For as long as I can remember I have struggled with my weight. Though, unknowingly I was fighting the voice inside myself, more than I was battling the scale. My striving to be perfect every time I tried, continually failed me. Nursing school is where everything came together for me, and my journey to better health began. Education was the key to my success and my experiences as a nurse fuel the ambition that keeps me going.
Academics were always an easy accomplishment for me, but then I met the beast called nursing school, where life just plain got messy. By messy I mean, sometimes my dishes would sit for over a week, and my organized, structured, clean life was occasionally disordered while juggling home life with school life. Life was anything but perfect. Nursing school humbled me, taught me how to be flexible, and showed me that there is always more than one way to accomplish a task.
Most importantly, as a child from poverty and a first-generation college student, nursing school taught me that I was capable of achieving anything I believed I could. I started working as soon as I had my LPN, was then hired right into an ICU after graduating with my RN and am currently in the middle of an RN to BSN program. Over the last three years, I have learned that very little that I accomplish in life will ever be perfect. If my accomplishments were perfect, there would never be any room for improvement, and I would never grow.
I learned that I can always be a better version of myself not only as a nurse but as a person, a mom, a friend, or whatever path I choose to walk down, or in my case, run down! I took this mindset of, moving forward is forward and progress is progress no matter how small or slow, and implemented it into my life and health.
Initially, my goal was to lose weight and be heart healthy, though it has evolved remarkably. Some of the patients I care for in the ICU are there due to repeated poor life choices. I try to imagine myself in their position. I read reports that the patient was warned over and over to make an appointment for a heart catheterization because a heart attack could happen any day, they refused to do so.
I imagine myself as the family member of that patient who suffered the heart attack, as the doctor informs them that their loved one's heart went without oxygen for too long, they are now brain dead, and will never recover. I thought to myself how can I possibly provide proper health education to my patients if I am not willing to take and implement my advice into my life? So, I set off into my journey, which is where I stumbled upon Healthy Nurse, Healthy Nation.
While being heart healthy and losing weight are still very much my goals, my aspirations and appetite for better health have grown! I took on the ketogenic diet; I eat veggies, healthy fats, and meats primarily. I absolutely love finding, cooking and then sharing healthy, delicious, recipes on Facebook, Twitter and with all my friends. I use an apple watch to track my activity and cheer on my friends when they complete exercises. I started running, something I have always wanted to do but when I was unable to run as fast or as far as I thought I should be able to, I gave up. Not only did I start running but I made a promise to myself I would run one mile, then two miles, then three miles, so I could compete in my first ever 5k! The mental clarity and overall boost of energy that accompanies this journey are so breathtakingly beautiful that I would not trade it for anything! I believe that I cannot wholeheartedly care for others unless I practice self-care. Being healthy makes me a better nurse!
With a new outlook my only goal is, try and keep trying! With that mindset, for the first time in my life, I ran my first three miles straight on September 16, 2018 and I ran just a little bit farther on September 18, 2018. My journey to better health is not perfect, and I am so thankful for that! I feel that I can better connect with every patient that I encounter who are trying but falls. I believe my journey expresses the message that it is okay to make a mistake, to fail, to fall but it is not okay to stay there. My mission is an example to my patients, not only to better connect with them but to improve their lives and health simultaneously. My health journey is also an example I hope my daughter proudly views someday. If I can do this, so can others and my purpose is to be an inspiration for all of those that follow behind me and allow me to support them on their journey!
I loved your story and I completely agree about accepting that sometimes we won't be perfect in our efforts to keep on track. It's the only way we can stay in it for the long haul. I think of it as not completely "falling off of the wagon", but keeping one hand on the wagon at all times. It's the pulling oneself back up that can be really hard, but the experience of doing it successfully over the years makes us realize that our minds are strong and we can always get back on track. I joined a running club and that really helped to keep me motivated.
I wish you all the best with your health, your career and your daughter!


Wood Group has been awarded a five-year contract in the UK sector of the North Sea by Nexen, valued at $150 million.
Extending a contract in place since April 2010, Wood Group PSN (WGPSN) will continue to deliver operations, maintenance and technical support across Nexen's North Sea offshore assets; the Buzzard, Scott and Golden Eagle platforms.
According to Wood Group, more than 190 jobs are retained under the contract, which is effective immediately and builds on Wood Group's 12 year relationship with Nexen in the UKCS.
This is the third contract extension WGPSN has secured in the UKCS this year.
In April, the company announced a two-year contract with TAQA, extending its provision of engineering, construction and maintenance services to five UKCS offshore assets.
In February, WGPSN secured an extension to continue to support Chevron Upstream Europe across four offshore assets in the North Sea.
Posted on April 21, 2016 with tags Nexen, North Sea, UK, Wood Group.


