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Are you looking to refine your geodesy knowledge and gain mapping skills? UNAVCO hosts technical short courses each summer that provide training on geodetic data processing techniques and tools.
This year, we currently have four different virtual short courses on the calendar:
Generic Mapping Tools (GMT) for Geodesy: This course utilizes UNIX shell scripting for the use of GMT 6. Through lectures, exercises, and labs, participants will learn GMT basic concepts, like plotting grids, images, and vector data.
Tuesday, July 5, 2022 – Friday, July 8, 2022
Optional graduate credit through the University of Montana
INSAR Processing and Theory with GMTSAR: This course covers the theory and application of InSAR processing with GMTSAR. Through lectures and exercises, participants will learn the basic theoretical concepts of InSAR. In addition to software installation, labs will involve running test data sets for standard interferogram formation and advanced processing for time series with Sentinel-1 A TOPS-mode data. Participants who are not familiar with the GMT software package are recommended to also attend the GMT for Geodesy short course.
Monday, July 11, 2022 – Friday, July 15, 2022
Optional graduate credit through the University of Montana
GNSS Data Processing and Analysis with GAMIT/GLOBK and track: This course covers the use of GNSS processing software suite GAMIT/GLOBK and kinematic module track at intermediate and advanced levels. Lectures will cover GAMIT/GLOBK commands and file structures, important UNIX command-line sequences, reference frame realization, error analysis, kinematic measurements, and efficient processing of continuous and survey-mode observations.
Monday, July 18, 2022 – Friday, July 22, 2022
Optional graduate credits through the University of Montana
Multi-GNSS Precise Point Positioning and Pride PPP-AR: This course covers the theory of Global Navigation Satellite System (GNSS) precise point positioning (PPP) and focuses on the necessary corrections (ionosphere, satellite altitude, etc.) that ensure millimeter precision in daily GNSS and centimeter precision in kinematic GNSS. Participants will utilize the PRIDE PPP-AR software for PPP and PPP ambiguity resolution at static and kinematic stations for tectonics, aerial photogrammetry, seismology, and airborne and shipborne gravimetry.
Monday, September 19, 2022 – Friday, September 23, 2022
Since 2020, we have pivoted to offer these short courses virtually, which allowed 510 participants to complete the short courses in 2021. In 2022, the short courses will also be held remotely.
Additionally, UNAVCO has partnered with the University of Montana to provide graduate credit for participants who are interested. Those aiming to receive graduate credit for the short course file paperwork and a small credit fee to the University of Montana. UNAVCO will relay course completion to the University of Montana, which will then issue a pass/fail grade.
Registration for the GMT, GMTSAR, and GAMIT/GLOBK short courses is open! Registration for the PPP short course will open in June. Follow our social media or join our email list to receive announcements on added courses and registration reminders.
Interested in viewing last year's short course sessions? Check out our Events page to see materials and recordings from UNAVCO's previous short courses.
Written by:
- Lucia Bellino
- Posted: 24 March 2022
- Last updated: 24 March 2022
- Tags: short course


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Half of families with young children will struggle to make ends meet in the next three months, according to new research on the effects of the coronavirus crisis.
A joint report by the Fawcett Society, the Women's Budget Group, Mile End Institute at Queen Mary University, and the London School of Economics, revealed that in the wake of the pandemic, 43 per cent of parents with children under 11 say their household has almost run out of money, 48 per cent are worried about how they will pay the mortgage or rent and 57 per cent face higher levels of debt in the aftermath of the lockdown.
The research, based on a survey of 1,783 people, found the impact is taking a greater toll on women. The pandemic magnifies existing inequalities that see women earning less, more likely to work part-time and in less secure roles, which in turn, causes higher anxiety levels. According to the report, "nearly half (46 per cent) of mothers of under-11s report anxiety above a seven on a scale of 0 – 10, compared with 36 per cent of fathers."
For single parents, 90 per cent of whom are women, the challenges become "so much tougher when you are on your own," says Sam Smethers, chief executive of The Fawcett Society. Jenna Norman of the Women's Budget Group adds: "As payment holidays begin to end and some parents are sent back to work without full-time childcare available, single parents face more impossible choices."
Read more
Coronavirus shows we need a revolution for working women – and it starts with trade unions
Here a single mother of three, 41, shares her coronavirus diary of the first three weeks of lockdown.
'It's tricky budgeting now'
Week 1
"I'm a single mother of a one, four and seven-year-old in my third year of a divorce with an abusive ex. I'm parenting 24/7. Their father didn't bother to see them on Sunday, which is meant to be his day. He isn't allowed overnight contact because of his excessive drinking.
I've been asking him to get us a weekly shop but he's refused. Taking three children round a supermarket is very stressful – they touch everything, run around, put their fingers up their noses. You get a lot of animosity, people think you're stupidly bringing kids with you who will spread the virus. Yet again it seems that single parents, usually mothers, are left to struggle. Or we are just not important enough?
I did finally get a Tesco slot this week though. I signed up on the Government website to the vulnerable list after my doctor told me I was at risk. The Government guidelines have been unclear. I've also been assigned a volunteer, we haven't worked with them yet but I'm concerned as she doesn't have a car. It means she wouldn't be able to do a full shop for us. I feel guilty as there may be others worse off than us but single-parent families who aren't co-parenting should be offered priority supermarket slots.
I have to budget very carefully. It's tricky not being able to hunt for the usual bargains and means that we will go without. I'm on Universal Credit, currently getting £1,432, £1,000 of which goes towards rent. I don't get any money for a third child. I also receive £192 in child benefits every four weeks. My ex has a court order to pay part of the rent.
I'm quite resourceful. I cook from scratch, make soup for the week. Sometimes friends drop off meals. We have also received support from charities who have provided food and stationery supplies.
I'm worried that I don't have enough energy and capacity to give my children what they need – emotionally, psychologically, mentally, physically, spiritually. It's totally overwhelming. I spend most of my time cooking and trying to keep the house in order but it's still chaos.
I suffer from a chronic sinus issue and I've got three autoimmune disorders. I worry about what would happen to the children if I became ill.
The house is a pit. The children are running riot but we have moments of calm and fun. We go on bike rides, build dens and do lots of drawing. Without our trampoline, I would have lost my mind!
Lots to say but I have to get back to the children.
'I'm exhausted and need support'
Week 2
I've felt absolutely exhausted this week.
I need some serious support. My ability to be a patient, kind and caring mother is being challenged. Feeling like I've failed when I end up shouting and roaring. I then beat myself up, feeling like a terrible parent.
I also feel seriously under pressure to home school. It's not possible to get my dyslexic kids to complete all of the tasks. I feel saturated with information and brain-fried with screens. I have headaches, feel panicked and can't think straight. As a family entitled to pupil premium, it feels like we're missing out even more now the children can't go to school. On top of that is the cost of homeschooling. I've had to spend a huge amount of our budget on printer ink and pencils etc. All these extras impact on our tight budget.
Pupil Premium
For each child who receives free school meals, the school receives around £1,000 from the Government to help close the attainment gap.
I also can't use the government vouchers replacing free school meals. The ones I've been provided with can only be used in-store, which I can't go to as a single-parent family because it's just too difficult at the moment. [Edenred, who provides the voucher service for the Government, suggests forwarding the vouchers onto someone who can go to the shops for you]. I wrote to the school who said it couldn't be changed but the vouchers were redeemable up to five years! This makes my blood boil. It just doesn't help.
However, there are many wonderful things happening with my small family. We are all getting to know each other better and having the opportunity to iron out behavioural issues, emotional issues and work on our relationships.
That's it. Writing this on my mobile in the dark with my sleeping one year old next to me. Got to take the opportunity to try to get some rest before the night torture begins!
'I'm trying not to be hard on myself'
Week 3
I haven't been well last week, coughing, headaches, exhaustion – could have been coronavirus.
It's been a struggle pushing through with the kids. We had a duvet day yesterday, all in bed with our pyjamas. I have to remember not to be hard on myself and just let things go.
I've been through worse. We were in a refuge when my youngest was 16 months. So I'm trying to stay positive and look for the benefits, like spending more time with my children. I think the boys have undiagnosed ADHD, and now I'm thinking that I might also – through watching them I can see myself.
I crave sugar because I'm so exhausted. I've put on weight. It's definitely taking its toll on my body. My hair has nearly all gone grey.
Worried about the children's education. Aware that because there's only me they are likely to be more disadvantaged. Aware that there's not much I can do about this but continue to try my best.
Thinking about what I'm going to do to get back into work and pull us out of the benefit trap. How am I going to manage to make money and improve our quality of life whilst giving my children the attention they need? How am I going to manage to do that during lockdown and in the new world after the lockdown begins to lift?"
What help do single parents need?
