after getting its first look at Maryland's new death penalty law, the state's highest court threw out a pretrial appeal Tuesday by a prisoner who is charged with murdering a correctional officer.
The order came four days after courtroom arguments in which an assistant attorney general asked the Court of Appeals to dismiss the bid by prisoner Lee Edward Stephens in the fatal stabbing of David McGuinn, a 42-year-old correctional officer.
In the ruling, the judges appear to agree with Assistant Attorney General James E. Williams, who argued that Stephens' appeal was premature. Stephens has not yet been tried in McGuinn's July 2006 homicide at the Maryland House of Correction, where Stephens, 31, and his co-defendant were then serving life sentences.
"Our argument was this was not allowed by law — it is not a final judgment," said Raquel Guillory, spokeswoman for the Attorney General's Office.
An Anne Arundel County judge had refused Stephens' request for a hearing on whether prosecutors have evidence that could meet the stringent restrictions that legislators enacted in 2009 in the death penalty law.
The changes limit prosecutors' authority to seek execution for first-degree murder convictions to killings in which there is DNA or other biological evidence, a videotaped confession or a video recording of the crime.
But, Williams had argued, they say nothing about a judge's deciding before the trial whether the evidence qualifies for a death penalty case. With rare exceptions, appeals do not start until after a verdict.
"The state made a large point that of the fact that this was not an appealable order," said H. Stephen Hut Jr., whose Washington law firm of Wilmer Cutler Pickering Hale and Dorr has been helping Stephens' attorneys. "It was not an insignificant part of the argument last Friday. … It is a disappointment, but is not a shock at all."
The ruling also lifted the Court of Appeals' stay on plans for a 10-week trial for Stephens. New trial dates will have to be selected.
Co-defendant Lamar Cornelius Harris, 41, has a pretrial appeal pending.
andrea.siegel@baltsun.com
Copyright © 2011, The Baltimore Sun


A ROAD safety alert has warned that roads across Limerick will remain 'treacherous' in the aftermath of Hurricane Ophelia.
The Road Safety Authority issued the alert late this Monday evening as the storm winds subsided in Limerick, leaving dozens, if not hundreds, of fallen trees on roads in its wake.
The RSA is advising people that they need to remain vigilant in the aftermath of the storm and "expect the unexpected as many roads will be treacherous".
"When storm Ophelia has passed the danger to road users will still be present. Many roads around the country will be blocked and treacherous with downed trees, branches and pylons.
"Council workers and emergency services will be out clearing these routes and people need to anticipate encountering these scenarios when using the roads," said a spokesperson.
"Pedestrians and cyclists should be aware of potential hazards. The severe winds may have loosened roof tiles and other objects from buildings and these may still pose a potential risk. Be aware of what's happening above you and stay clear from the edges of buildings.


text:
Q: como pasar una hoja .scss a .css? acabo de instalar sass con ruby installer cmd,ahora como deberia hacer para convertir mi codigo en css y aplicarlo con link en html? ? soy nuevo en los pre procesadores y no encuentro la opcion adecuada para lograr esto, en la pagina de sass econtre muypoca informacion ;(, alguna ayuda ? gracias ! ;D

A: si has instalado sass desde la consola solo tienes que tirar estas lineas en la consola :
sass input.scss output.css

Ahora vas a obtener el archivo css (output.css) y lo linkeas comunmente en tu archivo index.html. Te dejo un link
Has escuchado de gulp ?? Yo lo utilizo para correr tareas automaticas a la par que programo, por ejemplo obtener el css del codigo sass de mi codigo. gulpjs