The invention is directed to a transparent document pouch for carrying travel documents. The document pouch has a first pocket sized to receive an article of identification and a second pocket sized to receive larger travel documents such as an airline ticket or a boarding pass. In one embodiment, the document pouch is formed from a single sheet of transparent material folded substantially in half. The sheet of material is heat sealed along sides of the sheet and by a cross seal in a portion of the sheet to form the first and the second pocket. A slit is formed in the sheet of material to provide access to the second pocket. The document pouch is suspended from a lanyard wrapped around a neck of a traveler.
The application claims priority to Provisional Application U.S. Serial No. 60/370,869 filed Apr. 5, 2003.
With security expected to grow even tighter over the next few years, the need for devices to minimize inconvenience and the expenditure of time is readily apparent. It would therefore be beneficial to have a single device in which to store all travel-related documents such that the documents are readily visible and accessible to security personnel while, at the same time, permitting the traveler to maintain control of his or her luggage and to minimize confusion, inconvenience and delay at security checkpoints. It is to such a device that the present invention is directed.
FIG. 4 is a front elevation view of another embodiment of a transparent document pouch constructed in accordance with the present invention shown hanging around the neck of a traveler.
Referring now to the drawings and more particularly to FIG. 1, a transparent document pouch  10 constructed in accordance with the present invention is shown hanging about a neck 12 of a traveler 14. The transparent document pouch 10 has a first pocket 15, a second pocket 16, an aperture 17, a lanyard 18 and is made from a transparent sheet 20 of material, such as vinyl or plastic. While vinyl and plastic are herein specified, any material that is transparent and capable of bearing the weight of the documents of traveler 14, is suitable.
One suitable material for use when a photograph is to be kept in the document pouch is polypropylene. Polypropylene is well-known for use in archival storage of photographs. Other plastics such as vinyl may cause documents to turn yellow or may stick to the documents if the documents are left in the document pouch  10 for a long time.
Although the transparent document pouch  10 illustrated in FIG. 1 is constructed from a single sheet of transparent material, any number of sheets can be used. The use of more than one sheet causes the transparent document pouch 10 to have many layers, which increases the strength or carrying capacity of the transparent document pouch 10. It should be noted that in an embodiment having many layers, not all layers would need to be transparent.
The transparent sheet  20 is folded along a crease 22 and overlaid upon itself to form a front face 24 and a back face 26. Thus folded, the document pouch 10 has an upper end 28, a creased lower end 30 defined by the crease 22, a first side 32, a second side 33, the front face 24 and the back face 26. A first continuous heat seal 36 extends from near the upper end 28 to the creased lower end 30 of the transparent document pouch 10. The first continuous heat seal 36 joins the front face 24 to the back face 26 near the first side 32 of the transparent document pouch 10.
A second continuous heat seal  38 extending from near the upper end 28 to the creased lower end 30 of the transparent document pouch 10 joins the front face 24 to the back face 26 near the second side 33 of the transparent document pouch 10. The first continuous heat seal 36 and the second continuous heat seal 38 may, if desirable, extend to the upper end 28.
The crease  22 is used to support the weight of any travel documents. However, when more than one sheet of transparent material is utilized to construct a transparent document pouch, as illustrated in FIG. 2, the sheets need not be folded at all. Instead, a top sheet may be positioned on top of a bottom sheet with the upper surface of the top sheet defining a front face and the lower surface of the bottom sheet defining a back face. The sheets are heat sealed along the sides and the lower end, thus joining the front face to the back face.
Referring again to FIG. 1, a continuous cross seal  44 extends between the first continuous heat seal 36 and the second continuous heat seal 38 joining the front face 24 to the back face 26 so as to define the first pocket 15 so that the first pocket 15 is sized to receive an article of identification 48 between the front face 24 and the back face 26 of the transparent document pouch 10. Articles of identification, such as a driver's license, are typically sized to fit into a man's wallet and are typically on the order of two inches wide by three inches long and are less than one-tenth of one inch thick.
The transparent sheet  20 may be of any thickness so long as the front face 24 can be joined to the back face 26. While the front face 24 and the back face 26 are herein joined by continuous heat seals 36 and 38, any type of seal joining the front face 24 to the back face 26, such as spot sealing, dashed sealing or embossed sealing would be within the scope of this invention. Furthermore, heat sealing need not be used at all. Alternative means for joining the front face 24 to the back face 26 such as sewing, adhesives, clips, studs, magnets or any other equivalent methods would be within the scope of this invention. Also, should it be determined to be beneficial, the upper end 28 of the transparent document pouch 10 may be sealed in favor of some other opening without departing from the inventive concept.
The first pocket  15 is formed near the upper end 28 of the transparent sheet 20 and has an open upper end 50 through which the article of identification 48 is inserted between the front face 24 and the back face 26, thereby disposing the article of identification 48 into the first pocket 15. The open upper end 50 is defined by the unsealed portions of the front face 24 and the back face 26 at the upper end 28 of the transparent document pouch 10. The first pocket 15 has a lower end defined by the continuous cross seal 44, a first side 54 defined by the first continuous heat seal 36 and a second side 56 defined by the second continuous heat seal 38.
Also defined by the continuous cross seal  44 is an upper end 58 of the second pocket 16. While the upper end 58 of the second pocket 16 is defined by the continuous cross seal 44, the upper end 58 of the second pocket 16 could be defined by another seal entirely or have no seal at all, thereby being open.
The second pocket  16 is formed so as to be sized to receive a longer item such as a ticket, boarding pass, itinerary, or some other item too large to fit in the first pocket 15. In addition to having an upper end 58, the second pocket 16 also has a lower end 62, a first side 64 and a second side 66. The lower end 62 of the second pocket 16 is defined by the creased lower end 30 of the transparent document pouch 10. The first side 64 of the second pocket 16 is defined by the first continuous heat seal 36 of the transparent document pouch 10. The second side 66 of the second pocket 16 is defined by the second continuous heat seal 38 of the transparent document pouch 10.
Additional pockets, along with the openings to the pockets, could be disposed in the front face  24 or the back face 26. When multiple sheets of transparent sheet 20 are utilized, for instance three sheets, the transparent document pouch 10 could have pockets on both the front and the back of the transparent document pouch 10. Multiple sheets would also permit the formation of a transparent document pouch 10 with a transparent front face 24 and an opaque back face 26 or vice versa.
Furthermore, the pockets need not be configured as disclosed for the preferred embodiment. For instance, the second pocket  16 might be disposed closer to the top of the transparent document pouch 10, the second pocket 16 might be on one side with a plurality of smaller pockets on the other, the second pocket 16 might be configured to extend laterally, rather than vertically and so forth. Any configuration or layout of pockets is within the scope of the invention so long as the transparent document pouch 10 minimizes inconvenience for the traveler 14 by visibly storing travel documents in an easily accessible and secure location.
Disposed in the front face  24 of the transparent document pouch 10 in the area defined by the second pocket 16 is a slit 68. A boarding pass 70 is shown disposed through the slit 68 so that the boarding pass 70 is disposed between the front face 24 and the back face 26 of the transparent document pouch 10 in the area defined by the second pocket 16.
While the preferred embodiment specifies an open upper end  50 for the first pocket 15 and a slit 68 for the second pocket 16, so that travel papers may be disposed between the front face 24 and the back face 26 thus being contained by the transparent document pouch 10, any means may be used for an entry into a pocket. Such means may include slits, holes, slots, or overlapping portions of transparent sheet 20. The opening may be permanently open or securably closable using a snap, a non-metallic zipper, a continuous rail and clasp, a hook and loop surface, a mild adhesive, a flap, or any other means to temporarily secure an opening.
Near the upper end  28 of the transparent document pouch 10 is the aperture 17 through which the lanyard 18 is threaded. While the aperture 17 specified herein is a hole disposed through the front face 24 and the back face 26, the aperture 17 could be a slit, a fold of transparent sheet, a hole with a ring disposed thereabout, or like device. In fact, the aperture 17 could be eliminated altogether, so long as some other means for removably connecting the transparent document pouch 10 to the traveler 14 were employed in place of the aperture 17.
The lanyard  18 is disposed about the neck 12 of the traveler 14 so as to support the weight of both the transparent document pouch 10 and the contents of the transparent document pouch 10, and also to facilitate the flipping over of the transparent document pouch 10 to permit the personal information contained on the article of identification 48 and other travel documents to be obstructed from view when not presenting them to security or to airline personnel. The lanyard 18 could be a wire, string, shoe string, strap, rope, band or any other means by which the transparent document pouch 10 could be hung about the neck 12 of the traveler 14.
The lanyard  18 need not be threaded through the aperture 17, but might substitute some means for attaching the lanyard 18 to the transparent document pouch 10. Such means would include a hook, clasp, snap or any other suitable means for attaching a lanyard 18 to the transparent document pouch 10, so long as the attachment of the lanyard 18 to the transparent document pouch 10 allows the transparent document pouch 10 to be flipped to enable the viewing of both faces 24 and 26 of the transparent document pouch 10.
FIG. 2 shows the document pouch  10 flipped so that the back face 26 is facing outwardly. Having the document pouch 10 flippable, or rotatable, on the lanyard 18, enables the traveler 12 to selectively conceal or reveal information on fronts of the article of identification and the travel documents. If desirable, the back face 26 may be constructed of an opaque or translucent material to prevent a passerby from reading information on backs of the article of identification and the travel documents.
Other means to securely attach the transparent document pouch  10 to the traveler 14 could be utilized such as a non-metallic clasp or snap, a permanently attached band with a clip or a two-ended band, each end having a clip disposed thereon so long as said means permits the traveler 14 to attach the transparent document pouch 10 to the traveler 14 in a secure manner.
FIG. 3 shows another embodiment of a document pouch  80 suspended from the neck 12 of a traveler 14. The pouch 80 has a first pocket 82 and a relatively larger second pocket 84. The first pocket 82 is sized two to three inches (5.1 to 7.6 cm) long and three to four inches (7.6 to 10.2 cm) wide. The second pocket 84 is sized four to five inches (10.2 to 12.7 cm) wide and nine to ten inches (22.9 to 25.4 cm) long.
The first pocket  82 is formed by attaching a first sheet of material 86 to a second sheet of material 88. The second pocket 84 is formed by attaching a third sheet of material 90 to the second sheet of material 88. The first sheet of material 86 has a top 92, a bottom 94 and two sides 96. The second sheet of material 88 has a top 98, a bottom 100 and two sides 102. The third sheet of material has a top 104, a bottom 106, and two sides 108. The third sheet of material 90 is attached to the second sheet of material 88 by a substantially U-shaped heat seal 110 that leaves the second pocket 84 with an open top 112. The open top 112 permits using the second pocket 84 for storing documents that are longer than the third sheet of material 90. The U-shaped heat seal 110 supports the travel documents and define sidewalls of the second pocket 84.
The first sheet of material  86 is attached to the second sheet of material 88 by a heat seal 114 having four substantially vertical elements 116, 118, 120 and 122 and one horizontal element 124. The horizontal element 124 supports the article of identification and the inner vertical elements 118 and 120 define sidewalls of a receiving space for receiving the article of identification. The first pocket 82 has an open top 126. The open top 126 allows the traveler 12 to use an article of identification that is longer than first sheet of material 86.
An aperture  128 is disposed in the document pouch 80 for attachment of the document pouch 80 to a sliding clasp clip 130. The sliding clasp clip 130 is attached to a lanyard 132 which is suspended from the neck 12 of the traveler 14.
FIG. 4 shows a document pouch  130 that has three pockets 132, 134 and 136. The document pouch 130 is nearly identical to the document pouch 80 except for the presence of the third pocket 136. The third pocket 136 is shown holding a typical passport. The third pocket 136 has dimensions of about 3.5 inches (8.9 cm) in width and 4.0 inches (10.2 cm) in length.
a second open ended pocket sized to receive a travel document, the second pocket formed on the sheet of material below the first pocket and having at least one transparent side.
2. The document pouch of claim 1 wherein the first and second pockets are entirely transparent.
3. The document pouch of claim 2 wherein the document pouch is formed from a single sheet of transparent material.
4. The document pouch of claim 1 further comprising a third open ended pocket formed on the sheet of material near the lower end thereof.
5. The document pouch of claim 1 wherein the sheet of material is made of polypropylene.
6. The document pouch of claim 1 wherein the sheet of material is made of vinyl.
7. The document pouch of claim 1 further comprising a laynard connected to the upper end of the sheet of material to permit the document pouch to be suspended about the neck of a traveler.
8. The document pouch of claim 7 wherein the document pouch is rotatable on the lanyard to selectively conceal and reveal information on the front of the article of identification and the front of the travel document.
9. The document pouch of claim 1 wherein the document pouch has a front face and a back face and wherein the back face is constructed from a material chosen from a group of materials consisting transparent material, translucent material, and opaque material.
(d) forming a slit in the front face to provide access to the second pocket.
(e) forming a slit in the front face to provide access to the second pocket.
(g) disposing a lanyard through the aperture, wherein the lanyard is positionable about a neck of the traveler.
13. The method of claim 11 wherein, in step (c), the seal between the front face and the back face is a continuous heat seal.
14. The method of claim 11 wherein, in step (c), the seal between the front face and the back face is an embossed heat seal.
15. The method of claim 11 wherein, in step (c), the seal between the front face and the back face is formed by sewing.
16. The method of claim 11 wherein, in step (c), the seal between the front face and the back face is formed by adhering the front face to the back face with an adhesive.
(c) attaching the bottom and the two sides of the third sheet of material to the second sheet of material to form the second pocket, such that the second pocket has an open top.
18. The method of claim 17 wherein the first sheet of material is attached to the second sheet of material by a heat seal.
19. The method of claim 17 wherein the third sheet of material is attached to the second sheet of material by a heat seal.
20. The method of claim 17 wherein the first sheet of material is transparent.
21. The method of claim 17 wherein the second sheet of material is transparent.
22. The method of claim 17 wherein the third sheet of material is transparent.
(f) suspending the document pouch from a neck of a traveler by a lanyard.
24. The method of claim 17 further comprising a step of attaching the document pouch to the lanyard by a sliding clasp clip.
(d) rotating the document pouch on the lanyard so that a back face of the document pouch is displayed outwardly to conceal the front side of the article of identification and the front side of the travel document.
26. The method of claim 25 wherein the back face of the document pouch is made of a material chosen from a group of materials consisting of transparent materials, translucent materials, and opaque materials.
(d) rotating the document pouch on the lanyard so that a transparent back face of the document pouch is displayed outwardly to reveal the back side of the article of identification and the back side of the travel document.


text:
Canadian killed by police converted to Islam after troubled childhood
An image of Aaron Driver is projected on screens during a news conference with Royal Canadian Mounted Police (RCMP) Deputy Commissioner Mike Cabana (left) and Assistant Commissioner Jennifer Strachan in Ottawa, Ontario, Canada on Aug 11.PHOTO: REUTERS
Aug 12, 2016, 8:25 am SGT
http://str.sg/4TFE
WINNIPEG/TORONTO (Reuters) - Aaron Driver, the man killed during a Canadian police raid at his home this week as he was allegedly planning an attack, converted to Islam in his teens some time before his support for Islamic State attracted the attention of authorities.
Driver, who would have turned 25 next week, was living under a so-called peace bond that barred him from using social media and required him to undergo religious counseling when he was shot during a police raid in a small Ontario town.
He first came to the attention of police in December 2014 and was arrested in 2015 for supporting the militant group Islamic State on social media. He had openly spoken to Canadian media about his belief that Canada and its allies should expect retribution for their war against Islamic State.
Two suspects held in possible Rio Olympics plot: Police
"If a country goes to war with another country or another people or another community, I think that they have to be prepared for things like that to happen. And when it does happen, they shouldn't act surprised, they had it coming to them, they deserved it," Driver calmly told the Canadian Broadcasting Corp (CBC) last year.
His former lawyer, Leonard Tailleur, said on Thursday that he was surprised by police reports that Driver had planned an imminent rush-hour attack on a major Canadian city, noting that Driver was a devout Muslim but also a "passive individual."
FAMILY SCHISM
At the age of 7, Driver's mother died and he started growing apart from his father, who was then a soldier, Tailleur told Reuters. "He blames his father for the death," Tailleur said. "Ever since that time, there was a serious relationship issue."
He said that was part of what made Driver eventually abandon Christianity for Islam. "A bitterness that he had with his father - these are probably some of the undercurrents," Tailleur said.
"A lot of his problems, internal problems, I think, stem from that."
Driver attended Friday prayers off and on in 2014 at Winnipeg Central Mosque, arriving late and leaving early, executive director Idris Knapp told Reuters.
"He wasn't very connected with anyone. Just a person in his own bubble. Most of the time people come to connect with community, he never seemed like he was making an effort," Knapp said.
According to Knapp some Winnipeg Muslims took umbrage with Driver's online postings, especially those aggrandising Islamic State or other militant groups.
It was in Winnipeg that he was placed under a peace bond, required to stay off social media, have no contact with militant groups, observe a curfew and wear an electronic monitoring device, which Canadian police said he was later permitted to remove.
Earlier this year Driver moved to Strathroy to live with his sister, still alienated from his father.
Driver's father, Wayne Driver, told news outlets that he was saddened by his son's death but not surprised. He said his son had seemed beyond reason in his support for Islamic State.
He had hoped his son, who he sometimes feared, could be forced into a de-radicalisation programme.
"We lost him long ago, but I wasn't going to give up on him," Wayne Driver told CBC. "...And then yesterday, the light went out completely, forever. I knew he was lost, but I didn't know how far gone he was."
Amarnath Amarasingam, a researcher in religious studies, tweeted that Driver, who he knew, had changed since last emailing him in April.
At that time, Driver was radicalized, but "nuanced" in his views about violence, until attacks increased around the world, Amarasingam said.
Driver was in contact on social media with Elton Simpson, one of the shooters involved in a Texas attack in May 2015, Canadian police said. A few hours before that attack, Simpson sent a message to Driver and others, police said.
"People (that police) watch all the time never act,"Amarasingam tweeted. "And people they deem safe can be pushed to act based on changing circumstances around the world."

