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Tony & Gail Robles are proud to announce the launch of one of the newest companies in competitive pool, Silent Assassin Productions, Inc. (SAP). Based in New York, NY, SAP will be producing a variety of pool tournaments, including its first official event, the NYC 8-Ball Championships. The inaugural event will take place at Steinway Billiards in Astoria, NY this coming weekend June 6-8, 2014.
It's NYC's first ever BCAPL Regional Event and we are proud to offer 6 NYC Titles up for grabs. And the great part, there's a division for every level of player and you'll only play people similar in handicap to you! We also plan on running the NYC 9-Ball, 10-Ball and 14.1 Championships in the future!
Livestreaming: This event will be live-streamed by AZBtv and co-produced by NYC Grind! Wanna watch the live-stream, click here! Play starts Friday at 6pm. AZBtv has been on the scene for many of our events including the Ginky Memorial, Gotham City Billiards, Cue Bar, Raxx Pool Room, Mr. Cue Billiards and other rooms that host the Predator Pro/Am Tour.
There are still spots available but they are going quickly! For a list of tournaments and who's signed up, click here!
Entry Fees: All amateur events are $100 to enter. Our Grand Masters event is $150.
3-Day Event: The event will now take place Friday evening, June 6th through Sunday the 8th.
Men's Advanced starts Friday at 6pm.
Men's Open starts Friday at 8pm.
Men's & Women's Leisure start Saturday at 9am.
Men's Masters starts Saturday at 11am
Grand Masters starts Sunday at 11am.
Women's Open & Masters were cancelled.
Green Fees: $25 green fee which is already included in the entry above.
Not a BCAPL member? Ok. You can still play. There is an additional $25 BCAPL member fee to pay in addition to the $100 entry fee if you are not a current BCAPL member. This does not apply to Grand Masters.
Smaller Fields: We have lowered the number of entries in most of the divisions. This means an even bigger chance of becoming an NYC Champion so sign up now before spots fill up!
What are the Payouts? Payouts are subject to how many players we get in each division but the full entry fee less green fees will be added to each divisions payout. We have a confirmed $2,000 being added to the event and each division will get a portion of this added money!
This is a great way to warm-up for the BCAPL National Championships!
The 1st Annual NYC 8-Ball Championships will feature six NYC titles:
Men's Leisure
Women's Leisure
Men's Open
Men's Advanced
Men's Masters
Men's Grandmasters
For information on which division you'd be qualified for, please visit our website's events page for events information. EVENTS PAGE
Joining Tony & Gail, the NYC 8-Ball Championships will feature one of New York's hardest-working people in pool on its team: referee John Leyman (BCAPL National Referee & Amsterdam Billiards League director.)
Silent Assassin Productions is expecting well over 100 players, and strongly recommend all players sign up as soon as possible on the championship's newly-launched site, www.NYCPoolChampionships.com as all events are limited to a specific number of players.
To confirm your qualification, please contact Tony & Gail Robles at email@example.com before signing up.
For more information, please visit www.NYCPoolChampionships.com
Additional Information:
This is the first NYC Pool Championships, brought to you by Silent Assassin Productions, Inc. This BCAPL regional event is a singles only tournament and offers competition for every level of play from beginner to professional. SAP will also offer tournaments in 9-Ball, 10-Ball and 14.1 in the future so stay tuned!
Predator Cues, CSI, Steinway Billiards, NYC Grind, Delta-13 Racks, The National Amateur Pool League, Gotham City Technologies, AZ Billiards and Billiards Digest are the official sponsors of the NYC 8-Ball Championships.
Special thanks to Gail Robles & Ron Mason of Gotham City Technologies who put in an enormous amount of work in designing and creating the NYC 8-Ball Championships website.
Looking forward to seeing everyone at this historic event!


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On the Bookshelf
Faculty books
Palgrave Macmillan, $159.99
Though international education's impact on social, economic and leadership development has been thoroughly documented, data on the influence of international scholarship programs have been less plentiful. Dassin, professor of international education and development at Heller, steps in to fill the void, compiling current research and analysis to flesh out how international scholarships benefit individuals, countries, geographic regions and the globe.
Alumni books
Simon & Schuster, $37.50
Civil-rights historian Arsenault presents a compelling, exhaustively researched biography of Arthur Ashe, the African-American tennis star who splintered color barriers and fought hard to help disenfranchised people gain equality and respect. When Ashe died in 1993 at age 49 after contracting HIV from a blood transfusion, he had not only changed the face of tennis, he'd transformed our understanding of what a socially engaged sports hero can do.
Sarabande Books, $17.95
In this vivid, beautifully written historical novel, teenager Chaya and little brother Asher, Russian Jewish immigrants, flee their family's failing Wisconsin farm for a hard-knock life in Gilded Age Chicago. Chaya, who gets a job making cigars, is soon being courted by a rich family's socialist son. Asher, a street thief, moonlights as a child savant to entertain guests at fashionable parties. Against the backdrop of the 1893 World's Columbian Exposition, the siblings struggle for a foothold in a changing America.
Random House, $35
Kertzer, the Brown University social science professor who won a 2015 Pulitzer for "The Pope and Mussolini," again brings the complicated histories of the Vatican and Italy to life with an account of the pivotal 32-year reign of Pope Pius IX (1846-78). Alarmed by the revolutions that were ending aristocratic dynasties across Europe, Pius asserted his divine right to rule; worked against Italian unification; claimed the notion of papal infallibility; and opposed extending freedoms of speech, press, association and religion to all, steering the Roman Catholic Church into a deeply conservative stance.
Harvard University Press, $27.95
Remarkably, the "13 Keys to the White House" prediction model Lichtman created in 1981 has accurately foretold the outcome of every presidential election since then. Here, the political historian turns his attention to voting rights, which, when not protected, means every American will lose. Voter ID laws, gerrymandering and felon disenfranchisement are some of the assaults on the electoral process discussed.
Simon & Schuster, $27
Schneider, a former CNN senior political analyst, assesses the past 50 years of U.S. presidential politics, frayed by the pull of progressive reforms on the left and conservative backlash on the right. By explaining how Americans choose their issues and cast their votes, "Standoff" attempts to make sense of the candidates we elect, including the 2016 winner, whom, as Schneider points out, 61 percent of voters believed wasn't qualified to serve as president.
Rowman & Littlefield, $35
A companion piece to Shapiro's 2012 book, "The Last Great Senate," "Broken" dissects and diagnoses the U.S. Senate's accelerating decline from the early 1950s to now. Gridlock and hyper-partisanship have eroded the once-august body's promise and possibility, a sad state not lost on the senators themselves. "We have been miniaturized," observed Sen. Olympia Snow (R-ME) upon her retirement six years ago.
Wendy Lamb Books, $17.99
Growing up on Alcatraz Island in the 1930s is a mixed blessing for 13-year-old Moose, whose father is assistant warden at the prison. Like its predecessors, this fourth book in Choldenko's funny yet bittersweet Tales From Alcatraz series, for readers ages 8-12, includes encounters with Al Capone, the prison's most famous resident. At its heart, however, it's a warm family story, as Moose worries about his older sister, Natalie, who has autism; hones his baseball skills; and navigates the trials of adolescence.
Asking Matters, $29.95
You — yes, you — can learn to ask for the support that turns a great idea into a successful nonprofit, writes Saber, who is a fundraiser, coach and consultant. You just have to decide whether you're analytic or intuitive, introverted or extroverted, then adopt the appropriate asking style: rainmaker, go-getter, mission controller or kindred spirit. Saber offers clear, practical advice geared to each asker type.
Kar-Ben Publishing, $7.99
In Uganda, little Sara and her Auntie Sanyu celebrate Sukkot with a lion, a parrot, a camel and other friendly creatures. But will the avid wart hog who shows up first let go of the etrog so everyone else can enjoy it, too? A delightful story with winsome illustrations, for readers between the ages of 4-8.
Little, Brown Spark, $27
Social psychologist Tavris and her co-author make a well-argued case in favor of hormone replacement therapy after menopause. "Estrogen Matters" takes issue with the nearly-20-year-old finding that connected HRT with an increased risk for breast cancer, presenting scientific evidence that underscores HRT's benefits. As the authors assert, "The powerful belief that estrogen causes breast cancer has blinded otherwise reputable, serious investigators to what their own data actually reveals."
Clarion Books, $17.99
Written for history buffs ages 6-9, "Thirty Minutes Over Oregon" tells the true story of a Japanese pilot who, a few months after the Pearl Harbor attack, dropped bombs on a wooded area in coastal Oregon. Twenty years later, he returned to the U.S. to apologize. (A second 2018 book by Nobleman, "Fairy Spell: How Two Girls Convinced the World That Fairies Are Real," introduces young readers to another true story, about an infamous paranormal hoax cooked up by a couple of English girls in 1917.)
Wiley-Blackwell, $25.95
In fewer than 300 pages, this volume delivers a comprehensive, accessible introduction to Jerusalem, whose roots reach all the way back to the Middle Bronze Age, 2000-1500 BCE. The book's first section focuses on the present-day city; the second on its biblical past; the third on its imperial incarnations. The fourth and final part returns to modern Jerusalem, from late Ottoman times through its subsequent transformations. Zank is a professor of religion at Boston University and director of the Elie Wiesel Center for Jewish Studies.
