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A Successful First Fall for the ECNL Regional League Program
It was an exciting and challenging first season for the FC WI ECNL Regional League program as the players competed in the first season of the ECNL Girls Regional League - Heartland Conference against top teams from across the Midwest!
The ECNL Regional League - Heartland Conference is one of the best in the league, including nationally respected clubs like the Michigan Hawks, Eclipse Select, and St. Louis Scott Gallagher. The conference also features well-known clubs in the Midwest like the Rockford Raptors, Sporting Michigan, and more. The players knew it would be a great challenge and opportunity to face teams of this caliber - and the fall was exactly what was expected!
Each age group embraced the ECNL Regional League, and though they began the season as 5 individual teams, they ended the fall as a much more unified Program.
Development and skill growth is the focus of everything in the club, and the ECNL Regional League teams grew in understanding of the FC Wisconsin game model and the 4 moments of the game every week. Perhaps most impressive this fall was the huge improvement in communication, and the culture to compete every day that the players started to create. The teams took the club value of "Compliment and Coach" to heart by the end of the season.
Competition at the ECNL level is fierce, something these teams learned early in the season. With the speed of improvement shown this fall, the future for these players is bright.
Click HERE to learn more about the ECNL RL!


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Iatrogenic lipogranuloma of the penis : Asian Journal of Andrology
Archive 2014 Onwards
AJA Club
Thought you might appreciate this item(s) I saw in Asian Journal of Andrology.
Iatrogenic lipogranuloma of the penis
Zhang, Zheng-Chao1,2; Zeng, Tong-Wei1; Han, Jun-Ping3,; Xie, Yong-Qiang1,
1Department of Urology, The Third Affiliated Hospital of Gansu University of Traditional Chinese Medicine, Baiyin 730900, China
2Department of General Surgery, The Second Hospital of Lanzhou University, Lanzhou 730000, China
3Department of Pathology, The Third Affiliated Hospital of Gansu University of Traditional Chinese Medicine, Baiyin 730900, China
Correspondence: Dr. YQ Xie ([email protected]) or Dr. JP Han ([email protected])
Asian Journal of Andrology 25(1):p 147-148, Jan–Feb 2023. | DOI: 10.4103/aja202253
Lipogranuloma of the penis is defined as a foreign body reaction to lipids, hydrocarbons (paraffin tumors), silicones, methacrylate, hyaluronic acid, or collagen fillings.1 According to literature reports, some individuals attempt to augment their genitals with injections of illegal liquid paraffin oil.2,3 In recent years, increased reports of complications associated with lipogranuloma have led to a decline in the use of these paraffin oil injections.4,5 Iatrogenic lipogranuloma of the penis is caused by the infiltration of paraffin oil into the penis subcutaneously along the ruptured urethra after urethral trauma. The incidence of this disease is very low, and there are few reports about it at home and abroad, which has led to an insufficient understanding of this disease among urologists. We report here the case of a patient with iatrogenic lipogranuloma of the penis caused by paraffin oil. This study was approved by the Ethics Committee of The Third Affiliated Hospital of Gansu University of Traditional Chinese Medicine (Baiyin, China; ethics approval No. YL-X2-2016-027). The patient was informed of the content and significance of the study and signed an informed consent form.
A 75-year-old male patient was admitted due to "a penile mass found for more than half a month". More than half a month before admission, the patient inadvertently found multiple subcutaneous penile masses without pain, and the penis became significantly thicker. At that time, the patient did not seek any diagnosis or treatment, but the mass area increased significantly later. The patient had undergone transurethral resection of a bladder tumor in The Third Affiliated Hospital of Gansu University of Traditional Chinese Medicine on March 9, 2017, and the postoperative diagnosis was high-grade urothelial carcinoma. After surgery, pirarubicin hydrochloride was infused into the bladder regularly for chemotherapy. Three months after the operation, the patient developed dysuria and was diagnosed with urethral stricture. On September 13, 2017, direct visual internal urethrotomy was performed in our hospital, and urethral dilation was performed regularly after the operation.
After admission, the patient underwent physical examination, which showed that the penis diameter was significantly enlarged (Figure 1a), but the color was normal. A mass with a diameter of approximately 2 cm was visible at the junction of the penis root and the scrotum (Figure 1b) without ulceration. Diffuse, hard nodules were palpable under the penile skin, without tenderness, and the glans could not be completely exposed. The patient underwent a pathological biopsy of the penile mass. Surgical exploration revealed that the mass was white tissue, tough, and rich in blood supply, presenting a diffuse distribution with surrounding tissues (Figure 1c). Partial tissues were excised for pathological examination, and hematoxylin-eosin (H&E) staining was performed for the diagnosis. The pathological examination results were as follows: the mass was composed of fibrous connective tissue infiltrated by lymphocytes, plasma cells and a small number of mast cells, and cystic cavities of different sizes were observed. A large number of multinucleated giant cells were observed on the wall of the cystic cavity. The clinical history of the patient and the pathological examination results were consistent with a diagnosis of lipogranuloma (Figure 1d and 1e). The pathological diagnosis and pictures were made by JPH, chief physician of the Department of Pathology in The Third Affiliated Hospital of Gansu University of Traditional Chinese Medicine.
The patient's clinical data. (a–c) The patient's physical examination and surgical exploration on admission. (a) The diameter was significantly wider, and extensively diffuse subcutaneous tissue of the mass in the penis was palpable. (b) A mass with approximately 2 cm in diameter was visible at the junction of the root of the penis and the scrotum. (c) Surgical exploration showed that the mass was white, tough and rich in blood supply. (d and e) Pathological images (H&E staining). (d) The tissue was infiltrated by lymphocytes, plasma cells, and a small number of mast cells; and cysts of different sizes were observed. Scale bar = 100 µm. (e) A large number of multinucleated giant cells were observed on the walls of the cysts (arrows indicate multinucleated giant cells). Scale bar = 20 µm. (f–h) Follow-up results of the patient after 4 years. (f) The penis diameter was significantly smaller than that at admission. The extensive subcutaneous mass of the penis was still palpable, but the area was significantly smaller than before. (g) Due to the existence of the diffuse subcutaneous mass of the penis, the glans head still could not be completely exposed. (h) The mass at the junction of the root of the penis and the scrotum was also significantly reduced. H&E: hematoxylin-eosin.
Following admission, the patient received ceftriaxone sodium combined with dexamethasone and azithromycin. No significant reduction in penile mass was observed. The patient was discharged from the hospital with an unhealed condition, and he did not receive any treatment after discharge. Four years later, a follow-up examination showed that the mass was gradually healing. As shown in Figure 1f–1h, the patient's penile mass had significantly relieved after 4 years.
The patient had undergone urethrotomy for urethral stricture 1 month before the mass was noticed, and the surgery may have damaged the urethral mucosa. After surgery, regular urethral dilation was performed. During urethral dilation, liquid paraffin oil was used for urethral lubrication, and the paraffin oil may have penetrated into the penile subcutaneous connective tissue through the deep penile fascia along the damaged urethra. Thus, the paraffin oil entered the penile subcutaneous tissue as a foreign body, causing a tissue reaction and eventually mechanical damage and tissue necrosis, thereby leading to localized encapsulated granulomatous hyperplasia.
At present, urethrotomy is a minimally invasive routine method used by urologists in the treatment of simple urethral stricture.6,7 Due to the high recurrence of urethral stricture, some patients need regular urethral dilation. During urethral dilation, sterile paraffin oil is used as a lubricant to prevent urethral injury caused by the urethral dilator.8 There are two common application methods of paraffin oil as a lubricant in clinical urethral stricture dilation. One method is to spread paraffin oil on the urethral dilator for urethral dilation. The other method is to directly inject sterile paraffin oil into the urethra with a syringe and then apply pressure to the external urethral outlet for 1–2 min before urethral dilation. The latter method can easily cause paraffin oil infiltration of penile subcutaneous connective tissue in patients with urethral injury, resulting in the occurrence of fat granuloma. Therefore, the injection of paraffin oil into the urethra for urethral lubrication in patients with urethral injury should be carefully considered by clinicians.
More recently, Navarrete et al.9 reported a similar case. A 62-year-old male injected oil subcutaneously into his penis 10 years ago, which resulted in significant penile thickening. The diagnosis was confirmed by dermatoscopy combined with puncture biopsy. Because of our lack of experience in diagnosing the disease, the diagnosis of this case was directly made by incision biopsy. As early as 1984, Albers et al.10 also reported the cases of two patients with iatrogenic ureteral lipogranuloma. The authors injected paraffin oil through the ureteral catheter to facilitate the removal of calculi. Most such operations are successful, especially in patients with incarcerated stones. However, these two patients developed postoperative lipogranuloma of the ureter. Therefore, they strongly urged the use of water-soluble lubricants for the lubrication of ureters at that time. Furthermore, Hohaus et al.3 reported the case of a 30-year-old male with penile lipogranuloma caused by subcutaneous injection of paraffin oil who received medical treatment. The subcutaneous granuloma and the skin involved were removed, and the penile skin graft was taken from the prepuce, with a good prognosis.
Improper application of paraffin oil can cause lipogranuloma in the penis and ureter. Therefore, clinicians should avoid intraluminal injection of paraffin oil in patients with urethral injury. Furthermore, tetracaine colloidal slurry is also used in clinical practice as an alternative to paraffin oil for urethral stricture dilation lubrication. The auxiliary ingredient contained in tetracaine colloidal slurry is a kind of skeleton material that can increase the viscosity of the solution and play a role in lubrication.11 Due to its high viscosity, after injection into the urethra, there is no need to apply pressure to promote the mucosal penetration of narcotic drugs, so there is a certain advantage in preventing the lubricant from entering the penile subcutaneous connective tissue. Therefore, this tetracaine colloidal slurry is worth promoting.
In conclusion, urologists should be cautious about the use of paraffin oil during urethral operation, especially in patients with urethral injury, because improper use of paraffin oil in such patients may lead to penile lipogranuloma formation.
ZCZ was responsible for data collection, literature review, and manuscript drafting. TWZ contributed to data collection and follow-up and helped to revise the manuscript. YQX and JPH conceived the study, participated in its design and coordination, and helped to revise the manuscript. All authors read and approved the final manuscript.
All authors declare no competing interests.
This study was supported by the Science and Technology Project of Baiyin City in 2019 (project No. 2019-1-22Y).
1. Alcalde-Alonso M, Velasco-Albendea FJ, Soto-Díaz A, Gómez-Avivar P, Torres-Gómez FJ. Paraffinoma of the penis and scrotum (sclerosing granuloma of the male genitalia). Indian J Dermatol Venereol Leprol 2017;83:75–7.
2. Downey AP, Osman NI, Mangera A, Inman RD, Reid SV, et al. Penile paraffinoma. Eur Urol Focus 2019;5:894–8.
3. Hohaus K, Bley B, Köstler E, Schönlebe J, Wollina U. Mineral oil granuloma of the penis. J Eur Acad Dermatol Venereol 2003;17:585–7.
View Full Text | PubMed | CrossRef |
4. Cohen JL, Keoleian CM, Krull EA. Penile paraffinoma: self-injection with mineral oil. J Am Acad Dermatol 2002;47:S251–3.
5. Nyirády P, Kelemen Z, Kiss A, Bánfi G, Borka K, et al. Treatment and outcome of vaseline-induced sclerosing lipogranuloma of the penis. Urology 2008;71:1132–7.
PubMed | CrossRef |
6. Bullock TL, Brandes SB. Adult anterior urethral strictures: a national practice patterns survey of board certified urologists in the United States. J Urol 2007;177:685–90.
View Full Text | PubMed |
7. Hughes M, Blakely S, Nikolavsky D. Advancements in transurethral management of urethral stricture disease. Curr Opin Urol 2021;31:504–10.
8. Yildirim H, Hennus PM, Wyndaele MI, de Kort LM. Do previous urethral endoscopic procedures and preoperative self-dilatation increase the risk of stricture recurrence after urethroplasty?. Low Urin Tract Symptoms 2022;14:163–9.
9. Navarrete J, Cabrera R, Bunker CB, Agorio C. Dermoscopy of penile sclerosing granuloma. BMJ Case Rep 2021;14:e239846.
10. Albers DD, Hosty TA, Parkhurst JD, Mosca P, Barnes WF. Oil granuloma of the ureter. J Urol 1984;132:114.
11. Ali MS, McLaren CA, Rouholamin E, O'Connor BT. Ankle fractures in the elderly: nonoperative or operative treatment. J Orthop Trauma 1987;1:275–80.
Copyright: © The Author(s)(2022)
Asian Journal of Andrology25(1):147-148, Jan-Feb 2023.
Articles in PubMed by Zheng-Chao Zhang
Articles in Google Scholar by Zheng-Chao Zhang
Other articles in this journal by Zheng-Chao Zhang
Asian Journal of Andrology | Published by Wolters Kluwer – Medknow
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You are at:Home»News»Climate Measurement»NOAA allocates funding to support American Meteorological Society teaching programmes
NOAA allocates funding to support American Meteorological Society teaching programmes
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Teachers doing the 'hand-twist model' activity to better understand flow in high and low pressure systems The National Oceanic and Atmospheric Administration's (NOAA) Office of Education has earmarked US$2.3m to support a long-term partnership with the American Meteorological Society (AMS). NOAA's Environmental Literacy Programme provides grants for programmes that educate and inspire people to use Earth system sciences, and supports long-term partnerships that enable the education community to incorporate and deliver NOAA's latest scientific information on the topics of the ocean, coasts, weather and climate. Through this award, the AMS will provide professional development in atmospheric and ocean sciences to K-12 teachers from across the USA. The award will also enable the AMS to design curricula for introductory college-level Earth science courses, aimed to prepare pre-service teachers. In addition, it will allow the AMS to fund undergraduate scholarships and graduate fellowships that support workforce development and diversity in the fields of atmospheric, oceanic and hydrologic sciences.
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Helen has worked for UKi Media & Events for more than a decade. She joined the company as assistant editor on Passenger Terminal World and has since progressed to become editor of five publications, covering everything from aviation, logistics and automotive to meteorology. She has a love for travel and property and has redeveloped three houses in three years. When she's not editing magazines, she's running around after her two boys and their partner in crime, Pete the pug.
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Irish Tenors 25th Anniversary Tour Coming to Cleveland in March 2023
Cleveland Pops Orchestra will join the group for the performance
By Jeff Niesel on Tue, Dec 13, 2022 at 8:13 am
Courtesy of Playhouse Square
The Irish Tenors.
The Irish Tenors, a group that has performed at famous venues such as Radio City Music Hall, Madison Square Garden, Carnegie Hall and the Hollywood Bowl, will hit the road next year to celebrate its 25th anniversary.
As part of that tour, the Tenors will come to Cleveland on March 24 for a show at Connor Palace with the Cleveland Pops Orchestra.
A ticket presale begins at 10 a.m. on Thursday. Tickets for the Irish Tenors concert at Connor Palace go on sale to the general public at 10 a.m. on Friday.
Coming soon: Cleveland Scene Daily newsletter. We'll send you a handful of interesting Cleveland stories every morning. Subscribe now to not miss a thing. Follow us: Google News | NewsBreak | Instagram | Facebook | Twitter
Irish Tenors,
Connor Palace,
Cleveland Pops Orchestra
Jeff Niesel
Jeff has been covering the Cleveland music scene for more than 20 years now. And on a regular basis, he tries to talk to whatever big acts are coming through town, too. If you're in a band that he needs to hear, email him at [email protected]


