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I have very exciting news about the current “Wonderland” exposition at the Assumburg castle park in Heemskerk.
Since the end of April, six of my works from the “Florals” series can be seen in the castle garden. From September, 1, there is a great addition.
My photo of water lilies from Claude Monet’s garden in Giverny now decorates the fountain at the center of the park.
It is new to me to exhibit an artwork at a large format like that: 5 meters long! I say, big is beautiful!
During the first months of the exhibition, you could see a wonderful image by Desiree Dolron at the fountain. Unfortunately, severe storms this summer had damaged it.
It turned out a different printing technique was better for such windy conditions. It did not work optimally for that image, so it was decided to use another image for the last 2 months of the exposition.
I was honored and excited to hear the curators wanted to use my photos of Giverny for the new central piece!
We have selected a photograph of water lilies in Monet’s garden. Large vague purple spots add an “impressionistic” touch to the image.
I must say I am very pleased with how this photo looks in the middle of the pond. An image of water when presented directly against the water creates an interesting effect.
The material works well, it lets the wind through so that the canvas does not flap much (I was there on a windy day to see this). A little movement in the water at the lower edge, works beautifully for this image. The canvas lets the light pass through and that creates a glowing effect, a little like an LCD screen. That play of light makes the image even more vibrant. Sometimes, the reflection in the water seems to extend and double the image in a seamless flowing way. As the material is semi-transparent, the photo looks as good from its back side as from the front one.
I am happy to see even more of my work at this wonderful exposition, were art and nature are involved in a dialogue and co-creation.
The exposition will run till the end of October. You are most welcome to visit it, and the entrance to the park is free during the opening hours.


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Posted July 11, 2018 02:06:25 A young man has been celebrated after he received a “welcome” from a street photographer.
The man, who has not been named, was walking with a friend near the corner of the busy Kingsbridge intersection when he was attacked by a group of men in their early 20s.
“I saw him with his face covered, his clothes were down, he was just in shock, I said ‘what the f*** is going on’,” Mr Williams told the ABC.
“He just stood there and said ‘I don’t know who you are, I don’t like what you’re doing’.” The photographer told the man to run and Mr Williams helped him to safety.
“The police came and told us that they found him in the middle of the road with a knife and that’s when we went to get him and got him to the hospital.”
“I don.t know who that was.
I’ve never seen anyone who was dressed like that before,” Mr Williams said.
“They had a knife in his hand.
That’s a little bit upsetting.” “
A man has got his face on the ground.
That’s a little bit upsetting.”
Mr Williams is hopeful the incident will encourage people to be “cautious of these sorts of things”.
“I just hope it’s a wake up call to people who are walking down the street and are thinking ‘this is wrong’,” he said.
Mr Williams says he has been “caught off guard” by the incident.
“It’s not something that you would expect,” he said, adding that he is “surprised” that no one has been charged with a crime.
Mr Gildersleeve said he was shocked to hear of the attack. “
When you see a person that’s in a vulnerable state, they shouldn’t be doing this.”
Mr Gildersleeve said he was shocked to hear of the attack.
“We are talking about a young boy.
We are talking to a little boy who just lost his life and it is so sad and upsetting,” he told the station.
“If they feel unsafe, they need to go out and see somebody and get some help.””
If they feel unsafe, they need to go out and see somebody and get some help.”

domain: news


text:
We created this game modeled on space shooters running on old consoles from the 90'
Please test the the game, not completed.
Make sure that is working properly on your phone or tablet.
If you like games like: Tyrian, 1942, 1943, Flying Shark, iFighter, R-Type, Gradius,
Espgaluda, SWIV, Sky Force, Asteroids, Shogun, Warblade, Siberian Strike,
Air Attack, Radiant, DoDonPachi then this game is for you.
The purpose of the game is to destroy as much enemies.
The game is indie game .
This game is : action , twitch , hardest , minimal , reflex , speed , hard ,indie , survive , hd , graphics , stunning, survival , difficult , high speed , fast .

domain: game


DOVETAIL TO WEAVER / PICATINNY RAIL COMPRESSION ADAPTERS
Precision machined high quality aluminium alloy construction.
Less universal fitting than our other adapters.
LOW-PROFILE COMPRESSION TYPE ADAPTER RAILS
One piece low profile design.
131mm long overall.
15mm high overall.
Weighs approx 55 grams.
£3.50p per pair
SPECIAL OFFER PRICE
Two piece ultra-low profile design.
Each rail is 38mm long.
The pair weighs 24 grams.
Compression type fitting.
Dovetail to weaver / picatinny adapter.
Aluminium with a matte black anodised finish.
Converts 3/8" dovetail rail bases to standard Weaver bases, but will not fit all dovetail railed guns.
The one piece option;
Wont fit many standard dovetail rails as the clamping bolt shafts need slots to fit into which standard dovetail rails don't have.
The two piece compression pair option should fit;
Fits some airguns and .22 rifles such as Air Arms S410 TDR, Benjamin Marauder, Mauser 201, Marlin 795/60, Kimber 82G, Winchester 190, Beeman / Webley, Anschutz Pistol, CZ 452, Winchester Wildcat-22 and more.
Wont fit Remington 580 and others.
Reported as not fitting Weihrauch HW100.
Will accept a Nightforce ADI & 1913 MIL Spec Mount.
CZ models have either 11mm or 12.7mm wide rails at the top point, depending on the model and/ or calibre.
All CZ 452 'American' models 22lr/22wmr/17hmr are 12.7mm, so these adapters will not fit.
There is a similar adapter by Hawke, that is more adjustable and should fit.
These adapters are far less universal fitting than the other adapters.
Due to the compression type fitting rather than a moving clamp type.
There is very little adjustment so avoid trying to over tightening them.


"The OPCW Fact-Finding Mission (FFM) deployed in early January to further establish facts regarding the allegation. The FFM continues to independently collect and analyse information and will report its findings to States Parties to the Chemical Weapons Convention. All measures continue to be taken to ensure the safety and security of OPCW experts and personnel involved", the OPCW spokesperson said in a statement.
On 24 November, militants reportedly fired chlorine-filled shells at two neighbourhoods of Aleppo — al-Khalidiye and Al Zahraa — along with Nile Street. People were hospitalised with asphyxiation and other symptoms typical of poisoning. According to local media reports, 107 civilians were hospitalised.


text:
Though their bromance has come a long way since "Good Will Hunting," Matt Damon
appreciates how his friendship with Ben Affleck
has matured over the years.
Recently, the "Bourne Identity" star bought a house next-door to his friend of over 25 years. In an interview with E! News, the 41-year-old father of four explained, "Yeah, it's definitely not the life we had planned for ourselves in that way. It's great in a whole different way, like ...

domain: entertainment


He was detected speeding on the M7.
Laois gardaí have arrested a driver caught travelling at 221kmph.
The Road Policing Unit detected the driver during a speed check on the M7.
The vehicle was followed and stopped in Kildare where the motorist was arrested for dangerous driving.
He was taken to Portlaoise Garda Station and has been charged to appear before Portlaoise District Court.