Greenleaf Book Group Press, $23.95
Cloobeck, the founder and former CEO/chair of Las Vegas-based Diamond Resorts International, uses his hospitality-industry expertise to help you improve how you deal with people, problems and possibilities, in both your personal life and your work. Sample pieces of advice: "You learn more about a company from studying its culture than its strategy." "Spend your time obsessing over your customers, not your competition." "The language of hospitality is universal. The smallest gestures can leave the biggest impressions."
Princeton University Press, $24.95
"History is a novel for which the people is the author" goes the maxim. These essays by political historians, edited by Princeton historian Zelizer, begin the work of putting the Obama administration into its proper context, assessing its successes and failures within a year of the first African-American president's departure from the White House. For one thing, says Zelizer, despite his formidable wins as a policymaker "Obama did not leave behind a coalition that, at least in the short term, has the muscle to protect what he built."
Monthly Review Press, $19
The United States would do well to avoid repeating the mistakes of its 1945-91 Cold War with the Soviet Union, such as the "skewed priorities" that caused the U.S. government to spend 57 percent of its budget on the military and just 6 percent on education, health, labor and welfare programs. So argue Kuzmarov, assistant professor of American history at the University of Tulsa, and his co-author, who believe a new Cold War with Putin's Russia could prove similarly hysterical and dangerous.
Mascot Books, $16.95
In the space of two weeks during Deane's final semester at Brandeis, both his mother and his brother committed suicide. Deane's decades-long quest to understand this double tragedy and its effect on him is at the center of this moving, incisive memoir. "I trust my memory," he writes. "It has protected me from my greatest foe: ambiguity."
Ballantine Books, $28.99
The Kellermans roll out another crime thriller starring deputy coroner Clay Edison, as he travels a perilous path to identify a Jane Doe who's been brutally murdered in the Bay Area. No less an authority than Stephen King has praised the father-son writing team for their "brilliant, page-turning fiction."
Indiana University Press, $38
Sholes, assistant professor of music history at Central Connecticut State University, analyzes the musical references to works of earlier composers — J.S. Bach, Haydn, Mozart, Schubert and, especially, Beethoven — that Johannes Brahms wove throughout his own instrumental music. These influences, the author writes, both "inspired and in some cases burdened him as he emerged with his own unique artistic voice and established his own place in music history."
Platypus Media, $9.95
In this sweet bilingual book, accessible to speakers of English and Spanish, realistic illustrations show very young readers how various members of the animal kingdom care for their babies. Be they pandas, wolves, manatees or humans, attentive parents are shown looking after their little ones with tenderness and patience.
University of Chicago Press, $35
Rosen, professor emeritus of anthropology at Princeton and adjunct professor of law at Columbia, traces the intricacies of Islamic law by dispelling the misperceptions and oversimplifications often held by Westerners. These include the assumptions that Islamic law "invariably disadvantages women and minorities," "is predominantly characterized by brutal punishments" or "is concerned with enforceable rules not procedural justice."
Princeton University Press, $29.95
Can acts of violence ever be considered holy according to Jewish teachings? No, concludes Mittleman, who teaches Jewish philosophy at the Jewish Theological Seminary. In his journey to this answer, Mittleman ponders ethical principles as well as the meaning of "holiness" within the context of the Jewish tradition.
Ben Rose Creative Arts, $14.99
In his third performing-arts book for young actors, Kimmel presents 60 original monologues that can help them steal the spotlight at auditions; put together a stellar demo reel; or improve their film, television or theater performances. Monologues, Kimmel reminds his readers, are less a speech than "a way in which you're finally permitted to talk out loud to yourself in public." Throughout, the book encourages teens to be generous and selfless in their acting and their lives.
CreateSpace, $50
Educator Kahn helps struggling readers and writers gain a better understanding of syntax, organization and punctuation, and sharpen their reading and writing skills. This comprehensive text, which includes practice exercises, can be used as a resource for many learners, including high-school and college students, English as a Second Language students and people with dyslexia.
Yale University Press, $35
In 17th-century Massachusetts, John Wompas, a Nipmuc Indian, enrolled at Harvard, then left before graduating to embark upon a checkered career, becoming a sailor, a land speculator, a notable spendthrift and a defender of the Nipmucs' rights against the encroachment of English settlers. Pulsipher, associate professor of history at Brigham Young University, meticulously reconstructs the life of a fascinating man who drew upon his knowledge of polar-opposite worlds to chart a singular course in life.
Allworth Press, $29.99
Aspiring actors, English majors and matinee habitués will applaud the scope and clarity of this effort, written by a theater teacher/director/performer. From explanations of what actors, directors and producers do; to offering a new way to watch a play; to the 10 plays every theater student should see, "The Essentials of Theater" is a key to stellar stagecraft and drama knowledge.
Cambridge University Press, $34.99
Money changes everything, the English aristocracy discovered during the Tudor and Stuart dynasties. Merchants were amassing great wealth, and their daughters were marrying into blue-blood families, bringing, along with much-needed funds, new ideas and attitudes into the upper classes. Historian Peck details how upwardly mobile brides from merchant backgrounds — particularly those with an entrepreneurial or scholarly bent — helped spark lasting societal, economic and cultural changes within the old order.
Cornell University Press, $45
During the first decades of the 20th century, soon after the U.S. takeover of Puerto Rico, Puerto Ricans began coming to the U.S. mainland to look for jobs. The legal and labor disputes that resulted revolved around a difficult-to-answer question: Were the subjects of colonial territories "foreign" or "domestic"? McGreevey, an associate professor of history at the College of New Jersey, digs into this chapter of American history to map the tensions between colonialism, law and migration.
University of California Press, $85
An assistant professor of anthropology at Harvard who has worked as an emergency responder on the boundary between Arizona and Sonora, Jusionyte tells gripping stories of the professionals who serve in firefighting and rescue units at that border. As U.S. and Mexican authorities collaborate to handle life-or-death emergencies in unforgiving terrain, "wall" isn't part of the lexicon, and political topography means next to nothing. "When we are helping Mexico," one U.S. public-safety official is quoted as saying, "we are helping ourselves."
Seventh Street Books, $15.95
Novelist Cooper (a former TV reporter) adds a second volume to his Gus Parker/Alex Mills thriller series. Detective Mills and psychic Parker combine their talents to solve another Arizona crime mystery: Someone is leaving freshly dead bodies on the grounds of Phoenix cemeteries. Can the sleuths unmask the murderer before more Phoenicians meet a grisly end?
Brandeis University Press
He wrote the lyrics to "Me and My Shadow" and "It's Only a Paper Moon." He produced Broadway shows. He oversaw lavish spectacles showcasing stripper Sally Rand and "America's mermaid" Esther Williams, and an adaptation of Bizet's "Carmen" featuring an all-black cast. He was Billy Rose (1899-1966), whose outsized successes made him a household name, "a marvelously exaggerated example of the Jewish-American experience," writes Cohen. This is the first biography in 50 years of a man who supported Jewish war refugees and Israel as eagerly as he promoted himself and his entertainment empire.
The dean of Hebrew College's Rabbinical School, Judson "follows the money" to delineate the priorities of American synagogues from 1728 to the present day, analyzing how synagogues have raised and spent their funding. "The American embrace of free market principles in religion has influenced almost every aspect of synagogue life," he writes, "from the pay and quality of rabbis, to the size and architecture of synagogues, to even […] Jewish ritual itself." Part of the Brandeis Series in American Jewish History, Culture and Life.

subset: commoncrawl


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FG to Formalise Artisanal and Small Scale Miners
The Minister of Solid Minerals Development, Dr Kayode Fayemi has said that activities of artisanal and small scale miners in the country would be formalized to ensure sustainable development of the mineral resources and enhance the sector's contribution to national economic growth.
Fayemi said this in Ilorin at the Opening Ceremony of the 52nd Conference of the Nigerian Mining and Geosciences Society, NMGS, stressing that at the moment 80 percent of mining activities in some parts of the country is done by illegal artisanal miners.
He said his ministry will explore the possibilities of using information technology solutions to empower this group of miners through the establishment of ICT Tele Centres, as "it is important that miners have access to the most efficient supporting knowledge and technology".
The Minister disclosed that 300 men of the National Civil Defence Corps have been deplored as mines police to check the activities of illegal miners whom he described as menace to the growth of the mining sector, especially with respect to revenue generation and smuggling of mineral commodities".
Kwara State Governor, Dr Abdulfatah Ahmed in his speech said the mining sector can serve as a key driver in the quest for sustained economic growth and the development of the country.
Gov. Ahmed noted that it was unfortunate that with more than 40 different solid minerals spread across the country, that sector contributed only 0.34 percent to the country's GDP.
According to the Governor, with about five mining licenses in the state, his administration has established a special purpose vehicle to drive the exploitation and exploration of mineral resources for more earnings from the non-oil sector.
The governor identified mineral resources abundant in the state as including Gold, Kaolin, Marble, Dolomite, Tourmalines and Limestone that could be exploited for the industrial development of the State.
Dr Ahmed said the state government has set up the Midway Minerals Development Company Limited as a Special Purpose Vehicle to acquire mining licenses and promote investment in the sector.


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How to Make the Best Mexican Brownies
Just a touch of spice (and a special frosting) makes this Mexican brownie recipe extra nice.
Your friends and family might never guess that cinnamon is the secret ingredient in this Mexican brownie recipe. But a pinch of cayenne will add a warm spicy flavor to the recipe, too. To amp up the chocolate flavor, you can also try adding a pinch of instant coffee granules to the batter before baking.
Brush up on these tips for baking better brownies before you get started.