redpajama_set_name: RedPajamaCommonCrawl


Hands On Workshop (HOW) continues at Columbia Heights Education Campus. We had an even larger group for SY2017, with students ranging from 6th-8th grade for our teen HOW program. We also expanded our DCPS partnership to teach HOW at Takoma Education Campus. At Takoma we started a 4th graders class. It is amazing watching their development blossom from one project to the next, and they love to sew. Their confidence is growing and they�re coming up with great ideas on their own. They are also collaborating among themselves and take the initiative to clean up the tables and supplies. From Sy2016 to SY2017, HOW grew 400% in student numbers.
the D.C. Public Schools/Out of School Time Programs, and the District of Columbia Office of Youth Programs, Department of Employment Services.
and by participating as members of nationally recognized organizations such as Americans for the Arts.


text:
Cell, Tumor, and Stem Cell Biology
BRCA1 Promotes Induction of ssDNA by Ionizing Radiation
Brian P. Schlegel, Francine M. Jodelka and Rafael Nunez
Brian P. Schlegel
Francine M. Jodelka
Rafael Nunez
DOI: 10.1158/0008-5472.CAN-05-3209 Published May 2006
The BRCA1 tumor suppressor contributes to the repair of DNA double-strand breaks (DSB) through homologous recombination, but the mechanism is unknown. The rapid accumulation of BRCA1 into nuclear foci in response to induction of DNA breaks suggests that BRCA1 may function in an early step in the repair pathway. We examined the role of BRCA1 in one such early step, the resection of DSBs to generate ssDNA. The appearance of ssDNA in response to ionizing radiation is similar to that of BRCA1 foci formation, suggesting that the two processes are related. Furthermore, BRCA1 colocalizes to ssDNA sites induced by ionizing radiation. Overexpression of BRCA1 causes an increase in cells exhibiting ssDNA induced by ionizing radiation. Mutant BRCA1 that lacks the COOH-terminal BRCT domain also promotes ssDNA but fails to form nuclear foci. Knockdown of BRCA1 expression reduces ssDNA and Rad51 foci formation in response to ionizing radiation. These results indicate that BRCA1 promotes induction of ssDNA in response to ionizing radiation and accumulates at sites of ssDNA. (Cancer Res 2006; 66(10): 5181-9)
BRCA1
homologous recombination
ssDNA
Hereditary predisposition to breast and ovarian cancer has been associated with mutations of BRCA1 ( 1, 2). The BRCA1 tumor suppressor contributes to the repair of DNA double-strand breaks (DSB) through homologous recombination. Primary cells derived from BRCA1 knockout mice exhibit chromatid breaks and dicentric chromosomes, defects indicative of errors in recombination events ( 3). BRCA1-deficient mouse embryonic stem cells exhibit lower rates of DSB repair by homologous recombination ( 4, 5). Several activities have been ascribed directly to BRCA1 using in vitro functional assays, including transcriptional activation ( 6), E3 ubiquitin ligase activity ( 7, 8), and phosphopeptide-binding activity ( 9– 11). However, the mechanism by which BRCA1 contributes to homologous recombination is unknown.
The repair of DSBs is essential for cell survival, but some repair mechanisms are potentially mutagenic. Nonhomologous end joining involves direct ligation of DNA termini, but processing of DNA ends before ligation can lead to deletions or insertions. Alternatively, repair can proceed through homology-dependent mechanisms. Degradation of one strand at the break produces 3′-overhanging ssDNA ( 12). The resulting ssDNA can be up to several kilobases in length and can act as a substrate for two types of homology-dependent repair ( 13). Single-strand annealing can occur when strand resection reveals complimentary strands at both sides of the break site. These regions anneal, and the intervening sequences are removed. Homologous recombination takes place when the ssDNA invades a homologous dsDNA sequence. The complementary sequence acts as a template to extend the damaged strand and replace the lost sequence. Homologous recombination is potentially error-free because the identical sister chromatid sequence can act as the template for repair. In mammalian somatic cells, sequence exchange (crossover) between the damaged chromosome and the sister chromatid template is infrequent ( 14).
The rapid accumulation of BRCA1 into nuclear foci in response to agents that induce DSBs suggests that BRCA1 functions in an early step in DNA repair ( 15). Strand resection is an early step common to homology-dependent repair mechanisms. We present evidence that BRCA1 promotes induction of ssDNA in response to ionizing radiation. Furthermore, BRCA1 accumulation at ssDNA sites requires the COOH-terminal domain of BRCA1.
Cell culture. MCF10A and HCC1937 cells were obtained from the American Type Culture Collection (ATCC). Cells were maintained according to ATCC guidelines. Cells were exposed to 137Cs using a JL Shepard and Associates model 143-68 irradiator to deliver γ-irradiation at a dose of 10 Gy. The construction of recombinant adenoviruses that express 5′ HA-tagged BRCA1 (amino acids 1-1863) and 5′ HA-tagged BRCA1ΔCT (amino acids 1-1526) has been described previously ( 16, 17). Adenovirus that expresses LacZ was obtained from Quantum Biotechnologies, Inc. High titer stocks were generated by propagation of virus in 293 cells and purified by CsCl centrifugation according to standard protocols. Virus titer was determined by serial dilution using the Adeno-X Rapid Titer kit (BD Biosciences), which measures infectious particles using an anti-hexon antibody. Cells were infected with the HA-BRCA1 virus at a multiplicity of infection (MOI) of 175 infectious particles/mL and the HA-BRCA1ΔCT or LacZ virus at a MOI of 350 infectious particles/mL. This resulted in equal protein expression 48 hours after infection based on immunoblot analysis.
Short interfering RNA (siRNA) was obtained from commercial sources (Dharmacon and Ambion). siRNA transfection was done using DharmaFECT (Dharmacon). Cells were transfected using 150 μmol/L siRNA and harvested 24 hours later for immunoblot analysis. BRCA1 A siRNA targets the BRCA1 mRNA sequence AAGGUUUCAAAGCGCCAGUCA as described previously ( 18), and BRCA1 B siRNA targets the BRCA1 mRNA sequence GCGAGCAAGAGAAUCCCAG. The nonspecific siRNA sense sequence is UAGCGACUAAACACAUCAA and has no perfect match to known human genes.
BRCA1 antibody production. A cDNA sequence encoding amino acids 700-1110 of human BRCA1 was subcloned into pGEX-2TK (Pharmacia). DH5α Escherichia coli was transformed with this expression vector, the bacterial lysate was incubated with glutathione-agarose beads (Pharmacia), and the glutathione S-transferase (GST)-BRCA1 fusion protein was eluted with 10 μmol/L glutathione. The purified protein was sent to the University of Illinois at Chicago Protein Research Laboratory (Chicago, IL) for antibody antiserum production in rabbits. The IgG fraction was precipitated from the antiserum with a saturated ammonium sulfate solution; the resulting IgG pellet was dissolved in PBS and then dialyzed overnight to remove the ammonium sulfate. The IgG solution was precleared against GST bound to glutathione-agarose beads. The IgG solution was then applied to a CNBr-activated Sepharose column previously bound with purified GST-BRCA1 700-1110 protein and blocked in 1 mol/L ethanolamine (pH 8). The column was washed extensively with PBS, and the BRCA1-specific antibody was eluted at low pH [50 mmol/L glycine (pH 2.3), 150 mmol/L NaCl] and neutralized with one fourth of the elution volume using 0.5 mol/L sodium phosphate buffer (pH 7.7).
Fluorescence microscopy. Cells were grown on glass coverslips coated with poly-l-lysine. For ssDNA detection, MCF10A cells were cultured for 24 hours in the presence of bromodeoxyuridine (BrdUrd; 10 μg/mL; Invitrogen); HCC1937 cells were cultured 48 hours in the presence of BrdUrd due to a longer doubling time. To visualize mitochondria, cells were then incubated for an additional 1 hour in the presence of CytoTracker CMXRos (50 nmol/L; Invitrogen), a cell-permeable fluorescent dye specific for mitochondria. Cells were washed twice with PBS, fixed in methanol for 20 minutes at −20°C, and then briefly dipped in acetone at −20°C. A control sample was included in each experiment to determine the percentage of cells that incorporated BrdUrd. This was done after fixation by incubating the cells in 2 N HCl containing 0.2 mg/mL pepsin for 20 minutes. Alternatively, samples were incubated in 70% formamide with 2× SSC at 80°C for 1 minute and then dehydrated in an alcohol series. After fixation, cells were rehydrated in PBS for 5 minutes and incubated in blocking buffer (PBS, 0.02% NaN3, 0.2% Triton X-100, 0.05% Tween 20, 0.1% bovine serum albumin, 10% goat serum) for 30 minutes at room temperature. Samples were incubated with the RA81647 anti-BRCA1 rabbit polyclonal antibody (2.6 μg/mL) or an anti-Rad51 rabbit monoclonal antibody (mAb; Ab-1, Calbiochem) and anti-BrdUrd mouse mAb clone B44 (2.5 μg/mL; BD Biosciences) or the anti-BRCA1 mouse mAb Ab-1 (clone MS110, 1 μg/mL; Calbiochem) in blocking buffer overnight at 4°C. The coverslips were washed thrice in PBS for 5 minutes. The samples were incubated with an Alexa Fluor 488–conjugated goat anti-mouse antibody (8 μg/mL) and an Alexa Fluor 555–conjugated goat anti-rabbit antibody (8 μg/mL; Invitrogen) in blocking buffer for 1 hour at room temperature. Samples were mounted on glass slides with mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Vectashield).
Samples were viewed at ×1,000 magnification using a Labophot-2 upright fluorescence microscope. Cells were scored positive when ≥10 nuclear foci were visible by focusing through the cell. ssDNA-positive cells were normalized for BrdUrd incorporation. Images were acquired using a Carl Zeiss LSM 510 Meta Laser scanning confocal microscope equipped with a 63× water immersion objective. Beams of 488 nm from Ar laser, 543 nm from HeNe1 laser, and 405 nm from Diode were used for excitation. Green, red, and blue fluorescence emissions were detected through LP 505, 560, and 420 filters, respectively. The three different fluorochromes were scanned sequentially by using multitracking function to avoid any bleed through among those fluorescent dyes. Some images were scanned with zoom 2, resulting in a final ×1,260 magnification.
Immunoblot analysis. Cells were lysed directly on culture plates in 5% SDS containing 10 mmol/L sodium phosphate buffer (pH 7). The lysate was sonicated and then heated in a boiling water bath for 5 minutes. Samples were normalized for total protein, resolved on a 3% to 8% Tris-acetate SDS-PAGE, transferred to a polyvinylidene difluoride membrane, probed with specific antibody, and visualized by chemiluminescence.
Flow cytometry. Cells remaining on the culture plate after the coverslip was removed for immunofluorescence microscopy were trypsinized and fixed overnight in 70% ethanol at −20°C. Cells were resuspended in PBS containing 50 μg/mL propidium iodide (Sigma) and 100 μg/mL RNase A (Fermentas) and incubated for 2 hours at 4°C with rotation. Cell cycle profiles were determined by two-variable flow cytometry using a Becton Dickinson FACSCalibur flow cytometer and CellQuest software (Becton Dickinson). Acquired events were gated to eliminate cell aggregates and debris. A gated population of 2 × 104 single diploid cells was analyzed.
Visualization of ssDNA. To examine if BRCA1 contributes to the strand resection step, we used an assay that permits identification of sites of ssDNA >1 kb in situ ( 13). This assay is based on the observation that the BrdUrd nucleotide base analogue is recognized by an anti-BrdUrd antibody when incorporated into ssDNA but not dsDNA. The assay is similar in principle to the flow cytometric assays used to measure cells in S phase, except that the denaturant is omitted. This approach successfully showed that induction of DSBs promotes ssDNA formation followed by accumulation of replication protein A (RPA) and Rad51 at ssDNA sites ( 13). We applied this approach to study the appearance of ssDNA in MCF10A cells in response to ionizing radiation. MCF10A cells were chosen because they are an immortalized nontransformed human breast epithelial cell line that expresses wild-type BRCA1. BrdUrd incorporation was visualized by indirect immunofluorescence. In the absence of a denaturing agent, no nuclear staining of BrdUrd is apparent ( Fig. 1A ). After treatment of fixed cells with HCl to denature DNA, BrdUrd labeling appeared uniform throughout the nucleus, whereas staining with DAPI appeared reduced. The vast majority of cells was labeled by 24 hours ( Fig. 1B). A similar result was obtained when samples were denatured in formamide (data not shown), indicating that nuclear staining is specific for ssDNA.
