The p53 response in single cells is linearly correlated to the number of DNA breaks without a distinct threshold

Background: The tumor suppressor protein p53 is activated by cellular stress. DNA double strand breaks (DSBs) induce the activation of the kinase ATM, which stabilizes p53 and activates its transcriptional activity. Single cell analysis revealed that DSBs induced by gamma irradiation trigger p53 accumulation in a series of pulses that vary in number from cell to cell. Higher levels of irradiation increase the number of p53 pulses suggesting that they arise from periodic examination of the damage by ATM. If damage persists, additional pulses of p53 are triggered. The threshold of damage required for activating a p53 pulse is unclear. Previous studies that averaged the response across cell populations suggested that one or two DNA breaks are sufficient for activating ATM and p53. However, it is possible that by averaging over a population of cells important features of the dependency between DNA breaks and p53 dynamics are missed. Results: Using fluorescent reporters we developed a system for following in individual cells the number of DSBs, the kinetics of repair and the p53 response. We found a large variation in the initial number of DSBs and the rate of repair between individual cells. Cells with higher number of DSBs had higher probability of showing a p53 pulse. However, there was no distinct threshold number of breaks for inducing a p53 pulse. We present evidence that the decision to activate p53 given a specific number of breaks is not entirely stochastic, but instead is influenced by both cell-intrinsic factors and previous exposure to DNA damage. We also show that the natural variations in the initial amount of p53, rate of DSB repair and cell cycle phase do not affect the probability of activating p53 in response to DNA damage. Conclusions: The use of fluorescent reporters to quantify DNA damage and p53 levels in live cells provided a quantitative analysis of the complex interrelationships between both processes. Our study shows that p53 activation differs even between cells that have a similar number of DNA breaks. Understanding the origin and consequences of such variability in normal and cancerous cells is crucial for developing efficient and selective therapeutic interventions

Impacted maxillary canines and root resorptions of neighbouring teeth: a radiographic analysis using cone-beam computed tomography

The study analyses the location of impacted maxillary canines and factors influencing root resorptions of adjacent teeth using cone-beam computed tomography (CBCT). In addition, the interrater reliability between observers of two different dental specialties for radiographic parameters will be evaluated. CBCT images of patients who were referred for radiographic localization of impacted maxillary canines and/or suspicion of root resorptions of adjacent teeth were included. The study analysed the exact three-dimensional location of the impacted canines in the anterior maxilla, frequency and extent of root resorptions, and potential influencing factors. To assess interrater agreement, Cohen's correlation parameters were calculated. This study comprises 113 patients with CBCT scans, and 134 impacted canines were analysed retrospectively. In the patients evaluated, 69 impacted canines were located palatally (51.49 per cent), 41 labially (30.60 per cent), and 24 (17.91 per cent) in the middle of the alveolar process. Root resorptions were found in 34 lateral incisors (25.37 per cent), 7 central incisors (5.22 per cent), 6 first premolars (4.48 per cent), and 1 second premolar (0.75 per cent). There was a significant correlation between root resorptions on adjacent teeth and localization of the impacted canine in relation to the bone, as well as vertical localization of the canine. Interrater agreement showed values of 0.546-0.877. CBCT provides accurate information about location of the impacted canine and prevalence and degree of root resorption of neighbouring teeth with high interrater correlation. This information is of great importance for surgeons and orthodontists for accurate diagnostics and interdisciplinary treatment plannin

Complexity assessment and technical aspect of coronary angiogram and percutaneous coronary intervention following transcatheter aortic valve implantation

Background: Performing selective coronary angiogram (CA) and percutaneous coronary intervention (PCI) post transcatheter aortic valve implantation (TAVI) may be challenging with various success rates of coronary ostia engagement. Methods: Among all patients who underwent CA and/or PCI after TAVI from our single center TAVI registry, ostia cannulation success was reported according to the quality of ostia engagement and artery opacification, and was classified as either selective, partially selective or non-selective but sufficient for diagnosis. Results: Among the 424 consecutive TAVI procedures performed at the aforementioned institution, 20 (4.7%) CA were performed in 19 (4.5%) patients at a median time of 464 days post TAVI (25–75% IQ: 213–634 days). CA were performed in 7 CoreValve, 9 Evolut R, 1 Evolut PRO and 2 Edwards Sapien 3 devices. Transradial vascular approach was attempted in 9 procedures (45%, right n = 6 and left n = 3) and was successful in 8 (40%) patients. A total of 20 left main artery ostium cannulation were attempted leading to a diagnostic CA in all of them with selective engagement in 65%.  Engagement of the right coronary artery in 2 out of 15 attempted cases failed due to a low ostium in conjunction with a high implantation of a CoreValve prosthesis. 11 PCI (55% of CA) including 2 left main lesions were performed. In 4 patients (36.4% of the PCI), an extension catheter was required to engage the left main. All planned PCI were successful. Conclusions: Post TAVI CA and PCI are challenging but feasible even after supra-annular self-expandable valve implantation

Impacted maxillary canines and root resorptions of neighbouring teeth: a radiographic analysis using cone-beam computed tomography