Detective Inspector Sean Duffy returns for the incendiary sequel to The Cold Cold Ground.
"I'm ten years old. My Big Ben-style alarm clock has stopped working. Maybe I wound it too tightly, maybe it's just worn out. Whatever the reason, the alarm hasn't gone off in days.
The fourth in the DI Andy Horton marine mystery crime series, Dead Man's Wharf by Pauline Rowson, is published in China by Nanhai Publishing Company of Haikou.
Authors who are looking to increase their exposure should check out LosttoBooks. UK-based Sienna Logan's blog spotlights different genres.
I was one of the featured authors at a holiday party put on by the Reading Divas book club. The Wine & Cheese Holiday Gathering was more fun than business and a great way to end my writing year, and introduced me to the marvelous Overdue Recognition Art Gallery in Bowie, MD.
The new DI Andy Horton, number nine in the series, called Undercurrent, will be published in the UK and Commonwealth on 31 January and in the USA on 1 May 2013. Like all the DI Horton novelsit is set against the backdrop of the sea in the Solent area on the South Coast of England.
Tomorrow (Friday) I will crash the party on the Anything Goes blog talk radio show at 11pm (Eastern time). Bennett Pomerantz runs a fun and exciting show with literary guests. Set your alarm so you can tune in and join the fun at 11pm.