Detection of ssDNA in response to ionizing radiation. A, BrdUrd incorporation into DNA provides a marker for ssDNA. MCF10A cells were grown in the presence of BrdUrd (10 μg/mL) for 24 hours and then fixed and stained with a mouse monoclonal anti-BrdUrd antibody followed by an Alexa Fluor 488–conjugated anti-mouse antibody to visualize ssDNA. Untreated cells do not stain positive for BrdUrd (a), whereas cells treated with 2 N HCl after fixation to denature DNA are strongly positive (c). b and d, DAPI counterstain is specific for dsDNA. B, time course for BrdUrd incorporation into DNA. Points, mean of three or more independent experiments; bars, SD. A minimum of 100 cells was scored for each experiment. C, partial colocalization of ssDNA and Rad51 in response to ionizing radiation (IR). MCF10A cells were grown as in (A), treated with γ-irradiation (10 Gy), and incubated for an additional 8 hours. The cells were then fixed and stained for ssDNA and Rad51 using a polyclonal antibody specific for Rad51 and an Alexa Fluor 555–conjugated anti-rabbit antibody to visualize Rad51. ssDNA (a, e, i, and m; green) and Rad51 nuclear foci (b, f, j, and n; red) were only visible in response to ionizing radiation (e-p). c, g, k, and o, colocalization of ssDNA and Rad51. Arrows, specific foci.
We also observed extensive cytoplasmic staining that varied somewhat from cell to cell, suggestive of mitochondria. Replication of mitochondrial DNA (mtDNA) is not synchronized with nuclear DNA replication. Furthermore, mitochondria exhibit long stretches of ssDNA ( 19). Therefore, we might expect to see a cytoplasmic BrdUrd signal even in the absence of a denaturant. To confirm that the cytoplasmic BrdUrd signal represents mtDNA, we stained BrdUrd-labeled cells with a mitochondria-specific stain, MitoTracker Red CMXRos (Supplementary Fig. S1). This cell-permeable fluorescent dye is sequestered by live cells in metabolically active mitochondria and retained on fixation. Dual staining of BrdUrd and mitochondria in unirradiated cells resulted in extensive colocalization of cytoplasmic ssDNA and mitochondria. Virtually all of the ssDNA colocalizes with mitochondria. A few mitochondria do not exhibit ssDNA probably because they have not yet replicated their DNA. We conclude that the cytoplasmic BrdUrd signal reflects mitochondrial ssDNA and is not relevant to interpretation of the nuclear BrdUrd signal induced on exposure to ionizing radiation.
Next, we exposed cells labeled with BrdUrd to ionizing radiation (10 Gy) and incubated for an additional 8 hours. Cells were then fixed without a denaturization step and stained for BrdUrd. Similar to previous reports, we observed staining of BrdUrd as discrete nuclear foci only in a subset of irradiated samples ( Fig. 1C; refs. 13, 20, 21). Furthermore, when cells were stained concurrently for Rad51, partial colocalization of the two signals was observed ( Fig. 1C). This supports the possibility that at least some of the ssDNA observed in response to ionizing radiation represents an intermediate in homology-directed DNA repair.
Partial colocalization of BRCA1 and ssDNA in response to ionizing radiation. To stain cells concurrently for ssDNA and BRCA1, it is necessary to use a polyclonal antibody directed against BRCA1. We generated a rabbit polyclonal antibody directed against amino acid residues 700-1110 of BRCA1. The affinity-purified antibody recognizes in vitro–translated BRCA1 (Supplementary Fig. S2A). It produced an identical nuclear staining pattern in irradiated cells as mouse MS110 mAb, one of the first and best characterized antibodies directed against BRCA1 (Supplementary Fig. S2B; ref. 22). We again exposed cells labeled with BrdUrd to ionizing radiation (10 Gy), incubated for 8 hours, and stained for ssDNA and BRCA1 using our affinity-purified polyclonal antibody. We observed a subset of cells that exhibited ssDNA and BRCA1 nuclear foci. These cells also exhibited partial colocalization of ssDNA and BRCA1 ( Fig. 2A ), consistent with the putative role of BRCA1 in homology-directed repair.
Induction of ssDNA and BRCA1 foci by ionizing radiation. A, partial colocalization of BRCA1 and ssDNA in response to ionizing radiation. MCF10A cells were grown in the presence of BrdUrd (10 μg/mL) for 24 hours, treated with γ-irradiation (10 Gy), and incubated for an additional 8 hours. The cells were then fixed and stained for ssDNA and BRCA1 using mouse monoclonal anti-BrdUrd antibody and a polyclonal antibody specific for BRCA1 followed by an Alexa Fluor 488–conjugated anti-mouse antibody and an Alexa Fluor 555–conjugated anti-rabbit antibody to visualize ssDNA and BRCA1, respectively. ssDNA (a-c; green) and BRCA1 nuclear foci (d-f; red) were only visible in response to ionizing radiation. g to i, colocalization of ssDNA and BRCA1. j to l, DAPI counterstain is specific for dsDNA. Arrows, specific foci. B, kinetics of ionizing radiation induced ssDNA and BRCA1 foci formation. The appearance of ssDNA and BRCA1 foci follow a similar time course in response to ionizing radiation. MCF10A cells were treated as in (A) but fixed after irradiation at the indicated times. Cells exhibiting ≥10 foci per nuclei were scored as positive. A minimum of 100 cells was scored for each experiment. Points, mean of three or more independent experiments; bars, SD. C, progressive accumulation of BRCA1 in foci in response to ionizing radiation. MCF10A cells were treated as in (B) but without visualization of ssDNA. Scanning settings were held constant for each separate sample so that BRCA1 intensities could be compared directly.
Kinetics of ssDNA and BRCA1 foci formation in response to ionizing radiation. Next, we examined if ssDNA and BRCA1 foci form over a similar time course in response to ionizing radiation. To quantitate nuclear foci, we examined cells at ×1,000 magnification, making it easy to distinguish nuclear foci from the cytoplasmic background. Cells were scored as positive when ≥10 foci were observed while focusing through the nucleus. A minimum of 100 cells was scored for each of three or more independent experiments. We first observed ssDNA in a subset of cells as discrete nuclear foci 1 hour after irradiation ( Fig. 2B). The number of cells positive for ssDNA sites peaked at ∼40% between 4 and 8 hours after irradiation. Only ∼10% of the cells were positive for ssDNA 24 hours after irradiation.
Although a comparable trend was apparent for BRCA1-positive cells, there are some distinct differences. About 5% of cells exhibit BRCA1 nuclear foci even without treatment with ionizing radiation. After irradiation, BRCA1 nuclear foci appeared within 20 minutes. These foci were less intense than BRCA1 foci apparent before irradiation. Similar to ssDNA-positive cells, the number of cells positive for BRCA1 foci peaked between 4 and 8 hours after irradiation, and only ∼10% of the cells were positive for BRCA1 24 hours after irradiation ( Fig. 2B). The similar appearance of the kinetic profiles of ssDNA and BRCA1 foci formation suggests that the two processes are related. We observed that virtually all cells that were positive for ssDNA also exhibited some BRCA1 focus formation. However, ∼20% of cells positive for BRCA1 foci between 4 and 8 hours after irradiation did not exhibit ssDNA foci ( Fig. 2B). In addition, BRCA1 foci became more intense over the same time course ( Fig. 2C). Confocal microscope settings were optimized for the faint BRCA1 foci at the earliest time point, and images were acquired without changing settings between samples. Therefore, the progressively larger foci represent an increase in signal intensity. This suggests that BRCA1 may accumulate at sites of ssDNA over time.
Overexpression of BRCA1 promotes ssDNA formation in response to ionizing radiation. The partial colocalization of ssDNA and BRCA1 and the similar kinetic profiles of foci formation suggest an early role for BRCA1 in DNA repair. However, the observation that BRCA1 associates with Rad51 suggests that BRCA1 functions at a late step in homology-dependent repair ( 23). We examined ssDNA formation in response to ionizing radiation in HCC1937 cells. HCC1937 is a human breast epithelial tumor cell line that is homozygous for the BRCA1 5382insC mutation. The resulting protein product lacks a functional BRCT domain that is required for tumor suppressor activity ( 24). Hypersensitivity of these cells to ionizing radiation is abrogated by expression of full-length BRCA1 ( 25). However, these cells exhibit Rad51 formation in response to ionizing radiation ( 26), suggesting that resection of DSBs still occurs. We infected HCC1937 cells with a recombinant adenovirus that expresses full-length HA-tagged BRCA1 or a COOH-terminal deletion mutant ( Fig. 3A ). Infected cells were labeled with BrdUrd, exposed to ionizing radiation (10 Gy), fixed at various times, stained for ssDNA, and scored as described previously ( Fig. 3B). Surprisingly, ∼5% of uninfected HCC1937 cells exhibited ssDNA sites even without exposure to ionizing radiation. Treatment of cells with ionizing radiation further increased the number of ssDNA-positive cells ( Fig. 3B). Unlike MCF10A cells, the percentage of cells positive for ssDNA did not decrease by 24 hours, consistent with the observed hypersensitivity to ionizing radiation. Overexpression of full-length BRCA1 did not alter the early kinetics of ssDNA formation, but by 24 hours, there was a 2-fold increase in the percentage of ssDNA-positive cells in response to ionizing radiation ( Fig. 3B). This observation is consistent with a role for BRCA1 in strand resection in response to DSBs. Overexpression of the truncated form of BRCA1 that lacks the COOH-terminal BRCT domain also caused an increase in ssDNA-positive cells in response to ionizing radiation by 24 hours. This is not simply a result of adenovirus infection because infection of HCC1937 cells with a recombinant adenovirus alone does not result in an increase in ssDNA-positive cells in response to ionizing radiation. Overexpression of full-length or mutant BRCA1 does not alter the cell cycle profile of HCC1937 cells, indicating that the increase in ssDNA-positive cells 24 hours after irradiation is not a function of cell cycle progression (Supplementary Fig. S3). We conclude that the COOH-terminal domain of BRCA1 is not required for formation of ssDNA in response to ionizing radiation.
Overexpression of BRCA1 in HCC1937 cells promotes ssDNA formation in response to ionizing radiation. A, immunoblot analysis of HCC1937 cells infected with a recombinant adenovirus that expresses full-length HA-BRCA1 (amino acids 1-1863) or a truncated form of BRCA1 that lacks the COOH-terminal domain (HA-BRCA1ΔCT; amino acids 1-1527). Lysate protein was resolved by SDS-PAGE and visualized using an anti-HA or anti-β-actin antibody. B, overexpression of full-length and truncated BRCA1 promotes ssDNA formation in response to ionizing radiation. HCC1937 cells were infected with recombinant adenovirus (Ad) and grown in the presence of BrdUrd (10 μg/mL) for 48 hours. Cells were then treated with γ-irradiation (10 Gy), fixed at the indicated times, and stained for ssDNA. C, the COOH-terminal domain of BRCA1 is required for accumulation at sites of ssDNA. Uninfected HCC1937 cells (a-d) and cells infected with recombinant adenovirus that expresses full-length BRCA1 (e-h) or truncated BRCA1ΔCT (i-l) were treated as in (B), except that cells were also stained with a polyclonal antibody specific for BRCA1.
Endogenous truncated BRCA1 did not colocalize with sites of ssDNA on irradiation ( Fig. 3C). The overexpressed full-length protein formed foci and colocalized with sites of ssDNA, whereas the exogenous truncation mutant did not ( Fig. 3C). This observation is consistent with the previous finding that the BRCA1 COOH terminal is required for foci formation in response to ionizing radiation ( 27). We conclude that although the BRCA1 COOH-terminal deletion mutant cannot form nuclear foci in response to ionizing radiation, it may still promote ssDNA formation.
Knockdown of BRCA1 expression reduces ssDNA formation in response to ionizing radiation. To confirm the role of BRCA1 in ssDNA formation in response to ionizing radiation, we knocked down expression of BRCA1 in MCF10A cells by RNA interference (RNAi) and measured ssDNA foci formation in response to ionizing radiation. Exponentially growing cells were transfected with one of two BRCA1-specific siRNAs and labeled with BrdUrd. Immunoblot analysis of cell lysate indicates a substantial reduction in BRCA1 protein levels 24 hours after transfection of BRCA1-specific siRNAs compared with mock-transfected cells or cells transfected with a nonspecific siRNA ( Fig. 4A ). Cells were irradiated 24 hours after transfection and incubated for an additional 8 hours to allow BRCA1 foci to form. Immunofluorescence staining of irradiated cells indicated a substantial reduction in BRCA1 protein levels 24 hours after transfection ( Fig. 4B). The number of cells positive for BRCA1 foci was significantly reduced in cells transfected with BRCA1-specific siRNAs, whereas there was no significant change in the control samples ( Fig. 5A ). A similar effect was observed on ssDNA focus formation in response to ionizing radiation ( Fig. 5B). Indeed, entire fields of cells did not exhibit either ssDNA or BRCA1 focus formation in response to ionizing radiation ( Fig. 4C). Normally, we observed ssDNA and BRCA1-positive cells distributed throughout irradiated control samples.