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The book recounts the amazing tale of how the adored character Mario led to the rise of the quintessentially Japanese Nintendo in the American market.
Lawsuits, Hollywood, die-hard fans and face-offs with Sony and Microsoft… there’s a lot of drama!
Listen to the audio below for the audiobook snippet.
Recommendedby NEWSROOM AI
Venezuela – once as rich as Norway – is suffering an apocalyptic collapse without precedent.
The Money Show’s Bruce Whitfield interviews Fairtree Capital Portfolio Manager Jean-Pierre Verster.
The Money Show’s Bruce Whitfield interviews Truworths CEO, Michael Mark.
The Money Show’s Bruce Whitfield interviews Lonmin CEO Ben Magara.
Bruce Whitfield asks Personal Financial Advisor Warren Ingram for advice on how investors can safely navigate this hectic time.
The Money Show’s Bruce Whitfield interviews Pieter du Toit, the assistant editor at News24.
Swartz says he would have done anything to get the R1 million prize money.
Former police minister Adriaan Vlok has rejected accusations that former minister Malan was at the centre of a paedophile ring.
Entrepreneur Max Lichaba built his own empire and is the CEO of Lichaba Creations and several other businesses. This is his story.
The Clothing Bank gives women access to discounted merchandise and 500 hours of training in money management and business skills.

domain: economics


text:
Art and business: bridging the gulf
The four faces behind Draw Inc are attempting something tricky - trying to make a profitable business out of art.
And it just might work.
Deborah Duffield, Anne Challinor, Craig McClure and Lisa Benson make up Draw Inc, a collective of Hamilton artists who have established an art gallery and a name for themselves making art accessible for Hamiltonians.
All practising artists, they operate a philanthropic non-commercial business, exhibiting the work of emerging and established, local and international, artists and their exhibition openings are drawing crowds of 250 to 300 people.
The collective subsidises artist studio spaces and takes only a small commission on pieces sold through the gallery.
"But we're really aware that we actually need to be a sustainable business. We all have art practices and this takes a lot of time," Benson says.
"So we need to figure out how to get more value from the energy that we're putting in and still have time to be practising as well."
Benson taught the others while they were studying for arts degrees at Wintec. At graduation, Duffield came up with idea of setting up the collective and establishing a place that could serve as a gallery and studio space for arts graduates who she believes lack support after they finish studying.
Following the artist tradition they call "gentrifying abandoned buildings", the group took advantage of the impact of the economic downturn on the CBD.
They found an empty space in the former site of Pollock and Milne in Ward St and, with the support of Centre Place Shopping Centre, renovated to make it work as studio and gallery space.
The collective's name reflects its beginnings: from Ward to Draw.
Draw Inc used the spot for 10 months before moving to more empty commercial spaces in Alexandra St. They have set up permanent headquarters there, this time with the support of DV Bryant Trust, and are also utilising two other empty properties further up the street as temporary gallery spaces.
The collective won the Innes 48 Hours Startup challenge in April, a competition to help Waikato entrepreneurs develop innovative business ideas and, as part of the prize pack, received a mentorship opportunity with business growth centre Soda Inc.
The four have just finished the six-week long pre-incubation with Soda where they scrutinised their business models, value proposition and the underpinning values of the collective.
Draw has received funding and support from Wintec, Wintec Alumni and Creative Hamilton during the past 18 months. It has just started paying rent on its main property. The artists volunteer their time and no profit has been generated yet.
But with the conclusion of the mentorship with Soda, the four feel they are embarking on a new phase. They say they have undergone a shift in thinking and now see how artists can converse with the business world.
"It took us from a bunch of artists trying to make work and support other artists to actually going ‘this has got legs'. This is a valuable proposition for businesses, the city and for artists," Benson says.
There are other artist-run collectives in Hamilton but the point of difference for Draw Inc is its diversion from the traditional gallery business model and the way art is exposed in the community.
"We're trying to create a business model that's viable," Duffield says, "but whereas in your conventional galleries you are relying on commissions from sales to cover overheads, we're trying to pull on different revenue streams."
The group plans to diversify, supplementing the curation of exhibitions with corporate leases for artworks, workshops, scholarships and hosting artist talks.
They aim to form a charitable trust so that all earnings from Draw Inc will facilitate their goal of making art accessible to all people in the community.
- © Fairfax NZ News

domain: artistic


Ten years since the tragic helicopter crash in 2008, in which 11 ARM servicemen lost their lives returning from the ALTHEA peacekeeping mission in Bosnia and Herzegovina, were marked by laying fresh flowers on their Memorial.
On the occasion of marking the helicopter crash fresh flowers were laid on the Memorial in Blace-Katlanovo by delegations from: the Ministry of Defense, led by the Minister of Defense Radmila Shekerinska, the ARM General Staff, led by the Deputy Chief of General Staff, Major General Muhamed Racaj, the Cabinet of the President of the Republic of Macedonia, the Joint Operations Command (JOC), the Air Force Brigade (AFBde), the 1st Mechanized Infantry Brigade (1st MIB) and the family members of the dead soldiers.
On the memorial of the fallen ARM soldiers at the Butmir base in Bosnia and Herzegovina, delegations of MOD and ARM, members of the Macedonian contingent and the command of the ALTHEA mission laid fresh flowers, too.
Delegations from the MOD, GS of the ARM, JOC, and the 1st MIB also laid fresh flowers in front of the command of Air Force Brigade in Straso Pindzur barracks, in Petrovec.
In the helicopter crash that happened ten years ago in Blace-Katlanovo, Lieutenant Colonel Toni Spasovski, Major Goran Ristovski, Captains Brane Spasovski and Igor Gjoreski, Master Sergeants Slavcho Vasilev and Toni Davitkovski, Staff Sergeants Janko Shirikj, Miki Ivanovski, Aleksandar Taskovski and Aleksandar Vasikj and Corporal Zlatko Veljanovski lost their lives.


text:
|Product name||IdoBio Polydeoxyribonudeotide(PDRN)|
|Appearance||White crystal or powder|
|Packaging||1g/vial; 10/vial 100 g/bottle|
|Heat stability||be stable at 90℃ within 5 minutes|
|Ph||Stable at 5-9|
|Storage condition||2℃-8℃,keep out of the sun|
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What is PDRN?
PDRN is found in the human placenta and is one of the complexes in cells that produce DNA.With a 95% similarity to human DNA, there are no active proteins and peptides that trigger an immune response.
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1,Increases elasticity of epidermal and dermal layers,Stable against heat.
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3,Protect cells from UV-induced DNA damage.
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With 0.5% PDRN content, 20 healthy female volunteers (aged 18-36 years) were tested, and the results confirmed that the effect of removing scars and acne marks was obvious after 8 weeks of use.
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With 0.5% PDRN content, 20 healthy female volunteers (aged 18-36 years) were tested, and the results confirmed that after 4 weeks of use, the skin pore contractile was obvious and the anti-aging effect was obvious.
1, Regenerative anti-wrinkle:lotion, cream, essence, mask, etc
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3, Repair& anti-inflammatory: repair lotion, essence, anti - bacterial cream, mask, etc
4, Anti-alopecia: shampoo, lotion, essence,etc

domain: chemistry


This week's premiere broadcast of Mountain Stage was recorded on the campus of West Virginia University at the Canady Creative Arts Center. On this episode, we hear live performances from Duke Robillard Band, Cedric Burnside, Sam Weber, Las Cafeteras, and The Black Feathers.Continue Reading Take Me to More News
This is a developing story and may be updated.
Updated on Tuesday, April 2, 2024 at 3:20 p.m.
Gov. Justice has declared a State of Emergency for Fayette, Kanawha, Lincoln and Nicholas counties Tuesday due to severe thunderstorms with high winds, which caused flooding, downed trees, power outages, and road blockages.
The declaration is a proactive measure ensuring the allocation of necessary resources and expedition of emergency response efforts. The State of Emergency allows State and Local agencies to take swift and decisive actions to mitigate the impact of the storms and protect the lives and property of West Virginians.
Original Story: Sweeping Storms Spur Tornado Watch, Power Outages
A powerful line of storms rolled across West Virginia on Tuesday, knocking down trees and powerlines.
According to the National Weather Service, several rounds of thunderstorms are expected to continue into the night.
The storms could also bring “damaging winds, large hail, flash flooding and tornadoes,” the National Weather Service reported.
Storms will hit central West Virginia between 4 and 9 p.m. and move east.
In response to the storms, Marshall University closed its campuses for the remainder of the day Tuesday afternoon.
Emergency alerts and sirens sounded across Kanawha County Tuesday morning, as thirteen counties in southwestern West Virginia were placed under a tornado watch.
The Kanawha County Commission issued a state of emergency over the storms Tuesday afternoon. In a press release, the commission said 911 dispatchers had received more than 500 calls regarding the tornado warning that morning.
“Metro received calls regarding structure collapses, entrapments, vehicle wrecks, fires, fallen trees, and many medical calls,” the press release read. “There [have] been reported touch downs of tornadoes in the western portion of the county, but no confirmation at this time.”
As of 12:30 p.m., Appalachian Power reported 118,000 power outages, 53,000 of them in Kanawha County alone.
The commission urged Gov. Jim Justice to issue Kanawha County a state of emergency over the weather.
For additional updates on the storms, visit the National Weather Service website.