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Silicon Labs and Z-Wave Alliance opening Z-Wave to silicon and stack suppliers
December 23, 2019 By SP&T Staff
AUSTIN, Texas — Semi-conducter firm Silicon Labs and the Z-Wave Alliance have announced plans to open the Z-Wave Specification as a ratified, multi-source wireless standard available to all silicon and stack vendors for development.
With this change, semiconductor and software suppliers will be able to join the Z-Wave ecosystem, contribute to future advancements of the smart home standard, and develop and supply sub-GHz Z-Wave radio devices and software stacks. The Z-Wave Alliance will expand to a standards development organization for the Z-Wave Specification and will continue to manage the Z-Wave certification program, which will include software and hardware.
Expected to be available in the second half of 2020, the opened Z-Wave specification will include the ITU.G9959 PHY/MAC radio specification, the application layer, the network layer and the host-device communication protocol. Instead of being a single-source specification, Z-Wave will become a multi-source, wireless smart home standard developed by collective working group members of the Z-Wave Alliance.
The Z-Wave Alliance will maintain the certification program and expand the offering to provide technology vendors with both hardware and stack certification and product manufacturers with application layer certification.
"As a standards organization, the Z-Wave Alliance will help solve the interoperability challenges hindering the adoption of smart home devices," said Mitch Klein, executive director for the Z-Wave Alliance, in a prepared statement. "Members will work together on a single sub-GHZ connectivity solution that guarantees the forward-and-backward compatibility, interoperability, security and robustness needed to grow the IoT. The Z-Wave Alliance will collectively advance a fully realized smart home standard."
Both organizations say that by expanding access to Z-Wave as a standard supported by multiple vendors, the smart home ecosystem will benefit both from broader technology support as well as accelerated market adoption.
"Silicon Labs has worked to create positive alignment across the industry with the goal of advancing both security and compatibility in smart home devices," said Jake Alamat, vice-president and general manager of IoT home and consumer products at Silicon Labs, also in a statement. "Future success for the smart home industry relies on ecosystems getting closer, not farther apart. The smart home market opportunity is tremendous, and we want to help drive its success. When the ecosystems work together toward a common goal, the entire industry including manufacturers, developers, retailers and consumers benefit from this open collaboration."
Development on the opened Z-Wave specification will be managed by the new working groups in the Alliance in Q3 2020, and details on the silicon and stack platform certification program also will be announced in Q3.
Z-Wave Specification
Vunetrix promotes new director of sales
Genetec research shows cameras at risk of cyber attack due to outdated firmware


Good manufacturing practice" or "GMP" is part of a quality system covering the manufacture and testing of active pharmaceutical ingredients, diagnostics, foods, pharmaceutical products, and medical devices. GMPs are guidelines that outline the aspects of production and testing that can impact the quality of a product. Many countries have legislated that pharmaceutical and medical device companies must follow GMP procedures, and have created their own GMP guidelines that correspond with their legislation.
For more information, Kindly mail us on info@rbmcertifications.com or Contact us at +91 – 9870285089 to get your organization GMP certified in the most effective and efficient manner while realizing the true benefits of the certification using our specialized implementation methodology that is less time consuming, fast, easy to understand and implement, result oriented, time bound and cost effective.


Every day, staffing businesses send three million employees to work in America's offices, factories, hospitals, warehouses, and other work sites! Virtually every place that people work, staffing employees are on the job.
The staffing industry, comprised of more than 20,000 staffing firms and 16 million staffing employees, contributes nearly $136 billion to the U.S. economy through temporary and contract staffing, recruiting and permanent placement, outsourcing and outplacement, and human resource consulting.
Staffing is an essential industry that provides vital flexibility and support to businesses, and opportunities and flexibility for employees.Companies depend on staffing employees to get through busy seasons and to support major projects, new clients, and business growth. The flexibility staffing provides gives companies a competitive edge in matching resources and payroll to the needs at hand. More and more, companies are placing an increased premium on a more flexible and agile workforce and staffing plays a vital role in that strategy.
For employees, staffing offers a number of compelling benefits. At the top of that list is a bridge to permanent jobs?nearly nine out of 10 staffing employees say that temporary or contract work makes them more employable. One-half say it's a way to get a permanent job. One-third of temporary and contract employees were offered permanent positions by clients where they worked on assignments?two-thirds of those accepted the offers of employment.
Time has shown that staffing isn't just good for our economy; it is an essential industry that provides vital flexibility and support to businesses, and opportunities and flexibility for employees. And as the economy continues to evolve, with information and connectivity coming to dominate its core, the staffing, recruiting, and workforce solutions industry is prepared to provide the vital support businesses need to grow and prosper.


It's about where art comes from. How we can get it back if we've lost it. Or can we?
When you're writing a play about a real person, where do you start? At the day she's born? At the moment he becomes "significant?" The entry point not only dictates the shape of your script, but also underscores who you are as a playwright.
Consider Lauren Gunderson's Bauer, now at 59E59. Inspired by modernist painter Rudolf Bauer, it stages his final days, when his career and reputation had been negated by a contract dispute that left his work locked in the basement of the Guggenheim Museum.
"That's pretty horrible for any artist," Gunderson says. "So there was this terribly dark, natural ending. But I'm not a dark playwright. I don't want to leave the theatre feeling devastated. I want to feel like I'm leaning forward, not looking down."
So instead of wallowing in his failure, Gunderson's play uses the end of Bauer's life to test the tenacity of his creativity. "It's not a play about a contract," she says. "It's about where art comes from. Where it goes. How we can get it back if we've lost it. Or can we?"
Those questions are filtered through Bauer's connection to two real-life women: Hilla Rebay (a baroness and fellow artist who was both Bauer's lover and professional champion) and Louise Huber (who began as Bauer's maid but became his wife in 1944). In the play, Louise arranges a meeting between Hilla and Bauer after a long estrangement, and the result forces all of them to reckon with what they've become.
For Gunderson, those relationships capture how lust and love fuse with artistic impulses. "I got to play with the stickiness of jealousy and desire and people who still love despite a lifetime of battle," she says, adding that this reflects how Bauer and his contemporaries approached their work. "Their art was a spiritual undertaking. They thought of their job not as 'I'm gonna go paint this mountain' but as 'I'm gonna paint something unseen before.' That's an emotional state. That's fiery."
These collisions are fundamental to Gunderson's work, which has become a staple of theatres across the country. (She received five world-premiere productions last season alone.) "My plays often deal with the struggles around mortality and legacy," she says. "There's a sense, especially in my historic plays, of someone approaching their death---their end, their passing---and trying to find meaning or sense. And even trying to forgive themselves for what their lives were."
Crucially, her plays almost always have a fantastical element. Bauer's finale, for instance, leaps beyond reality when all three characters make a bold decision about their own creative output. Moments like that, Gunderson says, are "my dramatic way of ending plays that are impossible to end because they're about what happens next when you die. So it becomes, 'Alright, let's use the tools of the theatre to make something happen, something musical or dramatic or colorful. Something surprising."
She continues, "Ideally, the point is that you're hit with this 'whoa' feeling. It may be tinged with sadness or sorrow or levity or love, but there's a sense that something huge has happened. I'm certainly not successful all the time and not every production is successful, but usually at some point, you do get the sense of, 'Go big. Try to be bold.'"


