Human exonuclease 1 role in response to UV irradiation

DNA damage checkpoints are surveillance mechanisms that monitor the integrity of the genome. Nucleotide excision repair (NER) is a DNA repair mechanism that cells use to remove UV-induced DNA lesions. Previous publication from our laboratory demonstrated that recognition and processing of UV-induced damage by NER is required for proper activation of checkpoint through interactions between NER proteins and checkpoint factors in yeast and human primary fibroblasts. From a two hybrid screening in yeast exonuclease 1 (Exo1) was identified as a 9-1-1 complex interactor. Exo1 is a 5\u2019-3\u2019 exonuclease and 5'-flap-endonuclease with many different roles in DNA metabolism such as meiotic and mitotic recombination, mismatch repair and telomere processing. Characterization of an exo1 yeast deleted strain has shown that this protein is involved in the early steps of UV-induced DNA damage checkpoint. In human cells EXO1 is present as two isoforms named hEXO1a and hEXO1b genetarated by alternative splicing. We are analyzing the role of EXO1 in checkpoint activation in response to UV-C damage in human cells: using siRNA against both a and b isoform of hEXO1 in G1 cells we were able to observe a defect in Chk1 and p53 phosphorylation induced by UV-C irradiation

TLS Polymerases are involved in processing of EXO1-dependent lesions after UV-induced damage

UV light mainly damages DNA by generating CPDs and 6-4PP photoproducts, which are responsible for the pathological effects of sunlight. In a healthy organism, such DNA helix distorting lesions are removed by Nucleotide Excision Repair (NER), a multistep process. Mutations in NER genes cause the onset of severe pathologies. The principal symptom common to all diseases is the strong sensitivity to UV. A high predisposition to tumors development arises in xeroderma pigmentosum (XP) patients, while neurological dysfunctions have been observed in both XP and Cockayne syndrome patients. Upon DNA damage sensing, checkpoints are activated allowing a block or delay of cell cycle progression to ensure repair of the DNA lesions. Intriguingly, while in normal cells UV irradiation activates DNA damage checkpoints in all phases of the cell cycle NER yeast mutant strains and human fibroblasts derived from XP patients fail activate the checkpoint in G1 and G2. Recently, we demonstrated that the checkpoint response to UV light in cells that are not actively replicating their genome requires prior processing of the UV lesions. This involves NER factors but also the Exo1 nuclease. In particular, acting on NER intermediates, Exo1 generates structures containing long tracts of ssDNA in response to UV irradiation. This role of Exo1 is only observed at a subset of problematic lesions that cannot properly repaired by canonic NER. It is these Exo1-induced structures that provide the signal for checkpoint activation both in yeast and human non-replicating cells. The essential role of Exo1 in UV-induced checkpoint activation in vivo has been recently supported by in vitro reconstitution of the activation pathway. What are the problematic lesions that require EXO1 activity is still unknown. We hypothesized that Closely Opposing UV Lesions (COLs) on the two DNA strands could exist and may be a likely candidate. This scenario would require TLS polymerases bypass during repair synthesis step. Therefore, we are investigating Y-family polymerase recruitment at EXO1-positive local UV damage sites (LUDs). We found that Pol h is recruited at both EXO1-positive and EXO1-negative LUDs, while Pol \u3b9 and\uf020Pol \u3ba always co-localize with the nuclease\uf02e Using the CRISPR-Cas9 system, we generated EXO1 knock out cell lines that demonstrated a requirement for EXO1 in Pol \u3b9 and\uf020Pol \u3ba recruitment, consistently with our working model\uf02e Finally, when we silenced TLS polymerases we observed a hyper-activation of UV-induced DNA damage checkpoint, suggesting that EXO1 continues to process UV damaged DNA enlarging the gap and eventually producing DSBs. TLS polymerases, thus are crucial to prevent dangerous situations in non-replicating UV irradiated cells

Robotic therapy : Cost, accuracy, and times. New challenges in the neonatal intensive care unit