Knockdown of BRCA1 expression by siRNA. A, immunoblot analysis of lysate from MCF10A cells transfected with siRNA specific for BRCA1. Cells were mock transfected (lane 4), transfected with a nonspecific siRNA (lane 1), or transfected with one of two siRNAs specific for BRCA1 (lanes 2 and 3). Cells were harvested 24 hours after transfection. Lysate protein was resolved by SDS-PAGE and visualized using an anti-BRCA1 or anti-β-actin antibody. B, immunofluorescence analysis of cells transfected with BRCA1-specific siRNAs. Cells were transfected as in (A), treated with γ-irradiation (10 Gy) 24 hours later (except −IR), incubated for an additional 8 hours, and then fixed and stained for BRCA1. C, ssDNA foci formation is reduced in response to ionizing radiation after knockdown of BRCA1 expression. Samples were treated as in (B), except cells were also incubated in the presence of BrdUrd (10 μg/mL) immediately after transfection and stained to visualize BRCA1 and ssDNA foci after irradiation. High magnification is used to illustrate the decrease in ssDNA because nuclear ssDNA is difficult to visualize clearly under low magnification. Magnification, ×250 versus ×1,280.
Knockdown of BRCA1 expression reduces ssDNA formation in response to ionizing radiation. A, knockdown of BRCA1 levels in MCF10A cells. Cells were mock transfected (−IR and +IR) or transfected with a nonspecific siRNA or one of two BRCA1-specific siRNAs (BRCA1 A and BRCA1 B). Cells were treated with γ-irradiation (10 Gy) 24 hours later (except −IR), incubated for an additional 8 hours, and then fixed and stained for BRCA1. *, P ≤ 0.01, compared with irradiated control (+IR). Bars, SD. B, same as (A), except cells were grown in the presence of BrdUrd (10 μg/mL) immediately after transfection and stained for ssDNA after irradiation.
One explanation for the decrease in ssDNA-positive cells is that the decrease in BRCA1 expression alters the cell cycle profile in response to ionizing radiation. Cells that lack functional BRCA1 do not exhibit a mitotic checkpoint ( 28). Therefore, accumulation of cells in G1 after exposure to ionizing radiation may prevent strand resection because the nonhomologous end-joining pathway predominates during that phase of the cell cycle. Flow cytometric analysis indicated that knockdown of BRCA1 expression did not affect the cell cycle profile of irradiated cells (Supplementary Fig. S4). The decrease in ssDNA-positive cells cannot be attributed to changes in cell cycle progression in response to loss of BRCA1.
Consistent with the observed decrease in ssDNA formation, knockdown of BRCA1 also resulted in a decrease in Rad51 foci formation in response to ionizing radiation. MCF10A cells were transfected with one of two BRCA1-specific siRNAs and labeled with BrdUrd. Immunofluorescence staining of irradiated cells indicates a substantial reduction in Rad51 foci formation 24 hours after transfection of BRCA1-specific siRNAs compared with mock-transfected cells or cells transfected with a nonspecific siRNA ( Fig. 6A ). Cells were irradiated 24 hours after transfection and incubated for an additional 8 hours to allow Rad51 foci to form. The number of cells positive for Rad51 foci was significantly reduced in cells transfected with BRCA1-specific siRNAs, whereas there was no significant change in the control samples ( Fig. 6B). A similar effect was observed on ssDNA focus formation in response to ionizing radiation ( Fig. 6C). Immunoblot analysis of cell lysate from transfected cells did not show a change in Rad51 expression ( Fig. 6D), indicating that the decrease in Rad51 foci formation is not a result of decrease protein expression.
Knockdown of BRCA1 expression reduces Rad51 foci formation in response to ionizing radiation. A, decrease in Rad51 foci formation in response to ionizing radiation in cells transfected with BRCA1-specific siRNA. Cells were mock transfected (−IR and +IR) or transfected with a nonspecific siRNA or one of two BRCA1 specific siRNAs (BRCA1 A and BRCA1 B). Cells were treated with γ-irradiation (10 Gy) 24 hours later (except −IR), incubated for an additional 8 hours, and then fixed and stained for Rad51 (a-e) and counterstained with DAPI to visualize the nuclei (f-j). B, quantification of Rad51 foci formation. MCF10A cells were treated as in (A) and scored for Rad51 foci formation. Cells exhibiting ≥10 foci per nuclei were scored as positive. A minimum of 100 cells was scored for each experiment. Columns, mean of three or more independent experiments; bars, SD. *, P ≤ 0.01, compared with irradiated control (+IR). C, quantification of ssDNA formation. Same as (B), except cells were grown in the presence of BrdUrd (10 μg/mL) immediately after transfection and then fixed, stained, and scored for ssDNA after irradiation. D, knockdown of BRCA1 expression does not reduce Rad51 expression. Immunoblot analysis of Rad51 expression in cells transfected with BRCA1-specific siRNA as described in (A) but without irradiation. Lysate protein was resolved by SDS-PAGE and visualized using an anti-Rad51 or an anti-β-actin antibody.
Several lines of evidence support the conclusion that BRCA1 promotes formation of ssDNA in response to ionizing radiation. First, the appearance of BRCA1 foci exhibits a similar time course as ssDNA, suggesting that BRCA1 is involved in the formation and resolution of the ssDNA intermediate during DSB repair. Consistent with this observation, BRCA1 inhibits the 3′ to 5′ exonuclease activity of the Mre11-Rad50-Nbs1 complex in vitro, indicating that BRCA1 binds to the ends of DSBs ( 29). Second, a fraction of ssDNA foci colocalizes with BRCA1 as early as 1 hour after irradiation. Third, overexpression of BRCA1 increases the percentage of cells that form ssDNA in response to ionizing radiation. This is possibly due to artificially high levels of BRCA1 in cells during G1. The increase in BRCA1 may promote strand resection in a population of cells where nonhomologous end joining normally predominates. Fourth, knockdown of BRCA1 expression causes a decrease in cells that form ssDNA in response to ionizing radiation, indicating that BRCA1 promotes ssDNA foci formation in response to induction of DSBs. Analysis of homology-dependent repair frequencies in BRCA1-deficient cells supports a role for BRCA1 in strand resection. Repair of DSBs through both homologous recombination and single-strand annealing is reduced in BRCA1-deficient mouse embryonic stem cells ( 30). Strand resection is a step common to both pathways. However, our data do not rule out an indirect mechanism for promotion of strand resection, such as transcriptional regulation.
It is not certain that ssDNA, measured using the BrdUrd incorporation assay, represents an intermediate in homology-directed DNA repair. However, the ssDNA is observed in response to DNA-damaging agents that induce DSBs, such as ionizing radiation. Furthermore, several proteins involved in homologous recombination, such as Rad51, BLM, PLM, and TopBP1, have been shown to colocalize with ssDNA in response to ionizing radiation when visualized by this method ( 13, 20, 21). We have also shown that knockdown of BRCA1 expression reduces both ssDNA formation and Rad51 focus formation in response to ionizing radiation. Therefore, ssDNA, visualized using BrdUrd incorporation, likely reflects resection of DSBs.
Colocalization of BRCA1 with ssDNA requires the BRCT domain, whereas ssDNA formation does not, indicating that the two activities may be distinguishable. BRCA1-deficient mouse embryonic stem cells exhibit reduced Rad51 foci in response to ionizing radiation ( 31), possibly because strand resection is not as efficient in the absence of functional BRCA1. HCC1937 cells form RPA and Rad51 foci in response to ionizing radiation ( 26, 32) because the truncated form of BRCA1 present in these cells can still promote ssDNA formation.
A single report claims that BRCA1 is not required for Rad51 focus formation in human breast and ovarian tumor cells in response to ionizing radiation ( 33). Assuming that Rad51 foci represent ssDNA sites, this observation is not consistent with the data obtained using the BRCA1-deficient mouse embryonic stem cells or with our current findings. The inconsistency is likely due to the amount of functional BRCA1 present in the cells used in each study. Retroviral-mediated knockdown of BRCA1 by RNAi, the technique used in the aforementioned study, is not likely to decrease BRCA1 expression as completely as direct transfection of siRNA. We observe that BRCA1 focus formation is not necessary for strand resection. Hundreds or thousands of BRCA1 molecules must be present to be visible as foci, but as little as one BRCA1 molecule per DSB may be sufficient for ssDNA formation. Therefore, a modest decrease in BRCA1 expression may affect BRCA1 focus formation but not ssDNA or Rad51 focus formation.
We conclude that BRCA1 promotes ssDNA formation in response to ionizing radiation and then accumulates at ssDNA sites. A large number of functions have been attributed to BRCA1, such as roles in transcription ( 6, 34– 37), ubiquitination ( 7, 8, 38, 39), signaling of DNA damage ( 40– 42), and checkpoint control ( 28, 43– 45). However, it is unknown which, if any, of these functions contribute to the strand resection function. We propose a model where BRCA1 functions directly in resection of DSBs. BRCA1 is required for the exonuclease and/or helicase activities at DSBs that contribute to strand resection. This may be a result of direct recruitment of factors by BRCA1 to break sites or through indirect mechanisms, such as chromatin modification or transcriptional regulation. BRCA1 then accumulates at the resulting ssDNA sites, possibly contributing to a later step in homology-dependent repair.
Grant support: Illinois Division of the American Cancer Society grant 04-01, American Cancer Society Institutional Research grant 99-224, and University of Illinois Campus Research Board grant (B.P. Schlegel).
We thank M. Chen and the Research Resource Center (University of Illinois at Chicago) for assistance with confocal microscopy and B. Lee and the Protein Research Laboratory (University of Illinois at Chicago) for antibody production.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
Received September 6, 2005.
Accepted March 17, 2006.
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Paull TT, Cortez D, Bowers B, Elledge SJ, Gellert M. Direct DNA binding by Brca1. Proc Natl Acad Sci U S A 2001; 98: 6086–91.
Stark JM, Pierce AJ, Oh J, Pastink A, Jasin M. Genetic steps of mammalian homologous repair with distinct mutagenic consequences. Mol Cell Biol 2004; 24: 9305–16.
Bhattacharyya A, Ear US, Koller BH, Weichselbaum RR, Bishop DK. The breast cancer susceptibility gene BRCA1 is required for subnuclear assembly of Rad51 and survival following treatment with the DNA cross-linking agent cisplatin. J Biol Chem 2000; 275: 23899–903.
Choudhary SK, Li R. BRCA1 modulates ionizing radiation-induced nuclear focus formation by the replication protein A p34 subunit. J Cell Biochem 2002; 84: 666–74.
Zhou C, Huang P, Liu J. The carboxyl-terminal of BRCA1 is required for subnuclear assembly of RAD51 after treatment with cisplatin but not ionizing radiation in human breast and ovarian cancer cells. Biochem Biophys Res Commun 2005; 336: 952–60.
Chapman MS, Verma IM. Transcriptional activation by BRCA1. Nature 1996; 382: 678–9.
Monteiro AN, August A, Hanafusa H. Evidence for a transcriptional activation function of BRCA1 C-terminal region. Proc Natl Acad Sci U S A 1996; 93: 13595–9.
Schlegel BP, Green VJ, Ladias JA, Parvin JD. BRCA1 interaction with RNA polymerase II reveals a role for hRPB2 and hRPB10α in activated transcription. Proc Natl Acad Sci U S A 2000; 97: 3148–53.
Krum SA, Miranda GA, Lin C, Lane TF. BRCA1 associates with processive RNA polymerase II. J Biol Chem 2003; 278: 52012–20.
Mallery DL, Vandenberg CJ, Hiom K. Activation of the E3 ligase function of the BRCA1/BARD1 complex by polyubiquitin chains. EMBO J 2002; 21: 6755–62.
Starita LM, Horwitz AA, Keogh MC, Ishioka C, Parvin JD, Chiba N. BRCA1/BARD1 ubiquitinate phosphorylated RNA polymerase II. J Biol Chem 2005; 280: 24498–505.
Cortez D, Wang Y, Qin J, Elledge SJ. Requirement of ATM-dependent phosphorylation of brca1 in the DNA damage response to double-strand breaks. Science 1999; 286: 1162–6.
Chen J. Ataxia telangiectasia-related protein is involved in the phosphorylation of BRCA1 following deoxyribonucleic acid damage. Cancer Res 2000; 60: 5037–9.
Lee JS, Collins KM, Brown AL, Lee CH, Chung JH. hCds1-mediated phosphorylation of BRCA1 regulates the DNA damage response. Nature 2000; 404: 201–4.
Xu B, Kim S, Kastan MB. Involvement of Brca1 in S-phase and G2-phase checkpoints after ionizing irradiation. Mol Cell Biol 2001; 21: 3445–50.
Xu B, Kim ST, Lim DS, Kastan MB. Two molecularly distinct G2/M checkpoints are induced by ionizing irradiation. Mol Cell Biol 2002; 22: 1049–59.
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You are going to email the following BRCA1 Promotes Induction of ssDNA by Ionizing Radiation
Cancer Res May 15 2006 (66) (10) 5181-5189; DOI: 10.1158/0008-5472.CAN-05-3209
Diversity in Proadhesive Signaling Mechanisms in B-CLL Lymphocytes
Role of ATM Phosphorylation in Exon 11 of BRCA1
Methylated Genes in TGF-β Signaling in HNSCC
Show more Cell, Tumor, and Stem Cell Biology