The study recommends the Government offer more financial help to all parents, calling for an increase in child benefit by additional £50 per week per child, and amending Universal Credit, lifting the two-child limit and abolishing the five-week wait time. Norman also suggests parents should continue to be furloughed if they cannot get full-time childcare.
A Government spokesperson said: "This Government understands the challenges many are facing which is why we injected £6.5bn into the welfare system, including increasing Universal Credit and Working Tax Credit by up to £1,040 a year, as well as rolling out income protection schemes, mortgage holidays and additional support for renters.
"With Universal Credit, no one has to wait five weeks for their money as urgent advances are available – and around 970,000 people have requested and received one since 16 March."
For support and advice on being a single parent during the pandemic, visit Gingerbread. For support and information on domestic abuse, visit Women's Aid.

subset: commoncrawl


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The Bose Noise Cancelling Headphones 700 are good for phone calls. Their integrated microphone has a good recording quality, and it can separate speech from background noise even in crowded environments. If you take a lot of calls from your PC or Mac, you may notice a drop in sound quality.
Are Bose headphones good for audio editing?
Table of Contents hide
Its revolutionary noise cancellation technology and clean but durable build make the Bose QuietComfort 35 II a boon for mixing tracks and mastering recordings.
Which headphone has the best microphone?
How to choose the best headphones with a mic
- Bose Noise Cancelling Headphones 700.
- Cambridge Audio Melomania 1 Plus.
- Sony WF-1000XM4.
- Shure Aonic Free.
- Apple AirPods Pro 2.
- Shure Aonic 3. Award winner 2022.
- Sony WH-1000XM4. Award winner 2022.
- Panasonic RZ-S500W. A hugely talented and affordable pair of noise-cancelling earbuds.
Is Bose microphone good?
The Bose QuietComfort 35 II has some of the best noise cancelling in the business, and it's regarded as one of the most comfortable headsets. With a great default frequency response, travel-friendly design, and solid mic system, there's plenty to love about this headset.


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This website uses cookies so that we can provide you with the best user experience possible. Cookie information is stored in your browser and performs functions such as recognising you when you return to our website and helping our team to understand which sections of the website you find most interesting and useful.
Thu, 10 Jun. 2021, 08:30
Thu, 10 Jun. 2021, 09:30
UKABC is honoured to host Dan Chugg, UK Ambassador to Myanmar to an important roundtable discussion with UKABC partners. The aim of the conversation is to hear the latest developments in Myanmar and gain critical insight into what to expect in the coming months.
On 1 February, Myanmar's military seized control of the country following a landslide victory by Aung San Suu Kyi's NLD Party. The military has detained Ms Suu Kyi and arrested political opponents. The UK has condemned the coup and called for a peaceful return to democracy.
The ASEAN Secretary-General, Dato Lim Jock Hoi, and the Brunei Foreign Minister, Dato Erywan Yusof, as Chair of ASEAN, visited Myanmar to discuss the ASEAN Five-Point Consensus that aims to end violence; promote dialogue; deliver aid; appoint a special envoy, and send a delegation headed by the envoy to Myanmar to meet "with all parties concerned". Commentators have noted the growing divide among ASEAN member states in responding to the crisis.
Myanmar's economy is shrinking due to the coup and covid19. The World Bank forecasts Myanmar's GDP to fall by 10%. The UN Development Programme has predicted that almost half the 54million population could be living in poverty by 2022. Businesses are starting to write off their investments. Telenor, a Norwegian mobile operator in Myanmar for over ten years, has written off its £551m investment as it sees 'limited prospects of improvement'.
One area profiting is the drug trade as drug producers and traffickers take advantage of the crisis in the 'Golden Triangle' – Myanmar's border zone with Laos, Thailand and China.
Areas of discussion
- What to expect in Myanmar in the near term?
- ASEAN's Five-Point Consensus and the UK sanctions on Myanmar
- Is civil war a possibility?
- Population displacement and the impact it is having on its neighbours'
- What should UK business do?
This event is for UKABC board members and partners. To find out more about partnership opportunities contact firstname.lastname@example.com.
8.30 Welcome remarks
• Baroness Neville-Rolfe DBE CMG, Chair, UK-ASEAN Business Council
8.35 Partner conversation
• Dan Chugg, UK Ambassador to Myanmar
Chaired by Baroness Neville-Rolfe DBE CMG, Chair, UK-ASEAN Business Council
9.30 Roundtable concludes
Daniel Chugg took up his post as British Ambassador to Myanmar (Burma) at the end of May 2018.
His previous diplomatic roles include being the Head of the Counter-Daesh Communications Department and the Head of the ISIL Taskforce and Iraq Department. His experience in Asia includes posts in Beijing (Counsellor Political), New Delhi (First Secretary) and Hong Kong (Vice-Consul).
Dan is married with 2 children.
Further reading
EXCLUSIVE Amid divisions, ASEAN leaders plan Myanmar visit this week. Reuters.
Chairman's Statement on the ASEAN Leaders' Meeting. ASEAN Secretariat
UK announces sanctions on gemstone company linked to the military regime in Myanmar. Gov.UK
Myanmar's generals struggle to restart its stalled economy. The Economist.
Myanmar military fails to quell protests four months after coup. Reuters


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Probably Straubing, around 1460
With rests of its original, polychrome frame.
The picture of crowned Maria Iactans flanked by two angels is a common type of the picture of the Virgin Mary in devotional pictures since the Middle Age.
The Lower Bavarian area around Landshut and especially around Straubing is known for this kind of extraordinarily fine works of terracotta.
Height 31,8 cm, width 26 cm.


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Car manufacturers are great at building cars (who would've guessed, right?), but they aren't that great at developing nice media control interfaces. Besides, every car manufacturer has their own take on a media interface so there's no uniformity. Fortunately, the tech companies decided to step in a few years ago and launched extensions of their mobile operating systems for the car, namely CarPlay and Android Auto. Plex today announced its support for Android Auto. It will enable seamless media integration in the car.
There are plenty of new cars out there that come with support for Android Auto right off the production line. There are also many third-party units available with Android Auto support.
Plex for Android Auto is going to make accessing your music in the car easier as it seamlessly integrated with the car's display. The interface is simplified and the touch areas are larger so as to minimize distractions. All users need to get up and running is Android Auto and the new Plex for Android app.
They can then interact with Plex for Android Auto using the app on their phone, Google Voice, or the car's dashboard. Any car infotainment system that supports Android Auto will be able to run Plex for Android Auto. The Android Auto interface for the app can also be used on an Android smartphone or tablet.
Plex says that the Android Auto support will be rolled out to everyone in the next few days.
Filed in Android Auto and Plex. Source: plex.tv
. Read more about


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Following chemotherapy and relapse, high-risk neuroblastoma tumors harbor more genomic alterations than at diagnosis, including increased transcriptional activity of the Yes-associated protein (YAP), a key downstream component of the Hippo signaling network. Although YAP has been implicated in many cancer types, its functional role in the aggressive pediatric cancer neuroblastoma is not well-characterized. In this study, we performed genetic manipulation of YAP in human-derived neuroblastoma cell lines to investigate YAP function in key aspects of the malignant phenotype, including mesenchymal properties, tumor growth, chemotherapy response, and MEK inhibitor response. Standard cytotoxic therapy induced YAP expression and transcriptional activity in patient-derived xenografts treated in vivo, which may contribute to neuroblastoma recurrence. Moreover, YAP promoted a mesenchymal phenotype in high-risk neuroblastoma that modulated tumor growth and therapy resistance in vivo. Finally, the BH3-only protein, Harakiri (HRK), was identified as a novel target inhibited by YAP, which, when suppressed, prevented apoptosis in response to nutrient deprivation in vitro and promoted tumor aggression, chemotherapy resistance, and MEK inhibitor resistance in vivo. Collectively, these findings suggest that YAP inhibition may improve chemotherapy response in patients with neuroblastoma via its regulation of HRK, thus providing a critical strategic complement to MEK inhibitor therapy.
This study identifies HRK as a novel tumor suppressor in neuroblastoma and suggests dual MEK and YAP inhibition as a potential therapeutic strategy in RAS-hyperactivated neuroblastomas.
