Human DDK rescues stalled forks and counteracts checkpoint inhibition at unfired origins to complete DNA replication.

Eukaryotic genomes replicate via spatially and temporally regulated origin firing. Cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) promote origin firing, whereas the S phase checkpoint limits firing to prevent nucleotide and RPA exhaustion. We used chemical genetics to interrogate human DDK with maximum precision, dissect its relationship with the S phase checkpoint, and identify DDK substrates. We show that DDK inhibition (DDKi) leads to graded suppression of origin firing and fork arrest. S phase checkpoint inhibition rescued origin firing in DDKi cells and DDK-depleted Xenopus egg extracts. DDKi also impairs RPA loading, nascent-strand protection, and fork restart. Via quantitative phosphoproteomics, we identify the BRCA1-associated (BRCA1-A) complex subunit MERIT40 and the cohesin accessory subunit PDS5B as DDK effectors in fork protection and restart. Phosphorylation neutralizes autoinhibition mediated by intrinsically disordered regions in both substrates. Our results reveal mechanisms through which DDK controls the duplication of large vertebrate genomes.

A focus group study of Ontario dairy producer perspectives on neonatal care of male and female calves.

Providing optimal calf care remains a challenge on many dairy farms and has important implications for the future health, welfare, and productivity of male and female calves. Recent research suggests that male dairy calves receive a lower quality of care early in life than female calves, but further investigation is required to determine the factors that influence this disparity. The objectives of this study were to understand dairy producer perspectives on neonatal calf care practices and explore differences between male and female calf care. Overall, 23 dairy producers in Ontario, Canada, participated in 4 focus groups about calf care practices that were recorded and evaluated qualitatively using thematic analysis. Major barriers for good calf care included lack of knowledge about the best management practices for calf care and the prioritization of farm resources toward the milking herd. Some producers also noted that farm infrastructure (particularly during challenging weather) and employee training were important limitations. The economic cost of providing good neonatal calf care was important primarily for male calves and acted as a motivation or a barrier depending on the producer's beliefs about calf care and how they chose to market their calves. The primary source of knowledge producers used to develop calf care practices was their own experience, although many also relied on dairy-industry advisors, most often veterinarians. Producers were motivated by social norms, along with intrinsic pride and obligation to provide good calf care, and these motives were influenced by their emotional state. Producers expressed beliefs about which aspects of calf care are most important-notably colostrum management-and appreciated simple and economical solutions to calf-rearing challenges. Calf care practices were varied, and we identified a diversity of knowledge, motivations, and barriers to adopting best management practices, which sometimes differed between male and female calves. Some producers said that they did not know what happened to their male calves after they left the farm and tended to prioritize the care of female over male calves in subtle ways, such as less timely provision of colostrum. The infrastructure investment and other costs associated with caring for male calves often limited their care, but producers were still motivated to provide adequate care for male calves. These findings represent potential targets for additional research and intervention strategies to improve calf care practices on dairy farms.

Correlation of molecular expression with diel rhythm of oviposition in Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae) and implications for forensic entomology.

This study explored the molecular mechanisms potentially underlying blow fly nocturnal oviposition. A behavioral study revealed that Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae) possesses a diel rhythm of oviposition in light under 12:12 light/dark conditions. Reversal to 12:12 dark/light resulted in oviposition behavior changing to align with the adjusted regime in most females, but four of 59 experimental females lacked a diel rhythm of oviposition (were arrhythmic). Real-time PCR was used to monitor the molecular expression levels of known circadian genes per and tim in C. vicina to determine whether gene expression and behavior correlated. As with behavior, reversing light/dark conditions changed rhythmic gene expression to align with an adjusted light regime. This suggests that although it is unlikely that C. vicina will colonize dead bodies at night, arrhythmic females and oviposition in the dark was demonstrated.

Nocturnal colonization behavior of blowflies (Diptera: Calliphoridae) in southeastern Australia.

Worldwide research into nocturnal colonization by blowflies has produced many contradictory findings, prompting investigation specific to southeastern Australia. Initial experiments showed that blowfly colonization begins shortly after sunrise and continues until sunset; nocturnal colonization never occurred. Colonization peaks occurred at mid-morning, midday, and in the hours preceding sunset. In an additional experiment, wild blowflies were captured and placed in cages with colonization medium supplied nocturnally. Colonization occurred on four of five nights, and Calliphora augur (Fabricius) (Diptera: Calliphoridae) was the main species colonizing baits nocturnally. Results suggest that colonization is most likely to occur during warm weather and when flies are able to walk or crawl to bait. In particular, blowflies trapped within a confined space (such as a room or car) with warmer-than-ambient temperature may be stimulated to colonize nearby remains. Entomologists should consider these findings when estimating minimum postmortem interval under these environmental conditions.

Abiotic environmental factors influencing blowfly colonisation patterns in the field.