text:
Man gets jail sentence after major incident investigation in quiet cul-de-sac
The house in Rudham Avenue was the subject of a near week-long forensic examination
A major incident investigation which took over a quiet cul-de-sac for nearly a week in the run-up to Christmas has ended in a man being given a jail sentence for producing cannabis.
Christopher Young, 37, of Rudham Avenue, Grimsby, was also initially charged with blackmail, but that part of the case has been discontinued
A team of seven crime scene investigators carried out a painstaking search of the two-storey, semi-detached home where Young lived.
They spent over six days combing the property, removing items including computer equipment and floorboards to a huge container placed outside.
Young has now appeared before a Crown Court and was given a seven-week jail sentence for production of cannabis.
A large team of forensic officers investigated the property in Rudham Avenue, Grimsby (Image: Jon Corken/Grimsby Live)
Judge Caroline Wigin ordered proceedings in the blackmail charge to be discontinued.
Mr Young had already been in custody since his arrest on December 18, last year and had served the equivalent of a two-month custodial sentence, meaning he was eligible for immediate release.
The court heard a total of 34 cannabis plants, described as "immature," were discovered at the home.
Police had smashed in the front door in a dawn raid on the Grimsby property.
Neighbours were shocked by the number of police resources then dedicated to the investigation.
Computer equipment was taken away and loaded into the van in an investigation at the home on Rudham Avenue in Grimsby (Image: Jon Corken/Grimsby Live)
Humberside Police installed a six-metre long container on the driveway of the home and spent days removing items.
A number of belongings, including laptops, hard-drives and a computer printer were removed.
The team of investigators, wearing forensic suits, carried out a painstaking search of the property. Neighbours said floorboards and skirting were removed.
A metal door was fitted to the front of the house and metal sheets put up in front of the windows.
The windows have since been uncovered but the metal door remains in place at the unoccupied property.
The scene in Rudham Avenue, Grimsby (Image: Grimsby Live)
The large container has been removed from the driveway.
A neighbour said: "It still looks eerie with the metal door and the smashed up door in the driveway.
"No one has seen him since the incident. We still don't know what it was all about. It seems a lot of effort just for a few cannabis plants."
Another resident walking her dogs said: "I used to see him walking his two dogs but ever since the police came no one has seen him. I am just glad the street has returned to normal after all the police activity."
Neighbours in Rudham Avenue, Grimsby are pleased their quiet cul-de-sac has returned to normal following the investigation (Image: Grimsby Live)
Defence barrister, Adam Norris said: "He has been held in custody for the other matter which has been discontinued. He has no convictions. He's made full admissions from the start and it was for his own use."
Judge Wigin told Young: "You decided to grow cannabis for your own use. I've seen the photos and I would describe them more as large cuttings. You have spent time in custody in respect of this matter. That is effectively a two-month sentence. If I were sentencing you and you had not served it I would have suspended your sentence."
January in the courts
Boy's seizure after medication stolen
Deer poachers banned from region
Terrifying call made while in police car
Police pursuit ends in crash
For more top stories and breaking news see GrimsbyLive
Follow or contact Grimsby Live
Contact us : Email the reporter who wrote this story
Follow Grimsby Live on Facebook - Like our Facebook page to get the latest news in your feed and join in the lively discussions in the comments. Click here to give it or message us with a comment or story
Follow us on Twitter - For breaking news and the latest stories, click here to follow Grimsby Live on Twitter
Follow us on Instagram - On the Grimsby Live Instagram page we like to feature great pictures from our area - and if you tag us in your posts, we could repost your picture on our page! Click here to follow Grimsby Live on Instagram