text:
Full Line Of Succession To The British Throne
Succession to the British throne “is regulated not only through descent but also by Parliamentary statute,” according to the Royal Family’s website.
And Prince William and his wife, the Duchess of Cambridge, having their third child, the line of succession has changed.
Here’s a look at who currently is in line for the throne after Queen Elizabeth II, who is 92.
1. The Prince of Wales
Charles, the Prince of Wales, is the oldest child of Queen Elizabeth II. He is 69 years old and the heir apparent to the throne.
2. The Duke of Cambridge
3. Prince George of Cambridge

domain: celebrity


text:
- Open Access
Racial differences in RAD51 expression are regulated by miRNA-214-5P and its inhibition synergizes with olaparib in triple-negative breast cancer
Breast Cancer Research volume 25, Article number: 44 (2023)
Triple-negative breast cancer (TNBC) affects young women and is the most aggressive subtype of breast cancer (BC). TNBCs disproportionally affect women of African-American (AA) descent compared to other ethnicities. We have identified DNA repair gene RAD51 as a poor prognosis marker in TNBC and its posttranscriptional regulation through microRNAs (miRNAs). This study aims to delineate the mechanisms leading to RAD51 upregulation and develop novel therapeutic combinations to effectively treat TNBCs and reduce disparity in clinical outcomes.
Analysis of TCGA data for BC cohorts using the UALCAN portal and PrognoScan identified the overexpression of RAD51 in TNBCs. miRNA sequencing identified significant downregulation of RAD51-targeting miRNAs miR-214-5P and miR-142-3P. RT-PCR assays were used to validate the levels of miRNAs and RAD51, and immunohistochemical and immunoblotting techniques were used similarly for RAD51 protein levels in TNBC tissues and cell lines. Luciferase assays were performed under the control of RAD51 3’-UTR to confirm that miR-214-5P regulates RAD51 expression. To examine the effect of miR-214-5P-mediated downregulation of RAD51 on homologous recombination (HR) in TNBC cells, Dr-GFP reporter assays were performed. To assess the levels of olaparib-induced DNA damage responses in miR-214-5P, transfected cells, immunoblots, and immunofluorescence assays were used. Furthermore, COMET assays were used to measure DNA lesions and colony assays were performed to assess the sensitivity of BRCA-proficient TNBC cells to olaparib.
In-silico analysis identified upregulation of RAD51 as a poor prognostic marker in TNBCs. miRNA-seq data showed significant downregulation of miR-214-5P and miR-142-3P in TNBC cell lines derived from AA women compared to Caucasian-American (CA) women. miR-214-5P mimics downregulated RAD51 expression and induces HR deficiency as measured by Dr-GFP assays in these cell lines. Based on these results, we designed a combination treatment of miR-214-5P and olaparib in HR-proficient AA TNBC cell lines using clonogenic survival assays. The combination of miR-214-5P and olaparib showed synergistic lethality compared to individual treatments in these cell lines.
Our studies identified a novel epigenetic regulation of RAD51 in TNBCs by miR-214-5P suggesting a novel combination therapies involving miR-214-5P and olaparib to treat HR-proficient TNBCs and to reduce racial disparity in therapeutic outcomes.
Triple-negative breast cancer (TNBC) constitutes 15–20% of all breast cancers (BC), occurs at a young age (< 40 years), is more aggressive, and contributes to a relatively higher proportion of BC-related deaths in women. TNBCs are negative for both the estrogen receptor (ER) and progesterone receptor (PR) and do not overexpress epidermal growth factor receptor 2 (EGFR-2 or HER-2), which classify and define therapeutic modalities. Consequently, no targeted therapies are currently available for these patients [1,2,3,4]. Thus, most of these patients undergo cytotoxic chemotherapy; however, the responses are short-lived and the majority of patients develop resistance and undergo relapse [5,6,7]. Moreover, TNBC disproportionally affects women of African-American (AA) descent compared to other ethnicities and show the worst clinical outcomes in this population [8, 9]. The health statistics data indicate that the occurrence of TNBCs is over two times higher for AA women compared to women of European or Caucasian ancestry (EA) [10, 11]. Although socioeconomic  and comorbidity factors contribute to differences in clinical outcomes, the higher prevalence of TNBCs in young AA women, combined with higher death rates, suggests different molecular factors influence this disease .
RAD51 is a key downstream factor to BRCA1/2-mediated DNA repair  and facilitates the homology search to repair DNA double-strand breaks (DSBs)  in coordination with ATM/ATR-mediated DNA damage checkpoint responses . Moreover, RAD51 upregulation is evident in many cancers , including BCs , and is implicated in the development of tumor resistance to chemotherapeutics [19, 20]. Our previous studies showed that RAD51 is upregulated in TNBCs, and its expression is significantly higher in AA TNBCs compared to EA TNBC patients and correlates with a poor prognosis relative to EA TNBC patients . However, the mechanisms behind the upregulation of RAD51 in these patients have not been identified, particularly those contributing to the observed racial disparity.
The Food and Drug Administration (FDA) has approved PARP inhibitors (PARPi) olaparib  and talazoparib  for the treatment of BRCA-deficient BC [24, 25]. Although PARP inhibitors have shown promise, their use and effectiveness are limited to only 15–20% of TNBC patients, particularly those with BRCA mutations or homologous recombination (HR) DNA repair deficiency (HRD). In these patients, PARPi effectiveness is attributed to synthetic lethality , a process wherein the simultaneous perturbation of two compensatory genes or pathways causes cell death . Therefore, the majority (~ 80%) of the TNBC patients do not benefit from PARPi-targeted therapies. The increased expression of RAD51 observed in TNBCs, particularly in AA patients, may facilitate increased DNA repair and resistance, leading to therapeutic relapse and poor outcomes [9, 28]. Moreover, currently available data on biological and genetic differences do not explain the high mortality rate for AA women nor decode the genetic and epigenetic mechanisms that cause these discrepancies.
MicroRNAs (miRNAs) are short, non-coding RNAs with a length of 18–23 nucleotides that act as epigenetic regulators of gene expression . About 2000 miRNAs have been discovered in humans, and they regulate one-third of the human genome . miRNAs are associated with numerous human diseases and are being investigated as clinical diagnostics and therapeutic targets, particularly involving cancer progression and their influence on therapeutic responses . These observations prompted us to perform miRNA-seq analysis to find differentially regulated miRNAs in DNA repair-proficient TNBC cell lines representing both AA and EA ethnicities, particularly those targeting the RAD51 gene. Our present study identified the loss of miRNA-214-5P (miR-214-5P) in TNBCs, particularly in AA TNBC cell lines and tumors. Therefore, the goals of our current studies are to delineate miRNA-mediated epigenetic mechanisms leading to RAD51 upregulation and develop novel therapeutic combinations based on these biomarkers to effectively treat BRCA-proficient TNBCs and reduce disparity in outcomes.
In this study, our data show that miR-214-5P regulates RAD51 and suggests that this epigenetic regulation of RAD51 contributes to disparities in TNBC’s therapeutic outcomes. Additionally, our data demonstrate that miR-214-5P-mediated downregulation of RAD51 causes HRD, and synergistic lethality with the PARPi, olaparib, in HR-proficient TNBC cells.
Materials and methods
TNBC patient cohort and sample collection
To identify the RAD51 and miRNAs (miR-214-5P and miR-142-3P) differential expression in TNBCs of AA and EA women, we performed RT-PCR on a TNBC cohort. Formalin-fixed, paraffin-embedded (FFPE) TNBC samples were obtained from the Division of Anatomic Pathology in the University of Alabama at Birmingham (UAB). The UAB Institutional Review Board approved the collection and use of all samples in this study (IRB number: 060911009). Before RNA extraction, pathologists macro-dissected all tumors and corresponding normal regions.
Cell lines, culture method, and reagents
The human TNBC cell lines MDAMB231, MDAMB453, HCC1806, and MDAMB468 were purchased from ATCC, Manassas, VA. These cells were cultured in Dulbecco’s modified Eagle medium (Corning, Manassas, VA) supplemented with 10% fetal bovine serum (Omega Scientific Inc., Tarzana, CA) and 1% penicillin–streptomycin (50 U/mL, 50 μg/mL) (Invitrogen, Eugene, OR). Olaparib (Selleckchem, Houston, TX) was dissolved in DMSO and used at 25 µM for 24 h in western blot, COMET assay and immunofluorescence experiments. For the colony assay, 0–4 µM of Olaparib was used. Primary antibodies for the following were used for western blotting: pH2AX (Cat No: 05636, Millipore), RAD51 (Cat No: 8875, Cell Signaling) and GAPDH (Cat No: 32233, Santa Cruz).
Total RNA was isolated from AA (MDAMB468 and HCC1806) and EA (MDAMB231 and MDAMB453) TNBC cell lines and outsourced to Admera Health LLC for analysis. miRNA-seq data were trimmed with Cutadapt default parameters and then aligned with miRDeep2 and Bowtie. Quantification of miRNA expression was performed with miRDeep2. DE-Seq2 determined differentially expressed miRNAs. PCA plots and heatmaps were generated using R. miRNA sequencing was performed once, and RT-PCR further confirmed the results in three independent experiments.