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Best Driving Roads
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Brabus B45 – 450 | Review
3 April 2021	Redazione	News, Tuning
Words Christian Parodi / Photos Brabus
Halfway between being considered a very quick compromise and a practical sports car with five doors and five seats, the AMG A45 has retired its first generation and further moved its take on madness thanks to an increase in power to say the least astonishing. What is maintained is the compact look suitable for the family that in Comfort mode does not forget its origins and acts as a perfect partner for grocery and school runs as well as the trip back home, where you will surely not risk to arrive late. Starting from 360 horsepower and bringing the power to 381 with the restyling of the previous series, the second generation logically assumes the more sinuous shapes of the new A-Class, pulling out 421 horsepower from its 4-cylinder 2-liter.
An increase of 40 horsepower – for the A45 S – is pretty amazing, especially if we take into account that the body is basically that of a small-sized sedan and with a length of 4.4 meters and a weight of 1,550 kg, which combined with the permanent all-wheel drive also equipped with a self-locking differential that also promises some sort of "drift mode" and the 8-speed automatic transmission guarantees a more than realistic possibility of use, also and above all by less experienced drivers, than with AMGs of higher range would certainly have no way of reaching the same speeds with equal ease.
But where the AMG A45 S did not completely pleased us, at least in terms of driving involvement and for the promise kept only halfway in terms of traction and management of the same on the two axles, we are happy that the expert hands of Brabus, historic German tuner that for over forty years has left its mark on the cars of the three-pointed star, have worked their magic and given the smallest AMG in the list that grit that was lacking to be truly worthy of wearing its own badge. Called B45 – 450, where the "B" stands for Bottrop – the location where Brabus is based – the abbreviation leaves no room for imagination and reveals that the already overflowing power has been further increased and therefore brought to 450 horses. The 4-cylinder now has an even more impressive torque curve with an extra 50 Nm which therefore allows to deliver 550 Nm between 5,000 and 5,250 rpm. You will notice that despite the substantial increase in performance, the management of the control unit maintains the peaks at the original range, in such a way as not to distort the driving feel of the model that came out of the Affalterbach gates, but rather optimize the whole.
Of course, you can feel the extra horses and this definitely is the step that seemed insurmountable despite figures that on a hatchback sound exasperated. The violence during shifting in Sport mode and therefore Sport + becomes much more evident, with pleasant tugs that are discharged to the outside world through the new titanium exhaust system, another fundamental aspect that gives nerve to the sound that was really missing the point in a masterful 2-liters which, however, had to comply with the boring anti-pollution regulations that cut fun and decibels. We said that the delivery remains numerically similar to the original, obviously lowering the reaction times of the car once the throttle is pressed hard. Now you go from 0 to 100 kph in 3.7 seconds and no longer in 4, while top speed remains unchanged, 270 per hour electronically limited.
But the optimization made on the ECU, which comes home together with a 3-year or 100,000 km warranty, is not the only modification that makes the B45 – 450 the malignant doppelganger of the A45 S, because part of the dynamic improvement also goes to be attributed to the suspension, adjustable in a range between 0 and 30 mm, while the new 20-inch Brabus Monoblock Z wheels are beautiful to look at and transmit every slight bump or imperfection of the road straight to the steering. Not a package of improvements that make the AMG A45 S a better car, but a series of targeted actions that lead it to be what we would have expected, especially with an engine that today represents the absolute reference point in terms of performance-focused 4 cylinders.
The product offered by Brabus does not neglect the attention to details even in terms of aesthetics, with more carbon fiber, more marked spoilers and the classic B standing out in the middle of the tailgate and in the front grille, which has two red profiles, also taken up with the brake calipers. In the cockpit, regardless of whether the customer can really have it made according to each own taste, we find Brabus logos (not on the steering wheel though) illuminable in 64 different colors via the multimedia system, but in the best tradition of the German firm, nothing is upset and more generally the same mood of a traditional A45 is perceived, apart from the fact that once on the move, it is really easy to realize that you are dealing with a substantially evolved piece of kit.
Aesthetically more aggressive, dynamically sharpened and ultimately more convincing than the starting model has been able to do in 8 years, the B45 – 450 is what I would call the highest step in the hatchback universe, where the A45 S undresses the clothes of a small AMG and wears those of a small supercar. A subtle difference, you might argue, but Brabus has great experience and shows that for standing out in the immense tuning panorama you don't need absurd widebody kits, nor even obscene marble-like paint. What matters is precisely what has been infused into an upgrade that does not distort the image of a pleasing five-door suitable for young people and those who are beginning to have a pronounced receding hairline, but which lends supercar figures to the world of hot-hatches such as no one had ever dared to do until now and all this accompanied by more vivid driving feedback, abrupt if you like, but indispensable when dealing with a car to which the definition of "smallest AMG" is out of place. Gentlemen, we are now facing one of the best AMGs ever seen.
BRABUS B45 – 450
Engine 4 cylinder turbocharged, 1.991 cc Power 450 hp @ 6.750 rpm Torque 550 Nm @ 5.000-5.250 rpm
Traction All-Wheel-Drive Transmission 8-Speed Automatic Gearbox Weight 1.560 kg
0-100 kph 3,7 sec Top Speed 270 kph (limit.)
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The Rockland After School Program at Kakiat Elementary was facing shutdown but was saved just as students in East Ramapo were heading back to class.
A longtime after-school program facing shutdown was saved just as students in East Ramapo were heading back to class this week to start a new school year.
On Wednesday, the school board approved a lease agreement keeping the Rockland After School Program at Kakiat Elementary, which helped ease concerns from parents unsure of where they'd send their children each afternoon. Earlier that day, students across East Ramapo's 14 schools returned to class.
EARLIER COVERAGE: Will after school program be saved in time?
"We are grateful that (Superintendent Deborah) Wortham, the board and RASP were able to find a solution so it can remain open," Triny Hertzberg, the program's founder and director, said Thursday.
Last year, 80 children attended the East Ramapo RASP program.
For more than three decades, RASP has served thousands of children by offering them a place to spend time with friends, play games or get a jump start on homework. However, Hertzberg was uncertain what would happen in September after she was unable to pay the remaining balance of rent owed for the two rooms RASP uses at Kakiat.
Up until 2013, RASP was offered in Nanuet, but the district's family resource center took it over to help fill a void after the schools lost some county funding. Losing income from Nanuet was a blow to the East Ramapo program since it helped pay the rent at Kakiat, Hertzberg said.
Chris Bottini, a district spokesman, said Wortham, the school board and RASP worked together to create a plan "that would be both affordable and sustainable" going forward.
The agreement calls for a reduction in how much RASP must pay in order to lease the space, according to Hertzberg. She declined to provide an exact dollar figure.
"We hope that this is emblematic of increased understanding and cooperation among all members of our beloved community. We look forward to enrolling new students and welcoming home our wonderful children," she said.
"I'm happy," said Alexandria Manigo, one of the parents who called the district earlier this week, urging them to do whatever it could to keep RASP intact.
After her son was not selected during a lottery for a seat in the district's newly-expanded full-day kindergarten program, Manigo said she and her husband, both of whom work full-time, need RASP. Their son will be attending a half-day program until the district's planned roll-out of a universal full-day kindergarten later this month.
With so many families looking to plug holes left by not having access to a full-day program, Hertzberg said RASP is offering a "Kindergarten Club" from 8 a.m. to noon on school days.
"It can help fill some of the gaps for working families because it will give them a place to send their kids before afternoon sessions of kindergarten," Hertzberg said. "There are a lot of families struggling with what to do in the meantime."
Hertzberg anticipated that the program would return on Monday and said that registration is being accepted on a rolling basis. Over the last few weeks, many parents made other arrangements for after-school care since the program's fate was undetermined, Hertzberg said.
To register or receive more information about RASP, call 845-577-6141.