How to Make Mexican Brownies
For the Brownies:
- 3/4 cup baking cocoa
- 1/2 teaspoon baking soda
- 2/3 cup butter, melted, divided
- 1/2 cup boiling water
- 2 cups sugar
- 2 large eggs, beaten, room temperature
- 1 teaspoon vanilla extract
- 1-1/3 cups all-purpose flour
- 1-1/2 to 2 teaspoons ground cinnamon
- 1/4 teaspoon salt
- 1 cup (6 ounces) semisweet chocolate chips (See what brand our Test Kitchen recommends.)
For the Frosting:
- 6 tablespoons butter, softened
- 1/2 cup baking cocoa
- 2-2/3 cups confectioners' sugar
- 1 to 1-1/2 teaspoons ground cinnamon
- 1/3 cup evaporated milk
- 1 teaspoon vanilla extract
Forgot to soften butter ahead of time? Here's what to do in a pinch.
Step 1: Prepare the batter
In a bowl, combine cocoa and baking soda; blend in 1/3 cup melted butter. Then add boiling water, stirring until thickened. Stir in sugar, eggs, vanilla and remaining butter. Next, add your flour, cinnamon and salt. Gently fold in the chocolate chips.
Step 2: Bake your brownies
Pour mixture into a greased 13 x 9-in. baking pan. Bake at 350° until brownies are done, about 40 minutes.
Test Kitchen tip: Brownies continue cooking even after you remove them from the oven, so if you wait until there are no crumbs on the toothpick, you'll end up with an overcooked batch. For best results, stick to the time recommended in your recipe. See more mistakes everyone makes when baking brownies.
Step 3: Fix the frosting
Let the brownies cool completely. For frosting, cream butter in a bowl. Combine cocoa, confectioners' sugar and cinnamon; add alternately with the milk. Beat to a consistency that's easily spreadable and add vanilla. You can add more milk, if necessary.
Step 4: Finish and share
Spread the frosting over cooled brownies and cut into pieces. This recipe makes approximately 3 dozen brownies—and they'll all be gone before you know it!
Test Kitchen tip:To cut brownies more easily, warm the knife blade in hot water, dry and cut. Clean and rewarm the knife after each cut.
Want to try another Mexican dessert featuring cinnamon? Check out the recipe for reganadas—traditional Mexican cinnamon cookies.


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Chronic lymphocytic leukemia (CLL) arises from the clonal expansion of a CD5+ B lymphocyte that is thought not to undergo intraclonal diversification. Using VHDJH cDNA single strand conformation polymorphism analyses, we detected intraclonal mobility variants in 11 of 18 CLL cases. cDNA sequence analyses indicated that these variants represented unique point-mutations (1–35/patient). In nine cases, these mutations were unique to individual submembers of the CLL clone, although in two cases they occurred in a large percentage of the clonal submembers and genealogical trees could be identified. The diversification process responsible for these changes led to single nucleotide changes that favored transitions over transversions, but did not target A nucleotides and did not have the replacement/silent nucleotide change characteristics of antigen-selected B cells. Intraclonal diversification did not correlate with the original mutational load of an individual CLL case in that diversification was as frequent in CLL cells with little or no somatic mutations as in those with considerable mutations. Finally, CLL B cells that did not exhibit intraclonal diversification in vivo could be induced to mutate their VHDJH genes in vitro after stimulation. These data indicate that a somatic mutation mechanism remains functional in CLL cells and could play a role in the evolution of the clone.
Chronic lymphocytic leukemia (CLL)* is the most prevalent adult leukemia in Western countries, accounting for ∼30% of all leukemias (1). It is characterized by the clonal expansion of a CD5+ B cell (2) that was in the past viewed as having a poor propensity to undergo Ig V(D)J gene hypermutation (3–7). This view, however, changed when it was documented that the leukemic cells from ∼60% of CLL patients express Ig VH gene somatic point-mutations (8–13). The frequency of these mutations relates to the VH family expressed, being higher in those cells expressing VH3 genes than in those expressing VH1 and VH4 (13). However, even those studies that described CLL cases with a very high number of somatic mutations reported that these mutations were shared by all the CLL B cells, strongly suggesting that intraclonal diversification does not occur in these leukemic B cells (14–17).
The lack of intraclonal diversification in CLL B cells was considered consistent with the absence or scarcity of somatic mutations in human (2, 18) and murine (19–21) B1a cells. This, however, was not supported by the findings that human CD5+ B cells can undergo somatic hypermutation, antigen selection, and possibly affinity maturation (22–25). Rather, these findings supported the postgerminal center-like features that have been recently suggested to be characteristic of certain CLL cases such as V gene mutations (13), CD38 expression (26), and in vivo isotype class switching (6, 9, 11, 13, 27–29).
Prompted by these discrepancies and the recent advances in the understanding and induction of somatic hypermutation in human B cells (30–33), we decided to determine whether CLL B cells continue to develop V gene mutations after leukemic transformation and, therefore, intraclonally diversify. Using a highly sensitive and ad hoc devised approach (32) based on single-strand DNA conformational polymorphism (SSCP), we determined that intraclonal VHDJH gene diversification and clonal evolution are common features of the in vivo natural history of many CLL B cells. In addition, using appropriate stimuli, we induced somatic mutations in vitro in those leukemic B cells that did not show evidence of in vivo intraclonal diversification. These findings indicate that a hypermutation machinery is functional and active in certain CLL B cells and further point to the importance of a germinal center-like reaction in some of these B lymphocytes.
Materials And Methods
CLL Patients and B Cells.
The 18 patients (12 males and six females) in this study were diagnosed with typical B cell CLL based on clinical criteria and laboratory features. All cases represented clonal expansions of small lymphocytes with high nuclear/cytoplasmic ratios that coexpressed CD19, CD5, CD23, and CD27 along with monotypic H and L chain surface membrane Ig. The median age of the group at the time of analysis was 60 y with a distribution of clinical Rai stages as follows: 10 patients in stages O-II and eight patients in stages III-IV. PBMCs from these patients were obtained from heparinized venous blood by fractionation through Histopaque 1077® (Sigma-Aldrich). B lymphocytes were enriched from PBMCs by depletion of T cells and monocytes as described previously (31). All patients provided informed consent before giving blood samples.
PCR Amplification of V(D)J Transcripts.
RNA was extracted from 2 × 106 B cells using the RNeasy™ Total RNA kit (QIAGEN). mRNA was reverse transcribed using the SuperScript™ Preamplification System for first strand cDNA synthesis (Life Technologies, Inc.). VHDJH-CH cDNAs were amplified using sense primers specific for the sequences of the different VH families together with the antisense primers specific for the CH1-μ sequence (13, 32) and the Pfu Turbo® DNA polymerase (Stratagene). Each reaction consisted of 30 cycles (1 min denaturation at 94°C, 1 min annealing at 58°C, 1 min extension at 72°C, and 10 min extension at 72°C). The PCR cDNA products were purified, ligated into pCR-Blunt II-TOPO vector (Zero Blunt™ TOPO™ Cloning kit; Invitrogen), and transfected in TOP10 One Shot™ competent cells (Invitrogen). Bacterial colonies were screened by PCR and those positive for VHDJH-CH transcripts were selected for SSCP analysis.
Detection of Mutated VHDJH Transcripts by SSCP.
Mutated VHDJH transcripts were identified by SSCP analysis as described previously (32). In brief, cDNAs were amplified with Taq DNA polymerase (Life Technologies, Inc.) by PCR using the cloned cDNA inserted into pCR-Blunt II-TOPO vector as template, in the presence of 1 μCi [α-32P] dCTP (3,000 Ci/mmol; NEN Life Sciences). The internal VH leader sense primer and JH antisense primer (31) were used for VHDJH analysis. Samples were denatured and immediately loaded onto a 6% acrylamide gel (20:1 acrylamide:bis) with 1 × TBE containing 10% Glycerol. Electrophoresis was performed at room temperature for 18 h at 6 W. Gels were autoradiographed on Kodak X-Omat™ AR film (Kodak).
Sequencing Ig V(D)J-C Transcripts.
The Ig VHDJH cDNA clones displaying an altered electrophoretic mobility in SSCP gel as well as at least 5 clones from each patient with typical mobility were analyzed by sequencing to confirm and characterize the nature of the mutations (13, 32). Sequences were compared with the germline counterpart (34) and with the original CLL VHDJH sequence using MacVector v. 5.0 software (International Biotechnologies).
Mutational Analysis.
The census of the somatic point-mutations was determined by counting identical mutations in more than one transcript only once. Comparison of the observed with the expected frequency of replacement (R) and silent (S) point-mutations was performed using the inherent mutation rate of the CLL VHDJH sequences, calculated using the Inh. Sus. Calc. Program, version 1.0 for the Macintosh as reported by B. Chang and P. Casali (35). The expected frequency of mutations was calculated by taking into account the base composition of the unmutated CLL V(D)J sequence, i.e., it was corrected by the frequency of occurrence of the individual nucleotides, or di-, tri-, tetra-nucleotides considered within the CLL B cells V(D)J sequence assuming randomness. In the absence of negative or positive selective pressure on a gene product, nucleotide changes yielding amino acid R or S mutations are randomly distributed throughout the coding sequences. If a DNA segment displays a number of R mutations higher than that expected by chance alone, a positive selective pressure for variability is the likely cause. Conversely, if a DNA segment displays a number of R mutations lower than that expected by chance, it is likely that a negative pressure was exerted on the gene product to select against mutations, such that the protein structure is preserved.
T Cells.