Despite intense multimodal therapy, more than 50% of patients with high-risk neuroblastoma relapse with aggressive chemotherapy-resistant disease, stressing an exigent need for therapies targeting pathways that drive recurrence (1). Following treatment and relapse, high-risk neuroblastoma tumors display significantly more RAS/RAF/MAPK genetic alterations than at diagnosis (2, 3). Gene set enrichment analyses (GSEA) also show increased transcriptional activity of the Yes-associated protein (YAP) in posttherapy recurrent neuroblastoma tumors (4). YAP is a transcriptional coactivator that binds with TEA domain (TEAD) family transcription factors (TF) to regulate genes promoting organ growth, cell self-renewal, and survival (5–8). YAP, and its paralog TAZ, are negatively regulated by proteins of the Hippo pathway that phosphorylate YAP/TAZ, leading to their cytoplasmic retention and inactivation. When YAP is dephosphorylated, it translocates into the nucleus to bind TEAD and mediate transcription. YAP's aberrant transcription functionally contributes to many aspects of cancer formation and survival (8–12). YAP's transcriptional regulation of downstream target genes such as EGFR, SOX2, and OCT4 has been shown to promote the mesenchymal phenotype and resistance to therapy (8–13). In addition, YAP regulates genes associated with epithelial-to-mesenchymal transition, thus promoting metastasis in many cancers (14). In an in vivo metastatic mouse model of neuroblastoma, neuroblastoma tumors that had metastasized to the brain displayed increased levels of YAP, suggesting a potential microenvironmental and metastatic role for YAP (15). While YAP "genetic signatures" are associated with tumor recurrence and independently predict poor neuroblastoma patient outcome, the functional role for YAP itself in neuroblastoma has not been characterized (3, 15). Furthermore, given the lack of YAP mutations or upstream Hippo pathway mutations in relapsed neuroblastoma tumors, it is unclear how YAP transcriptional activity increases at or before tumor recurrence and whether it contributes to neuroblastoma relapse. We now show that standard cytotoxic chemotherapy induces both YAP expression and transcriptional activity in murine models of neuroblastoma. We also demonstrate that YAP promotes a mesenchymal phenotype to affect in vivo neuroblastoma tumor growth and therapy response, and further inhibits apoptosis in response to nutrient deprivation through a novel regulation of the proapoptotic protein, Harakiri (HRK).
Materials and Methods
Cell lines and patient-derived xenograft models
Human-derived neuroblastoma cell lines were obtained from the Children's Oncology Group Childhood Cancer Repository and the ATCC. Neuroblastoma cell lines were cultured in RPMI1640 (Sigma) with 10% FBS (Gemini) and 1% penicillin–streptomycin (Gemini) and incubated at 37°C with 5% CO2 (16). All cells used were maintained at low passage, which did not exceed 15 passages. Cell lines and transduced cell models underwent short tandem repeat–based genotyping (Texas Tech Cancer Cell Repository) and identities were verified using the Children's Oncology Group cell line genotype database (www.cccells.org). Cells were routinely tested for Mycoplasma with MycoAlert Mycoplasma Detection Kit (Lonza). Patient-derived xenografts (PDX) were created from fresh deidentified human tumor tissue obtained from patients at diagnosis or at relapse under a Children's Healthcare of Atlanta Biorepository (Atlanta, GA) protocol approved by the institutional review board (IRB00034535). Written informed consent was obtained from patients/families for tumor tissue procurement, including cell line and PDX derivation, and deidentification for research investigations. Studies were conducted in accordance with recognized ethical guidelines (Declaration of Helsinki).
RNA was purified from neuroblastoma cell lines and snap-frozen xenograft tumors using the RNeasy Mini Kit (Qiagen) and TRIzol (Ambion) and Chloroform (Sigma), respectively. cDNA was created using the high-capacity cDNA Reverse Transcription Kit (Applied Biosystems). Power SYBR Green PCR Master Mix (Applied Biosystems) and the Bio-Rad System were utilized for quantitative PCR, with samples run in triplicate. Relative gene expression was normalized to GAPDH and HPRT using the CFX Manager Software (Bio-Rad). Primer sequences are detailed in Supplementary Table S1.
Western blot analysis
Snap-frozen xenograft tumors were dissociated with mortar and pestle. Neuroblastoma cell lines were consistently harvested at 80% confluency, as YAP expression can be influenced by cell contact (17). Neuroblastoma cells were lysed with CHAPS buffer (10 mmol/L HEPES, 150 mmol/L NaCl, and 2% CHAPS) with protease inhibitors (Roche Protease Inhibitor Cocktail 1×, 2 mmol/L sodium orthovanadate, and 2 mmol/L PMSF), run on Nu-PAGE 4%–12% Bis-Tris Gels (Invitrogen), transferred to polyvinylidene difluoride Membranes (Invitrogen), and detected for proteins by chemiluminescence as described previously (18).
Antibodies from Cell Signaling Technology used include: YAP (#4912, RRID:AB_2218911), phospho-YAP (Ser127) (#4911, RRID:AB_2218913), TAZ (V386) (#4883, RRID:AB_1904158), pan-TEAD (D3F7L) (#13295, RRID:AB_2687902), EGFR (C74B9) (#2646, RRID:AB_2230881), phospho-EGFR (Tyr1068) (#3777, RRID:AB_2096270), CYR61 (E5W3H) (#39382, RRID:AB_2799154), GAPDH (14C10) (#2118, RRID:AB_561053), BCL-XL (#2762, RRID:AB_10694844), BIM (#2933, RRID:AB_1030947), and cytochrome c (#11940, RRID:AB_2637071). Other antibodies used were: β-Tubulin (Santa Cruz Biotechnology, #sc-53140, RRID:AB_793543), BCL2 (Agilent, t# M0887, RRID:AB_2064429), and MCL1 (Enzo Life Sciences, #ADI-AAP-240, RRID:AB_10997659).
Stable transduction of YAP genetic inhibition and expression
YAP gene expression was stably knocked down in SK-N-AS (RRID:CVCL_1700) and NLF (RRID:CVCL_E217) using MISSION YAP shRNA Lentiviral Particles (Sigma, SHCLNV-NM_006106). Cells were transduced with shYAP or control vector (CV) containing lentiviral particles with 8 μg/mL polybrene for 48 hours, and then, transfectants were selected for and maintained in puromycin. For YAP expression in IMR5 (RRID:CVCL_1306) and NGP (RRID:CVCL_2141), pGAMA-empty (Addgene, plasmid #74755; RRID: Addgene_74755) and pGAMA-YAP (Addgene, plasmid #74942; RRID:Addgene_74942) were gifts from Miguel Ramalho-Santos (University of Toronto, Toronto, Canada). HEK293T (RRID:CVCL_0063) cells were transfected with 3 μg of plasmid DNA, pMD2.G (gift from Didier Trono, EPFL, Lausanne, Switzerland; Addgene, plasmid #12259; RRID:Addgene_12259) and psPAX2 (gift from Didier Trono; Addgene, plasmid # 12260; RRID:Addgene_12260) using Fugene 6 (Promega) transfection reagent protocol at ratio of 1:3 (plasmid DNA:Fugene 6). Plasmid containing viral particles were collected from the supernatant and used to transduce neuroblastoma cells.
Cell proliferation assays
Cells were plated in triplicate at 10,000 cells/well and CellTiter-Glo (Promega) was used to quantify live cells at different timepoints.
Colony formation assays
Cells were plated at 2,000 cells/well and cultured for 7 days. Cells were fixed with formaldehyde (1%) and stained with crystal violet (0.1%, w/v) in 20% methanol. Wells were imaged with a Lionheart FX Microscope (BioTek) and quantification was performed with ImageJ (NIH; RRID:SCR_003070).
Neurosphere assays
Cells were plated at 15,000 cells/mL in neurosphere media (50/50 F12/DMEM, Thermo Fisher Scientific; 1× B27, Thermo Fisher Scientific; 1× N2, Thermo Fisher Scientific; 0.1 mmol/L BME, Sigma; 2 μg/mL heparin, Stemcell Technologies; 1% penicillin–streptomycin, Gemini; 20 ng/mL EGF, Corning; and 40 ng/mL FGF, Corning) and cultured in Ultra-Low Attachment Plates (Corning). Neurospheres were harvested after 7 days for qRT-PCR. For quantification assays, cells were plated in triplicate at a density of 2,000 cells/100 μL in each well and grown for 7 days, and then imaged.
In vitro drug studies
Cells were plated in triplicate at 10,000 cells/well. After 24 hours, cells were treated with vehicle (DMSO), etoposide (Sigma), irinotecan (Sigma), topotecan (Sagent Pharmaceuticals), and melphalan (Sigma) for 48 hours, or trametinib (Selleck Chemicals) for 72 hours and evaluated by CellTiter-Glo (Promega).
Cytosolic/mitochondrial fractionation
Harvested cells were suspended in subcellular fractionation buffer (250 mmol/L sucrose, 20 mmol/L HEPES, 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, and 1 mmol/L EGTA) with protease inhibitors (Roche Protease Inhibitor Cocktail 1×, 2 mmol/L sodium orthovanadate, and 2 mmol/L PMSF) and lysed. Lysate was incubated on ice for 20 minutes, and then centrifuged at 720 × g for 5 minutes. The resulting supernatant was centrifuged at 10,000 × g for 5 minutes to produce cytosolic (supernatant) and mitochondrial (pellet) fractions. The mitochondrial fraction was washed and resuspended in subcellular fractionation buffer supplemented with 10% glycerol and 0.1% SDS. The resulting fractions were immunoblotted for cytochrome c.