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Cell Signaling Mediating Critical Radiation Responses View all 4 Articles
Mary Helen Barcellos-Hoff
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Front. Oncol., 14 November 2019 | https://doi.org/10.3389/fonc.2019.01244
FAM53A Affects Breast Cancer Cell Proliferation, Migration, and Invasion in a p53-Dependent Manner
Jie Zhang, Mingfang Sun, Miaomiao Hao, Kexin Diao, Jian Wang, Shiping Li, Qixue Cao and Xiaoyi Mi*
Department of Pathology, College of Basic Medical Sciences, First Affiliated Hospital, China Medical University, Shenyang, China
Family with sequence similarity 53-member A (FAM53A) is an uncharacterized protein with a suspected but unclear role in tumorigenesis. In this study, we examined its role in breast cancer. Immunohistochemical staining of specimens from 199 cases of breast cancer demonstrated that FAM53A levels were negatively correlated with p53 status. In the p53 wild-type breast cancer cell line MCF-7, FAM53A overexpression inhibited cell migration, invasion, and proliferation, downregulated the expression of Snail, cyclin D1, RhoA, RhoC, and MMP9, and decreased mitogen-activated protein kinase kinase (MEK) and extracellular-signal regulated kinase (ERK) phosphorylation. Concurrently, it upregulated E-cadherin and p21 expression levels. Interestingly, opposite trends were observed in the p53-null breast cancer cell line MDA-MB-231. The MEK inhibitor PD98059 reduced the biological effects of FAM53A knockdown in MCF-7 cells and FAM53A overexpression in MDA-MB-231 cells, suggesting that FAM53A affects breast cancer through the MEK-ERK pathway. Silencing TP53 in MCF-7 cells and stably expressing wild-type p53 in MDA-MB-231 cells confirmed that the effects of FAM53A signaling through the MEK/ERK pathway depended on the p53 status of the cells. These results suggest that FAM53A acts as a tumor suppressor in p53-positive breast cancer by modulating the MEK-ERK pathway, but may be a potential candidate for targeted anticancer therapies in p53-negative breast cancer.
The mitogen-activated protein kinase kinase (MEK)-extracellular-signal regulated kinase (ERK) pathway is an evolutionarily conserved signal transduction pathway of crucial importance in tumorigenesis. It is often aberrantly activated in malignant tumors, resulting in signal amplification during tumor invasion and metastasis (1, 2). Abnormal activation leads to loss of differentiation and apoptosis and increased proliferation and invasion, causing tumorigenesis and eventual metastasis (3–6). The MEK-ERK pathway is activated at the cell membrane by Ras, which activates Raf, starting a phosphorylation cascade that results in the sequential activation of MEK1/2 and ERK1/2 (7–12). ERK expression is significantly higher in breast cancer tissue than in benign hyperplastic breast tissue, and ERK phosphorylation is increased in severe atypical hyperplasia and breast cancer tissues compared with that in benign proliferating breast tissue, suggesting that abnormally increased ERK activation plays an important role in the development of atypical hyperplasia into cancer, and can stimulate the proliferation of breast cancer cells (13). Phosphorylated ERK (p-ERK) enters the nucleus to phosphorylate specific transcription factors such as c-Myc and c-Jun (14). Decreased p-ERK reduces the expression of matrix metalloprotease (MMP)1 and MMP9, significantly inhibiting breast cancer invasiveness (14, 15). In breast cancer cells, ERK inactivation is accompanied by the inactivation of cyclin D1 and BCL2, leading to apoptosis (15).
Family sequence similarity (FAM) genes are families of uncharacterized genes of similar protein sequence. Several of these families have been linked to the development of tumors, including breast cancer, non-small cell lung cancer, lung adenocarcinoma, renal cell carcinoma, prostate cancer, colorectal cancer, and esophageal squamous cell carcinoma, where they are thought to play important roles in proliferation, apoptosis, migration, and invasion (16–23). FAM53 is a vertebrate-specific family of proteins that bind to transcriptional regulators of proliferation and neural tube development, encoded by three homologous genes: FAM53A, FAM53B, and FAM53C (24–27). FAM53A, also known as dorsal neural tube nuclear protein, is thought to play an important role in neurodevelopment by specifying the fate of dorsal cells within the neural tube (28, 29). Expression quantitative trait loci variants of FAM53A identified in TP53-based interaction analysis are associated with the use of therapeutic doxorubicin in breast cancer (27). In the triple-negative TP53-missense mutant breast cancer cell line MDA-MB-231, downregulation of FAM53A increased doxorubicin resistance. However, in the luminal B p53-truncated mutant line MDA-MB-361 and the luminal A p53-wild-type line MCF7, downregulation of FAM53A resulted in increased sensitivity to doxorubicin (27). The role of FAM53A in breast cancer is unclear, and its relevance to the clinical pathology of breast cancer has not been reported. In this study, we examined the expression and localization of FAM53A in breast cancer tissues and cell lines. We then altered FAM53A expression in two breast cancer cell lines to explore its effects on the cells and gain mechanistic insight into how FAM53A affects cancer.
Patients and Specimens
Primary tumor specimens were obtained from 199 patients diagnosed with invasive ductal carcinoma who underwent complete resection in the First Affiliated Hospital of China Medical University between 2011 and 2013. Patients whose tissue samples were used in this research provided written informed consent. This study was approved by the local institutional review board of China Medical University.
MCF-10A, MCF-7, T47D, MDA-MB-231, BT-474, and BT-549 cell lines were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and identified by short tandem repeat (STR) DNA analysis. The cells were cultured and frozen, and experiments were performed after 10 passages. MCF-10A cells were cultured in 1:1 Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco, Waltham, MA, USA) supplemented with 5% horse serum, 10 μg/mL insulin (Sigma-Aldrich Co, St. Louis, MO, USA), and 20 ng/mL epidermal growth factor (EGF). MDA-MB-231 cells were cultured in L15 (Gibco) supplemented with 10% fetal bovine serum (FBS). MCF7 and T47D cell lines were cultured in DMEM (Gibco) supplemented with 10% FBS. BT-474 and BT549 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 (Gibco) supplemented with 10% FBS. Cells were cultured in sterile culture flasks in a 5% CO2 incubator at 37°C and subcultured every 2 days by trypsinization.
Surgically removed tumor specimens were fixed in 10% neutral formalin, embedded in paraffin, and continuously cut into 4-μm thick sections. The tissue slices were baked in an oven at 70°C for 2 h; then the sections were dewaxed in xylene, absolute ethanol, gradient alcohol, and distilled water, and boiled in 0.01 M citrate buffer (pH 6.0) at high temperature and high pressure for 2 min. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide and sections were incubated with 5% normal goat serum for 30 min at 20°C to reduce non-specific binding. Tissue sections were then incubated with FAM53A antibody (1:100 dilution; see Table S1 for information on antibodies used in the study) overnight at 4°C. The reaction was observed using an Elivision super HRP (mouse/rabbit) immunohistochemistry kit (Maixin-Bio, Shenzhen, China) and 3,3′-diaminobenzidine (DAB). The nuclei were counterstained with hematoxylin.
FAM53A expression levels were evaluated based on the percentage of positive cells (PP) and the staining intensity (SI) within the whole tissue section. FAM53A staining intensity was evaluated semi-quantitatively using the immune response score (IRS) and calculated as follows: IRS = PP × SI; where PP: 0, no dye; 1, 1–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%; and SI: 0, no staining; 1, weak staining; 2, medium staining; and 3, strong staining. The scores for each tumor sample were multiplied to give a final score of 0–9; tumor samples with scores >3 were classified as having high FAM53A expression, while samples with scores ≤3 were classified as having low FAM53A expression.
Plasmid Transfection, siRNA Interference, and Inhibitor Treatments
Transfection was performed using Xfect Transfection Reagent (Takara Bio USA, Mountain View, CA, USA) according to the manufacturer's instructions. The plasmids pCMV6-ddk-myc and pCMV6-ddk-myc-FAM53A were purchased from Origene (Rockville, MD, USA). One day before transfection, cells were plated in 2 mL of complete growth medium, aiming for 50–70% confluency at the time of transfection. Plasmid DNA (5 μg) was added to Xfect Reaction Buffer to a final volume of 100, and 1.5 μL Xfect Polymer was added. After a 10 min incubation at room temperature (15–25°C), the solution was added dropwise to the cell culture medium. Cells were incubated at 37°C for 4 h, then the medium was replaced with 2 mL of fresh complete growth medium and the cells were incubated at 37°C for 48 h. FAM53A (sc-88998) and non-targeted control (NC; sc-37007) siRNAs were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and transfected into cells using HiPerFect Transfection Reagent (Qiagen, Hilden, Germany) according to the manufacturers' protocols. Cells were plated in 2 mL of complete growth medium, aiming for 30–50% confluence at the time of siRNA transfection. The next day, 37.5 ng of siRNA was added to 100 μL of serum-free culture medium, 3 μL of HiPerFect Transfection Reagent was added, and the solution was mixed by vortexing and incubated for 5–10 min at room temperature to allow the formation of transfection complexes. The complexes were then added to the cells, which were incubated for 48 h prior to analysis.
PD98059, a MEK inhibitor, was purchased from Selleck Chemicals (Houston, TX, USA). A stock solution was generated by dissolving PD98059 in dimethyl sulfoxide (DMSO). This solution was added to MCF-7 and MDA-MB-231 cells at final concentrations of 10 μM for 1 h and 25 μM for 2 h, respectively, prior to analysis.
Breast cancer cells cultured in 24-well plates for 24 h were fixed in 2% paraformaldehyde for 15 min, blocked in 5% BSA for 2 h, and incubated with anti-FAM53A antibody (1:100) at 4°C overnight. Cells were then incubated with a tetramethylrhodamine-labeled secondary antibody at room temperature for 2 h in the dark; the nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Images were captured using an Olympus FV1000 laser scanning confocal microscope (Olympus, Tokyo, Japan).
Cells and tumor tissues were lysed in lysis buffer (Thermo Fisher Scientific), and total protein was quantified using the Bradford method. Proteins (80 μg/lane) were separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were incubated overnight at 4°C with the appropriate primary antibody (Table S1) and for 2 h at room temperature with horseradish peroxidase-labeled secondary antibody, then visualized with ECL (Thermo Fisher Scientific, Waltham, MA, USA) on a BioImaging System (UVP Inc., Upland, CA, USA). Protein levels were analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA), with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control.