Background: The medication process in the Neonatal Intensive Care Unit (NICU), can be challenging in terms of costs, time, and the risk of errors. Newborns, especially if born preterm, are more vulnerable to medication errors than adults. Recently, robotic medication compounding has reportedly improved the safety and efficiency of the therapeutic process. In this study, we analyze the advantages of using the I.V. Station\uae system in our NICU, compared to the manual preparation of injectable drugs in terms of accuracy, cost, and time. Method: An in vitro experimental controlled study was conducted to analyze 10 injectable powdered or liquid drugs. Accuracy was calculated within a 5% difference of the bottle weight during different stages of preparation (reconstitution, dilution, and final product). The overall cost of manual and automated preparations were calculated and compared. Descriptive statistics for each step of the process are presented as mean \ub1 standard deviation or median (range). Results: The median error observed during reconstitution, dilution, and final therapy of the drugs prepared by the I.V. Station\uae ranged within \ub15% accuracy, with narrower ranges of error compared to those prepared manually. With increasing preparations, the I.V. Station\uae consumed less materials, reduced costs, decreased preparation time, and optimized the medication process, unlike the manual method. In the 10 drugs analyzed, the time saved from using the I.V. Station\uae ranged from 16 s for acyclovir to 2 h 57 min for teicoplanin, and cost savings varied from 8% for ampicillin to 66% for teicoplanin. These advantages are also capable of continually improving as the total amount of final product increases. Conclusions: The I.V. Station\uae improved the therapeutic process in our NICU. The benefits included increased precision in drug preparation, improved safety, lowered cost, and saved time. These advantages are particularly important in areas such as the NICU, where the I.V. Station\uae could improve the delivery of the high complexity of care and a large amount of intravenous therapy typically required. In addition, these benefits may lead to the reduction in medication errors and improve patient and family care; however, additional studies will be required to confirm this hypothesis

Maternal views on facilitators of and barriers to breastfeeding preterm infants

Background: The supply of breast milk to preterm infants tends to occur at a lower rate than that recorded among term infants. We aimed to investigate the facilitators of and barriers to breastfeeding during hospital stay according to the experiences of mothers that gave birth to premature infants requiring admission to neonatal intensive care unit. Methods: A cross-sectional questionnaire survey was conducted. Mothers who had delivered a newborn with a gestational age 6433 weeks requiring intensive care, entered the study. Basic subjects' characteristics and infant feeding practices were also recorded. Results: A total of 64 mothers were enrolled, leading to a total of 81 infants. At discharge, any breastfeeding was recorded in 66% of infants, with 27% of those infants being exclusively breastfed. Any infant was exclusively fed directly at the breast. Most mothers experienced adequate support during their infant's hospitalization and reported satisfaction with breastfeeding. Almost all mothers felt that feeding their infant human milk was beneficial for the infant's health. Thirty percent of the mothers reported that they had experienced some obstacles to breastfeeding. Specifically, infants born to mothers who experienced difficulties in pumping breast milk (OR = 4.6; CI 1.5-13.9) or in providing an adequate amount of milk to the infant (OR = 3.57; CI 1.1-11.5) were at higher risk of being fed with formula at discharge. Conclusions: On the basis of the present results, health care professionals should target their efforts to optimize breastfeeding support for mothers of premature infants admitted to level III care, especially by improving breast milk production and endorsing direct breastfeeding

Complementary feeding practices in a cohort of Italian late preterm infants

Limited data are available on complementary feeding in preterm infants, who show increased nutritional needs and are at risk of altered postnatal growth. The aim of this study was to investigate the timing and content of complementary feeding in a cohort of late preterm infants. We conducted a prospective, observational study, including mothers who had given birth to infants admitted to level I or II of care with a gestational age between 34 and 36 weeks. Mothers were contacted at 3, 6 and 12 months after delivery by phone calls and were asked about their infant\u2019s mode of feeding and the timing and schedule of the introduction of different solid foods types. A total of 49 mothers and 57 infants completed the study. The mean postnatal age of the introduction of complementary foods was 5.7 \ub1 0.7 months. Low energy and/or low protein-dense foods were first introduced in most infants. Fruit as the first type of complementary food in the infant\u2019s diet was associated with a 1.6-month advance in initiating complementary feeding. The present findings provide further insight into complementary feeding practices in late preterm infants and underline the need for specific recommendations addressing this vulnerable population

Start a neonatal extracorporeal membrane oxygenation program: A multistep team training