Apoptosis assays
For serum starvation experiments, cells were grown in RPMI with 10% FBS for 24 hours, media were replaced with RPMI containing 10% FBS or 0.1% FBS, and cells were incubated for 48 hours. For etoposide (Sigma) treatments, cells were exposed to either vehicle (DMSO) or etoposide (5 μmol/L) for 48 hours. BD ViaProbe/7-AAD (Thermo Fisher Scientific) and APC Annexin V (BioLegend) were added and detected by Flow Cytometry (Cytek Aurora). Analysis was performed using FlowJo software (v10, RRID:SCR_008520). Caspase Glo 9 (Promega) was used per protocol to quantify caspase-9 activity.
Ethics statement
All animal studies were conducted in accordance with policies set forth by the Emory University (Atlanta, GA) Institutional Animal Care and Use Committee (IACUC). Our protocol was approved by the Emory IACUC (PROTO201700089). Euthanasia was performed by CO2 and cervical dislocation.
Mouse xenograft in vivo studies
Mice were approximately 6 weeks of age at time of injection and male/female mice were distributed equally among experimental arms. For all tumor growth and drug treatment studies, mice were randomized on the basis of tumor size and gender into the different treatment arms to maintain equivalent median tumor sizes per arm of each study. We measured binary endpoints (i.e., tumor growth or tumor regression), 4–6 mice per group were used to detect 50% differences in group means, assuming sample SDs < 50% of the mean (α = 0.05 and power = 0.8). For PDX studies, low passage (≤3rd) PDX tumors were removed from one NOD scid gamma (NSG; The Jackson Laboratory; IMSR catalog no., ARC:NSG, RRID:IMSR_ARC:NSG) mouse, dissociated, mixed 1:1 in Matrigel (Corning) as a single-cell suspension with 4 × 106 cells/injection, and reinjected into the subcutaneous flank of additional NSG mice. Tumor volumes were measured with calipers and calculated using formula L × W × H × π/6. When tumors reached approximately 150 mm3, mice were treated with vehicle control (PBS) or 0.05 mg/kg topotecan (Sagent Pharmaceuticals) and 20 mg/kg cyclophosphamide (Jiangsu Hengrui Medicine) via intraperitoneal injections once daily for 5 days. Mice were sacrificed on day 6 and tumor was harvested and evaluated. For tumor formation studies, SK-N-AS CV, shYAP#1, and shYAP#2 cells (4 × 106 cells/injection) were injected into the subcutaneous flanks of NSG mice (n = 4 per arm). Mice were sacrificed when tumor volumes reached 1,500 mm3. For cyclophosphamide treatment studies, SK-N-AS xenografts were established as above in nu/nu athymic mice (The Jackson Laboratory; MGI #3848172, RRID:MGI:3848172). Mice (n = 5 per arm) were treated with vehicle (PBS) or 75 mg/kg cyclophosphamide by intraperitoneal injection twice weekly for four doses and sacrificed when tumor volumes reached 1,500 mm3. For trametinib studies, SK-N-AS xenografts established subcutaneously in NSG mice (n = 6 per arm) were treated with vehicle (35% Kollisolv PEG E 400, 60% Phosal, and 5% DMSO) or 3 mg/kg Trametinib (Selleck Chemicals) for 14 days once daily via oral gavage and mice were sacrificed when tumor volumes reached 1,500 mm3.
SK-N-AS CV, shYAP#1, and shYAP#2 pellets in triplicates were harvested, RNA was isolated, and quality checked at the Emory Integrated Genomics Core. RNA-sequencing (RNA-seq; poly-A selection, 20 M paired-end 150 bp reads on the Illumina NovaSeq Platform) was performed at Novogene, with data analyzed by the Emory Integrated Computational Core. Sequencing data were first checked by FastQC (RRID:SCR_014583) for quality control, and then aligned to the human "indexed" reference genome (hg38) using STAR (RRID:SCR_015899; ref. 19). Gene quantification was performed using HTSeq-count (RRID:SCR_011867; ref. 20) and gene count data were filtered before analysis to remove lowly expressed genes. Counts per million (CPM) data were generated after normalization using trimmed method of M-values, as implemented in the Bioconductor package (RRID:SCR_006442), EdgeR (RRID:SCR_012802; ref. 21). To determine genes important for a case–control, a t test followed by FDR (Benjamini–Hochberg method) on normalized log2-transformed CPM was performed. A gene was defined as differentially expressed gene (DEG) if its FDR ≤ 0.05. To characterize expressed genes, a preranked permutation-based GSEA was performed with weighted Kolmogorov–Smirnov statistical test (22). A gene set from the Molecular Signatures Database (v7.0) was used as input data for this analysis. The identified DEGs were used in pathway analysis.
Statistical analysis
A Student t test or a one-way ANOVA was used to assess statistical significance. A log-rank (Mantel–Cox) test was performed on the Kaplan–Meier survival curves for xenograft studies. Statistical significance was defined as P < 0.05. GraphPad Prism (v8.0; RRID:SCR_002798) was used for all analyses.
YAP tumor expression and transcriptional activity increase in response to in vivo standard chemotherapy treatment of neuroblastoma tumors
Given previous reports demonstrating increased YAP transcription in primary recurrent neuroblastomas, we first sought to determine YAP expression in neuroblastoma PDXs from the same patient's prechemotherapy diagnostic (NBX-4) and post-chemotherapy recurrent (NBX-4R) tumor. Results demonstrate that YAP expression and transcriptional activity increased in the relapsed PDX compared with the pretherapy PDX from the same patient (Fig. 1A). In keeping with this finding, paired isogenic cell lines derived from the same patient at diagnosis prechemotherapy (SK-N-BE1) and at relapse following chemotherapy (SK-N-BE2) showed increased YAP expression in the relapsed SK-N-BE2 (Supplementary Fig. S1). To gain a sense for the timing of YAP increase during or after therapy, we established in vivo tumors using chemotherapy-naïve NBX-4 cells and subsequently treated mice with 5 days of vehicle or topotecan and cyclophosphamide at doses and regimen comparable with a standard cycle of chemotherapy used for neuroblastoma patient induction (23). The tumors grew through chemotherapy or vehicle treatment and were harvested on day 6. Notably, YAP gene and protein expression increased in the chemotherapy-treated tumors and remained absent in the vehicle-treated control and expression of YAP transcriptional targets, CTGF and EGFR, increased in the chemotherapy-treated tumor, but not in the vehicle control (Fig. 1B and C). We treated a second neuroblastoma PDX derived from another patient's diagnostic chemotherapy-naïve tumor (NBX-1a) with topotecan and cyclophosphamide. Tumors grew through the topotecan and cyclophosphamide treatment, and again we observed that YAP protein expression increased by day 6 in the chemotherapy-treated PDX, but not in the vehicle control (Fig. 1D). We then evaluated a panel of human-derived neuroblastoma cell lines and found YAP protein to be heterogeneously expressed (Fig. 1E). Phosphorylated and inactive YAP (pYAP) was present in some cell lines. TAZ was neither always coexpressed with YAP nor was it increased in YAP-null cells. TEAD protein was present throughout all cell lines. Expression of known downstream targets of YAP-mediated transcription, EGFR and CYR61 (24, 25), was increased in neuroblastomas with YAP expression, suggesting that YAP transcription may be active despite the concurrent presence of pYAP in certain cells. We also found that YAP expression does not correlate with MYCN amplification status and YAP was commonly seen in neuroblastoma cell lines harboring RAS/MAPK mutations, but not ALK mutations (Fig. 1E; Supplementary Table S2).
YAP genetic inhibition suppresses neuroblastoma tumor growth in vivo
A prime role for YAP in both normal development and in cancer is to promote growth through transcriptional as well as posttranslational regulation of genes involved in cell proliferation (26, 27). To test the effects of YAP on neuroblastoma growth, we used both gain- and loss-of-function approaches to perturb YAP expression. Western blot analysis of these cell lines confirmed YAP knockdown (Fig. 2A) and overexpression (Fig. 2B; Supplementary Fig. S2A). TAZ and TEAD expression were not altered by YAP knockdown (Fig. 2A). While EGFR and phosphorylated EGFR (pEGFR) did not significantly change, YAP targets, CTGF and CYR61, decreased with YAP knockdown and increased with YAP overexpression, suggesting these models efficiently modulate YAP transcriptional activity (Fig. 2A and B). In vitro, YAP perturbation does not significantly alter cell proliferation rates (Fig. 2C; Supplementary Fig. S2B). Yet, in the in vivo setting, time to sacrifice occurred later in the SK-N-AS shYAP xenografts compared with the SK-N-AS CV tumors (Fig. 2D). Importantly, YAP remained knocked down at time of sacrifice (Fig. 2D). These results suggest that YAP affects neuroblastoma proliferation within the heterotypic tumor microenvironment (TME) and may promote the initiation of neuroblastoma growth.
