DNA damage induced during mitosis undergoes DNA repair synthesis.

Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNA-damaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase.

Dynamic optical contrast imaging as a novel modality for rapidly distinguishing head and neck squamous cell carcinoma from surrounding normal tissue.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) are debilitating diseases for which a patient's prognosis depends heavily on complete tumor resection. Currently, the surgeon's fingers determine the location of tissue margins. This study evaluated the diagnostic utility of a novel imaging modality, dynamic optical contrast imaging (DOCI), in the detection of HNSCC. This system generates contrast by illuminating the tissue with pulsed light and detecting variations in endogenous fluorophore lifetimes. METHODS: A total of 47 fresh ex vivo samples from 15 patients were imaged with the DOCI system immediately after surgical resection. DOCI maps were analyzed to determine the statistical significance of contrast between tumors and adjacent nonmalignant tissue. Pilot intraoperative clinical data were also acquired. RESULTS: Statistical significance (P < .05) between muscle and tumor was established for 10 of 10 emission wavelengths, between collagen and tumor for 8 of 10 emission wavelengths, and between fat and tumor for 2 of 10 wavelengths. The system extracted relative fluorescence decay information in a surgically relevant field of view in <2 minutes. CONCLUSIONS: This study demonstrates the feasibility of using DOCI to rapidly and accurately distinguish HNSCC from surrounding normal tissue. An analysis of DOCI images revealed microscopic characterization sufficient for tissue-type identification consistent with histology. Such an intraoperative tool would be transformative by allowing the rapid delineation of tumor tissue from nontumor tissue and thus maximizing the efficacy of resection and improving patient outcomes. Cancer 2017;123:879-86. © 2016 American Cancer Society.

Assessment of Apical Extrusion during Root Canal Irrigation with the Novel GentleWave System in a Simulated Apical Environment.

INTRODUCTION: This study assessed apical extrusion during treatment with GentleWave (GW; Sonendo Inc, Laguna Hills, CA), a conventional open-ended 30-G needle (CN), or Endovac (EV; SybronEndo, Orange, CA) in root canals enlarged to different dimensions with and without apical constriction. METHODS: Sixteen mandibular molars were mounted in an in vitro apparatus. Roots were immersed in a pressure-regulated chamber containing distilled water with pressure kept at 5.88 ± 0.15 mm Hg to simulate periapical back pressure. Mesiobuccal (curved ≤30°) and distal (straight) canals were instrumented to the working length (WL) as follows: minimal instrumentation (MI, #15/.04), traditional instrumentation (#35/.06), or overinstrumentation (OI, #35/.06, to the WL + 1 mm). Canals were tested 5 times each with distilled water using GW, CN (at WL-3 mm), or EV and the mass (g) of extruded water recorded. Extrusion frequency and mean extruded mass were compared for each canal, irrigation group, and canal instrumentation mode (Wilcoxon t test, P < .05). RESULTS: No extrusion occurred with GW and EV, whereas the frequency of extrusion with CN was 33%. Mean extruded water mass using CN ranged in mesial canals from 0.000 ± 0.000 g (OI) to 0.047 ± 0.098 g (MI) and in distal canals from 0.123 ± 0.191 g (MI) to 0.505 ± 0.490 g (OI). With traditional instrumentation and OI instrumentation, extruded mass in distal canals was significantly higher than in mesial canals (P < .002) and distal canals with MI (P < .020). CONCLUSIONS: Within this study's limitations, root canal treatment with GW and irrigation with EV was not associated with extrusion. Extruded irrigation mass using the open-ended 30-G needle depended on the canal type and enlargement. These results have to be interpreted with caution, and further investigations are warranted to evaluate the possibility of extrusion using GW in different tooth types and clinical situations.

Optical imaging for brain tissue characterization using relative fluorescence lifetime imaging.

An autofluorescence lifetime wide-field imaging system that can generate contrast in underlying tissue structures of normal and malignant brain tissue samples with video rate acquisition and processing time is presented. Images of the investigated tissues were acquired with high resolution (∼35 µm) using an algorithm to produce contrast based on differences in relative lifetimes. Sufficient contrast for delineation was produced without the computation of fluorescence decay times or Laguerre coefficients. The imaged tissues were sent for histological analysis that confirmed the detected imaged tissues morphological findings and correlations between relative lifetime maps and histology identified.

Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation.

In this study the femtosecond near-IR and nanosecond green lasers are used to induce alterations in mitotic chromosomes. The subsequent double-strand break responses are studied. We show that both lasers are capable of creating comparable chromosomal alterations and that a phase paling observed within 1-2 s of laser exposure is associated with an alteration of chromatin as confirmed by serial section electron microscopy, DAPI, γH2AX and phospho-H3 staining. Additionally, the accumulation of dark material observed using phase contrast light microscopy (indicative of a change in refractive index of the chromatin) ∼ 34 s post-laser exposure corresponds spatially to the accumulation of Nbs1, Ku and ubiquitin. This study demonstrates that chromosomes selectively altered in mitosis initiate the DNA damage response within 30 s and that the accumulation of proteins are visually represented by phase-dark material at the irradiation site, allowing us to determine the fate of the damage as cells enter G1. These results occur with two widely different laser systems, making this approach to study DNA damage responses in the mitotic phase generally available to many different labs. Additionally, we present a summary of most of the published laser studies on chromosomes in order to provide a general guide of the lasers and operating parameters used by other laboratories.