How can your gift to Indiana University change the world? It empowers IU students, faculty, and researchers across all campuses who are working to address major global concerns. For example, in the School of Public and Environmental Affairs, they are tackling an issue that affects us all: the availability of clean water—now and for future generations.
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SPEA's water resources program equips students with the skills and experiences to address water quality treatment, pollution prevention, sustainable urban growth, and more.
Graduates become scientists, advocates, and policymakers with global influence.
SPEA's network of 30,000 alumni includes graduates who have worked at the Environmental Protection Agency, U.S. Geological Survey, and other influential agencies and companies.
Faculty members are empowered to conduct critical research in their fields.
SPEA faculty bring diverse backgrounds and interests to their classrooms and research, addressing such topics as sustainability, water utilities, pesticides, and lakes and rivers.
Whether you choose to support SPEA or another great cause, your gift to IU can do a world of good.


COSMETIC ORTHODONTIC SYSTEMS LAB, LLC is corporation filed with the New York State Department of State (NYSDOS). The DOS ID is 5146193. The business entity is incorporated in Onondaga County. The entity type is DOMESTIC LIMITED LIABILITY COMPANY. The initial DOS filing date is 05/31/2017. The address is 7555 Morgan Road, Liverpool, New York 13090. The city is Liverpool, New York. The zip code is 13090.


text:
Beata Sokołowska-Kulesza (born 10 January 1974 in Gorzów Wielkopolski) is a Polish sprint canoer who competed from 1999 to 2004. Competing in two Summer Olympics, she won two bronze medals in the K-2 500 m event (2000, 2004).

Sokołowska-Kulesza has also been successful at the ICF Canoe Sprint World Championships, winning ten medals. This includes a gold (K-2 500 m: 1999), four silvers (K-2 200 m: 1999, 2001; K-2 500 m: 2001, K-4 200 m: 2005), and five bronzes (K-2 200 m: 2003, K-2 500 m: 2003, K-4 200 m: 1999, K-4 500 m: 1999, 2003).

For her sport achievements, she received:
 Golden Cross of Merit in 2000;
 Knight's Cross of the Order of Polonia Restituta (5th Class) in 2004.

References
DatabaseOlympics.com profile

Pkol.pl profile

1974 births
Canoeists at the 2000 Summer Olympics
Canoeists at the 2004 Summer Olympics
Living people
Olympic canoeists of Poland
Olympic bronze medalists for Poland
Polish female canoeists
Olympic medalists in canoeing
Knights of the Order of Polonia Restituta
Recipients of the Gold Cross of Merit (Poland)
Sportspeople from Gorzów Wielkopolski
ICF Canoe Sprint World Championships medalists in kayak
Medalists at the 2004 Summer Olympics
Medalists at the 2000 Summer Olympics
21st-century Polish women

redpajama_set_name: RedPajamaWikipedia


Ljubljana, 21 March - The Constitutional Court has ruled that personal circumstances of refugees need to be taken into account as it decided in favour of a Roma family from Kosovo whose request for extension of temporary residence had been rejected.
The headquarters of Slovenia's Constitutional Judge.
The news item consists of 1.047 characters (without spaces) or 201 word words.


text:
namespace DTTD.Engine
{
    using Contracts;
    using Factory;
    using DuelToTheDeath.Class;
    using System;
    using System.Collections.Generic;
    using System.Linq;
    using System.Text;

    public class GameEngine : IEngine
    {
        private const string PlayerAlreadyExist = "Player {0} already exist. Choose a different username!";
        private const string PlayerRegisterеd = "Player {0} registered successfully! \r\nTo equip a player, enter: Equip (PlayerName) (Weapon)";
        private const string PlayerEquiped = "Player {0} equipped successfully with {1}!\r\nTo cast a player, enter: Cast (PlayerName) (SkillName) (EnemyPlayer)";
        private const string PlayerCast = "Player {0} has successfuly casted {1}!";
        private const string InvalidCommand = "{0} is not a valid command!";

        private static IEngine SingleInstance;

        private ICommandReader reader;
        private ILogger logger;

        private ICollection<IPlayer> players;
        private ICollection<IMageSkills> mages;
        private ICollection<IRogueSkills> rogues;
        private ICollection<IWarriorSkills> warriors;
        private IPlayerFactory playerFactory;
        private IItemFactory itemFactory;

        private GameEngine(ICommandReader reader, ILogger logger)
        {
            if (reader == null)
            {
                throw new NullReferenceException("CommandReader should not be null!");
            }
            this.reader = reader;

            if (logger == null)
            {
                throw new NullReferenceException("Logger should not be null!");
            }
            this.logger = logger;

            this.playerFactory = new PlayerFactory();
            this.itemFactory = new ItemFactory();
            this.players = new List<IPlayer>();
            this.mages = new List<IMageSkills>();
            this.rogues = new List<IRogueSkills>();
            this.warriors = new List<IWarriorSkills>();
        }

        public static IEngine Initialise (ICommandReader reader, ILogger loger)
        {
            if (SingleInstance == null)
            {
                SingleInstance = new GameEngine(reader, loger);
            }

            return SingleInstance;
        }

        public void Start()
        {

            var currentCommand = this.reader.ReadCommand();

            while (!string.IsNullOrEmpty(currentCommand))
            {
                var command = this.CommandParse(currentCommand);
                var commandResult = this.ProcessSingleCommand(command);
                this.logger.Log(commandResult);
                currentCommand = this.reader.ReadCommand();
            }
        }

        private IComand CommandParse(string currentCommand)
        {
            return new Command(currentCommand);
        }

      /*private List<string> ProcessCommands(IEnumerable<IComand> commands)
        {
            var reports = new List<string>();

            foreach (var command in commands)
            {
                try
                {
                    var report = this.ProcessSingleCommand(command);
                    reports.Add(report);
                }
                catch (Exception ex)
                {
                    reports.Add(ex.Message);
                }
            }

            return reports;
        }*/

        private string ProcessSingleCommand(IComand command)
        {
            string nameOfPlayer = command.Parameters[1];

            switch (command.Parameters[0])
            {
                case "Create":
                    if (command.Parameters.Count < 3)
                    {
                        return "Create command should have 3 parameters"; // We could change this
                    }

                    string typeOfPlayer = command.Parameters[2];

                    return this.CreatePlayer(nameOfPlayer, typeOfPlayer);

                case "Equip":
                    if (command.Parameters.Count < 2)
                    {
                        return "Equip command should have 2 parameters"; // We could change this
                    }

                    string typeOfItem = command.Parameters[2];

                    return this.EquipPlayer(nameOfPlayer, typeOfItem);

                case "Cast":
                    if (command.Parameters.Count < 4)   // We do not need enemy player for some of the methods
                    {
                        return "Cast command should have 4 parameters"; // We could change this
                    }

                    string skillName = command.Parameters[2];
                    string enemyPlayer = command.Parameters[3];

                    return this.CastPlayer(nameOfPlayer, skillName, enemyPlayer);
            }

            return string.Format(InvalidCommand, command.Parameters[0]);
        }
        private string CreatePlayer(string nameOfPlayer, string typeOfPlayer)
        {
            if (this.players.Any(p => p.Name.ToLower() == nameOfPlayer.ToLower()))
            {
                return string.Format(PlayerAlreadyExist, nameOfPlayer);
            }

            switch (typeOfPlayer)
            {
                case "Warrior":

                    var userWarrior = this.playerFactory.CreateWarrior(nameOfPlayer);

                    this.players.Add(userWarrior);
                    this.warriors.Add(userWarrior);

                    return string.Format(PlayerRegisterеd, nameOfPlayer);
                case "Mage":

                    var userMage = this.playerFactory.CreateMage(nameOfPlayer);

                    this.players.Add(userMage);
                    this.mages.Add(userMage);

                    return string.Format(PlayerRegisterеd, nameOfPlayer);
                case "Rogue":

                    var userRogue = this.playerFactory.CreateRogue(nameOfPlayer);

                    this.players.Add(userRogue);
                    this.rogues.Add(userRogue);

                    return string.Format(PlayerRegisterеd, nameOfPlayer);
            }

            // We could change this swith-case construction to something more appropriate
            return "You don't have the ability to create such a player in this version of the game";
        }

        private string EquipPlayer(string nameOfPlayer, string typeOfItem)
        {
            if (typeOfItem == "Axe")
            {
                var axe = this.itemFactory.EquipAxe(50);

                this.players.Where(p => p.Name == nameOfPlayer).Select(p => p.AttackPoints += axe.AttackPoints);
                return string.Format(PlayerEquiped, nameOfPlayer, typeOfItem);
            }

            else if (typeOfItem == "Bow")
            {
                var bow = this.itemFactory.EquipBow(30);