YAP is increased by stem-like conditions and regulates genes promoting a mesenchymal phenotype
In other tumor models, YAP regulates tumor cell growth by mediating contact-independent cell proliferation and expression of stem-like genes (9, 10, 28). We, therefore, evaluated the colony formation potential of NLF shYAP cells and show a significant decrease in the number of colonies formed compared with NLF CV cells (Fig. 3A). To further investigate for mesenchymal properties of YAP, we grew human-derived neuroblastoma cell lines as neurospheres in neurobasal media, thus mirroring the stem-like environment. YAP gene expression was significantly increased upon neurosphere formation (Fig. 3B). OCT4 and SOX2, known mesenchymal genes regulated by YAP (28, 29), increased under neurosphere conditions, compared with regular culture conditions (Fig. 3B). We evaluated for changes in mesenchymal gene expression in the NLF shYAP model versus NLF CV cells grown in neurobasal media and noted that YAP knockdown leads to decreased levels of these genes (Fig. 3C). Furthermore, RNA-seq of SK-N-AS shYAP models compared with SK-N-AS CV shows genes associated with a mesenchymal and stem-like state in other tumor types, such as JAK1, IL6ST, TBX3, and PPP2R2B (30, 31), decreased when YAP was knocked down in neuroblastoma (Supplementary Fig. S3). We also noted fewer neurospheres in the NLF shYAP cells, and an increased number of neurospheres in the YAP-overexpressed cells (Fig. 3D). Together, these results suggest that YAP promotes a dedifferentiated mesenchymal phenotype.
YAP does not influence chemotherapy-induced apoptosis in neuroblastoma in vitro
Neuroblastoma cells with a mesenchymal phenotype represent the most chemotherapy-resistant population (29, 32–34). Given YAP is increased at relapse and under stem-like media conditions, we evaluated whether YAP expression and transcriptional activity affected neuroblastoma response to chemotherapy. We treated neuroblastoma cells harboring YAP knockdown or CV in vitro with cytotoxic agents used in high-risk neuroblastoma first-line therapy or in relapsed salvage therapy, melphalan, irinotecan, topotecan, and etoposide, and evaluated for cell death. YAP genetic knockdown or YAP overexpression neither sensitizes nor promotes resistance to cytotoxic therapy, respectively, in vitro (Fig. 4A; Supplementary Fig. S4A–S4D). Short hairpin RNA (shRNA) inhibition of YAP also fails to augment apoptosis in NLF shYAP cells treated with etoposide in vitro (Supplementary Fig. S5). YAP has been shown to transcriptionally upregulate prosurvival multidomain BCL2 family members (BCL2 and BCL-XL) in other cancers (35, 36), however, YAP knockdown does not affect baseline BCL2 family prosurvival protein expression in neuroblastoma (Supplementary Fig. S6A). Although YAP knockdown leads to an increase in proapoptotic BIM protein expression, it does not alter BIM binding to prosurvival BCL2 members that are known to prevent apoptosis (Supplementary Fig. S6B; ref. 37).
YAP genetic inhibition sensitizes neuroblastoma xenografts to cytotoxic and MEK inhibitor therapy in vivo
Given the heterogeneity of solid tumors due to their TME (38), we ascertained whether YAP affects cytotoxic response in vivo. We treated SK-N-AS CV and shYAP xenografts with cyclophosphamide and monitored tumor growth. SK-N-AS shYAP xenografts were significantly more sensitive to cyclophosphamide in vivo compared with control xenografts (Fig. 4B). Because of strong evidence in other cancers for an interaction between YAP and oncogenic RAS (39), we queried whether YAP expression also affects MEK inhibitor therapy response. We treated the NRAS-mutated SK-N-AS shYAP and CV cells with the MEK inhibitor, trametinib, both in vitro and in vivo. Similar to chemotherapy responses, there was no change in trametinib-induced cell death in vitro with YAP genetic inhibition in neuroblastoma cell lines harboring NRAS (SK-N-AS) and NF1 (NLF) mutations (Fig. 4C). In contrast, SK-N-AS shYAP xenografts were significantly more sensitive to trametinib in vivo with more tumor regression in YAP shRNA–transduced cell line xenografts compared with SK-N-AS CV xenografts (Fig. 4D). Importantly, for both the cytotoxic and trametinib xenograft studies, the decrease in tumor growth in response to therapy in the YAP-knockdown models remained significant after considering the delay in normal tumor growth imparted by YAP knockdown alone. Therefore, YAP knockdown has an increased effect on cytotoxic and trametinib responses in situ, supporting a potential role for YAP-influencing responses to stresses imparted by the 3-dimensional microenvironment in neuroblastoma.
YAP regulates BH3 genes involved in intrinsic apoptosis in neuroblastoma
To understand genes regulated by YAP that may be contributing to the more profound effects seen on tumor growth and treatment response in vivo, we performed RNA-seq on SK-N-AS shYAP#1 and #2 models and SK-N-AS CV cells. Hierarchical clustering analysis revealed that the gene expression profiles of the SK-N-AS shYAP#1 and SK-NAS shYAP#2 were more similar than SK-N-AS CV cells (Fig. 5A). While the direction of gene expression (increased or decreased) in the two YAP-knockdown models was not identical, this was likely due to the higher degree of YAP genetic inhibition in shYAP#1 compared with shYAP#2 (Fig. 5B). We, therefore, focused on the shYAP#1-knockdown model and compared it with SK-N-AS CV RNA-seq results to prioritize our evaluation of genes affected by changes in YAP expression, and then confirmed those genes were expressed similarly in SK-N-AS shYAP#2. As expected, YAP was one of the downregulated genes in the Hippo signaling pathway analysis, confirming effective knockdown in this model (Fig. 5C). Hippo pathway genes that are YAP transcriptional targets, such as SMAD1, and the stemness gene, PPP2R2B, were downregulated with YAP knockdown, again supporting inhibition of YAP transcription (Fig. 5C). We evaluated for changes in apoptosis genes and confirmed that BCL2 antiapoptotic members were not altered upon YAP knockdown and that proapoptotic BIM (BCL2L11) expression increased, as was seen at the protein level (Fig. 5D; Supplementary Fig. S6A).
Given that tumor environmental factors, such as hypoxia and nutrient deprivation, can contribute to solid tumor therapy resistance in situ, and that increased BIM in shYAP neuroblastoma cells failed to enhance in vitro apoptosis from chemotherapy, we honed in on other YAP-regulated genes that mediate apoptosis in response to TME stress. Notably, HRK, a BH3-only prodeath protein that activates mitochondrial apoptosis in response to cytokine deprivation (40, 41), had one of the highest fold increases in expression of all genes altered between the YAP shRNA neuroblastoma models compared with CV (Fig. 5D; Supplementary Tables S3 and S4). We confirmed upregulated HRK in both shYAP#1 and #2 models compared with CV (Fig. 5E). We queried a high-risk neuroblastoma primary tumor gene expression dataset (R2 database: http://r2.amc.nl) and found that patients with high-risk neuroblastoma with decreased tumor expression of HRK had significantly worse overall survival, supporting a potential tumor suppressive role for HRK in neuroblastoma. In that same dataset, high YAP expression portended a worse prognosis, supporting the reciprocal relationship (Fig. 5F). HRK tumor expression did not correlate with MYCN status, but was noted to have lower expression in higher staged tumors (Supplementary Fig. S7).