CD4+ T cells were positively selected from PBMCs by fractionation through Histopaque 1077® (Sigma-Aldrich) using CD4-conjugated magnetic beads® (Miltenyi Biotec). Selected cells were cultured in FCS-RPMI 1640, and expanded by weekly stimulation with a feeder cell mixture containing irradiated (1,200 rads) PBMCs, 100 μg/ml of phytohemagglutinin (Life Technologies Inc.), and 100 U/ml of human recombinant IL-2 (Genzyme). For T/B cell coculture experiments, CD4+ T cells were used at least 2 wk after their last activation, and were incubated for 6 h with 20 ng/ml of 13-phorbol 12-myristate acetate (Sigma-Aldrich), and 500 ng/ml of ionomycin (Calbiochem-Novabiochem) before culture with B cells.
B/T Cell Cocultures.
B/T cell cocultures were performed as described previously (31). In brief, CLL B cells were cultured at 0.5 × 105 cells per well in the presence of 2.5 × 105 irradiated (4,000 rads) CD4+ T cells, 106 irradiated (4,000 rads) human CD40L-transfected 293 cells (CD40L-293 cells) and cytokines, including IL-4 (100 U/ml) and IL-2 (100 U/ml) in a U-bottomed, 96-well plate (200 μl FCS-RPMI 1640 vol). To cross-link the BCR, CLL B cells were incubated for 2 h at 4°C with Sepharose®-conjugated rabbit Abs to human IgM and Ig (H + L) chain (2 μg/ml; Irvine Scientific), and then washed with cold PBS. After 7 d of culture, CLL B cells were collected, freed of dead cells and debris by fractionation through Histopaque® 1077 (Sigma-Aldrich), exposed again to anti-BCR Abs, washed, and reseeded over a new layer of irradiated T cells and CD40L-293 cells, in the presence of cytokines. At day 14 of culture, CLL cells were harvested for total RNA extraction.
CLL VHDJH Gene Diversification Determined by SSCP Analyses.
The VH, D, and JH genes expressed by the 18 CLL cases included in this study are listed in Table I. Of the VH genes identified, VH3 family members were the most frequent, and JH4 was the most represented JH gene, as reported for other CLL cases (13). The origin of two D gene segments could not be determined.
The Ig VHDJH cDNAs amplified by PCR using high-fidelity Pfu Turbo® DNA polymerase were cloned into appropriate vectors for nested PCR amplification in the presence of [α-32P] dCTP. The amplified [α-32P] labeled VHDJH cDNAs were then subjected to SSCP analysis. The cDNAs from seven cases were homogeneous in electrophoretic mobility, as exemplified by case 216 (Fig. 1 A). In the remaining 11 cases, the VHDJH cDNA transcripts displayed inconstant patterns of electrophoretic mobility, suggesting a variable degree of intraclonal diversification, as exemplified by case 105 (Fig. 1 B).
DNA Sequence Analyses Confirm that CLL B Cells Can Accumulate New Ig V Gene Mutations and Thereby Intraclonally Diversify.
The sequences of the VHDJH cDNAs that differed in their SSCP mobility profiles from the majority of the VHDJH cDNAs and the sequences of at least five cDNAs representative of the dominant mobility profile were determined. In each case, all the VHDJH cDNAs were collinear, strengthening the monoclonality of the leukemic cells. However, as expected, intraclonal nucleotide differences were detected in those VHDJH cDNAs that displayed altered electrophoretic mobility (Table I). These findings were consistent, regardless of how many transcripts were analyzed. This suggests that the data provided by our SSCP analyses accurately reflected the nature of the in vivo process.
The nucleotide differences detected in these sequence analyses were distributed randomly throughout VH. In most instances, these differences were single base substitutions, resulting in both S and R mutations. The frequency of R mutations in the complementarity determining regions (CDR) and framework regions (FR) of the VH gene segments were not different from those expected by chance alone, suggesting the lack of a selective pressure applied to these Ig VH gene products.
Table I lists the number and characteristics of the cDNAs analyzed in each CLL case. These transcripts are noted as containing either "shared" (mutations present in all the Ig VHDJH gene transcripts analyzed), "partially shared" (mutations exhibited by some but not all VHDJH transcripts), or "unique" point-mutations (mutations found only in distinct VHDJH transcripts) as compared with the respective germline template. The number of partially shared and unique point-mutations differed between the different cases analyzed, ranging from 1 (case 270) to 35 (case 105).
The Presence of Intraclonal Diversification Is Not Related to the Initial Load of Shared Mutations.
CLL cases can be divided into two subgroups based on the presence of Ig V gene mutations (13). To determine whether the degree of leukemic B cell intraclonal diversification was related to this original mutational load, we analyzed by linear regression the relationship of the number of partially shared and unique mutations to the total number of mutations (Table I). When all the cases were included in this analysis, the degree of intraclonal diversification was found to be dependent on the overall load of point-mutations (P < 0.05). However, when we excluded from the analysis the two most intraclonally diversified cases (105 and 261) that were atypical in their extent and patterns of diversification (vide infra), the relationship was no longer significant (P > 0.1). Thus, intraclonal diversity appears to occur in CLL B cells regardless of their original Ig V gene mutational load.
The High Degree of Intraclonal Diversification in Certain CLL Cases Allows the Construction of Genealogical Trees.
To outline the evolution of the changes that occurred within each CLL clone, we aligned all the VHDJH cDNAs to the closest germline gene sequence and analyzed them on the assumption that the shared mutations occurred due to single rather than independent events. This allowed us to identify in each case the putative progenitor VHDJH gene sequence and to assess the level of intraclonal complexity. Of the 11 cases that exhibited intraclonal diversification, nine (four originally unmutated CLL 141, 156, 258, and 270, and five originally mutated CLL 113, 169, 175, 178, and 249) displayed only one level of diversification, i.e., they expressed only unique mutations and no partially shared mutations. However, two cases, CLL 105 and 261, displayed different levels of complexity with several partially shared mutations that allowed us to construct genealogical trees, as reported previously (36, 37).
CLL 261 showed 14 unique and eight partially shared point-mutations, compared with the progenitor VHDJH gene sequence (Fig. 2 A). These mutations identified a genealogical tree with three branches that spanned 1–3 generations. CLL 105 showed 24 unique point-mutations and 11 partially shared mutations, compared with the progenitor VHDJH gene sequence (Fig. 2). There were five branches to this genealogical tree, spanning 1–3 generations.
Induction of Somatic Mutation in CLL B Cells In Vitro.
To determine whether CLL B cells could be induced to mutate the expressed VHDJH genes in vitro, the leukemic cells from CLL cases 136 and 216 were reacted with immobilized Abs to human Ig, and then cultured in the presence of activated normal allogeneic human CD4+ T cells and IL-2 and IL-4. After 14 d of culture, VHDJH-CH cDNAs were analyzed as illustrated in Fig. 3. These cases were chosen for these studies because their B cells did not exhibit evidence for in vivo intraclonal variability (40 independent bacterial clones containing VHDJH cDNAs screened by SSCP and cDNA sequencing revealed that all cDNAs were identical; Table I).
After in vitro stimulation, 3 of 32 (9%) VHDJH cDNAs in CLL 136 and 5 of 34 (14.7%) in CLL 216 displayed a gel mobility different from that of the corresponding cDNAs from the unstimulated CLL cells (Fig. 4). All of the clones with a different gel mobility pattern in SSCP contained new point-mutations. In CLL 136, we detected three mutations. These were independent point-mutations confined to the VHDJH sequence (375 bp) and comprised three transversions (clone 11: 4 G > C; clone 20: 23 G > T; clone 9: 120 T > G). Thus, CLL 136 was induced to undergo mutation with an overall frequency of 2.7 × 10−4 changes/base, >sixfold the PCR amplification error rate with high-fidelity Pfu Turbo® DNA polymerase mentioned above. In CLL 216, a total of nine mutations were found in VHDJH transcripts. These were also independent point-mutations that were confined to the VHDJH sequence and consisted of nine transitions (clone 2: 25T > C, 36T > C, 88G > A; clone 4: 75T > C; clone 23: 87G > A, 21G > A; clone 32: 21T > C, 157G > A; clone 18: 75A > G). These mutations occurred at a frequency of 6.86 × 10−4 changes/base. Thus, these CLL cells were triggered to mutate their expressed VHDJH genes with the same modalities that induce mutations in normal B cells and monoclonal B cell lines (30, 31).
Comparison of the Mutations Induced In Vitro with Those Occurring In Vivo.
Randomly occurring somatic point-mutations are expected to be one-third transitions and two-thirds transversions (38). Among the 128 original in vivo shared VHDJH point-mutations, transitions exceeded transversions, 72:56 (Table II). As in normal B cells, the original shared mutations targeted A nucleotides at a frequency (46/128 total mutations) that was ∼48% higher than expected by chance alone, after correcting for base composition, i.e., normalizing for the relative occurrence of A in the unmutated VHDJH sequence. A > G mutations accounted for 59% of the total A mutations and 38% of the total transitions observed (Table II).
The 74 in vivo partially shared and unique point-mutations showed a similar, albeit lesser bias for transitions over transversions (39:35). However, these mutations lacked an A base preference since only 15/74 mutations (20%) targeted A nucleotides. Among the two cases with the more extensive partially shared and unique mutations, CLL 261 demonstrated some evidence for A targeting (9/22 mutations involving A), whereas CLL 105 exhibited minimal evidence for this tendency (2/35 mutations involving A). These two cases also differed in the ratio of transitions to transversions detected among these mutations (CLL 261–14:8 and CLL 105–15:20).
