Not to change the topic exactly:
Exonuclease VII repairs quinolone-induced damage by resolving DNA gyrase cleavage complexes |
To understand the role of ExoVII in the repair of quinolone-induced DNA damage, we first confirmed that inactivation of ExoVII in E. coli leads to hypersensitivity to ciprofloxacin, which traps type IIA TOPcc ( 8 ). Compared to wild-type (WT) strains, deficiency in either subunit of ExoVII (Δ xseA or Δ xseB ) decreased the minimum inhibitory concentration (MIC) of ciprofloxacin by 60 to 70% ( Fig. 1A and fig. S1, A and B). ExoVII-deficient strains are not hypersensitive to the non–quinolone topoisomerase catalytic inhibitor novobiocin ( 15 ), suggesting that ExoVII is specifically involved in repairing trapped DNA gyrase rather than damages stemming from the loss of DNA gyrase activity.
By contrast, loss of ExoVII does not sensitize E. coli to trapped type IA topoisomerase ( A ). Average MIC values for ciprofloxacin (Cipro) in WT strain or strains deficient in either subunit of ExoVII (Δ xseA and Δ xseB ) ( n ≥ 4). Statistical significance was calculated using two-tailed Student's t test in GraphPad Prism. ** P < 0.01. *** P < 0.001. ( B ) Same as in (A), except that the E. coli strains express the quinolone-resistant mutation in the catalytic subunit of DNA gyrase, GyrA-S83L. ( C ) Transformants with p Y. pestis topA (p YtopA , encoding Y Top1) or p Y. pestis topA-R327W (p YtopA-R327W , encoding Y Top1-R327W) were established in either the WT, Δ xseA , or Δ xseB strains. Left panel shows the spotted growth of serial dilutions of pYtopA transformants after induction with 0.2% arabinose for 2 hours. Middle and right panels show the growth of the uninduced or induced p YtopA-R327W transformants, respectively. The dilution of each sample is indicated above the images. Columns of equal spotting densities are indicated by red circles. Representative plates of duplicates are shown. ( D ) Growth of p YtopA-R327W transformants under continuous induction with the indicated concentrations of arabinose. Representative plates of duplicates are shown.
( A ) The upper strands of all three DNA constructs were internally radiolabeled. The constructs differ only by the terminal chemical group of the 5′-overhangs, phosphate (X = P), hydroxyl (X = OH), or phosphotyrosine (X = Y). Concentrations of ExoVII ranged from 0.033 to 0.1 U/μl. The DNA cleavage products were resolved on a 20% denaturing polyacrylamide gel electrophoresis (PAGE). A representative gel of three independent experiments is shown. ( B ) The tyrosyl nuclease activity of ExoVII processes the Y-40 construct [same as in (A)] to generate a 39-nt product bearing a 5′-phosphate. Treatment of ExoVII-generated product with alkaline phosphatase (CIP) removes the 5′-phosphate, as demonstrated by the two 39-nt markers bearing either a 5′-phosphate or 5′-hydroxyl group. Note that the oligo bearing an additional phosphate group travels faster in PAGE. ( C ) Scheme of the specific cleavage activity of ExoVII for 5′-tyrosine adducts. The single-stranded overhang regions and the site of ExoVII cleavage are indicated (red arrow). ( D ) The length of the 5′-overhangs influences the tyrosyl nuclease activity of ExoVII. Constructs differed only in the complementary lower strands, resulting in 5′-overhangs with different lengths. Concentrations of ExoVII ranged from 0.033 to 0.1 U/μl. A representative gel of five independent experiments is shown.
To compare ExoVII's tyrosyl nuclease activity with its single-stranded exonuclease activity, we tested the activity of ExoVII for 5′-tyrosine on a 4-nt overhang versus a substrate with 5-nt overhang (fig. S3B), where the single-stranded regions of the two substrates are comparable in length. ExoVII processed both substrates similarly, with a slightly higher efficiency for the tyrosine adducts (fig. S3, B and C). We also compared DNA constructs with varying lengths in their duplex regions. The tyrosyl nuclease efficiency of ExoVII increased with DNA constructs containing longer duplex regions (fig. S4, A and B). The fact that longer duplex regions lead to higher cleavage efficiencies of ExoVII suggests a mechanism where ExoVII likely could bind and scan the duplex DNA before locating its single-stranded cleavage substrates at the end.