Background: Extracorporeal membrane oxygenation (ECMO) is a complex life-saving support for acute cardio-respiratory failure, unresponsive to medical treatment. Emergency events on ECMO are rare but require immediate and proficient management. Multidisciplinary ECMO team members need to acquire and maintain over time cognitive, technical and behavioral skills, to safely face life-threatening clinical scenarios. Methods: A multistep educational program was delivered in a 4-year period to 32 ECMO team members, based on guidelines from the Extracorporeal Life Support Organization. A first traditional module was provided through didactic lectures, hands-on water drills, and laboratory animal training. The second phase consisted of a multi-edition high-fidelity simulation-based training on a modified neonatal mannequin (SimNewB\uae). In each session, participants were called to face, in small groups, ten critical scenarios, followed by debriefing time. Trainees underwent a pre-test for baseline competency assessment. Once completed the full training program, a post-test was administered. Pre- and post-test scores were compared. Trainees rated the educational program through survey questionnaires. Results: 28 trainees (87.5%) completed the full educational program. ECMO staffskills improved from a median pre-test score of 7.5/18 (IQR = 6-11) to 14/18 (IQR = 14-16) at post-test (P < 0.001, Wilcoxon rank test). All trainees highly rated the educational program and its impact on their practice. They reported high-fidelity simulations to be beneficial to novice learners as it increased self-confidence in ECMO-emergencies (according to 100% of surveyed), theoretical knowledge (61.5%) and team-work/communicative skills (58%). Conclusions: The multistep ECMO team training increased staff' knowledge, technical skills, teamwork, and self-confidence, allowing the successful development of a neonatal respiratory ECMO program. Conventional training was perceived as relevant in the early phase of the program development, while the active learning emerged to be more beneficial to master ECMO knowledge, specific skills, and team performance

Do a Few Weeks Matter? Late Preterm Infants and Breastfeeding Issues

The late preterm infant population is increasing globally. Many studies show that late preterm infants are at risk of experiencing challenges common to premature babies, with breastfeeding issues being one of the most common. In this study, we investigated factors and variables that could interfere with breastfeeding initiation and duration in this population. We conducted a prospective observational study, in which we administered questionnaires on breastfeeding variables and habits to mothers of late preterm infants who were delivered in the well-baby nursery of our hospital and followed up for three months after delivery. We enrolled 149 mothers and 189 neonates, including 40 pairs of twins. Our findings showed that late preterm infants had a low rate of breastfeeding initiation and early breastfeeding discontinuation at 15, 40 and 90 days of life. The mothers with higher educational levels and previous positive breastfeeding experience had a longer breastfeeding duration. The negative factors for breastfeeding were the following: Advanced maternal age, Italian ethnicity, the feeling of reduced milk supply and having twins. This study underlines the importance of considering these variables in the promotion and protection of breastfeeding in this vulnerable population, thus offering mothers tailored support

Sensing of Replication Stress and Mec1 Activation Act through Two Independent Pathways Involving the 9-1-1 Complex and DNA Polymerase ε

Following DNA damage or replication stress, budding yeast cells activate the Rad53 checkpoint kinase, promoting genome stability in these challenging conditions. The DNA damage and replication checkpoint pathways are partially overlapping, sharing several factors, but are also differentiated at various levels. The upstream kinase Mec1 is required to activate both signaling cascades together with the 9-1-1 PCNA-like complex and the Dpb11 (hTopBP1) protein. After DNA damage, Dpb11 is also needed to recruit the adaptor protein Rad9 (h53BP1). Here we analyzed the mechanisms leading to Mec1 activation in vivo after DNA damage and replication stress. We found that a ddc1Δdpb11-1 double mutant strain displays a synthetic defect in Rad53 and H2A phosphorylation and is extremely sensitive to hydroxyurea (HU), indicating that Dpb11 and the 9-1-1 complex independently promote Mec1 activation. A similar phenotype is observed when both the 9-1-1 complex and the Dpb4 non-essential subunit of DNA polymerase ε (Polε) are contemporarily absent, indicating that checkpoint activation in response to replication stress is achieved through two independent pathways, requiring the 9-1-1 complex and Polε

Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway

Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends

Histone methyltransferase Dot1 and Rad9 inhibit single-stranded DNA accumulation at DSBs and uncapped telomeres

Cells respond to DNA double-strand breaks (DSBs) and uncapped telomeres by recruiting checkpoint and repair factors to the site of lesions. Single-stranded DNA (ssDNA) is an important intermediate in the repair of DSBs and is produced also at uncapped telomeres. Here, we provide evidence that binding of the checkpoint protein Rad9, through its Tudor domain, to methylated histone H3-K79 inhibits resection at DSBs and uncapped telomeres. Loss of DOT1 or mutations in RAD9 influence a Rad50-dependent nuclease, leading to more rapid accumulation of ssDNA, and faster activation of the critical checkpoint kinase, Mec1. Moreover, deletion of RAD9 or DOT1 partially bypasses the requirement for CDK1 in DSB resection. Interestingly, Dot1 contributes to checkpoint activation in response to low levels of telomere uncapping but is not essential with high levels of uncapping. We suggest that both Rad9 and histone H3 methylation allow transmission of the damage signal to checkpoint kinases, and keep resection of damaged DNA under control influencing, both positively and negatively, checkpoint cascades and contributing to a tightly controlled response to DNA damage