Control miRNA (CN-001000-01-20), miR-214-5P (C-301153-01-0020), and miR-142-3P (C-300610-03-0005) were procured from Horizon Discovery Biosciences Limited. miRNAs were transfected using Lipofectamine RNAiMAX (Life Technologies, Eugene, OR) based on the protocol supplied by the manufacturer.
HR Dr-GFP assay
As previously described , the Dr-GFP reporter assay measures HR activity. pDR-GFP and pCBASceI were gifts from Maria Jasin (Addgene plasmids # 26477 and # 26475, respectively), Addgene (Watertown, MA). In brief, MDAMB468 cells were stably transfected with pDr-GFP and selected for puromycin resistance (2 μg/mL). Upon 60% confluence, the stably transfected cells were transfected with the plasmid I-Sce1 and a miR-control or miR-214-5P. Restriction enzyme I-Sce1 cuts the reporter plasmid and initiates GFP expression when the damage is repaired by HR. After transfection of miRNAs for 24 h, GFP-positive cells were measured by flow cytometry using a BD Accuri (BD Biosciences) flow cytometer. A total of three independent experiments were performed.
Protein expression by western blot
The cells were placed on ice and washed twice with ice-cold PBS, and cell lysates were collected using cytoskeletal (CSK) buffer (10 mM PIPES at pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.1 mM ATP, 0.1% Triton X-100 freshly supplemented with 1 mM dithiothreitol, 1 × protease, and phosphatase inhibitors with EDTA). Protein content was measured with Bradford reagent, and the proteins were equilibrated using CSK buffer with 6 × Laemmli buffer and heated at 100 °C for 15 min. The proteins were resolved on gradient polyacrylamide gels and then transferred onto nitrocellulose membranes using the BioRad Trans-Blot Turbo system. The membranes were blocked with a 2.5% blocking grade blocker (BioRad, USA) in 1 × Tris-buffered saline in 0.1% Tween 20 (TBST) and incubated with the primary antibody overnight on a rocking platform at 4 °C. Membranes were then washed three times with 1 × TBST before being incubated for an hour at room temperature with the secondary antibody. The membranes were then washed three times with 1 × TBST, exposed to Western lightning plus ECL (PerklinElmer, USA), and developed in a dark room with Konica Minolta equipment.
Cell cycle analysis
After transfection with miR-control or miR-214-5P, cells were trypsinized, collected in tubes, spun down, and washed with ice-cold PBS. Cells were then suspended in ice-cold ethanol and incubated overnight at − 20 °C. After incubation, cells were washed with PBS, stained with propidium iodide (PI) (Invitrogen, Eugene, OR) in the presence of RNAse A (Thermo Scientific), and processed for cell cycle profiles by flow cytometry using a BD Accuri (BD Biosciences) flow cytometer. ModFit LT 5.0 software was used to analyze the cell cycle profiles. A total of three independent experiments were performed.
Tissue sections were incubated with RAD51 (Cat No: 8349, Santa Cruz), followed by a specific biotinylated secondary antibody (1:250 dilution) using the kit from Vectastain (PK-6101), and then with conjugated horse radish peroxidase-streptavidin and 3′3-diaminobenzidine HCl chromogen, and tissues were counterstained with hematoxylin. Stained sections were imaged with an AxioCam camera, and RAD51 intensities were analyzed with a Zeiss Axioscope microscope as described previously . A total of three independent experiments were performed.
RNA isolation and real-time PCR
Using a ZYMO Research (Irvine, CA) Quick-RNA MiniPrep RNA isolation kit, total RNA was extracted from cells transfected with miR-control or miRNA-214-5P. RNA (1 µg) was reverse-transcribed using a high-capacity cDNA reverse transcription kit (Invitrogen, Eugene, OR), as per the manufacturer’s protocol and as described previously . The primers for amplified genes (RAD51 and GAPDH) were purchased from Bio-Rad. Amplification of PCR products was quantified using SYBR green dye (ABI), and fluorescence was monitored on a QuantStudio 12 K Flex detection system. Melting curve analysis was accomplished for each amplicon. Quantitation was done using the 2−ΔΔCt technique with GAPDH as an endogenous control. A total of two independent experiments in triplicate were performed in patients’ samples. A total of three independent experiments were performed in cell lines.
First, the cells were seeded into FluoroDishes (World Precision Instruments) and incubated overnight for adherence. After treatment, cells were fixed with 4% formaldehyde for 10 min at room temperature. The cells were permeabilized with PBS containing 0.2% Triton X-100 for 3 min. The cells were washed and blocked with 10% goat serum in PBS for 40 min. After three PBS washes, cells were incubated overnight at 4 °C with primary antibodies (RAD51: Cat No: 8349, Santa Cruz or pH2AX: Cat No: 05636, Millipore) in PBS, then incubated with a fluorescent secondary antibody (Molecular Probes, Eugene, OR) for 2 h at room temperature. The cells were mounted with Vectashield containing 4′,6-diamidino-2-phenylindole and analyzed for foci formation using a Nikon Eclipse TE confocal microscope. A total of three independent experiments were performed.
Comet chip assay
As previously reported  and following the manufacturer’s instructions, comet assays were performed under alkaline conditions using Comet Chip Assay Kits (Trevigen, Gaithersburg, MD). The cells transfected with miR-control or miR-214-5P were treated for 24 h with DMSO or 25 μM olaparib, harvested, suspended at a concentration of 1 X 105 cells/ml in PBS combined with 1% low melting point agarose at a ratio of 1:10 (v/v). The Comet chip was immersed in a lysis solution for 30 min and electrophoresed in a horizontal electrophoresis apparatus containing an alkaline solution. The chip was then neutralized with 0.4 M and 20 mM Tris–HCl, pH 7.4, and stained with SYBR gold overnight. The chip was again de-stained with 20 mM Tris–HCl, pH 7.4, to visualize cellular DNA with a Zeiss Axio fluorescence microscope. Fluorescence images were analyzed using the ImageJ comet plugin to demarcate each comet's “head” and “tail” regions. The comet tail areas were measured, and calculations were averaged from three independent experiments.
Clonogenic survival assay
For high-density and low-density colony formation assays, 5 × 103 cells per well and 5 × 102 cells per well, respectively, were seeded into 6-well culture plates and incubated overnight for adherence. Cells transfected with miR-control or miR-214-5P were then treated with DMSO or various concentrations of olaparib and cultured for colony formation over 7–10 days. After colony formation, the growth medium was removed, and cells were washed with ice-cold PBS three times and then fixed in ice-cold methanol for 5 min. Methanol was removed, and 1% w/v crystal violet (Invitrogen, Eugene, OR) was added for staining. After 10 min, the wells were washed under tap water, and the plates were allowed to dry at room temperature. Colonies were then imaged and counted using ImageJ software. A total of three independent experiments were performed.
Luciferase promoter assay
Individual GoClone (Luciferase with a RAD51 3’-untranslated region (3’UTR) (Cat No: S807477, Active Motif) constructs were purchased from Switch Gear Genomics. Luciferase assays were performed for MDAMB468 and HCC1806 cells using a LightSwitch Assay System kit (Cat No: LS010, Switch Gear Genomics) according to the manufacturer’s protocol. In brief, TNBC cells were transfected with miR-control or miR-214-5P for 48 h and, for the last 24 h, co-transfected with the vector expressing luciferase mRNA with the RAD51 3’UTR. After transfection/treatment, luciferase activity in the cells was measured using LightSwitch Assay reagents with a Tecan microplate reader. A total of three independent experiments were performed.
The prognostic landscapes of RAD51 in BCs were identified from the PrognoScan database. The expression profiles of RAD51 in BCs were identified from analysis of the TCGA database using the UALCAN portal.
Student’s t-test was performed to estimate statistical significance using GraphPad Prism 8.0 software.
RAD51 is overexpressed in TNBCs, particularly those of AA women, and its high expression correlates with a poor prognosis