YAP suppresses HRK to inhibit apoptotic responses to serum deprivation
To confirm RNA-seq data, we evaluated SK-N-AS and NLF shYAP models for HRK expression and found that HRK gene expression was indeed upregulated when YAP was inhibited genetically. We complemented this analysis by showing HRK gene expression decreases significantly in the IMR5 cell line with YAP overexpression (Fig. 6A). HRK is a BH3-only prodeath protein that is activated by environmental stresses such as cytokine depletion and severe hypoxia in neurons, conditions that are often found in aggressive solid tumors such as neuroblastoma (40, 41). We, therefore, evaluated neuroblastoma cells with and without YAP knockdown for their survival in both hypoxic and normoxic conditions and found no difference in apoptosis (Supplementary Fig. S8). Given the role of metabolic stress in the tumor environment, we evaluated neuroblastoma cells with and without YAP knockdown for their responses to serum starvation. Upon 48 hours of serum starvation, NLF shYAP showed increased apoptosis in low serum (0.1% FBS) conditions compared with YAP-expressing NLF CV cells in low serum conditions (Fig. 6B). Importantly, under serum starvation, HRK gene expression increased significantly in both NLF shYAP and SK-N-AS shYAP cells compared with their control counterparts (Fig. 6C). Furthermore, cytochrome c release and caspase-9 activation were increased in serum-starved shYAP neuroblastoma cells, confirming that apoptosis proceeds through the intrinsic apoptosis pathway, where HRK-mediated apoptosis occurs (Fig. 6D; Supplementary Fig. S9; ref. 42). Most importantly, apoptosis in response to etoposide treatment in vitro was significantly increased in NLF shYAP and SK-N-AS shYAP cells, but not in control cells treated with etoposide under serum-starved conditions (Fig. 6E).
We initially showed that YAP expression increases in relapsed PDXs compared with matched diagnostic tumors and YAP also increases in response to a single cycle of chemotherapy in vivo (Fig. 1A and B). We, therefore, evaluated for HRK expression in these same models. The inverse correlation for YAP and HRK holds true in vivo, as HRK expression was significantly decreased in NBX-4R PDX compared with the NBX-4 PDX (Fig. 6F). Furthermore, following topotecan and cyclophosphamide treatment in vivo, not only did YAP expression increase, but HRK gene expression concurrently decreased in the chemotherapy-treated NBX-4 PDX (Fig. 1B and F). Thus, YAP may be driving the aggressive nature of recurrent neuroblastoma through modulation of in vivo stress conditions, promoting tumor growth and therapy resistance via inhibition of HRK.
Despite the finding of increased YAP transcriptional activity (4), the role of YAP in relapsed neuroblastoma has not been well-characterized. Here, we describe a role for YAP in regulating neuroblastoma tumor growth and therapy response in vivo that may, in part, be due to YAP repression of the proapoptotic protein, HRK. We have confirmed that YAP transcriptional activity increases in relapsed neuroblastoma models, and now show that YAP expression and transcriptional activity immediately increase in response to standard high-risk neuroblastoma chemotherapy in vivo. Given the brief period of drug exposure and the continued growth of tumors throughout chemotherapy or vehicle treatment, our findings suggest that increased YAP expression in the chemotherapy-treated tumors may represent a cell intrinsic response to treatment rather than a therapy-induced cell death and selection of a YAP-expressing drug-resistant clone.
YAP is necessary for organ growth during the embryonic stages of development (43) and is involved in normal neural crest migration and fate (44). YAP also contributes to the mesenchymal state in pediatric solid tumors like rhabdomyosarcoma (39). Our results concur with such findings in neuroblastoma, with both neurosphere studies and RNA-seq showing the transcription of stem-like genes to be dependent on YAP expression in neuroblastoma. Importantly, we have uniquely shown that mesenchymal properties such as colony formation, tumor growth, and stem cell gene expression are negatively affected upon YAP genetic knockdown, supporting a potential tumor-initiating role for YAP in neuroblastoma.
A hallmark of the mesenchymal phenotype in neuroblastoma and other cancers is extreme therapy resistance (29, 45, 46). This may be attributed to YAP's transcriptional regulation of mesenchymal genes that are also known to impart therapy resistance, such as EGFR. For example, YAP transcriptionally upregulates EGFR in esophageal cancer to mediate resistance to chemotherapy (13). Our results also show that EGFR is overexpressed in YAP-expressing neuroblastoma cells and upregulated in PDXs in response to chemotherapy treatment. Yet, EGFR expression does not change in response to YAP knockdown or overexpression, perhaps because EGFR can be influenced by many additional pathways (47). Despite the persistence of EGFR in SK-N-AS shYAP xenografts, the shYAP tumors regress more in response to trametinib treatment compared with control, suggesting that alternative YAP-regulated genes are responsible for in vivo therapy resistance, such as the suppression of HRK. YAP has also been shown to promote therapy resistance through inhibition of mitochondrial apoptosis through upregulation of prosurvival BCL2 family genes (35, 36). In neuroblastoma, however, we show that YAP knockdown does not affect prosurvival BCL2 family gene or protein expression and it does not restore apoptosis in response to multiple cytotoxic agents in vitro. Despite the increased expression of BIM with YAP knockdown, BIM remains sequestered to and inactivated by MCL1, which may explain the lack of in vitro chemosensitization. BIM overexpression has also been a marker of trametinib sensitivity in other cell models (48, 49), yet the lack of in vitro sensitization to trametinib with YAP knockdown again negates BIM as the etiology for in vivo trametinib sensitization by YAP genetic inhibition.
YAP regulates MEK inhibitor resistance in multiple adult cancers (50, 51). Coggins and colleagues recently demonstrated that YAP transcription is activated in response to trametinib therapy in RAS-mutated neuroblastoma cell lines in vitro and YAP reciprocally induces MEK inhibitor resistance through transcriptional activation of MYCN and E2F in an NF1-mutated MYCN-amplified neuroblastoma cell treated with trametinib (52). We extend this to show that in neuroblastoma PDXs without RAS-hyperactivating mutations, YAP expression and transcriptional activity also increase in response to cytotoxic chemotherapy. Coggins and colleagues also show that complete knockout of YAP in NLF sensitized the cells to trametinib in vitro (52). While complete knockdown of YAP in NLF was not achieved by us, we provide data showing that complete knockdown of YAP in NRAS-mutated SK-N-AS and overexpression of YAP in NF1-mutated IMR5 and NGP did not significantly affect cell proliferation, chemotherapy response, or trametinib response in vitro. Importantly, we show enhanced tumor regression to trametinib in MYCN-nonamplified NRAS-mutated SK-N-AS xenografts when YAP was genetically inhibited in vivo. Our results, therefore, both build upon others' findings and add new dimensions by confirming that this combination holds true in vivo and highlighting the therapeutic potential of YAP and MEK coinhibition in all RAS/RAF/MAPK-mutated neuroblastomas, regardless of MYCN status.
The differences in our cell culture and xenograft responses to YAP knockdown lead us to conclude that YAP's influence on cytotoxic and targeted therapy resistance in vivo may be due to its effect on tumor environmental factors that notably contribute to solid tumor therapy resistance in situ (38). Apoptosis pathway analyses showed that gene expression of HRK significantly increased upon YAP knockdown. Rather than being activated in response to chemotherapy or radiation, HRK instead is activated and promotes apoptosis in response to environmental stresses such as cytokine withdrawal, nutrient deprivation, and hypoxia, tumor properties that have all been shown to promote therapy resistance in vivo (38, 40, 41). Therefore, we focused on HRK and its potential effects on neuroblastoma therapy response. Under serum deprivation conditions, we demonstrate that HRK expression increases, mitochondrial apoptosis ensues, and cells are more sensitive to chemotherapy in vitro, but only in neuroblastoma cells with YAP knocked down. These environmental stresses may explain the more profound differences in tumor growth and xenograft response to therapy seen in vivo, but not in vitro (in normal media conditions) when YAP is genetically inhibited. Importantly, the reciprocal relationship of HRK inhibition in the setting of YAP expression was maintained in vivo. When YAP expression was increased, HRK was downregulated in the relapsed compared with diagnostic PDX paired tumors. HRK also decreases in response to chemotherapy treatment of a chemotherapy-naïve PDX when YAP increases.
While HRK has not been extensively investigated in neuroblastoma or other cancers, primary tumor gene expression data confirm a significantly negative impact on high-risk neuroblastoma patient survival when HRK is low, legitimizing HRK as a potential tumor suppressor in neuroblastoma. HRK is inactivated in other cancers via epigenetic silencing through promoter methylation (53, 54). Preliminary methylation analysis of the HRK promoter region following PCR amplification reveals that this region is not methylated at baseline in YAP-expressing neuroblastoma cells (Supplementary Fig. S10). We have also interrogated TF binding site databases (JASPAR, TFBIND, and ALGGEN), which do not show putative TEAD binding sites (CATTCCA/T-3′) on the HRK promoter. YAP and TEAD1–4 family members have been described to bind to other transcriptional cofactors (such as MEF2, AP-1, and SRF) that can bind to the promoter and enhancer regions of target genes and recruit YAP/TEAD to regulate these genes (55). While TEAD might not bind to the HRK promoter directly, there may be a potential for other transcriptional cofactors to directly interact with HRK and recruit YAP/TEAD to suppress HRK transcription (55, 56). Therefore, investigations are ongoing to deduce the direct versus indirect regulation of HRK by YAP/TEAD and other potential coregulators.
