Rad51 Nucleoprotein Filament Disassembly Captured Using Fluorescent \u3cem\u3ePlasmodium falciparum\u3c/em\u3e SSB as a Reporter for Single-Stranded DNA

Single-stranded DNA binding (SSB) proteins coordinate DNA replication, repair, and recombination and are critical for maintaining genomic integrity. SSB binds to single-stranded DNA (ssDNA) rapidly and with very high affinity making it a useful molecular tool to detect free ssDNA in solution. We have labeled SSB from Plasmodium falciparum (Pf-SSB) with the MDCC (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)-carbonyl)coumarin) fluorophore which yields a four-fold increase in fluorescence upon binding to ssDNA. Pf-SSBMDCC binding to DNA is unaffected by NaCl or Mg2+ concentration and does not display salt-dependent changes in DNA binding modes or cooperative binding on long DNA substrates. These features are unique to Pf-SSB, making it an ideal tool to probe the presence of free ssDNA in any biochemical reaction. Using this Pf-SSBMDCC probe as a sensor for free ssDNA, we have investigated the clearing of preformed yeast Rad51 nucleoprotein filaments by the Srs2 helicase during HR. Our studies provide a rate for the disassembly of the Rad51 filament by full length Srs2 on long ssDNA substrates. Mutations in the conserved 2B domain in the homologous bacterial UvrD, Rep and PcrA helicases show an enhancement of DNA unwinding activity, but similar mutations in Srs2 do not affect its DNA unwinding or Rad51 clearing properties. These studies showcase the utility of the Pf-SSB probe in mechanistic investigation of enzymes that function in DNA metabolism

Cholecystectomy Deferral in Patients with Endoscopic Sphincterotomy

BACKGROUND: Cholecystectomy is not required in up to 64% of patients who adopt a wait-and-see policy after endoscopic clearance of common bile duct stones. Although reports of retrospective cohort series have shown a higher mortality among patients who defer cholecystectomy, it is not known if this is due to the patients\u27 premorbid health status or due to the deferral of cholecystectomy. Randomised clinical trials of prophylactic cholecystectomy versus wait-and-see have not had sufficient power to demonstrate differences in survival. OBJECTIVES: To evaluate the beneficial and harmful effects of cholecystectomy deferral (wait-and-see) versus elective (prophylactic) cholecystectomy in patients who have had an endoscopic biliary sphincterotomy. SEARCH STRATEGY: We searched The Cochrane Hepato-Biliary Group Controlled Trials Register, the Cochrane Controlled Trials Register (CENTRAL) in The Cochrane Library, MEDLINE (1966 to 2007), EMBASE (1980 to 2007), and Science Citation Index Expanded without language restrictions until April 2007. SELECTION CRITERIA: Randomised clinical trials comparing patients whose gallbladder was left in-situ after endoscopic sphincterotomy (wait-and-see group) versus patients who had cholecystectomy with either endoscopic sphincterotomy or common bile duct exploration (prophylactic cholecystectomy group), irrespective of blinding, language, or publication status. DATA COLLECTION AND ANALYSIS: We assessed the impact of a wait-and-see policy on mortality. Secondary outcomes assessed were the incidence of biliary pain, cholangitis, pancreatitis, need for cholangiography, need for cholecystectomy, and the rate of difficult cholecystectomy. We pooled data using relative risk with fixed-effect and random-effects models. MAIN RESULTS: We included 5 randomised trials with 662 participants out of 93 publications identified through the literature searches. The number of deaths was 47 in the wait-and-see group (334 patients) compared to 26 in the prophylactic cholecystectomy group (328 patients) for a 78% increased risk of mortality (RR 1.78, 95% CI 1.15 to 2.75, P = 0.010). The survival benefit of prophylactic cholecystectomy was independent of trial design, inclusion of high risk patients or inclusion of any one of the five trials. Patients in the wait-and-see group had higher rates of recurrent biliary pain (RR 14.56, 95% CI 4.95 to 42.78, P \u3c 00001), jaundice or cholangitis (RR 2.53, 95% CI 1.09 to 5.87, P = 0.03), and of repeat ERCP or other forms of cholangiography (RR 2.36, 95% CI 1.29 to 4.32, P = 0.005). Cholecystectomy was eventually performed in 35% (115 patients) of the wait-and-see group. AUTHORS\u27 CONCLUSIONS: Prophylactic cholecystectomy should be offered to patients whose gallbladders remain in-situ after endoscopic sphincterotomy and common bile duct clearance

Monitoring Replication Protein A (RPA) Dynamics in Homologous Recombination Through Site-specific Incorporation of Non-canonical Amino Acids

An essential coordinator of all DNA metabolic processes is Replication Protein A (RPA). RPA orchestrates these processes by binding to single-stranded DNA (ssDNA) and interacting with several other DNA binding proteins. Determining the real-time kinetics of single players such as RPA in the presence of multiple DNA processors to better understand the associated mechanistic events is technically challenging. To overcome this hurdle, we utilized non-canonical amino acids and bio-orthogonal chemistry to site-specifically incorporate a chemical fluorophore onto a single subunit of heterotrimeric RPA. Upon binding to ssDNA, this fluorescent RPA (RPAf) generates a quantifiable change in fluorescence, thus serving as a reporter of its dynamics on DNA in the presence of multiple other DNA binding proteins. Using RPAf, we describe the kinetics of facilitated self-exchange and exchange by Rad51 and mediator proteins during various stages in homologous recombination. RPAf is widely applicable to investigate its mechanism of action in processes such as DNA replication, repair and telomere maintenance