Cell Proliferation and Colony Formation Assays
Cell viability was measured by the mitochondrial reduction of 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Cells (3,000/well) were seeded in 96-well plates in medium containing 10% FBS and have used a separate blank (100 ml of fresh medium). MTT solution (10 μL/well) was added and samples were incubated for 4 h. Then, the medium was aspirated from each well, and the obtained MTT formazan was dissolved in 150 μL DMSO. Finally, the absorbance was measured at 490 nm using a microplate reader daily, and the OD value of the blank was subtracted from the absorbance at a given time. We calculated the relative ratio and plotted the cell curve.
For colony formation experiments, cells (1,000/dish) were seeded in 40-mm dishes and incubated for 10–15 days in medium containing 10% fetal calf serum, which was changed every 3 days. After incubation, the cells were washed with PBS and stained with hematoxylin, and the number of colonies with >50 cells were counted. At least three independent experimental replicates were performed.
Cell Migration and Invasion Assays
Cell migration and invasion experiments were performed using 24-well Transwell chambers (Costar, Cambridge, MA, USA) with a pore size of 8 μm. For the invasion assay, the upper chamber of the Transwell chamber was coated with 100 μL Matrigel (1:9 dilution; BD Biosciences); for cell migration assays, no Matrigel was added. Approximately 24 h after transfection, the cells were trypsinized, and 1 × 105 cells in 100 μL of medium supplemented with 2% FBS were transferred to the upper chamber. Medium supplemented with 10% FBS was added to the lower chamber as a chemoattractant. After 18 h of incubation, the chamber was removed and stained with hematoxylin, and cells that migrated through the chamber membrane were counted using an Olympus FV1000 laser-scanning confocal microscope (Olympus, Tokyo, Japan). At least three independent experimental replicates were performed.
Correlations between FAM53A expression and clinicopathological features of breast cancer were analyzed using the chi-squared test. For experiments involving cells, differences between the control and experimental groups were compared by paired t-test. P < 0.05 was considered statistically significant.
FAM53A Expression in Breast Cancer Cells Is Associated With p53
We examined the localization of FAM53A in the breast cancer cell lines MCF-7, T47D, MDA-MB-231, and BT-549 and the non-malignant human mammary epithelial cell line MCF-10A by immunofluorescence and observed its presence in the cytoplasm and nucleus (Figure 1A). FAM53A was expressed at significantly lower levels in the p53-wild-type breast cancer cell line MCF-7 compared with the normal human mammary epithelial cell line MCF-10A, whereas in the p53-mutant breast cancer cell lines T47D, MDA-MB-231, BT-549, and BT-474, FAM53A was highly expressed, particularly in MDA-MB-231 cells (Figure 1B). We therefore selected MCF-7 and MDA-MB-231 cells for subsequent experiments. To investigate the association between FAM53A expression and clinicopathological features of breast cancer, we selected 199 breast cancer tissues for immunohistochemical staining. As shown in Table 1, FAM53A levels were negatively correlated with wild-type p53 (P < 0.001; Figures 1C,D), but had no significant correlation with age (P = 0.781); tumor size (P = 0.110); TNM stage (P = 0.056); lymph node metastasis (P = 0.996); or estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2 (HER-2) status (P = 0.069). Therefore, we next examined the effects of modulating FAM53A levels in the presence and absence of functional p53.
Figure 1. FAM53A expression in breast cancer cell lines and tissues. (A) FAM53A expression in breast cancer cell lines was analyzed by immunofluorescence. (B) FAM53A protein levels in five breast cancer cell lines and a normal human mammary epithelial cell line (MCF-10A) were assessed by western blotting. (C,D) The relationship between FAM53A expression and p53 levels in breast cancer tissues was analyzed by immunohistochemistry.
Table 1. Correlation between FAM53A expression and clinicopathological characteristics of invasive breast cancer.
FAM53A Inhibits Proliferation, Migration, and Invasion in the p53-Wild-Type Breast Cancer Cell Line MCF-7
To examine the effects of FAM53A expression on tumorigenesis-related processes in a wild-type p53 cell line, FAM53A was both overexpressed and depleted in MCF-7 cells (Figure 2A). When FAM53A was overexpressed, colony formation ability and proliferation were significantly decreased; accordingly, FAM53A depletion resulted in increased colony formation ability and proliferation (Figures 2B,C). We also performed western blot analysis to measure the levels of several proteins related to proliferation. When FAM53A was overexpressed, cyclin D1, cyclin-dependent kinase 4 (CDK4), and c-Myc expression was downregulated, while p21 expression was increased; FAM53A depletion resulted in the opposite effects on these proteins (Figure 2D). FAM53A overexpression inhibited the migration and invasion of MCF-7 cells, while FAM53A depletion promoted these processes (Figure 2E). Changes in FAM53A protein levels also affected the expression of proteins involved in cell migration and invasion, with decreased RhoA, RhoC, Rho kinase 1 (ROCK1), and MMP9 expression and increased RhoB expression after FAM53A overexpression. Expression of RhoA, RhoC, ROCK1, and MMP9 correspondingly increased after FAM53A depletion, along with decreased RhoB expression (Figure 2F). These results suggest that FAM53A inhibits proliferation, migration, and invasion in the presence of wild-type p53.
Figure 2. FAM53A inhibits proliferation, migration, and invasion in p53-wild-type breast cancer cells. (A) MCF-7 cells were transfected with FAM53A-specific (si-FAM53A) or control (NC) siRNA, or with a FAM53A expression (p-FAM53A) or vehicle (p-NC) plasmid. Western blotting was performed to evaluate transfection and silencing efficiency. (B) Representative images and quantification of colony formation assays. (C) MTT assay results. (D) Expression changes in proliferation-related proteins. (E) Cell invasion and migration assays. (F) Expression of proteins associated with cell migration and invasion. For all panels, *P < 0.05; **P < 0.01.
FAM53A Promotes Proliferation, Migration, and Invasion in the p53-Mutant Breast Cancer Cell Line MDA-MB-231
FAM53A overexpression and depletion was also performed in MDA-MB-231 cells (Figure 3A). In this cell line, overexpression of FAM53A increased colony formation ability and proliferation, while depletion had the opposite effects (Figures 3B,C). The expression of proteins important for proliferation showed opposite trends with FAM53A modulation to those observed in MCF-7 cells; namely, FAM53A overexpression increased cyclin D1, CDK4, and c-Myc levels, while decreasing p21 levels. The depletion of FAM53A expression resulted in decreased cyclin D1, CDK4, and c-Myc and increased p21 (Figure 3D). FAM53A overexpression promoted migration and invasion in MDA-MB-231 cells, while depletion inhibited these processes (Figure 3E). FAM53A overexpression increased RhoA, RhoC, ROCK1, and MMP9 expression and decreased RhoB expression, and the opposite trends were observed with FAM53A depletion (Figure 3F). Taken together, the above results indicate that FAM53A has opposing effects on proliferation, migration, and invasion of breast cancer cell lines with wild-type and mutated p53.
Figure 3. FAM53A promotes proliferation, migration, and invasion in p53-mutant breast cancer cells. (A) MDA-MB-231 cells were transfected with FAM53A-specific (si-FAM53A) or control (NC) siRNA, or with a FAM53A expression (p-FAM53A) or vehicle (p-NC) plasmid. Western blotting was performed to evaluate transfection and silencing efficiency. (B) Representative images and quantification of colony formation assays. (C) MTT assay results. (D) Expression changes in proliferation-related proteins. (E) Cell invasion and migration assays. (F) Expression of proteins associated with cell migration and invasion. For all panels, *P < 0.05; **P < 0.01.
FAM53A Differentially Affects the Expression of Epithelial-Mesenchymal Transition (EMT)-Related Proteins
EMT has dramatic effects on tumor cell behavior, as it results in changes in cell polarity and the formation of tight junctions between cells, a gradual loss of adhesion leading to cell invasiveness and migration, and the expression of a large number of extracellular matrix components that affect proliferation, migration, and invasion. As the above experiments demonstrated that FAM53A affects the proliferation, migration, and invasion of breast cancer cell lines, we next explored whether FAM53A regulation is a potential inhibitory mechanism against EMT by examining the expression of EMT-related proteins after modulating FAM53A levels. FAM53A overexpression in MCF-7 cells increased ZO-1 and E-cadherin expression and decreased zinc finger E-box binding homeobox 1 (ZEB1), N-cadherin, and vimentin expression; depletion of FAM53A had the opposite effects on these proteins (Figure 4A). We also examined the expression of EMT-related proteins in MDA-MB-231 cells. FAM53A overexpression decreased the expression of ZO-1 and E-cadherin and increased the expression of ZEB1, N-cadherin, and vimentin, while FAM53A depletion had the opposite effects (Figure 4B).
Figure 4. FAM53A regulates the levels of EMT-related proteins. (A,B) Expression changes in EMT-related proteins in MCF-7 and MDA-MB-231 cells, respectively. E-Ca, E cadherin; N-Ca, N cadherin. *P < 0.05; **P < 0.01.
FAM53A Regulates Breast Cancer Cells Through the MEK/ERK Signaling Pathway
We next sought to gain mechanistic insight into the effects of FAM53A on breast cancer cells. FAM53B, a homolog of FAM53A, is involved in the regulation of Wnt/β-catenin signaling and β-catenin nuclear localization (26). However, a Wnt/β-catenin signaling pathway-related protein assay found no significant changes upon modulation of FAM53A levels (Figure S1). The MEK/ERK signaling pathway is crucial for the proper regulation of proliferation, migration, and invasion, so we next examined the effects of FAM53A on MEK/ERK activation. In MCF-7 cells, FAM53A overexpression resulted in decreased p-MEK1/2 and p-ERK1/2, indicating decreased pathway activation. Conversely, FAM53A depletion significantly increased the phosphorylation of MEK1/2 and ERK1/2 (Figure 5A). Modulation of FAM53A levels had no effect on EGF receptor (EGFR), Ras, Raf, MEK1/2, or ERK1/2 levels, nor did it affect EGFR phosphorylation.
Figure 5. FAM53A regulates MCF-7 cells via the MEK/ERK signaling pathway. MCF-7 cells transfected with control or FAM53A siRNA were treated with or without PD98059 and analyzed for (A) changes in the expression and activation of MEK/ERK signaling proteins, (B) colony formation ability, (C) migration and invasion, and (D) expression of proteins involved in cell proliferation, migration, and invasion. For all panels, *P < 0.05; **P < 0.01. E-Ca, E cadherin; N-Ca, N cadherin.
