Impact of clinical guidance and rapid molecular pathogen detection on evaluation and outcomes of febrile or hypothermic infants.

OBJECTIVE: To quantify the impact of clinical guidance and rapid respiratory and meningitis/encephalitis multiplex polymerase chain reaction (mPCR) testing on the management of infants. DESIGN: Before-and-after intervention study. SETTING: Tertiary-care children's hospital. PATIENTS: Infants ≤90 days old presenting with fever or hypothermia to the emergency department (ED). METHODS: The study spanned 3 periods: period 1, January 1, 2011, through December 31, 2014; period 2, January 1, 2015, through April 30, 2018; and period 3, May 1, 2018, through June 15, 2019. During period 1, no standardized clinical guideline had been established and no rapid pathogen testing was available. During period 2, a clinical guideline was implemented, but no rapid testing was available. During period 3, a guideline was in effect, plus mPCR testing using the BioFire FilmArray respiratory panel 2 (RP 2) and the meningitis encephalitis panel (MEP). Outcomes included antimicrobial and ancillary test utilization, length of stay (LOS), admission rate, 30-day mortality. Outcomes were compared across periods using Kruskal-Wallis and Pearson tests and interrupted time series analysis. RESULTS: Overall 5,317 patients were included: 2,514 in period 1, 2,082 in period 2, and 721 in period 3. Over the entire study period, we detected reductions in the use of chest radiographs, lumbar punctures, LOS, and median antibiotic duration. After adjusting for temporal trends, we observed that the introduction of the guideline was associated with reductions in ancillary tests and lumbar punctures. Use of mPCR testing with the febrile infant clinical guideline was associated with additional reductions in ancillary testing for all patients and a higher proportion of infants 29-60 days old being managed without antibiotics. CONCLUSIONS: Use of mPCR testing plus a guideline for young infant evaluation in the emergency department was associated with less antimicrobial and ancillary test utilization compared to the use of a guideline alone.

APE2 is required for ATR-Chk1 checkpoint activation in response to oxidative stress.

The base excision repair pathway is largely responsible for the repair of oxidative stress-induced DNA damage. However, it remains unclear how the DNA damage checkpoint is activated by oxidative stress at the molecular level. Here, we provide evidence showing that hydrogen peroxide (H2O2) triggers checkpoint kinase 1 (Chk1) phosphorylation in an ATR [ataxia-telangiectasia mutated (ATM) and Rad3-related]-dependent but ATM-independent manner in Xenopus egg extracts. A base excision repair protein, Apurinic/apyrimidinic (AP) endonuclease 2 (APE2, APN2, or APEX2), is required for the generation of replication protein A (RPA)-bound single-stranded DNA, the recruitment of a checkpoint protein complex [ATR, ATR-interacting protein (ATRIP), and Rad9] to damage sites, and H2O2-induced Chk1 phosphorylation. A conserved proliferating cell nuclear antigen interaction protein box of APE2 is important for the recruitment of APE2 to H2O2-damaged chromatin. APE2 3'-phosphodiesterase and 3'-5' exonuclease activity is essential for single-stranded DNA generation in the 3'-5' direction from single-stranded breaks, referred to as single-stranded break end resection. In addition, APE2 associates with Chk1, and a serine residue (S86) in the Chk1-binding motif of APE2 is essential for Chk1 phosphorylation, indicating a Claspin-like but distinct role for APE2 in ATR-Chk1 signaling. Our data indicate that APE2 plays a vital and previously unexpected role in ATR-Chk1 checkpoint signaling in response to oxidative stress. Thus, our findings shed light on a distinct mechanism of how an ATR-Chk1-dependent DNA damage checkpoint is mediated by APE2 in the oxidative stress response.

WD40-repeat protein WDR18 collaborates with TopBP1 to facilitate DNA damage checkpoint signaling.

The genomes of all living organisms are exposed to a wide spectrum of insults. To maintain genomic integrity, eukaryotes have evolved an elaborate surveillance mechanism - DNA damage checkpoint signaling - to detect damaged DNA and to arrest cell cycle progression, allowing time to process and repair DNA damage. TopBP1 plays multiple roles in the regulation of DNA damage checkpoint signaling. However, the molecular mechanism of how TopBP1 regulates ATR-mediated Chk1 phosphorylation is poorly understood. In this communication, we demonstrate (1) that the Chk1 activation domain of TopBP1 is critical in response to several different types of DNA damage; (2) that WD40-repeat protein WDR18 associates with the C-terminus of TopBP1 in vitro and in vivo; (3) that the association between WDR18 and TopBP1 is required for AT70-induced Chk1 phosphorylation; (4) and that WDR18 itself is required for AT70-triggered Chk1 phosphorylation. In addition, WDR18 associates with Chk1 in vitro. The data suggest that WDR18 facilitates ATR-dependent Chk1 phosphorylation via interacting with both C-terminus of TopBP1 and Chk1. Our findings indicate that WDR18 is a bona fide checkpoint protein and that WDR18 works together with TopBP1 to promote DNA damage checkpoint signaling.

Study of the DNA damage checkpoint using Xenopus egg extracts.

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop. Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle. The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures. DNA damage can be induced by ultraviolet (UV) irradiation, γ-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling. Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.