We recently showed that RAD51 is upregulated in BCs, particularly in TNBCs . To determine whether RAD51 could be a prognostic marker for BCs, we utilized RAD51 expression data from PrognoScan for a meta-analysis. PrognoScan uses the minimum p-value approach for the grouping of patients. Patients are separated into two groups (high and low expression) at each cutpoint, and the risk differences between the two groups are assessed using the log-rank test. The best cutpoint with the most pronounced p-value is then chosen to determine high and low expression . Our analysis of RAD51 expression data for BC showed that patients with high RAD51 levels (n = 110) had low overall survival compared to the patients with low RAD51 (n = 88) (Fig. 1A). Similarly, patients with high RAD51 expression (n = 100) also had lower disease-free survival compared to the patients with low RAD51 expression (n = 149) (Fig. 1B). Consistently, BC patients with high RAD51 levels (n = 192) showed lower metastasis-free survival and relapse-free survival (n = 72) compared to the patients with low RAD51 (n = 94 and n = 87), respectively (Fig. 1C, D). We expanded our analysis to explore the expression of RAD51 protein levels in BC using the UALCAN portal. Interestingly, RAD51 levels were upregulated in the TNBC subtype (n = 16), compared to normal breast tissues (n = 18) and other subtypes (luminal: n = 64; HER2-positive: n = 10) of BC (Fig. 1E). Additionally, these data indicate relatively high RAD51 protein levels in TNBCs of AA (n = 18) patients compared to those of EAs (n = 80) (Fig. 1F). To validate CPTAC data, we analyzed RAD51 expression levels in TNBCs of our bio-specimen repository using RT-PCR and immunohistochemistry (IHC). Consistently, our results show that RAD51 transcript levels were significantly higher in AA TNBC (n = 26) samples compared to EA TNBC (n = 26) (Fig. 2A). Age at diagnosis and stage of TNBC for the samples used in Fig. 2A are listed in Table 1, showing no distinctive difference between the AA (n = 26) and EA (n = 26) TNBCs. Similarly, RAD51 protein levels were also significantly higher in AA TNBC (n = 5) specimens compared to EA TNBC (n = 5) as evidenced by the IHC data (Fig. 2B, C). These data indicate an important role for RAD51 in TNBC progression and prognosis. Additionally, RAD51’s distinct expression in TNBCs of AA and EA ethnicity implicates a role in racial disparities in these patients. However, the mechanisms that influence RAD51 expression in TNBCs, and its distinctive regulation in AA TNBC patients are not known.
RAD51 is epigenetically regulated in TNBCs, and loss of miR-214-5P and miR-142-3P leads to RAD51 upregulation
Recent studies from non-coding RNAs (ncRNAs), including miRNAs, indicate their differential regulation in different cancers and ethnicities, including TNBCs, and their attribution to disease progression and prognosis [35, 36]. To identify differentially regulated miRNAs, particularly those that target and epigenetically regulate RAD51, we performed miRNA-seq in two pairs of cell lines from each AA (MDAMB468 and HCC1806) and EA (MDAMB231 and MDAMB453)-derived TNBCs. A list of the top 30 miRNAs that were differentially regulated in AA vs EA TNBC cell lines are presented in Fig. 2D. The cell line-wise miRNA expression heatmap is shown in supplementary Fig. 1 (Additional file 1: Figure S1) and the list of miRNAs that are differentially expressed in AA TNBCs (MDAMB468 and HCC1806), compared to EA (MDAMB231 and MDAMB453) TNBCs, is presented in Table 2. To distinguish the upregulated and downregulated miRNAs based on the fold change and p-values, we presented a volcano plot analysis of the miRNA-seq data (Fig. 2E). Analysis of these data using the miRNA Target Base portal identified miR-214-5P and miR-142-3P as RAD51-targeting miRNAs based on the presence of their seed sequences (Fig. 2F). To validate these cell line data, we analyzed the expression of these miRNAs (miR-214-5P and miR-142-3P) in TNBC samples using RT-PCR. As shown in Fig. 2G, H, the expression of miR-142-3P and miR-214-5P was significantly downregulated in AA TNBCs (n = 16) compared to EA TNBCs (n = 16), respectively. Additionally, analysis of the TCGA database using the UALCAN portal further validated that miR-214 is significantly downregulated in AA TNBCs (n = 160) compared to EA TNBCs (n = 578) (Fig. 2I).
miR-214-5P downregulates RAD51 in BRCA wild-type/proficient TNBC cells
We first determined whether expression levels of miR-214-5P and miR-142-3P negatively correlate with RAD51 expression in racially distinctive TNBC cell lines by performing RT-PCR analysis. As shown in Fig. 3A, the RAD51 levels were significantly higher in two pairs of AA in TNBC cell lines compared to their respective EA counterparts, except the comparison between MDAMB453 and MDAMB468. On the contrary, miR-142-3P (Fig. 3B) and miR-214-5P (Fig. 3C) were downregulated in AA TNBC cell lines compared to the EA TNBC cell lines. Collectively, these data indicate a negative correlation between specific miRNAs (miR-214-5P and miR-142-3P) and RAD51 levels in two pairs of AA and EA TNBC cell lines (Fig. 3A–C). To investigate whether miR-214-5P and miR-142-3P target RAD51 and downregulate its expression, MDAMB468 cells were transfected with miR-control, miR-214-5P, or miR-142-3P and analyzed for RAD51 protein levels by western blots at different time points after the transfection. As expected, miR-214-5P-transfected cells showed 70% to 90% downregulation of RAD51 in the time points evaluated (Fig. 3D; Additional file 2: S2A). However, miR-142-3P was less efficient in RAD51 downregulation than miR-214-5P (Fig. 3E; Additional file 2: S2B). This could be due to multiple factors, including the presence of binding sites for miR-214-5P in two different regions (1522–1528 and 1702–1708) of RAD51 3’UTR (shown by miRNA Target Base analysis), which might influence efficient targeting. Therefore, further studies were focused only on using miR-214-5P. To rule out that these results were not cell line-specific, we further examined multiple TNBC cell lines. Consistent with MDAMB468 cells’ data (Fig. 3D), miR-214-5P downregulated the expression of RAD51 in MDAMB453 and HCC1806 TNBC cell lines (Fig. 3F). RAD51 is a critical factor in BRCA and associated protein complex mediated repair of DNA double-strand breaks (DSBs) and maintains the integrity of chromosomal DNA in replicating cells [37,38,39,40]. Since non-replicating cells tend to have low expression of the HR proteins, it is essential to rule out the contribution of any cell cycle discrepancies in miR-214-5P-transfected cells to RAD51 downregulation. In fact, our flow cytometry results from miR-214-5P-transfected MDAMB468 cells showed increased accumulation of cells in the S-phase compared to the miR-control-transfected cells (Fig. 3G; Additional file 2: S2C and S2D), which rules out the contribution of any cell cycle discrepancies to RAD51 downregulation.
miR-214-5P targets 3’UTR region of RAD51 and regulates its mRNA levels
To confirm that miR-214-5P downregulates RAD51 by targeting the 3’UTR region of the transcript, we performed luciferase assays (Promega). A luciferase reporter was designed to include the luciferase ORF under a CMV promoter, followed by the RAD51 3’UTR region, which has the predicted seed sequences (1522–1528 and 1702–1708) for miR-214-5P (Fig. 4A). Co-transfection of the luciferase reporter with either miR-control or miR-214-5P showed > 50% decreased luciferase activity in miR-214-5P-transfected MDAMB468 and HCC1806 TNBC cells compared to the miR-control (Fig. 4B). Consistently, RT-PCR data showed downregulation of RAD51 in miR-214-5P-transfected cells compared to control miRNAs (Fig. 4C), which confirms that miR-214-5P targets the 3’UTR region of RAD51 mRNA and regulates RAD51 posttranscriptionally. Additionally, this seed sequence is conserved in RAD51 mRNA (1522–1528) in different primates (Additional file 3: Figure S3A), indicating a conserved mechanism of epigenetic regulation.
miR-214-5P downregulates RAD51 and induces HR deficiency (HRD) in BRCA wild-type/proficient TNBC cells
RAD51 is an essential factor of a multi-protein complex of BRCA and related proteins that most accurately repair replication-associated DSBs [37,38,39,40]. Thus, deficiency or inhibition of RAD51 affects cells’ ability to repair DSBs due to HR deficiency [21, 40]. We performed Dr-GFP reporter assays to determine whether miR-214-5P-mediated downregulation of RAD51 affects TNBC cells’ efficiency to repair DSB by HR [41, 42]. We used BRCA-proficient MDAMB468 cells to transfect with the Dr-GFP plasmid and selected them in a puromycin selection medium. These Dr-GFP-expressing cells were then co-transfected with an ISCE-1 expression plasmid to induce DSBs and either miR-control or miR-214-5P and then analyzed for GFP expression using a flow cytometer. Consistent with RAD51 downregulation, miR-214-5P-treated MDAMB468 cells showed > 50% reduction in HR efficiency (p < 0.001) compared to the miR-control-transfected cells (Fig. 5A, B).
To further examine whether miR-214-5P-induced HRD in DNA repair-proficient TNBC cells causes increased DNA damage in response to PARPi olaparib, we transfected two RAD51 upregulated AA-derived TNBC cell lines (MDAMB468 and HCC1806) with miR-control or miR-214-5P and treated them with vehicle or olaparib. Consistent with the DNA repair deficiency, miR-214-5P-transfected MDAMB468 and HCC1806 cells showed downregulation of RAD51 and increased expression of pH2AX (p < 0.0001) protein levels in response to olaparib when compared to their respective miR-control-transfected cells treated with olaparib (Fig. 5C, D).
To further confirm that miR-214-5P-mediated downregulation of RAD51 also affects its repair foci formation in olaparib-induced DNA damage, we examined for the levels of repair foci of RAD51 and pH2AX in HCC1806 cells (Fig. 6A, C). In miR-control-transfected cells, RAD51 and pH2AX foci were undetectable. Consistent with the western blot data (Fig. 5C, D), olaparib treatment increased the number of RAD51 and pH2AX foci compared to DMSO-treated cells. Furthermore, for the miR-214-5P and olaparib combination, miR-214-5P attenuated olaparib-induced RAD51 foci formation compared to the miR-control and olaparib combination (Fig. 6B). Contrarily, pH2AX foci levels were elevated upon olaparib treatment in control cells and were further elevated by the miR-214-5P and olaparib combination, indicating increased DNA damage in these cells (Fig. 6D). These data confirm that the combination of miR-214-5P and olaparib downregulates RAD51 and increases DNA damage as measured by pH2AX.
miR-214-5P synergizes with PARPi olaparib in BRCA wild-type/proficient TNBC cells