The in vitro–induced mutations also favored transitions over transversions (9:3). However, the small number of these mutations did not allow us to draw firm conclusions regarding their nature.
The generation of high affinity–specific antibodies usually occurs in the germinal centers of organized lymphoid tissues and depends on a complex series of interactions involving antigen activated B cells, specific T cells, and follicular dendritic cells (39). This process usually introduces point-mutations into Ig V genes and thus increases receptor diversity (40, 41). Subsequently, certain B cells are selected for survival and expansion based on the affinity for antigen of the BCR encoded by the newly mutated V genes (42, 43). Recently, it has been suggested that somatic mutations may occur via another pathway that does not require the participation of T cells and may occur outside of classical germinal center structures (44, 45).
CLL B cells have been viewed as B lymphocytes that cannot sustain effective somatic hypermutation of their Ig VHDJH genes. However, analyses of the expressed Ig V genes in CLL indicate that as many as 50% of IgM+ CLL and 75% of non-IgM+ (IgG and IgA) CLL cases show evidence of somatic mutations with a subset of these displaying R mutations in a pattern consistent with antigen selection (13). Thus, although the traditional views about the presence of Ig V gene mutations in the leukemic cells of this disease have been modified, the literature has remained consistent in characterizing these V gene mutations as static, without evidence for intraclonal instability and the accumulation of diversity. Occasional studies have reported the detection of somatic variants over time in typical CLL cases and in a CD5− case (11, 46, 47). However, these and other studies did not demonstrate detailed evidence that significant intraclonal diversification occurred in the leukemic cells of many CLL B cases, leading to the conclusion that somatic mutation ceases as a consequence of leukemic transformation (15–17). In this report, we document that intraclonal VHDJH gene diversification and clonal evolution do occur, albeit at variable degrees, in >50% (11/18) of CLL B cases. Additionally, in those cases in which this phenomenon is not detected in vivo, diversity can be induced in vitro after providing physiologically relevant signals.
It is unlikely that these findings represent PCR errors. First, the frequency of unique nucleotide changes ranged from 3.5-fold (case 270) to 58-fold (case 105) higher than the expected frequency of misinsertions calculated based on the error rate of the high-fidelity Pfu Turbo® DNA polymerase provided by the manufacturer (10−6 base/cycle, i.e., 0.01 base changes in a 375 bp DNA sequence after 30 cycles). This published error rate is actually somewhat higher than that calculated by us using the sequence data generated for the Ig CH region from the exact same cDNA used for the VH data presented above and from other cDNA prepared in an identical manner (0.6 × 10−6 base/cycle). Nevertheless, we have used the manufacturer's standard in order to be conservative about our estimations. However, even if we are extremely conservative in our calculations and exclude those cases with ≤2 point-mutations, a significant number of the remaining cases (7/18; ∼35%) still exhibit from 3–21 mutations, which is inconsistent with these PCR error rates. Second, when we subjected a subclone of the Burkitt's lymphoma cell line RAMOS (clone 1; references 48 and 49) to the same RT-PCR-SSCP cloning and sequencing procedure that we used for the CLL cases, a mutation frequency of 1.3 × 10−4/bp was identified, which compares favorably with the published mutation frequencies determined for this RAMOS subclone (0.8–0.93 × 10−4/bp; references 48 and 49). These two comparisons also suggest that the reverse transcription step was not a major source of error in our analyses, since these calculations of potential error are very similar despite the fact that our analyses were based on RNA as the starting material and the others on DNA.
Thus, the calculated rate of IgV gene mutations in these B cells is clearly greater than that occurring spontaneously in vivo elsewhere in the genome or to that attributable to in vitro PCR error with high-fidelity Pfu Turbo® DNA polymerase (Table I). It is also considerably lower than that seen in other B cell lymphomas thought to originate in the germinal center such as follicular lymphoma and Burkitt's lymphoma (50–54). These mutation frequency differences are in line with the in vivo proliferative differences of the malignant cells in CLL and these two subtypes of B cell lymphoproliferative disorders.
Our findings indicate that a somatic mutation process is active in at least certain members of the CLL clone, indicating that the leukemic cells are not functionally inert, but are in this regard functionally active. What is unclear is the extent to which this mutational activity is mediated by the classic IgV gene hypermutation mechanism, and also whether it is induced by external stimuli or is inherent to the B cells due to genetic alterations that occurred as a consequence of the leukemic transformation. Although our data do not directly indicate that the observed in vivo intraclonal diversification in CLL B cells is externally mediated, there is evidence from these studies to suggest that this could occur. In vitro stimulation through the BCR and other critical costimulatory molecules expressed on activated T cells induced mutations in those CLL B cells that did not display intraclonal diversity in vivo (Fig. 4). These mutations occurred at similar frequencies to those identified in vivo. We cannot formally exclude that mutated VHDJH transcripts predated the in vitro induction and were, in some way, positively selected in our culture conditions. However, the complete absence of mutating Ig VHDJH transcripts among the large number of B cells analyzed before in vitro stimulation (Table 1) makes this possibility unlikely. Thus, in these cases the BCR was still effective in inducing somatic mutation in vitro implying that in vivo external triggering could result in the observed Ig V gene mutational heterogeneity and suggesting a potential role for ongoing receptor stimulation in the evolution of the CLL clone. The identification of mutations in only a small subset of the CLL B cells might be consistent with this view, since presumably not all members of the clone would have access to the relevant antigen(s) or other BCR cross-linkers. Furthermore, the occurrence of intraclonal variation in most, but not all, CLL B cells would be consistent with differences in BCR-mediated signaling among CLL cases, as has been suggested (13, 55–59). Alternatively, these point-mutations could occur spontaneously or result from ongoing stimulation via other receptors in leukemic subclones made competent to receive them by genetic alterations. The apparent lack of selection for R mutations in the CDR and against R mutations in the framework regions among the partially shared and unique new mutations may support this interpretation.
In addition, the lack of targeting of R mutations to the CDR and the apparent different nature of the originally shared (A preference as in normal B cells) versus unique point-mutations (non-A preference) favor the idea that the unique point-mutations detected in the leukemic cells might occur via mechanisms different from the canonical Ig V gene hypermutation machinery. However, other features (Tables I and II) are compatible with a canonical process of somatic hypermutation (individual point-mutations resulting in more transitions than would be predicted for a stochastic event; reference 38). Further studies will be necessary to identify more precisely the mechanisms responsible for this intraclonal V gene diversity. In this regard, we have begun to study the relationship between the intraclonal VHDJH gene diversification and the downregulation of DNA pol η/pol ζ expression ratio. BCR engagement and subsequent Ig V(D)J gene mutation can be associated with downregulation of the translesion DNA pol η, and the maintenance or upregulation of the translesion DNA pol ζ (33), although studies in mice differ in this regard (60). In preliminary studies, we have analyzed the expression of DNA pol η and pol ζ in B cells of six CLL cases with and four without intraclonal diversification. DNA pol η was expressed at a normal level in all four cases without in vivo intraclonal diversification, but was significantly downregulated in all but one of the cases with intraclonal diversification (unpublished data). DNA pol ζ was expressed at comparable levels in all cases, yielding normal and low pol η/pol ζ expression ratios in the nondiversified and diversified CLL cases, respectively. Furthermore, preliminary studies suggest that activation-induced cytidine deaminase, that appears to play an essential role in somatic hypermutation (61, 62), can be detected by RT-PCR in some CLL cases, although a correlation between enzyme mRNA expression and intraclonal diversification is not clear at this point (unpublished data).
The CLL cases studied differed in the extent of intraclonal diversification identified. In most instances, the Ig V gene mutations detected were unique to individual clonal members within a specific CLL patient. However, in two cases (nos. 105 and 261) mutations were shared among clonal members (Table I), making it possible to construct genealogical trees outlining the clonal evolution of the CLL B cells (Fig. 2). The reason(s) for the occurrence of such sublineages among certain, but not all, patients is not clear. Several possibilities should be considered. First, if the mutational machinery is not active at all points throughout the "life" of the leukemic clone, then those cases in which the machinery was active earlier and for a longer time interval would be more likely to have shared mutations than those cases in which the machinery was active later and for a shorter time interval. In addition, differences in the inherent proliferative rates of CLL cases or in individual members of the clone could affect the number and frequency of point mutations, and therefore the sharing of point-mutations. Alternatively, additional genetic damage, induced by this or other mutational processes, could have affected the potential for somatic hypermutation, causing its acceleration or termination in individual subclones. This latter issue could be especially important not only for the diversification of the Ig V gene repertoire, but also for the potential level of "malignancy" of an individual subclone since in normal B cells and in other B cell lymphoproliferative disorders a somatic hypermutation process can target non-Ig genes (32, 63–67). Finally, it is conceivable that the two patients with the extensive mutational lineages (CLL nos. 261 and 105) differ from the others that exhibit unique mutations but do not display this feature. For instance, the somatic mutation processes could have occurred by different mechanisms and/or at different anatomic sites in these two patients than those cases with solely unique point mutations or even possibly with the original shared mutations. As mentioned earlier, recent data suggest that human B cells can accumulate Ig V gene mutations outside of classical germinal center structures and adequate T cell help (44, 45, 68).
