Nucleoside analogues are a valuable experimental tool. Incorporation of these molecules into newly synthesized DNA (i.e. pulse-labeling) is used to monitor cell proliferation or to isolate nascent DNA. Some of the most common nucleoside analogues used for pulse-labeling of DNA in cells are the deoxypyrimidine analogues 5-ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC). Click chemistry enables conjugation of an azide molecule tagged with a fluorescent dye or biotin to the alkyne of the analog, which can then be used to detect incorporation of EdU and EdC into DNA. The use of EdC is often recommended because of the potential cytotoxicity associated with EdU during longer incubations. Here, by comparing the relative incorporation efficiencies of EdU and EdC during short 30-min pulses, we demonstrate significantly lower incorporation of EdC than of EdU in noninfected human fibroblast cells or in cells infected with either human cytomegalovirus or Kaposi's sarcoma-associated herpesvirus. Interestingly, cells infected with herpes simplex virus type-1 (HSV-1) incorporated EdC and EdU at similar levels during short pulses. Of note, exogenous expression of HSV-1 thymidine kinase increased the incorporation efficiency of EdC. These results highlight the limitations when using substituted pyrimidine analogues in pulse-labeling and suggest that EdU is the preferable nucleoside analogue for short pulse-labeling experiments, resulting in increased recovery and sensitivity for downstream applications. This is an important discovery that may help to better characterize the biochemical properties of different nucleoside analogues with a given kinase, ultimately leading to significant differences in labeling efficiency of nascent DNA.

Nucleoside analogues are a valuable experimental tool. Incorporation of these molecules into newly synthesized DNA (i.e. pulse-labeling) is used to monitor cell proliferation or to isolate nascent DNA. Some of the most common nucleoside analogues used for pulse-labeling of DNA in cells are the deoxypyrimidine analogues 5-ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC). Click chemistry enables conjugation of an azide molecule tagged with a fluorescent dye or biotin to the alkyne of the analog, which can then be used to detect incorporation of EdU and EdC into DNA. The use of EdC is often recommended because of the potential cytotoxicity associated with EdU during longer incubations. Here, by comparing the relative incorporation efficiencies of EdU and EdC during short 30-min pulses, we demonstrate significantly lower incorporation of EdC than of EdU in noninfected human fibroblast cells or in cells infected with either human cytomegalovirus or Kaposi's sarcoma-associated herpesvirus. Interestingly, cells infected with herpes simplex virus type-1 (HSV-1) incorporated EdC and EdU at similar levels during short pulses. Of note, exogenous expression of HSV-1 thymidine kinase increased the incorporation efficiency of EdC. These results highlight the limitations when using substituted pyrimidine analogues in pulse-labeling and suggest that EdU is the preferable nucleoside analogue for short pulse-labeling experiments, resulting in increased recovery and sensitivity for downstream applications. This is an important discovery that may help to better characterize the biochemical properties of different nucleoside analogues with a given kinase, ultimately leading to significant differences in labeling efficiency of nascent DNA.

Mikheil Saakashvili, the former president of Georgia, vowed on Friday to help his new boss, Ukrainian President Volodymyr Zelenskiy, clean out a political "swamp" of oligarchs' interests that he said were preventing Ukraine prospering. Twice president of Georgia, Saakashvili had a brief but stormy spell in Ukrainian politics five years ago under Zelenskiy's predecessor Petro Poroshenko in which he once clambered onto a roof to avoid law enforcement.

The SHV β-lactamases (BLs) have undergone strong allele diversification that changed their substrate specificities. Based on 147 NCBI entries for SHV alleles, in silico mathematical models predicted five positions as relevant for the β-lactamase inhibitor (BLI) resistant (2br) phenotype, 12 as relevant for the extended-spectrum BL (ESBL) (2be) phenotype, and two positions were related to solely the narrow spectrum (2b) phenotype. These positions and additional 6 positions described in other studies (including one promoter mutation), were systematically substituted and investigated for their substrate specificities in a BL-free E. coli background, representing, to our knowledge, the most comprehensive substrate and substitution analysis for SHV alleles to date. An in vitro analysis confirmed the essentiality of the positions 238 and 179 for the 2be phenotype and 69 for the 2br phenotype. The substitutions E240K and E240R, which do not occur alone in known 2br SHV variants, led to a 2br phenotype, indicating a latent BLI-resistance potential of these substitutions. The substitutions M129V, A234G, S271I and R292Q conferred latent resistance to cefotaxime. In addition, 7 positions that were found to be not always associated with the ESBL phenotype resulted in increased resistance to ceftaroline. We also observed that coupling of a strong promoter (IS26) to a A146V mutant with the 2b phenotype resulted in a highly increased resistance to BLIs, cefepime and ceftaroline but not to 3^rd generation cephalosporins, indicating that SHV enzymes represent an underestimated risk for empirical therapies that use piperacillin/tazobactam or cefepime to treat different infectious diseases caused by gram-negatives.

After weeks of rumor and speculation, Mikhail Saakashvili, the former Georgian president who is wanted for embezzlement at home, has been appointed as head of Ukraine’s executive council on reforms.
Read Full Article at RT.com

Tbilisi is recalling its ambassador from Kiev for consultations, a day after Ukraine elevated Mikhail Saakashvili, the fugitive ex-president of Georgia who faces embezzlement charges at home, to the head of Ukraine’s reform body. 
Read Full Article at RT.com

Read Full Article at RT.com

The U.S. Army Corps of Engineers has signed a memorandum agreement with the Southeastern Power Administration, Tennessee Valley Authority and Tennessee Valley Public Power Association, Inc., to perform a variety of work at Corps hydropower projects.