Overall, these data demonstrate a novel role for YAP in suppressing HRK to restrict stress-induced apoptosis in vitro and inhibit therapy response and promote tumor growth in vivo. This strongly supports YAP as a logical therapeutic target to combine with MEK inhibitors or chemotherapy, and HRK as a novel tumor suppressor worthy of further study in neuroblastoma and perhaps other aggressive solid malignancies.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
J. Shim: Conceptualization, resources, data curation, software, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing-original draft, project administration, writing-review and editing. J.Y. Lee: Data curation, formal analysis, methodology. H.C. Jonus: Data curation, formal analysis, methodology. A. Arnold: Data curation. R.W. Schnepp: Conceptualization, writing-review and editing. K.M. Janssen: Data curation. V. Maximov: Data curation. K.C. Goldsmith: Conceptualization, resources, supervision, funding acquisition, validation, visualization, methodology, project administration, writing-review and editing.
This work was supported by CURE Childhood Cancer Foundation and Hyundai Hope on Wheels (to K.C. Goldsmith) and Hyundai Hope on Wheels and Emory Pediatrics Fellow Research Fund (to J. Shim). We are grateful for the thoughtful advice and reviews on our work by Drs. Lawrence Boise and Anna Kenney.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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The following discussion is part of an occasional series, "Ask the Automation Pros," authored by Greg McMillan, industry consultant, author of numerous process control books, and 2010 ISA Life Achievement Award recipient. Program administrators will collect submitted questions and solicits responses from automation professionals. Past Q&A videos are available on the ISA YouTube channel. View the playlist here. You can read all posts from this series here.
Looking for additional career guidance, or to offer support to those new to automation? Sign up for the ISA Mentor Program.
Erik Cornelsen's Question
In a continuous process, the operators have to manually adjust the actuators of an extrusion machine to get the product within spec. However, the width and thickness gauges are located hundreds of meters further down the line, due to process related reasons, and cannot be moved. The dead time for the width and thickness control is between four and eight minutes, depending on the line speed.
What would be a good control strategy to fully automate this dead time dominant process?
I saw in the literature some mentions to model predictive controllers (MPC) in dead time dominant applications; however, I did not find an MPC function block integrated into modern, state-of-the-art programmable logic controllers (PLCs). In this case, what would be the implementation steps of a MPC into a modern PLC (not a distributed control system (DCS))? In addition, how can we empirically get the required MPC parameters (e.g., manipulated variables, control variables, disturbance variables, prediction time, mathematical nodel definition, etc.) that are used by the algorithm?
Russ Rhinehart's Answer
Many of our simpler control algorithms (for instance the Smith predictor and internal model control (IMC)) were designed for processes with delays. But implementation for even these simple controllers requires an array to store the variables from now until the end of the delay. This might not be possible in your PLC. Hopefully, however, your PLC has delay, lead, lag, and proportional integral derivative (PID) functions that could be structured as either the Smith predictor or IMC. But if it is possible, since your delay is variable and depending on line speed, the delay in the controller needs to be scaled with speed. The Smith predictor and IMC are relatively simple solutions, but require a good estimate of the total loop delay including measurements.
Another solution would be to implement human logic as the controller. Unfortunately, the classic name for this is "fuzzy logic," which sounds like faulty thinking, but it is just a way to automate human intuitive action. Humans are effective in complex tasks such as driving a car and catching a ball. If continual operator adjustment is an effective way to control the process, then implementing that logic will be both relatively simple and very effective.
In batch processes, the control action to regulate the end-of-batch results cannot be implemented until after the end of the batch. The control action cannot affect what was made, but can affect the next batch. Extreme dead time processes are very similar, even if the process is continuous. You might investigate batch-to-batch recipe control methods.
If there are uncontrolled disturbances that continually affect the delayed results, then you might consider using algorithmic statistical process control (automated SPC) to temper either the measurement or the controller output. Do not make changes until the evidence gives 95% confidence that a change is warranted. See Rhinehart, R. R. "A Statistically Based Filter", ISA Transactions, Vol. 41, No. 2, April 2002, pp 167-175, or Muthiah, N., and R. R. Rhinehart, "Evaluation of a Statistically-Based Controller Override on a Pilot-Scale Flow Loop", ISA Transactions, Vol. 49, No. 2, pp 154-166, 2010.
Greg McMillan's Answer
There are many misconceptions about the use of controllers for dead time dominant loops. Here are some prominent ones.
- Both dead time compensating PID controllers (e.g., Smith predictor) and model predictive control (MPC) for dead time dominant loops are more traumatically affected by an overestimate rather than underestimate of the dead time. A model dead time just 20% greater the actual dead time can cause rapid oscillations. An underestimate of the model dead time just results in a more sluggish response. This is the opposite situation for a conventional PID controller where underestimates and overestimates of dead time used for tuning cause an oscillatory and sluggish response, respectively. Tests discussed in the upcoming June Control Talk column show that for dead time compensation of lag dominant loops, a model dead time much larger than the actual dead time gives better control.
- The potential improvement in performance by dead time compensating PID is much greater for lag dominant loops. In fact, both Greg Shinskey and I have confirmed that the benefit of dead time compensation diminishes becoming negligible in nearly pure dead time loops.
The better dead time compensating controller (PIDθ ) found by me and Shinskey is not the Smith predictor but a PID with a dead time block simply inserted in the external-reset feedback because it eliminates the need and consequential error of setting the open loop gain and time constant and to create a separate operator interface to restore the controlled variable as the process variable and its setpoint. The PIDθ requires more aggressive tuning especially in terms of a smaller reset time approaching nearly zero for a model dead time exactly equal to the total loop dead time. The dead time block should be updated particularly for changes in production rate. You can insert a small filter on the PID output to smooth out the high frequency oscillations for a model dead time slightly larger than actual. However, you need a PID standard form with the positive feedback implementation of integral action creating external-reset feedback, which is only available by a few suppliers.
- Dead time dominance is not the reason to go MPC instead of PID even though this statement is pervasive in the literature. The dead time compensating PID can do a better job than MPC for unmeasured process input (load) disturbances. The use of feedforward and ratio control can also do a better job than disturbance variables in an MPC unless the dynamics are complex (e.g., inverse response) or there are interactions and constraints.
One supplier offers a small standalone DCS that has as its default a PID Standard Form with positive feedback implementation of integral action. You simply need to turn on the dynamic reset limit option to get external-reset feedback. The DCS also includes a small MPC eliminating the need for external software and interface. A Simulate-Pro package allows you to try out and develop the PID and its tuning for your application on your laptop.
It has been shown in tests that a PID with structure options offers better performance than fuzzy logic control (FLC) when properly tuned for load response with setpoint weights for best setpoint response. Several of the mentors have implemented FLC decades ago when it was all the rage but concluded a PID controller could have done the job and would have been more understandable and maintainable. If the measurement response is not reproducible and a dynamic model for tuning a PID not possible, which apparently occurs in pulp and paper industry, FLC may be the solution.
The simplest and most reliable and maintainable solution in my book is the use of a dead time compensating PID controller with feedforward, ratio, and override control or an MPC with disturbance and constraint variables for more complex applications. For sheet line cross sectional thickness and optical clarity, a series of MPC was found to perform better than a series of Smith predictors from extensive simulation study and pilot plant sheet line tests done decades ago.
If the dead time dominance was due to an analyzer cycle time, an enhanced PID per Annex E of ISA-TR5.9 could be used without the need to model the dead time or even update tuning settings if the time between updates increases. The PID gain can approach the inverse of the open loop gain and the reset time approach twice the process dead time (excluding the larger dead time from the analyzer cycle). This enhanced PID is also useful for slow wireless updates on fast loops.
Not commonly discussed is the possible use of first principle and data driven model to provide an inferential measurement of the controlled variable for the PID or MPC without the dead time. A combination of the models is increasingly being considered. The estimated dead time is inserted into the estimator whose output is compared to the measurement of the actual controlled variable and a fraction of the error (e.g., 0.4) is used to provide a bias correction of the estimator output and the PID or MPC inferential measurement.
Mark Darby's Answer
I concur with the responses above about use of PID on dead time dominant processes, and the need to get a good dead time estimate if dead time compensation is used. If it varies, it is important to update the dead time as a function of process operation (such as throughput or hold-up) to ensure good performance.
Some DCS vendors do provide MPC capability in the DCS that could be an option, particularly for a multivariable problem with constraints. Note, though, a DCS–based MPC will normally have less features and capabilities compared to MPCs that run in a separate, networked computer, so a DCS-based MPC would need to be evaluated. If you did not have a multivariable problem or constraints, I would recommend a PID-based scheme.