Consistent with its heterogeneity in terms of incidence and magnitude, the occurrence of ongoing somatic hypermutation in CLL B cells could also depend on the microenvironment. This dependency could lead either to an induction or abolishment of the mutational process during the course of the disease. It is becoming increasingly clear that the natural history and behavior of CLL B cells reflect not only intrinsic defects of the leukemic cells, but also extrinsic factors. For instance, bidirectional lymphocyte–nonlymphocyte cell interactions may lead to the inhibition of apoptosis in neoplastic B cells (69–72). Similarly, the presence or absence of proliferation centers that resemble germinal centers and alterations in the B/T cell network could affect the ability of CLL B cells to undergo somatic hypermutation (73, 74). Our finding that CLL B cells from cases 136 and 216, in which no intraclonal diversification was found, effectively mutated the VHDJH genes in vitro upon application of appropriate stimuli supports the notion that external factors can overcome the putative "differentiation block" of these leukemic cells. This is consistent with previous studies indicating that activated normal T cells or polyclonal B cell activators can induce terminal differentiation of CLL B cells (75, 76).
CLL cases can be segregated into two subgroups based on the mutational load of the expressed VH sequence (13), as defined by the number of shared mutations, and these subgroups differ very significantly in clinical course and outcome (26, 77). It is surprising that the occurrence and frequency of intraclonal Ig V gene variants is independent of the original mutation status of the individual CLL cases. This suggests several points about the relatedness of these two subgroups of CLL cases. First, these data indicate that all CLL cells, regardless of their initial V gene mutation status, retain the capacity to develop V gene mutations. If these mutations occur via the normal Ig V gene hypermutation process, then it is unlikely that the differences in V gene mutations between the two subgroups is inherent and a consequence of the leukemic process. Indeed, this might support the notion that all CLL cases derived from antigen-experienced (78) or memory (79) B cells. Second, these data may provide some insight into whether the differences in the load of shared mutations reflect distinct maturation stages of mature B cells at which these CLL cells arose, e.g., pregerminal center B cells versus post-germinal center B cells (10, 13, 14, 26, 77), or activation pathways that the precursor B cells followed, e.g., T cell–dependent versus T cell–independent triggers (13, 26). Finally, the occurrence of significant intraclonal diversification in a subset of CLL patients (nos. 261 and 105; Fig. 2) may occasionally impact on the utility of IgV gene mutation status as a prognostic marker in CLL. Recent studies indicate that the presence of significant numbers of V gene mutations (≥2% VH difference from the most similar germline gene) correlates with a relatively benign clinical course, whereas the absence of such mutations is associated with a more accelerated and unfavorable clinical course and outcome (26, 77). It remains to be seen whether the extent of V gene changes that converted the VH gene mutation status of CLL 105 from the "unmutated" to the "mutated" subgroup, will impact on the predictive value of this marker in such cases, although this seems unlikely since this type of "conversion" appears to be relatively infrequent.
We thank Dr. Kozaburo Yamaji and Dr. Andras Schaffer for their most helpful discussion. We thank Shefali Shah and Patricia Dramitinos for their help with tissue culture and SSCP analysis.
National Institutes of Health (NIH) grants AG13910, AR40908, AI45011, and AI07621 to P. Casali, and NIH grants CA81554, CA87956, and AI10811 to N. Chiorazzi supported this work. Support was also provided by the Joseph Eletto Leukemia Research Fund, the Jean Walton Fund for Lymphoma & Myeloma Research, the Jerry and Cecile Shore Fund for Immunologic Research, and the Richard and Nancy Leeds Fund.
P. Casali and N. Chiorazzi contributed equally to this work.
Abbreviations used in this paper: CDR, complementarity determining region; CLL, chronic lymphocytic leukemia; SSCP, single-strand conformation polymorphism.


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Nora Naughton nee Doherty
Portacloy, Carrowtigue, Ballina, Co. Mayo - peacefully at her residence on the 26th of
June 2022.
Predeceased by her parents Anthony and Moria, her husband Edward, infant twins John and Ellen, brothers Pat and Tom, sons- in-law Tom Ruane and Bill Johnston and grandson Michael Edward Tomas.
Nora will be sadly missed by her loving and devoted family, son John (Portacloy), daughters Helen Johnston (London), Mary Ruane (Crossmolina), Bridie Tomas (Ballycastle), Una Hopkins (Ballycastle) Noreen Gernon (Dundalk) and Jacinta Healy (Ballinaboy), daughter-in-law Mary, sons-in- law Micheal, James, Johnny and Declan, grandchildren, great grandchildren, great great grandchild, beloved family pet Messi, extended family, neighbours and friends.
May Nora's Kind and Gentle Soul Rest In Peace.
Remains will repose at Healy's Funeral Home, Barnatra on Tuesday evening the 28th of June from 5 o'clock until 8 o'clock.
Funeral cortège will depart from her residence in Portacloy on Wednesday at 12.30 to arrive at The Church of the Immaculate Conception, Carrowtigue for Funeral Mass as 1 o'clock.
Interment will take place afterwards in Kilgalligan Cemetery.
Streaming of Nora's Funeral Services will be available for viewing on Healy's Funeral Directors Facebook page on Wednesday evening.
Face coverings must be worn, if possible for Nora's Funeral Services.
Family flowers only please. Donations if desired in lieu of flowers to Pieta West, c/o Healy's Funeral Directors, Barnatra.
Donations can also be made online by clicking on the link below:
Messages of condolence for Nora's family can be left by clicking on the "Condolences" button at the bottom of this webpage.
All enquiries to Healy's Funeral Directors, Barnatra on 087-6259911 / 087-6815481.


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- The three Chinese astronauts arrived at the Tiangong (Heavenly Palace) space station about six and a half hours after liftoff.
- As the Chinese Space Agency announced on Saturday morning, the capsule and crew docked on the "Tianhe" (Heavenly Peace) core module of the space station, which is still under construction.
- The astronauts will live and work there for about six months.
It is by far the longest running space mission in China. The astronauts blasted off Friday evening in a Long March 2F rocket from the Jiuquan Space Station on the edge of the Gobi Desert. In addition to the 55-year-old commander Zhai Zhigang and 41-year-old Ye Guangfu, Wang Yaping is the first Chinese female astronaut since 2013.
The 41-year-old posted on Chinese social media shortly before her second flight into space after 2013: "On a business trip for the next six months." Wang will also receive a very special honor on the mission: she will be the first Chinese woman to tour in space.
The three-man crew is also slated to test the Tianhe core module systems on their long-haul flight, work outside the spaceship and conduct science experiments. Prior to their stay, six tons of supplies, fuel and equipment were brought to the base unit on a cargo flight at the end of September.
"Tv specialist. Friendly web geek. Food scholar. Extreme coffee junkie."


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How to Get More Out of Your Meal
By: Danielle
Do you know where energy comes from?
Don't worry…this isn't a trick question. The answer: movement and food.
But for the purpose of this post, let's focus on the latter. Good ol' food.
In order to have energy, we need to create it. And fueling our bodies with the right foods is one way to do that. Where people get stuck is knowing what to eat. Eating healthy and building energy is easier once you know what to put on your plate. So this is one of my favourite tips for people who want to keep things simple and doable. Include these 3 things at every meal and snack:
- Fiber (50% of your plate)
- Fat
- Protein
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So what are you going to put on your plate tonight for dinner to keep things balanced and create some healthy energy?


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And we've reached our final top 20 recap for the infield, and, if you exclude the catchers, 3rd base is by far the shallowest position. 3rd base ain't great, y'all! This is the first position where there's guys at the tail end that I actively wouldn't have wanted on my fantasy teams, and guys like Yoan Moncada, Bobby Dalbec, Alex Bregman, KeBryan Hayes and Alec Bohm all stunk, and didn't even come within a sniff of the top 20 3rd basemen. To recap my recap before the recap, this final ranking is from our Fantasy Baseball Player Rater with my comments. This is not for next year. Anyway, here's the top 20 3rd basemen for 2021 fantasy baseball and how they compare to where I originally ranked them:
1. Jose Ramirez – Funny (not haha) that people acted like Jo-Jo-Ram was a product of Lindor being in Cleveland when Ramirez has now been top three at his position four of the last five years. Lindor didn't make Jo-Ram; Jo-Ram made Lindor. Absolutely guarantee you he won't be drafted like he's a top 3rd baseman next year, and people will be like, "Sigh, I guess, whatever, ho-hum, shucks, I guess I have to, like, I suppose, draft Jose Ramirez? But I don't wanna," then they will cry for 45 minutes. The biggest difference between a top option at a position and the runner-up was Salvador Perez, but Jo-Ram is the runner-up to not having a closer runner-up. Roughly nine dollars in value separated him from Rafael Devers, who ain't too bad himself! Preseason Rank #1, 2021 Projections: 91/29/101/.268/23 in 564 ABs, Final Numbers: 111/36/103/.266/27 in 552 ABs
2. Rafael Devers – Shame he doesn't have great speed, because, while I do think he can have a Vlad Jr.-type year at some point, Devers won't find it easy becoming the top hitter in baseball. A top 10 hitter? Think he's already there and no one expects much. For whatever reason, Devers is looked at like he's Jose Abreu or someone. Devers is 24 years old (for a few more days at least), a top hitter, and still getting better! Devers is a sneaky future Hall of Famer that no one talks about. He actually could go to a different team in a few years, and become a 40+ homer guy for another five years, because the Green Monster, while helping his average, is knocking down some of his power. He has to pull anything he wants to hit out, and, unlike a 13-year-old me, he doesn't pull everything. Preseason Rank #3, 2021 Projections: 106/31/109/.284/8 in 602 ABs, Final Numbers: 101/38/113/.279/5 in 591 ABs
3. Manny Machado – His overall power numbers were down, but he was also pretty unlucky. Quite a few balls hit deep that should've been out, just wrong park or wrong spot in said park. For unstints, he should've hit five more homers if his home park was Petco, and, this is crazy part, his home park is Petco. In 2019, he hit 32 homers, and had fewer No Doubt homers. Finally, and I don't know how to check this, but he must have the slowest Sprint Speed with that many steals ever. Sprint Speed is "hustle" in his case, excuse me, I meant to say "head case." Preseason Rank #2, 2021 Projections: 97/34/104/.278/10 in 584 ABs, Final Numbers: 92/28/106/.278/12 in 564 ABs
4. Austin Riley – Around July/August, I begin to enjoy the ride. The ebbs, the flows, the baseball grind. It's glorious. While ebbing and flowing and grinding like Urban Meyer, I lose track of what guys are doing. If someone asks me to look it up, I will, but I don't think anyone asked if what Austin Riley was doing was "real." So, I just looked now and, um, I thought he was breaking out. I thought. Hmm, so he's striking out more and has a BABIP .88 points above previous season when he hit .239. That's gonna be a yikes from me, dawg. I wrote an Austin Riley sleeper last year, he might jump right to a schmohawk. Preseason Rank #22, 2021 Projections: 84/31/96/.277/2 in 582 ABs, Final Numbers: 91/33/107/.303 in 590 ABs