To validate that increased pH2AX is due to increased DNA lesions, we performed alkaline COMET assays in these cell lines (MDAMB468 and HCC1806) (Fig. 7A; Additional file 3: S3B). In agreement with the pH2AX levels, miR-214-5P-transfected MDAMB468 and HCC1806 cells showed significantly elevated levels of COMET tails (p < 0.0001) in response to olaparib when compared to their respective miR-control-transfected cells treated with vehicle or olaparib (Fig. 7B, C). Together, these results suggest that miR-214-5P downregulates the HR protein RAD51, causes HR repair deficiency, and potentiates olaparib-induced DNA damage in BRCA-proficient TNBC cells. Furthermore, these data also propose a novel synergistic lethality mechanism involving miR-214-5P and olaparib combination therapy, similar to BRCA-mutant or HR-deficient TNBCs.
To confirm that the miR-214-5P-mediated downregulation of RAD51 causes synergistic lethality with the PARPi olaparib, we first performed a high-density colony assay using HCC1806 cells transfected with miR-control or miR-214-5P, and treated them with various concentrations of olaparib. As shown in Fig. 7D, HCC1806 cells transfected with miR-control showed a dose-dependent decrease in the colony-forming ability of these cells. Consistent with the DNA repair deficiency, miR-214-5P-transfected cells showed decreased colony formation capacity relative to miR-control-treated cells in response to olaparib at all the tested drug concentrations. To further confirm, we performed low-density colony assays using MDAMB468 (Fig. 7E) and HCC1806 cells transfected with miR-control or miR-214-5P, treated with or without various concentrations of olaparib and plotted the survival fractions (Fig. 7F, G). The miR-214-5P-transfected cells showed significantly decreased colony formation compared to miR-control-treated cells in response to olaparib at all the tested drug concentrations. Together, these data suggest a novel synergistic lethality combination of miR-214-5P and olaparib to effectively treat BRCA-proficient TNBCs that could aid in decreasing disparities in the therapeutic outcomes of TNBCs.
Epidemiological and other population-based studies show the existence of racial disparities relating to BC progression and therapy outcomes in AA ethnic population . In particular, the occurrence of TNBC, the most lethal form of BC, is 2.21 times higher for AA women compared to EA women, and it affects them at a younger age and has a worse prognosis [10, 11, 44, 45]. Several biological and non-biological factors have been identified to influence racial disparities . Biologically, obesity and waist–hip ratio (WHR) have been identified as increased risk for AA TNBC patients. The prevalence of TNBC in overweight and obese AA women is twofold compared to that in normal-weight AA women , and these studies are further supported by many studies implicating WHR as a strong risk factor for TNBC in AA women . However, specific endpoints like somatic mutations in prominent genes like TP53 (46% of AA patients, compared to 27% in EA patients) have been documented as the racial disparity in the overall cancer incidence but not in TNBC . These observations confirm the need to identify more potential mechanistic underpinnings that cause a racial disparity in TNBC.
MiRNAs have been identified as a potential regulator of racial disparity in multiple cancers [50,51,52], including TNBC [53, 54]. Our present study identifies that the loss of miR-214-5P, which epigenetically regulates RAD51, could be a marker for a poor prognosis in TNBC patients. It could contribute to our understanding of racial disparities in TNBCs. Particularly, miR-214-5P’s role has been documented in proliferation , migration, invasion , aggressive tumor growth  and resistance  in multiple cancers. Our studies identified a novel role for miR-214-5P in regulating critical DNA repair gene RAD51 and suggested that loss of it in AA TNBCs upregulates RAD51 and affects their response to therapeutics. As miR-214-5P is significantly decreased in AA TNBCs compared to EA TNBCs, it indicates its contribution to racial disparities in TNBC therapeutic outcomes and could serve as a biomarker for these patients. Notably, the mechanism identified in our study paves a way to treat BRCA wild-type TNBC patients who do not benefit from single-agent PARPi-targeted therapies.
Upon treatment with PARPi, cells with HRD display extensive DNA lesions and synthetic lethality . PARP inhibitors are a class of agents approved first for BRCA-deficient ovarian cancer and BC and later for other cancers . The biochemical effects of deleterious germline mutations in BRCA1 and BRCA2 compromise their essential role in maintaining functional, high-fidelity DNA repair via homologous recombination, indicating their potential clinical utility [61, 62]. However, later studies established that not only BRCA genes but proteins with loss-of-function mutations involved in HR (including RAD51, FANCD2, and PALB2) also synergize with PARPi [63,64,65]. However, PARP inhibitors are currently effective only in 15–20% of TNBC patients with genetically compromised HR. Several researchers have identified alternative ways to induce HR non-genetically by targeting various proteins involved in HR, including RAD51 [66,67,68]. Accordingly, our previous study identified that CHK1 inhibitor prexasertib inhibits RAD51 and synergizes with olaparib . Similarly, inhibition of Myc , glycosylase OGG1 , nicotinamide phosphoribosyltransferase , CDK  and topoisomerase 1  have been shown to combine with PARPi in TNBCs synergistically. However, none of these combinations addresses the potential racial disparity observed in AA TNBC patients. Based on our results, we propose a novel synergistic lethality mechanism involving the combination of miR-214-5P and PARPi to effectively treat TNBC, especially in AA TNBC patients, as they express significantly low miR-214-5P and increased RAD51 compared to EA TNBCs. Additionally, a recent clinical study evaluating olaparib against metastatic BCs in patients with BRCA mutations shows promising results . Since olaparib is already FDA-approved, targeting RAD51 either by miRNAs (e.g., miR-214-5P) or by drugs that promote its degradation would be an effective combination to treat HR-proficient TNBCs to overcome therapeutic resistance.
Our studies provide preclinical evidence for targeting RAD51 by miR-214-5P to induce HR deficiency in BRCA-wild-type or HR-proficient TNBCs and shows synergistic lethality with an FDA-approved PARPi olaparib. Since significantly decreased expression of miR-214-5P and compensatory upregulation of RAD51 were observed in AA TNBCs compared to EA TNBCs, these observations could serve as biomarkers for racial disparity in TNBC. Importantly, these results also suggest that a novel combination of miR-214-5P and olaparib could be an effective therapy for BRCA-wild-type TNBCs and reduce racial disparities in TNBC outcomes.
Availability of data and materials
All data generated or analyzed during this study are included in this research article and its supplementary information files.
Triple-negative breast cancer
The Cancer Genome Atlas
The University of Alabama at Birmingham Cancer Data Analysis Portal
Green fluorescent protein
Human epidermal growth factor receptor 2
Breast cancer susceptibility protein
Breast cancer gene 1/2
Ataxia telangiectasia and rad3-related
DNA double-strand break
Food and Drug Administration
Poly-ADP ribose polymerase (PARP) inhibitors
Homologous recombination deficiency
Real-time polymerase chain reaction
Ethylenediamine tetra acetic acid
Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetra acetic acid
Tris-buffered saline in 0.1% tween 20
Cancer stem cell
Checkpoint kinase 1
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This work was supported by grants from Weitlauf Endowment for Cancer Research and partly by cancer health disparities supplements to NIH grant CA219187 (KP), and by CPRIT: Texas Regional Excellence in Cancer Award RP210154 to CM.
Ethics approval and consent to participate
The UAB Institutional Review Board approved collecting and using all samples in this study (IRB number: 060911009).
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The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Additional file 1.
Column heat map analysis of miRNA sequencing between EA (MDAMB231 and MDAMB453) and AA (MDAMB468 and HCC1806) cells.
Additional file 2.
miR-214-5P regulates the expression of RAD51 in a cell cycle independent manner. (A) Densitometry analysis for RAD51 expression in MDAMB468 cells transfected with miR-214-5P in three independent experiments are represented in the histogram with standard deviation as error bars. (B) Densitometry analysis for RAD51 expression in MDAMB468 cells transfected with miR-142-3P in three independent experiments isrepresented in the histogram with standard deviation as error bars. Cell cycle profile of MDAMB468 cells treated with miR-control (C) or miR-214-5P (D).
Additional file 3.
Bioinformatic analysis of protein sequence conservity. (A) Protein alignment analysis using UNIPORT shows a highly conserved seed sequence 1522- 1528 in RAD51 mRNA. (B) Comet assay representative images in HCC1806 cells transfected with miR-control or miR-214-5P and treated with or without 25M olaparib for 24 h.
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Mani, C., Acharya, G., Saamarthy, K. et al. Racial differences in RAD51 expression are regulated by miRNA-214-5P and its inhibition synergizes with olaparib in triple-negative breast cancer. Breast Cancer Res 25, 44 (2023). https://doi.org/10.1186/s13058-023-01615-6
- Racial disparity
- HR deficiency