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text:
Genetics Link Height to Heart Disease
4927 Views July 31, 2015 No Comments Health admin
The New England Journal of Medicine recently published research linking height and coronary artery disease through the study of genetics. Doctors have known since the 1950s about the link between short stature and coronary artery disease, "but the reason behind this really hasn't been completely clear," said Nilesh Samani, a cardiologist at the University of Leicester in the U.K.
Samani and his colleagues looked at the genes of nearly 200,000 people, "and we found a very striking relationship," Samani said. "We've been rather simplistic in our view of what causes coronary artery disease. We thought about traditional risk factors and then genes that might cause coronary disease. But what the study highlights is that developmental processes are going on that probably have an influence on height, and they probably also have an influence on blood vessels of the heart in a way that predisposes you to getting coronary artery disease."
The challenge now is to ferret out the actual genetic variations that underlie both height and heart disease. "Eventually, of course, there may be some treatments that could emerge from this, but I wouldn't want to say that's a short-term possibility," Samani said.
For someone 2.5 inches shorter than average, the risk of coronary artery disease increases by about 13.5 percent. And the shorter you are, the larger the effect; however, the risk is much smaller than the risk posed by smoking or high cholesterol. Most of the height genes had no obvious connection to heart disease, though a few of them did, such as a trait related to LDL cholesterol and another that influences triglycerides. Those account for less than a third of the effect, leaving the root cause of this link mainly a mystery.
The study also found that people who had more of the height-increasing genetic markers were at lower risk for coronary artery disease. People who had the most height-increasing genetic markers were 26 percent less likely to have coronary artery disease than those with the fewest height-increasing genetic markers.
Shared biologic processes that determine achieved height and the development of atherosclerosis may explain some of the association, notes the study. "We observed significant associations only with levels of low-density lipoprotein cholesterol and triglycerides, accounting for approximately thirty percent of the association," researches said.
David Goldstein, Director of the Institute for Genomic Medicine at Columbia University is enthusiastic about the opportunities the study brings to the medical community. "As we begin to systematically characterize the genetic bases of these traits, it's going to open up a whole bunch of brand new windows into biology," Goldstein says. "And that's really what I find exciting."
The study of height and heart disease is also a reminder that traits are usually the result of many different genes acting in concert, so it's not so simple to alter these traits, for example, to treat or prevent disease.
One major problem with the study is that it looked mostly at white males. With an increase in the diversity of subjects, perhaps scientists could have a better understanding of these genetic traits and markers.
See more at: http://www.lpwonline.com/news/page/2/#sthash.MTEvicpI.dpuf
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Trump News TV from Michael_Novakhov (11 sites): FoxNewsChannel's YouTube Videos: Do you feel gymtimidated?
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Trump News TV from Michael_Novakhov (11 sites): FoxNewsChannel's YouTube Videos: Does this finally settle the debate over death metal?
The end of the Russia probe has brought out the hypocrites and crazies.
WASHINGTON — The end of a sprawling investigation that has riveted the nation usually calls for a news conference from the person in charge, but Robert S.
The post The big clue that SDNY is going to indict Donald Trump's family appeared first on Palmer Report.
Google Alert – Elections Israel: POTUS Recognized Israel's Rights to the Golan. What's Next ?


The Thermal Combustor System is a market-driven answer to a unique set of vapor combustion air pollution control questions. Highly concentrated vent gas emissions are common within the natural gas processing and production, petrochemical, and chemical processing industries. These emissions are commonly low in volume, but very high in concentration.
Historically, answers to these emissions challenges have included inefficient and dangersous open flame flare technology.
The Thermal Combustor System is a much safer, more efficient, and more versatile abatement technology that is guaranteed to meet your air pollution needs. It is designed to safely burn or combust hydrocarbon vapor mixtures that have varying energy content where enclose flares may fail. It combines the simplicity of an Enclosed Flare with the robustness and proven performance of a thermal oxidizer. It is widely flexible and confidently handle the vent gas, which may or may not be within the flammable range. It provides an extraordinary level of safety, when compared to traditional flaring systems.
Thermal Combustor Systems are designed to burn safely or combust hydrocarbon vapor mixtures that have varying energy content where enclosed flares may fail. The TCS combines the simplicity of an enclosed flare with the robustness and proven performance of a thermal oxidizer.


Naltóoshgán, below, was home to the great southern clan the Ganaax.ádi, ancestral to the Aanx'aakhittaan and Deisheetaan. Entering Southeast via Nass River, the Ganaax.ádi established villages at Naltóoshgán, the good place (Whitewater) and Chayéek, arm goes way up inside (Chaik Bay) long before the founding of Aangóon. In places like Naltóoshgán, academic categories—Nature, Culture—relax and shake hands. For the past decade or so, my role as a naturalist has been to facilitate that meeting.
In August, 2012, Brenda Campen led Bob Christensen and me quietly through the ancient village of Naltóoshgán, on a raised, formerly tidal bench only a few feet up from the active drift line. A wall of vigorous spruces fronts the landing beach today. None were present in 1948 aerials. Branch whorls confirmed growth of over a foot per year on well-drained, culturally enriched soils, full of shell midden according to Frederica deLaguna's test pits. The mossy turf resembled a tended golf course, typical of many reforesting village sites where tightly interlocking tree crowns discourage vascular vegetation.
Sketched from a visit to AMNH in 1989.
This structure is by no means comprehensive, only reflecting several fairly narrow topics that appeal to me as a landscape-level naturalist. At Discovery, we have no pretensions toward expertise in—for example—Native language, customs, oral history, or spirituality. Our hope, however, is to support in any way we can, the exciting cultural resurgence that's happening everywhere you turn these days: in education, in art, and on the radio.