The accuracy of minimum post-mortem interval (mPMI) estimates usually hinges upon the ability of forensic entomologists to predict the conditions under which calliphorids will colonise bodies. However, there can be delays between death and colonisation due to poorly understood abiotic and biotic factors, hence the need for a mPMI. To quantify the importance of various meteorological and light-level factors, beef liver baits were placed in the field (Victoria, Australia) on 88 randomly selected days over 3 years in all seasons and observed every 60-90 min for evidence of colonisation. Baits were exposed during daylight, and the following parameters were measured: barometric pressure, light intensity, wind speed, ambient temperature, relative humidity and rainfall. Collected data were analysed using backward LR logistic regression to produce an equation of colonisation probability. This type of analysis removes factors with the least influence on colonisation in successive steps until all remaining variables significantly increase the accuracy of predicting colonisation presence or absence. Ambient temperature was a positive predictor variable (an increase in temperature increased the probability of calliphorid colonisation). Relative humidity was a negative predictor variable (an increase in humidity decreased the probability of calliphorid colonisation). Barometric pressure, light intensity, wind speed and rainfall did not enhance the accuracy of the probability model; however, analysis of species activity patterns suggests that heavy rainfall and strong wind speeds inhibit calliphorid colonisation.

Mps1 directs the assembly of Cdc20 inhibitory complexes during interphase and mitosis to control M phase timing and spindle checkpoint signaling.

The spindle assembly checkpoint (SAC) in mammals uses cytosolic and kinetochore-based signaling pathways to inhibit anaphase. In this study, we use chemical genetics to show that the protein kinase Mps1 regulates both aspects of the SAC. Human MPS1-null cells were generated via gene targeting and reconstituted with either the wild-type kinase (Mps1(wt)) or a mutant version (Mps1(as)) sensitized to bulky purine analogues. Mps1 inhibition sharply accelerated anaphase onset, such that cells completed mitosis in 12 min, and prevented Cdc20's association with either Mad2 or BubR1 during interphase, i.e., before the appearance of functional kinetochores. Furthermore, intramitotic Mps1 inhibition evicted Bub1 and all other known SAC transducers from the outer kinetochore, but contrary to a recent study, did not perturb aurora B-dependent phosphorylation. We conclude that Mps1 has two complementary roles in SAC regulation: (1) initial cytoplasmic activation of Cdc20 inhibitors and (2) recruitment of factors that promote sustained anaphase inhibition and chromosome biorientation to unattached kinetochores.

Effect of morphine on the growth rate of Calliphora stygia (Fabricius) (Diptera: Calliphoridae) and possible implications for forensic entomology.

Insect specimens collected from decomposing bodies enable forensic entomologists to estimate the minimum post-mortem interval (PMI). Drugs and toxins within a corpse may affect the development rate of insects that feed on them and it is vital to quantify these effects to accurately calculate minimum PMI. This study investigated the effects of morphine on growth rates of the native Australian blowfly, Calliphora stygia (Fabricius) (Diptera: Calliphoridae). Several morphine concentrations were incorporated into pet mince to simulate post-mortem concentrations in morphine, codeine and/or heroin-dosed corpses. There were four treatments for feeding larvae; T 1: control (no morphine); T 2: 2 microg/g morphine; T 3: 10 microg/g morphine; and T 4: 20 microg/g morphine. Ten replicates of 50 larvae were grown at 22 degrees C for each treatment and their development was compared at four comparison intervals; CI 1: 4-day-old larvae; CI 2: 7-day-old larvae; CI 3: pupae; and CI 4: adults. Length and width were measured for larvae and pupae, and costae and tibiae were measured for adults. Additionally, day of pupariation, day of adult eclosion, and survivorship were calculated for each replicate. The continued presence of morphine in meat was qualitatively verified using high-performance liquid chromatography with acidic potassium permanganate chemiluminescence detection. Growth rates of C. stygia fed on morphine-spiked mince did not differ significantly from those fed on control mince for any comparison interval or parameter measured. This suggests that C. stygia is a reliable model to use to accurately age a corpse containing morphine at any of the concentrations investigated.

Polo kinase and separase regulate the mitotic licensing of centriole duplication in human cells.

It has been proposed that separase-dependent centriole disengagement at anaphase licenses centrosomes for duplication in the next cell cycle. Here we test whether such a mechanism exists in intact human cells. Loss of separase blocked centriole disengagement during mitotic exit and delayed assembly of new centrioles during the following S phase; however, most engagements were eventually dissolved. We identified Polo-like kinase 1 (Plk1) as a parallel activator of centriole disengagement. Timed inhibition of Plk1 mapped its critical period of action to late G2 or early M phase, i.e., prior to securin destruction and separase activation at anaphase onset. Crucially, when cells exited mitosis after downregulation of both separase and Plk1, centriole disengagement failed completely, and subsequent centriole duplication in interphase was also blocked. Our results indicate that Plk1 and separase act at different times during M phase to license centrosome duplication, reminiscent of their roles in removing cohesin from chromosomes.