To confirm that FAM53A functions through the MEK/ERK signaling pathway, we employed the specific MEK/ERK pathway inhibitor PD98059. While FAM53A depletion significantly activated MEK/ERK signaling, PD98059 blocked this activation. PD98059 treatment also blocked the increased colony formation activity observed after FAM53A depletion, and rescued the increased migration and invasion, confirming that FAM53A regulates MCF-7 cells through the MEK/ERK signaling pathway (Figures 5B,C). We also examined the expression of proliferation- and EMT-related proteins with PD98059 treatment and found that MEK/ERK inhibition restored protein levels to those observed without FAM53A depletion (Figure 5D). These experiments demonstrate that FAM53A inhibits proliferation, migration, and invasion and negatively regulates the EMT in p53 wild-type breast cancer cells through the MEK/ERK pathway.
In MDA-MB-231 cells, MEK1/2 and ERK1/2 phosphorylation increased with FAM53A overexpression and decreased with its depletion, while EGFR, Ras, Raf, MEK, and ERK levels did not significantly change (Figure 6A). PD98059 rescued the increased colony formation caused by FAM53A overexpression, as well as the increased migration and invasion, confirming that the effects of FAM53A are mediated through MEK/ERK signaling in this cell line as well (Figures 6B,C). PD98059 also reversed the effects of FAM53A overexpression on the expression of proliferation- and EMT-related proteins (Figure 6D).
Figure 6. FAM53A regulates MDA-MB-231 cells via the MEK/ERK signaling pathway. MDA-MB-231 cells were transfected with control or FAM53A overexpression plasmids, treated with or without PD98059, and analyzed for (A) changes in the expression and activation of MEK/ERK signaling proteins, (B) colony formation ability, (C) migration and invasion, and (D) expression of proteins involved in cell proliferation, migration, and invasion. For all panels, *P < 0.05; **P < 0.01. E-Ca, E cadherin; N-Ca, N cadherin.
The Opposing Effects of FAM53A on MCF-7 and MDA-MB-231 Cells Depend on Their p53 Status
Interestingly, our results indicated that FAM53A has opposite effects on the MCF-7 and MDA-MB-231 cell lines. As the only positive correlation between FAM53A expression and the clinicopathological characteristics of invasive breast cancer was p53 status (Table 1), we next investigated whether the differential effects of FAM53A in these lines was due to the presence or absence of functional p53. An MCF-7 cell line with stable expression of a p53 shRNA was generated, which displayed decreased FAM53A expression compared with the parental line (Figure 7A). Depletion of p53 reversed the trends observed upon FAM53A depletion in the parental line, decreasing MEK/ERK activation (Figure 7B), colony formation (Figure 7C), and migration and invasion (Figure 7D).
Figure 7. The opposing effects of FAM53A on MCF-7 and MDA-MB-231 cells depend on their p53 status. (A) An MCF-7 cell line with stable depletion of wild-type p53 (shWTp53-7) was constructed and evaluated for (B) changes in the expression and activation of MEK/ERK signaling proteins, (C) colony formation ability, and (D) migration and invasion. (E) An MDA-MB-231 cell line stably expressing wild-type p53 (WTp53-231) was constructed, transfected with control or FAM53A overexpression plasmids, and evaluated for (F) changes in the expression and activation of MEK/ERK signaling proteins, (G) colony formation ability, and (H) migration and invasion. For all panels, *P < 0.05; **P < 0.01.
Next, we constructed an MDA-MB-231 cell line stably expressing wild-type p53 (Figure 7E) and subjected it to the same analysis. FAM53A overexpression in WTp53-231 cells inhibited MEK/ERK activation (Figure 7F), colony formation (Figure 7G), and migration and invasion (Figure 7H).
While FAM53A has yet to be directly linked to tumorigenesis, studies have shown that members of the FAM53 protein family bind to transcriptional regulators that regulate cell proliferation, suggesting potential effects on cancer development. To the best of our knowledge, this is the first demonstration of the relevance of FAM53A expression in cancer. The results indicate that FAM53A levels are negatively correlated with wild-type p53, suggesting a link between the role of FAM53A in breast cancer and p53 status.
FAM53A was shown to affect the sensitivity of breast cancer cell lines to doxorubicin, with opposing effects on breast cancer cell lines with different p53 status (27). However, the effects of FAM53A on breast cancer cells and their related mechanisms were unknown. Our study demonstrates that overexpression of FAM53A reduces proliferation, migration, and invasion ability in the p53-wild-type breast cancer cell line MCF-7, but promoted these abilities in the p53-mutant line MDA-MB-231. We had also selected the p53-wild-type lung cancer cell line A549 and the p53-mutant breast cancer cell line SK-BR3, the results are consistent with the previous conclusions (Figure S2). Mechanistically, the results indicate that FAM53A affects cell proliferation by regulating the p21-cyclin D1-CDK4 signaling axis. In addition, FAM53A may affect the migration and invasion of breast cancer cells by regulating the expression of RhoA, RhoB, RhoC, ROCK1, and MMP9. Through these proteins, FAM53A could drastically affect the behavior of malignant cells.
EMT occurs during tumor invasion and metastasis and represents an important milestone in tumor progression. EMT is mainly characterized by loss of epithelial cell polarity, loss of adhesion between cells, and acquisition of mesenchymal cell characteristics, accompanied by enhanced migration ability (30, 31). Our results indicate that FAM53A affects several key proteins in EMT. Moreover, these effects were consistently opposite between the p53-positive and -negative cells.
Our result demonstrated that FAM53A inhibits MEK and ERK activity in p53 wild-type breast cancer cells, but activates these enzymes in p53-mutant cells. The use of PD98059 to antagonize ERK activation after FAM53A overexpression or depletion indicated that FAM53A affects breast cancer proliferation, migration, and invasion via the MEK/ERK signaling pathway. Further mapping of this pathway will allow more precise determination of the mechanism of FAM53A action.
As a key tumor suppressor gene involved in the regulation of the cell cycle, apoptosis, senescence, and DNA repair, somatic TP53 mutations are estimated to occur in 20–30% of cancer cases, including breast cancer (31, 32). The prognosis of breast cancer is closely related to lymph node metastasis, which TP53 mutations can be used clinically to predict (32). Importantly for breast cancer, TP53 status may be associated with estrogen receptor (ER), HER-2, and Ki-67 status, with important synergistic or regulatory effects (33, 34). When TP53 is mutated, normal regulation of these biomarkers is lost, promoting lymph node metastasis. The effects of FAM53A levels on the sensitivity of breast cancer to doxorubicin is correlated with p53 status (27), and our previous immunohistochemical staining demonstrated that FAM53A colocalizes with p53. In this study, FAM53A inhibited proliferation, migration, and invasion by inhibiting MEK/ERK signaling in p53 wild-type cells, while in p53 negative breast cancer cells, FAM53A activated MEK/ERK signaling, promoting these behaviors. This suggests that the role of FAM53A is reversed with loss of p53. We used immunoprecipitation experiments to test for protein-protein interaction between FAM53A and p53 and did not detect one (data not shown), suggesting an indirect regulatory link between FAM53A and p53. This relationship will require further exploration. It is important to note that in addition to their p53 status, MCF-7 and MDA-MB-231 cells differ in the expression of the ER, K-Ras, and other important cancer signaling proteins, and whether these factors affect the biological behavior of FAM53A in breast cancer remains to be determined.
Our understanding of the role of FAM53A in cancer tumorigenesis and progression is limited. Taken together, our study demonstrates for the first time that FAM53A affects the proliferation, migration, and invasion of breast cancer cells in a p53-dependent manner. These findings suggest that FAM53A has broad prospects in cancer research, particularly in P53 wild-type and P53 mutant breast cancer.
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.
Patients whose tissue samples were used in this research provided written informed consent. This study was approved by the local institutional review board of China Medical University.
XM, MS, MH, KD, and JZ contributed conception and design of the study. JZ, MS, MH, JW, SL, and QC performed experiment and the statistical analysis. JZ and MS wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.
This work was supported by the National Natural Science Foundation of China (grant no. 81572615 to XM).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2019.01244/full#supplementary-material
Table S1. Antibodies used for western blotting.
Figure S1. FAM53A does not regulate MCF-7 and MDA-MB-231 cells via the Wnt/β-catenin pathway. (A) In MCF-7 cells, up- or down-regulation of FAM53A levels did not significantly change β-catenin and glycogen synthase kinase 3β levels. (B) In MDA-MB-231 cells, changes in Wnt/β-catenin pathway-related proteins did not show any clear trends.
Figure S2. FAM53A has the opposite effect on A549 and SK-BR3. In the p53-wild-type lung cancer cell line A549(ABC), FAM53A inhibits the ERK pathway and inhibits its proliferation, migration and invasion. While in the p53-mutant breast cancer cell line SK-BR3(ABC), FAM53A promotes the ERK pathway and promotes its proliferation migration and invasion.
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Keywords: breast cancer, cell migration, epithelial-mesenchymal transition (EMT), invasion, metastasis, p53, extracellular-signal-regulated kinase (ERK)
Citation: Zhang J, Sun M, Hao M, Diao K, Wang J, Li S, Cao Q and Mi X (2019) FAM53A Affects Breast Cancer Cell Proliferation, Migration, and Invasion in a p53-Dependent Manner. Front. Oncol. 9:1244. doi: 10.3389/fonc.2019.01244
Received: 10 August 2019; Accepted: 29 October 2019;
Published: 14 November 2019.
Mary Helen Barcellos-Hoff, University of California, San Francisco, United States
Hanumantha Rao Madala, Harvard Medical School, United States
Ali Vaziri-Gohar, Case Western Reserve University, United States
Copyright © 2019 Zhang, Sun, Hao, Diao, Wang, Li, Cao and Mi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Xiaoyi Mi, xymi@cmu.edu.cn
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Q: Pausing a loop to run another in Arduino I'm working on a circuit that has two separate 4-bit binary counters with LEDs. I press a button and one counter begins counting to 15 in binary. I press a second button and the first counter pauses where it is and the second group of LEDs begin counting to 15 in binary. I got both counters working, but I can't get the first group to pause and the second to begin. I've tried using if statements with a boolean flag, but it messes up the first group of LEDs. How can I get ledPins1[] to pause when button2 is pressed, then resume when ledPins2[] finish?
int ledPin1[] = {2,3,4,5};
int ledPin2[] = {7,8,9,10};
int button1 = 11;
int button2 = 12;
boolean button1Last = LOW;
boolean button1Current = LOW;
boolean button2Last = LOW;
boolean button2Current = LOW;