5. Max Muncy – Already went over him in the recap of the top 20 1st basemen for 2021 fantasy baseball.
6. Nolan Arenado – He maintained the torso of his power, but his average did plummet in the move to The Louie by the River. In retrospect, it's so obvious. A guy who has the speed of a tractor that is being pulled by a giant tortoise who hits everything in the air loses 70 points on BABIP when he leaves one of the best parks for BABIP? You don't say! Not to freak anyone out but Torenado looks like he might have the career trajectory of Mostsuckass, who's currently circling the drain. Preseason Rank #4, 2021 Projections: 88/32/106/.279/1 in 592 ABs, Final Numbers: 81/34/105/.255/2 in 593 ABs
7. Tommy Edman – Already went over him in the recap of the top 20 2nd basemen for 2021 fantasy baseball.
8. Jonathan India – Already went over him in the recap of the top 20 2nd basemen for 2021 fantasy baseball.
9. Justin Turner – Because I was just looking at Torenado and Mostsuckass, I was curious why Toomgis Turner wasn't circling the drain like Mostusckass or about to get sucked up in the Torenado. Line drives, from what I gather. Turner doesn't hit for only power, so he's managed to be a 25/.270 vs. a 32/.245, because 32/.245 turns into 32/.225, then 17/.197 and, finally, "Wha' happened?" And those changes come at varying levels of speed. Might start calling this the Metamorphosis of Mostsuckass. Preseason Rank #23, 2021 Projections: 71/23/80/.277/2 in 491 ABs, Final Numbers: 87/27/87/.278/3 in 533 ABs
10. Kris Bryant – Already went over him in the recap of the top 20 1st basemen for 2021 fantasy baseball.
11. Ryan McMahon – Already went over him in the recap of the top 20 1st basemen for 2021 fantasy baseball.
12. Eduardo Escobar – Already went over him in the recap of the top 20 2nd basemen for 2021 fantasy baseball.
13. Kyle Seager – On the chart of Metamorphosis of Mostsuckass, Seager is at 32/.225 or one step beyond Torenado and one step until a fully blossomed Mostsuckass. Sounds like a future Met! Preseason Rank #32, 2021 Projections: 67/24/78/.231/5 in 581 ABs, Final Numbers: 73/35/101/.212/3 in 603 ABs
14. Luis Urias – Already went over him in the recap of the top 20 2nd basemen for 2021 fantasy baseball.
15. Jean Segura – If it wasn't clear, Bryant was the point where the 3rd basemen fell off to another tier. Not a terrible cliff-off-into-the-unknown-type fall-off, but a drop. Segura is interesting in that he's almost entirely driven by counting stats. Take away his runs/ribbies, and he's Jed Lowrie with a better average. Or I guess Alex Verdugo in the infield. Call him VerJedgo. Actually, don't. That's terrible. Preseason Rank #29, 2021 Projections: 72/15/77/.272/13 in 581 ABs, Final Numbers: 76/14/58/.290/9 in 514 ABs
16. Isiah Kiner-Falefa – His splits are comical and also confusing. How does one become less fast? Because in the 2nd half, he hit for an average that was ~.40 points higher and he stopped running. Israeli Diner Falafel went from sexy batter to cooked seemingly overnight, which might explain the stale oil taste. Preseason Rank #26, 2021 Projections: 78/8/61/.283/18 in 569 ABs, Final Numbers: 74/8/53/.271/20 in 635 ABs
17. Josh Donaldson – Starting to not like these backend 3rd basemen — and I do mean backend. Speaking of backend, Pujols was better than some of these guys at the age of 68 — Donaldson wasn't that bad. Not great, don't get me wrong. But take Donaldson for 457 ABs and waivers for 170 ABs and that's not bad. Unlike the Israeli Diner Falafel for 635 ABs, which was so painful, unless at catcher, but this is not catcher. This is 3rd base. What? 2nd base. Preseason Rank #20, 2021 Projections: 62/22/74/.242/2 in 412 ABs, Final Numbers: 73/26/72/.247 in 457 ABs
18. Jeimer Candelario – To give you an idea of how shallow 3rd base is, Jeimer was the 27th best 1st baseman, so there's nine more 1st basemen than 3rd basemen. Not great! Jeimer really wasn't good, and shouldn't be in the top 20 of anything, except the Top 20 Hitters Who Aren't Very Good. Preseason Rank #27, 2021 Projections: 71/23/82/.263/3 in 553 ABs, Final Numbers: 75/16/67/.271 in 557 ABs
19. Matt Chapman – This guy's stats fell off the ugly tree and hit every branch on the way down. Not quite as bad, er, as Bader, but for a guy like Chapman, who used to be the apple of my eye, he's now flinging poop at the wall and hoping something sticks, and it stuck to his Statcast page. His xBA on fastballs and breaking pitches respectively is .209 and .183. You can throw this guy anything and get him out. Is he blinking for five seconds at a time when at the plate? It's hard to imagine a guy with his eyes open hitting this bad. Preseason Rank #9, 2021 Projections: 91/36/93/.239/1 in 574 ABs, Final Numbers: 75/27/72/.210/3 in 529 ABs
20. DJ LeMahieu – Holy shizz, his season sucked. He has no business being in the top 20 of any position, let alone seeing 679 plate appearances! Jesus Sanchez burnt on a piece of toast, that is awful. No wonder the Yankees' offense was sneaky bad. They had DJ putting on his greatest hits, DJ's Got No Hits, Vol #1. Preseason Rank #5, 2021 Projections: 112/20/74/.312/6 in 604 ABs, Final Numbers: 84/10/57/.268/4 in 597 ABs


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Johnson promises 'can-do spirit' in delivering Brexit after Tory Leadership victory
- Gibraltar Government and opposition parties react to result
Boris Johnson began putting together his Cabinet last night and preparing for government after a landslide victory in the Tory leadership contest.
The new Conservative Party leader used his victory speech to promise that he would meet the October 31 Brexit deadline with a "new spirit of can-do", releasing the country's "guy ropes of self-doubt and negativity".
The Chief Minister, Fabian Picardo, has written to Mr Johnson to congratulate him on becoming the Leader of the Conservative and Unionist Party. He will make a further statement when Mr Johnson takes over from Theresa May as prime minister today.
Mr Picardo, who also wrote to Jo Swinson to congratulate her on being elected as the Leader of the UK's Liberal Democrat party, met with Mr Johnson just last week to discuss Brexit.
He tweeted a photo of himself with the future prime minister and Deputy Chief Minister Dr Joseph Garcia, congratulating Mr Johnson and adding: "Looking forward to working together in the best interests of #Gibraltar and the #UK."
Gibraltar's opposition parties also moved to welcome Mr Johnson as the new prime minister, as they expressed hope that Gibraltar's interests would be taken into account when considering Brexit.
The GSD said: "This task claimed Theresa May who unsuccessfully attempted to navigate those very complicated waters between the various Parliamentary factions and shades of public opinion on the most divisive of issues."
GSD Leader Keith Azopardi said: "We hope that Mr Johnson will ensure that he also has the wishes and interests of the people of Gibraltar in mind when considering Brexit."
"We are the only British territory with a land frontier with mainland Europe. We have decisively expressed a wish to have a close relationship with the European Union and while Mr Johnson was one of the foremost advocates of the Vote Leave campaign we trust he will be sympathetic to the economic and political aspirations of the people of Gibraltar."
"A free flowing border and market access to the EU and the UK are clear goals for Gibraltar and it is important he does his utmost to ensure our political and economic rights are protected and fully respected by Spain," Mr Azopardi said.
In welcoming Mr Johnson in his new role, Independent MP Marlene Hassan Nahon wished him well navigating these "hard political times ahead".
She told the Chronicle that the overwhelming message from the people of Gibraltar in connection to Brexit had always been to stay in the European Union.
"We are also united in the reality that there is no mandate, across the board, for a hard Brexit. As such, and as a people, we are a community of remainers, and support the prospect of a second referendum."
"Regardless of this possibility, we hope that our goals and interests will be duly represented by the new premiership going forward," Ms Hassan Nahon said.
Mr Johnson secured more than two-thirds of the votes in the contest, comfortably defeating Foreign Secretary Jeremy Hunt.