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Here is a picture of my mystery quilt. I managed to finish it in spite of the gremlins who kept messing with my machine and hiding things from me. Now the quilt top is in my pile of tops waiting to be quilted.
I was given a prize because I was the first one to finish my quilt top. Here is my cute little cupcake pincushion.
When I got home from the retreat, Mr.Frogg started complaining about how cold he was without a quilt of his own. I went right to work making him a quilt. I used embroidered blocks of his family members so he would sleep well, knowing his family was surrounding him with hugs.
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1 August, 2016
Fine Chicken Liverpate
A great chicken only pâté
Last week I got fresh chicken liver in a bulk pack and decided to make some liver pâté out of it. I decided to make a smooth pâté with only chicken in it.
I came across several recipes and ended up making my own one based on what I found.
Making pate is sometimes a long and complicated process, but I want to make it easy and quick.
This is what it looks like at the end.
Fist I cooked the chicken breast. In most recopies I found it says you need to take legs and a bit of breast. I took only breast, knowing this is less fat and more dry.
I compensated this with more liquid.
First I poached the breast in whole for about 15-20 minutes. I added some onions and leak to my poaching water, but no salt.
Then I let this cool down and cut it in smaller pieces about 2cm large.
During poaching I measured my spices and mixed them together.
Next I cleaned the liver, pulled out any fat or veins. I added the liver to my blender and mixed it for about 15 seconds until it was smooth and started building bubbles.
Then I added the spices, the poaching water and cream and gave it an initial mix 2-3 seconds before adding 1/3 of the chicken breast. I stared mixing on high speed and added the rest of the breast pieces. After about 30-50 seconds it was all mixed well together and smooth.
Using a blender was the best choice. When you use a food processor you would need to mince the meat first and the cutting would take several minutes. My food processor would never get it that smooth.
I filled the pate in glasses and placed them for 45 minutes at 90C (190F) in the oven using a tray filled with hot water up the 3/4 of the height of the glasses. It is important the pate does not get heated up over 80 C. So you need to have an eye on this.
Once cooled down put the pate in the fridge for at least a day before tasting. But It should rest for 1-2 weeks, if possible, to build up all flavors.
This pate is not very fat and because of that it is not as soft as most other pates, but the taste is great.
- 600g chicken breast, organic
- 400g chicken liver , organic
- ½ onion
- some leak
- 50g double cream
- 150ml poaching water
- 2.8g curing salt / also called pink salt (or add 2g normal salt to the rest of the salt)
- 16g salt
- 2g ground white pepper
- 0.5g ground ginger
- 0.5g ground macis
- 0.3g ground cardamom
- 2g raw sugar
- poach the chicken breast with the onion and leak for 20 min in some water, covering the meat.
- clean the liver from veins
- let the meat cool down and cut in small pieces
- Add the liver to the blender and mix for some seconds until smooth and bubbly
- Add 150ml of the sifted poaching liquid, the cream and spices and just mix 1-2 seconds
- Add ⅓ of the chicken breast pieces and put your blender on full speed. When incorporated add the rest of the meat and mix until well mixed and smooth. This should not take longer than 40-60 seconds.
- fill in glasses and close them with a lid. Place a pan with hot water (not boiling) in your oven and place the glasses in there.
- heat up the oven to about 90C (190F) and once the water has reached 80C (176F) keep it at that temperature for 45 minutes (1 minute per cm diameter of your glass, my are 4.5 cm)
- let cool down and them place in fridge before serving.