The other major class of topoisomerase, type IB topoisomerases, is largely absent from the bacterial domain with the exception of a handful of species ( 27 , 28 ). While type IA and type IIA topoisomerases form cleavage complexes on the 5′-end of DNA, type IB topoisomerase forms cleavage complex on the 3′-end of DNA. To further define the substrate specificity of ExoVII, we tested the tyrosyl nuclease activity of ExoVII for tyrosine adducts on 3′-DNA overhangs. As expected, because of its exonuclease activity, ExoVII degraded DNA substrates with 3′-overhangs but not the substrates with blunt ends. Furthermore, ExoVII degraded substrates with tyrosine adducts on 3′-overhangs to the same degree as the substrates bearing a phosphate or a hydroxyl group on the 3′-overhangs (fig. S5A). We conclude that the presence of a tyrosine adduct on the 3′-overhangs has no impact on the 3′-single-stranded exonuclease activity of ExoVII.
To establish the tyrosyl nuclease activity of ExoVII in bacteria, we reasoned that inactivating ExoVII would lead to increased accumulation of trapped DNA gyrase on genomic DNA. To facilitate the detection of trapped DNA gyrase in vivo, we introduced a C-terminal His-tagged GyrA on a single-copy plasmid (p gyrA His ) into WT, Δ xseA , and Δ xseB E. coli strains. As expected, only transformants of p gyrA His showed detectable GyrA His expression (fig. S6A). Immunoblots probed with anti-GyrA antibodies confirmed that the exogenous GyrA His was expressed at levels close to that of endogenous GyrA to minimize any potential perturbations caused by exogenous expression of GyrA (fig. S6B). All transformants displayed similar levels of sensitivity to quinolones and comparable MIC for ciprofloxacin as the nontransformed strains (figs. S6, C and D, S1, A and B; and Fig. 1A ).
Using these transformant strains in a modified rapid approach to DNA adduct recovery (RADAR) assay (fig. S7A) ( 32 – 34 ), we detected trapped DNA gyrase on genomic DNA in vivo upon ciprofloxacin treatment in a dose-dependent manner (fig. S7B). Having optimized the RADAR assay, we measured trapped DNA gyrase levels in different transformant strains after a 6-hour treatment with clinical plasma concentration of ciprofloxacin (~0.5 μg/ml) ( 35 ). ExoVII-deficient transformant strains (Δ xseA -p gyrA His and Δ xseB -p gyrA His ) accumulated significantly more trapped DNA gyrase than the WT transformant strain (WT-p gyrA His ) ( Fig. 3, A and B ). The levels of DNA in each sample probed with anti-DNA antibody served as loading controls. The enhanced accumulation of trapped DNA gyrase correlated with the decreased survival rates of the ExoVII-deficient transformant strains (fig. S8A). The ExoVII-deficient strains also showed hypersensitivity to ciprofloxacin even with relatively short treatments (fig. S8B). Combined, these results support the conclusion that ExoVII is directly involved in the repair of trapped DNA gyrase in vivo.
( A ) E. coli WT and strains deficient in either subunit of ExoVII, Δ xseA or Δ xseB were transformed with a single-copy plasmid bearing gyrA with a C-terminal His-tag (p gyrA His ). Each transformant strain was grown to log phase and subjected to 6-hour treatment with ciprofloxacin at 0.5 μg/ml. Bacteria were lysed to extract their genomic DNA. Equal amounts of DNA were spotted onto polyvinylidene difluoride (PVDF) membrane and probed with indicated antibodies. Immunoblotting with antibodies for DNA served as a loading control. ( B ) Quantification of trapped GyrA His . The intensity of anti–His-tag band from each sample in (A) was corrected for the amount of input DNA, measured by the intensity of the respective anti-DNA band. The adjusted GyrA His signals were then normalized to the signal of the WT strain, set as 1. Statistical significance was calculated using two-tailed Mann-Whitney test in GraphPad Prism. * P < 0.05. ** P < 0.01. A.U., arbitrary units. ( C ) Immunoblotting of purified E. coli (p gyrA His ) genomic DNA from RADAR assays after ciprofloxacin treatment. E. coli genomic DNA samples were either untreated, treated with benzonase or with ExoVII (1 U/μl) at 37°C for 2 hours, and then resolved by tris-glycine–SDS-PAGE and probed with anti–His-tag antibodies. A representative blot of three independent experiments is shown.
The eukaryotic TDP enzymes cannot efficiently excise TOPcc unless the TOPcc is first unfolded or digested ( 10 , 36 – 41 ). Hence, we examined whether the tyrosyl nuclease activity of ExoVII is capable of excising native trapped DNA gyrase generated with recombinant DNA gyrase and radiolabeled DNA substrates. In the presence of ciprofloxacin, the majority of DNA substrate (30 nt long) became retained in the wells of sequencing gels as a result of covalent attachment to the DNA gyrase (fig. S9). The addition of ExoVII failed to resolve the DNA gyrase TOPcc and to release the DNA from the wells (fig. S9), indicating that ExoVII is unable to process native intact DNA gyrase cleavage complexes.
Look: Paige Spiranac's New Golf Towel Is Going Viral
Paige Spiranac unveiled on Wednesday a line of golf towels. Unsurprisingly, the golf towels are going viral on social media.