As Greg points out, a PID can usually out-perform MPC for load disturbances due to its (typically) faster execution and due to the simple bias model update that can be slow to adapt to unmeasured load disturbances. In practice, MPC applications often incorporate well-functioning PID regulatory controllers. So, such a cascade approach may also be useful for the dead time dominant process. If dead time is modeled in the MPC and the dead time changes, the MPC controller would need to be able need support an online update of the dead time and be able to execute sufficiently faster than the dead time.
A final comment on the traditional MPC model updating scheme: there are ways to improve the disturbance rejection capability through a different MPC updating scheme that can attribute the model error to an unmeasured disturbance that is assumed to enter at the process input. With such a model, the MPC prediction will continue to evolve in the future, causing the MPC to take more aggressive, anticipatory action. Not all MPC controllers support this capability natively, but there may be custom ways to implement this. This is a good topic to take up with MPC suppliers.


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Scottish actor Ewan McGregor, who got his big break in Danny Boyle's 'Trainspotting,' has been a fixture in popular culture for the better part of the previous three decades. His career trajectory after that point was nothing short of amazing. McGregor's parents strongly supported his early acting ambitions, which is how he got his start in the business so young.
Ewan's first acting role was in the 1993 series "Lipstick on your collar," and he began studying drama at London's Guildhall School of Music and Drama in 1988. It wasn't until Danny Boyle cast him in the starring part of 'Trainspotting' two years later that he really broke out.
McGregor's big break in Hollywood came when George Lucas cast him as a youthful Obi-Wan Kenobi in the Star Wars prequel trilogy.
From then on, he collaborates with numerous A-list directors like Baz Luhrmann, Tim Burton, Guillermo del Toro, Ron Howard, Ridley Scott, Michael Bay, Roman Polanski, and Steven Soderbergh. He's been very close to Disney in recent years and collaborated with them on several projects. But he famously came back to the series as the main character, Obi-Wan Kenobi.
Ewan Mcgregor Net Worth
Scottish actor Ewan McGregor is worth an estimated $25 million. Ewan McGregor's most recognizable part to date is perhaps that of Obi-Wan Kenobi in the Star Wars prequels. Also well-known are his performances in such films as "Moulin Rouge!" and "Trainspotting."
In addition to the Golden Globe, Ewan has received numerous other honors throughout his career. He was ranked as the UK's fourth most important cultural figure in 2004. McGregor is as well-known for his work in the theater, in addition to film and television.
Ewan McGregor's $1.9 Million LA Home Split
Ewan McGregor and his ex-wife Eve Mavrakis spent over $1.9 million on a home in Los Angeles in 2002. After relocating their family to the Brentwood house from the 1930s, everything altered in 2018 when Mcgregor and Mavrakis divorced. McGregor initially filed for divorce from Mavrakis, and under California law, the couple's assets are to be divided evenly.
It was stated in 2019 that since Mavrakis had primary custody of the children, she would likely receive the entire family home. The parent who has primary physical custody of the children in a California divorce procedure will typically receive the marital home. Ewan was granted joint custody despite his request. This verdict was predicted to cost McGregor $4 million.
During this time, Ewan McGregor and his new girlfriend, Mary Elizabeth Winstead, were out looking at houses. McGregor's separation from his wife in 2017 followed the publication of incriminating photos of him with Winstead and the revelation of their secret romance. The pair reportedly bought a new house in Topanga Canyon in 2019.
Ewan and Eve's Divorce Settlement: Eve Gets $6.6 Million Mansion, $500K Cash, and More
Ewan and Eve, who had been married for 22 years, finally called it quits in August of 2020. Eve received a $6.6 million mansion, her jewelry, $500,000 in cash, and five vehicles as part of the deal. In addition, she will receive a share of Ewan's future movie revenues from films in which he appeared during their marriage.
That number includes several major motion pictures, including three "Star Wars" films. Ewan's monthly obligations amount to $51,000 because of the $15,000 in child support and the $36,000 in spousal support. That works out to roughly $1.2 million in pre-tax income per year for Ewan, if he wants to cover child support obligations. Ewan gets to retain thirty cars for himself!
If you wanted to check out more celebrities' wealth and real estate details then you can join us on our Twitter account.
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Pressure continues to grow for the phasing out of fossil fuels ahead of the COP28 UN Climate Summit being staged at the end of November.
According to a report in the Financial Times, France, Spain, Ireland and Kenya have joined together as part of a group of 15 nations, known as the High Ambition Coalition – part of "a global accord to dump oil and gas."
Following pre-Summit preliminary talks in Abu Dhabi, a statement signed by 15 ministers, including from poorer nations like Ethiopia, Vanuatu and Samoa, declared: "Fossil fuels are at the root of this crisis. We must work together to develop a comprehensive global clean energy access approach, to accelerate the transition away from fossil fuels."
Pushing for a tripling of renewable energy capacity by 2030, the Coalition said that the production and use of fossil fuels needed to be wound down. That meant an "urgent phase-out of coal-fired power generation."
And they warned that the scaling up of carbon capture and storage technology – advocated by industries dependent upon fossil fuels – should not be used as a climate action delaying tactic.
The UN scientific body, the Intergovernmental Panel on Climate Change, has said that carbon capture and storage techniques only have a minor role to play in removing carbon from the atmosphere – despite major fossil fuel producers wanting it to be given far greater priority in global climate change policies.
According to the Coalition, these technologies should not be relied upon to cut emissions in "hard to abate" sectors, such as steel and cement, or applied in power generation.
"They should not be used to delay climate action in sectors such as electricity generation where feasible, effective and cost-efficient mitigation alternatives are available, particularly in this critical decade when emissions need to be reduced urgently and dramatically," the Coalition said.
Their views are at odds with those of Sultan al-Jaber, President-designate of COP28 and the head of the UAE's state-owned oil company, who has repeatedly called for emissions to be reduced, rather than any phasing out of production.
Nevertheless, he has also recognised that fossil fuels needed to be wound down by an as-yet undefined mid-century deadline.
The FT also reported that the role of emissions-capturing technologies had resulted "in fraught recent discussions among EU bloc countries, as Brussels sought to agree its negotiating position ahead of COP28."
Meanwhile, Danish global climate policy minister, Dan Jørgensen, who will be leading discussions on the so-called global stocktake for COP28, said that discussions about an unabated end to fossil fuels would be at the centre of negotiations at the forthcoming Summit.
Expressing a degree of optimism about what could be achieved at COP28, he said: "We are definitely not where we need to be on any of the issues yet. There's a lot of work still to be done, but I do honestly feel that there is some momentum that this COP will be an important COP, where important decisions will be made and that it needs to be very ambitious."
Future of fossil fuels leaves nations at odds ahead of UN climate summit (ft.com)


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Commercial Real Estate Made Easy
Investing in commercial real estate may seem intimidating, but with the proper guidance and knowledge, it can be a lucrative and satisfying venture.
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When it comes to purchasing or leasing a commercial property, several crucial factors must be taken into account. One of the most critical is location, as it can have a significant impact on the success of your business. Other factors to keep in mind include the size and layout of the property, its condition, and any zoning or regulatory restrictions that may apply.
Selling a commercial property can also be a complex process, but with the right approach, it can be extremely profitable. By collaborating with a skilled real estate agent with expertise in commercial transactions, you can navigate the process and ensure that you obtain the best possible price for your property. Overall, commercial real estate can be an excellent opportunity for investment, provided that you're willing to put in the effort and time to do it correctly. With the right guidance and support, it can be a straightforward and gratifying process that can result in long-term financial success.
Greg Palmquist recently SOLD a large medical facility located at 5050 Powderhouse Road in Cheyenne with a list price of $11.1 million to another Wyoming investor! Congratulations to the sellers and new owners. It's a win-win when we are able to meet the needs of investors wanting to keep their investments close to home. Greg recently collaborated on this blog titled: Why Cheyenne's Commercial Real Estate is a smart investment for your business.
Watch the video below to learn more about commerical real estate investing. RE/MAX Capitol Properties, agent, Brenton Leavitt, defined commercial real estate (CRE) for us; this refers to any property used for business purposes. There are five main sectors: industrial, office, retail, multifamily, and special purpose. There are many other property types, such as self-storage, medical, elder care, land or hotel. The supply and demand, yield, and overall profitability of each sector vary greatly. Currently, industrial is the top-performing CRE asset class, while retail space is the lowest performing sector.
Brenton suggests investing in commercial real estate, where you can diversify your portfolio and potentially earn passive income through rent. Plus, commercial real estate can provide a hedge against inflation and a potential tax advantage. For more information about Cheyenne's commercial real estate market call us at 307-635-0303 or stop by our office at 4000 Central Ave, Suite #1, in Cheyenne, Wyoming. Be sure to subscribe to our YouTube channel for more tips on real estate and learning about our community.


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