Fluorescent Probes to Investigate Homologous Recombination Dynamics

There are multiple mechanisms by which DNA can become damaged. Such damage must be repaired for the cell to avoid ill-health consequences. Homologous recombination (HR) is a means of repairing one specific type of damage, a double-strand break (DSB). This complex pathway includes the Rad51-DNA nucleoprotein filament as its primary machinery. Current methodology for studying HR proteins includes the use of fluorescently labeled DNA to probe for HR dynamics. This technique limits the number of proteins that can be involved in experimentation, and often only works as an end reporter. The work here aims at improving upon standard techniques by creating two fluorescent protein probes. The first probe was developed by directly attaching a fluorophore to Saccharomyces cerevisiae Rad51 with the use of click chemistry and the incorporation of unnatural amino acids. This probe could function as a primary reporter on the formation and dissociation of the Rad51-DNA filament in the presence of pro- and anti- HR mediator proteins. The second probe was created by labeling the exterior cysteine residues of Plasmodium falciparum single strand DNA binding protein (SSB) with a fluorophore via maleimide chemistry. This probe acts as a secondary reporter for HR dynamics by signaling for when free single stranded DNA (ssDNA) is available

Death of a cluster: the destruction of M67 as seen by the SDSS

We probe the spatial and dynamical structure of the old open cluster M67 using photometric data from the Sloan Digital Sky Survey's sixth data release. Making use of an optimal contrast, or matched filter, algorithm, we map the distribution of high probability members of M67. We find an extended and elongated halo of likely members to a radius of nearly 60'. Our measured core radius of Rcore = 8.'24+/-0.'60 is somewhat larger than that of previous estimates. We attribute the larger core radius measurement to the SDSS probing lower mass main sequence stars than has been done before for similar studies of M67, and the exclusion of post main sequence M67 members in the SDSS sample. We estimate the number of M67 members in our SDSS sample to be 1385+/-67 stars. A lower limit on the binary fraction in M67 is measured to be 45%. A higher fraction of binary stars is measured in the core as compared to the halo, and the luminosity function of the core is found to be more depleted of low-mass stars. Thus the halo is consistent with mass segregation within the cluster. The galactic orbit of M67 is calculated from recent proper motion and radial velocity determinations. The elongated halo is roughly aligned to the proper motion of the cluster. This appears to be a result of mass segregation due to the galactic tidal field. Our algorithm is run on 2MASS photometry to directly compare to previous studies of M67. Decreasing core radii are found for stars with greater masses. We test the accuracy of our algorithm using 1000 artificial cluster Monte Carlo simulations. It is found that the matched filter technique is suitable for recovering low-density spatial structures, as well as measuring the binary fraction of the cluster.Comment: 20 figures, ApJ Accepte

Chromospheric Activity of HAT-P-11: an Unusually Active Planet-Hosting K Star

Kepler photometry of the hot Neptune host star HAT-P-11 suggests that its spot latitude distribution is comparable to the Sun's near solar maximum. We search for evidence of an activity cycle in the CaII H & K chromospheric emission $S$-index with archival Keck/HIRES spectra and observations from the echelle spectrograph on the ARC 3.5 m Telescope at APO. The chromospheric emission of HAT-P-11 is consistent with a $\gtrsim 10$ year activity cycle, which plateaued near maximum during the Kepler mission. In the cycle that we observed, the star seemed to spend more time near active maximum than minimum. We compare the $\log R^\prime_{HK}$ normalized chromospheric emission index of HAT-P-11 with other stars. HAT-P-11 has unusually strong chromospheric emission compared to planet-hosting stars of similar effective temperature and rotation period, perhaps due to tides raised by its planet.Comment: 16 pages, 8 figures; accepted to the Astrophysical Journa

Spatiotemporal relationships of coyotes and free-ranging domestic cats as indicators of conflict in Culver City, California

As habitat generalists, urban coyote (Canis latrans) populations often utilize an abundance of diverse food sources in cities. Within southern California, domestic cats (Felis catus) comprise a higher proportion of coyote diets than in other studied urban areas throughout the United States. However, it is unclear which ecological factors contribute to higher rates of cat depredation by coyotes in this region. While previous research suggests that coyote presence may have a negative effect on free-ranging domestic cat distributions, few studies have determined whether urban green spaces affect coyote or free-ranging domestic cat occurrence and activity within a predominantly urbanized landscape. We placed 20 remote wildlife cameras across a range of green spaces and residential sites in Culver City, California, an area of Los Angeles County experiencing pronounced coyote-domestic cat conflict. Using data collected across 6 months from 2019–2020, we assessed the influence of green space and prey species (i.e., cottontail rabbits (Sylvilagus spp.) and domestic cats) on coyote habitat use and activity. Coyotes exhibited a preference for sites with higher amounts of green space, while domestic cat habitat use was high throughout our study region. Although cottontail rabbit habitat use was also highly associated with urban green space, neither cottontails nor domestic cats appeared to temporally overlap significantly with coyotes. Unlike other cities where coyotes and domestic cats exhibit strong habitat partitioning across the landscape, domestic cats and coyotes spatially overlapped in green space fragments throughout Culver City. We suggest that this pattern of overlap may be responsible for the frequent cases of domestic cat depredation by coyotes in Culver City