The former collegiate golfer turned sports media personality has developed a massive following . Spiranac has more than 3 million followers on Instagram and more than 400,000 followers in Twitter. She continues to build on her brand and is currently working with PointsBet.
“No one will ever take you seriously if you keep posting pictures with cleavage,” she wrote in quotation marks on Instagram earlier today.
The towels are currently being sold on her website. The “Paige in Green” towel is currently going for about $25.
Celebs Are Obsessed With This Bizarre Top Trend—It Shows SO Much Cleavage! - SHEfinds
Spring is only a few weeks away but celebs are already stripping down. From already breaking out the bikinis to wearing sheer everything, there has been plenty of skin on display.
But one seriously bizarre trend that celebs like Beyoncé and Kim Kardashian are loving shows A LOT of cleavage!
READ MORE: Kim Kardashian Can't Stop Wearing These 'Bare' Bikinis—They're Going To Be Everywhere!
Beyoncé is known as Queen Bey to her adoring fans because she is so multi-talented. She’s not only an epic musician, but also an incredible businesswoman and designer. She also happens to be truly stunning. So it’s no surprise fans went WILD when Beyoncé shared this super sexy look with her 167m followers on February 19th in anticipation of her Adidas x Ivy Park collab.
The post might have been captioned, “ICY PARK TOMORROW” but there was no ice in the comments—they were filled with fire emojis.
Bey’s blue track suit is ultra sexy and sleek, and of course she turned up the heat by unzipping the jacket and showing off some serious cleavage.
READ MORE: These Before And After Photos Of Beyoncé Are Insane–She Looks So Different Now!
This kind of cleaveage is what we’re seeing celebs show off more and more. Who knew underboob would be such a thing—especially before spring is even here? With 2.9m likes, we wouldn’t be surprised to see Bey fans trying out this trend for themselves!
Great minds must think alike because Kim Kardashian also shared a snap on February 19th channeling the same sexy energy as Beyonce while showing off her curves.
While you're here...
The best adhesive bra - Baltimore Sun
Tops and dresses with strapless or backless designs look best when worn with adhesive bras. Their convenient self-stick cups are virtually undetectable beneath even sheer or flimsy garments.
There are countless adhesive bras on the market now, including designs available in different colors or unique shapes. We introduce you to these styles in our buying guide on adhesive bras, and we share a few of our favorite picks. Our top choice, Fashion Forms Women's NuBra Ultralite Bra , has a front snap closure for a customizable fit.
The most popular styles of adhesive bras include silicone and fabric stick-ons. Silicone types are seamless and smooth, while fabric styles offer more support. These styles typically come with bottles of glue or specialty tape. Laced adhesive bras are similar in construction, only they have centerline lacing to offer a customizable fit.
There are some U-plunge backless strapless bras, many of which have underwires. They're made for deep-cut backless tops and dresses. Other options for adhesive bras include lift tape and pasties, both of which cover the nipples.
Given their unique shape, adhesive bras are only sold in bra cup sizes ranging from AA to DD. Some wearers recommend going up a cup size to achieve additional coverage or a better fit. Another detail to keep in mind is that cup sizing isn't very consistent across manufacturers, so be prepared to try more than one.
Adhesive bras are available in neutral colors like nude, white, black and pale pink. If possible, opt for an adhesive bra whose color is the same as your top or dress. If not, using a skin-colored adhesive bra is a suitable alternative.
Many styles of adhesive bras have front closures. Silicone and fabric bras may have snap closures, while laced styles feature a corset-inspired laced closure. All these styles offer a customizable fit to some degree. However, wearers interested in emphasizing cleavage are better served by laced styles.
Many adhesive bras are noticeably thinner than regular lined bras, since they're intended to be low-profile and undetectable. A few adhesive bras have padded cups to enhance the bust. Some wearers feel these styles also provide a bit more support.
Cut refers to how low the bra sits on the bust. The average silicone or fabric adhesive bra works well for most strapless and backless garments. Deep-cut tops and dresses are better served by U-plunge designs, which are contoured in such a way that they aren't noticeable.
Rihanna flashes cleavage as she arrives at LA eatery in clingy ivory satin dress and aviator
The star, 33, stepped out for dinner at Nobu in West Hollywood with friends on Tuesday night looking sensational in a clingy ivory satin dress.
Her hair was styled in a high topknot and she donned a black face mask as she arrived at the eatery.
Mix and match: The singer and businesswoman, 33, added a shearling-lined aviator jacket to keep out the spring chill
Night out: Rihanna, who recently dropped $13.8 million on a stunning Beverly Hills mansion, paired the sexy frock with matching crumpled boots with pointy toes
There are many classics when it comes to outerwear: the puffer, the trench, the teddy. But a plethora of our favorite stars can't resist an aviator jacket.