The study analyses the location of impacted maxillary canines and factors influencing root resorptions of adjacent teeth using cone-beam computed tomography (CBCT). In addition, the interrater reliability between observers of two different dental specialties for radiographic parameters will be evaluated. CBCT images of patients who were referred for radiographic localization of impacted maxillary canines and/or suspicion of root resorptions of adjacent teeth were included. The study analysed the exact three-dimensional location of the impacted canines in the anterior maxilla, frequency and extent of root resorptions, and potential influencing factors. To assess interrater agreement, Cohen's correlation parameters were calculated. This study comprises 113 patients with CBCT scans, and 134 impacted canines were analysed retrospectively. In the patients evaluated, 69 impacted canines were located palatally (51.49 per cent), 41 labially (30.60 per cent), and 24 (17.91 per cent) in the middle of the alveolar process. Root resorptions were found in 34 lateral incisors (25.37 per cent), 7 central incisors (5.22 per cent), 6 first premolars (4.48 per cent), and 1 second premolar (0.75 per cent). There was a significant correlation between root resorptions on adjacent teeth and localization of the impacted canine in relation to the bone, as well as vertical localization of the canine. Interrater agreement showed values of 0.546-0.877. CBCT provides accurate information about location of the impacted canine and prevalence and degree of root resorption of neighbouring teeth with high interrater correlation. This information is of great importance for surgeons and orthodontists for accurate diagnostics and interdisciplinary treatment planning

Stimulus-dependent dynamics of p53 in single cells.

Many biological networks respond to various inputs through a common signaling molecule that triggers distinct cellular outcomes. One potential mechanism for achieving specific input-output relationships is to trigger distinct dynamical patterns in response to different stimuli. Here we focused on the dynamics of p53, a tumor suppressor activated in response to cellular stress. We quantified the dynamics of p53 in individual cells in response to UV and observed a single pulse that increases in amplitude and duration in proportion to the UV dose. This graded response contrasts with the previously described series of fixed pulses in response to γ-radiation. We further found that while γ-triggered p53 pulses are excitable, the p53 response to UV is not excitable and depends on continuous signaling from the input-sensing kinases. Using mathematical modeling and experiments, we identified feedback loops that contribute to specific features of the stimulus-dependent dynamics of p53, including excitability and input-duration dependency. Our study shows that different stresses elicit different temporal profiles of p53, suggesting that modulation of p53 dynamics might be used to achieve specificity in this network

Stimulus‐dependent dynamics of p53 in single cells

Many biological networks respond to various inputs through a common signaling molecule that triggers distinct cellular outcomes. One potential mechanism for achieving specific input–output relationships is to trigger distinct dynamical patterns in response to different stimuli. Here we focused on the dynamics of p53, a tumor suppressor activated in response to cellular stress. We quantified the dynamics of p53 in individual cells in response to UV and observed a single pulse that increases in amplitude and duration in proportion to the UV dose. This graded response contrasts with the previously described series of fixed pulses in response to γ-radiation. We further found that while γ-triggered p53 pulses are excitable, the p53 response to UV is not excitable and depends on continuous signaling from the input-sensing kinases. Using mathematical modeling and experiments, we identified feedback loops that contribute to specific features of the stimulus-dependent dynamics of p53, including excitability and input-duration dependency. Our study shows that different stresses elicit different temporal profiles of p53, suggesting that modulation of p53 dynamics might be used to achieve specificity in this network

Site-Specific Chemical Modification of a Cytokine Mimic for Small Molecule-Based Tumor Targeting

The targeted delivery of bioactive proteins, such as cytokines, for cancer immunotherapy approaches mostly relies on antibodies or antibody fragments. However, fusion proteins may display low tissue penetration due to a large molecular size. Small molecule ligands with high affinity toward tumor-associated antigens provide a promising alternative for the selective delivery of cytokines to tumor lesions. We developed a one-pot procedure for the site-specific thiazolidine formation between an aldehyde bearing small molecule and the in situ generated N-terminal cysteine of a bioactive protein. Thereby, neoleukin-2/15 (Neo-2/15), a computationally engineered interleukin-2 and -15 mimic, was chemically conjugated to acetazolamide plus, a potent carbonic anhydrase IX (CAIX) ligand. The conjugate retained the biological activity of Neo-2/15 and revealed its ability to accumulate in renal cell carcinoma (SK-RC-52) xenografts upon systemic intravenous administration. The results highlight the potential of small molecule targeting moieties to drive the accumulation of a protein cargo to the respective disease site while conserving the small construct size.ISSN:1043-1802ISSN:1520-481

Recurrent initiation: a mechanism for triggering p53 pulses in response to DNA damage.

DNA damage initiates a series of p53 pulses. Although much is known about the interactions surrounding p53, little is known about which interactions contribute to p53's dynamical behavior. The simplest explanation is that these pulses are oscillations intrinsic to the p53/Mdm2 negative feedback loop. Here we present evidence that this simple mechanism is insufficient to explain p53 pulses; we show that p53 pulses are externally driven by pulses in the upstream signaling kinases, ATM and Chk2, and that the negative feedback between p53 and ATM, via Wip1, is essential for maintaining the uniform shape of p53 pulses. We propose that p53 pulses result from repeated initiation by ATM, which is reactivated by persistent DNA damage. Our study emphasizes the importance of collecting quantitative dynamic information at high temporal resolution for understanding the regulation of signaling pathways and opens new ways to manipulate p53 pulses to ask questions about their function in response to DNA damage

p53 dynamics control cell fate.

Cells transmit information through molecular signals that often show complex dynamical patterns. The dynamic behavior of the tumor suppressor p53 varies depending on the stimulus; in response to double-strand DNA breaks, it shows a series of repeated pulses. Using a computational model, we identified a sequence of precisely timed drug additions that alter p53 pulses to instead produce a sustained p53 response. This leads to the expression of a different set of downstream genes and also alters cell fate: Cells that experience p53 pulses recover from DNA damage, whereas cells exposed to sustained p53 signaling frequently undergo senescence. Our results show that protein dynamics can be an important part of a signal, directly influencing cellular fate decisions