void setup()
{
  pinMode(button1, INPUT);
  pinMode(button2, INPUT);
  for(int i=0; i<4; i++)
  {
    pinMode(ledPin1[i], OUTPUT);
  }
  for(int i=0; i<4; i++)
  {
    pinMode(ledPin2[i], OUTPUT);
  }
}

boolean waitForButtonPush1 (boolean lastStartSwitchState1)
{
  boolean currentStartSwitchState1 = digitalRead(button1);
  if(lastStartSwitchState1 != currentStartSwitchState1) delay(20);
  currentStartSwitchState1 = digitalRead(button1);
  return currentStartSwitchState1;
}

boolean waitForButtonPush2 (boolean lastStartSwitchState2)
{
  boolean currentStartSwitchState2 = digitalRead(button2);
  if(lastStartSwitchState2 != currentStartSwitchState2) delay(20);
  currentStartSwitchState2 = digitalRead(button2);
  return currentStartSwitchState2;
}

void loop()
{
  button1Current = waitForButtonPush1(button1Last);
  if(button1Last == LOW && button1Current == HIGH)
  {
    for (byte counter =0;counter<=15; counter++)
  {
    displayBinary(counter);
    delay(500);
  }
  }
  button2Current = waitForButtonPush2(button2Last);
  if(button2Last == LOW && button2Current == HIGH)
  {
    for (byte counter =0;counter<=15; counter++)
  {
    displayBinary2(counter);
    delay(500);

  }
  }
}

void displayBinary(byte numToShow)
{
  for (int i =0;i<4;i++)
  {
    if (bitRead(numToShow, i)==1)
    {
      digitalWrite(ledPin1[i], HIGH);
    }
    else
    {
      digitalWrite(ledPin1[i], LOW);

    }
  }

}

void displayBinary2(byte numToShow)
{
  for (int i =0;i<4;i++)
  {
    if (bitRead(numToShow, i)==1)
    {
      digitalWrite(ledPin2[i], HIGH);
    }
    else
    {
      digitalWrite(ledPin2[i], LOW);
    }
  }

}

A: Welcome to the world of embedded devices!
Getting a small microprocessor to do several things at the same time is a bit tricky.
The key is to never block.  No calls to delay(), no sending large buffers on the serial port at 9600 bauds in one go, etc...
There are some simple techniques to do it, one of the most commonly used is finite state machines.
Let's analyse your app a bit.

*

*2 similar dssplay counters, with delay

*2 buttons, buttons usually need to be debounced, that also involves a delay.

Some code, for you to tinker with:
// ****************************
// pinout

static const byte ledPin1[] = { 2, 3, 4, 5 };
static const byte ledPin2[] = { 7, 8, 9, 10 };

constexpr byte button1 = 11;   // using constexpr for these saves 2 bytes of RAM.
constexpr byte button2 = 12;

// ****************************
// Counter data

static constexpr unsigned int led_delay = 500;  // 500 ms, for all counters.

// constexpr ??  arduino supports c++17.  Not all features in the main .ino
// module and all features in .cpp modules.

// Hint: you could have a member variable in the structure below for delay,
// this would allow for counters running at different speeds, or add buttons
// to increase/decrease speed.

// we have only 2 states, but you could add more, like running
// backwards, or run a different chase pattern maybe?

enum class led_counter_state : byte
{
    stopped,
    running,
};

struct led_counter_data_t
{
    led_counter_state state; // STATE
    byte counter;            // counter current value
    unsigned int timestamp;  // used for timing.
    const byte* leds;        // LED pins.
};

static led_counter_data_t led_counter[2];

void led_display_init()
{
    for (byte i = 0; i < 2; ++i)
    {
        led_counter[i].state     = led_counter_state::stopped;
        led_counter[i].counter   = 0;
        led_counter[i].timestamp = 0;
    }
   led_counter[0].leds = ledPin1;
   led_counter[1].leds = ledPin2;
}

// ****************************
// LED cotrrol

static void leds_display_value(const led_counter_data_t& cntr)
{
    for (byte i = 0, val = cntr.counter; i < 4; ++i, val >>= 1)
         digitalWrite(cntr.leds[i], val & 0x01);
}

static void leds_control(led_counter_data_t& cntr)
{
    const auto now = (unsigned int)millis();   // keep track of time.

    switch(cntr.state)
    {
    default:   // something is wrong.. stop.
        cntr.state = led_counter_state::stopped;

        // fall through ...

    case led_counter_state::stopped:
        return;                       // if not running, do nothing

    case led_counter_state::running:
        if (now - cntr.timestamp >= led_delay) // check delay
        {
            if (++cntr.counter > 15)           // advance counter.
                cntr.counter = 0;

            leds_display_value(cntr);           // show value.
            cntr.timestamp = now;              // keep track of time.
        }
        break;
    }
}

static void leds_start(led_counter_data_t& cntr)
{
    if (cntr.state != led_counter_state::stopped)
        return;

    cntr.state = led_counter_state::running;

    if (++cntr.counter > 15)  // advance counter.
        cntr.counter = 0;

    led_display_value(cntr);  // show value.

    cntr.timestamp = (unsigned int)millis();
}

static void leds_stop(led_counter_data_t& cntr)
{
    cntr.state = led_counter_state::stopped;
}

// ****************************
//  switch inputs data

static constexpr byte switch_debounce_delay = 30;  // 30ms is a good value for
                                                   // debouncing
struct switch_data_t
{
    byte sw1_state : 1;  // no need to waste more than 1 bit per switch
    byte sw2_state : 1;
    byte timestamp;      // we'll only count to 30 ms, so 1 byte timestamp will do
};

static switch_data_t switch_data;

// ****************************
//  switch inputs code

static void control_inputs()
{
    const auto now = (byte)millis();

    if (now - switch_data.timestamp < switch_debounce_delay)
        return;

    switch_data.timestamp = now;

    // All switch control logic is regrouped here, and isolated
    // form other control code, this makes the logic easier to
    // write, read, and debug.

    bool b = digitalRead(button1);
    if (b & !switch_data.sw1_state)   // button was pushed right now.
    {
        if (led_counter[0].state == led_counter_state::stopped)
        {
            leds_start(led_counter[0]);   // start counter 1
            leds_stop(led_counter[1]);    // stop counter 2
        }
        else
        {
            leds_stop(led_counter[0]);    // stop counter 1
        }
    }
    switch_data.sw1_state = b;

    b = digitalRead(button2);
    if (b & !switch_data.sw2_state)   // button was pushed right now.
    {
        if (led_counter[1].state == led_counter_state::stopped)
        {
            leds_start(led_counter[1]);   // start counter 2
            leds_stop(led_counter[0]);    // stop counter 1
        }
        else
        {
            leds_stop(led_counter[1]);    // stop counter 2
        }
    }
    switch_data.sw2_state = b;
}

// ****************************

void setup()
{
    pinMode(button1, INPUT);
    pinMode(button2, INPUT);

    for (byte i = 0; i < 4; ++i)
    {
        digitalWrite(ledPin1[i], LOW);
        pinMode(ledPin1[i], OUTPUT);

        digitalWrite(ledPin2[i], LOW);
        pinMode(ledPin2[i], OUTPUT);
    }

    led_display_init();
}

// ****************************
// The goal, always, is to exit loop() as fast as possible, so
// everything will run smoothly, and appear to run simultaneously.

void loop()
{
    control_inputs();
    leds_control(led_counter[0]);
    leds_control(led_counter[1]);
}

I do not have an arduino with me, so I did not comppile nor ran this, but it should be pretty close.  Let me know if you're having issues or have any questions.

redpajama_set_name: RedPajamaStackExchange


For its successful application. Model Driven Security relies heavily on a series of tools. We have implemented our approach as a proof-of-concept framework. The framework consists of prototypical tools, we developed ourselves and integrates various third party components. It supports the development of all models and the generation of systems for their implementation in local runtime environments. We first introduce the set of tools the stakeholders planning the realization of a Global Workflow may need (Section 10.1) and give an overview of how the deployment process may be managed (Section 10.2).