Katie Holmes, Jennifer Lopez and Alexa Chung are all fans. Rihanna has also jumped on the bandwagon by donning R13's AW20 design multiple times. This is the latest look; we love how she's thrown it over a slip dress and boots - just proof they can be worn with anything.
However, if the $3395 price tag is a bit too high, peruse the carousel for cheaper look-alikes from Mango, UGG and Monki.
Epitope antibody testing is effective in detecting immune responses to SARS-CoV-2
The authors of the study note their epitope antibody test could be helpful and more effective than conventional screening methods to identify people with the most effective plasma and antibodies for antibody therapy in the absence of a vaccine.
The researchers then searched for the exact targets of SARS-CoV-2 antibody responses. They created peptides with 89 epitopes for the S, N and membrane (M) proteins and exposed them to 15 donated plasma samples, including 14 samples from positive infection and one control.
The team found antibody responses honed in on 54 epitopes from the S and N protein. To assess the strength of an antibody response to treatment, the researchers created a ‘master epitope array’ with 16 epitopes that corresponded with strong IgG signaling.
The major 16 epitopes were screened with donated plasma from 89 COVID-19 patients and 9 controls. Findings showed that antibodies from people in the severe group in the ICU responded to more epitopes than the group with moderate infection.
The coronavirus spike protein has undergone the most mutations out of the whole coronavirus, and is the reason for the emergence of variants. To further understand this, the team created 28 mutations to the S or N proteins, which relates to the B.1.1.7 and B.1351 variant.
Results showed the mutations helped reduce or eliminate IgG binding to epitopes. Specifically, a P681H mutation changed the S-671 epitope and made it undetectable by antibodies.
The team developed a test that would measure the epitope-antibody response for coronavirus infections. When tested against donated plasma, agglutination was only found in plasma that had a positive diagnosis of COVID-19.
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
Jocelyn Solis-Moreira graduated with a Bachelor's in Integrative Neuroscience, where she then pursued graduate research looking at the long-term effects of adolescent binge drinking on the brain's neurochemistry in adulthood.
Platelet-rich plasma may restore ovarian function in early menopause
An injection of platelet-rich plasma and gonadotropins near the ovarian follicles may help restore ovarian function for early menopausal women, according to data from a pilot study published in Menopause .
“The retained immature follicles in ovaries of early menopausal women can be enhanced or stimulated to grow to mature follicles employing our new method, the whole dimension subcortical administration of platelet-rich plasma and gonadotropins,” Chao Chin Hsu, MD, PhD , of Taiwan United Birth-Promoting Experts (TUBE) Fertility Clinic in Tainan, Taiwan, told Healio. “We have proven successful oocyte retrieval to obtain mature oocytes for in vitro fertilization and embryo transfer in six early menopausal women, and one reached clinical pregnancy.”
Hsu and colleagues enrolled 12 ethnic Han Chinese women aged 40 to 50 years who had amenorrhea for more than 1 year and were referred to the TUBE Fertility Clinic between November 2018 and November 2019 (mean age, 44.4 years). All participants underwent routine preoperative evaluation before the procedure. The platelet-rich plasma was prepared from 40 mL of peripheral blood by the buffy coat protocol. A laparoscopic operation was performed in which 5 mL of platelet-rich plasma was combined with 300 IU of recombinant follicle-stimulating hormone. Injections of 0.2 mL were performed at each point of the superficial layer of the ovarian cortex and 15 injection sites in each ovary.
After treatment, participants were asked to have regular follow-up for 6 months or longer, including vaginal ultrasound scanning at weekly intervals to detect ovarian follicle growth. Participants who resumed ovarian function had in vitro fertilization carried out through the natural ovulatory cycle or under conventional controlled ovarian stimulation.
All 12 participants completed the procedure without specific complaints other than a mild pain over the lower abdomen. Due to the COVID-19 pandemic, most participants were restricted from attending scheduled visits and follow-up, and the cohort had a mean follow-up time of 125 days.
There were 10 oocyte retrieval procedures performed on six participants, with a mean interval of 65.2 days between treatment and first oocyte retrieval. Thirteen mature eggs were retrieved and fertilized through intracytoplasmic sperm injection to obtain 10 normally fertilized two pronuclei oocytes. Two participants had cleavage stage embryos transferred, and one achieved clinical pregnancy.
“This administration mode provides another treatment scenario for women of early menopause and those of impending ovarian failure to have better opportunity to conceive using their own eggs,” Hsu said. “There are also several devastating menopausal symptoms in women of early menopause, such as hot flashes, sweating and genitourinary syndrome, due to the dramatic decrease of ovarian hormones. Our study showed the regain of follicle growth with elevated serum estradiol level in most menopause women receiving our treatment. Thus, this application might help to relieve the symptoms and signs of early menopause.”
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