He said it was an "extraordinary honour and privilege" and insisted that "we are going to unite this amazing country and we are going to take it forward".
At the leadership announcement event in central London, Mr Johnson said: "We are going to get Brexit done on October 31, we are going to take advantage of all the opportunities that it will bring in a new spirit of can-do.
"And we are once again going to believe in ourselves and what we can achieve, and like some slumbering giant we are going to rise and ping off the guy ropes of self-doubt and negativity."
Improved education, infrastructure, more police and full-fibre broadband are among the ways Mr Johnson said this would be achieved.
But Mr Johnson's main task will be fulfilling his "do or die" promise to deliver Brexit on October 31, which he has said he will do with or without a deal.
Theresa May, who will resign as Prime Minister today, offered her congratulations but stressed that Mr Johnson should work "to deliver a Brexit that works for the whole UK", a clear warning against a no-deal departure.
She promised Mr Johnson "my full support from the back benches".
US President Donald Trump, who repeatedly praised Mr Johnson even while visiting Mrs May, said he would be a "great" prime minister.
Michel Barnier, the EU's chief Brexit negotiator, said Brussels looked forward to working with the new prime minister on ratifying the Withdrawal Agreement - the deal which Mr Johnson has already declared dead.
Mr Barnier said the EU was ready to "rework" the Political Declaration on the future relationship.
Mr Johnson will be appointed as prime minister today by the Queen after Mrs May formally resigns.
He secured 92,153 votes - 66.4% - to defeat Mr Hunt in the leadership ballot.
Despite the resounding victory, Mr Johnson's share of the vote was slightly lower than that achieved by David Cameron in the 2005 Conservative leadership election, when he took 67.6%.
Mr Johnson faces a daunting in-tray at Number 10, not only the tight Brexit deadline but also the diplomatic crisis in the Gulf, where tensions have been heightened following Iran's seizure of the British-registered Stena Impero tanker.
The challenge facing Mr Johnson is made even more difficult by a wafer-thin Tory-DUP majority of two in the Commons, with the prospect of it being reduced to just one if the Conservatives fail to win the Brecon and Radnorshire by-election on August 1.
The incoming premier has been left in no doubt about the opposition he will face from his own benches if he attempts to force through a no-deal Brexit.
Sir Alan Duncan quit as a Foreign Office minister on Monday and Anne Milton as education minister on Tuesday, rather than serve under Mr Johnson.
Cabinet ministers Philip Hammond, David Gauke and Rory Stewart are expected to join them on the back benches after the leadership change.
Mr Stewart congratulated Mr Johnson, and confirmed he would return to the back benches on Wednesday, tweeting: "Backbench tomorrow serving Cumbria. Thank you all. More walking!"
Chancellor Philip Hammond, who will resign from the Government on Wednesday afternoon, also congratulated Mr Johnson and said he had his "wholehearted support" in seeking a deal with Brussels.
Labour leader Jeremy Corbyn urged Mr Johnson to call a general election.
He said: "Boris Johnson has won the support of fewer than 100,000 unrepresentative Conservative Party members by promising tax cuts for the richest, presenting himself as the bankers' friend, and pushing for a damaging no-deal Brexit. But he hasn't won the support of our country."
Mr Johnson is expected to make Cabinet appointments this evening.
Mark Spencer will become his chief whip - a key position as Mr Johnson attempts to govern with a working Tory-DUP majority of just two.
Mr Hunt said Mr Johnson would make a "great prime minister" and conceded he had had a "big mountain to climb" in the contest.
"I never felt it was too much of an uphill struggle but I always knew that it was going to be a big mountain to climb," the Foreign Secretary told the BBC.
"I think Boris fought a very, very good campaign. It was a very clear campaign, his messages were absolutely clear and I think, as I say, he will now be a great prime minister.
"It's a challenging moment for our country - this is perhaps the most difficult legacy that a prime minister has inherited in peacetime. It's going to be very challenging.
"But he has got the confidence and the optimism and the energy to get us through this and that's why I think he will be a great prime minister."


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Tell your friends where you are going.
Black Fair September Edition - September 2022
Start Date:
Sep. 11th 2022
End Date:
Sep. 25th 2022
Starting at: 5 PM SLT
Black is the new Black at Black Fair September Edition! This timeless color made it through all epochs and never went out of fashion.
It is not only a sophisticated, modern, urban color, as with imagination and creativity any combinations are possible.
Discover the power of black at Black Fair 2022!
Our friends at: Black Fair September Edition - September 2022


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Tools Used
- Derwent Graphic Hard graphite pencils
- Staedtler 100% Rag Vellum paper
Number four in my "People Who Kick Ass" series, Ed Norton is a talented actor and was really impressed by his versatility when he did "American History X", "Fight Club" and "Keeping the Faith" in succession. The trickiest part about this was the 5 o' clock shadow – I had to not make it look like I just shaded in an area and not make the stubble to pronounced so as to end up giving him a beard.
The second version is after some styling in Photoshop.


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Mercedes-Benz Canada announces comprehensive Mercedes-EQ charging strategy
Canada Mercedes-Benz today announced the next phase of its electric offensive-a comprehensive electric vehicle charging strategy that will ensure that Mercedes-EQ drivers can enjoy clean, digitally integrated , Fast and convenient charging experience, no matter in the home or anywhere in Canada.
Together with its charging partners, Mercedes-Benz Canada is committed to raising the bar on EV charging with Mercedes me Charge, the official industry-leading charging ecosystem enabled by ChargePoint. This charging ecosystem will benefit drivers of the new all-electric EQS and other future electric mobility products.
"Mercedes-Benz Canada is pairing the exciting upcoming Mercedes-EQ product lineup with the very best electric ecosystem, so Mercedes-EQ drivers can experience peace of mind and convenience on their electric driving journey," said Eva Wiese, president and CEO, Mercedes-Benz Canada. "Through partnerships with established charging experts, Mercedes me Charge offers drivers widespread charging coverage from coast-to-coast, along with seamless navigation and green charging options. Whether at-home or on Canadian roads, Mercedes-EQ drivers will experience modern electric luxury every step of the way."
At launch, Mercedes-EQ drivers will have access to more than 4,900 public charging ports across Canada, making up more than half of the country's networked public charging ports. Through a strategic partnership with ChargePoint, drivers will have access to places to charge with ChargePoint, FLO and other charging providers in Canada.
In an industry first, the Mercedes me Charge ecosystem can also provide drivers access to an additional network of more than 2,400 semi-public ports on the ChargePoint network that reside in access-controlled environments like workplaces, shopping malls and hotels. This will ensure that Mercedes-EQ drivers have easy access to charging stations for daily commutes as well as for longer road trips. This combination of public and semi-public access spread across multiple partners will give Mercedes-EQ drivers seamless access to the Mercedes me Charge network throughout Canada, making cross-country trips easy and worry-free. Additional network partners will join the service after launch, ensuring coverage will continue to grow as Mercedes-EQ expands its electric portfolio. This charging infrastructure will also serve future Mercedes-Benz PHEV drivers.
By the end of 2021, Mercedes me Charge customers will benefit from Plug & Charge functionality, allowing for a seamless, convenient charging process. Simply open the flap, plug in and the current immediately starts flowing without any further customer interactions. Plug & Charge will be a significant enhancement to the EQS charging feature-set, which also allows drivers to enable charging via MBUX (Mercedes-Benz User Experience) on the vehicle's media display, through the Mercedes me App or by using the Mercedes me Charge card.
"In terms of the new Mercedes-EQ sub-brand, Canada is an important and highly anticipated market for Mercedes-Benz," Mercedes-Benz Business Development Charges Nico Dettmer, Global Head of Partnerships and Partnerships, said. "This is why our Canadian charging strategy is so comprehensive. Canadian Mercedes-EQ drivers can look forward to a large and rapidly expanding public and semi-public charging station infrastructure that will allow them to travel across Canada easily. With many cutting-edge innovations in Mercedes me Charge, customers will also be able to enjoy a convenient and sustainable experience. "
Dodge Charger SRT Hellcat Octane Edition: Black is the new black
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July 19, 2021, Southern District of Texas – Trustee Daniel H. Golden of the DFC Liquidating Trust for the estate of Deans Food Company, et al (DFC) recently commenced about three hundred and twenty-one (321) clawback actions against several creditors to avoid and recover alleged preferential transfers of property that occurred during the ninety (90) day period before the commencement of the bankruptcy proceedings of the Debtor and its affiliates (collectively, the "Debtors") according to sections 547 and 550 Bankruptcy Code. The Trustee also seeks to avoid the transfers that may have been alleged fraudulent conveyances under sections 548 and 550 of the Bankruptcy Code.
Some of the defendants sued by the Trustee in the Debtors' bankruptcy case include; The Dennis Group, LLC, Unilever United States, Inc., Winn – Dixie Stores, Inc., Truist Equipment Finance Corp, Ruan Logistics Corporation. Tampico Beverages Inc. By way of background, the Debtors manufactured, marketed, and distributed a wide variety of branded and private label dairy and dairy case products, including fluid milk, ice cream, cultured dairy products, creamers, ice cream mix, and other dairy products to retailers, distributors, foodservice outlets, educational institutions, and governmental entities across the United States.
On November 12, 2019, DFC and affiliated debtors filed a voluntary petition for relief under Chapter 11 of the Bankruptcy Code in the United States Bankruptcy Court for the Southern District of Texas. The cases are pending before the Honorable Judge David R Jones and are jointly administered under Case No. 19 -36314. ASK LLP is representing the Trustee in the bankruptcy case.


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