                this.players.Where(p => p.Name == nameOfPlayer).Select(p => p.AttackPoints += bow.AttackPoints);
                return string.Format(PlayerEquiped, nameOfPlayer, typeOfItem);
            }

            else if (typeOfItem == "Hammer")
            {
                var hammer = this.itemFactory.EquipHammer(20);

                this.players.Where(p => p.Name == nameOfPlayer).Select(p => p.AttackPoints += hammer.AttackPoints);
                return string.Format(PlayerEquiped, nameOfPlayer, typeOfItem);
            }

            else if (typeOfItem == "Knife")
            {
                var knife = this.itemFactory.EquipKnife(15);

                this.players.Where(p => p.Name == nameOfPlayer).Select(p => p.AttackPoints += knife.AttackPoints);
                return string.Format(PlayerEquiped, nameOfPlayer, typeOfItem);
            }

            else if (typeOfItem == "Sword")
            {
                var sword = this.itemFactory.EquipSword(60);

                this.players.Where(p => p.Name == nameOfPlayer).Select(p => p.AttackPoints += sword.AttackPoints);
                return string.Format(PlayerEquiped, nameOfPlayer, typeOfItem);
            }

            else if (typeOfItem == "Armor")
            {
                var armor = this.itemFactory.EquipArmor(100);

                this.players.Where(p => p.Name == nameOfPlayer).Select(p => p.DeffencePoints += armor.DefencePoints);
                return string.Format(PlayerEquiped, nameOfPlayer, typeOfItem);
            }

            return string.Format("There's no such item like {0}", typeOfItem);
        }

        private string CastPlayer(string nameOfPlayer, string skillName, string enemyPlayer)
        {
            // We do not need enemy player for some of the methods
            IPlayer enemy = this.players.SingleOrDefault(p => p.Name == enemyPlayer);

            //Mage Skills
            if (skillName == "Heal")
            {
                this.mages.SingleOrDefault(m => m.Name == nameOfPlayer).Heal();

                return string.Format(PlayerCast, nameOfPlayer, skillName);
            }

            else if (skillName == "BlackMagicAttack")
            {
                this.mages.SingleOrDefault(m => m.Name == nameOfPlayer).BlackMagicAttack(enemy);

                return string.Format(PlayerCast, nameOfPlayer, skillName);
            }

            else if (skillName == "DodgeAttackDefense")
            {
                this.mages.SingleOrDefault(m => m.Name == nameOfPlayer).DodgeSingleAttack();

                return string.Format(PlayerCast, nameOfPlayer, skillName);
            }

            else if (skillName == "EnergyShieldDefense")
            {
                this.mages.SingleOrDefault(m => m.Name == nameOfPlayer).EnergyShieldDefense();

                return string.Format(PlayerCast, nameOfPlayer, skillName);
            }

            else if (skillName == "WhiteMagicAttack")
            {
                this.mages.SingleOrDefault(m => m.Name == nameOfPlayer).WhiteMagicAttack(enemy);

                return string.Format(PlayerCast, nameOfPlayer, skillName);
            }

            //Warrior Skills
            else if (skillName == "CutByAxeAttack")
            {
                this.warriors.SingleOrDefault(m => m.Name == nameOfPlayer).CutByAxeAttack(enemy);

                return string.Format(PlayerCast, nameOfPlayer, skillName);
            }

            else if (skillName == "SpearAttack")
            {
                this.warriors.SingleOrDefault(m => m.Name == nameOfPlayer).SpearAttack(enemy);

                return string.Format(PlayerCast, nameOfPlayer, skillName);
            }

            else if (skillName == "BerserkMode")
            {
                this.warriors.SingleOrDefault(m => m.Name == nameOfPlayer).BerserkMode();

                return string.Format(PlayerCast, nameOfPlayer, skillName);
            }

            else if (skillName == "UseShieldDefense")
            {
                this.warriors.SingleOrDefault(m => m.Name == nameOfPlayer).UseShieldDefense(enemy);

                return string.Format(PlayerCast, nameOfPlayer, skillName);
            }

            else if (skillName == "BlockAttackDefense")
            {
                this.warriors.SingleOrDefault(m => m.Name == nameOfPlayer).BlockAttackDefense();

                return string.Format(PlayerCast, nameOfPlayer, skillName);
            }

            //Rogue Skills
            else if (skillName == "BowAttack")
            {
                this.rogues.SingleOrDefault(m => m.Name == nameOfPlayer).BowAttack(enemy);

                return string.Format(PlayerCast, nameOfPlayer, skillName);
            }

            else if (skillName == "SwordAttack")
            {
                this.rogues.SingleOrDefault(m => m.Name == nameOfPlayer).SwordAttack(enemy);

                return string.Format(PlayerCast, nameOfPlayer, skillName);
            }

            else if (skillName == "DeadAttack")
            {
                this.rogues.SingleOrDefault(m => m.Name == nameOfPlayer).DeadAttack(enemy);

                return string.Format(PlayerCast, nameOfPlayer, skillName);
            }

            else if (skillName == "ShieldDefense")
            {
                this.rogues.SingleOrDefault(m => m.Name == nameOfPlayer).ShieldDefense(enemy);

                return string.Format(PlayerCast, nameOfPlayer, skillName);
            }

            else if (skillName == "DodgeAttackDefense")
            {
                this.rogues.SingleOrDefault(m => m.Name == nameOfPlayer).DodgeAttackDefense();

                return string.Format(PlayerCast, nameOfPlayer, skillName);
            }

            else if (skillName == "CollectHealthPointsFromCorpses")
            {
                this.rogues.SingleOrDefault(m => m.Name == nameOfPlayer).CollectHealthPointsFromCorpses();

                return string.Format(PlayerCast, nameOfPlayer, skillName);
            }

            else if (skillName == "TakeRestHealthRestorationk")
            {
                this.rogues.SingleOrDefault(m => m.Name == nameOfPlayer).TakeRestHealthRestoration();

                return string.Format(PlayerCast, nameOfPlayer, skillName);
            }

            return string.Format("There's no such skill for you to cast.");
        }

      /*private void PrintReports(IList<string> reports)
        {
            var output = new StringBuilder();

            foreach (var report in reports)
            {
                output.AppendLine(report);
                output.AppendLine(new string('#', 20));
            }

            Console.Write(output.ToString());
        }*/
    }
}

redpajama_set_name: RedPajamaGithub


All that stands between Celtic and a place in the Europa League group stages is a home game against Lithuanian side Suduva. Celtic have a slight advantage going into the game having drawn the away leg 1-1, meaning a clean sheet guarantees them a place in the group stages. Here's the XI that we think Brendan Rodgers will select for the game: 1/12 SLIDES © Ian MacNicol/Getty Images Sport Celtic Predicted XI: Suduva (H) All that stands between Celtic and a place in the Europa League group stages is a home game against Lithuanian side Suduva.
Scott Brown (born 25 June 1985) is a Scottish professional footballer who plays as a central midfielder for Scottish Premiership club Celtic . Brown started his career with Hibernian, breaking into their first team in 2002.
Celtic Park will host 'A Match for Cancer' tomorrow, sponsored by Dafabet and Laduma, that will see Brendan Rodgers and Liverpool boss Jurgen Klopp lead teams of former footballers and Celtic legends. Stiliyan Petrov will captain the Celtic side, while James Milner will line-up as Liverpool's skipper. The returning faces to Paradise include Hoops legends Henrik Larsson and Jackie McNamara, as well as current captain Scott Brown. The Liverpool side also features a number of former Reds, including Dirk Kuyt, jamie Redknapp and Jason McAteer. Former Republic of Ireland goalkeeper Shay Given, who played for Celtic in his younger years, will play a half for both sides. All funds raised from the match and connected events will be donated to fighting cancer through the Celtic FC Foundation, The Stiliyan Petrov Foundation and The James Milner Foundation. The full teams are as follows: Petrov (Celtic) Team Stilyan Petrov, Shay Given, Artur Boruc, Henrik Larsson, Scott Brown, Hristo Stoichkov, Lubo Moravchik, Paddy McCourt, Tosh McKinlay, Jackie McNamara, Simon Donnelly, Gareth Barry, Luis Garcia, Gaizka Mendieta, Joos Valgaeren, Johan Mjallby, John Harrison, Robbie Keane, Tom Boyd, Paulo Ferreira, Martin Compston. Milner (Liverpool) Team James Milner, Shay Given, Chris Kirkland, Stephen Warnock, John Arne Riise, Micah Richards, Joleon Lescott, Jason McAteer, Jamie Redknapp, Marcos Senna, Emile Heskey, Owen Hargreaves, Dirk Kuyt, Joan Capdevilla, Jeremy Lynch, John Terry, Jermain Defoe.
Celtic Football Club (/ˈsɛltɪk/) is a Scottish professional football club based in Parkhead, Glasgow. The club was founded in 1887 and played their first match in May 1888, a friendly match against Rangers.
© Ian MacNicol/Getty Images Sport The captain is almost indispensable to Celtic, with his leadership skills invaluable.
Rodgers believes Celtic don't deserve Champions League football .
Celtic boss Brendan Rodgers says that his side don't deserve to be playing Champions League football this season, as per a report on Sky Sports. Celtic fell at the first hurdle in qualifying for the UEFA Champions League this season as they suffered defeat to AEK Athens in the play-off round. The defeat has resulted in the Hoops playing Europa League football this campaign and Rodgers believes it is where his side deserves to be. Rodgers was asked if he was disappointed after not qualifying for the Champions League group stages. "No, it's where we deserve to be." "We are not entitled to be in the Champions League, we didn't qualify – simple as that. We are right in the competition that we deserve to be in." "We respect that and it is our duty to get as far as we possibly can." Celtic will likely be without Kristoffer Ajer tomorrow with the player reportedly suffering from a virus.
Scott Brown insists his playing career is not over and wants to land a new deal. The 33-year-old is Yet the Parkhead captain is clear that day remains a long way in the future But Brown is clear he won't allow his long-term ambitions to impede his desire to play for Celtic as long as he possibly can.
Brendan Rodgers believes Scott Brown has earned the right to seal a lucrative move to Australia as speculation over the Celtic skipper's future intensifies. Celtic captain Scott Brown takes part in a training session.


text:
Q: The wireless internet does not work and the internet driver does not update The wireless internet does not work and the internet driver does not update. I have been trying for over 3 hours.

A: What is your wireless card name details
You can find by running this
sudo lshw
Mostly default drivers should work , which driver you trying to install manually
System Settings > Software and Update > Additional drivers
Does it show any options for additional drivers?

redpajama_set_name: RedPajamaStackExchange