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August 16, 2004
"True sanity entails in one way or another the dissolution of the normal ego, that false self competently adjusted to our alienated social reality...and through this death a rebirth, the ego now being the servant of the divine, no longer its betrayer."
(Swans - August 16, 2004) The pathology of American culture is as ubiquitous as its strip-mall ugliness. It is abundantly evident, in almost every aspect of contemporary life: From the predatory (to the point of psychopathic) practices of its captains of the corporate boardroom, down to the pandemic enervation and proliferate anomie of its morally scurvy, gallery slaves languishing in their soulless cubicles -- from the genitalia-devoid mascots at DisneyWorld, to the genitalia-obsessed torturers of Abu Ghraib prison -- the soul-sickness spreads before us like George W. Bush's taunting, executioner's smirk.
Ronnie Laing's profound dictum leaves us with many poignant questions regarding the true nature of the psychic lives of so-called ordinary Americans and our ability to function in this corrupt and crumbling empire. In short, is it sane to be able to adapt to an insane culture?
And: If conforming to group, cultural, and national pathology is rewarded (therefore encouraging the formation of the "false self") -- how might an individual, stranded within the dysfunctional dynamic, resist it all, and begin to work towards a more meaningful awareness of their own essential nature -- and then come to a similar reckoning regarding how to live in the mists of the life-defying demands of the present era -- and, despite it all -- retain some measure of meaning, inner depth, and human resonance?
Laing apprehended: When we were children, authority (in the form of parents, teachers, clergy) loomed before us... Alternatively menacing and comforting, these powerful figures could just as easily have crushed us as comforted us. (Tragically, all too often, they perpetrated the primary.)
Hence, we learned how to curry favor from these baffling, seemly implacable, forces by the creation of a cipher persona, "a false self," a tricky, tap-dancing, little suck-up -- who garnered approval and acceptance -- and thereby avoided punishment and scorn by the reflexive subjugation of his true nature, all the while, showing an agreeable face towards mindless authority which masked a mind seething with misplaced resentments and shallow subterfuge -- the perfect corporate citizen/consumer.
In childhood, the presence of these huge, confounding forces created irreconcilable anxieties within us: the actions and activities of authority figures seemed as overwhelming and unpredictable as nature, itself; they were perceived as implacable forces that we could not fight nor resist... They always confounded us: to our young, fragile psyches, these all-powerful creatures might nurture us -- but -- they could just as easily -- have devoured us.
-- Is it any wonder that children are obsessed with monsters? And childish adults see God as a wrathful father?
At Multiplexes, day and night, across the United States, cinematic imagery, with stories containing the thematic complexities rarely rising above leveling of understanding of a dim nine-year-old, flicker over the passive faces of moviegoers.... (The same can be said of the psychotic story lines of the EndTime sermons raved in fundamentalist churches.)
In these dramas (whether promulgated by mountebanks from Hollywood or home-grown, fundie jihadist) -- monsters rise and then are vanquished by bland heroes. On a metaphoric level, we are replaying the trauma of having our essential humanness devoured by the monsters of corporate hegemony.
Corporate Capitalism has left Americans psychically stuck in a pathetic simulacrum of childhood and its inchoate fears of being preyed upon by their (alleged) protectors (who they internally and accurately recognize as monsters)... of having their individual uniqueness stolen by enslavement to an mindless system that lives for no other reason than it lives -- a system that eats its young (giving new mean to the term "'consumer' economy") -- that exists only to perpetuate itself -- a system that has become a soul-devouring monster.
-- Is it any wonder we grow to accept monstrous acts committed in our names?
We endlessly replay the trauma of the devouring of own true selves to the insatiable appetite of the corporate/consumer empire: from mirroring it in our ceaseless cravings for the empty spectacles offered up by Hollywood movies and the lurid B-movie imagery of the Book of Revelations, to our addiction to junk food, to the relentless banalities of our political discourse, to the ugliness of our ad-hoc architecture -- it has become the mask and mantle of our existence. Worse, the exterior falseness of the American landscape has become internalized -- the lines of demarcation have disappeared -- the outer phoniness has merged with the inner phony (and vice versa) -- becoming a hall of mirrors where distorted images mirror distorted images -- where any semblance of an non-distorted image is taken for freakish and must be scorned, ignored, or shattered to shards.
This is how the American people can dismiss the torture committed at Abu Ghraib prison as simply "having some fun."
Those sorts of pathological distortions start in childhood.
Is it any wonder America has become a nation inhabited by emotionally-stunted, infantilized consumers? How passive and apathetic so many Americans have become...with mass media spoon-fed mouths agape, sitting in our own polluted poop, ignorant of the world (but mollified by the dazzle of its petty lights) as chubby fingers point at consumer goods, screaming -- "MINE!"
It is what the corporate/consumer empire has demanded we become: for it does not take long for children (most of us anyway) to learn what actions on our part are accepted and rewarded -- and, conversely, which are punished; what aspects of our "character" are acceptable, and which are scorned; and what roles we are expected to play in exchange for being loved, fed, clothed, sheltered, and educated.
This exchange insured that we're given a "safe" place within the community -- not cast out into the wilderness and fed to the wolves. (This fear is not such an outrageous fantasy: it is, in fact, a primal memory, because various forms of infanticide were once common practices nearly all cultures, including the act of abandoning outcast children to die in the wilderness. And the knowledge still lingers within our psyches, where the memories of such terrors still howl just beyond the tree-line of our awareness, instilling within us the terror of ridicule, of failure, of being ostracized... Far too many people succumb to these fears and begin playing the roles circumscribed by their families, communities, and cultures: Tragically, their true selves, for all practical purposes, were smothered in their cribs.)
So what becomes of those who are "thrown to the wolves?"
America's prisons bristle with them.
As for those of us who are less criminally inclined: our "cast out" selves find provisional relief in places like Las Vegas, where "What happens here, stays here," in the words of that city's latest advertising campaign.)
Las Vegas and prisons are the fastest growing population centers in the United States...a landscape now comprised of jails and clip-joints... a land of suckers and criminals... It is a natural progression: for capitalism has always depended on a predatory class of sociopaths...has always relied upon thievery and murder...and needs an endless supply of suckers and victims.
(It would seem: Most of those who languish in the American penal system lacked the forethought and initiative to have first been born into the privileged class -- thereby ensuring they could have committed their crimes with impunity.)
But there is another way, for those who have been cast out into the wildness: There are some (non-conformists, creative thinkers, artists) who welcome (rather than fear) the metaphorical wolves (who are recognized, each to each, as fellow outcasts). Instead of being eaten by them -- they are suckled by the wolves...
Nourished by their outsider status, the creative spirit thrives when freed from the constraints of a mindless adherence to groupthink... The wilderness becomes their natural habitat: ...they howl at the moon; they reject the daylight world of bland consensus... they learn to see in the dark... The wilderness being their home: they don't clean-up nicely for polite company... they don't let themselves be bred down (as a few domesticated wolves did) to yapping Chihuahua, in exchange for a few food scraps...
Yes, when your looking at a Chihuahua -- you're looking at a former wolf.
One day, you're loping through the woods, snout held high, smelling the scent of fresh game on the wind...and the next thing you know, you're being led around on a leash and collar, encrusted with tacky rhinestones, and you're salivating at the sound of an electric can-opener... One moment, you're a child and you're entranced in play, hard-wired to eternity -- the next thing you know, you're sitting at work and your passions, hopes, and yearnings have been shrunk down to Chihuahua-sized cravings... You're truckling for your boss's approval... You're counting the minutes until your breaktime when you can devour some junk food... Like a domesticated pet, or an unfortunate animal incarcerated in a zoo, you are no longer a noble animal -- you are a thing that waits for lunch.
To resist, we must cast off the fear of being an outcast.
Though we may never achieve the independence and nobility of wolves, there is no call for us to spend our lives being habitually obedient to our corporatist masters whose treatment of us rarely rises above the level of neglect and contempt.
I remain hopeful: There is yet a molecule or two of the wild wolf left within us cringing Chihuahuas. Because within us lies an indomitable self, encoded with the grace and fury of the natural world, and, if acknowledged and respected, it will awaken and rise -- imbrued with perennial vitality and natural appetites -- and it, at the very least, will make a quick meal of the paltry flesh of our false selves.
Then the fight begins: The fur will fly -- as we fight tooth and nail to retake our own essential natures, and, by extension, begin the struggle to restore the health, imagination, and dignity, that is inherent, in those who struggle to achieve individual freedom to a nation of cage-accepting, sick puppies.
· · · · · ·
US Elections & Democracy on Swans
America the 'beautiful' on Swans
Phil Rockstroh on Swans (with bio).
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