Georgia summoned its ambassador to Ukraine back to Tbilisi in a sign of its displeasure over Kyiv’s decision to name Mikheil Saakashvili to a government reform body. 

Ashvin Kumar recently took a trip down memory lane and reminisced upon Irrfan Khan’s professionalism

The Department today announced a settlement resolving allegations that the Metropolitan Government of Nashville and Davidson County (Metropolitan Government) violated the Fair Housing Act (FHA) and the Religious Land Use and Institutionalized Persons Act (RLUIPA) by discriminating against Teen Challenge, a Christian substance abuse treatment program.

Cristina Andres Perfecto of Nashville, Tenn., was sentenced late yesterday in federal court in Memphis to 190 months in prison for sex trafficking by force, fraud and coercion, sex trafficking of a juvenile and conspiracy.

The Department has entered into a settlement agreement with the Metropolitan Government of Nashville, Tenn., and Davidson County, Tenn., to enhance the security of students with disabilities on public school buses.

The Department has reached an agreement with Argosy University in Nashville, Tenn., and its parent company, Education Management Corporation of Pittsburgh, to settle allegations that Argosy refused to hire a lawful permanent resident because he is a non-United States citizen and intimidated him for reporting the school’s refusal to hire.

A Nashville, Tenn., man pleaded guilty today to conspiring to participate in racketeering activity related to his membership in the Bloods criminal enterprise.

Antonio Washington, 22, aka “T.O.,” of Nashville, also was ordered by U.S. District Judge Aleta Trauger to serve five years of supervised release following his prison term.

The United States has asked a federal court to shut down a Mo’ Money Taxes tax preparation office in Nashville.

A federal court permanently barred Toney Fields and Trumekia Shaw, who do business as Fields Mo’ Money Taxes in Nashville, Tenn., from preparing federal tax returns.

After a storm hit Tennessee last Sunday and Nashville saw record-shattering rainfall, the Cumberland River swelled to overflowing and the city experienced an epic flood. As soon as it was safe, residents were out

Backyard chickens may be trendy, but in some cities they are still illegal. But that doesn't stop some would-be chicken keepers.

To make an extra bit of income and to have a smaller home to live in for the future, this Nashville couple decided to build a modernist micro-home.

St. Jude Rock ‘n’ Roll Nashville Marathon

Charles Esten :60 TV PSA

St. Jude Rock ‘n’ Roll Nashville Marathon

IN OUR CASE, THE CIT(A) ORDER HAS BEEN PASSED ON 17.12.2019 AND ON THE DAY OF 31.01.2020, THE TIME LIMIT FOR ITAT APPEAL IS STILL PENDING. BUT TILL TODAY THE ASSESSEE HAS NOT FILED ANY APPEAL.
CAN THE ASSESSEE OPT FOR VSV SCHEME ? AND IF YES, THEN GUIDE US THE PROCEDURE FOR THAT.

As there are restrictions to move around because of the nationwide COVID-19 lockdown, actress Sonam K. Ahuja says she is missing her siblings -- Rhea Kapoor and Harshvardhan Kapoor. Sonam took to Instagram, where she shared a monochrome photograph, which seems to be from her wedding celebrations.

"See you soon brats.. miss you more than you guys can imagine," she wrote.

 

 

 

View this post on Instagram

A post shared by Sonam K Ahuja (@sonamkapoor) onApr 30, 2020 at 8:58pm PDT

Sonam tied the knot with her long-time beau Anand Ahuja in 2018. The grand wedding celebrations were attended by celebrities like Salman Khan, Shah Rukh Khan, Kareena Kapoor Khan and Karan Johar.

She was last seen on screen in "The Zoya Factor" (2019), an adaptation of Anuja Chauhan's 2008 novel of the same name, which follows the story of a girl, Zoya Solanki, who becomes a lucky charm for the Indian Cricket team during the 2011 Cricket World Cup.

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Georgian once mooted as next deputy prime minister, takes on top co-ordinating role

Miranda Lambert and her band the Pistol Annies are out celebrating award season. Ashley Monroe Angaleena Presley and Miranda all looked elegant as they arrived at the Grammy Awards party.

The 29-year-old pop star was once told to give up any hope of making it in Nashville, she revealed while serving as a 'mega-mentor' on Tuesday's episode of The Voice

American Idol winner Taylor Hicks was one of many Nashville residents affected by the monstrous tornado that tore a 2-mile-long path through Putnam County early Tuesday.

The 28-year-old recently parted ways with the boxer Wladimir Klitschko, and has moved to LA with the conclusion of her CMT series Nashville, leading to the decision to put the home up on the market.

Ashvin Kumar recalls he had no budget to pay Irrfan Khan, but the actor voluntarily supported his film.

Singapore : Springer, c2019.

Cham : Springer, [2019]

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Dewey Library - QD172.S93 H43 2019

Symposium on Biotechnology for Fuels and Chemicals (28th : 2006 : Nashville, Tenn.)

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Rotch Library - DK679.35.T35 2019

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